Welcome to JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION)

2020 Vol. 51, No. 6

Advances in Oral Science
Abstract:
  Obejective   To investigate the role of a novel chemically defined medium (CDM) in the regulation of dental papilla cells (DPCs) functional phenotype in vitro and periodontal bone regeneration in vivo.  Methods  DPCs were isolated and cultured in conventional medium (CM) or CDM. The surface makers, and the proliferation, migration and osteogenic differentiation abilities of DPCs were evaluated. In vivo, the DPCs that mixed with collagen gel were implanted into the model rats in the defect of periodontal to repair the periodontal tissue. Regeneration of the tissues was examined by microcomputed tomography and histological observation.  Results  DPCs in the CM group and CDM group showed similar surface markers. Compared to the CM group, the CDM significantly enhanced the proliferation, colony-forming efficiency and migration of DPCs in vitro. In addition, real time PCR showed that the expression levels of osteogenesis-related genes, Runx2, Alp and Opn. were significantly enhanced in DPCs in the CDM group. DPCs cells treated with CDM also exhibited higher alkaline phosphatase activity and stronger ability of formation of mineralized nodules in vitro. In vivo, DPCs from CDM group significantly enhanced the periodontal bone regeneration and the reconstruction of periodontal bone tissues in rat periodontal defect model.  Conclusion  CDM is a suitable medium to culture DPCs for periodontal bone regeneration. This research provided a substitute for basic research and set the stage for future clinical application of stem cell transplantation.
Abstract:
  Objective   To investigate the effect of Candida albicans (C. albicans) on proliferation and virulence of Streptococcus mutans (S. mutans), and to verify the role of the ergosterol pathway in it.  Methods  After single- and co-cultivation of C. albicans wild-type 5314 and S. mutans UA159, the absorbance value of OD600 and colony-forming units (CFU) were detected to reflect the influence of C. albicans on the growth of S. mutans. To reflect the influence of C. albicans on the virulence of S. mutans, the production of extracellular polysaccharides was detected by anthrone-vitriol method, and the acid production capacity was detected by a pH meter. After single- and co-cultivation of C. albicans mutant strains and S. mutans, the growth of S. mutans was evaluated by CFU. After inhibiting the ergosterol pathway by 0 mg/L, 0.012 5 mg/L, and 0.025 mg/L fluconazole for 24 h, the CFU of single- and co-cultivated C. albicans wild-type and S. mutans were detected.  Results  The OD600 absorbance value and CFU in theco-cultivation of C. albicans wild-type and S. mutans was higher than that in the single culture (P<0.05), and the production of extracellular polysaccharides in S. mutans was increased when S. mutans was co-cultured with C. albicans (P<0.05), accompanied with a more obvious decrease of pH (P<0.05). Fourteen strains in whole 42 C. albicans mutant strains lost the growth-promoting effect on S. mutans, including 6 ergosterol synthesis-related mutant strains. After co-cultivation of the 6 ergosterol synthesis-related mutant strains and S. mutans, the CFU of S. mutans remained unchanged or decreased. After inhibiting the ergosterol pathway by 0.012 5 mg/L and 0.025 mg/L fluconazole, the CFU of S. mutans in the co-cultivation of C. albicans wild-type and S. mutans was lower than that without fluconazole treatment (P<0.05), while the CFU of C. albicans and S. mutans single-cultivations did not change significantly (P>0.05) and the CFU of C. albicans in the co-cultivation of C. albicans wild-type and S. mutans did not change significantly (P>0.05).  Conclusion  C.albicans can enhance the growth ability and virulence of S. mutans through the ergosterol-related pathway. This process can be inhibited by fluconazole, which is expected to become a novel strategy to prevent and treat dental caries.
Abstract:
  Objective  To study the antibacterial effect of berberine combined with amylmetacresol on Enterococcus faecalis.  Methods  Both dilution method and live bacteria CFU were used to determine the minimum inhibitory concentration (MIC) of berberine and amylmetacresol on E. faecalis. The killing effect of berberine and amylmetacresol on planktonic E. faecalis was detected by suspension quantitative germicidal test and live/dead bacteria staining. The effects of berberine and amylmetacresol on the structure of mature biofilm of E. faecalis was observed by scanning electron microscopy (SEM). The toxicity of berberine and amylmetacresol on human oral keratinocytes (HOK) was determined by CCK-8 cell proliferation and cytotoxicity assay and cytotoxicity LDH assay.  Results  The MIC of berberine was 512 μg/mL, and the MIC of amylmetacresol was 0.023 3%. 512 μg/mL berberine and 0.002 33% amylmetacresol had a weak killing effect on planktonic E. faecalis alone, while they showed a synergistic antibacterial effect in combination. Cell survival in the biofilm was only slightly changed by berberine and amylmetacresol. The structure of biofilm was obviously changed by berberine and amylmetacresol. 512 μg/mL berberine and 0.002 33% amylmetacresol alone or in combination showed the survival rate was much higher than the injury rate, suggesting berberine and amylmetacresol had a low cytotoxicity.  Conclusion  Berberine and amylmetacresol had synergism against E. faecalis, and the biological safety of the combination use was better.
Abstract:
  Objective  This study aimed to investigate the expression level of protein kinase D (PKD) in oral squamous cell carcinoma (OSCC) and its relationship with differentiation of OSCC.  Methods  Sample was collected from 10 healthy control subjects and 40 OSCC confirmed by histopathological diagnosis, and the immunohistochemical staining was adopted to detect the expression of PKDs in OSCC tissues. The proportion of stained cell and staining intensity were evaluated to get a score, which used to analyze the difference among PKD1, PKD2 and PKD3 in various differentiation OSCC tissues. The correlations between the staining score of PKDs and differentiation of OSCC were further analyzed.  Results  PKD1 and PKD3 were high expression in OSCC tissues. There were statistical significance among the staining score of PKD1, PKD2 and PKD3 in various differentiation OSCC tissues (P<0.001). In addition, there was a significantly negative correlation between the staining score of PKD1 and PKD2 with the differentiation of OSCC (r=−0.574, −0.341, P<0.001).  Conclusion  In OSCC tissues with different degree of differentiation, there might be some differences among PKDs which play a major role. The expression of PKD1 and PKD2 was correlated with the differentiation of OSCC, the poor differentiation of OSCC, the higher expression of PKD1 and PKD2.
Abstract:
The oral environment provides suitable conditions for the colonization of various microorganisms. However, the oral microbials could be the initial factors of some kinds of oral infectious diseases, therefore the treatment against oral microbial pathogens has become an effective strategy. Artemisinin, a kind of sesquiterpene lactone extracted from Traditional Chinese Medicine Artemisia annua L, is the first-line therapy to treat tertian malaria, subtertian malaria and anti-chloroquine malaria for its high efficiency and low toxicity. In recent years, artemisinin and its derivatives have also been proven to be effective against bacteria, fungi, viruses, parasites, and tumors, some of which are closely related to oral diseases. In this review, we summarize the potential effects of artemisinin and its derivatives on oral microorganism by analyzing previous research and latest progress to provide the evidence for further improvement, and look forward to the new research directions. Further studies are needed to improve existing technologies and standards to clarify the effects of artemisinin and its derivatives on microorganisms with controversial effects, to expand the detection of microorganisms associated with oral infectious diseases, and to clarify the interaction with existing antifungal agents in the field of antifungal diseases. In addition, in the study of anti-oral infectious diseases, artemisinin and its derivatives’ administration scheme, potential drug interactions, toxic and side effects and other aspects are necessary conditions for further research, which is also a new direction of research. With the maturity of the production process, the improvement of relevant research and the potential demand for the treatment of oral infectious diseases, artemisinin and its derivatives have a broad prospect in the field of oral microorganisms, and provide a new opportunity for the research and development of oral drugs.
Abstract:
Peri-implantitis is the most common and intractable complication of dental implant-supported prothesis affecting its long-term success, and is one of the main reasons for implant failure. Due to the limitation of the research methods, the pathogenesis and pathological processes of peri-implantitis remain unclear. Animal models are indispensable tools to study the pathogenesis of diseases. With the advances of the dental implants, the peri-implantitis mouse model has been used in experimental research. This paper summarized recent studies from the following five aspects: the advantages of the mouse model, the influence of mouse strain, the design of micro-implant, the way of implant insertion, as well as the induction of peri-implantitis, aiming to provide references and help for researchers. Compared with the large animal models of peri-implantitis, the mouse model of peri-implantitis is more flexible in use. Lower costs can better control the sample number and shorter induction time can better control experimental duration. The completion of mouse genome sequencing and the progress of the genetic operating system also make the pathogenetic study possible. However, the mouse model of peri-implantitis still has some limitations. Limited by the small size of mouse oral cavity, implant insertion surgery is technically demanding, and complex surgeries are even more challenging. Moreover, due to short history of the peri-implantitis mouse model, its corresponding technical theories such as implantation methods, peri-implantitis induction methods and so on are not unified yet and still need further research and development.
Abstract:
Connexins and Pannexins play important roles in osteocytes and osteoblasts differentiation, intracellular signal transduction, maintenance of bone balance, and bone regeneration. This article reviews the progress and limitations of Connexins-mediated gap junctions and Pannexins mediated hemichannel in bone. Current research has shown that these molecules, in the form of gap junctions or separate hemichannels, deliver external stimuli to the skeletal system. However, little is known about the role of other cell types in bone development and homeostasis, such as pre-osteoblasts and bone marrow mesenchymal stem cells, in maintaining normality. In addition, at present, the most well-studied member of the Connexins family is Connexin43 (Cx43), while the roles and mechanisms of other members in bone development are still behind the veil. Gene-edited animal models provide basic information on the role of Connexins and Pannexins in the skeletal system, but the similarities and differences between Connexins and Pannexins remain to be discovered. Targeting a specific function of Connexins or Pannexins for bone stimulation and bone disease remains a challenge, with pharmacological selective overlap between channels, compensation of other subtypes, differences in methods for assessing channel function, and genetic changes associated with transgenic mouse models. Therefore, better tools and research pathways are needed to understand the role of these pathways in bone and cartilage. An essential task for future research will be to identify specific compounds that regulate Connexins or Pannexins subtypes to enable them to be used as pharmaceutical agents in the treatment of bone diseases, providing the possibility to develop new therapeutic strategies for improving bone health and treating diseases of the skeletal system.
Abstract:
Osteogenesis of mesenchymal stem cells to differentiate between bone marrow by multiple signaling pathways that control, directly or indirectly affect small related transcription factor 2 (runt-related transcription factor 2, Runx2) and osteoblast specific transcription factor (osterix, Osx), the expression of osteogenesis key transcription factors, such as in the development and regeneration of the bone, bone repair has played a key role in the process of reconstruction. These pathways play their mechanism of action, but also intertwined associated constitute a complex signal transduction network, but due to the limitations of research methods, the osteogenic differentiation related signaling pathways of the specific mechanism is still unclear, if you can clarify these different signaling pathways play to the role of their relevant mechanism and the relationship between various pathways and the mechanism study of osteogenesis differentiation is of great importance. This article will review the progress of various signaling pathways in the regulation of osteogenic differentiation of bone marrow mesenchymal stem cells.
Abstract:
  Objective  The effect of Smad7 on epithelial-Mesenchymal Transition (EMT) of keloid keratinocytes was studied.  Methods  Culture formed keloid cutin cells (KK) and normal skin cutin cell (NK cells), built the Smad7 too slow virus slow virus vector and Smad7 interference expression vector, screening the best expression and interfering with the slow virus infection NK and KK cells respectively, and contrast carrier puro screening stable expression cell lines, stem cells can be divided into 8 groups: NK-Control (normal training of NK cells); NK-NC (NK cells screened against lentivirus); NK-shSmad7 (NK cells that interfere with lentivirus screening); NK-mSmad7 (NK cells screened for overexpression of lentivirus); KK-control (normal cultured KK cells); KK-NC (KK cells screened against lentivirus); KK-shSmad7 (KK cells that interfere with lentivirus screening); KK-mSmad7 (KK cells screened for overexpression of lentivirus). Cell proliferation was observed by the CCK-8 method, cell apoptosis was detected by flow cytometry, cell migration ability was detected by Transwell chamber, and expression of key proteins (N-cadherin and Occludin) in epithelium-interstitial transform was detected by Western blot.  Results  The Smad7 interfering lentivirus vector and Smad7 overexpressing lentivirus vector were successfully constructed. Interference with Smad7 can promote NK cell and KK cell proliferation and migration, and inhibit KK cell apoptosis, but it has no significant effect on NK cell apoptosis (P>0.05). Overexpression of Smad7 inhibited the proliferation and migration of NK cells and KK cells, and promoted their apoptosis. After interfering with lentivirus infection, NK cells and KK cells showed decreased expression of Occludin protein compared with NC group (P<0.01), increased N-cadherin protein expression in KK cells (P<0.01), but there was no significant change in N-cadherin protein expression in NK cells (P>0.05); After lentivirus overexpression, NK and KK cells showed increased expression of Occludin protein (P<0.05), the expression of N-cadherin protein in NK cells decreased (P<0.05), but there was no significant change in N-cadherin protein expression in KK cells (P>0.05).  Conclusion  The regulation of Smad7 gene can affect the EMT in normal skin keratinocytes and keloid keratinocytes, and further regulate the ability of cell proliferation, migration and apoptosis. The effect of Smad7 gene regulation on EMT in keloid keratinocytes was greater than that on normal skin keratinocytes.
Abstract:
  Objective  To explore the role of Kruppel-like factor 4 (KLF4) in the regulation of Keratin 17 (KRT17) expression, and to reveal the molecular mechanism of overexpression of KRT17 in psoriatic lesions.  Methods  The skin lesions of 18 patients with psoriasis vulgaris were taken as experimental group and 10 healthy persons as control group. Real time-PCR and Western blot were used to detect the expression of KLF4 in psoriasis and normal skin samples, and the changes of KRT17 expression in HaCat cells after transfection of KLF4 overexpression and EP300 interfering plasmid. ChIP-qPCR was used to detect KLF4 binding and histone H3 acetylation levels in the promoter region of KRT17 in psoriasis and normal skin samples, and the changes of KLF4 binding and histone H3 acetylation levels in the promoter region of KRT17 in HaCat cells after transfection of KLF4 overexpression and EP300 interfering plasmid. Co-IP detects the interaction between KLF4 and EP300.  Results  The expression level of KLF4, KLF4 binding level and histone H3 acetylation level in the promoter region of KRT17 in psoriasis group were significantly higher than those in normal group (P<0.01). Compared with the control group, the expression level of KRT17 was significantly higher after KLF4 overexpression (P<0.01). After KLF4 overexpression combined with EP300 interference, the expression level of KRT17 was significantly lower than that of KLF4 overexpression group (P<0.01), slightly lower than that of control group (P<0.05). Compared with the control group, the histone H3 acetylation level in KRT17 promoter region in KLF4 over-expression group was increased significantly (P<0.01). After KLF4 over-expression combined with EP300 interference, the acetylation level of histone H3 in KRT17 promoter region was significantly lower than that in KLF4 overexpression group (P<0.01) and control group (P<0.01). Co-IP confirmed that KLF4 and EP300 could form protein complexes.  Conclusion  Excessive KLF4 increases the level of histone H3 acetylation in KRT17 promoter region by synergistic EP300, and mediates the over-expression of KRT17 in psoriatic lesions.
Abstract:
  Objective  To investigate the effects of atorvastatin calcium (ATR) on the survival of ultra-long dorsal random skin flaps in rats.  Methods  Thirty SD rats were divided into five groups (n=6) according to the random number table: normal saline control group (CON group), ATR 10 mg/kg group (P10 group), ATR 20 mg/kg group (P20 group), ATR 30 mg/kg group (P30 group), and ATR 40 mg/kg group (P40 group). After pretreatment with ATR or 0.9% saline for 3 days, an ultra-long dorsal random skin flaps with size of 8 cm×2 cm was made on the back of each rat and replanted in situ. After the operation, the ATR or saline treatment lasted for 3 d. Seven days after operation, the appearance of skin flaps was observed with naked eyes, the survival rate of skin flaps was calculated. The pathological changes in the surviving areas of skin flaps were observed by HE staining, the number of microvessels in skin flaps was observed by immunohistochemistry staining, the mRNA expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were tested by quantitative real-time PCR, and the contents of superoxide dismutase, nitrogen monoxide and malonaldehyde were tested by enzyme linked immunosorbent assays (ELISA).  Results  On the 7th day after operation, the skin flap of the CON group showed a large area of necrosis, and the necrotic part formed crusts. Crusts were hard and inelastic, and a large amount of tissue fluid exudated. The fascial layer showed dark purple. No exudation of tissue fluid was observed in the flaps of P10, P20, P30 and P40 groups. The scab shell fell off naturally and the fur grew normally. HE staining of CON group showed that a large number of inflammatory cell infiltration, epidermal loss and necrosis in skin flaps, but the pathological changes in skin flaps were significantly improved after treatment with ATR. Compared with the CON group, the survival rate of skin flaps, the number of microvessels in skin flaps and the levels of VEGF mRNA, bFGF mRNA, SOD, NO in skin flaps also increased with the dose of ATR, which reached a peak at 30 mg/kg ATR (P<0.05). However, the level of MDA in skin flaps decreased with the dose of ATR, which reached the lowest at 30 mg/kg ATR (P<0.05).  Conclusion  ATR can enhance the survival of ultra-long dorsal random skin flaps in rats, which may be related to promoting microangiogenesis and inhibiting inflammatory and oxidative stress.
Abstract:
  Objective  This study was designed to show the time-dependent changes in the expression profiles of long non-coding RNAs (lncRNAs) in rat vein grafts by using the high-throughput sequencing technique, and subsequently figure out lncRNAs related to vein graft failure.  Methods  The whole SD rats were randomly classified into four groups according to different sampling time points (0, 7, 14 and 28 days after surgery, respectively; n=3 per group). The day 0 group was set as a control, and the other three groups were set as experimental groups (the model of external jugular vein-carotid artery bypass was prepared in the experimental group). We identified the differentially expressed lncRNAs of the vein graft sample at different sampling time points with high-throughput sequencing, and verified these results using the technique of real-time quantitative PCR (RT-qPCR). Meanwhile, we used Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to screen lncRNAs which may play roles in the restenosis process of vein grafts. The function of lncRNA-mRNA pairs was predicted. We subsequently used RT-qPCR to verify these lncRNAs.  Results  A total of 2 572 sustained differentially expressed lncRNAs were identified in our study. We showed the top ten differentially expressed lncRNAs at each post-operative time point. Through GO analysis and KEGG pathway analysis, we revealed the sustained differentially expressed genes which may be involved in VGF-related biological process, cellular component, molecular function and biological pathways. Finally, we screened 15 pairs of lncRNA-mRNA, including MRAK083052-Nrp1, which may play roles in mediating the restenosis of vein graft. And the results of RT-qPCR were consistent with the results of the high-throughput sequencing.  Conclusion  The present study investigated the time-dependent changes in the expression profiles of lncRNAs in vein grafts. We also screened 15 pairs of lncRNA-mRNA which may paly roles in vein graft failure.
Abstract:
  Objective  To investigate the expression of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) in the ovarian tissue of polycystic ovary syndrome (PCOS) rat model and obese PCOS rat model, and the possible mechanism of PCOS.  Methods  Thirty SD rats were randomly divided into control group, PCOS rat model group and obese PCOS rat model group. DHEA dissolved in 0.2 mL soybean oil was injected daily into the rats of two PCOS groups for 21 d. Rats in obese PCOS model group were added with high-fat diet based on DHEA modeling, and each group had 10 rats. Body mass were measured before and on the 22nd day after modeling. The serum testosterone (T) levels were measured by abdominal aortic blood, and the ovarian tissues of rats were taken for histological changes were observed by HE staining, immunohistochemical staining and Western blot to detect PGC-1ɑ protein expression.  Results  The body mass of rats in each group increased after modeling, and the body mass of rats in PCOS group and obese PCOS group increased significantly (P<0.05). The serum T concentration of two PCOS model groups was higher than that of control group (P<0.01). The serum T concentration in obese PCOS model group was higher than that in the PCOS group (P<0.05). The results of HE staining of rat ovarian tissue showed that there were follicles and a small amount of corpus luteum at different developmental stages in the control group, and the granulosa cells were arranged in 4-6 layers. The number of immature small follicles in the two PCOS groups was significantly increased. The granulosa cells were arranged in 1-3 layers, relatively looser, and some follicles were atresia. In the obese PCOS group, the diameter of ovarian atretic follicles increased, the number of granulocyte layers decreased, and oocytes disappeared more obviously. Immunohistochemical staining showed that the PGC-1ɑ protein was mainly expressed in the cumulus and granulosa cells of ovarian tissue in the control group. The mean gray level of PGC-1ɑ protein expression in PCOS group (0.53±0.06) and obese PCOS group (0.36±0.03) was lower than that of the control group (0.75±0.03), with the statistical difference (P<0.05). PGC-1ɑ expression in the obese PCOS group was lower than that in the PCOS group (P<0.01). The results of Western blot were consistent with those of immunohistochemical staining.   Conclusion  PGC-1ɑ is associated with damage of ovarian granulosa cells in high-fat environment. The decrease of PGC-1ɑ expression in granulosa cells of ovarian follicles may be an important cause of PCOS development.
Abstract:
  Objective  To study dietary patterns during the second trimester of pregnancy and to investigate the relationship between dietary patterns and gestational weight gain (GWG).  Methods  A prospective cohort study was conducted to select healthy singleton pregnant women at 8-14 weeks of gestation in a maternal and child health care institution in Chengdu city. Food items and quantities were collected at 8-14, 24-28, 32-36 weeks of gestation by using the 3-day 24-hour dietary recall and energy intakes were calculated. Dietary patterns during the second trimester were established by factor analysis and factor scores were calculated. The weight of pregnant women was measured at 8-14, 24-28 weeks of gestation and 1 week before delivery, and the total GWG and the GWG rates in the second and third trimesters were calculated. Multiple linear regression analyses were used to analyze the association between dietary patterns and GWG.  Results  A total of 1 004 samples were included. Three dietary patterns were identified: Milk-egg-whole grain pattern, Beverage-dessert pattern and Traditional pattern. The average total GWG was (13.2±4.5) kg. The average weight gain rate was (0.4±0.2) kg/week in the second trimester. The average weight gain rate was (0.5±0.3) kg/week in the third trimester. After adjusting for confounding factors including maternal age, body mass index before pregnancy, dietary energy intake, physical activity, multiple linear regression analysis showed that the factor score of Beverage-dessert pattern was positively associated with the total GWG and the weight gain rate in the third trimester (β=0.370, 95% confidence interval (CI): (0.103, 0.636), P=0.007; β=0.014, 95%CI: (0.000, 0.027), P=0.049, respectively), and the factor score of Traditional pattern was negatively associated with the total GWG (β=−0.285, 95%CI: (−0.555, −0.015), P=0.039). There was no association between the Milk-egg-whole grain pattern and GWG.  Conclusion  Dietary patterns during the second trimester of pregnancy are associated with GWG. The Beverage-dessert pattern may increase the total GWG and weight gain rate in the third trimester. The traditional pattern may help control the total GWG.
Abstract:
  Objective  In order to provide guidance for clinical use of four-dimensional cone-beam CT (4D CBCT), the accuracy of image registration and its influencing factors were analyzed using the automatic registration method when 4D CBCT was used as an image guidance strategy for patients with chest tumors.  Methods  The respiratory motion model and two kinds of lung plug-ins were used to simulate two types of tumors and their movements in the chest. 4D CT was scanned for each kind of simulated tumor, and 4D CBCT was scanned under various artificial positioning errors. For the registration of 4D CBCT, the manual and automatic registration methods were used for each group.  Results  There were more obvious mismatches in the intrapulmonary adhesion tumor group. When the masks were created based on the size of the target area or expanding the target area by 0.5 cm, the results between the automatic registration and manual registration were statistically different. There were no significant mismatches in the isolated lung tumor group, and there was no statistical difference between the results of automatic registration and manual registration.  Conclusions  When 4D CBCT is used as an image guidance strategy for patients with chest tumors, the automatic registration procedure should not be used for tumors adhering to chest wall and mediastinum. For solitary lung tumors, the automatic registration method and the manual registration method have similar registration accuracy, but significant mismatches need to be excluded.
Abstract:
  Objective  To investigate the feasibility of measuring pulmonary blood volume (PBV) by ultrasound microbubble angiography, which may provide a feasible method for further detection of PBV changes.  Methods  Ultrasound microbubble angiography was used to calculate the PBV by detecting pulmonary transit time (PTT) and heart rate-normalized pulmonary transit time (nPTT). To evaluate the consensus degree based on the intra-, inter-observer and within-day variation in order to determine the repeatability. The method was used for acute left ventricular dysfunction models to determine the feasibility as well.  Results  The Bland-Atlman plots showed good intra-observer, within-day, and inter-observer consistency of measurement results. Application in acute left ventricular dysfunction models showed that, compared with the control, left heart failure models had higher PTT, nPTT and PBV (P<0.05).  Conclusion  Detection of PTT/nPTT to calculate PBV by ultrasound microbubble angiography is simple and feasible, it is not easy to produce miscarriage of justice, with good intra- and inter-observer consistency of repeatability test results. The method has certain feasibility.
Abstract:
  Objective  To compare the application of fluorescein videoangiography (FL-VA) and indocyanine green videoangiography (ICG-VA) in intracranial aneurysm surgery.  Methods  A total of 65 patients who underwent aneurysm clipping in our hospital from January 2019 to January 2020 were included in the study. FL-VA and ICG-VA were used during the surgery to determine whether the aneurysm is completely clipped and the artery bearing the aneurysm and the perforating artery around the aneurysm are unobstructed.  Results  All 65 patients underwent both FL-VA and ICG-VA intraoperatively after aneurysm clipping. FL-VA was applied first. In 30 cases, FL-VA and ICG-VA provided the same results. In 10 cases, FL-VA performed obviously better over ICG-VA in visualizing small perforating arteries (2 cases of internal carotid artery-posterior communicating artery aneurysms and 3 cases of anterior communicating artery aneurysm) and evaluating whether the aneurysm was completely clipped (3 cases of middle cerebral artery aneurysm, 1 case of internal carotid artery-posterior communicating artery aneurysms and 1 case of distal anterior cerebral artery aneurysm). In the remaining 25 cases, ICG-VA was repeatedly applied in a short period of time due to quick clearance of indocyanine green from the blood vessels, but this couldn’t be done with FL-VA.  Conclusions   Compared with ICG-VA, FL-VA can provide better visualization of perforating artery, and can determine whether the aneurysm was completely clipped more accurately. However FL-VA couldn’t be repeatedly applied during a short period of time.
Abstract:
  Objective  To observe the postoperative outcomes of the scleral fixation of posterior chamber intraocular lens (SF-PCIOL) using the modified Yamane’s technique with the aid of swept-source optical coherence tomography (SS-OCT).  Methods  Prospective observational case series. This study involved 20 patients who underwent SF-PCIOL with the modified Yamane’s technique, from December 2017 to November 2019. All patients had routine preoperative examinations, including biometric measurement by IOL master, measurements of uncorrected distance visual acuity (UDVA) and best corrected distance visual acuity (BCDVA). The SRK/T formula was used to calculate the power of intraocular lens (IOL). After the surgery, UDVA and intraocular pressure were evaluated for 1 d, UDVA, BCDVA, spherical equivalent (SE) and corneal endothelial cell density were measured for 1 week, 1 month, 3 and 6 months, respectively. The IOL tilt and the symmetry of hepatitis in the scleral tunnel were measured by SS-OCT.  Results  The mean follow-up duration was (7.20±6.56) months (range, 3-26 months). The mean preoperative UDVA was (1.70±0.38) LogMAR, and it improved to (0.48±0.50) LogMAR (P=0.001). There was no statistically significant difference between the pre- and post-operative BCDVA, i.e. (0.44±0.50) LogMAR and (0.32±0.48) LogMAR, respectively (P=0.08). The mean spherical equivalent was (−0.53±0.86) diopter (D) and the postoperative refractive error was (0.27±0.82) D. Seventeen patients underwent SS-OCT examinations. The mean IOL tilt was (3.28±3.00)°. There was no significant difference between the horizontal and vertical tilt (P=0.326). The IOL tilt did not show a significant correlation with spherical and cylindrical refractive error (P=0.532, P=0.241). There was no statistically significant difference in the HL (the length of haptics fixed in the scleral tunnel) of nasal and temporal haptic, which were (2.24±0.20) mm and (2.17±0.23) mm, respectively (P=0.193). And there were no statistically significant differences between the HD (the distance between the center of haptic flange and scleral spur) of nasal and temporal haptic, (1.58±0.07) mm and (1.66±0.08) mm, respectively (P=0.338). The changes of IOL haptics in the scleral tunnel were tracked by 10 patients. The HL (nasal: HL-N; temporal: HL-T) and the HD (nasal: HD-N; temporal: HD-T) of haptics in the tunnel were measured and recorded at three time points, including 1 week, 1 and 3 months after surgery. There was no significant difference in HL-N, HL-T, HD-N and HD-T at the three time points (P=0.931, P=0.091, P=0.175, and P=0.505, respectively). All patients underwent uneventful surgery. The postoperative complications included transient corneal edema in 6 eyes, transient IOP elevation in 3 eyes, vitreous hemorrhage in 1 eye, cystoid macular edema in 2 eyes, and macular hole in 1 eye.  Conclusion  The SF-PCIOL using modified Yamane’s technique, is capable of producing satisfactory and consistent postoperative outcomes for patients with few postoperative complications. SS-OCT is a powerful tool for measuring optic tilt and the IOL hepatic symmetry in scleral tunnel.
Abstract:
  Objective  To analyze the clinicopathological characteristics and prognostic factors of multifocal lung cancer (MFLC) patients.  Methods  From January 2012 to January 2018, 187 MFLC patients whose largest lesion diameter was ≤4 cm and without lymphatic involvement or systemic metastases, were retrospectively reviewed. All the patients received surgical treatment. The Kaplan-Meier method was used for survival analysis, and a multivariable Cox proportional hazards regression model was used to assess the independent prognostic factors.  Results  Among 187 cases, 173 were simultaneous MFLC (SMFLC) and 14 were metachronous MFLC (MMFLC). The 5-year disease-free survival (DFS) and overall survival (OS) rates of this group MFLC patients were 63.5% and 89.1%, respectively. In the SMFLC group, according to the American College of Chest Physicians (ACCP) guidelines (3rd edition), 133 patients were defined as synchronous multiple primary lung cancer (SMPLC) while 40 patients had intrapulmonary metastases, there was no statistical difference in DFS between the two subgroups (P=0.531). EGFR mutation status (same mutations, different mutations, all wild-type) had no statistically significant effect on DFS of SMFLC (P=0.388). Univariate and multivariate regression analysis revealed that radiographic feature of solid nodules (hazard ratio (HR)=7.4, P=0.008) and MMFLC (HR=5.6, P=0.001) were independent risk factors for poor prognosis.  Conclusion  MFLC can achieve a favorable prognosis with early surgical treatment. Tumor density and metachronous lesions are two important prognostic predictors.
Abstract:
  Objective  To retrospectively analyze the symptoms and characteristics of nervous system damage in severe/critically severe patients with coronavirus disease 2019 (COVID-19) in Sichuan province, with a view to providing basic references for the prevention and treatment of COVID-19.  Methods  A total of 90 patients with severe/critically severe COVID-19 were included, who were diagnosed and treated in COVID-19 designated hospital of Sichuan province from 11 January 2020 to 20 March 2020. Clinical features, test results, treatment options and clinical outcomes were analyzed retrospectively.  Results  Of 90 patients, there were 54 males and 36 females, with an average age of (53.90±16.92) years. In addition to the classic symptoms such as fever and/or respiratory symptoms, 53 patients also had various degrees of neurologic manifestations, including 33 cases of fatigue, 21 muscle soreness, 12 dizziness, 8 headaches, 3 mental disorders, and 1 consciousness disorders and 1 case of neck pain. Compared with the patients without neurologic manifestations, those with neurologic manifestations took a longer time from admission to diagnosis of COVID-19 (P<0.05), and received more antifungal treatment (P<0.05).  Conclusions  Neurological symptoms are not uncommon in severe/critically severe patients with COVID-19, and it’s relatively difficult in the treatment. It should be paid attention in order to avoid misdiagnosis.
Abstract:
A 41 years old female patient experienced enlarged right nipple more than 6 years ago. Physical examination showed enlarged and hardened right nipple (about 2 cm) but no tenderness. Mammographic results showed the mixed calcification pattern, which was presenting multiple sizes of circular and granular calcification of the nipple areola complex. Ultrasound revealed large calcification in the nipple areola area with posterior acoustic shadow. This patient underwent extensive excision of the nipple areola complex. The surgical margin was confirmed to be negative. Postoperative pathology confirmed an infiltrating syringomatous adenoma with ossification in some areas. Tumor cell infiltration was not found at the surgical margin. After 4 years of follow-up, no recurrence was found. The mammography and ultrasound findings of this benign tumor were significantly different from those reported in previous literatures. We should improve the understanding of the variousness of clinical imaging manifestations of this disease.
Abstract:
A 5+-day-old male patient was hospitalized due to a significant increase of urine protein for 5+ d. A 36+4 weeks preterm male infant was found with III° polluted amniotic fluid and excessive placenta, presented with proteinuria, hypoproteinemia, and progressive edema after birth. Two heterozygous mutations of NPHS1 gene, c.3325C>T (p.Arg1109*) and c.2479C>T (p.Arg827*), were found through the whole exon gene detection. The latter has not been reported domestically and the diagnosis of congenitalnephrotic syndrome of the Finnish type (CNF) is definite. The report of c.2479C>T mutation gene will expand the mutation spectrum of CNF gene data in China. Early genetic testing is recommended for cryptogenic congenital nephrotic syndrome (CNS) and early genetic diagnosis of CNF is important for prognostic evaluation, genetic counseling and clinical management.
Original Articles
Abstract:
  Objective  To investigate the influence of the protamine sulfate on endocytosis and intracellular stability of tetrahedral framework nucleic acid (tFNA).  Methods  Articular cartilage cells were collected from 3-day-old C57BL mice. Cells at passage 1-2 were used in the experiments. 4 single-strand DNAs (S1 was marked by Cy5) were utilized to synthesize tFNAs via annealing process and ultrafiltration for purification. High-performance capillary electrophoresis (HPCE) was used to verify synthesis of tFNAs and transmission electron microscope was used to photo morphological characteristics. The 1 mg/mL protamine sulfate solution was slowly dropped into newly synthesized tFNAs (N/P=5/1). Then, Zeta potential was detected. Cells were treated with 100 nmol/L tFNAs with protamine sulfate in Dulbecco’s Modified Eagle’s medium (DMEM) (Exp.1), 100 nmol/L tFNAs in DMEM (Exp.2), and DMEM (Control), respectively. Flow cytometry was used to quantitatively detect intracellular Cy5 fluorescence after 6 h and 12 h treatments. Immunofluorescence staining was used to qualitatively observe internalized Cy5 fluorescence after 12 h treatment by laser confocal microscope. Lysosome of living cells were stained with lysosome probe. Colocalization between lysosome and tFNAs was observed by laser confocal microscope.  Results  After incubating protamine sulfate, negative potential was transformed into positive one ( (−1.567±0.163) mV to (4.700±0.484) mV). The fluorescence intensity of tFNAs in the Exp.1 group was higher than that of the Exp.2 group in 6 h and 12 h (P<0.05). This was consistent with the results of immunofluorescence staining after 12 h. Colocalization of Cy5 fluorescence and lysosome in the Exp.1 group was more rare than that in the Exp.2 group at 6 h and 12 h. Furthermore, a large amount of Cy5 fluorescence was still seen in the Exp.1 group at 12 h, while Cy5 fluorescence of the Exp.2 group was less.  Conclusion  Protamine sulfate can effectively enhance endocytosis, and to some extent it can achieve lysosome escape of tFNAs.
Techniques and Methods
Abstract:
  Objective  To evaluate the application value of CT metal artifact correction technology (MACTM) in CT review after total hip replacement.  Methods   A total of 72 patients who underwent CT re-examination after total hip replacement from December 2018 to March 2020 were enrolled, and the original data were reconstructed by filter backup projection (FBP) and MAC. Select three identical levels in the two sets of reconstructed images and place the same ROI. The selected levels were the initial level, central level, and lower edge of acetabulum. Measure the CT and noise (SD) of metal high and low density artifacts of the three levels area, as well as metal hip joint space, metal para-bone tissue, muscle, bladder and subcutaneous fat, and calculate the average value. Subcutaneous fat value was used as a reference to calculate the SNR and CNR of metal implant para-bone tissue, muscle and bladder. Two radiologists scored the two groups of reconstructed images using blinded method, Kappa’s test was used to compare the homogeneity.  Results  There were differences between the two groups of reconstructed images in high- and low-density artifact areas, joint gap CT values, and image noise. Compared with the FBP group, the CT value of the high-density area and the joint space of the MAC group decreased, the CT value of the low-density area increased, and the noise value of each area decreased. The SNR and CNR of metal adjacent bone tissue, muscle and bladder were higher in the MAC group than those in the FBP group, and the difference was statistically significant (P<0.05). The difference in subjective scores between the two groups was statistically significant (Z=−6.564, P<0.05). 2 radiologists had moderate consistency with Kappa value of 0.72 on FBP group, and good consistency with Kappa value of 0.85 on MAC group.   Conclusion  MACTM in CT review after total hip replacement can reduce metal artifacts, make the joint space more clear, and improve the quality of CT images.
Clinical Medicine
Abstract:
  Objective  To investigate the effect of trunk control training on trunk function, balance, mobility and functional independence in hemiplegia patients after acute stroke, and to compare the therapeutic effects with conventional stroke rehabilitation.  Methods  The study was designed as a randomized controlled trial. The subjects and evaluators were blinded and the therapist was unblinded. Subjects were randomly assigned to either the experimental group or the control group using a computer-generated random number table, the experimental group (n=15) received30 min of high-intensity trunk control training plus 15 min of low-intensity conventional stroke rehabilitation (45 min, once1 d for 5 d). The control group (n=15) received only 45 min low-intensity conventional stroke rehabilitation, once 1 d for 5 d. Trunk function was the primary outcome, which was evaluated by the Trunk Impairment Scale (TIS). The secondary outcome included balance, mobility and functional independence, which were assessed by the Brunel Balance Assessment (BBA), the Modified Rivermead Mobility Index (MRMI) and the modified Barthel Index (MBI). Subjects were assessed before and after the intervention.  Results  After 5 d of treatment, the scores of TIS general table, TIS static sitting balance sub table, TIS coordination sub table, BBA, MRMI and MBI were increased, but there was no statistically significant difference between the two groups (all P>0.05). The trunk control training group improved dynamic sitting balance more significantly, the score of TIS dynamic sitting balance sub table increased more than that in the control group, with a statistically significant difference (P<0.05).  Conclusion  The results of this study indicate that trunk control training has improved trunk function, balance, mobility, and activities of daily living in hemiplegia patients after acute stroke, and trunk control training improves dynamic sitting balance more effectively than conventional stroke rehabilitation.