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YKL-40促进Ⅱ型肺泡上皮细胞A549炎症因子的表达

YKL-40 Promotes the Expression of Inflammatory Factors in Type Ⅱ Alveolar Epithelial Cell Model of A549 Cell Line

    摘要
  • 摘要:
      目的  YKL-40是一种人软骨糖蛋白39或几丁质酶-3样蛋白1，N端由酪氨酸（Y)、赖氨酸（K）、亮氨酸（L）组成，因此被命名为YKL-40。本研究探索YKL-40能否促进Ⅱ型肺泡上皮细胞炎症因子的表达。
      方法  体外培养A549细胞，加入IL-1β（20 ng/mL） 、IL-6（20 ng/mL）、TNF-α（20 ng/mL）、IFN-γ（20 ng/mL），RT-qPCR检测YKL-40 转录水平的表达；A549细胞中加入5、10、20 ng/mL的IL-1β培养，Western blot检测YKL-40蛋白水平的表达。A549细胞中加入0、100、500、1000 ng/mL人源重组YKL-40蛋白培养，RT-qPCR检测IL-6、IL-8的表达水平。设计三对靶向YKL-40小干扰RNA（si-YKL-40-1/2/3）和阴性对照（negative control，NC），分别转染A549细胞，RT-qPCR和Western blot鉴定YKL-40的表达，筛选出si-YKL-40-3用于后续实验。在A549细胞中，转染si-YKL-40-3和si-NC后，加入IL-1β（20 ng/mL）培养，RT-qPCR检测细胞YKL-40、IL-6、IL-8表达，QAH-INF-1试剂盒检测上清液中多种因子的表达。
      结果  RT-qPCR 结果表明，与对照组相比，IL-1β可以上调YKL-40蛋白的转录水平，差异有统计学意义（P<0.01），而IL-6、TNF-α、IFN-γ则不能上调； Western blot结果表明，IL-1β（20 ng/mL）可以显著促进YKL-40的表达，与对照组相比，不同质量浓度IL-1β组差异均有统计学意义（P<0.01）。A549细胞中加入人源重组YKL-40蛋白后，炎症因子IL-6和IL-8的表达明显升高，与对照组相比，差异有统计学意义（P<0.05）；转染si-YKL-40-3降低YKL-40的表达后，与对照组相比，IL-6（P<0.05）、IL-8（P<0.05）等炎症因子表达受到抑制。
      结论  YKL-40能够促进Ⅱ型肺泡上皮细胞A549表达并分泌IL-6、IL-8等急性期炎性因子，从而加重炎症反应。通过靶向抑制YKL-40的表达可有效抑制炎症反应。

     

    Abstract:
      Objective  YKL-40, also known as chitinase-3-like-1 (CHI3L1), is a human cartilage glycoprotein-39, with its N-terminus consisting of tyrosine (Y), lysine (K), and leucine (L), hence the name YKL-40. In this study, we explored whether YKL-40 could promote the expression of inflammatory factors in type Ⅱ alveolar epithelial cells.
      Methods  A549 cells were cultured in vitro with interleukin (IL)-1β (20 ng/mL), IL-6 (20 ng/mL), tumor necrosis factor-alpha (TNF-α) (20 ng/mL), and interferon-gamma (IFN-γ) (20 ng/mL). The expression of YKL-40 transcription was determined by RT-qPCR. A549 cells were cultured with IL-1β at 5, 10, and 20 ng/mL and the expression of YKL-40 protein was determined by Western blot. A549 cells were cultured with recombinant YKL-40 protein at 0, 100, 500, and 1000 ng/mL and the expression levels of IL-6 and IL-8 were measured by RT-qPCR. Three pairs of small interfering RNAs targeting YKL-40 (si-YKL-40-1/2/3) and the negative control (NC) were designed and used to transfect A549 cells, respectively, and the expression of YKL-40 was determined by RT-qPCR and Western blot. si-YKL-40-3 was screened out for subsequent experiments. In A549 cells, si-YKL-40-3 and si-NC were transfected and, then, IL-1β (20 ng/mL) was added in for culturing. The expression of YKL-40, IL-6, and IL-8 was determined by RT-qPCR and the expression of multiple factors in the supernatant was measured with the QAH-INF-1 kit.
      Results  RT-qPCR results showed that IL-1β could up-regulate YKL-40 protein transcription level compared with that of the control group and the difference was statistically significant (P<0.01), but IL-6, TNF-α, and IFN-γ could not up-regulate YKL-40 protein transcription level. Western blot results showed that IL-1β (20 ng/mL) could significantly promote the expression of YKL-40 and, compared with that of the control group, the differences showed by groups treated with different concentrations of IL-1β were all statistical significant (P<0.01). After adding human recombinant YKL-40 protein to A549 cells, the results showed that the expression of inflammatory factors IL-6 and IL-8 was significantly increased and the difference was statistically significant compared with that of the control group (P<0.05). After the expression of YKL-40 was decreased by si-YKL-40-3 transfection, the expression of IL-6 (P<0.05), IL-8 (P<0.05), and other inflammatory factors was inhibited compared with that of the control group.
      Conclusion  YKL-40 can promote the expression and secretion of IL-6, IL-8, and other acute inflammatory factors in A549 cell line, a type Ⅱ alveolar epithelial cell model, thus aggravating the inflammatory response. Targeted inhibition of YKL-40 expression may effectively inhibit inflammatory response.

     

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