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龙世棋, 吴翠芳, 曾柱. 不同刚度Ⅰ型胶原凝胶三维环境调控NK细胞免疫功能[J]. 四川大学学报(医学版), 2024, 55(1): 81-86. DOI: 10.12182/20240160401
引用本文: 龙世棋, 吴翠芳, 曾柱. 不同刚度Ⅰ型胶原凝胶三维环境调控NK细胞免疫功能[J]. 四川大学学报(医学版), 2024, 55(1): 81-86. DOI: 10.12182/20240160401
LONG Shiqi, WU Cuifang, ZENG Zhu. The Three-dimensional Environment of Type Ⅰ Collagen Gels With Varying Stiffness Modulates the Immunological Functions of NK Cells[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(1): 81-86. DOI: 10.12182/20240160401
Citation: LONG Shiqi, WU Cuifang, ZENG Zhu. The Three-dimensional Environment of Type Ⅰ Collagen Gels With Varying Stiffness Modulates the Immunological Functions of NK Cells[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(1): 81-86. DOI: 10.12182/20240160401

不同刚度Ⅰ型胶原凝胶三维环境调控NK细胞免疫功能

The Three-dimensional Environment of Type Ⅰ Collagen Gels With Varying Stiffness Modulates the Immunological Functions of NK Cells

  • 摘要:
    目的 构建不同刚度Ⅰ型胶原凝胶并探讨其三维(three-dimensional, 3D)培养环境对自然杀伤 (natural killer, NK) 细胞形态、自由迁移能力以及细胞杀伤功能的影响。
    方法 分离Sprague Dawley(SD)大鼠鼠尾Ⅰ型胶原蛋白并制备不同刚度的胶原凝胶,通过激光共聚焦显微镜观察其微结构,流变仪测量平台期的储能模量表征其刚度。将NK-92MI细胞培养于不同刚度Ⅰ型胶原凝胶中,倒置显微镜观察NK-92MI细胞的形态,高内涵成像系统记录NK-92MI细胞的自由迁移过程,分析迁移速度和距离。不同刚度的Ⅰ型胶原凝胶培养NK-92MI细胞24、48 h,再与人结直肠腺癌上皮细胞(DLD-1)共培养后,用CCK8法检测DLD-1细胞增殖率,分析NK-92MI细胞的细胞杀伤能力。
    结果 成功制备出刚度为(10.97±2.10) Pa的低刚度Ⅰ型胶原凝胶和刚度为(114.50±3.40) Pa的高刚度Ⅰ型胶原凝胶。与在低刚度Ⅰ型胶原凝胶中相比,在高刚度Ⅰ型胶原凝胶中,NK-92MI细胞呈现更细长的形态(P<0.05),细胞平均面积减少〔(69.88±26.97) μm2 vs.(46.59±21.62) μm2 P<0.05 〕,细胞圆度减小(0.82±0.12 vs. 0.78±0.18, P<0.05),细胞迁移速度降低〔(2.50±0.91) μm/min vs. (1.70±0.72) μm/min,P<0.001〕,迁移距离缩短〔(147.10±53.74) μm vs. (98.03±40.95) μm,P<0.0001〕,差异均有统计学意义。与低刚度Ⅰ型胶原凝胶相比,高刚度Ⅰ型胶原凝胶培养24 h的NK-92MI细胞可促进DLD-1细胞增殖〔增殖率(46.39±12.79)% vs. (65.87±4.45)%,P<0.05〕,降低细胞杀伤能力,48 h的比较结果类似〔增殖率(31.36±2.88)% vs. (74.57±2.16)%,P<0.05〕,差异均有统计学意义。
    结论 不同刚度Ⅰ型胶原凝胶3D培养环境可改变NK-92MI细胞的形态、迁移能力以及杀伤功能。该研究为探索和理解生物力学微环境影响NK细胞免疫应答机制提供了研究基础,为优化免疫治疗方案提供理论依据。

     

    Abstract:
    Objective To construct type Ⅰ collagen gels with different stiffness and to investigate the effects of three-dimensional (3D) culture environments of the gels on the morphology, free migration ability, and cell killing function of natural killer (NK) cells.
    Methods Type Ⅰ collagen was isolated from the tails of Sprague Dawley (SD) rats and collagen gels with different levels of stiffnesses were prepared accordingly. The microstructure of the collagen gels was observed by laser confocal microscopy. The stiffness of the collagen gels was assessed by measuring the plateau modulus with a rheometer. NK-92MI cells were cultured in collagen gels with different levels of stiffness. The morphology of NK-92MI cells was observed by inverted microscope. High content imaging system was used to record the free migration process of NK-92MI cells and analyze the migration speed and distance. NK-92MI cells were cultured with type Ⅰ collagen gels with different levels of stiffness for 24 h and 48 h and, then, co-cultured with human colorectal DLD-1, a human adenocarcinoma epithelial cell line. CCK8 assay was performed to determine the proliferation rate of DLD-1 cells and analyze the cell killing ability of NK-92MI cells.
    Results Low-stiffness type Ⅰ collagen gel and high-stiffness type Ⅰ collagen gel with the respective stiffness of (10.970±2.10) Pa and (114.50±3.40) Pa were successfully prepared. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, the NK-92MI cells in the high-stiffness type Ⅰ collagen gel showed a more elongated shape (P<0.05), the mean area of the cells was reduced (69.88±26.97 μm2 vs. 46.59±21.62 μm2, P<0.05), the roundness of the cells decreased (0.82±0.12 vs. 0.78±0.18, P<0.05), cell migration speed decreased (2.50±0.91 μm/min vs. 1.70±0.72 μm/min, P<0.001) and the migration distance was shortened (147.10±53.74 μm vs. 98.03± 40.95 μm, P<0.0001), with all the differences being statistically significant. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, NK-92MI cells cultured with high-stiffness type Ⅰ collagen gel for 24 h could promote DLD-1 cell proliferation, with the proliferation rate being (46.39±12.79)% vs. (65.87±4.45)% (P<0.05) and reduce the cell killing ability. Comparison of the cells cultured for 48 h led to similar results, with the proliferation rates being (31.36±2.88)% vs. (74.57±2.16)% (P<0.05), and the differences were all statistically significant.
    Conclusion The 3D culture environment of type Ⅰ collagen gels with different levels of stiffness alters the morphology, migration ability, and killing function of NK-92MI cells. This study provides the research basis for exploring and understanding the mechanisms by which the biomechanical microenvironment affects the immune response of NK cells, as well as laying the theoretical foundation for optimizing immunotherapy protocols.

     

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