新生鼠肺成纤维细胞原代培养方法的比较与体外肌成纤维细胞分化模型的建立

发布时间：2018-04-20 07:38

本文选题：成纤维细胞 + 原代培养　；参考：《医学研究生学报》2014年02期

【摘要】：目的肌成纤维细胞(fibroblasts,FB)是器官纤维化形成过程中关键的靶细胞,以特异表达α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和细胞基质沉积为特征。文中比较胰蛋白酶消化法和组织贴壁法原代培养乳鼠肺FB的优缺点,并报道体外肌FB分化模型的建立。方法分别采用胰蛋白酶消化法和组织贴壁法原代培养乳鼠肺FB,倒置相差显微镜观察原代培养,MTT法检测细胞增殖,免疫细胞化学染色法鉴定细胞来源,采用转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导FB向肌FB分化。结果采用胰蛋白酶消化法和组织块贴壁法均可以成功原代培养鼠胚肺FB,2种方法无论在形态、细胞增殖水平还是在细胞鉴定方面均无差异,但胰蛋白酶消化法在相同条件下能够快速获得较组织贴壁法更多的细胞,且2种方法获得的FB在4代之内均不表达α-SMA,需经TGF-β1诱导分化为肌FB。随TGF-β1诱导时间的延长,α-SMA和Ⅰ型胶原蛋白表达上调。结论胰酶消化法较组织贴壁法能快速、大量获得纯度较高的FB,4代以内的乳鼠FB适合建立肌FB分化模型。
[Abstract]:Objective myofibroblast fibroblast (FB) is a key target cell in the process of organ fibrosis. It is characterized by the specific expression of 伪 -smooth muscle actin (伪 -SMA) and cell matrix deposition. In this paper, the advantages and disadvantages of trypsin digestion method and tissue adherence method in primary culture of FB in neonatal rat lung were compared, and the establishment of FB differentiation model of extracorporeal muscle was reported. Methods using trypsin digestion method and tissue adherence method, the primary cultured neonatal rat lung FB was cultured, the primary culture cell proliferation was detected by inverted phase contrast microscope, and the cell origin was identified by immunocytochemical staining. Transforming growth factor 尾 1(transforming growth factor- 尾 1 (TGF- 尾 1) was used to induce the differentiation of FB into muscle FB. Results there was no difference in morphology, cell proliferation and cell identification between the two methods of trypsin digestion and tissue mass adherence. However, more cells could be obtained by trypsin digestion under the same conditions than by tissue adherence method, and the FB obtained by both methods did not express 伪 -SMAwithin 4 passages, and it was necessary to differentiate into muscle FB by TGF- 尾 1. The expression of 伪 -SMA and type I collagen was up-regulated with the prolongation of TGF- 尾 1 induction time. Conclusion Pancreatic enzyme digestion is more rapid than tissue adherent method, and a large number of FB with high purity can be obtained in the fourth generation of FB, which is suitable for the establishment of muscle FB differentiation model.
【作者单位】：河北联合大学医学实验研究中心;河北联合大学中医学院;河北联合大学病理学系;
【基金】：国家自然科学基金(81202162) 唐山市科技计划项目(1313021092) 河北联合大学博士科研项目(35757299)
【分类号】：R135.2;R563.9

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