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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。
Objective Fibroblasts (FBs) are the key target cells in the process of organ fibrosis. They are characterized by α-smooth muscle actin (α-SMA) and cell matrix deposition. The advantages and disadvantages of primary cultures of lung fibroblasts from neonatal rats were compared by trypsin digestion and tissue adherent method, and the establishment of FB differentiation model in vitro was reported. Methods Lung fibroblasts (FBs) were primarily cultured by trypsin digestion and tissue adherent method respectively. Primary cultured cells were observed by inverted phase contrast microscope. Cell proliferation was detected by MTT assay. Cell origin was identified by immunocytochemical staining. Transforming growth factor - transforming growth factor-β1, TGF-β1) inducing FB to muscle FB differentiation. Results Both trypsin digestion and tissue adhesion were successful in primary culture of mouse embryo lung FB. There was no difference in morphology, cell proliferation or cell identification between the two methods, but trypsin digestion method under the same conditions Under the second generation method, more cells could be rapidly obtained than the tissue-adherent method. The FBs obtained by the two methods did not express α-SMA within the 4th generation, and were induced to differentiate into muscle FBs by TGF-β1. With the prolongation of TGF-β1 induction, the expression of α-SMA and type Ⅰ collagen was up-regulated. Conclusion The trypsin digestion method can rapidly and massively acquire FBs with high purity, and the FBs in the fourth generation can be used to establish the muscle FB differentiation model.