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  

Trypsin digestion versus tissue adherence in primary culture of lung fibroblasts from newborn rats and establishment of a myofibroblast differentiation model in vitro

作　　者：孙月[1] 徐洪[1] 徐丁洁[2] 魏中秋[3] 于婉莹[1] 邓海静[1] 薛新新 杜世璞 杨方[1] 

机构地区：[1]河北联合大学医学实验研究中心,唐山医学硕士研究生063000 [2]河北联合大学中医学院,唐山063000 [3]河北联合大学病理学系,唐山063000

出　　处：《医学研究生学报》2014年第2期129-132,共4页Journal of Medical Postgraduates

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)

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。Objective Myofibroblasts, characterized by specific expressions of α-smooth muscle actin （ct-SMA） and extra-cellular matrix （ECM） proteins, are critical components of organ fibrosis, The aim of this study is to compare trypsin digestion with tis-sue adherence in the primary culture of lung fibroblasts isolated from newborn rats and establish a myofibroblast differentiation model in vitro. Methods We primarily cultured lung fibroblasts from newborn rats by trypsin digestion and tissue adherence respectively. For in vitro studies, we observed cell morphology under the inverted phase contrast microscope and determined cell proliferation and mark-ers by MTT assay and immunocytochemistry. Myofibroblast differentiation was induce by TGF-1β. Results Lung fibroblasts from the newborn rats were successfully cultured by both trypsin digestion and tissue adherence, with no difference in cell morphology, prolifera- tion and markers between the two methods. However, under the same condition, more cells were rapidly obtained by trypsin digestion than by tissue adherence. The lung fibroblasts cultured by the two methods did not express α-SMA within 4 passages and needed to be induced to myofibroblast differentiation by TGF-1β. The expressions of α-SMA and collagen I were up-regulated with the prolonging of TGF-β induction. Conclusion Compared with the method of tissue adherence, trypsin digestion can rapidly obtain more fibroblasts with a higher purity, and the fibroblasts from newborn rats within 4 passages are suitable for the establishment of the myofibroblast dif-ferentiation model.

关 键 词：成纤维细胞 原代培养 肌成纤维细胞 

分 类 号：R813.11[医药卫生—放射医学]