大鼠脂肪组织来源干细胞的分离、培养和生物学特性的研究

目的:建立一种分离和培养大鼠脂肪干细胞的方法,并探讨获得的脂肪干细胞的部分生物学特性。方法:取SD大鼠腹股沟处的皮下脂肪,I型胶原酶消化法获取原代脂肪干细胞,接种至含10%胎牛血清的DMEM培养液,培养并适时传代,每日在倒置显微镜下观察记录细胞形态和增殖特征,测定其生长曲线,进行冻存复苏实验。第3代细胞使用成骨诱导液诱导其向成骨细胞分化,进行VonKossa染色;使用成脂诱导液诱导其向成脂细胞分化,进行油红O染色。结果:从大鼠脂肪组织中分离出脂肪干细胞,其在体外生长增殖迅速,呈成纤维样细胞生长。生长曲线表明第3代脂肪干细胞增殖能力最强,可以在地塞米松、维生素C、β-甘油磷酸钠的诱导下,表现出成骨细胞特性,VonKossa染色出现矿化结节;在IBMX(3-异丁基-1-甲基黄嘌呤)、吲哚美辛、胰岛素的诱导下,表现出脂肪细胞特性,油红O染色后发现胞浆内有脂肪滴。脂肪干细胞在液氮中冻存1个月后,其生长增殖活性和多向分化能力没有明显降低。结论:成功建立了一种简单有效的分离和培养大鼠脂肪干细胞的方法,获得的脂肪干细胞生长增殖旺盛,具有多向分化能力,可以方便地保存,有望作为细胞治疗和组织工程的种子细胞。在分离大鼠的脂肪干细胞时,NH4Cl裂解红细胞这一步可以省略,地塞米松在成脂诱导过程中不是必需的。

An Experimental Study of the Cultrure, Isolation and Biological characteristics of Rat Adipose Tissue-derived Stromal Cells in vitro

OBJECTIVE: To establish a method for the isolation and culture of rat adipose tissue-derived stromal cells (ADSCs) and explore some biological characteristics of the acquired ADSCs. METHODS: Adipose tissues were isolated from the inguinal fat of SD rats. Primary ADSCs were obtained by the method of collagenase I digestion, inoculated, cultured in the Dulbecco modified Eagle medium with 10% fetal bovine serum, and subcultured at the right moment. The morphology and proliferation characteristics of the cells were observed under the inverted phase contrast microscope every day. Their growth curves were detected and experiments of freezing and resuscitation performed. The third passage ADSCs were induced into osteoblasts by osteogenic inducing fluid and into adipocytes by adipogenic inducing fluid. The osteogenic phenotypes were examined by Von Kossa staining and the adipocytes by Oil Red O staining. RESULTS: ADSCs were successfully obtained and cultured from the rat adipose tissue. They appeared fibroblast-like and could proliferate rapidly in vitro, the third passage having the most active proliferative ability. Calcium nodes characteristic of osteoblasts were observed in the ADSCs on Von Kossa staining after induction with dexamethasone, ascorbic acid and beta-sodium glycerophosphate, and red-stained fats characteristic of adipocytes were noted in the cytoplasm on Oil Red O staining after induction with IBMX, indomethacin and insulin. The ADSCs showed no significant decrease in their proliferation activity and capability of differentiating into diverse cell types after cryopreserved in liquid nitrogen for a month. CONCLUSION: A simple and effective method for the isolation and culture of rat ADSCs was successfully established. The ADSCs obtained could grow and proliferate rapidly in vitro, capable of differentiating into diverse cell types, easy to be preserved and promising to be seed cells for cell therapy and tissue engineering. The procedure of schizolysising erythrocytes with NH4Cl could be omitted in the isolation of the rat ADSCs and dexamethasone is not indispensable in the induction of ADSCs into adipocytes.