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目的:探讨微重力环境对肝干细胞增殖及分化的影响。方法:从人肝组织中分离、培养肝干细胞,进行肝干细胞鉴定后,分别进行平面培养、微载体培养、模拟微重力下三维培养。利用倒置相差显微镜、扫描电镜观察不同培养条件下细胞形态学变化;检测上清液中葡萄糖消耗量和乳酸脱氢酶含量以比较细胞增殖和活性情况;荧光定量PCR法检测细胞ALB、EpCAM、CK19、AFP、HNF6、CYP3A4、CYP3A7 mRNA表达变化。结果:模拟微重力环境下,肝干细胞以微载体球面为基底呈三维立体结构生长,形成大小不等细胞团;扫描电镜下见细胞团表面有丰富的细胞外基质及微绒毛;从9 d开始葡萄糖消耗量明显增加,至17 d达高峰,而LDH含量始终保持低水平;培养至21 d细胞仍高表达EpCAM、ALB、CK19、HNF6、CYP3A7,不表达AFP、CYP3A4,符合肝干细胞表型特征,与微载体培养结果相比有统计学差异(P<0.05)。结论:模拟微重力环境能促进肝干细胞呈三维立体结构生长,有利于细胞快速增殖并维持细胞活性和表型。
Objective: To investigate the effects of microgravity on the proliferation and differentiation of hepatic stem cells. Methods: Hepatic stem cells were isolated and cultured from human liver tissue. After identification of hepatic stem cells, they were cultured in vitro and in microcarriers, respectively. The morphological changes of cells under different culture conditions were observed by inverted phase contrast microscope and scanning electron microscope. The content of glucose and lactate dehydrogenase in the supernatant were measured to compare the cell proliferation and activity. The expression of ALB, EpCAM, CK19 , AFP, HNF6, CYP3A4, CYP3A7 mRNA expression changes. Results: Under the simulated microgravity environment, the liver stem cells grew in a three-dimensional structure with microcarrier spheres as basal cells to form cell clusters with different sizes. The surface of cell masses was rich in extracellular matrix and microvilli under scanning electron microscope. From the 9th day Glucose consumption increased significantly and peaked on day 17, but LDH content remained low. EpCAM, ALB, CK19, HNF6, CYP3A7, AFP and CYP3A4 were still highly expressed on day 21 , Compared with the results of microcarrier culture (P <0.05). CONCLUSION: Simulated micro-gravity environment can promote the growth of hepatic stem cells in three-dimensional structure, which is conducive to rapid cell proliferation and maintain cell viability and phenotype.