人脐动脉平滑肌细胞体外培养的生物学特性及其永生株的初步建立

Biological Characteristics of Human Umbilical Artery Smooth Muscle Cells Cultured in vitro and the Pre-establishment of Immortalized Cell Line

目的 建立人脐动脉血管平滑肌细胞 (HU ASMC)原代、传代培养的最佳方法 ,并初步建立其永生株。方法 采用人脐动脉血管中膜组织块贴壁法原代培养 ,比较不同培养基对原代及传代细胞增殖的影响 ;选择标记为新霉素 (G4 18)的带有端粒酶催化亚基 (h TERT)基因的重组质粒导入人脐动脉血管平滑肌细胞。结果 经 α-肌动蛋白鉴定 ,组织块贴壁法可获得高纯度的 HUASMC。原代培养的 HU ASMC1～ 4代细胞增殖能力强、生长旺盛 ,此后细胞的增殖能力逐渐降低 ,到第 10代几乎丧失增殖力。原代培养以 MCDB131为最佳培养基。 h TERT转染细胞克隆由长梭形变为短梭形 ,接触抑制消失 ,生长速度快 ,目前已传至 2 0代 ,增殖能力与第 1代无差别。转染前后平滑肌特异的α-肌动蛋白表达阳性。结论 h TERT转染的 HU ASMC保留了 HU ASMC的基本生物学特征 。

Objective To establish the best primary and propagated culture approach of the human umbilical artery smooth muscle cells (HUASMC) and immortalized the primary cells. Methods The primary culture of HUASMC was performed using the primary explant method, and the effects on the cells' growth characteristic by different culture mediums were conducted. Primary cultures of HUASMC derived from normal human umbilical arteries were transfected with recombination plasmid containing human telomerase reverse transcriptase (hTERT) open reading frame. The transfected HUASMC was analyzed for cell biological characteristics, and growth characteristics, and specific α actin. Results Highly purified primary HUASMC were gained and could be propagated about 10 population doublings(PD). After PD 4, the cells lost the proliferation ability gradually. However, the transfected HUASMC could be propagated at least 20 population doublings and could express specific α actin. Conclusion The hTERT transfected HUASMC could keep the basic biological characteristic of smooth muscle cells and prolonged life span at least 20 PD. It might be a good cell model for the study of cardiovascular disease, myoma of uterus and chronic graft disfunction. It might also bring the hope to engineered vascularization in the future.