端粒酶反转录酶在体外培养的内皮祖细胞中的表达  

Expression of telomerase reverse transcriptase in endothelial progenitor cell cultured in vitro

作　　者：何小解[1] 许自川[1] 易著文[1] 党西强[1] 

机构地区：[1]中南大学湘雅二医院小儿肾脏病研究室湖南省小儿肾脏病临床中心,410011

出　　处：《中华肾脏病杂志》2009年第10期755-760,共6页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30500546）

摘　　要：目的探讨体外培养的大鼠内皮祖细胞增殖活性与端粒酶反转录酶表达的关系。方法原代培养大鼠骨髓源性内皮祖细胞。在培养7、14、21和28d时，用MTT法检测内皮祖细胞在不同时间点的增殖活性；流式细胞术检测内皮祖细胞早期凋亡率；免疫细胞化学方法、RT—PCR和Western印迹技术检测内皮祖细胞端粒酶反转录酶mRNA和蛋白表达的变化。结果大鼠骨髓单个核细胞可以被诱导成内皮祖细胞。内皮祖细胞增殖活性随着培养时间的延长呈现单峰曲线，在培养14d时增殖活性最高（P〈0．01）。内皮祖细胞凋亡率随培养时间的延长而增加，在培养7d、14d、21d和28d时分别为0．28％、0．66％、1．38％、1．52％；衰老率分别为3．04％、20．28％、24．36％、16．52％，14d内皮祖细胞衰老率显著增加（P〈0．01），21d衰老率最高，但二者之间差异无统计学意义。端粒酶反转录酶mRNA和蛋白表达呈单峰曲线，在培养14d时表达最高，以后表达随培养时间的延长逐渐降低（P〈0．05）。结论大鼠骨髓内皮祖细胞增殖活性下降可能与端粒酶反转录酶活性下降有关。Objective To explore the relationship between the expression of telomerase reverse transcriptase （TERT） and the proliferation of endothelial progenitor ceils （EPCs）. Methods The bone marrow-derived EPCs form SD rats were cultured in vitro. At the end of week 1, 2, 3 and 4 of culture, MTT assay was used to detect the EPCs proliferation rate in the growth duration; Annexin-V-FITC/PI asaay was applied to examine the apoptosis rate in early stage of EPCs; RT-PCR, immunocytochemistry and Western blotting were employed to detect the TERT mRNA and protein expression. Results The mononuclear cells from rats bone marrow could be induced into endothelial progenitor cells in vitro. The proliferation rate of EPCs from different culture duration took on a singlet curve, with the peak at day 14 （P〈 0.01）. The apoptosis rate in EPCs was 0.28%, 0.66%, 1.38%, 1.52% respectively at week 1 to 4, increasing along with the growth duration within 28 days. Aging rate of EPCs was 3.04%, 20.28%, 24.36%, 16.52% respectively at the end of lth, 2th, 3th and 4th weeks, which significantly increased at day 14 （P〈 0.01） and was highest at day 21, but no significant difference was found. The TERT mRNA and protein expression took on a single curve with its peak at day 14 as well, then reduced with the culture time. Conclusion The down-regulated TERT expression may contribute to the increasing apoptosis rate in early stage and the decreasing proliferaton rate of rat bone marrowderived EPCs.

关 键 词：端粒 末端转移酶 细胞增殖 细胞凋亡 内皮祖细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]