【摘 要】
：
目的 将TERT基因转染后的内皮祖细胞(endothelial progenitor cells,EPCs)移植到5/6肾切除大鼠体内,观察其对模型大鼠肾功能、肾小管周围微血管密度和肾间质纤维化的影响.方法 用RT-PCR方法从幼鼠肝脏总RNA中扩增出TERT片段,通过双酶切方法将其与pZsGreen1-C1载体连接,得到pZsGreen1-C1-TERT重组质粒.密度梯度离心法体外分离培养获得大
【机 构】
：
中南大学湘雅二医院小儿肾脏病研究室,湖南 长沙 410011
【出 处】
：
庆祝中国当代儿科杂志创刊15周年大会暨当代儿科论坛
论文部分内容阅读
目的 将TERT基因转染后的内皮祖细胞(endothelial progenitor cells,EPCs)移植到5/6肾切除大鼠体内,观察其对模型大鼠肾功能、肾小管周围微血管密度和肾间质纤维化的影响.方法 用RT-PCR方法从幼鼠肝脏总RNA中扩增出TERT片段,通过双酶切方法将其与pZsGreen1-C1载体连接,得到pZsGreen1-C1-TERT重组质粒.密度梯度离心法体外分离培养获得大鼠骨髓来源的EPCs,利用脂质体LipofectamineTM 2000法将空质粒pZsGreen1-C1和pZsGreen1-C1-TERT体外转染EPCs,RT-PCR检测转染后TERT mRNA、Western-blot检测转染后TERT蛋白表达,MTT法检测细胞增生,SA-β-半乳糖苷酶(β-gal)活性试验检测细胞衰老,流式细胞术检测细胞凋亡.5/6肾切除术后一周尾静脉注射EPCs(EPCs-N组)、空质粒转染的EPCs(pZ-EPCs-N组)、TERT基因转染的EPCs (pZ-TERT-EPCs-N组)及等体积培基(模型组和假手术组),分别于注射后第4 w、8 w、12w时杀鼠留取标本,检测血生化改变、肾小管周围微血管密度和肾间质纤维化.结果 成功构建了pZsGreen1-C1-TERT质粒；转染TERT基因的EPCs细胞中TERT基因和蛋白表达较EPCs组和空质粒转染组显著增高(P ＜0.05)；SA-β-半乳糖苷酶(β-gal)活性与凋亡细胞显著降低(P＜0.05),增生能力明显提高(P＜0.05).EPCs-N组、pZ-EPCs-N组和pZ-TERT-EPCs-N组大鼠血清尿素氮和肌酐水平较模型组均明显下降(P＜0.05),肾小球滤过率增高(P均＜0.05),肾小管周围微血管密度明显增加、肾间质纤维化程度明显减轻(P均＜0.05),且pZ-TERT-EPCs-N组改善效果最明显.结论 将TERT基因导入EPCs,能够使EPCs持续稳定表达TERT,延缓其衰老、凋亡,提高其增生能力.将其移植到5/6肾切除肾纤维化模型大鼠体内,可改善肾脏功能,减少肾小管周围微血管的丢失,阻抑肾间质纤维化的进展.
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