重组腺相关病毒介导人端粒酶逆转录酶基因转染对人髓核细胞功能的影响

Effect on function of human nucleus pulposus(HNP) cells transfected by the recombinant adeno-associated virus vector-mediated human telomerase reverse transcriptase gene

目的:研究重组腺相关病毒介导人端粒酶逆转录酶基因(recombinant adenoassociated virus vector-me-diated human telomerase reverse transcriptase,rAAV-hTERT)转染对体外培养人髓核细胞功能的影响。方法:利用机械与酶消化的方法获得均一性的人椎间盘髓核细胞,将构建好的rAAV-hTERT转染P2代体外单层培养的髓核细胞。用重组腺相关病毒-增强型绿色荧光蛋白(recombinant adenoassociated virus vector-mediatedenhanced green fluorescent protein,rAAV-EGFP)作为标记基因观察细胞转染效率,按感染复数(multiplicity ofinfection,MOI)103、104、105病毒基因组拷贝数/细胞(vector genomes/cell,v.g/cell)设组,流式细胞仪检测,筛选病毒转染的最佳MOI;设立rAAV-hTERT转染组、AAV空病毒转染组及空白细胞对照组,用反转录聚合酶链反应(reverse transcription polymerase chain reareaction,RT-PCR)及免疫印迹(Western-blot)方法检测rAAV-hTERT转染后hTERT基因在髓核细胞内的表达,实时定量聚合酶链反应(real-time quantitative PCR)及酶联免疫法(enzyme linked immunosorbent,ELISA)检测髓核细胞合成Ⅱ型胶原及蛋白多糖能力的变化。结果:rAAV-EGFP转染细胞后第7天时,MOI为105v.g/cell组转染效率可达73.9%,明显高于MOI 103、104v.g/cell组(P<0.05);rAAV-hTERT转染组在转染后的第7、60、90、120天均可检测到hTERT基因的表达,而其他两组则无表达;转染rAAV-hTERT后120d之前,rAAV-hTERT转染组分泌蛋白多糖及Ⅱ型胶原较其他两组均明显增高(P<0.05),而两对照组之间则始终无明显差异(P>0.05)。结论:rAAV-hTERT能成功转染人椎间盘髓核细胞并正确表达,rAAV-hTERT转染能有效延长髓核细胞分泌Ⅱ型胶原及蛋白多糖功能。

Objectives： To investigate the effects on the function of HNP cells transfected by the rAAV- hTERT. Methods： The cultured homogeneous HNP cells were obtained and released by mechanical dissection and enzyme digestion. The second generation of HNP cells cultured in monolayer cuhure was transfected by rAAV-hTERT, rAAV-EGFP was firstly used as mark gene to detect the efficiency of the transduction at MOI 103, 104, 105 vector genomes/cell（v.g/cell） by flow cytometry. Three groups were designed for the experiment： （1）group h HNP cells transfected by rAAV-hTERT; （2）control h HNP cells transfected by AAV; （3） control 2： HNP cells as noviral transduction group. The expression of the hTERT gene was determined by RT-PCR and Western-blot. Cellular matrix transcript/translated levels were determined by real-time quantitative PCR/Elisa, respectively. Results： The expression of the rAAV-EGFP was 73.9% for MOI（I@ v.g/cell） group which was much higher than the MOI（10^3, 10^4 v.g/cell） groups at 7 days after transfection. The expression of the rAAV-hTERT was successfully detected at 7, 60, 90, 120 days after transfection in rAAV-hTERT group, while not present in two controls. The expressional levels of collagen 2 and aggrecan of rAAV-hTERT group were much higher than two controls in 120 days after transfeetion（P〈0.05）, while the two controls showed no difference from the beginning to the end（P〉0.05）. Conclusions： rAAV-hTERT can infect the HNP cells and also express in the transfeeted cells. The transfection of rAAV-hTERT can improve the cell potency to produce collagen 2 and aggrecan.