利用慢病毒介导hTERT基因建立永生化髓核细胞系

CONSTRUCTION OF AN IMMORTALIZED NUCLEUS PULPOSUS CELL LINE USING THE LENTIVIRUS-MEDIATED HUMAN TELOMERASE REVERSE TRANSCRIPTASE GENE

目的以慢病毒介导人端粒酶逆转录酶(hTERT)基因转染体外培养的人髓核细胞,建立永生化髓核细胞系,为后期转基因与动物实验提供种子细胞。方法利用机械与酶消化的方法获得正常的人椎间盘髓核细胞,将构建好的重组慢病毒-hTERT(pLVX-hTERT)转染第2代体外单层培养的髓核细胞。用重组慢病毒-增强型绿色荧光蛋白作为标记物,在荧光电镜下观察细胞转染效率,按感染复数(MOI)40、60、80、100设组,选择出病毒转染的最佳MOI;设立pLVX-hTERT转染组、空病毒转染对照组及空白细胞对照组。在倒置显微镜下观察髓核细胞的形态,用反转录聚合酶链反应、细胞免疫荧光技术检测转染后hTERT基因在髓核细胞内的表达情况,用反转录聚合酶链反应检测髓核细胞Ⅱ型胶原及蛋白多糖的表达情况。结果慢病毒转染细胞后第6天,MOI为100时转染效率最高,可达82.9%。pLVX-hTERT转染组在转染后第7、15、30、60、90天均可检测到hTERT基因的表达,而两对照组则未见表达。pLVX-hTERT转染组保持了髓核细胞的形态,两对照组的细胞则出现返祖现象。pLVX-hTERT转染组细胞的蛋白多糖及Ⅱ型胶原基因表达水平较其他两组均明显增高(F=173.5、111.1,P<0.05),而两对照组之间则无明显差异(P>0.05)。结论pLVX-hTERT成功转染人椎间盘髓核细胞,转染pLVX-hTERT后的髓核细胞持续表达hTERT,获得永生化的能力,且分泌Ⅱ型胶原及蛋白多糖的量增加。

Objective To construct an immortalized nucleus pulposus cell line using the human nucleus pulposus cells cultured in vitro and transfected with the lentivirus-mediated human telomerase reverse transcriptase(hTERT)gene,and to provide seed cells for transgenic and animal experiments in future.Methods The mechanical and enzymatic digestion method was used to obtain normal human nucleus pulposus cells of the intervertebral disc,and the second-generation monolayer nucleus pulposus cells cultured in vitro were transfected with recombinant lentivirus-hTERT(pLVX-hTERT).With the recombinant lentivirus-enhanced green fluorescent protein as a marker,a fluorescent electron microscope was used to observe transfection efficiency.The cells were divided into groups based on a multiplicity of infection(MOI)of 40,60,80,and 100 to screen out the optimal MOI.The pLVX-hTERT transfection group,the empty virus transfection group,and the blank control group were established.An inverted microscope was used to observe the morphology of nucleus pulposus cells;RT-PCR and cell immunofluorescence were used to measure the expression of the hTERT gene in nucleus pulposus cells after transfection,and RT-PCR was used to measure the expression of type Ⅱ collagen and proteoglycan in nucleus pulposus cells.Results On day 6 after lentiviral transfection,the transfection efficiency reached the highest level of 82.9%at the MOI of 100.The expression of the hTERT gene was observed in the pLVX-hTERT transfection group on days 7,15,30,60,and 90 after transfection,while such expression was not observed in the two control groups.The morphology of the nucleus pulposus cells was maintained in the pLVX-hTERT transfection group,while atavism was observed in the two control groups.The pLVX-hTERT transfection group had significantly higher mRNA expression of proteoglycan and type Ⅱ collagen than the other two groups(F=173.5,111.1;P<0.05),while there were no significant differences between the two control groups(P>0.05).Conclusion Human nucleus pulposus cells of the intervertebral disc are successfully transfected with pLVX-hTERT,and after pLVX-hTERT transfection,nucleus pulposus cells have persistent expression of hTERT and thus acquire the ability for immortalization,with increases in the secretion of type Ⅱ collagen and proteoglycan.