短发夹RNA对人骨髓间充质干细胞生长增殖的影响

Targeting hTERT gene influenced the viability on hMSCs by small hairpin RNA

目的观察靶向人端粒酶逆转录酶(hTERT)的短发夹RNA(shRNA)对人骨髓间充质干细胞(hMSCs)生长增殖的影响。方法根据RNA干扰原理,利用构建的表达shRNA的靶向hTERTmRNA的真核表达质粒(shRNA1),非特异性的shRNA真核表达质粒(shRNA2),转染试剂和正常培养液处理细胞。24h后在共聚焦显微镜下检测荧光表达情况;24h、48h、72h用MTT法检测细胞增殖活性;48h后行HE染色、RT-PCR及端粒酶活性检测。结果(1)共聚焦显微镜下见shRNA1及shRNA2组有大量的细胞表达荧光。(2)相应时间点各组细胞生长增殖无明显差异。HE染色发现,同等培养条件下,各组细胞生长密度及形态无明显变化。(3)RT-PCR显示各组细胞均无hTERTmRNA的表达;端粒酶活性均为阴性表达。结论靶向hTERTmRNA的shRNA对正常hMSCs的生长增殖无明显影响,该方法对不表达hTERT的正常体细胞是安全的。

Objective To observe the effect of shRNA by targerting human telomerase reverse transcriptase （hTERT） mRNA in the human mesenchymal stem cells（hMSCs）lines and explore the safety of RNAi by targerting hTERT mRNA in clinical application, Methods According to the strategy of RNAi, which determined the specific base sequences to design shRNA plasmid, Plasmid shRNA1 involved in fluorescein gene and homologous to hTERT were designed, control shRNA2-a random sequences heterogenous to mankind＇ s gene were also constructed. METAFECTENE was used as the transfection reagent. The experiment was divided into 4 groups： A （shRNA1）,B （shRNA2）, C （metafectene） and D （normal culture medium ）. At 24h, fluorescence expression was detected by confocal microscopy after administration of plasmid. At 24h, 48h and 72h cells viability were determined using the MTT assay. At 48h, the morphological change were examined by HE stain. At 48h, hTERT mRNA were detected by reverse transcription polymerase chain reaction （RTPCR） and the telomerase activity of hMSCs were examined by PCR ELISA. Results Many cells gave out green fluorescent after treated by shRNA1 and shRNA2. It was observed that the viability of hMSCs hadn＇t change after treatment with different methods within 72h （P 〉 0.05）. Cells, morphology and density had not change after treated by different factor. All cells didn＇t express hTERT mRNA and the telomere activity of hMSCs was negative. Conclusions The results suggest that shRNA plasmid directed against hTERT can not influence the viability on hMSCs. The treatment with RNA interference may be a safety strategy for gene therapy in vivo.