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 Vability 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，探讨靶向hTERT的RNA干扰技术临床应用的安全性。方法：根据RNA干扰原理，利用构建的表达shRNA的靶向hTERT mRNA的真核表达质粒（shRNAl），非特异性的shRNA真核表达质粒（shRNA2），转染试剂（德国metafectene）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后进行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（P〉0．05）；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。③RT—PCR显示各组细胞均无hTERT mRNA的表达；端粒酶活性为阴性。结论：靶向hTERT mRNA的shRNA对正常hMSCs的生长增殖无明显影响，该方法对不表达hTERT的正常体细胞可能是安全的。

Abstract 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 shRNAI involved in fluorescein gene and homologous to hTERT were designed, control shR- NA2 - 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 24 h, fluorescence expression was detected by confocal microscopy after administration of plasmid. At 24 h,48 h,and 72 h ceils viability were determined using the MTT assay. At 48 h, the morphological change were examined by HE stain. At 48 h, hTERT mRNA were detected by reverse transcription polymerase chain reaction （ RT - PCR） and the telomerase activity of hMSCs were examined by PCR ELISA. Results： Many ceils gave out green fluo- rescent after treated by shRNA1 and shRNA2. It was observed that the viability of hMSCs hadn＇t change after treatment with different methods within 72 h （P 〉 0.05）. Ceils, morphology and density had not change after treated by different factor. All ceils 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.