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背景:通过RNA干扰沉默特异性的肿瘤相关基因可以抑制肿瘤细胞的增殖,诱导肿瘤细胞凋亡,为RNA干扰治疗恶性肿瘤提供了一条新的途径。但RNA干扰治疗的安全性如何?是否在沉默特定基因的同时对正常的机体产生危害?单位:三峡大学仁和医院耳鼻咽喉科。材料:实验于2005-11/2006-03在武汉大学医学部中心实验室(BSL-1)完成。主要实验材料:hMSCs(第3代)由北京科宇联合干细胞生物技术有限公司提供并授权使用。以metafectene(德国)为转染试剂。目的:观察靶向人端粒酶反转录酶(human telomerase reverse tran-scriptase,hTERT)的短发夹RNA(short hairpin RNA,shRNA)对人骨髓间充质干细胞生长增殖的影响,探讨靶向hTERT的RNA干扰技术临床应用的安全性。设计:观察对比实验。方法:①shRNA质粒的设计、构建及实验分组:根据RNA干扰原理,实验分4组:利用构建的表达shRNA的靶向hTERT mRNA的真核表达质粒(shRNA1组),非特异性的shRNA真核表达质粒(shRNA2组),转染试剂组和正常培养液组处理细胞。②质粒转染:hMSCs细胞常规培养1d后进行转染。③MTT法测定细胞增殖活性:培养24,48,72h后采用酶联免疫监测仪测定492nm处各组吸光度A值。④反转录-聚合酶链反应:培养48h后用TRIZOL-Reagent一步法分别提取各组的总RNA。以常规培养的喉鳞癌Hep-2细胞为对照。以紫外分光光度计测量各组RNA的A260及A280的吸光度值。将RNA稀释后进行反转录,以凝胶成像系统对电泳产物进行照相。⑤端粒酶活性检测:细胞培养48h后用Roche Molecular Biochemicals公司端粒酶PCR ELISA试剂盒进行活性测定。主要观察指标:①转染24h后在共聚焦显微镜下检测荧光表达情况。②培养24,48,72h后测定492nm处各组吸光度A值。③倒置相差显微镜下观察各组细胞生长情况;苏木精-伊红染色进行形态学分析。④反转录-聚合酶链反应结果。⑤端粒酶活性检测。结果:①共聚焦显微镜下见shRNA1组、shRNA2组及转染试剂组有大量的细胞表达绿色荧光。正常培养液组未见表达绿色荧光的细胞。②相应时间点各组细胞生长增殖无明显差异(P>0.05)。③倒置显微镜下观察各组细胞呈长梭形纤维细胞样贴壁生长,未见明显形态变化。苏木精-伊红染色见各组细胞生长密度及形态无明显变化。④反转录-聚合酶链反应显示各组细胞均无hTERT mRNA的表达。⑤端粒酶活性检测为阴性。结论:靶向hTERT mRNA的shRNA对正常人骨髓间充质干细胞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。
BACKGROUND: Silencing specific tumor-associated genes by RNA interference can inhibit the proliferation of tumor cells and induce the apoptosis of tumor cells, providing a new way for RNAi to treat malignant tumors. However, the safety of RNA interference treatment? Whether the silence of specific genes at the same time on the normal body harm? Unit: Renhe Hospital of China Three Gorges University otolaryngology. Materials: The experiment was performed at the Central Laboratory of Medical Sciences of Wuhan University (BSL-1) from November 2005 to March 2006. Main experimental materials: hMSCs (3rd generation) were provided by Beijing Keyu Joint Stem Cell Biotechnology Co., Ltd. and authorized for use. Metafectene (Germany) as a transfection reagent. OBJECTIVE: To observe the effect of short hairpin RNA (shRNA) targeting human telomerase reverse tran-scriptase (hTERT) on the proliferation and proliferation of human bone marrow mesenchymal stem cells hTERT RNA interference technology for clinical application of safety. Design: observe the contrast experiment. Methods: ①shRNA plasmid design, construction and experimental grouping: According to the principle of RNA interference, the experiment was divided into 4 groups: the eukaryotic expression plasmid (shRNA1 group) targeting hTERT mRNA, the non-specific shRNA eukaryotic expression plasmid (shRNA2 group), transfection reagent group and normal culture medium group. ② plasmid transfection: hMSCs were cultured 1d conventional transfections. ③ MTT assay of cell proliferation activity: cultured 24,48,72 h after enzyme-linked immunosorbent assay 492nm at each absorbance A value. ④ Reverse transcription - polymerase chain reaction: After cultured for 48h, the total RNA of each group was extracted by TRIZOL-Reagent one-step method. The routine culture of laryngeal squamous cell carcinoma Hep-2 cells as a control. The absorbance values of A260 and A280 of each group of RNA were measured by UV spectrophotometer. The RNA was diluted and reverse transcribed, and the electrophoresed product was photographed using a gel imaging system. ⑤ telomerase activity test: 48h after cell culture using Roche Molecular Biochemicals telomerase PCR ELISA kit for activity determination. MAIN OUTCOME MEASURES: ① Fluorescence expression was detected by confocal microscopy 24h after transfection. ② After 24, 48 and 72h incubation, the absorbance A of each group at 492nm was determined. ③ inverted phase contrast microscope to observe the growth of cells in each group; hematoxylin-eosin staining for morphological analysis. ④ reverse transcription - polymerase chain reaction results. ⑤ telomerase activity test. Results: (1) A large number of cells expressing shRNA1, shRNA2 and transfection reagent groups expressed green fluorescence under the confocal microscope. Normal culture fluid group did not express green fluorescent cells. At the corresponding time points there was no significant difference in the proliferation of each group (P> 0.05). ③ Inverted microscope observation of each group of cells spindle fibroblast-like adherent growth, no obvious morphological changes. Hematoxylin - eosin staining showed no significant changes in cell density and morphology of each group. ④ Reverse transcription - polymerase chain reaction showed no expression of hTERT mRNA in each group of cells. ⑤ telomerase activity was negative. CONCLUSIONS: shRNA targeting hTERT mRNA has no significant effect on the proliferation of normal human bone marrow mesenchymal stem cells. This method may be safe for normal somatic cells that do not express hTERT.