慢病毒介导的大鼠前强啡肽基因在骨髓间充质干细胞中的表达

发布时间：2018-06-02 06:35

本文选题：前强啡肽 + 强啡肽　；参考：《医学研究生学报》2017年02期

【摘要】：目的强啡肽镇痛效应强、安全性好、无呼吸抑制和成瘾性等优势,是目前生物镇痛研究的重点。探讨慢病毒介导的大鼠前强啡肽(PDYN)基因在骨髓间充质干细胞(BM-MSCs)中的表达,为后续研究大鼠癌痛模型的生物镇痛提供条件。方法采用贴壁筛选法分离和扩增BM-MSCs,流式细胞学和体外成脂、成骨细胞诱导分化实验鉴定。构建大鼠PDYN慢病毒载体并感染BM-MSCs,倒置荧光显微镜下观察绿色荧光蛋白的表达和Western blot法筛选最适感染复数(MOI);实验分为空白组(BM-MSCs)、实验组(PCDH-CMV-PDYN-EF1-cop GFP)、空载体组(PCDH-CMV-MCS-EF1-cop GFP),采用实时荧光定量聚合酶链反应(qPCR)和Western blot检测PDYN表达变化,采用免疫细胞化学染色法检测强啡肽蛋白表达。结果BM-MSCs呈长梭形纤维细胞样贴壁生长,多数表达CD29、CD44、CD90,少数表达CD45。成脂诱导培养后油红O染色为阳性;成骨诱导培养后出现矿化结节,经茜素红染色呈橘红色。流式细胞仪检测示BM-MSCs细胞中CD29、CD90、CD44、CD45阳性率分别为(99.80±0.19)%、(99.62±0.24)%、(96.86±1.27)%、(0.82±0.06)%,转染PDYN基因后BM-MSCs表面标记CD29、CD90、CD44、CD45阳性率分别为(99.59±0.34)%、(98.06±1.51)%、(95.23±0.71)%、(10.23±0.59)%,转染前后BM-MSCs干细胞特性差异无统计学意义(P0.05)。经PCR扩增克隆测序鉴定慢病毒载体PCDH-CMV-PDYN-EF1-cop GFP构建成功,重组慢病毒滴度为5×106IU/m L。绿色荧光蛋白的表达和Western blot结果示慢病毒MOI=100为最佳病毒感染复数。qPCR、Western blot检测到经慢病毒载体感染后的BM-MSCs中PDYN基因表达均上调(P0.05),免疫细胞化学染色检测显示强啡肽蛋白表达显著增强。结论成功培养BM-MSCs和构建大鼠PDYN基因过表达慢病毒载体,在BM-MSCs中能高效稳定表达PDYN基因和分泌强啡肽蛋白。
[Abstract]:Objective dynorphin has strong analgesic effect, good safety and no advantages of respiratory inhibition and addiction, so it is the focus of biological analgesia research. To investigate the expression of rat prodynorphin (PDYN) gene mediated by lentivirus in bone marrow mesenchymal stem cells (BM-MSCs), and to provide the conditions for further study on the biological analgesia of rat cancer pain model. Methods BM-MSCs were isolated and amplified by adherent screening, flow cytology and adipogenesis in vitro, and osteoblast induction and differentiation were identified. The rat PDYN lentivirus vector was constructed and infected with BM-MSCs, the expression of green fluorescent protein was observed under fluorescence microscope and the optimum number of infected moi was screened by Western blot method. The experiment was divided into blank group, experimental group, PCDH-CMV-PDYN-EF1-cop GFPV, empty vector group, PCDH-CMV-MCS-EF1-cop GFPA, and real-time fluorescence was used. The changes of PDYN expression were detected by light quantitative polymerase chain reaction (QPCR) and Western blot. The expression of dynorphin protein was detected by immunocytochemical staining. Results BM-MSCs grew like long fusiform fibroblasts, most of them expressed CD29, CD4 and CD90, and a few expressed CD45. Oil red O staining was positive after adipogenic culture, and mineralized nodules appeared after osteogenesis induction culture, which was orange by alizarin red staining. Flow cytometry showed that the positive rates of CD29, CD90, CD44, CD45 in BM-MSCs cells were 99.80 卤0.19 and 99.62 卤0.24, respectively. There was no significant difference in the characteristics of BM-MSCs stem cells before and after transfection of PDYN gene. The positive rates of CD2990, CD44 and CD45 were 99.59 卤0.34, 98.06 卤1.51 and 10.23 卤0.59, respectively. There was no significant difference in the characteristics of BM-MSCs stem cells before and after transfection. The construction of lentivirus vector PCDH-CMV-PDYN-EF1-cop GFP was confirmed by PCR amplification cloning and sequencing. The recombinant lentivirus titer was 5 脳 106IU/m L. The expression of green fluorescent protein and Western blot showed that lentivirus MOI=100 was the best viral infection complex number. QPCRN Western blot detected that the expression of PDYN gene in BM-MSCs infected by lentivirus vector was up-regulated (P0.05), and immunocytochemical staining showed dynorphin eggs. White expression was significantly enhanced. Conclusion BM-MSCs and rat PDYN gene overexpression vector can express PDYN gene and secrete dynorphin protein efficiently and stably in BM-MSCs.
【作者单位】：汕头大学医学院;广东省人民医院(广东省医学科学院)麻醉科;
【基金】：广东省省级科技计划项目(2013B021800206)
【分类号】：R96

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