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  

Expression of lentivirus-mediated rat prodynorphin gene in bone marrow mesenchymal stem cells

作　　者：张创强 孙柯[2] 马珏[2] 赵国栋[2] 

机构地区：[1]汕头大学医学院,汕头医学硕士515041 [2]广东省人民医院(广东省医学科学院)麻醉科,广州510080

出　　处：《医学研究生学报》2017年第2期138-144,共7页Journal of Medical Postgraduates

基　　金：广东省省级科技计划项目(2013B021800206)

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(P>0.05)。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(P<0.05),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。Objective Dynorphins have advantages in powerful analgesic effect, high safety, no respiratory depression and no addiction, which is the emphasis of analgesic research at present. The aim of the article was to explore the expression of lentivirusmediated rat prodynorphin gene in bone marrow mesenchymal stem cells（BM-MSCs） and contribute to the subsequent studies on bio- logical analgesia in cancer pain of rat model. Methods BM-MSCs were isolated and proliferated using the adherence screening method, and further identified by flow cytometry, adipogenic and osteogenic differentiation experiments. The PDYN lentiviral vectors in rats were transfected into BM-MSCs after construction. The expression of green fluorescent protein （GFP） was detected under inversion fluorescence microscope and the best multiplicity of infection （MOI） of virus was screened by western blot. There are three groups in the ex- periment： blank group, experimental group （PCDH-CMV-PDYNEFI-copGFP） and empty vector group （PCDH-CMV-MCS-EF1- copGFP）. PYDN gene was determined by qPCR and western blot, while DYN protein was detected by immunochemical method.Results BM-SMCs were in longspindle-shape and fibrocyte-like adherent growth, most in expression of CD29, CD44 and CDg0, and a few in CD45. The oil red-O staining of the induced cells by adipogenic differentiation was positive. The mineralized nodules formed in the induced cells by osteogenic differentiation were orange after alizarin red staining. Flow cytometry detection showed the positive rates of CD29, CD90, CD44 and CD45 were respectively （99.80±0.19） %, （99.62±0.24） %, （96.86±1.27） %, （0.82±0.06） %, while after transfection the positive rates were （99.59±0.34） %, （98.06±1.27） %, （95.23±0.71） %, （10.23±0.59） %, representing no significant difference before and after PDYN transfection. Lentiviral vector of PCDH-CMV-PDYN-EFI-copGFP was successfully construc- ted after the identification of PCR amplification, cloning and sequencing. The

关 键 词：前强啡肽 强啡肽 骨髓间充质干细胞 载体构建 慢病毒 

分 类 号：Q78[生物学—分子生物学]