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目的研究抗肌萎缩蛋白微基因(micro-dystrophin)体外转染成肌细胞后的表达,探讨成肌细胞移植联合基因治疗Duchenne型肌营养不良症的可行性。方法制备感受态大肠杆菌JM109,将pSL139质粒转化感受态大肠杆菌,挑取阳性克隆菌落,培养过夜后按碱裂解法提取质粒,并用PvuⅠ和ClaⅠ双酶切后琼脂糖凝胶电泳。取5～7天龄C57/BL10雄性小鼠10只,体重4～5g,采用多步酶消化与差速贴壁法行成肌细胞原代和传代培养,采用结蛋白(Des-min)免疫荧光检测鉴定。以pSL139质粒转染第3代成肌细胞为实验组,未转染为对照组。转染48h后行RT-PCR检测和细胞免疫荧光检测两组成肌细胞内micro-dystrophinmRNA和蛋白的表达,并计算转染效率。结果 pSL139质粒酶切后琼脂糖凝胶电泳40min,即观察到3.75kb的目的基因和载体部分。倒置相差显微镜观察示,培养24～48h后细胞完全贴壁,形态为三角形或菱形,大小基本一致;4～6d后细胞生长至融合,可以传代。第2代成肌细胞爬片经Desmin免疫荧光检测,多数细胞胞浆可见绿色荧光,纯度在90%以上。pSL139质粒转染成肌细胞48h后,RT-PCR检测示实验组和对照组成肌细胞cDNA在300bp左右均出现条带,且实验组条带亮度高于对照组。细胞免疫荧光检测显示实验组部分成肌细胞胞浆中有绿色荧光,micro-dystrophin阳性细胞表达效率为45%～55%,对照组成肌细胞胞浆中无绿色荧光。结论脂质体能介导pSL139质粒转染成肌细胞并在其胞浆中转录和表达。
Objective To investigate the expression of dystrophin micro-dystrophin after transfection into myoblasts in vitro and to explore the feasibility of combined gene therapy of myoblast transplantation for Duchenne muscular dystrophy. Methods The competent Escherichia coli JM109 was prepared. The pSL139 plasmid was transformed into competent E. coli and the positive clones were picked. After overnight culture, the plasmids were extracted by alkaline lysis and were digested with Pvu I and Cla I for agarose gel electrophoresis. Ten C57 / BL10 male mice, 5 to 7 days old, weighing 4-5 g were used. Primary and subcultured myoblasts were obtained by multi-step enzymatic digestion and differential adherent method. Des-min immunization Fluorescence detection and identification. The third generation of myoblasts transfected with pSL139 plasmid were used as the experimental group and were not transfected into the control group. Forty-eight hours after transfection, the expression of micro-dystrophin mRNA and protein in myoblasts were detected by RT-PCR and immunofluorescence, and the transfection efficiency was calculated. Results After pSL139 plasmid digestion agarose gel electrophoresis 40min, 3.75kb of the gene of interest and the carrier part was observed. Inverted phase contrast microscopy showed that after 24h ~ 48h cells completely adherent, the shape of a triangle or diamond, the size of the same; 4 ~ 6d after the cells grow to confluence, you can pass. The second passage of myoblasts was detected by Desmin immunofluorescence, and the majority of cytoplasm showed green fluorescence with a purity of over 90%. After transfection of pSL139 plasmid into myoblasts for 48h, the mRNA of myoblasts in the experimental group and the control group showed bands around 300bp, and the brightness of the experimental group was higher than that of the control group. Cell immunofluorescence assay showed that some myoblasts in the experimental group had green fluorescence in the cytoplasm, the expression efficiency of micro-dystrophin positive cells was 45% -55%, and the control group had no green fluorescence in the cytoplasm of myoblasts. Conclusion The liposome can mediate the transfection of pSL139 plasmid into myoblasts and its transcription and expression in the cytoplasm.