课题背景：课题得到广东省深圳市科技局赞助，项目编号200304016，赞助经费2.0万元；此外还有深圳市人民医院院配基金2.5万元，暨南大学给3个研究生3年配置的经费1.89万元。相关课题研究目前已发表4篇文章，目前已向深圳市科技局申报成果。

术语解析：骨骼肌卫星细胞是成体骨骼肌中位于肌细胞膜和基膜之间、具有增殖分化潜能的肌源性干细胞，平时处于未分化状态，当骨骼肌受损时能被激活进入细胞周期，然后互相之间或与受损的肌纤维融合，恢复骨骼肌的连续性及功能。

偏倚或不足：①实验在骨骼肌卫星细胞原代取材过程中，存在步骤多、操作时间长、易造成污染等缺点。今后可以将胰蛋白酶和胶原酶两者放在一起消化，摸索出两者联合消化的最佳时间，即最大产出量，最少的时间与步骤。②电转染死亡率过高，且效率低，需继续寻找最佳的电转染参数。③关于转染后的检测略显简单，如果在流式细胞仪检测方面再加上凋亡率可能更具说服力。

摘要
目的：电穿孔的原理是应用短暂高压电脉冲使细胞膜形成纳米级的微孔，外源DNA通过这些微孔或者伴随微孔关闭时膜成分的再分布而直接进入细胞内。实验拟验证电穿孔法介导外源性基因转染大鼠骨骼肌卫星细胞的可行性和转染效率，以获得基因治疗自体移植的载体。
方法：实验于2006-10/2007-09在暨南大学医学院中心实验室完成。①材料：清洁级1~3 d龄SD大鼠3只，购于广东省医学实验动物中心，实验过程中对动物的处置符合动物伦理学标准。基因转染过程应用的pEGFP-N1质粒由本课题组保存。②实验方法：大鼠颈椎脱臼处死，在无菌条件下分离四肢肌组织，剪碎成1 mm×1 mm×1 mm组织块，采用改良酶消化法分离骨骼肌卫星细胞，再以差速贴壁和非连续梯度离心法分两步进行纯化，细胞均匀增殖至培养面80%或局部增殖过度密集时胰酶消化传代。取第3代处于对数生长期的骨骼肌卫星细胞，消化离心后调整细胞数为2.0×107 L-1，取400 μL注入4 mm电击杯中，再加入pEGFP-N1质粒15 μg混匀，将电击杯放入电转槽静置5 mim。电击条件为电容1 050 μF，电压180 V，脉冲时间20 ms，脉冲数2次，脉冲间隔时间1 min，电击缓冲液为不含钙镁的磷酸盐缓冲液。放电后电击杯冰浴，并用不含血清的DMEM清洗，将清洗液转入培养瓶中，4 h后换用体积分数为0.1胎牛血清的完全培养基，于37 ℃、体积分数为0.05的CO2无菌细胞培养箱中培养。同时设不加质粒的空白对照。③实验评估：倒置显微镜下观察细胞生长状况。
免疫细胞化学检测肌动蛋白的表达。随机计数多个低倍视野，计算电穿孔pEGFP-N1质粒转染卫星细胞的效率。绘制细胞生长曲线，并用流式细胞仪测量细胞周期。
结果：①骨骼肌卫星细胞的形态：原代培养48~72 h细胞完全贴壁，呈梭形或纺垂形，长轴平行排列，胞核圆形，核仁明显。5 d时细胞可伸出一个或数个突起。随培养时间的延长，卫星细胞连接成网状。当延迟分瓶或使用分化培养基，相邻的细胞会融合成肌管，继而形成串联，且能看见肌管的跳动。传代后细胞形态呈更加一致的梭形，6 h即完全贴壁，细胞增殖速度明显加快，生长更为旺盛。传至8代卫星细胞出现老化现象。②免疫细胞化学检测：骨骼肌肌动蛋白呈阳性表达。③转染效率：pEGFP-N1质粒电转染8 h后即可看见散在发绿色荧光的卫星细胞，48~72 h达高峰，阳性表达率约35%。④细胞生长曲线及细胞周期检测：电转染细胞接种后第3天进入对数生长期，第7天由于接触抑制导致增殖速度减慢，81.5%处于G0/G1期，19.3%处于S+G2+M期。未转染细胞生长曲线、细胞周期均与其相似。
结论：电穿孔法介导外源性基因表达于大鼠骨骼肌卫星细胞具有较高的转染效率，操作简便，重复性好，且对卫星细胞生物学行为无明显影响。
关键词：骨骼肌卫星细胞；基因转染；绿色荧光蛋白；电穿孔；原代培养

董少红，李江华，罗林杰，高虹，梁新剑，庞新利，张鹏.电穿孔介导外源性基因转染大鼠骨骼肌卫星细胞的可行性[J].中国组织工程研究与临床康复，2008，12(12):2206-2210 [www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2206(ps).pdf]

Feasibility of transfecting exogenous genes into rat skeletal myoblasts by electroporation

Abstract

AIM：The principle of electroporation is to use the high pressure pulsed electrical currents to permeabilize cell membranes into nanometeric micropore and thereby enhance the uptake of the exogenous genes into the cells through these micropores or the redistribution of membrane component by exogenous DNA. This study aimed to verify the feasibility and transfection efficiency of exogenous genes into skeletal myoblasts by electroporation, in order to get genetically modified cells for autograft.

METHODS: The experiment was completed in the Central Laboratory of Medical College of Jinan University from October 2006 to September 2007.①Materials: Three 1-3 days old clean SD rats were bought from the Medical Experimental Animal Centre of Guangdong Province, and the disposal to animals fit the animal ethical standard. The pEGFP-N1 plasmid was stored by this laboratory.②Methods: Rats were sacrificed by luxatio of the cervical vertebra. Under sterile conditions, the skeletal muscles of the extremities were excised from the rats, and were cut into pieces that the volume within 1 mm × 1 mm×1 mm. Rat skeletal myoblasts were obtained by modified enzyme digestion, and were purified by preferential attachment method and discontinuous percoll gradient centrifugation. When the cells grew to 80% confluence or proliferated densely in local site, digestion of the cells was performed with trypsin. The skeletal myoblasts of the third generation at logarithmic growth phase were obtained by digestion and the density were adjusted into 2.0×107 L-1. 400 μL cells were removed into the 4-mm-wide electrode, then mixed with 15 μg pEGFP-N1 plasmid. The electrode was put into the bucket for 5 minutes. Shock conditions were: electric capacity 1 050 μF, voltage 180 V, pulse duration 20 ms, 2 electric pulses, interval pulses 1 minute. The electroporation buffer was phosphate buffer saline without Ca and Mg. After discharge, the electrode with cells and plasmid were put into the ice, and were washed by DMEM without fetal bovine serum. The washed solution was removed into the culture flask, and the DMEM was changed into the complete medium with 10% fetal bovine serum. The cells were cultured in the sterile incubator with 0.05 volume fraction of CO2 at 37 ℃. Meanwhile the blank contrast without plasmid was set up.③Assessments: The morphological character of cells was observed in the inverted microscope. The expression of actin was tested by immunocytochemistry. Several low power fields were selected at random to count the efficiency of transfecting pEGFP-N1 plasmid into skeletal myoblasts. The growth curve of skeletal myoblasts was drawn, and the cell cycle was tested by the flow cytometry.

RESULTS: ①Morphology of skeletal myoblasts: After 48-72 hours primary culture, the cells were completely attached and gradually extended in the shape of spindle with the round nucleus, paralleled along their longitudinal axis and appeared chromatospherite. After 5-day culture, the cells stretched out one or several apophyses. As the culture time prolonged, the cells interconnected into a net. The closed cells could fuse, form the myotubes and then cascaded if delaying the passage or culturing with differential medium. The well-differentiated myotubes gave rise to spontaneous pulsation and contraction. Six hours after cell passaging, the cells completely attached to the wall of the flask, showing obviously accelerated proliferation and more coherent spindle. The cells began aging when passaging to the eighth generation.②Immunocytochemistry showed sarcomeric actin positive expression.③Transfection efficiency: Eight hours after pEGFP-N1 plasmid electroporation, several cells showed green fluorescence, which achieved a peak 48-72 hours after transfection. The positive expression rate was about 35%.④Cell growth curve and cell cycle: On the third day after electroporation, the cells entered logarithmic growth phase but the rat of proliferation slowed down at the 7th day due to contact inhibition. 81.5% in the G0/G1 phase and 19.3% in the S+G2+M phase. The growth curve and cycle of the untransfected cells were similar to the that of the transfected cells.

CONCLUSION: The efficiency of transfecting exogenous genes into skeletal myoblasts is high, with convenient operation and good reproducibility, moreover the electroporation has no obvious influences on the biological behavior of skeletal myoblasts.

Dong SH, Li JH, Luo LJ, Gao H, Liang XJ, Pang XL, Zhang P.Feasibility of transfecting exogenous genes into rat skeletal myoblasts by electroporation.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2206-2210(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2206(ps).pdf]