成年动物骨骼肌细胞原代培养及转染外源基因的初步研究

1、    成年动物骨骼肌细胞原代培养及转染外源 基因的初步研究        摘要：目的观察损伤对成年动物骨骼肌卫星细胞增殖的影响，以获取基因治疗自体移植的载体。方法以阳离子脂质体介导法将pRSV-LacZ或pRch-TH质粒转入培养肌细胞。结果骨骼肌损伤后，分离出的肌卫星细胞及组织块游离成肌细胞数均显著增高；外源基因能在细胞内表达。结论损伤可诱导成年骨骼肌卫星细胞增殖并促进体外培养肌细胞成活。关键词：肌卫星细胞；骨骼肌；损伤；成年大鼠；猴；LacZ基因；TH基因分类

2、号：Q813文献标识码：A文章编号：0529-1356(2000)01-87PRIMARY CULTURE OF ADULT SKELETAL MUSCLE CELLS ANDEXPRESSION OF TRANSFECTED FOREIGN GENESLÜ Jie(Beijing Institute for Neurosciences, Capital University of Medical Sciences,Beijing 100054,China)ZHAO Chun-li(Beijing Institute for Neurosciences, Capital Univer

3、sity of Medical Sciences,Beijing 100054,China)LU Qiang(Beijing Institute for Neurosciences, Capital University of Medical Sciences,Beijing 100054,China)ZHANG Jin-lu(Beijing Institute for Neurosciences, Capital University of Medical Sciences,Beijing 100054,China)XU Qun-yuan(Beijing Institute for Neur

4、osciences, Capital University of Medical Sciences,Beijing 100054,China)Abstract：ObjectiveTo stimulate the proliferation of the muscle cells from adult animals with a slight injury for primary culture, in order to get the genetically modified cells for autografting. MethodsThe injury of the gastrocne

5、mius muscle of the animal was repeatedly made by a short period of blockage of the popliteal artery with a rubber band and an electro-stimulation was simultaneously given to the muscle. This injured muscle was then taken out from the leg and cultured for 72 hrs. These muscle cells were then transfec

6、ted by a plasmid containing a report gene, LacZ gene, or objective gene, TH gene, by using the method of lipofection in vitro. ResultsThe primary muscle cells were survived properly and grew luxuriantly. An appearance of proliferation of muscle cells was also seen in the culture. Quite a few of thes

7、e muscle cells showed a good expression of LacZ or TH gene with X-gal or TH staining after transfecting. ConclusionThe results indicate that the skeletal muscle cell from an adult animal, including human being, can be cultured in vitro after a slight injury and this provides a useful way to practice

8、 gene therapy.Key words：Muscle satellite cell; Skeletal muscle; Injury; Adult rat; Monkey; LacZ; TH不少研究证明，骨骼肌细胞可以用来作为转基因治疗某些中枢神经系统疾病的运载细胞，因为骨骼肌细胞具有取材方便、免疫源性低和移植后存活时间长等优点1。以往多采用胚胎及新生动物的骨骼肌细胞，很少采用成体骨骼肌细胞作为转基因载体。然而，由于成体骨骼肌细胞可以直接取自病人自身，这就可以避免载体细胞的致瘤性及异种或异体载体的免疫源性。本研究根据损伤骨骼肌激活肌卫星细胞的原理，采取刺激成年大鼠及猴骨骼肌的方法，将骨

9、骼肌卫星细胞激活为成肌细胞2，使之能成为良好的基因治疗的自体细胞移植载体。材料和方法1.损伤取SD大鼠(体重0.40.5kg)及成年恒河猴(体重5kg)，雌雄不限。以橡皮套压迫一侧后肢<"04 (128 bytes)" src="/med/cano/201003/20100308201354828" 15 13>动脉，继以电极连续刺激腓肠肌10 min(8HZ,20V)，松开橡皮套，使血流再灌注2min，重复3次。未受损伤的对侧腓肠肌设为对照组。2.骨骼肌细胞培养损伤72h后，取损伤处及对侧(对照组)的相应腓肠肌各约1mm3，分别剪碎，一部分

10、直接铺于培养瓶中作组织块培养，另一部分以0.2%胰酶(Gibco)、0.1%胶原酶()(Gibco)消化30min，分散成单个细胞，以1×105密度种于培养皿。培养基为DMEM(Gibco)，含10%胎牛血清(fetal calf serum,FCS)(Gibco)和2%鸡胚浸出液(Gibco)。分离细胞培养1周，组织块培养2周后，作肌动蛋白(actin,Sigma)免疫组织化学染色。3.转染分离细胞培养23d后，以阳离子脂质体(lipofectin, Gibco)介导法将携带LacZ基因的pRSV质粒(美国Wisconsin大学提供)或携带TH基因的pRch质粒(本研究室重组)转入

11、培养的成肌细胞内，方法同前3。继续培养约1周后，作X-gal(5-Bromo-4-chloro-3-indoly-b-Dgalactoside,Sigma)组织化学染色或TH免疫组织化学染色。结果和讨论酶消化分离结果显示，损伤组肌纤维松散，有大量肌卫星细胞游离(1)，每条肌纤维上附有约2030个，呈串珠状。分离的细胞数约为3×105/mm3组织。培养细胞贴壁后由圆形变为梭形，胞体饱满，两端尖细(2)，72h后融合为肌管，1周后形成成熟的肌纤维(3，4)，并可见收缩活动。而对照组肌纤维完整，只有少量卫星细胞游离(5)，每条肌纤维上约110个，分离的单细胞数约为1×103/mm

12、3组织。培养细胞贴壁后2d内即死亡。损伤组组织块培养12d后，开始有肌卫星细胞游出，继而由圆形变为梭形，逐渐融合和成熟，且较消化培养的细胞生长旺盛(6)；对照组组织块培养1周仍未见有卫星细胞游离。    1成年猴骨骼肌细胞的酶消化分离(损伤组)：肌纤维松散，有大量游离卫星细胞。标尺示50mFig.1The dissociation of skeletal muscle cell in adult monkey with enzyme:more free satellite cells were found in injury group. Bar=50m

13、    2消化后培养的猴成年骨骼肌细胞：细胞贴壁，排列成排。标尺示50mFig.2The cultured monkey muscle cells after dissociation:the adhesion of satellite cells to the plate. Bar=50m    3成年大鼠骨骼肌细胞：成熟的肌纤维(Actin染色)标尺示25mFig.3The mature muscle fibers in cultured rat muscle cells(Actin staining). Ba

14、r=25m    4成年猴骨骼肌细胞：成熟的肌纤维(Actin染色)标尺示25mFig.4The mature muscle fibers in cultured monkey muscle cells (Actin staining). Bar=25m    5成年猴骨骼肌细胞的酶消化分离(对照组)：肌纤维完整，只有少量卫星细胞游离。标尺示50mFig.5The dissociation of skeletal muscle cells in adult monkey with enzyme: few free

15、satellite cells were found in control group. Bar=50m    6成年大鼠骨骼肌组织块培养：游离细胞多，较消化后培养生长旺盛。标尺示50mFig.6The tissue culture of rat muscle: the free satellite cells were more than that of post-dissociation. Bar=50m转染pRSV LacZ的大鼠肌细胞经X-gal染色呈胞浆蓝染的多核细胞，(7)；转染pRch TH的猴肌细胞经TH染色呈胞质棕染的多核细胞(8)。两

16、者为转染基因表达的结果。    7pRSV-LacZ在lipofectin介导下被转入培养的大鼠肌细胞中，经X-gal组织化学染色使表达LacZ基因的细胞深染，箭头所示为深染的肌细胞，可见多个细胞核。标尺示50mFig.7The cultured rat muscle cells were lipofected with plasmid pRSV-LacZ, the intense stained cells showing LacZ gene expression with X-gal histochemical staining; the arrow

17、 showed an intense stained myotube with three nuclei. Bar=50m    8pRch-TH在lipofectin介导下被转入培养的猴肌细胞中，经TH免疫组织化学染色使表达TH基因的细胞深染，箭头所示为深染的肌细胞，可见多个细胞核。标尺示50mFig.8The cultured monkey muscle cells were lipofected with plasmid pRch-TH, the intense stained cells showing TH gene expression wi

18、th TH immunohistochemical staining; the arrow showed an intense stained myotube with multi-nuclei. Bar=50m本实验结果表明，在同样的消化条件下，损伤组与对照组的骨骼肌细胞培养结果有明显差异。损伤组可见大量肌卫星细胞，其数量约为对照组的300倍，并呈现一定的发育过程。而对照组的肌卫星细胞很少，并很快死亡，这可能与细胞成活需要一定的密度相关。同样，损伤组与对照组的组织块培养情况也有所不同。损伤组组织块周围很快游离出卫星细胞，并迅速增殖成片，还可传代培养，并能分化成熟；而对照组组织块始终处于休眠状

19、态。这种组织块培养较酶消化培养生长更旺盛的现象，可能是组织块本身能提供更多的成纤维细胞而对肌卫星细胞具有营养作用的结果4。发育期的成肌细胞是骨骼肌细胞的前体细胞，其中80%分化为成熟的多核肌细胞；20%进入G0期，在一定条件下可重新进入细胞周期进行分裂增殖5。关于骨骼肌再生的体内外研究表明，缺血、缺氧、挤压、切割和运动疲劳等因素可激活肌卫星细胞，引起骨骼肌再生，形成新的肌纤维4。本实验所使用的电刺激-缺血再灌注损伤看来也可引起肌卫星细胞增殖，并能分化为成熟的肌细胞。培养细胞转染pRSV-LacZ及pRch-TH的结果表明，报告基因或目的基因可融入宿主细胞中，并表达生物学性状，说明体外培养的成年骨骼肌细胞有可能作为基因治疗转基因的载体。作者简介：吕捷(1971)，男，辽宁朝阳人，医学硕士，助教作者单位：吕捷（首都医科大学北京神经科学研究所，北京 100054）赵春礼（首都医科大学北京神经科学研究所，北京 100054）鲁强（首都医科大学北京神经科学研究所，北京 100054）张进禄（首都医科大学北京神经科学研究所，北京 100054）徐群渊（首都医科大学北京神经科学研究所，北京 100054）参考文献