摘要
目的:观察低氧促进大鼠骨骼肌成肌细胞增殖的作用,并分析CoCl2对成肌细胞增殖的影响。方法:实验于2005-05/2006-07在解放军军事医学科学院基础医学研究所神经肌肉发育研究室完成。①低氧CO2温箱(Forma Scientific,美国);CoCl2(北京化工厂)。②选取4～5周龄Wistar雄性大鼠5只,脱颈处死无菌切取后腿肌群,剪除脂肪和筋膜,制备单细胞悬液。以连续贴壁法筛选大鼠骨骼肌成肌细胞,接种于96孔板进行单克隆培养。采用成肌细胞特异性标志抗原desmin免疫化学染色,鉴定成肌细胞标志蛋白——结蛋白的表达,弃去desmin阴性的细胞克隆,继续培养desmin阳性的细胞克隆,隔天换液1次,7d进行酶消化传代,获得大量扩增的细胞,并可冻存复苏,用于实验。③以1×107L-1接种于20个35mm培养皿中,接种细胞3h后,将培养皿随机数字表法分为5组:常氧对照组、轻度低氧组、中度低氧组、CoCl2组、轻度低氧+CoCl2组,4皿/组。常氧对照组置于体积分数为0.2的O2常规氧气环境中;轻、中度低氧组分别于低氧温箱中维持体积分数为0.1与0.03的O2低氧环境中;CoCl2组向培养皿中加入终浓度为15μmol/L的CoCl2;轻度低氧+CoCl2组向细胞培养皿中加入终浓度为15μmol/L的CoCl2后,放入体积分数为0.1的O2低氧温箱。低氧培养24,48,72h时,各组取出培养皿进行细胞消化离心,倒置相差显微镜下观察细胞生长情况,血球计数板计数法进行细胞计数。④以1×108L-1接种于8个60mm培养皿中,接种细胞3h后,将培养皿随机数字表法分为2组:常氧对照组、轻度低氧组,4皿/组。两组干预措施同细胞计数过程。低氧培养48h时,两组取出培养皿进行细胞消化离心,流式细胞仪检测细胞周期。结果:①骨骼肌成肌细胞的单克隆培养结果:成肌细胞可在体外存活6个月,增殖旺盛期约为3个月,可传代15次以上。单克隆培养2周后,可以由单个细胞长满96孔板中的1个孔,并不断扩增,最终可以得到细胞类型专一单克隆化的成肌细胞。②成肌细胞特异性标志抗原desmin鉴定结果:镜下不同视野desmin阳性率达100%,即培养的成肌细胞单克隆纯度达100%。③细胞计数结果:与常氧对照组比较,低氧培养24,48,72h时轻、中度低氧组均可明显促进大鼠骨骼肌成肌细胞体外增殖,且轻度低氧组作用尤为明显(t=4.98,P<0.001);CoCl2组无明显变化,但轻度低氧+CoCl2组细胞数量显著增加(t=4.62,P<0.001)。④细胞周期分布:与常氧对照组比较,低氧48h时轻度低氧组处于S期的细胞明显增多[(26.67±0.89)%,(65.43±0.23)%,t=2.36,P<0.01],且增殖指数亦显著上升(33.4%,67.1%,t=2.15,P<0.01)。结论:①轻中度低氧可以促进大鼠骨骼肌成肌细胞的增殖,为体外大量扩增细胞提供了新思路。②CoCl2对骨骼肌成肌细胞没有促增殖作用。
AIM： To observe the effects of hypoxia in promoting the proliferation of rat skeletal myoblasts, and analyze the effect of COCI2 on it. METHODS： Th＇e experiment was conducted in the institute of Basic Medical Sciences of Academy of Military Medical Sciences from May 2005 to July 2006. （1） Hypoxic incubator of CO2 （Forma Scientific, American）; COCI2 （provided by Beijing Chemical Plant）. （2） Five male Wistar rats of 4-5 weeks old were executed by cutting off the heads to sterilely obtain the muscle group in hind limbs, remove the fat and the fascia, so as to prepare for single cell suspension. Rat skeletal myoblasts were selected with continuous adherence method for monoclonal cultivation by inoculating into a 96-pore plate. Desmin, the marker of myobiasts, was adopted to identify the expression of specific marker protein of sarcoblast desmin with immunohistochemistry. Desmin negative cell clones were removed, and desmin cell clones were cultured continuously with the culture fluid replaced once every other day. Enzymatic digestion was conducted 7 days later for passage, and massive proliferated cells were obtained, which could be frozen for revitalization and used for the experiment. （3） Cells were seeded in 20 35-mm plates at 1×10^7/L. Three hours after inoculation, the culture plate of cells were randomly divided into 5 groups： Normoxic control group, slight hypoxic group, medium slight hypoxic group, CoCl2 group and slight hypoxia ＋ CoCl2 group with 4 plates in each group. Cells in the normoxic control group were placed in 0.2 02; Cells in slight and medium hypoxic groups were kept in hypoxic incubators of 0.1 and 0.03 02 respectively; Cells in COCI2 group were cultured with COCI2 at 15 μmol/L; Cells in slight hypoxic ＋ CoCl2 group were cultured in COCI2 plate at 15 μmol/L, and then placed 0.1 in hypoxic incubator of 02. At 24, 48 and 72 hours after intervention, cells were digested and centrifuged, and cell growth was observed under inverted phase contrast microscope, and cells were counted with blood cell counting. （4 Cells were inoculated at 1×10^8/L in eight 60-mm plates. After 3 hours, the culture plates were randomly divided into 2 groups： Normoxic control group and slight hypoxic group with 4 plates in each group. The interventions and cell counting were the same in both groups. At the 48^th hour of hypoxic culture, cells were collected, digested and centrifuged in both groups, and the ceil cycle was detected with flow cytometer RESULTS： （1） Monoclonal culture of skeletal myoblasts： Myoblasts could survive for 6 months in vitro with 3 months of high-proliferation and more than 15 times of passages. The stage of vigorous proliferation is 3 months. Two weeks after monoclonal culture, cells could overgrow one well of 96-well from one cell and proliferate continuously, and stabile monoclonal skeletal myoblasts were finally obtained. （2） Identification of the marker protein of myoblasts： The positive rate of desmin of different fields of view was 100% under the microscope. That is to say the purity of monoclonal myoblasts was 100%. （3） Cell counting： Compared with the normoxic control group, in vitro proliferation of rat skeletal myoblasts could be significantly promoted at 24, 48 and 72 hours after culture in hypoxia in both slight and medium hypoxic groups. Moreover, the effect in the slight hypoxic group was much more remarkable （t=4.98,P〈 0.001）. （4） Distribution of cell cycle： Compared with the normoxic control group, cells of S stage at the 48^th hour of hypoxia in the slight hypoxic group were markedly proliferated [（26.67±0.89）%, （65.43±0.23）%, t =2.36,P 〈 0.01], and the proliferation indexes were obviously enhanced （33.4%, 67.1%, t =2.15, P 〈 0.01 ）. CONCLUSION： （1） Slight hypoxia can promote the proliferation of rat skeletal myoblasts and provide a new way for abundantly cultivating cells in vitro. （2） CoCl2 has no effect on the proliferation of skeletal myoblasts.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第3期419-422,426,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
