不同氧环境及葡萄糖浓度对骨骼肌卫星细胞生长的影响

Effect of Oxygen and Glucose Concentration on In-vitro Development of Skeletal Muscle Satellite Cells

研究目的:通过离体实验,观察不同氧环境及葡萄糖浓度对其增殖、损伤的影响,分析相关原因,为骨骼肌的损伤修复提供进一步的实验依据。研究方法:原代骨骼肌卫星细胞取自4周龄雄性SD大鼠双侧的腓肠肌与比目鱼肌。经0.1%II型胶原酶结合0.25%胰蛋白酶消化法,两次差速贴壁纯化得到骨骼肌卫星细胞,使用横纹肌肌动蛋白鉴定骨骼肌卫星细胞的纯度。取第三代骨骼肌卫星细胞,分别置于常氧低糖(21%O2+5.5mM Glu)、常氧高糖(21%O2+25mM Glu)、低氧低糖(1%O2+5.5mM Glu)及低氧高糖(1%O2+25mM Glu)环境下进行培养,3h后采用CCK-8法和LDH法进行增殖、损伤程度的测定。结果:CCK-8的结果显示,常氧高糖实验组的A值显著高于常氧低糖实验组(P<0.01),低氧高糖实验组的A值显著高于低氧低糖实验组(P<0.01),低氧低糖实验组的A值显著高于常氧低糖实验组(P<0.01);LDH的结果显示,低氧低糖实验组的A值显著高于常氧低糖实验组(P<0.01),低氧高糖实验组的A值显著高于常氧高糖实验组(P<0.01)。结论:1)相同氧气浓度下,高葡萄糖浓度较低葡萄糖浓度的培养液更能促进骨骼肌卫星细胞的增殖。2)相同葡萄糖浓度下,低氧浓度可以促进骨骼肌卫星细胞的增殖。3)相同葡萄糖浓度下,低氧会使骨骼肌卫星细胞的LDH释放量增加。

Objective：The effects of oxygen and glucose on the development of skeletal muscle salellite cells were evaluated in vitro, to analyze the reasons for proliferation and injury and repair of SCs. Methods：The primary passage SCs were derived from gastrocnemius muscle and soleus muscle of the Sprague-Dawley rats （4 weeks of age, male）. The SCs were digested with 0. 1% Collagenase II and 0.2 5 % Trypsin, purified with means of differential attachment technique and identified with α-sarcometricactin. The third passage SCs were treated with different doses both of oxygen and glucose ： 21%O2 ＆ 5.5 mM Glu, 21%O2 ＆ 25 mM Glu, 1%O2 ＆ 5.5 mM Glu, 1%O2 ＆25mM Glu. The cell proliferation rate was measured by CCK-8 cell proliferation assay after 3h and the levels of lactate dehydrogenase （LDH） released to the extracellular bathing media had been used for assessing SCs injury. Results：The experimental group of 21% O2 ＆25mM Glu was with a higher proliferation rate than the experimental group of 21%O2 ＆5. 5mM Glu （P〈0. 01）. The experimental group of 1% O2 ＆25mM Glu was with a higher proliferation rate than the experimental group of 1%O2 ＆5.5mm Glu（P〈0.01）. The experimental group of 1%O2 ＆5. 5mM Glu was with a higher proliferation rate than the experimental group of 21%O2 ＆5. 5ram Glu （P〈0. 01 ）. （2）The LDH level of experimental group of 1%O2 ＆5. 5mM Glu was higher than experimental group of 21%O2 ＆5. 5mM Glu （P〈0.0 1）. The LDH level of experimental group of 1%O2 ＆25 mM Glu was higher than experimental group of 21%O2 ＆25 mM Glu （ P 〈 0. 0 1 ）. Conclusion ： Under the same oxygen condition, SCs＇ proliferation rate in DMEM of high-glucose was higher than that in DMEM of low-glucose. Under the same glucose concentration, SCs＇ proliferation rate in hypoxia was higher than that in normal oxygen. Under the same glucose concentration, the level of LDH released was higher in hypoxia than that in normal.