C2C12成肌细胞体外诱导分化为肌管的实验

In Vitro Inducement of C2C12 Myoblasts into Differentiation of Myotubes

【目的】观察低浓度马血清体外诱导成肌细胞C2C12向成熟肌细胞分化、形成肌管的作用,探索定向诱导肌分化的途径及其分子机制。【方法】将C2C12成肌细胞用含不同浓度马血清的高糖DMEM培养基培养,收集细胞,在相差显微镜下观察形态变化,并通过免疫细胞化学(Imminocytochemistry,ICC)及RT-PCR方法分别检测肌性相关基因表达的改变。【结果】①20mL/L和50mL/L的马血清均能促使C2C12细胞形成肌小管,20mL/L浓度马血清诱导效率最好(第7天时肌小管形成率分别为31.4%±2.1%和19.0%±1.6%,P<0.001)。②20mL/L马血清诱导3～4d开始有肌管形成,之后肌管越来越多,到8～9d达高峰(第9天40.2%±1.3%),往后细胞逐渐衰老死亡。③20mL/L马血清诱导C2C12细胞向成熟肌细胞分化过程中,肌相关蛋白分子表达发生变化:结蛋白(desmin)、成肌分化抗原(MyoD)、生肌素(myogenin)和肌球蛋白(myosin)等表达逐渐增强(未刺激时分别约为49.6%±1.1%、23.4%±1.1%、4.8%±1.6%和2.6%±1.5%;第6天时分别提高为80.4%±1.8%、85.4%±1.1%、22.2%±1.1%和26.0%±1.6%;P<0.001);desmin、MyoD和myogenin相应的mRNA分子也可以通过RT-PCR检测到。【结论】①低浓度的马血清能够有效刺激C2C12成肌细胞向成熟细胞分化,以20mL/L浓度效果最好;②C2C12细胞是研究肌细胞发育分化的理想模板;③肌分化发育过程比较复杂,许多因素都可能起影响,研究设计要尽量标准化。

[Objective] To observe the role of low concentration horse serum on differentiation inducement of C2C12 myoblasts into mature myocytes and consequent formation of myotubes, exploring a method for committed myogenic differentiation, and its related molecular mechanisms. [Methods] The C2C12 myoblasts were cultured in high glucose-containing DMEM complemented with various concentration horse serum, then harvested and observed under the phase contrast microscope for the alteration of their appearance, and detected for the expression changes of muscle-associated genes by immunocytochemistry and RT-PCR assays respectively. [Results] （1）Both 20 mL/L and 50 mL/L horse serum concentrations were capable of stimulating myotube formation of C2C12 myoblasts, of which the 20 mL/L horse serum had the higher inducing effect （on day 7, the ratios of myotube formation in both groups are 31.4% ± 2.1% and 19.0% ± 1.6%, respectively, P 〈 0.0001）. （2）Induced with 20 mL/L horse serum for 3-4 days, myotubes started to form. Later on, more and more myotubes appear, and at the peak on 8-9 days （day9, the myotube formation ratio was 40.2% ± 1.3%）. After that, the cells gradually got senile and end in death. （3）In the maturation process from C2C12 to myocytes triggered by 20 mL/L horse serum, expression of muscle-related protein molecules changed： expressions of desmin, MyoD, myogenin, and myosin increased gradually（prior to simulation, their expressions ratios were 49.6% ± 1.1%, 23.4% ± 1.1%, 4.8% ± 1.6%, and 2.6% ± 1.5%, respectively; on day 6, up to 80.4% ± 1.8%, 85.4% ± 1.1%, 22.2% ± 1.1%, and 26.0% ± 1.6%, respectively; P 〈 0.001）, of which the counterpart mRNAs of desmin, MyoD, and myogenin could also be detected by RT-PCR. [Conclusions] （1）Low concentration horse serum is capable of effectively inducing C2C12 myoblats to differentiate into mature myocytes, of which the 20 mL/L horse serum bears the higher effect. （2）C2C12 cells are an optimal model for research on myogenic development and differentiation. （3）The process of muscle development and differentiation is complicated, in which too many factors are involved with. So, the design should be made in a standard way.