IGF-1促原代人胚骨骼肌成肌细胞体外增殖与分化的研究

EFFECT OF IGF-1 ON PROLIFERATION AND DIFFERENTIATION OF PRIMARY HUMAN EMBRYONIC MYOBLASTS

目的为更好地将成肌细胞应用于组织工程构建,探讨IGF-1对原代人胚骨骼肌成肌细胞体外增殖与分化的作用与机制。方法通过核素3H-TdR掺入法测定不同浓度IGF-1(1、2、4、8、16和32ng/mL)作用下成肌细胞的每分钟射线脉冲数(count per minute,CPM)值,根据浓度-CPM值量效曲线确定IGF-1促增殖的适宜浓度。在生长培养基中加入该浓度的IGF-1用于实验组细胞,对照组细胞仅接受生长培养基。分别观察实验组与对照组生长曲线,比较两组细胞增殖周期的变化。运用流式细胞技术及核素掺入法测定适宜浓度IGF-1作用下,成肌细胞增殖周期的变化。另选取不同浓度IGF-1(0、5、10、15、20、25、30ng/mL)作用于成肌细胞,观察4d后成肌细胞融合率,通过融合率-IGF-1浓度-量效曲线确定IGF-1促进成肌细胞融合的最佳浓度,并将其用于实验组,而未接受该浓度IGF-1的成肌细胞作为对照组,分别测定两组细胞融合率与磷酸肌酸激酶(creatine kinase,CK)含量并进行比较。结果5ng/mL的IGF-1为促成肌细胞增殖最佳浓度。对照组成肌细胞倍增时间为96h;实验组成肌细胞生长曲线左移,倍增时间缩短为60h。对照组DNA G1、S期与G2M期各亚周期所占时间分别是70.03、25.01与0.96h,实验组为22.66、16.47与20.87h。实验组与对照组成肌细胞CPM峰值出现的时间分别是48h与96h,与生长曲线流式细胞技术测定结果一致。IGF-1促进分化研究显示20ng/mL IGF-1为促成肌细胞分化最佳浓度。实验组成肌细胞融合率较对照组提高30%,CK合成量提高2000mU/mL。结论IGF-1对成肌细胞的增殖与分化均具有促进作用,是通过减少G1期成肌细胞数量,增加S期与G2M期细胞实现的。20ng/mL IGF-1能明显增加成肌细胞融合率CK合成。

Objective To investigate the effect of IGF-1 on the growthof primary human embryonic myoblasts. Methods The method of incorporation of 3H-TdR was used to evaluate the ability of proliferation of myoblasts. The count per minute （CPM） values of myoblasts at different concentrations（1, 2, 4, 8, 16 and 32 ng/mL） of IGF-1 were measured, and dose-effect curves were drawn to choose the optional concentration of IGF-1 to promote the proliferation. Then the experimental group of myoblasts received the addition of the optional concentration of IGF-1 in the growthmedium,the control group just received the growth medium. The flow cytometry was used to detect the cell cycle. The method of incorporation of ^3H-TdR was used to measure the peak-CPM. The myotube fusion rate was measured in myoblasts withdifferent concentrations（0,5, 10,15,20,25 and 30 ng/mL） of IGF-1 in fusion medium, the dose-effect curves were also drawn, so as to decided the optional concentration of IGF- 1 in stimulating differentiation. Fusion medium with optional concentration of IGF- 1 was used in experimental group, and the control group just with fusion medium. The fusion rate of myotube and the synthesis of creatine kinase（CK） were detected in both groups. Results The optional concentration of 5 ng/mL IGF-1 was chosen for stimulating proliferation. It was shown that the time of cell cycle of control was 96 hours, but that of the experimental group was reduced to 60 hours. The results of flow cytometry showed that the time of G1 phase, S phase and G2M phase was 70.03, 25.01 and 0.96 hours respectively in control group, and were 22.66, 16.47 and 20.87 hours respectively in experimental group. The time-CPM value curves showed that the peak-CPM emerged at 96 hours in control group and 48 hours in experimental group, whichwas in agreement with the results of the flow cytometry. The optional concentration stimulating proliferation was 20 ng/mL IGF-1. Compared with control, the quantity of CK was increased by 2 000 mU/mL and the fusion rate was elevated by 30% in experimental group. Conclusion The concentrations of 20 ng/mL IGF-1 can elevat obviously the fusion rate and the quantity of CK. IGF-1 can enhance the proliferation and differentiation of myoblasts via inducing the number of myoblasts at G1 phase and increasing the number of myoblasts at S and G2M phases.