硫酸软骨素促进成肌细胞增殖作用的研究

PROMOTION EFFECT OF CHONDROITIN SULFATE ON PROLIFERATION OF MYOBLASTS

目的探讨硫酸软骨素(chondroitin sulfate,CS)对成肌细胞增殖以及肌管形成的影响。方法取第5代成肌细胞制成浓度为1×108个/L的细胞悬液,依据在成肌细胞培养基中添加不同浓度CS,将实验分为A组(0μg/m L)、B组(50μg/m L)、C组(100μg/m L)和D组(200μg/m L)。干预4、5、8 d于倒置显微镜下观察各组细胞形态学变化及肌管形成情况,6 d采用MTT法检测各组细胞增殖情况,8 d行HE染色检测各组细胞肌管形成数。结果倒置显微镜观察示,细胞贴壁后呈单核梭形,细胞核呈卵圆形,细胞质致密;贴壁90%时整体肌母细胞呈漩涡状走行,长梭状生长。继续培养成肌细胞细胞核出现融合现象,形成多核肌管。8 d,A组细胞多数已融合形成肌管,B、C组细胞有部分未融合,D组细胞融合形成肌管数最少。MTT检测示,A、B、C、D组活细胞吸光度(A)值分别为0.045 2±0.004 4、0.540 4±0.096 7、0.660 9±0.143 4、1.069 0±0.039 0,A组显著小于B、C、D组,B、C组小于D组,差异均有统计学意义(P<0.05);B、C组间差异无统计学意义(P>0.05)。HE染色示,A组细胞大多数已融合形成肌管;B、C组有少部分细胞未融合形成肌管;D组细胞融合形成肌管数相对最少,未融合细胞数稍多。A、B、C、D组肌管形成数分别为(222.01±30.02)、(193.13±42.46)、(170.26±11.96)、(136.88±16.78)根,各组间比较差异无统计学意义(F=1.658,P=0.252)。结论 CS显著促进成肌细胞增殖,200μg/m L CS促成肌细胞增殖能力显著优于其他浓度。

Objective To research the effect of chondroitin sulfate （CS） on the proliferation of myoblasts and the formation of myotube. Methods The myoblasts at passage 5 were used to prepare the cells suspension （1×10^8 cells/mL）, and the experiment was divided into 4 groups based on CS concentration in the medium： group A （0μg/mL）, group B （50 lag/mL）, group C （100μg/mL）, and group D （200μg/mL）. The cell morphology and myotube formation were observed by inverted microscope at 4, 5, and 8 days after treatment; MTT was used to detect the cell proliferation at 6 days, and the number of myotube was calculated by HE staining at 8 days. Results Cells showed spindle shape after adherent, with ovoid nuclei and dense cytoplasm under inverted microscope. When the cell adherent rate was 90%, cells arranged in whorls swirled and showed long fusiform adherent growth; and then nuclei fusion resulted in formation of multincleated myotubes. At 8 days, most myoblasts fused to form myotube in group A, but less myotube was observed in groups B and C, and the least myotube in group D. The absorbance （A） values of groups A, B, C, and D were 0.045 2±0.004 4, 0.540 4±0.096 7, 0.660 9±0.143 4, and 1.069 0±0.039 0 respectively, showing significant difference between other groups （P〈0.05） except between groups B and C （P〉0.05）. HE staining observation showed that most myoblasts fused to form myotube in group A, but less myotube in groups B and C, and the least myotube in group D. The number of myotube of groups A, B, C, and D were 222.01±30.02, 193.13±42.46, 170.26±11.96, and 136.88±16.78 respectively, showing no significant difference among groups （F=1.658, P=0.252）. Conclusion CS can significantly promote the proliferation of myoblast, the promotion is the biggest when CS concentration is 200μg/mL.