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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 investigate the effects of chondroitin sulfate (CS) on myoblast proliferation and myotube formation. Methods The myoblasts of the 5th passage were made into a cell suspension with a concentration of 1 × 108 cells / L. The cells were divided into group A (0 μg / ml), B Group (50μg / m L), group C (100μg / m L) and group D (200μg / m L). The morphological changes and the formation of myotubes in each group were observed under inverted microscope on day 4, 5 and 8 after intervention. The proliferation of each group was detected by MTT method on day 6 and the number of myotubes in each group was detected by HE staining on the 8th day. Results Inverted microscope showed that the cells were mononuclear fusiform after adherent, the nucleus was oval and the cytoplasm was compact. When the adherent cells were 90%, the whole myoblasts were swirling and grew long fusiform. Continue to develop myoblast nuclei fusion phenomenon, the formation of multi-nuclear myotubes. On the 8th day, most of the cells in group A had been fused to form myotubes. Some cells in group B and C were partially unfused, and the cells in group D were fused to form the least number of myotubes. The MTT results showed that the absorbance (A) values of living cells in 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, while those in group A were significantly lower than B (P <0.05). There was no significant difference between group B and group C (P> 0.05). Hematoxylin and eosin staining revealed that most of the cells in group A had been fused to form myotubes. A small number of cells in group B and C were not fused to form myotubes. The number of myotubes in group D was the least, and the number of unfused cells was slightly larger. The number of myotubes in groups A, B, C and D was (222.01 ± 30.02), (193.13 ± 42.46), (170.26 ± 11.96) and (136.88 ± 16.78), respectively. There was no significant difference between groups = 1.658, P = 0.252). Conclusion CS significantly promoted the proliferation of myoblasts. The proliferative capacity of CSCs stimulated by 200 μg / mL of CS was significantly better than that of other concentrations.