不同浓度地黄低聚糖对离体培养成年大鼠骨骼肌成肌细胞增殖的影响

Effects of various concentrations of rehmannia (地黄) glutinosa oligosaccharides on proliferation of adult rat skeletal myoblasts in vitro

目的观察不同浓度地黄低聚糖(RGOs)对离体培养成年大鼠骨骼肌成肌细胞(SMs)增殖的影响。方法分离成年大鼠SMs,每日观察细胞的生长形态。第3代(培养10 d后)的SMs行α-骨骼肌肌动蛋白免疫组化染色。用无血清培养液[Dulbecco改良Eagle培养基(DMEM)/F12]孵育原代培养3 d的SMs 24 h使细胞同步化,分别以RGOs终浓度为0(对照)、0.156、0.625、2.500和10.000 g/L含体积分数为15%胎牛血清(FBS)的DMEM/F12培养液培养,连续计数6 d。从原代培养到细胞计数,整个过程分别重复3次,取平均值,观察不同浓度RGOs对SMs增殖的影响。结果本研究方法分离的SMs经锥虫蓝染色显示成活率均在90%以上。原代分离提纯的SMsα-肌动蛋白(α-actin)染色阴性;传代2次后(距原代分离10 d),α-actin染色阳性。RGOs可明显促进原代分离的SMs增殖,这种效应反映在指数增长期提前,细胞分裂数量增加,肌管融合速度增加,较低浓度(0.156～2.500 g/L)时,促增殖效应呈剂量依赖性(P均<0.05);但给予高浓度(10.000 g/L)时并不能进一步促进SMs增殖。结论RGOs可以明显改变SMs生长特性,最适干预浓度为0.625 g/L。有望为临床上心肌梗死的中西医结合细胞学治疗提供实验依据。

Objective To evaluate the effects of different concentrations of rehmannia （地黄） glutinosa oligosaccharides （RGOs） on proliferation of autologous skeletal myoblasts （SMs） of adult rats in vitro. Methods SMs were procured by a modified method from adult Sprague-Dawley （SD） rats. The α-actin protein of the 3rd generation SMs was examined by immunohistochemistry. The primary generation was cultured for 3 days, and the SMs had been incubated in Dulbecco＇s modified Eagle＇s medium （DMEM）/F12 culture fluid without serum for 24 hours to synchronize the cells. Afterwards, the SMs were divided into 5 groups, and they respectively contained 0 （control group）, 0. 156, 0. 625, 2. 500, 10. 000 g/L RGOs in DMEM/F12 and 15% fetal bovine serum （FBS）. Cell count had been consecutively done for 6 days to observe the effects of various concentrations of RGOs on proliferation of SMs, the whole course from primary culture to cell count was repeated for 3 times, and the average value of each time was calculated. Results The viability of SMs was more than 90% as assessed by trypan blue staining. The α-actin was negative in the primary cultured SMs, but it turned positive after 10 days （the 3rd generation）. After the stimulation of RGOs, the results suggested SMs showed a proliferative property manifesting a higher cell number, increase of cell mitotic number, increase of myotube fusion rate and increase of growth rate. The effects depended on the RGOs concentration between 0. 156 - 2. 500 g/L （all P〈0. 05）. But high concentration （10. 000 g/L） of RGOs couldn＇t promote the proliferation of SMs. Conclusion The results suggest that RGOs promote the proliferation of SMs significantly. The optimal concentration is 0. 625 g/L. It is expected that this study may provide an experimental basis for the clinical treatment of myocardial infarction treated by cytological method of combination of traditional Chinese and western medicine.