Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (26): 4130-4136.doi: 10.12307/2021.110
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Chen Shi1, Du Chao1, He Xuemei1, 2, Zhou Xiangyu1
Received:
2020-09-12
Revised:
2020-09-15
Accepted:
2020-10-22
Online:
2021-09-18
Published:
2021-04-26
Contact:
Zhou Xiangyu, MD, Professor, Department of Thyroid Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
E-mail:xiangyuzhou971@163.com
About author:
Chen Shi, Master candidate, Physician, Department of Thyroid Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
Supported by:
CLC Number:
Chen Shi, Du Chao, He Xuemei, Zhou Xiangyu. Effect of proanthocyanidins on proliferation and differentiation of skeletal muscle satellite cells under hypoxic-ischemic condition[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(26): 4130-4136.
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2.1 细胞实验结果 2.1.1 原花青素对缺血缺氧条件下人骨骼肌卫星细胞的影响 首先将人骨骼肌卫星细胞置于生理盐水或不同质量浓度原花青素(5,10,15,20 mg/L)的培养基中培养1 d。当原花青素质量浓度增加到5 mg/L以上时,人骨骼肌卫星细胞的细胞状态变差,提示原花青素质量浓度不超过5 mg/L可能是合适的诱导浓度。 2.1.2 原花青素对缺血缺氧条件下人骨骼肌卫星细胞增殖的影响 骨骼肌肌卫星细胞处于缺氧缺血状态时,细胞被激活,进入细胞周期,进行增殖。为了探索不同质量浓度原花青素对细胞增殖的影响,用生理盐水(对照组)、2.5 mg/L或5 mg/L原花青素(实验组)分别处理细胞,并置于缺血缺氧条件下培养,于不同时间点观察各组细胞增殖情况。 (1)细胞增殖实验:缺血缺氧第1,2,3,4天,原花青素组A值明显高于对照组,差异有显著性意义(P < 0.05);其中5 mg/L原花青素组A值高于2.5 mg/L原花青素组,但差异无显著性意义(P > 0.05),见图1A,说明原花青素组细胞增殖明显高于对照组。 (2)增殖蛋白的表达:缺血缺氧第1天,原花青素组增殖细胞核抗原蛋白表达明显高于对照组,差异有显著性意义(P < 0.05);其中5 mg/L原花青素组中蛋白表达高于2.5 mg/L原花青素组,但差异无显著性意义(P > 0.05),见图1B,C。 (3)细胞周期:缺血缺氧第1天,原花青素组S期所占细胞数量明显多于对照组,说明原花青素组DNA复制活跃;其中5 mg/L原花青素组中蛋白表达高于2.5 mg/L原花青素组,但差异无显著性意义(P > 0.05),见图1D,E,说明原花青素组细胞增殖较对照组活跃。 以上结果提示,原花青素可能促进骨骼肌卫星细胞增殖。"
2.1.3 原花青素对缺血缺氧条件下人骨骼肌卫星细胞分化的影响 当人骨骼肌卫星细胞处于缺氧缺血状态时,细胞被激活,诱导细胞融合及多核肌管形成。为了探索不同质量浓度原花青素对缺血缺氧条件下人骨骼肌卫星细胞分化的影响,用生理盐水(对照组)、2.5 mg/L或5 mg/L原花青素(实验组)分别处理细胞,并且置于缺血缺氧条件下培养3-5 d,观察各组细胞分化程度。 (1)肌原性标志蛋白的表达:3组细胞均有肌原性标志蛋白的表达,且随时间增加表达增加。与对照组相比较,两种质量浓度的原花青素组肌原性相关蛋白MyF5、myoD和myogenin的表达更高(P <0.05);其中2.5 mg/L原花青素组中蛋白表达略高于5 mg/L原花青素组,但差异无显著性意义(P > 0.05),见图2A,B。 (2)细胞免疫荧光观察肌管的形成:Myogenin作为肌源性分化的标志主要于细胞核内表达,见图2C。在正常条件下,3组均无肌管形成。在缺血缺氧条件下,3组细胞均有肌管形成,肌管数目随时间增加而增加,但各组细胞分化程度不一致:与对照组相比,原花青素组多核肌管数量明显增加,生成肌管数目更多(P < 0.05);2.5 mg/L原花青素组较5 mg/L原花青素组肌管数目略多,但差异无显著性意义(P > 0.05)。 综上,说明在缺血缺氧条件下,原花青素可促进骨骼肌卫星细胞肌原性标志基因的表达,从而促进更多肌管形成。"
2.1.4 缺血缺氧条件下参与人骨骼肌卫星细胞分化的信号通路 结果显示,原花青素组p-P38-MAPK信号通路蛋白与肌原性标志基因表达水平较对照组明显增高,见图3A,说明p-P38-MAPK信号通路可能参与了原花青素促进人骨骼肌卫星细胞成肌分化过程。为进一步明确P38-MAPK信号通路在其间的作用,分别设置4组,分别为生理盐水组、原花青素组、生理盐水+P38-MAPK通路抑制剂组、原花青素+P38-MAPK通路抑制剂组,结果显示,原花青素组p-P38-MAPK信号通路蛋白与肌原性标志基因表达水平较生理盐水组明显增高 (P < 0.05),而后两组无明显差异(P > 0.05),见图3B,提示38-MAPK通路抑制剂不仅能抑制原花青素对P38-MAPK 信号通路的激活,还能抑制肌原性标志基因表达,进一步说明原花青素可能通过促进P38-MAPK信号通路的激活,进而诱导骨骼肌卫星细胞的成肌转化过程。"
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