中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (14): 2228-2234.doi: 10.3969/j.issn.2095-4344.1661

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

筛选适合酸中毒小鼠骨骼肌组织蛋白提取的裂解液

罗旭光1，藏好晶1，孙 鹏1，曹锡梅2   

  1. 山西医科大学，1微生物与免疫学教研室，2组织学与胚胎学教研室，山西省太原市 030001
  • 收稿日期:2019-01-10 出版日期:2019-05-18 发布日期:2021-04-28
  • 通讯作者: 曹锡梅，副教授，硕士生导师，山西医科大学组织胚胎学教研室，山西省太原市 030001
  • 作者简介:罗旭光，男，1979年生，山西省大同市人，汉族，2006年山西医科大学毕业，硕士，实验师，主要研究领域为真核细胞基因调控机制。
  • 基金资助:

    山西省自然科学基金青年基金(2014021028-1)，项目负责人：曹锡梅；山西医科大学科技创新基金(01201401)，项目负责人：曹锡梅；山西医科大学基础医学院331基础医学科技培植基金计划(201413)，项目负责人：曹锡梅

Choosing appropriate lysis buffers for protein extraction from acidotic mouse skeletal muscles

Luo Xuguang1, Zang Haojing1, Sun Peng1, Cao Ximei2   

  1. 1Department of Microbiology and Immunology, 2Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Received:2019-01-10 Online:2019-05-18 Published:2021-04-28
  • Contact: Cao Ximei, Associate professor, Master’s supervisor, Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • About author:Luo Xuguang, Master, Experimentalist, Department of Microbiology and Immunology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Supported by:

    the Natural Science Foundation of for the Youth of Shanxi Province, No. 2014021028-1 (to CXM); the Science and Technology Innovation Foundation of Shanxi Medical University, No. 01201401 (to CXM); the 331 Early Career Researcher Grant of Basic Medical School of Shanxi Medical University, No. 201413 (to CXM)

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摘要：

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文题释义：
裂解液：裂解是使样品中的核酸游离在裂解体系中的过程，纯化则是使核酸与裂解体系中的其他成分，如蛋白质、盐及其他杂质彻底分离的过程。目前常用的裂解液配方基本都包括缓冲液体系、表面活性剂及蛋白酶抑制剂，上述各成分协同作用保证高效抽提蛋白质的同时尽量避免对蛋白质的破坏。
核糖体蛋白S6(rpS6)：调节细胞增殖和葡萄糖稳态，是核糖体40S亚单位的组成部分，其磷酸化参与抑制细胞增殖，可能是通过降低蛋白质合成速率的途径。
 
 
背景：RIPA Buffer对不同组织细胞总蛋白的提取效率有差异，并不通适于所有的组织样本。
目的：筛选适合酸中毒小鼠骨骼肌组织蛋白提取的裂解液，为研究骨骼肌萎缩奠定基础。
方法：3月龄健康雄性C57BL/6小鼠20只，体质量25-30 g，由山西医科大学实验动物中心提供。麻醉后脱臼处死小鼠，分离获取下肢腓肠肌。实验分为2组：HCl诱导酸中毒组每日定时供给10 g混合10 mL 0.4 mol/L HCl的饲料；对照组饲料中混合同等量的H2O，连续7 d。比较RIPA Buffer、Original Buffer和JP Buffer对酸中毒小鼠骨骼肌组织蛋白的提取效果；蛋白免疫印迹实验检测AKT、p-AKT (Thr308)、rpS6和p-rpS6 (Ser235/236)的表达；RT-qPCR定量分析GLUT4的表达。

结果与结论：①3种裂解液对骨骼肌组织蛋白的提取能力有差异，JP Buffer提取蛋白产量高，但是目的蛋白信号并不强；RIPA Buffer提取蛋白产量相对偏低；Original Buffer提取足量骨骼肌组织蛋白，蛋白免疫印迹显示目的蛋白条带清晰；②蛋白免疫印迹实验评分显示，Original Buffer总分高于其他2种裂解液；③蛋白免疫印迹实验结果显示，酸中毒组和对照组AKT、rpS6磷酸化程度均无变化；④RT-qPCR定量分析显示，酸中毒组和对照组RNA水平GLUT4表达无变化；⑤结果提示，Original Buffer是提取骨骼肌组织蛋白的最佳裂解液；短期HCl诱导酸中毒组AKT信号通路未激活，延长酸中毒时间是否会激活该信号通路值得研究。针对不同实验样本选择合适的裂解液是保证蛋白免疫印迹实验结果可信度的前提。

ORCID: 0000-0001-9700-1680(罗旭光)

 

关键词: 裂解液, SDS, RIPA Buffer, Original Buffer, JP Buffer, Western blot, RT-qPCR, 骨骼肌, 小鼠

Abstract:

BACKGROUND: RIPA Buffer exhibits different extraction efficiencies of proteins of cells and tissues, which is not appropriate for all samples. 

OBJECTIVE: To achieve an optimal lysis buffer for skeletal muscle protein extraction in mice of acidosis, and to provide basis for studies on skeletal muscle atrophy.
METHEDS: Twenty male healthy C57BL/6 mice, aged 3 months, weighting 25-30 g, were provided by Laboratory Animal Center of Shanxi Medical University. The mice were sacrificed after anesthesia, and the gastrocnemius muscle of lower extremity was isolated. There were two groups: acidosis group was given 10 g of feed mixed with 0.4 mol/L hydrochloric acid (10 mL), and control group received 10 g of feed mixed with same volume of water, for 7 consecutive days. The effect of RIPA Buffer, Original Buffer and JP Buffer on the skeletal muscle protein extraction in mice of acidosis was compared. The expression levels of AKT, p-AKT (Thr308), rpS6 and p-rpS6 (Ser235/236) were detected by western blot assay. GLUT4 mRNA expression was examined by RT-qPCR.
RESULTS AND CONCLUSION: (1) Different buffers generated different protein-yields. The protein yield was highest in JP Buffer, but the target protein signal was not high. The protein yield was low in RIPA Buffer. Original Buffer could extract sufficient proteins, and had clear band detected by western blot assay. (2) Western blot assay scores in Original Buffer were higher than those of other two buffers. (3) Western blot assay results showed that the extent of phosphorylation in both groups showed no significant changes. (4) GLUT4 mRNA expression level examined by RT-qPCR showed no significant changes in both groups. (5) These results indicate that Original Buffer is optimal lysate of skeletal muscle protein extraction. Inactivated AKT signaling pathway is seen in the short-term hydrochloric acid-induced acidosis group, so whether lengthening acidosis time can activate the signaling pathway. Selecting the optimal lysis buffer for different samples is premise to ensure western blot assay results.  

Key words: Acidosis, Blotting, Western, Muscle, Skeletal, Tissue engineering

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