牦牛骨骼肌中特异基质金属蛋白酶及其功能相关基因的表达分析

doi:10.16431/j.cnki.1671-7236.2023.05.005

摘要/Abstract

摘要： 【目的】筛选并鉴定在牦牛骨骼肌中特异表达的基质金属蛋白酶(matrix metalloproteinases,MMPs)家族成员及其功能相关基因,以期为研究牦牛骨骼肌结构形成机制提供依据。【方法】采集成年公牦牛臀肌和胸肌,利用组织化学染色法(HE和Masson)分析肌纤维特征和胶原纤维数量;通过实时荧光定量PCR鉴定MMPs和基质金属蛋白酶抑制因子(TIMPs)的mRNA表达量;采用Western blotting技术和免疫组织化学法分析MMPs、Ⅰ和Ⅲ型胶原蛋白的表达量和组织分布;通过STRING网站分析MMPs及其功能相关基因之间的蛋白质互作网络;采用EnzChek^TM明胶酶/胶原酶试剂盒检测牦牛臀肌和胸肌中明胶酶/胶原酶的酶活性差异。【结果】Masson染色和实时荧光定量PCR分析结果显示,牦牛臀肌中胶原纤维面积、MMP-2和MMP-14基因表达量显著低于胸肌(P＜0.05),TIMP-3基因在牦牛臀肌中表达量显著高于胸肌(P＜0.05)。Western blotting鉴定发现,MMP-3、MMP-14、Ⅰ和Ⅲ型胶原蛋白在牦牛臀肌中的表达量极显著或显著低于胸肌(P＜0.01;P＜0.05)。免疫组织化学法分析显示,MMP-2、MMP-9、MMP-14和Ⅰ型胶原蛋白在牦牛臀肌中的表达量显著或极显著低于胸肌(P＜0.05;P＜0.01),而MMP-16在牦牛臀肌中的表达量显著高于胸肌(P＜0.05)。蛋白质功能相关性聚类分析表明,MMP-2、MMP-14、MMP-16、TIMP-2、TIMP-3、COL1A2和COL3A1可聚为一簇,MMP-3、MMP-9和TIMP-1可聚为一簇;臀肌和胸肌中明胶酶/胶原酶的酶活性变化趋势一致,且差异不显著(P＞0.05)。【结论】MMPs家族成员及其功能相关基因在牦牛骨骼肌中广泛表达,且在臀肌和胸肌中的表达量存在差异,为进一步研究MMPs调控牦牛骨骼肌结构形成的作用机制提供了参考依据。

关键词: 牦牛; 骨骼肌; 基质金属蛋白酶(MMPs); 胶原蛋白

Abstract: 【Objective】 This study was aimed to screen and identify the members of matrix metalloproteinases (MMPs) family and their functional related genes specifically expressed in skeletal muscle of yak,in order to provide a basis for studying the formation mechanism of skeletal muscle structure in yak.【Method】 Glutei and pectoral muscle of adult male yaks were collected.The characteristics of muscle fibers and the number of collagen fibers were analyzed by histochemical staining (HE and Masson).The mRNA expression of MMPs and matrix metalloproteinase inhibitors (TIMPs) were detected by Real-time quantitative PCR.Western blotting and immunohistochemistry were used to analyze the protein expression and tissue distribution of MMPs,types Ⅰ and Ⅲ collagen.Protein-protein interaction networks analysis of MMPs and their functionally related genes was constructed by STRING website.EnzChek^TM Gelatinase/Collagenase kit was used to detect the differences in enzyme activities of gelatinase/collagenase between gluteus and pectoral muscles.【Result】 Masson staining and Real-time quantitative PCR analysis results showed that the area of collagen fiber,the expression of MMP-2 and MMP-14 genes in gluteus of yak was significantly lower than that in pectoral muscle (P＜0.05),whereas the expression of TIMP-3 gene was the opposite (P＜0.05).Western blotting results showed that the expression of MMP-3,MMP-14,types Ⅰ and Ⅲ collagen in gluteus were extremely significantly or significantly lower than those in pectoral muscle (P＜0.01 or P＜0.05).Immunohistochemical analysis results showed that the expression of MMP-2,MMP-9,MMP-14 and type Ⅰ collagen in gluteus were significantly or extremely significantly lower than that in pectoral muscle (P＜0.05 or P＜0.01),but the expression of MMP-16 was the opposite (P＜0.05).Protein function correlation clustering analysis showed that MMP-2,MMP-14,MMP-16,TIMP-2,TIMP-3,COL1A2 and COL3A1 could cluster together,and MMP-3,MMP-9 and TIMP-1 could cluster together.The enzyme activities of gelatinase/collagenase in gluteus and pectoral muscle showed a similar tendency,and the difference was not significant (P＞0.05).【Conclusion】 Members of the MMPs family and their functionally related genes were widely expressed in skeletal muscle of yak,and the expression were different between gluteus and pectoral muscle,which provided reference basis for further study on the mechanism of MMPs regulating the structure formation of skeletal muscle in yak.

Key words: yak; skeletal muscle; matrix metalloproteinases (MMPs); collagen

中图分类号:

Q786

武志娟, 柴志欣, 王吉坤, 王嘉博, 钟金城, 信金伟. 牦牛骨骼肌中特异基质金属蛋白酶及其功能相关基因的表达分析[J]. 中国畜牧兽医, 2023, 50(5): 1774-1784.

WU Zhijuan, CHAI Zhixin, WANG Jikun, WANG Jiabo, ZHONG Jincheng, XIN Jinwei. Expression Analysis of Specific Matrix Metalloproteinases and Functionally Related Genes of Skeletal Muscles in Yak[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1774-1784.

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