| 摘要: |
| 【目的】从牦牛和犏牛睾丸组织中分离培养支持细胞，检测骨形态发生蛋白4基因（BMP4）及其受体基因在牦牛、犏牛睾丸组织及支持细胞中的表达情况，为深入研究BMP4对犏牛精子发生过程的作用提供理论基础。【方法】采集12月龄牦牛和犏牛睾丸组织，使用Trizol法提取各睾丸组织总RNA，利用RT-PCR技术检测BMP4及其受体基因（骨形态发生蛋白受体1B型(ALK6)、骨形态发生蛋白受体2型(BMPR2)、激活素A受体2A型(ACVR2A)、激活素A受体2B型(ACVR2B)）的mRNA表达水平。通过两步酶法消化睾丸组织制备细胞悬液，经差速贴壁、饥饿处理和胰酶消化分离纯化牦牛和犏牛睾丸支持细胞。采用HE染色、油红O染色、碱性磷酸酶（ALP）染色、免疫荧光染色鉴定支持细胞，用Trizol法提取细胞总RNA，RT-PCR检测支持细胞、生精细胞、间质细胞、类肌细胞标志基因和BMP4及其受体基因的mRNA表达水平。采用实时荧光定量PCR检测BMP4及其受体基因在牦牛和犏牛睾丸支持细胞中的差异表达情况。【结果】HE染色结果显示，第3代（F3）支持细胞呈长条形或梭形。油红O染色结果显示，体外培养的细胞胞质中有明显脂滴积累，且在细胞核内可观察到支持细胞特有的双极小体结构。ALP染色结果显示，细胞不着色。免疫荧光染色结果显示，支持细胞标志蛋白GATA 结合蛋白4(GATA4）呈阳性表达。RT-PCR检测结果显示，支持细胞标志基因BMP4、SRY-box转录因子9基因（SOX9）、波形蛋白基因(Vimentin)、Wilm肿瘤抑制基因(WT1)、FAS细胞表面死亡受体基因(FAS）和胶质细胞源性神经营养因子基因(GDNF)存在明显表达，而生精细胞标志基因出局区解旋酶4(DDX4)和类肌细胞标志基因半胱氨酸的血管生成诱导剂61(CYR61)无明显表达，间质细胞标志基因细胞色素P450家族11亚家族A成员1(CYP11A1)则有少量表达。BMP4在牦牛和犏牛睾丸支持细胞中都有表达，且在牦牛支持细胞中的表达量显著高于犏牛支持细胞，而其受体基因ALK6、BMPR2、ACVR2A和ACVR2B在牦牛支持细胞中的表达量显著或极显著低于犏牛支持细胞。【结论】从牦牛和犏牛睾丸组织中分离获得支持细胞，BMP4在牦牛支持细胞中表达量较高，暗示BMP4对犏牛精子发生具有潜在调控作用。 |
| 关键词: 牦牛 犏牛 骨形态发生蛋白4基因 支持细胞 动物遗传育种 |
| DOI： |
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| 基金项目:西南民族大学研究生创新型科研项目（CX2021SZ19）；西南民族大学中央高校基本科研业务费专项资金项目（2020-NZD04）；国家肉牛牦牛产业技术体系项目（CARS-37） |
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| Differential expression of BMP4 in testicular tissue and sertoli cells of yak and cattleyak |
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WANG Mingxiu,ZHANG Peng,CHEN Xuemei,et al
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| Abstract: |
| 【Objective】This study isolated and cultured sertoli cells from yak and cattleyak testicle tissues and detected the expression of bone morphogenetic protein 4 gene (BMP4) and its receptors gene in sertoli cells to provide basis for further research on effect of BMP4 on spermatogenesis of cattleyak.【Method】Testicular tissues of 12-month-old yak and cattleyak were collected,and total RNA of testicular tissues was extracted by Trizol method.The mRNA expression levels of BMP4and its receptor genes including bone morphogenetic protein receptor type 1B（BMPR1B,ALK6）,bone morphogenetic protein receptor type 2（BMPR2）,activin A receptor type 2A（ACVR2A） and activin A receptor type 2B（ACVR2B） were detected by RT-PCR.Cell suspension was prepared by two step enzymatic digestion of testicular tissue.The sertoli cells of yak and cattleyak testis were isolated and purified by differential adhesion,starvation and trypsin digestion.Sertoli cells were identified by HE staining,oil red O staining,alkaline phosphatase (ALP) staining and immunofluorescence staining.Total RNA was extracted by Trizol method,and mRNA expression levels of sertoli cells,spermatogenic cells,mesenchymal cells,myocytoid marker genes,BMP4 and their receptors were detected by RT-PCR.The differential expression of BMP4 and its receptor genes in sertoli cells of yak and cattleyak testis was detected by real-time quantitative PCR.【Result】HE staining showed that the third generation (F3) cells were in elongated or fusiform.The results of oil red O staining showed that lipid droplets accumulated in the cytoplasm of cultured cells,and the characteristic biminimal structure of sertoli cells could be observed in the nucleus.ALP staining showed no staining of medium cells.Immunofluorescence staining showed that GATA binding protein 4（GATA4） was positive.RT PCR results showed that sertoli cell marker genes BMP4,SRY-box transcription factor 9 (SOX9),Vimentin,Wilm’s tumor suppressor gene (WT1),FAS cell surface death receptor (FAS) and glial cell derived neurotrophic factor（GDNF） were significantly expressed,spermatogenic cell marker genes DEAD box helicase 4（DDX4） and myocytic cell marker gene Cysteine rich angiogenic inducer 61 （CYR61） were not significantly expressed,while mesymal cell marker gene cytochrome P450 family 11 subfamily A member 1（CYP11A1） was slightly expressed.BMP4 and its receptor genes were expressed in sertoli cells of yak and cattleyak testis.The results of qPCR showed that the expression of BMP4 in yak sertoli cells was higher than that in cattleyak sertoli cells.BMP4 receptor genes ALK6,BMPR2,ACVR2A and ACVR2B were significantly down-expressed.【Conclusion】The sertoli cells were successfully isolated from the testicular tissues of yak and cattleyak.The expression of BMP4 gene was higher in yak sertoli cells,suggesting that BMP4 had potential regulation effects on cattleyak spermatogenesis. |
| Key words: yak cattleyak BMP4 sertoli cells animal genetics and breeding |
