中文摘要：

冷诱导RNA结合蛋白（cold-inducible RNA-binding protein,CIRP）是目前哺乳动物中被广泛研究的冷应激蛋白之一.本研究通过分析CIRP在牦牛（Bos grunniens）睾丸不同发育阶段的表达变化,探究其在牦牛睾丸发育过程中所发挥的功能,为进一步阐明CIRP在高原哺乳动物精子发生调节过程的作用机制提供相关数据.本实验采用RT-PCR法克隆牦牛CIRP基因序列（KF682140）,通过实时荧光定量RTPCR （quantitative Real-time,qRT-PCR）、Western blot及免疫组织化学链霉亲和素-生物素-过氧化物酶复合物（strept avidin-biotin complex,SABC）技术检测1日龄、5月龄、1岁及3岁牦牛睾丸CIRP mRNA及蛋白的表达.RT-PCR反应扩增得到牦牛CIRP mRNA序列编码区长为642 bp,编码213个氨基酸,编码蛋白大小约为23 kD,与家牛有较高的同源性.qRT-PCR和Westem blot实验结果显示,在mRNA及蛋白水平CIRP在各年龄牦牛睾丸中均有表达,且随着年龄的增长呈上升趋势.免疫组织化学染色结果表明,1日龄牦牛睾丸曲精小管上皮观察到较弱免疫阳性反应,阳性细胞为精原细胞;5月龄曲细精管上皮CIRP表达较丰富,阳性细胞仍为精原细胞;1岁曲细精管上皮出现初级精母细胞,并与精原细胞表达阳性;3岁牦牛睾丸发育成熟,曲精小管内精原细胞和初级精母细胞的胞核强阳性表达.综上所述,牦牛睾丸中CIRP表达量随年龄增长而变高,这提示CIRP可能对牦牛睾丸发育及生精细胞的增殖分化具有调控和保护作用.

英文摘要：

Among a significant number of described cold shock proteins, CIRP （cold-inducible RNA binding protein） has been widely researched. In order to explore the function of CIRP in the development of yak testes, and further provide datas for elucidating the regulatory mechanism by which of CIRP acts in spermatogenesis and testicular function of plateau mammalian, we investigated the dynamic expression of CIRP in yak testis during postnatal development by means of Real-time RT-PCR, Western blot and its cellular localization by the immunohistochemical SABC （strept avidin-biotin complex） method. In this study, whole testes were collected from 1-day-old, 5-month-old, 1-year-old and 3-year-old male yaks. The results showed yak CIRP open reading frame contained 642 bp, encoded for 213 amino acids and their molecular weight was estimated as 23 kD. The eDNA sequences of yak CIRP revealed significant homology of 99.8% with Bos taurus, qRT-PCR （quantitative Real-time PCR） and Western blot results showed that CIRP expressed in the testis of different age yaks in mRNA and protein levels, and increased with age. The immunostaining of CIRP was weakly expressed in the seminiferous tubules and occurred in the spermatogonium of 1-day-old yak testes. Then the immunostaining of CIRP was widely localized in the spermatogonium of 5-month-old yak testes. And in 1- year-old yak testes, primary spermatocyte was observed and positive substances were located in the nucleus of spermatogonium and primary spermatocyte. Additionally, in 3-year-old yak testes which developed well, CIRP expressed strongly in seminiferous tubules, and the positive immunostaining occurred in nucleus of spermatogonium and primary spermatoeyte. Nothing was observed in the negative controls. This research suggested that CIRP might play an important role in regulating testieular development and maintaining the proliferation and differentiation of spermatogenic cells.