The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed d...The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells weresuccessfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase Ⅱ and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.展开更多
文摘The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells weresuccessfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase Ⅱ and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.
文摘目的探讨照射剂量和照射时间对电离辐射诱导L02人正常肝细胞(简称"L02细胞")的线粒体编码基因细胞色素c氧化酶(COX)Ⅱ基因表达、三磷酸腺苷(ATP)水平和细胞活力变化的影响。方法 采用2×7析因设计方法。0、1、3、5、8、10、15 Gy剂量60Coγ射线分别照射L02细胞24、48 h后,采用逆转录聚合酶链反应(PCR)和实时荧光定量PCR法检测COXⅡmRNA表达水平,蛋白质印迹法检测COXⅡ蛋白表达水平,ATP发光检测试剂盒和CCK-8试剂盒检测细胞内ATP水平和细胞活力。结果 照射时间与照射剂量对L02细胞的COXⅡmRNA及蛋白表达水平上调、ATP水平升高和细胞活力下降的影响存在交互作用(F值分别为92.43、267.40、6.99、116.11,P<0.05或P<0.001)。在一定照射剂量范围内,照射后24 h COXⅡ蛋白表达水平、照射后24、48 h ATP水平和细胞活力分别与照射剂量存在剂量-效应关系(P<0.05或P<0.01)。结论 照射剂量和照射时间的交互作用影响60Coγ照射诱导的L02细胞COXⅡ基因表达和细胞内ATP水平与细胞活力。
