人/山羊造血干细胞嵌合模型的分子检测

Molecular identification of the human/goat xenogeneic model

目的 使用分子生物学技术对人 /山羊造血干细胞嵌合模型进行检测。方法 从 11只胚胎期移植了人造血干细胞的山羊外周血中分别抽提DNA和总RNA ,然后分别对 11只山羊DNA进行人CD3 4 基因、GPA基因、SRY基因的PCR扩增 ,对其中 5只山羊的人CD3 4 基因及另 6只山羊的GPA基因表达进行了RT PCR检测 ,并用人α 微卫星序列片段作为探针对 8只实验山羊DNA进行了Southernblot杂交 ,同时还用人Y染色体探针进行荧光原位杂交 (FISH)。结果 经PCR检测 ,所有 11只实验山羊的DNA中均可扩增出人CD3 4 基因和GPA基因 ,5只实验山羊的DNA中可扩增出人SRY基因。South ernblot、RT PCR和FISH的结果亦均显示阳性。结论 通过分子检测证实人造血干细胞已在山羊体内存活。用该方法对人 /山羊造血干细胞嵌合模型进行检测是可行的。

Objective To identify the human hematopoietic stem cells from the human/goat xenogeneic model with molecular techniques. Methods DNA and total RNA were extracted from 11 transplanted goat periphe ral blood cells. Human CD 34 , GPA and SRY genes were amplified with PCR in these samples, and CD 34 , GPA mRNA transcripts were detected using RT PCR in 5 and 6 goat peripheral blood cells, respectively. Southern blot analysis was performed in 8 goat DNAs to detect the human specific α satellite sequence. Meanwhile FISH was also performed to detect the human cells in goat blood with a probe of human Y chromosome. Results Human CD 34 and GPA genes could be detected with PCR in all the 11 goats, and SRY gene did in 5 goats transplanted with hematopoietic stem cells derived from male human babies. Southern blot showed that human specific α satellite sequence was present in 8 goats. By RT PCR,human CD 34 mRNA was detected in 5 experimental goats, GPA mRNA was found in the other 6 experimental goats and FISH assay showed that some peripheral blood cells of the human/goat xenogeneic model were positive. Conclusion Existance of human cells in the recipient goats was identified by molecular detection, which was feasible for the examination of human/goat xenogeneic models.