免疫组织化学方法在人/山羊嵌合体人源细胞鉴定中的应用

Immunohistochemical analysis of the human cellsin human/goat chimeras

目的 在胎山羊宫内移植人造血干细胞 (hHSC)获得人 /山羊嵌合体基础上 ,应用免疫组织化学技术分析嵌合体实验山羊肝、肺和肾组织中人源细胞的生长及分化情况。方法 对 7头经荧光激活的细胞分选 (FACS)、荧光原位杂交 (FISH)和分子生物学方法鉴定为阳性的实验山羊 ,在出生后不同时间取其肝、肺和肾组织 ,制作石蜡切片 ,然后采用免疫组织化学的EnVision系统 ,用特异性人增殖期细胞核抗原 (PCNA)单抗、人肝细胞特异性抗原 (HSA)单抗及人 β 2微球蛋白 (β 2M )单抗鉴定这些组织中的人源细胞。结果 7头实验山羊的肝组织中有一定比例的PCNA和HSA抗体阳性染色细胞 ,肺和肾组织内有PCNA及 β 2M抗体阳性染色细胞 ,正常人的肝、肺和肾细胞也呈阳性染色 ,而非hHSC移植的正常山羊的肝、肺和肾组织中未见有阳性染色细胞。结论 胎山羊宫内移植hHSC所形成的嵌合体的肝脏中有人源细胞存活 ,并检测到表达人肝细胞特异抗原的人肝样细胞。免疫组化鉴定的阳性结果与分子生物学等方法的鉴定结果相符。此外 ,在嵌合体山羊肺和肾组织中也发现有人源细胞。

Objective To characterize the proliferation and differentiation of human cells in liver, lung and kidney tissues of the human/goat chimeras produced by in utero transplantation of human hematopietic stem cells (hHSC) into fetal goats using immunohistochemical methods Methods The liver, lung and kidney tissues were obtained from seven positive experimental goats that were previously identified by FACS (fluorescent activated cell sorting), FISH(fluorescent in situ hybridization) and molecular biological analyses at different intervals after birth, and paraffin sections were made Envision system was used for the immunohistochemical analysis Human proliferating cell nuclear antigen (PCNA), human hepatocyte specific antigen (HSA) and human β 2 microglobulin (β 2M) were respectively applied as primitive monoclonal antibodies to characterize the human cells in the above tissues Results PCNA and HSA positive cells with a certain proportion were present in the livers tissue of the seven chimeras PCNA as well as β 2M positive human cells were also found in the lung and kidney tissues of the chimeras Positive stained cells were shown in human liver, lung and kidney tissues No human cells were present in the liver, lung and kidney tissues of the control normal goats Conclusion After the production of human/goat chimeras with the method of in utero transplantation of hHSC into fetal goats, the human cells were subjected to survival in the liver of the chimeras Particularly, human liver like cells that expressed human hepatocyte specific antigen were also identified The results of immunohistochemical analysis were in accordance with that of molecular biological detection In addition, human cells were found in the lung and kidney tissues of the chimeras