造血干细胞移植建立血小板整合素β3胞内段截短体转基因小鼠模型  

Transgenic Mouse Models of the Truncated Platelet Integrin β3 Cytoplasmic Tail Established by Stem Cell Transplantation

作　　者：崔雄鹰[1] 施小凤[1] 黄建松[1] 刘萍[1] 陶岚岚[1] 周玉兰[1] 阮铮[1] 奚晓东[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院,上海血液学研究所,医学基因组学国家重点实验室,上海200025

出　　处：《中国实验血液学杂志》2013年第3期667-673,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目;编号81070414

摘　　要：本研究通过逆转录病毒感染整合素β3缺陷小鼠(β3-/-)骨髓造血干细胞再进行骨髓移植建立β3及其胞内段不同截短体的转基因小鼠模型,为进一步研究整合素β3胞内段在血小板双向信号转导途径中的作用提供基础。将整合素β3野生型、β3-Δ759(缺失R760GT762氨基酸片段)和β3-Δ754(缺失T755NITYRGT762氨基酸片段)的cDNA分别克隆至带有GFP编码基因的MSCV MigR1逆转录病毒载体质粒中,用质粒转染BOSC23包装细胞株包装出带有β3全长及不同突变体的逆转录病毒,再感染β3-/-供体小鼠骨髓细胞,尾静脉注射移植入经致死剂量辐照的野生型C57BL/6小鼠体内,移植后6-8周流式细胞术检测GFP和β3的表达率以评估转基因效率。结果表明,成功建立了空载(Vector)、β3野生型和截短突变β3-Δ759、β3-Δ754等4种小鼠模型,血小板转基因成功的比率(即GFP阳性率)为18%-66%,转基因血小板β3表达可达到β3+/-水平。结论:利用逆转录病毒感染的造血干细胞移植建立血小板转基因小鼠模型是一种高通量快速有效的建立转基因小鼠模型的方法,这为进一步研究整合素β3胞内不同区段对血小板整合素信号转导途径的影响及体内对血小板进行基因操作和基因治疗奠定了基础。This study was purposed to establish the transgenic mouse models of the truncated platelet integrin 133 by retrovirus-infected hematopoietic stem cells （HSCs） transplantation and to provide the basis for further study of the role of integrin 133 cytoplasmic domain in platelet bi-directionai signaling pathways. Wild-type 133, 133-A759 （R76GT762 truncated 133） and 33-A754 （T755NITYRGT762 truncated 133 ） cDNAs were subcloned into MSCV MigR1 retrovirai vector bearing a GFP gene and packaged into infective retrovirus with BOSC23 cell strain. The bone marrow HSCs of the 33 deficient mice were infected by the retroviruses, and transplanted into lethaily-irradiated wild type C57BL/6 mice. GFP positive rate and surface f33 expression of the recipients＂ platelets at 6 to 8 weeks after transplantation were detected by flow cytometry to evaJuate the transgenic efficiency. The results showed that four kinds of transgenic mouse models in- cluding vector, wild-type 133, 133-A759 and 133-A754 were established successfully. GFP positive rates of transgenic mouse platelets ranged from 18% to 66% and the 133 expression of transgenic mouse reached heterozygote （ 133 ＋/- level of mouse）. It is concluded that establishment of transgenic mouse models mediated by retrovirus-infected HSCs trans- plantation is a feasible, fast, and high throughput transgenic approach and laid a solid foundation for further research on the role of integrin β3 cytoplasmic domain for bi-directional signaling of platelets in vivo, and for the gene therapy of platelet disorders.

关 键 词：血小板整合素β3 截短突变 逆转录病毒 转基因小鼠模型 造血干细胞移植 

分 类 号：R331[医药卫生—人体生理学]