HLA—DR基因反义RNA导入脐血干／祖细胞对HLA—DR抗原表达的影响

目的：探讨组织相容性抗原Ⅱ类（ＭＨＣ－Ⅱ）基因反义ＲＮＡ是否可调控脐血细胞ＭＨＣ－Ⅱ类基因的表达及降低脐血细胞的免疫原性和脐血细胞移植时发生移植物抗宿主反应（ＧＶＨＲ）的程度。方法：应用分子克隆技术构建了含人ＨＬＡ－ＤＲβ基因的逆转录病毒表达载体，经过狭缝杂交、电泳、酶切分析鉴定，获得了ＨＬＡ－ＤＲβ基因反义ＲＮＡ重组体，用ｌｉｐｏｆｅｃｔｉｎ（脂质体）将重组体导入包装细胞ＰＡ３１７。结果：产重组病毒的细胞ＰＡ３１７的病毒滴度可达１×１０５ｃｆｕ／ｍｌ。用逆转录多聚酶链反应（ＲＴ－ＰＣＲ）可从转染的脐血干细胞中扩增出ＤＲ－β基因ｃＤＮＡ，说明反义ＲＮＡ重组体目的基因已有效表达。用流式细胞仪测定转染的脐血细胞ＨＬＡ－ＤＲ抗原阳性细胞率为２８％（对照组为４５％），其抑制率为３８．２％。结论：导入脐血干细胞的ＨＬＡ－ＤＲ基因反义ＲＮＡ重组体成功地降低了其ＨＬＡ－ＤＲ抗原的表达程度，为临床上降低脐血移植的ＧＶＨＲ提供了理论依据和实验基础。

Transduction of antisense RNA of MHC-II gene into CD3+ cord blood stem/progenitor cells inhibits the expression of HLA-DR antigen]

OBJECTIVE: To explore whether the antisense RNA of HLA-DR gene could modulate the expression of the gene and reduce the immunogenicity of cord blood (CB) cells. METHODS: A retroviral vector encoding antisense RNA of HLA-DR gene was constructed and transducted into packaging cell line PA317 by lipofectin. CD34+ hematopoietic stem cells/progenitors were sorted and enriched by MACS. RESULTS: A high titer (1 x 1095) cfu/ml) helper-free,virus producing cell PA317 was obtained. The vector was identified by blot hybridization and mapping of restriction points. The CD3+ CB cells were infected by the supernatant of the viral cultures. The G418-resistant clone was demonstrated to express the antisense RNA of HLA-DR gene by RT-PCR technique. The expression rate of HLA-DR antigen on CB cells was 28%, which was apparently lower than that of the control (45%) with an inhibition rate of 38.25%. CONCLUSION: The introduction of antisense RNA of HLA-DR gene into CB hematopoietic stem cells can successfully decrease the expression of HLA-DR antigen. These studies provide evidences for the prevention and treatment of GVHR in clinical CB transplantation.