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目的:探讨2型重组腺相关病毒(recombin antadeno-associated virus 2,rAAV2)载体介导人β-珠蛋白基因转染地贫患者造血细胞治疗β-地中海贫血的可行性。方法:分离β41-42/β654杂合子型重型β-地贫流产胎儿造血细胞,经rAAV2-β-globin病毒转染(MOI=50)后移植入经X射线照射的BALB/c裸小鼠体内,分别于移植后14d、21d处死受体小鼠,RT-PCR及等位基因特异性PCR法检测人珠蛋白基因在受体小鼠体内的表达。结果:RT-PCR方法于3只受体小鼠骨髓样本中成功检测到人β-actin和人β-珠蛋白基因的表达,而21d处死的转染与对照小鼠外周血样本中未检测到人β-珠蛋白基因表达;等位基因特异性PCR方法在所有受体鼠体内同时检测到β41-42和β654突变基因,以及正常β-珠蛋白基因的表达,但rAAV2-β-globin转染组小鼠体内正常人β-珠蛋白基因表达水平明显高于对照组。结论:rAAV2可有效转染β41-42/β654杂合子型重型地中海贫血患者造血细胞,并通过exvivo途径介导β-珠蛋白转基因的体内表达,提高红系细胞内正常β-珠蛋白基因的表达水平。
Objective: To investigate the feasibility of treatment of β-thalassemia with hematopoietic cells from type 2 thalassemia gene transduced by recombin antadeno-associated virus 2 (rAAV2) vector. Methods: Fetal hematopoietic progenitor cells from β-thalidomide beta-thalassemia major were isolated and transplanted into BALB / c nude mice irradiated by X-ray after transfected with rAAV2-β-globin virus (MOI = 50) The recipient mice were sacrificed 14 days and 21 days after transplantation. The expression of human globin gene in recipient mice was detected by RT-PCR and allele-specific PCR. RESULTS: The expression of human β-actin and human β-globin gene was successfully detected in 3 recipient mouse bone marrow samples by RT-PCR method. However, the expression of human β-actin and human β-globin gene was not detected in peripheral blood samples of 21d- Human β-globin gene expression; allele-specific PCR method simultaneously detected in all recipient rat β41-42 and β654 mutant gene, and normal β-globin gene expression, but rAAV2-β-globin transfection The normal human β-globin gene expression level in the group of mice was significantly higher than that in the control group. CONCLUSION: rAAV2 can effectively transfect the hematopoietic cells of patients with β41-42 / β654 heterozygous thalassemia major and enhance the expression of normal β-globin gene in erythroid cells by mediating the expression of β-globin transgene in exvivo pathway Level.