目的 探讨2型重组腺相关病毒（rAAV2）能否有效转染重型β-地中海贫血患者造血细胞，通过体外途径基因治疗地中海贫血。方法 6只BALB/c裸小鼠分为转染组（n=4）和未转染组（n=2），经X射线照射后，分别移植入经rAAV2 β-珠蛋白病毒转染（MOI=50）或未转染的β41-42/β654杂合子型重型β-地中海贫血流产胎儿造血细胞，转染后第28天和第70天分别处死rAAV2转染小鼠（n=2）和未转染受体小鼠（n=1）。采用 RT-PCR、等位基因特异性PCR（ASPCR）法检测rAAV2介导的人β-珠蛋白基因在小鼠骨髓中的表达，并以高压液相色谱法量化分析受体小鼠外周血中人β-珠蛋白肽链的水平。结果 ①RT-PCR于所有受体小鼠样本中均可检测到人β-actin和人β-珠蛋白基因的表达。②ASPCR检测中，β41-42突变基因稳定表达于所有受体小鼠，β654突变基因仅在移植后第70天受体小鼠样本中被检测到；移植后第28天，rAAV2转染及未转染小鼠体内均可检测到主要来自于β654突变基因的正常β-珠蛋白基因表达；但至移植后第70天，仅rAAV2转染小鼠骨髓样本中仍可检测到rAAV2-β-globin载体介导的正常β-珠蛋白基因表达。③移植后第70天处死的转染小鼠外周血中人β链/α链分别为0.328和0.325，相对未转染组0.135的比值，两者的增加百分比高达144.78%和142.54%。结论 经rAAV2介导基因修饰后的重型β-地中海贫血患者造血细胞在体内可长期稳定表达正常人β-珠蛋白基因，而其分化产生的外周血人红系细胞内β-珠蛋白肽链的合成增加。

Objective To investigate the transfection of recombinant adeno-associated virus 2 (rAAV2) in human hematopoietic stem cells of patients with βthalassemia, and explore the feasibility of ex vivo gene therapy for β-thalassemia. Methods Six BALB/c nude mice pretreated with X-ray were divided into rAAV2-transfected group (n=4) and mock-transfected group (n=2). Isolated human hematopoietic cells from fetus with β-thalassemia major of β41-42/β654 heterozygote were infected or mock infected with rAAV2-β-globin (MOI＝50), and were transplanted into BALB/c nude mice. After transfection for 28 d and 70 d, mice were sacrificed, RT-PCR and allele-specific PCR (ASPCR) were applied to detect the expression of rAAV2-mediated β-globin gene in mouse bone marrow cells, and the levels of β-globin chains in peripheral blood of recipient mice were quantified by HPLC. Results ①The expression of human β-actin and β-globin gene was detected in all recipient mice by RT-PCR. ②By ASPCR, the expression of β41-42 was found in all recipient mice, while β654 in recipient mice on day 70. There was expression of normal β-globin gene mainly from β654 gene in both groups of mice on day 28. However, on day 70,  rAAV-2 mediated normal β-globin gene expression was found only in transfected mice.  ③The human β/α in peripheral blood of transfected mice were 0.328 and 0.325 on 70 d, 144.78% and 142.54% higher than that of mock-transfected mice (0.135). Conclusion rAAV2 vectors genetically modified hematopoietic cells of patients with β-thalassemia major could mediate long-term stable expression of normal β-globin gene in vivo, with significantly elevated expression of β-chains in human erythroid cells in peripheral blood.