摘要
目的探讨反转录病毒载体介导的携带小片段５′ＨＳ２增强子的β珠蛋白基因在小鼠体内整合与表达状态，对能否应用于临床地中海贫血基因治疗作出初步的评价。方法用“乒乓”超感染方法筛选具有较高病毒滴度的单向性产病毒细胞系，采用回体（ｅｘｖｉｖｏ）基因转移策略，分析人β珠蛋白基因在小鼠体内的整合及表达。结果病毒重组体的病毒滴度在“乒乓”超感染后有明显提高，最高病毒滴度约为１×１０６ＣＦＵ／ｍｌ；在小鼠ＣＦＵＳ１２（第１２天脾结节）中检测到２个重组体中人β珠蛋白基因的整合，表达水平约为内源性鼠α珠蛋白基因的０５％～５％。在２只长期造血重建小鼠的骨髓、脾、胸腺及另一只小鼠的骨髓及脾中整合有人β珠蛋白基因，在一只重建小鼠的骨髓和脾中有人β珠蛋白基因表达，约为鼠α珠蛋白基因的７％。结论反转录病毒载体介导的人β珠蛋白基因可稳定整合在小鼠骨髓造血干细胞中。
Objective To examine the in vivo properties of retroviral recombinants carrying partially deleted human β globin gene (Δβ) and truncated erythroid enhancer (292 bp and 341 bp of 5′HS2) at the mRNA levels following short and long term reconstitution in mice with infected marrow cells.Methods First ecotropic virus producer cell lines with higher virus titers were isolated using “ping pong” procedures. Then the human β globin gene was transferred into murine hematopoietic progenitor cells and the integration and expression of transferred gene were analyzed by southern blot and RNase protection assay or RT PCR. Results The virus titers of both recombinants increased obviously after “ping pong” procedures. The transferred human β globin gene was detected in murine CFU S 12 and the expression level was about 0.5%～5% of endogenous mouse α globin gene. In 3 of 14 mice surviving long term transplanted with bone marrow cells transduced with high titer virus, bone marrow, spleen and thymus from two mice and bone marrow and spleen from another mouse contained the intact proviral genome. Long term expression of the transferred gene was seen in one mouse at level of 7% of endogenous murine α globin gene.Conclusions The transferred human β globin gene can stably integrate into murine hematopoietic stem cells mediated by retroviral vectors and express in an erythroid specific manner.
出处
《中华医学杂志》
CAS
CSCD
北大核心
1999年第2期121-124,共4页
National Medical Journal of China
基金
国家"八六三"高技术发展计划资助
