猕猴造血嵌合体检测方法的建立与应用

Establishment and Application of a Method for Assessing Hemopoietic Chimerism in Rhesus after Allogeneic Stem Cell Transplantation

在受体内检测到供体细胞嵌合是判断动物移植模型成功建立的关键。为了研究猕猴移植模型中的造血细胞嵌合状态,本研究利用多聚酶链反应(PCR)扩增猕猴Y特异性序列,建立检测性别不同供受者猕猴移植模型嵌合体的方法;用体外雄性、雌性DNA混合稀释实验研究该方法的敏感性;用染色体分析法验证该方法的准确性,并对1例异基因外周血造血干细胞移植的猕猴和1例间充质干细胞(MSC)异体输注的猕猴进行嵌合监测。结果表明:雄性猕猴DNA扩增产物长度为176bp,雌性无扩增产物,敏感性达0.05%(雄性/雌性DNA比例)。对异基因外周血造血干细胞移植的猕猴,用Y特异性序列分析法和性别染色体检测法于移植后7及14天均检测到雄性供者嵌合。输注非亲缘雄性MSC的雌性猕猴,输注后1小时的外周血和30天的骨髓用本方法检出雄性供体嵌合,结果性别染色体检测未发现雄性供体嵌合。结论:采用PCR法扩增猕猴Y特异性序列检测猕猴移植模型嵌合体方法简便,样本需要量少,敏感性高。该法可用于猕猴各种异体器官移植和细胞输注实验的嵌合状态评价。

Monitoring engraftment of donor cells after allogeneic transplantation 1s the key of assessing successful establishment of animal transplantation model. The purpose of this study was to establish a method for analysis of chimerism in rhesus transplantation model. Y-specific sequence in rhesus was amplified by the polymerase chain reaction（ PCR）, method for analysis of chimerism in rhesus after sex-mismatched transplantation was established ; the feasibility and sensitivity of the approach were tested by using serial DNA mixtures of sex-mismatched individuals ; the accuracy of results was confirmed by chromosome karyotype analysis simultaneously; Chimerisms of one rhesus received allogeneic stem cell transplantation and the other received mesenchymal stem cells （MSC） transfusion were detected by this method. The results showed that a 176 bp long sequence of PCR product was gained in male rhesus,while no product was gained in female rhesus. The sensitivity of this method was up to 0.05% （male/female DNA ratio）. Male donor chimerism were found on day 7 and 14 after allogeneic stem cell transplantation by Y-specific sequence and chromosome karyotype analysis. Otherwise, male donor chimerism was found in peripheral blood at 1 hour and in bone marrow on day 30 after MSC transfusion by this method, but no male donor chimerism was found after MSC transfusion using chromosome karyotype analysis. In conclusion, this rapid, sensitive approach can used to assess chimerism in experiments of rhesus alloorgan transplantation and cell transfusion.