mFasL基因转移诱导异基因骨髓移植受者获得免疫赦免的实验研究  

Transfected BMNC Induce Syngeneic BMT Recipient Become Immunity Tolerence

作　　者：江端[1] 黄星原[1] 徐之良[1] 刘凌波[2] 邹萍[2] 李爱香[2] 马艳萍[2] 

机构地区：[1]武汉大学人民医院儿科,武汉430060 [2]华中科技大学同济医学院血液病研究所,武汉430022

出　　处：《武汉大学学报（医学版）》2003年第3期205-207,221,共4页Medical Journal of Wuhan University

基　　金：国家自然科学基因资助项目 (No.3 970 76)

摘　　要：目的 :通过导入外源FasL cDNA ,诱导异基因骨髓移植物中针对受者MHC的淋巴细胞凋亡 ,从而防止移植物抗宿主病 (GVHD)的发生。方法 :pBillneo mFasL质粒经酶切鉴定后 ,采用脂质体转移法导入Balb c鼠骨髓单个核细胞 (BMNC) ,体外与BAC鼠骨髓移植物混合培养后 ,输注给经致死量照射的Balb c小鼠 ,通过观察受者存活率、GVHD的表现、肠、肝、皮肤病理组织学检查 ,以观察受者鼠GVHD的发生情况 ,同时观察受者鼠的脾结节以了解其造血功能重建。受者鼠骨髓细胞Y染色体分析 ,以查明供者来源的造血干细胞是否植入。结果 :pBillneo mFasL内mFasL cDNA酶切鉴定提示可以按顺序合成出功能蛋白质 ,采用脂质体法传导入BMNC后 ,Westen Blot显示出抗FasL抗体识别的条带。转基因BMNC与BAC鼠脾单个核细胞 (SMNC)混合培养后 ,经流式细胞仪检测 ,SMNC凋亡率明显高于对照组 [(14 .76 %± 0 .36 ) %vs(1.8± 0 .4 2 ) % ,t=3.0 1,P <0 .0 0 1]。骨髓移植 (BMT)后 ,受者GVHD的发生率实验组明显低于对照组 (4 0 %vs10 0 % ,χ2 =6 .0 0P <0 .0 5 )。Y染色体分析 ,提示有供者来源的造血干细胞植入并增殖活化。移植 10d后受者即有脾结节形成 ,2 0d达正常水平。结论 :采用脂质体转移法 ,能将mFasL cDNA转入鼠BMNC并能有效地表达 。Objective: To prevent the occurrence of graft versus host disease (GVHD),and to transfect mouse Fas Ligand cDNA (mFasL cDNA) into recipient's bone marrow mononecdeit cells (BMNC) to induce the apoptosis of lymphocytes from donor's bone marrow graft. Methods: pBillneo mFasL plasmid was identified to be satisfied with our need,then we tranfected it into Balb/c mouse BMNC by liposomes and incubated the BMNC together with donor BAC mouse bone marrow graft. Six days later, the mixiag cells into Balb/c mouse through the tail vein were infused. The occurrence of GVHD of the recipient mouse according the magnefestion of GVHD was observed. The pathomornhic chang of the nut,liver and skin was also observed. The CFU S were assayed and the Y chromosome from donor BAC mouse were analysed. Results: The mFasl cDNA in pBillneo mFasL plasmid was satisfied with our need. Western Blot showed the stripe from the mFasL cDNA transfected BMNC was identified by Fas ligand antibody. The flow cell meter (FCM) showed that the apoptosis rate of sleen monuleit cells (SMNC) was higher than that of the control[(14.76±0.36)% vs(1.8±0.42)%, [WTBX]t=3.01,P<0.001). After bone marrow transplantation (BMT) the incidence of recipient mouse GVHD was lower than that of the control (40%vs100%,χ 2=6.00,P<0.05). The hematopoietic stem cells was planted in recipient body by Y chromosome analysis. Ten days after BMT,the CFU S was observed in recipient spleen. Conclusion: mFasL cDNA could be transfected into mouse BMNC and could express its products efficiently. The transfected BMNC could induce syngeneic reactive lymphocyte to apoptosis and prevent the GVHD.

关 键 词：mFasL 基因转移 诱导 异基因 骨髓移植 免疫赦免 实验研究 

分 类 号：R457.7[医药卫生—治疗学]