FasL基因转移诱导同种异体反应性T细胞凋亡的实验研究  

Transfect mFasL- cDNA into mouse bone marrow mononucleate cells by liposomes: an efficient way to induce the apoptosis of allogeneic reactive lymphocytes

作　　者：徐之良[1] 邹萍[2] 刘凌波[2] 江钟炎[1] 黄星原[1] 

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

出　　处：《中华儿科杂志》2002年第8期485-488,共4页Chinese Journal of Pediatrics

基　　金：国家自然科学基金资助 (3 9770 767)

摘　　要：目的　研究FasL基因转染鼠骨髓单个核细胞 (BMMNC)后 ,诱导同种异体反应性T淋巴细胞凋亡的效率。方法　采用脂质体法将mFasL cDNA转入Balb/C小鼠造血细胞 ,Western Blot检测其是否表达 ,然后将转染mFasL cDNA的BMMNC与BAC鼠 (Balb/C×C5 7BL/6 ,H 2d×b ,F1)脾单核细胞 (SMNC)混合培养 ,通过混合淋巴细胞反应 (MLR)及流式细胞仪 (FCM)检测SMNC的凋亡效率。结果　基因转移后Western Blot分析Balb/C鼠BMNC显示明显抗FasL抗体识别的条带 (约 4 0 0 0 0 ) ,未转基因的BMMNC无阳性条带发现 ;转基因组进行MLR其每分钟脉冲数 (CPM)值为 5 6 2 8± 186 ,明显低于对照组 (96 74± 78) (t=4 4 86 ,P <0 0 0 1) ;FCM检测转基因组诱导SMNC凋亡率为 (14 8± 0 4 ) % ,明显高于对照组 [(1 8± 0 4 ) % ](t=3 0 1,P <0 0 1)。结论　采用脂质体法能将mFasL cDNA转入鼠BMMNC ,并能有效表达 。Objective The activation and proliferation of the T lymphocytes from the donor bone marrow graft are the cause of graft versus host disease (GVHD) The clearance of the activated T lymphocytes from the bone marrow graft may reduce the morbidity of GVHD In order to find out a new way to clear the activated T lymphocyte from the bone marrow graft, the authors transfected the mouse Fas ligand (mFasL) gene into the Balb/c mouse bone marrow mononuclear cells (BMMNC) Then the transfected cells were incubated with BAC mouse spleen mononucleate cells (SMNC) in order that the activated T lymphocytes became apoptotic Methods The mFasL cDNA in pBillneo mFasL plasmid was identified and transfected into the Balb/C mouse BMMNC via the liposomes The expression products were detected by Western blot The BMMNC carrying mFasL cDNA was incubated with the BAC mouse SMNC Six days later, the mixed cell suspension was collected and labeled with annexin V and propidium iodine (PI), and their apoptosis rate was detected with the flow cytometry (FCM, n =8). By using the Balb/c mouse BMMNC as the stimulator cells and BAC mouse SMNC as the responder cells, the proliferative ability of T lymphocytes from SMNC with the mixed lymplocyte reaction (MLR) was observed Results Western blot showed a distinct band (about 40kd) that came from transfected BMMNC and recognized by Fas ligand antibody, and no band from untransfected BMMNC MLR revealed that the counts per minute (CPM) of transfected cells was 5628±186, which was lower than that of the control (9674±78, t =44 86, P <0 01) The FCM analysis showed the apoptosis rate of T lympholytes induced by the transfected BMMNC was much higher than that of the control (14 7%±0 4% vs. 1 8%±0 4%, t =3 01, P <0 01) Conclusion The mFasL cDNA could be transfected into the mouse BMMNC and could express efficiently The BMMNC transfected with mFasL cDNA could induce apoptosis of the allogeneic reactive T lymphocytes

关 键 词：FASL基因 CD95抗原 膜糖蛋白类 基因转移 T淋巴细胞 脱噬作用 同种异体骨髓移植 

分 类 号：R392[医药卫生—免疫学]