诱导小鼠同种异体反应性T细胞凋亡

Apoptosis Induction of Mouse Alloreactive T Cells

通过 Fas L - Fas途径诱导小鼠同种异体反应性 T细胞 (alloreactive T cells,ARTC)凋亡 ,为减轻异基因骨髓移植 (allo- BMT)后的移植物抗宿主病 (GVHD)探索新的手段。采用磁性细胞分离系统 (MACS)分离 BAL B/ c小鼠 (H- 2 d)干细胞抗原 (Sca) - 1+ 早期造血细胞 (early hematopoietic cells,EHC) ,然后与经丝裂霉素 C去增殖的能产生高滴度表达 m Fas L假病毒的逆转录病毒包装细胞 (PA317)共培养 ,进行 m Fas L c DNA基因转移 ;用含干细胞因子(SCF)、白细胞介素 - 3(IL- 3)和白细胞介素 - 6 (IL- 6 )的完全培养基培养 1周后 ,借助 PCR、RT- PCR、FCM技术检测外源 m Fas L c DNA在扩增的造血细胞 (HC)中的整合与表达效率 ;或与异基因 BAC小鼠 (H- 2 dxb)脾细胞进行单向混合淋巴细胞培养 (OWML C) ,6 d后用 Annexin V/ PI标记和 FCM检测 BAC脾细胞凋亡。结果显示 :用 MACS法成功分离出 BAL B/ c小鼠 Sca- 1+ EHC (1× 10 5个 /只 ) ,纯度为 (89.0± 6 .1) % ;经逆转录病毒法对它进行外源 m Fas Lc DNA转染并扩增 1周后 ,细胞总数达 2× 10 6 个 /只 ;基因组 DNA PCR和 RT- PCR证实 HC整合有 m Fas L c DNA和Neor基因 ,并表达外源基因 m RNA;FCM发现 m Fas L+ HC占 (43.9± 5 .6 ) % ,而用表达

To induce apoptosis of mouse alloreactive T cells by FasL Fas way, the stem cell antigen 1 (sca 1) + early hematopoietic cells (EHC) of BALB/c mouse (H 2 d ) were isolated from bone marrow by using a highgradient magnetic cell sorting system, then cocultured with retroviral packing cells (PA317) that can produce higher titers pseudovirus expressing mouse Fas ligand (mFasL) and was treated by mitomycin C to transfer exogenous mFasL gene. After these mFasL transferred EHCs were cultured in a complete liquid medium containing stem cell factor (SCF), interleukin (IL) 3 and IL 6 for one week, the integration and expression of exogenous mFasL cDNA gene in these expanded hematopoietic cells (HC) were assayed by genomic DNA PCR, mRNA RT PCR and flow cytometry (FCM). After the mitomycin C treated HC were cocultured with the spleen cells of BAC mouse (H 2 dxb ) as a one way mixed lymphocyte culture (OWMLC ) for 6 days, the mixed cell were labeled by Annexin V/PI kit and the apoptosis rate was assayed by FCM.The results showed Sca 1 + EHC from the bone marrow of BALB/c mouse were successfully isolated by MACS (1×10 5 cells per mouse) with the purity being (89 0±6 1)%. After these early HC were transferred with exogenous mFasL gene by retrovirus method and expanded for one week, numbers were increased to 1×10 6 per mouse (about 20 fold). The genomic integration and the mRNA expression of exogenous mFasL and Neor gene in HC were found by genomic DNA PCR and mRNA RT PCR. The mermbrane mFasL expression rate of the HC was(43 9±5 6)% as assayed by FCM, whereas the expression rate of the HC transferred by the pseudovirus expressing neor was only (2 7±1 3) % ( P <0 01). After the mitomycin C treated HC higher expressing mFasL were cocultured with the allogeneic spleen cells from BAC for 6 days, the apoptosis rate of these mixed cells was(51 9±4 7)%, but after the spleen cells were cocultured with allogeneic mitomycin C treated HC having lower mFasL expressing level, the apoptosis rate was only (19 6±4 3)% ( P <0 01). These results sugget the HC transferred exogenous mFasL gene and higher expressing mFasL membrane protein can induce apoptosis of alloreactive T lymphocyte in vitro .