转IL-2基因的骨髓基质细胞对骨髓移植后免疫功能重建的促进作用

Improved effect of bone marrow stromal cell line transfected IL-2 cDNA on the im-mune reconstitution in allogeneic bone marrow transplantation

目的：探讨了用逆转录病毒载体转入小鼠IL-2基因的基质细胞系QXMSCI对异基因骨髓移植后免疫功能重建的促进作用。方法：将小鼠IL-2cDNA片段连接到逆转录病毒载体PLXSN上，构建重组逆转录病毒载体PL2SN（含小鼠IL-2c-DNA）。用磷酸钙共沉淀法将PMSN转入单嗜性包装细胞系CRE，获得G418抵抗细胞株后，以上清感染双嗜性包装细胞系CRIP，G418筛选后获得高滴度的包装细胞系CRIPIL-2。感染NIH3T3细胞测定CRIPIL-2培养上清病毒滴度为3．4 x10^5cfu/ml。感染骨髓基质细胞系QXMSC1（H－2d），C418筛选，有限稀释法得10个单克隆细胞株，选择表达IL-2最高的细胞林为实验用细胞QXMSC1IL-2，用于以后的实验。供体小鼠BALB/c（H-2d）骨髓以抗T细胞单抗anti-Thy1．2加补体去除骨髓中T细胞，受体小鼠C57BL/6（H－2b）经γ射线致死剂量照射后，输入供体骨髓1×10~7/只，同时输入基质细胞QXMSClIL-2 5×10~5/只。在第30天、60天检测BMT小鼠脾细胞对LPS、ConA的反应，脾细胞产生IL-2的能力，BMT小鼠产生抗体生成细胞（PFC）的能力及产生DTH的能力。流式细胞仪检测了 QXMSClIL-2对BMT小鼠 T细胞亚群的影响。结果：电泳及酶切鉴定证实构建的PL2SN质粒。QXMSC1IL-2细胞系IL-2的分泌量为857 U（1×10~6·24h）。异基因骨髓移植，

Abstract Objective:To observe whether bone marrow stromal cell line QXMSC1 engineered to secreted IL-2 (QXMSC1IL-2) can accel-erate the immune reconstitution in T cell depleted allogeneic bone marrow transplantation. Methods-.The mIL-2 retrovirus vector PL2SN was construction with the vector pCDmIL-2, which contains mIL-2 cDNA, and a retrovirus vector PLXSN. The ecotropic virus-packaging cell, CRE, was transduced with recombined retrovirus vector PL2SN by calcium phosphate coparticipation and G418-resistance clone was selected. The amphotropic virus packaging cell line, CRIP, was transfected using CRE supernatant. CRIP G418-resistance clone was selected. The PL2SN retrovirus tilers were analyzed with transduced NIH3T3 cells. Clone CRIPIL-2 (mIL-2/CRIP) had retrovirus tilers 3.4 x 10~5 cfu/ml. QXMSC 1 IL-2 cell was constructed with transfected using the CRIPIL-2 cell supernatant, subclone was selected by limiting dilution ways and the highest secreted cells was selected by IL-2 activity assay and used in followed experiments. The QXMSC 1IL-2 was cotransplanted with T cells depleted allogeneic bone marrow. Lymphocyte proliferation to ConA and IPS, the IL-2 produced of splenocytes, plaque-forming cell (PFC), delayed type hypersensitivity (DTH) were examined in post transplantation 30,60 days, respectively. T subset cells, the radio of CD4+ /CD8+ was also detected by flow cytometer. Results: The results showed that lymphocytic proliferation to ConA and LPS, the IL-2 produced of spleno-cytes, the ability of DTH and PFC were increased by coinfused stromal cells QXMSC 1 IL-2 on 30,60 days after bone marrow transplantation. The radio of CD4+/CD8+ was improved at same time. Conclusion: These data demonstrated that the bone marrow stromal cell line QXMSC1 transduced with IL-2 (QXMSClIL-2) can improved the immune reconstitution in allogeneic bone marrow transplantation.