转染白介素18的骨髓基质干细胞对脑荷瘤大鼠的免疫调节作用

Immunoregulation of bone marrow stromal cells transfected interleukin 18 on intracranial glioma rats

目的:研究转染白细胞介素18的骨髓基质干细胞移植入胶质瘤荷瘤大鼠体内后的免疫调节作用。方法:培养骨髓基质干细胞,转染白细胞介素18,以未转染的骨髓基质干细胞为对照。ELISA方法检测BMSCs/IL-18培养上清液不同时间点的IL-18含量。建立荷瘤模型,进行BMSCs/IL-18移植,而后ELISA方法检测血清IFN-γ、IL-2、IL-10浓度。将BMSCs/IL-18、C6细胞、荷瘤鼠淋巴细胞共同培养观察体外细胞毒作用。流式细胞术检测荷瘤鼠脾细胞淋巴细胞亚群。TUNEL法检测肿瘤内部细胞凋亡情况,抗CD34染色观察微血管密度。结果:BMSCs/IL-18可长期稳定地分泌IL-18,移植到荷瘤大鼠体内后,其IL-2、IFN-γ血清浓度明显增高,IL-10明显下降。流式细胞术检测显示CD4+、CD8+T淋巴细胞比例均有增高。接受BMSCs/IL-18移植的荷瘤大鼠再次种植C6细胞后,可诱发快速的免疫反应。体外细胞毒试验显示,BMSCs/IL-18可明显激活淋巴细胞裂解C6细胞。TUNEL检测显示,在Group2大鼠中,肿瘤内部每视野凋亡细胞数为15.74±6.23,高于其他各组。微血管密度检测显示,Group2大鼠脑肿瘤微血管密度(6.51±2.71)明显低于Group1(13.52±3.06)、Group3(12.67±2.61)及Controlgroup(14.84±1.47)。结论:BMSCs/IL-18可通过诱导Th1细胞因子,抑制Th2细胞因子,激活细胞毒性T淋巴细胞,产生明显的抗肿瘤效果。

Objective：To clarify the mechanism of immunoregulation of bone marrow stromal cells（BMSCs） transfected interleukin 18（IL-18） after their transplantation into glioma bearing rats.Methods：Cultured BMSCs from SD rats were transfected with rmIL-18（BMSCs/IL-18）.Untransfected BMSCs were used as control.Culture supernatant medium was collected for IL-18 examination at different time point by ELISA kit.After establishment of glioma bearing rats followed by BMSCs/IL-18 transplantation,serum concentration of IFN-γ,IL-2 and IL-10 were examined by means of ELISA kit.And their splenocytes were cultured with C6 cells and BMSCs/IL-18 for in vitro cytotoxicity assay,and subsets of splenocyte were detected by flow cytometry.TUNEL was used to clarify apoptosis cells inside glioma and anti-CD34 staining was performed to observe microvessel density（MVD）.Results：BMSCs/IL-18 could secret IL-18 long term and stably.After being transplanted with BMSCs/IL-18,serum concentration of IL-2,IFN-γ in glioma bearing rats＇ increased obviously and serum concentration of IL-10 decreased.Flow cytometry results showed that CD4^＋ and CD8^＋ T lymphocytes increased in the splenocytes.And rechallenge with C6 cells induced a rapid immuno-reaction.In vitro cytotoxicity assays.It was showed that BMSCs/IL-18 could stimulate splenocytes to kill C6 cells obviously.TUNEL assay showed that there were 15.74±6.23 apoptosis cells inside glioma in each view in Group 2,which was much more when compared with other groups.Microvessel density inside glioma in group 2（6.51±2.71） was lower than in group 1（13.52±3.06）,group 3（12.67±2.61） and control group（14.84±1.47）.Conclusion：By means of inducing Th1 cytokine and suppressing Th2 cytokine and activating cytotoxic T lymphocyte,BMSCs/IL-18 induces obviously anti-tumor activity.