CAJ | 학술논문

目的探讨转染CD40L cDNA的卵巢癌细胞株OVHM对树突状细胞(DC)成熟、分化的影响及诱导其分泌细胞因子的机制.方法采用脂质体转染法将鼠全长CD40L基因转染人小鼠卵巢癌细胞株OVHM中,用G418筛选出表达CD40L基因的抗药性克隆细胞(CD40L-OVHM).应用免疫磁珠分选并纯化小鼠骨髓DC.将DC与转染和未转染CD40L cDNA的OVHM细胞混合培养,流式细胞术检测DC细胞表面人主要组织相容性复合物Ⅰ类分子(MHC-Ⅰ)、Ⅱ类分子(MHC-Ⅱ)、CD80、CD86和CCR7的表达,逆转录聚合酶链反应(RT-PCR)检测共培养细胞中白细胞介素(IL)-12、IL-23、IL-27、IL-18、γ干扰素(IFN-γ)、Mig基因的表达.结果 DC与CD40L-OVHM细胞混合培养后可形成集落,且上调了DC细胞MHC-Ⅰ、MHC-Ⅱ、CD80、CD86、CCR7的共刺激分子和黏附分子的表达.在DC+CD40L-OVHM组可检测到细胞因子IL-12、IL-23、IL-27、IL-18、IFN-γ、Mig基因的表达,而在DC组、DC+OVHM组、CD40L-OVHM组、OVHM组未检测到这些基因的表达.结论表达CD40L的卵巢癌细胞促进了DC的成熟,诱导了Th1型细胞因子的分泌,这可能是转染CD40L基因的卵巢癌细胞产生抗肿瘤效应的机制之一.
목적 탐토전염CD40L cDNA적란소암세포주OVHM대수돌상세포(DC)성숙、분화적영향급유도기분비세포인자적궤제.방법 채용지질체전염법장서전장CD40L기인전염인소서란소암세포주OVHM중,용G418사선출표체CD40L기인적항약성극륭세포(CD40L-OVHM).응용면역자주분선병순화소서골수DC.장DC여전염화미전염CD40L cDNA적OVHM세포혼합배양,류식세포술검측DC세포표면인주요조직상용성복합물Ⅰ류분자(MHC-Ⅰ)、Ⅱ류분자(MHC-Ⅱ)、CD80、CD86화CCR7적표체,역전록취합매련반응(RT-PCR)검측공배양세포중백세포개소(IL)-12、IL-23、IL-27、IL-18、γ간우소(IFN-γ)、Mig기인적표체.결과 DC여CD40L-OVHM세포혼합배양후가형성집락,차상조료DC세포MHC-Ⅰ、MHC-Ⅱ、CD80、CD86、CCR7적공자격분자화점부분자적표체.재DC+CD40L-OVHM조가검측도세포인자IL-12、IL-23、IL-27、IL-18、IFN-γ、Mig기인적표체,이재DC조、DC+OVHM조、CD40L-OVHM조、OVHM조미검측도저사기인적표체.결론 표체CD40L적란소암세포촉진료DC적성숙,유도료Th1형세포인자적분비,저가능시전염CD40L기인적란소암세포산생항종류효응적궤제지일.
Objective To examine whether the enhanced expression of CD40L cDNA on murine ovarian cancer(OVHM)cells could induee the secretion of Th1 cytokines from dendritic cells(DC).Methods OVHM cells were transfected with the full-length mouse CD40L cDNA by lipofectamineTM 2000and then G418 resistant cells as positive cells were selected.They were examined for their expression of CD40L with flow cytometry.Bone marrow cells were firstly depleted of erythrocytes,maerophages,T and B cells with PE-conjugated magnetic beads,and then cultured in 10% FCS RPMI 1640 medium supplemented with recombinant mouse GM-CSF and IL-4 for 10 days.PKH67-labeled tumor cells were cultured with DC,and then the stained cells were analyzed for the expression of MHC-Ⅰ,MHC-Ⅱ,CD80,CD86,CCR7 in DC with flow cytometry.The expression of p40,p19,p35,p28,EBI3 subunits,IL-18,IFN-γ,Mig gene in cocultured DC-tumor cells were detected by RT-PCR.Resuits The CD40L cDNA was Successfully transfected into OVHM cells.Bone marrow-derived DCs.when cultured with CD40L/OVHM,formed clusters with the tumors and showed an upregulated expression of MHC-Ⅰ,MHC-Ⅱ,CD80,CD86,CCR7.Expression of the IL-12,IL-23,IL-27,IL-18,interferon-γ(IFN-γ)and Mig(monokine induced by IFN-γ)genes was induced in the DCs that were cultured with CD40L/OVHM but not with OVHM cells.Conclusion These data directly showed that the expression of CD40L on ovarian cancer cells facilitates the interaction between DCs and tumors,enhances the maturation of DCs,induces secretion of Th1 cytokines,especially for IL-12,IL-23 and IL-27,which maybe one of the possible antitumor mechanism for CD40L-transfected ovarian cancer cell line.