【摘要】 目的探讨粒细胞巨噬细胞集落刺激因子(GM-CSF)与IL-21基因共转染的体外抗肿瘤效应。方法制备pRSC-GM-CSF-IL21重组质粒,再用脂质体将质粒pRSC-GM-CSF、pRSC-IL21和pRSC-GM-CSF-IL2分别转染人卵巢癌SKOV3细胞(依次命名为SKOV3/GMCSF、SKOV3/IL21、SKOV3/GM-CSF-IL21细胞)。以RTPCR方法检测GM-CSF与IL-21表达;倒置相差显微镜观察各组细胞形态学变化;噻唑蓝法测定各组细胞增殖活性;流式细胞仪检测各组细胞细胞周期分布,细胞表面HLA-ABC、主要组织相容性复合体I类相关基因(MICA/B)、细胞间黏附分子1(ICAM-1)的表达,以及各组细胞培养上清液对NK细胞活性的影响。以未转染的SKOV细胞和转染空质粒pRSC的细胞(SKOV3/Neo)为对照。结果经酶切与测序鉴定,pRSC-GM-CSF-IL21重组体构建正确,共转染SKOV3细胞中有目的基因GM-CSF、IL-21的表达。各组细胞的形态学、增殖特性、细胞周期分布及HLA-ABC表达无明显变化。SKOV3、SKOV3/Neo、SKOV3/GM-CSF、SKOV3/IL21和SKOV3/GM-CSF-IL2细胞的MICA/B表达量分别为19.24%±2.31%、31.11%3.76%、37.27%±3.04%、54.97%±4.77%和63.94%±5.90%ICAM-1表达量分别为35.88%±3.06%、37.75%±4.09%、68.59%±4.84%、78.53%±5.78%和84.10%±3.98%;加入各组细胞培养上清液后NK细胞活性分别为31.8%±3.6%42.7%±3.3%、54.7%±7.4%、55.9%±4.4%及63.4%6.4%。SKOV3/GM-CSF-IL21细胞分别与SKOV3、SKOV3Neo细胞比较,3项指标的差异均有统计学意义(P<0.01)结论GM-CSF与IL-21基因共转染可上调SKOV3细胞表面MICA/B与ICAM-1表达,表达产物可增强NK细胞活性,这有助于免疫细胞激活,增强抗肿瘤效应。更多还原

【Abstract】 Objective To explore the in vitro anti-tumor effect of cotransfection of GM-CSF and IL-21 genes. Methods The IL-21 gene was amplified from recombinant pRSC-IL21 by PCR and cloned directly into the recombinant pRSC-GM-CSF to establish a new recombinant plasmid, pRSC-GM-CSF-IL21. And then, pRSC-GM-CSF, pRSC-IL21, and pRSC-GM-CSF-IL21 were respectively transfected into SKOV3 cells by using lipofectamine (SKOV3/GM-CSF, SKOV3/IL21, and SKOV3/GM-CSF-IL21 groups). The proliferation activity of the transfected SKOV3 cells was determined using MTT method. The expressions of GM-CSF and IL-21 genes, cell surface molecular of human leucocyte antigen-ABC (HLA-ABC), MHC class-I-chain-related protein (MIC A/B), and intercellular adhesion molecule-1 (ICAM-1), and distribution of cell cycle were detected by RT-PCR and flow cytometry respectively. The effect of GM-CSF and IL-21 secreted by SKOV3 cells on the cytotoxicity of NK cells was also assayed. Inverted phase contrast microscope was employed to observe the morphology of the cells. Non-transfected SKOV3 cells (SKOV3 group) and the cells transfected with blank plasmid pRSC (SKOV3/Neo group) were set as controls. Results The recombinant pRSC-GM-CSF-IL21 plasmid was identified by endonuclease digestion and DNA sequencing. The expression of GM-CSF and IL-21 genes was detected in the SKOV3 cells after cotransfection. No significant differences among morphology, proliferation activity, distribution of cell cycle, and HLA-ABC expression were detected among the SKOV3, SKOV3/Neo, SKOV3/GM-CSF, SKOV3/IL21, and SKOV3/GM-CSF-IL21 cells. The expressios of MICA/B in the 5 groups of cells were 19.24% ± 2.31%, 31.11% ± 3.76%, 37.27% ± 3.04%, 54.97% ± 4.77%, and 63.94% ± 5.90%, respectively; the expression of ICAM-1 were 35.88% ± 3.06%, 37.75% ± 4.09%, 68.59% ± 4.84%, 78.53% ± 5.78%, and 84.10 ± 3.98%; and the NK cytotoxicity were 31.8% ± 3.6%, 42.7% ± 3.3%, 54.7% ± 7.4%, 55.9% ± 4.4%, and 63.4% ± 6.4% after been treated with the supernatant of the 5 groups (P < 0.001 for all). Conclusion The expressions of MIC A/B and ICAM-1 on the surface of SKOV3 cells can be upregulated by cotransfection of GM-CSF and IL-21 gene, which can increase the cytotoxicity of NK cells, activating immune cells and enhancing anti-tumor effect.更多还原