CADM1基因修饰骨髓间充质干细胞对口腔癌细胞增殖和免疫因子的影响及机制  

Effects of CADM1gene-modified bone marrow mesenchymal stem cells on the proliferation and immune factors of oral cancer cells

作　　者：王静[1] 许小婷[1] 王守儒[1] WANG Jing;XU Xiao-ting;WANG Shou-ru(Department of Stomatology,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou450002,China)

机构地区：[1]河南省中医院口腔科,河南郑州450002

出　　处：《中华肿瘤防治杂志》2022年第20期1452-1459,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20190603)。

摘　　要：目的探讨细胞粘附分子1(CADM1)修饰的骨髓间充质干细胞(BMSC)对口腔癌细胞生长与免疫相关指标的影响及机制。方法分离培养5只BALB/c小鼠BMSC,分为腺病毒感染CADM1-BMSC组及阴性对照腺病毒感染NC-BMSC组;收集病毒感染BMSC的培养上清液,将人口腔癌细胞HSC3分为空白对照组、BMSC组、NC-BMSC组和CADM1-BMSC组,CCK-8法、EdU染色和细胞克隆形成实验检测细胞生长情况,乳酸脱氢酶(LDH)释放法检测NK-92MI对细胞的杀伤作用,实时荧光定量PCR(qRT-PCR)检测细胞免疫因子肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)、转化生长因子-β1(TGF-β1)表达水平,蛋白质印迹检测细胞负性调节区域因子1(NCR1)与自然杀伤因子蛋白46(NKP46)的表达水平,流式细胞术检测细胞NKP46表达水平。结果成功分离到小鼠源BMSC,经CADM1腺病毒感染的BMSC可见明显的绿色荧光蛋白表达。对照组、NC-BMSC组、CADM1-BMSC组的CADM1mRNA相对表达量分别为1.00±0.05、1.01±0.06和3.65±0.32,CADM1-BMSC组上升,差异有统计学意义,F=32.145,P<0.001;各组CADM1蛋白相对表达量分别为0.42±0.04、0.40±0.03和1.28±0.09,CADM1-BMSC组上升,差异有统计学意义,F=25.674,P<0.001。空白对照组、BMSC组、NC-BMSC组和CADM1-BMSC组的细胞增殖活性、EdU标记的阳性细胞比例及细胞克隆形成数目之间差异均有统计学意义,均P<0.05;CADM1-BMSC组细胞增殖活性下降,且EdU标记的阳性细胞比例与细胞克隆形成数目均减少,F值分别为248.334和357.214,均P<0.001;各组细胞内TNF-α、IFN-γ及TGF-β1mRNA相对表达量之间差异均有统计学意义,均P<0.05;CADM1-BMSC组的细胞内TNF-αmRNA相对表达量和IFN-γmRNA相对表达量均下调,TGF-β1mRNA相对表达量上调,F值分别为32.019、40.127和76.454,均P<0.001。此外,NK-92MI细胞对肿瘤细胞的杀伤率升高,NCR1蛋白和NKP46相对表达量分别上调为1.22±0.09、2.21±0.19,差异均有统计学意义,F值分别为49.517、64.668,均P<0Objective To explore the effect of cell adhesion molecule 1(CADM1)-modified bone marrow mesenchymal stem cell(BMSC)on the growth and immune-related indicators of oral cancer cells.Methods BMSC from BALB/c mice were isolated and cultured,and then divided into adenovirus-infected CADM1-BMSC group and negative control adenovirus-infected NC-BMSC group;the culture supernatant of virus-infected BMSC was collected,and human oral cancer cells HSC3 were randomly divided into control group,BMSC group,NC-BMSC group and CADM1-BMSC group.Cell growth was detected by CCK-8method,EdU staining and cell colony formation assay;lactate dehydrogenase(LDH)release assay detected cell killing by NK-92MI;the expression levels of intracellular immune factors tumor necrosis factor-α(TNF-α),γ-interferon(IFN-γ)and transforming growth factor-β1(TGF-β1)were detected by real-time quantitative PCR(qRT-PCR);the expression levels of negative regulatory region factor 1(NCR1)and natural killer protein 46(NKP46)were detected by western blotting,and the expression levels of NKP46were detected by flow cytometry.Results The mouse-derived BMSC were successfully isolated,and the BMSCs infected with CADM1adenovirus showed obvious expression of green fluorescent protein.The relative expressions of CADM1mRNA in the control group,NC-BMSC group and CADM1-BMSC group were 1.00±0.05,1.01±0.06and 3.65±0.32,respectively,and the relative expression of CADM1 mRNA in the CADM1-BMSC group was significantly decreased,the difference was statistically significant(F=32.145,P<0.001).The relative expressions of CADM1protein in the control group,NC-BMSC group and CADM1-BMSC group were 0.42±0.04,0.40±0.03and 1.28±0.09,respectively,and the relative expression of CADM1protein in the CADM1-BMSC group was significantly decreased,and the difference was statistically significant(F=25.674,P<0.001).There were statistically significant differences in cell proliferation activity,EdU-labeled positive cell ratio and number of cell clones among blank control group,BMSC group,N

关 键 词：口腔癌 细胞粘附分子1 骨髓间充质干细胞 肿瘤细胞生长 免疫 

分 类 号：R739.8[医药卫生—肿瘤]