摘要
目的探讨广藿香醇(PA)对巨噬细胞吞噬人胃癌细胞的影响及其机制。方法采用RT-PCR和Western blot检测人正常胃黏膜上皮细胞GES-1及胃癌MKN45、SNU-1、HGC-27和MKN74等胃癌细胞系中CD47的mRNA和蛋白表达水平。以MKN45细胞作为研究对象,CCK-8检测不同浓度(0,0.1,0.25,0.5,1,10,20 mmol/L)PA处理下MKN45细胞的增殖活性;再采用不同浓度(0,0.25,0.5,1 mmol/L)PA处理MKN45细胞24 h,采用RT-PCR和Western blot检测细胞中CD47的mRNA和蛋白表达水平。将人单核细胞THP-1诱导为M1型巨噬细胞,采用流式细胞术检测M1型巨噬细胞表面标志物CD86和诱导型一氧化氮合酶(iNOS)的水平。将MKN45细胞分为blank组、vector组和oe-CD47组,将CD47过表达质粒(oe-CD47)及其阴性对照质粒(vector)分别转染至MKN45细胞48 h后采用RT-PCR和Western blot检测细胞中CD47的mRNA和蛋白表达水平以验证转染效率。经1 mmol/L PA处理,采用羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)对MKN45细胞进行标记,再与M1型巨噬细胞共培养6 h,将MKN45细胞分为对照组、PA组、PA+vector组和PA+oe-CD47组。采用流式细胞术检测细胞中CD11b+/CFSE+双阳性率以评估巨噬细胞对MKN45细胞的吞噬作用。结果与GES-1细胞系比较,MKN45、SNU-1、HGC-27和MKN74等胃癌细胞系中CD47 mRNA和蛋白表达水平显著升高,其中在MKN45细胞系中表达最高(均P<0.05)。经不同浓度PA处理的MKN45细胞增殖活性以剂量依赖性显著降低(不同浓度PA组与0 mmol/L PA组比较均P<0.05)。与0 mmol/L PA组比较,0.25,0.5,1 mmol/L PA组细胞中CD47 mRNA和蛋白表达水平均显著降低(均P<0.05),其中1 mmol/L PA浓度效果最佳。THP-1细胞经诱导后,CD86和iNOS阳性率分别为96.54%和97.46%,确定M1型巨噬细胞诱导成功。与blank组比较,oe-CD47组细胞中CD47的mRNA和蛋白表达水平均显著升高(均P<0.05),而vector组差异无统计学意义(P>0.05),确定CD47过表达细胞模型构建成功(转染效率为82%)。与对照组比较,PA组细胞中CD11b+/CFSE+双阳性率显著升高(P<0.05);与PA组比较,PA+oe-CD47组细胞中CD11b+/CFSE+双阳性率显著降低(P<0.05),而PA+vector组差异无统计学意义(P>0.05)。结论广藿香醇通过下调CD47表达,增强巨噬细胞对胃癌细胞的吞噬作用。
Objective To investigate the effect of patchouli alcohol(PA)on the phagocytosis of human gastric cancer cells by macro⁃phages and its underlying mechanism.Methods The mRNA and protein expression levels of CD47 were first assessed in the human normal gastric mucosal epithelial cell line GES-1 and a panel of gastric cancer cell lines(including MKN45,SNU-1,HGC-27,and MKN74)using RT-PCR and Western blot analysis.Next,MKN45 cell lines were selected as the research subject and its proliferative activity treated with different concentrations of PA(0,0.1,0.25,0.5,1,10,20 mmol/L)was evaluated by CCK-8 assay.Subse⁃quently,MKN45 cells were treated with different concentrations of PA(0,0.25,0.5,1 mmol/L)for 24 h,and the mRNA and protein expression levels of CD47 were detected by RT-PCR and Western blot.M1-type macrophages were generated by differentiating human monocytic THP-1 cells,and then the expression of the M1 macrophage surface markers CD86 and inducible nitric oxide synthase(iNOS)was analyzed by flow cytometry.After transfecting MKN45 cells with a CD47 overexpression plasmid(oe-CD47)or its negative control plasmid(vector),cells were divided into blank,vector,and oe-CD47 groups.Transfection efficiency was verified by detecting CD47 mRNA and protein expression levels via RT-PCR and Western blot 48 hours post-transfection.To assess the effect of PA on phagocytosis,MKN45 cells were first treated with 1 mmol/L PA and then labeled with carboxyfluorescein diacetate succinimidyl ester(CFSE).The labeled cells were co-cultured with M1-type macrophages for 6 hours.The experiment included four groups:control was quantified by flow cytometry,measuring the proportion of double-positive(CD11b+/CFSE+)cells.Results Compared with the GES-1 cell line,the mRNA and protein expression levels of CD47 were significantly elevated in gastric cancer cell lines MKN45,SNU-1,HGC-27,and MKN74,with the highest expression observed in MKN45 cell line(all P<0.05).The proliferation of MKN45 cells was significantly inhibited by PA in a dose-dependent manner(all P<0.05 compared to the untreated control).Compared with 0 mmol/L PA group,the mRNA and protein expression levels of CD47 were significantly decreased in 0.25,0.5,1 mmol/L PA groups(all P<0.05),especially in 1 mmol/L group.After induction,the positive rates for CD86 and iNOS in THP-1 cells were 96.54%and 97.46%,respectively,confirming successful induction of M1-type macrophages.Compared with blank group,CD47 mRNA and protein expression levels were significantly increased in oe-CD47 group(both P<0.05),while no significant difference was observed in vector group(P>0.05),confirming the successful construction of the CD47 overexpression cell model(transfection efficiency=82%).Compared with control group,the CD11b+/CFSE+double-positive rate was significantly increased in PA group(P<0.05).Compared with PA group,the CD11b+/CFSE+double-positive rate was significantly decreased in PA+oe-CD47 group(P<0.05),while no significant difference was found in PA+vector group(P>0.05).Conclusion Patchouli alcohol can enhance macrophage phagocytosis of gastric cancer cells by down-regulating CD47 expression.
作者
皇甫晨瑶
高雁
王娜
侯坤
HUANGFU Chenyao;GAO Yan;WANG Na;HOU Kun(Department of Pharmacy,Peking University Cancer Hospital Inner Mongolia Hospital,Hohhot 010102,China)
出处
《山西医科大学学报》
2026年第2期113-120,共8页
Journal of Shanxi Medical University
基金
内蒙古自治区自然科学基金项目(2023QN08003)
内蒙古医科大学青年项目(YKD2024QN014)。
关键词
广藿香醇
CD47
巨噬细胞
吞噬
胃癌
肿瘤免疫治疗
patchouli alcohol
CD47
macrophages
phagocytosis
gastric cancer
tumor immunotherapy
