摘要
目的:研究人肝癌相关成纤维细胞(hCAFs)的原代培养及其分泌蛋白聚糖谱。方法:组织块贴壁法与酶消化联合组织块贴壁法进行hCAFs和正常成纤维细胞(NFs)的原代培养,观察两种方法对组织周围爬出细胞天数的影响;差速消化法和反复贴壁法进行成纤维细胞的纯化;实时荧光定量PCR(RT-qPCR)、免疫印迹法(western blotting)、间接免疫荧光结合形态学观察对成纤维细胞进行鉴定;利用凝集素芯片检测hCAFs和NFs分泌蛋白聚糖谱。结果:与组织块贴壁法相比,酶消化法联合组织块贴壁法细胞爬出天数减少(P<0.01);经纯化后hCAFs的胞体较大,胞体突起较多,而NFs胞体较小,胞体突起较少;两种成纤维细胞中均有FSP、FAP、α-SMA、Vimentin蛋白和mRNA的表达,而在hCAFs中表达较高(P<0.05)。hCAFs的分泌蛋白对CALSEPA、JAC、MPL、SSA、STL等5种凝集素的亲和性增强,而对IRA、IAA、PSA、CSA、GSL-IA4的亲和性减弱。结论:酶消化联合组织块贴壁法可加速细胞爬出,更适于原代培养成纤维细胞;通过比较hCAFs和NFs的分泌蛋白聚糖谱,提示T/Tn抗原、唾液酸、N-乙酰氨基葡萄糖结构在hCAFs的分泌蛋白中增加;而N-乙酰半乳糖胺和α-甘露糖的结构减少。
Objective:To study the primary culture of human hepatocellular carcinoma associated fibroblasts(hCAFs)and their secreted proteoglycan profiles.Methods:The primary culture of hCAFs and normal fibroblasts(NFs)was performed by tissue block adhesion method and enzyme digestion combined with tissue block adhesion method and the effect of the two methods on the number of days of cell emergence around the tissue was observed.Fibroblasts were purified by differential digestion and repeated adherence.The fibroblasts were identified by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR),western blotting and indirect immunofluorescence combined with morphological observation.The secreted proteoglycan profiles of hCAFs and NFs were detected by lectin microarray.Results:The number of days of cell emergence was reduced by enzyme digestion combined with tissue block adherence method compared with tissue block adherence method(P<0.01).After purification,hCAFs had larger cell bodies and more cell processes,while NFs had smaller cell bodies and fewer cell processes.The protein and mRNA expressions of FSP,FAP,α-SMA and Vimentin were found in both types of fibroblasts,and those in hCAFs were higher(P<0.05).The affinity of secreted proteins of hCAFs to five lectins increased,including CALSEPA,JAC,MPL,SSA and STL,while the affinity of secreted proteins of HCAFs to IRA,IAA,PSA,CSA and GSL-IA4 decreased.Conclusion:Enzyme digestion combined with tissue block adhesion method can accelerate cell emergence,and is more suitable for primary culture to form fiber cells.By comparing the secreted proteoglycan profiles of hCAFs and NFs,it shows that T/Tn antigen,sialic acid and N-acetylglucosamine structure increase in the secreted proteins of hCAFs while the structure of N-acetylgalactosamine andα-mannose decrease.
作者
卢钊露
覃斯梦
莫翠菊
张林艳
区莹莹
李山
Lu Zhaolu;Qin Simeng;Mo Cuiju;Zhang Linyan;Ou Yingying;Li Shan(The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)
出处
《广西医科大学学报》
CAS
2023年第3期413-419,共7页
Journal of Guangxi Medical University
基金
国家自然科学基金项目(No.81560447)
广西自然科学基金青年基金项目(No.2018GXNSFBA281160)
关键词
成纤维细胞
肿瘤微环境
凝集素芯片
分泌蛋白聚糖谱
fibroblasts
tumor microenvironment
lectin microarray
secreted proteoglycan profiles
