人肾上腺组织单细胞悬液制备方法的研究  

Preparation of single cell suspensions from human adrenal gland tissue

作　　者：郭冰倩 王逸夫 郭雅婕 叶雨[3] 黄燕[3] 陈虹霏 蒋永华[1,2] 莫曾南[1,2] Guo Bingqian;Wang Yifu;Guo Yajie;Ye Yu;Huang Yan;Chen Hongfei;Jiang Yonghua;Mo Zengnan(Center for Genomic and Personalized Medicine;Collaborative Innovation Centre of Regenerative Medicine and Medical BioResource Development and Application,Guangxi Medical University,Nanning 530021,China;The Second Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学基因组与个体化医学研究中心,南宁530021 [2]广西医科大学再生医学与医用生物资源开发应用协同创新中心,南宁530021 [3]广西医科大学第二附属医院,南宁530021

出　　处：《广西医科大学学报》2022年第1期164-171,共8页Journal of Guangxi Medical University

基　　金：国家重点研究开发项目资助(No.2017YFC0908000);国家自然科学基金资助项目(No.81770759);广西基因组与个体化医学研究重点实验室资助项目(No.16-380-54,No.17-259-45,No.19-050-22,No.19-185-33,No.20-065-33)。

摘　　要：目的:探讨人肾上腺组织单细胞悬液制备的最佳消化方法。方法:收集来自广西医科大学第二附属医院手术终止妊娠的16~18孕周胎儿肾上腺组织3例,分别使用3种消化方案(A方案:HBSS溶液中加入1 mg/mLⅠ型胶原酶,0.1 mg/mL DNaseⅠ;B方案:HBSS溶液中加入1 mg/mLⅡ型胶原酶,0.1 mg/mL DNaseⅠ;C方案:HBSS溶液中加入1 mg/mLⅣ型胶原酶,0.1 mg/mL DNaseⅠ)进行消化。采用显微镜计数得到单细胞总数和细胞存活率;通过mRNA测序获得不同消化方案的差异基因表达并对其功能进行分析;使用实时荧光定量PCR检测不同消化方案得到的细胞的标志基因相对表达量;使用流式细胞术检测CYP17A1^(+)类固醇细胞和CHGA+嗜铬细胞的比例。结果:使用A方案和B方案消化得到的单细胞悬液结团少、形态完整、边界清楚、杂质及细胞碎片较少、背景较干净,而使用C方案得到的单细胞悬液有少量结团;使用B方案和C方案消化得到的细胞存活率差异均无统计学意义(均P>0.05);类固醇细胞标志基因CYP11A1,CYP17A1,CYP11B1,类固醇调控基因SF-1,MC2R和成熟的嗜铬细胞标志基因CHGA的表达水平均在使用B方案消化时较高。结论:使用B方案消化是较优选的单细胞悬液制备方法。Objective:To investigate the best digestion method for the preparation of single cell suspensions from human adrenal gland tissues.Methods:Three cases of adrenal gland tissues from fetuses of 16-18 gestational weeks terminated by operation were collected from The Second Affiliated Hospital of Guangxi Medical University.They were digested using three schemes(scheme A:1 mg/mL typeⅠcollagenase and 0.1 mg/mL DNase I were added to HBSS solution;scheme B:1 mg/mL typeⅡcollagenase and 0.1 mg/mL DNase I were added to HBSS solution;scheme C:1 mg/mL typeⅣcollagenase and 0.1 mg/mL DNase I were added to HBSS solution).The total number of single cells and cell viability were counted by microscope.After mRNA sequencing,differential gene expression and function of different digestion regimens were obtained through bioinformatics analysis.The expressions of marker genes in cells obtained by different digestion regimenswere detected by RT-qPCR.The proportion of CYP17A1+steroid cells and CHGA+chromaffin cells was determined by flow cytometry.Results:The single cell suspensions obtained by digestion scheme A and digestion scheme B have fewer clusters,complete morphology,clear boundary,less impurities and cell debris,and clean background.However,the single cell suspension obtained by digestion scheme C had a small amount of agglomeration.There was no statistical difference in the cell viability between digestion scheme B and digestion scheme C(P>0.05).The expression levels of steroid cell related marker genes CYP11A1,CYP17A1,CYP11B1,steroid regulating gene SF-1,MC2R and mature chromaffin cell marker gene CHGA were higher in digestion scheme B.Conclusion:Digestion scheme B is the preferred method for preparation of single-cell suspension in the study of adrenal tissue.

关 键 词：肾上腺组织 单细胞悬液 酶消化法 差异表达基因 流式细胞术 

分 类 号：R361[医药卫生—病理学]