摘要
目的使用不同浓度消化酶制备大鼠膈肌单细胞悬液,筛选最佳消化酶浓度组合。方法将胰蛋白酶稀释为0.125%、0.250%、0.375%、0.500%、0.625%,胶原酶Ⅰ型稀释为0.050%、0.100%、0.150%、0.200%、0.250%,胶原酶Ⅳ型稀释为0.050%、0.100%、0.150%、0.200%、0.250%,将三种消化酶按照浓度从低到高依次组合为五个浓度组,即胰蛋白酶0.125%+胶原酶Ⅰ0.050%+胶原酶Ⅳ0.050%、胰蛋白酶0.250%+胶原酶Ⅰ0.100%+胶原酶Ⅳ0.100%、胰蛋白酶0.375%+胶原酶Ⅰ0.150%+胶原酶Ⅳ0.150%、胰蛋白酶0.500%+胶原酶Ⅰ0.200%+胶原酶Ⅳ0.200%、胰蛋白酶0.625%+胶原酶Ⅰ0.250%+胶原酶Ⅳ0.250%。取SD大鼠新鲜膈肌组织,分成A、B、C、D、E组,采用机械—酶消化法制备膈肌单细胞悬液,分别加入上述五种浓度组合的消化酶进行消化;另设对照组,加入等量PBS。单细胞悬液经台盼蓝染色,观察悬液中细胞的形态,计数活细胞数、死细胞数、细胞团块数,计算细胞存活率和单细胞悬液浓度;经Anti-RatCD11b PE标记后进行流式细胞术检测,获得目标细胞比值,比较各组检测结果。结果A~E组获得的活细胞数分别为156.00×10^(5)、200.92×10^(5)、182.08×10^(5)、160.00×10^(5)、140.02×10^(5)/mL,细胞存活率分别为87.01%、90.02%、91.86%、90.75%、86.94%,单细胞悬液浓度分别为19.50×10^(5)、30.25×10^(5)、22.75×10^(5)、20.00×10^(5)、17.50×10^(5)/mL,均高于对照组(P均<0.05),其中B、C组活细胞数量和单细胞悬液浓度高于其他三组(P均<0.05)。经CD11b标记后,C组D11b阳性的中性粒细胞数、CD11b阳性细胞占活细胞的百分比均高于其他各组(P均<0.05)。结论采用机械—酶消化法制备大鼠膈肌单细胞悬液,不同消化酶浓度组合均能获得优质的单细胞悬液,其中以胰蛋白酶0.375%+胶原酶Ⅰ0.150%+胶原酶Ⅳ0.150%获得的单细胞悬液细胞存活率最高、死细胞数最少。
Objective To prepare the rat diaphragm single-cell suspension by using different concentrations of diges⁃tive enzymes and to screen the optimal digestion enzyme concentration.Methods Trypsin was diluted to 0.125%,0.250%,0.375%,0.500%,and 0.625%,collagenase typeⅠwas diluted to 0.050%,0.100%,0.150%,0.200%,and 0.250%,and collagenase typeⅣwas diluted to 0.050%,0.100%,0.150%,0.200%,and 0.250%,respectively.The three digestive enzymes were combined into five concentration groups in order of concentrations from low to high,namely:trypsin 0.125%+collagenaseⅠ0.050%+collagenaseⅣ0.050%,trypsin 0.250%+collagenaseⅠ0.100%+collagenaseⅣ0.100%,trypsin 0.375%+collagenaseⅠ0.150%+collagenaseⅣ0.150%,trypsin 0.500%+collage⁃naseⅠ0.200%+collagenaseⅣ0.200%,trypsin 0.625%+collagenaseⅠ0.250%+collagenaseⅣ0.250%,respec⁃tively.Fresh diaphragm tissues of SD rats were taken,and then were divided into groups A,B,C,D and E,and the dia⁃phragm single cell suspension was prepared by mechano-enzymatic digestion method,and the digestive enzymes of the above five concentration combinations were added for digestion.A control group was also set up,and an equal amount of PBS was added.The single-cell suspension was stained with trypan blue,and the morphology of the cells in the suspension was observed,and the number of living cells,dead cells,cell masses,cell survival rate,and single-cell suspension con⁃centration were counted.After Anti-Rat CD11b PE labeling,flow cytometry was performed to obtain the target cell ratio and we compared the detection results of each group.Results The number of living cells obtained in the groups A-E was 156.00×10^(5),200.92×10^(5),182.08×10^(5),160.00×10^(5),140.02×10^(5)/mL,the cell survival rates were 87.01%,90.02%,91.86%,90.75%,and 86.94%,respectively,and the single-cell suspension concentrations were 19.50×10^(5),30.25×10^(5),22.75×10^(5),20.00×10^(5),17.50×10^(5)/mL,which were higher than those in the control group(all P<0.05).Among them,the number of viable cells and single-cell suspension concentrations in group B and group C were higher than those in the groups A,D and E(all P<0.05).After CD11b labeling,the ratio of the number of cells labeled in the group C was higher than that of the other four groups;the number of D11b-positive neutrophils and the percentage of CD11b-positive cells in live cells in the group C were higher than those in other groups(all P<0.05).Conclusions We use mechano-en⁃zymatic digestion method to prepare rat diaphragm muscle single-cell suspension,and different combinations of digestive enzyme concentrations can obtain high-quality single-cell suspension,of which,trypsin 0.375%+collagenaseⅠ0.150%+collagenaseⅣ0.150%-obtained single-cell suspension cell survival rate is the highest with the lowest number of dead cells.
作者
何杰
王宁
张杉杉
刘青铃
周舟
陈谡
吴学东
HE Jie;WANG Ning;ZHANG Shanshan;LIU Qingling;ZHOU Zhou;CHEN Su;WU Xuedong(Department of General Surgery,The Affiliated Hospital of Panzhihua College,Panzhihua 617000,China;不详)
出处
《山东医药》
CAS
2022年第26期45-48,共4页
Shandong Medical Journal
基金
云南省卫生健康委员会医学领军人才培养计划(L-2017026)
云南省教育厅科学研究基金项目(2019Y0277)。
关键词
膈肌
单细胞悬液
消化酶
最佳作用浓度
流式细胞术
动物实验
diaphragm
single cell suspension
digestive enzymes
optimal concentration of action
flow cytome⁃try
animal experiment
作者简介
通信作者:吴学东(E-mail:xuedong3288@sina.con)。
