摘要
该文旨在研究三叶青总提取物(TH-t)对人γδT细胞功能的影响。用80%的乙醇提取三叶青干块根中的三叶青总提取物;利用Ficoll分离液分离人静脉血单个核细胞(PBMC)。用异戊烯焦磷酸法进行γδT细胞定向诱导培养。应用流式细胞分析技术检测PBMC培养前后的γδTCR表面标记率,用荧光标记的单克隆抗体检测γδT细胞表面CD107a、granzymeB和Perforin的百分数。用乳酸脱氢酶释放法检测TH-t诱导后的γδT细胞对肿瘤细胞株杀伤活性。用CCK8法检测TH-t对γδT细胞增殖能力。用MTT法检测TH-t对肝癌(HepG2)细胞株、胃癌(SGC-7901)细胞株和乳腺癌(MCF-7)细胞株的抑制率。PBMC培养前γδTCR表达率为3.12%,定向培养10天后为90.46%。在TH-t对γδT细胞增殖能力影响的实验中,经过诱导72 h后,浓度为0.62 μg/mL的TH-t促使γδT细胞增殖最明显(增长率为44.50%),显著高于对照组(3.50%)(P<0.05)。当TH-t浓度为0.15 μg/mL时,诱导的γδT细胞表面Perforin和granzymeB的阳性表达率可达到最高值,分别为76.90%±2.30%和30.50%±1.30%),显著高于对照组(65.40%±1.29%和25.10%±2.30%),组与组之间的比较均存在统计学差异(P<0.05)。经浓度为0.125 μg/mL TH-t诱导后的γδT细胞对肿瘤HepG2、SGC-7901和MCF-7细胞株的杀伤活性最高(分别为72.10%、52.30%和79.10%),显著高于对照组(分别为38.50%、30.50%和41.20%),各组间的比较存在统计学差异(P<0.05)。当Th-t浓度≥9.75 μg/mL时,对3株肿瘤细胞均有抑制作用。TH-t即能促进γδT细胞增殖又能提高其杀伤肿瘤细胞活性;一定浓度的TH-t能抑制肿瘤HepG2、SGC-7901和MCF-7细胞株的生长。
The purpose of this paper was to study the effect of Tetrastigma hemsleyanum total extraction (TH-t) on the function of human γδT cells.Tetrastigma hemsleyanum total extraction (TH-t) was extracted from dried roots of Tetrastigma hemsleyanum with 80% ethanol.Human venous blood mononuclear cells (PBMC) were isolated using Ficoll separation solution.PBMCs were in orientational culture of γδT cells with the isopentenyl pyrophosphate method.Flow cytometry was used to detect the surface labeling rate of γδT CR before and after PBMC culture.And the percentage of CD107a,Granzyme B and Perforin on the surface of γδT cells was detected by fluorescently labeled monoclonal antibody.The effect of γδT cells induced by TH-t on the killing activity of tumor cell lines was examined by lactate dehydrogenase release assay.The CCK8 method was used to detect the effect of TH-t on the proliferation of γδT cells.MTT assay was used to detect the inhibition rate of TH-t on liver cancer (HepG2) cell line,gastric cancer (SGC-7901) cell line and breast cancer (MCF-7) cell line.The expression rates of γδTCR before PBMC culture were 3.12% and 90.46% after 10 days of directional culture.In the experiment of the effect of TH-t on the proliferation of γδT cells,TH-t at a concentration of 0.62 μg/mL induced the most obvious proliferation of γδT cells (44.50%) after 72 h of induction,which was significantly higher than that of the control group (3.50%) (P<0.05).When the TH-t concentration was 0.15 μg/mL,the positive expression rates of Perforin and granzymeB on the surface of induced γδT cells reached the highest values (76.90%±2.30% and 30.50%±1.30%,respectively),which was significantly higher than that of the control group was 65.40%±1.29% and 25.10%±2.30%,and there was a statistically significant difference between the groups (P<0.05).After induction by TH-t at a concentration of 0.125μg/mL,γδT cells had the highest cytotoxic activity against tumor HepG2,SGC-7901 and MCF-7 cells (72.10%,52.30% and 79.10%,respective
作者
许青
骆晓梅
杨阳
孙香香
冯莉亚
郭妙妙
刘军权
XU Qing;LUO Xiaomei;YANG Yang;SUN Xiangxiang;FENG Liya;GUO Miaomiao;LIU Junquan(Department of Clinical Laboratory,the 71st Group Army Hospital of CPLA Army,Xuzhou 221004,China;Department of Pharmacy,the 71st Group Army Hospital of CPLA Army,Xuzhou 221004,China;Hangzhou Golden Field Medical Laboratory Co.,Ltd.,Hangzhou 310053,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2020年第3期461-468,共8页
Chinese Journal of Cell Biology
基金
南京军区医学科技创新基金(批准号:14MS032)资助的课题。
关键词
三叶青
黄酮
ΓΔT细胞
增殖
杀伤活性
抑制率
Tetrastigma hemsleyanum
flavonoids
γδT Cells
proliferation
cytotoxicity
inhibition rate
