汉黄芩素提高人CD3AK细胞杀伤肝癌细胞的实验研究和机理探讨  

Mechanism research of proliferation and killing hepatoma cell of CD3AK cell by Wogonin

作　　者：李晓楠[1,2] 姬会春[3] 周燏[3] 郑璐[3] 刘军权[3] 朱月华[3] 

机构地区：[1]徐州医学院附属医院西院 [2]中煤五公司职工医院,徐州221006 [3]解放军第97 医院,徐州221006

出　　处：《中国免疫学杂志》2015年第3期347-353,共7页Chinese Journal of Immunology

基　　金：南京军区医学科技创新课题(MS039)

摘　　要：目的：观察汉黄芩素对人CD3AK细胞增殖及对肝癌细胞SMMC-7721杀伤活性的影响,并探讨其发生的机制。方法：分离健康者外周血PBMC;在体外用多种细胞因子联合诱导培养CD3AK细胞。收集培养第7天的CD3AK细胞给予不同浓度汉黄芩素诱导48 h：CCK-8法检测人CD3AK细胞增殖率;MTT法检测汉黄芩素对SMMC-7721细胞生长的影响;流式细胞术（FCM）检测汉黄芩素作用前后CD3AK细胞穿孔素（PFP）、颗粒酶B（Gr B）、CD107a的表达;乳酸脱氢酶（LDH）释放法测定汉黄芩素对CD3AK细胞杀伤肝癌细胞SMMC-7721活性;Western blot检测药物诱导前后CD3AK细胞胞外信号调节激酶（ERK1/2）蛋白表达。用transwell chambers小室进行药物诱导前后肝癌细胞SMMC-7721迁移率检测。划痕愈合实验观察药物诱导后肝癌细胞SMMC-7721生长融合情况。结果：汉黄芩素能明显促进CD3AK细胞增殖,汉黄芩素浓度为3.2 mg/L时,细胞增殖率比对照组（0 mg/L）高23%,两者比较有统计学差异（P〈0.05）。在汉黄芩素为3.2 mg/L时诱导的CD3AK细胞对靶细胞SMMC-7721杀伤活性最高（60.4%）,与对照组（42.7%）比较差异有统计学意义（P〈0.05）。经汉黄芩素诱导后的CD3AK细胞PFP、Gr B、CD107a表达显著高于对照组（P〈0.05）。汉黄芩素作用48h后的CD3AK细胞其ERK1/2蛋白表达较对照组均有所上调,浓度在12.5~0.8 mg/L时,ERK1/2蛋白表达高于对照组（P〈0.05）。经浓度为50、12.5、3.2、0.8、0.2mg/L汉黄芩诱导48 h后,肝癌细胞SMMC-7721通过transwell小孔细胞数以汉黄芩浓度为12.5 mg/L时最低、肝癌细胞SMMC-7721的融合率以3.2 mg/L时最低,与对照组比较有统计学意义（P〈0.05）。结论：汉黄芩素在一定浓度范围内能够促进CD3AK细胞增殖,增强其对肝癌细胞SMMC-7721的杀伤活性,抑制SMMC-7721细胞生长、迁移和细胞的融合。汉黄芩素促进CD3AK细胞增殖和功能改变可能与活化细胞ERK1/2蛋白,上调CD3Objective：To investigate the effect and mechanism of Wogonin on CD3AK cell proliferation and cytotoxicity to SMMC-7721. Methods：CD3AK cells were cultured from peripheral blood mononuclear cells （PBMC） in vitro by a variety of cytokines for 7 d, and treated with different concentrations of Wogonin for 48 h. CD3AK cells proliferation was measured by CCK-8 assay. SMMC- 7721 cell growth was detected by MTY. The expression of perforin （PFP）, granzyme B （GrB） and CD107a on CD3AK cellswere measured by flow cytometry （FCM）. The cytotoxicity to SMMC-7721 cells was detected by LDH release assay. The expression of ERK1/ 2 on CD3AK cells was detected by Western blot. The mobility of SMMC-7721 cells was detected with transwell chambers. The merge of SMMC-7721 cells were measured with Wound healing assay. Results：Wogonin could significantly promote CD3AK cells proliferation, especially at 3.2 mg/L （23% higher than that of control group,P〈0. 05 ）. The highest cytotoxieity to SMMC-7721 was also at the con- centration 3.2 mg/L （60. 4% ）. The expression of PFP, GrB, CD107a were significantly higher than that of control group （P〈0. 05 ）. The expression of ERK1/2 was obviously improved,especially at 12. 5-0. 8mg/L After treated with Wogonin 50,12. 5,3.2,0. 8,0. 2 mg/L for48 h,the lowest transwell cell was at 12. 5 mg/L and lowest merge rate was at 3.2 mg/L. Conclusion： Wogonin could promote CD3AK cell proliferation and enhance the cytotoxicity to SMMC-7721. Wogonin could also inhibit SMMC-7721 cell growth, migration and cell merge. The mechanism may be related to activated ERK1/2 and increase the expression of PFP, GrB, CD107a.

关 键 词：汉黄芩素 肝癌/SMMC-7721 CD3AK细胞 

分 类 号：R392[医药卫生—免疫学]