葛根素对γδT细胞杀伤肝癌SMMC-7721细胞的影响

Puerarin Influences the Cytotoxic Activity of γδT Cells Against Liver Cancer Cell Line SMMC-7721

关键词:  葛根素  γδT细胞  肝癌SMMC-7721细胞  杀伤活性

Key words:  puerarin  γδT cells  liver cancer cell line SMMC-7721  cytotoxic activity

摘要:

目的 探讨葛根素对人γδT细胞杀伤肝癌SMMC-7721细胞的影响及机制。方法 异戊烯焦磷酸法(isopentenylpyrophosphate，IPP)体外扩增人外周血γδT细胞，不同浓度葛根素作用于γδT细胞24，48，72 h，CCK8法检测葛根素对γδT细胞增殖率的影响，FCM检测γδT细胞穿孔素(perforin，PFP)、颗粒酶B(granzyme B，GraB)及CD107a的表达情况，LDH释放法检测γδT细胞对肝癌SMMC-7721细胞的杀伤活性，Western blot法检测γδT细胞中P-ERK1/2和Bcl-2的表达情况。结果 葛根素作用人γδT细胞48 h后，葛根素1.6~100 μmol?L^-1浓度组的γδT细胞增殖明显(P<0.05)，葛根素12.5~50 μmol?L^-1浓度组人γδT细胞PFP、GraB及CD107a表达明显高于对照组(P<0.05)，葛根素3.125~50 μmol?L^-1浓度组人γδT细胞P-ERK1/2和Bcl-2表达显著增高(P<0.05)，且葛根素3.125~50 μmol?L^-1浓度组人γδT细胞对肝癌SMMC-7721细胞杀伤活性较对照组明显增强(P<0.05)。结论 葛根素能够促进人γδT细胞增殖，并能够提高γδT细胞对肝癌SMMC-7721细胞的杀伤活性，其机制可能与上调γδT细胞表面的PFP，GraB和CD107a的表达及活化P-ERK1/2和Bcl-2的表达有关。

Abstract:

OBJECTIVE To explore the effect and mechanism of puerarin on γδT cells and their cytotoxicity on human liver cancer cell line SMMC-7721. METHODS Amplification γδT cells of human peripheral blood in vitro by using isopentenylpyrophosphate(IPP). γδT cells were cultured with different concentrations of puerarin in vitro. CCK8 assay was used to measure the growth curve of γδT cells. The expression of perforin(PFP), Granzyme B(GraB) and CD107a on γδT cells were detected by FCM. The cytotoxic activity of γδT cells to SMMC-7721 cells was detected by lactate dehydrogenase release(LDH). The expression of phosphorylation of kinase1/2(P-ERK1/2) and Bcl-2 of γδT cells was measured by Western blot. RESULTS After cultured with puerarin for 48 h, the proliferation rate of human γδT cells increased signi?cantly with puerarin at concentrations from 1.6 μmol?L^-1 to 100 μmol?L^-1(P<0.05); the expression of GraB, PFP, CD107a and P-ERK1/2, p-AKT of human γδT cells were significantly higher than that of control group with puerarin at concentrations from 12.5 to 50 μmol?L^-1(P<0.05) and 3.125 to 50 μmol?L^-1(P<0.05), respectively; the cytotoxic activity of human γδT cells against SMMC-7721 was remarkably higher than that of the control group with puerarin at concentrations from 3.125 to 50 μmol?L^-1 (P<0.05). CONCLUSION Puerarin at a certain range of concentration could promote the growth of γδT cells and enhance the cytotoxic activity of γδT cells to SMMC-7721 cells. The possible mechanism may be that puerarin could increase the expression of PFP, GraB, CD10707a, P-ERK1/2 and Bcl-2. KEY WORDS: puerarin; γδT cells; liver cancer cell line SMMC-7721; cytotoxic activity