Mechanism of Liuwei Dihuangwan in Enhancing Hepatocarcinoma Suicide Gene Therapy from Cx32
10.13422/j.cnki.syfjx.20190113
- VernacularTitle: 基于Cx32探讨六味地黄丸增效自杀基因抗肝癌的缝隙连接机制
- Author:
Hua YI
1
;
Jun-fang SU
1
;
Xue LI
1
;
Shao-feng WU
1
;
Ting-xiu ZHAO
1
;
Qiao LI
1
;
Yi-dan ZOU
1
;
Guo-li LIN
2
;
Biao-yan DU
1
Author Information
1. School of Basic Medicine, School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
2. The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China
- Publication Type:Research Article
- Keywords:
hepatocarcinoma cell;
Liuwei Dihuangwan;
serum pharmacology;
gene therapy;
bystander killing effect;
gap junction
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2019;25(1):76-81
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To investigate the effect of Liuwei Dihuangwan on connexin 32 (Cx32) in hepatoma cell line CBRH7919 and its gap junction intercellular communication (GJIC), and furthermore study its mechanism of enhancing the bystander killing effect of suicide gene therapy. Method: Liuwei Dihuangwan (32 g·kg·d-1) and the same volume of normal saline were given to the rats by intragastrical administration. Blood was taken to prepare the medicated serum of Liuwei Dihuangwan and blank control serum, respectively. The hepatoma cell line CBRH7919 were treated by control serum and medicated serum of Liuwei Dihuangwan in different concentrations. There were four groups in experiment:the blank control group (volume fraction of 10%), medicated serum high dose group of Liuwei Dihuangwan (the volume fraction of 10%), medicated serum middle dose group of Liuwei Dihuangwan (the volume fraction of 5%), and medicated serum low dose group of Liuwei Dihuangwan (the volume fraction of 2.5%). The expression levels of Cx32 protein and mRNA in hepatoma cell line CBRH7919 were detected by indirect immunofluorescence assay (ⅡA) and Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) assay. The fluorescence redistribution after photobleaching (FRAP) method was used to detect the function of GJIC of hepatoma cell line CBRH7919. Result: ① The indirect immunofluorescence assay (ⅡA) analysis indicated that as compared with the blank control group, the cx32 expression of CBRH7919 cells was up-regulated in a concentration-dependent manner in each dose group of the serum containing Liuwei Dihuangwan (P<0.05 in low dosage group; P<0.01 in middle dosage and high dosage group), especially on cell membrane.② The results of Real-time PCR assay indicated that as compared with the blank control group, the expression of Cx32 mRNA was increased in each dose group (P<0.05 in low dose group, P<0.01 in middle and high dose group) in a dose-effect relationship. ③ The result of fluorescence redistribution after photobleaching (FRAP) analysis indicated that the average fluorescence recovery rates of drug groups were significantly higher than that in blank control group, and also showed a certain concentration-dependent trend. Conclusion: The mechanism of medicated serum of Liuwei Dihuangwan in enhancing the bystander killing effect of suicide geneis related to gap junction. Liuwei Dihuangwan may enhance the function of GJIC by increasing the localization of cx32 on the cell membrane of CBRH7919 cells and increasing the expression of cx32 mRNA and protein to achieve the synergistic action.
