Effect of electronic stimulation at Neiguan(PC 6) acupoint on gene expression of adenosine triphosphate-sensitive potassium channel and protein kinases in rats with myocardial ischemia

OBJECTIVE:To investigate the effects of electronic stimulation at acupoints Neiguan(PC 6) and Lieque(LU 7) on the gene expression of the adenosine triphosphate(ATP)-Sensitive potassium channel(KATP:Kir6.1,Kir6.2,SUR2 A,and SUR2B) and protein kinases(PKA,PKG,and PKCβ2) in myocardial cells of rats with myocardial ischemia(Ml) induced by isoproterenol(ISO).METHODS:Rats were randomly divided into a control,model,Neiguan(PC 6),Lieque(LU 7),and non-acupoint groups.The Ml model was established by injecting rats with ISO.Electro-acupuncture treatment was given to the acupuncture groups,once a day for 7 days.Gene expression was analyzed with real-time PCR.RESULTS:The gene expression of KATP and protein kinases in the model group was higher than those in the control group(P < 0.05).After acupuncture treatment,the KATP and protein kinase expression levels were significantly lower in the Neiguan(PC 6)and Lieque(LU 7) groups compared with the model group[P < 0.05).The Neiguan(PC 6) group lowered these levels significantly more than that of the Lieque(LU 7) group(P < 0.05).No significant differences were observed between the model and non-acupoint groups(P > 0.05).CONCLUSION:Our findings suggest that electronic needling of Neiguan(PC 6) can both reduce the gene expression of KATP and protein kinases in rats with ISO-induced Ml.

Characteristics of ovarian cancer cells transduced by the bicistronic retroviral vector containing GM-CSF and HSV-TK genes

Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells.Methods Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the “ping-pong' technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells.Results The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6×105?cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7?μg/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4?ng*ml-1*106 cells-1*2 days-1.Conclusion The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.