Enhancing Effect Of Active Component Of Salviae Miltirrhize On Suicide Gene Therapy System And Its Mechanism On Gap Junction Function

ObjectiveTo verify the enhancing effect of effective components of traditional Chinese medicine on suicide gene therapy system and search its mechanism, human umbilical vein epithelial cells’ recombinant herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV1-tkGFP/GCV) suicide gene system with green fluorescent protein GFP)monitoring function was constructed. To study effect of TanshinoneⅡA (Tan ⅡA) and Salvianic acid A sodium(SAAS) on this system and its mechanism. Methods to study effect of effective components of tradi-tional Chinese medicine on suicide gene therapy system in Anti-tumor vascular therapy.Methods1 construct HUVEC-tkGFP/GCV suicide gene therapy system(1)The plasmid both pLV-tkGFP and pLV-Cl were propagated and extracted by the Ultra Pure QIAGEN Plasmid Midi Kit.293T cells were infected to improve the recombinant plasmids packaged virus, and then human umbilical vein epithelial cells were infected. After puromycin separation, limited dilution assay, Limited dilution after fluorescent tracer and flow cytometer sorting green fluorescent cells,stable anti-puromycin cell lines transfected with the recombinant plasmid.(2)The effects of plasmid on HUVEC was tested by MTT assay.(3)The killing effects of GCV on HSV-tkGFP/GCV suicide gene therapy system was examined by MTT assay. Bystander effect of mixed HUVEC-tkGFP and HUVEC in certain ratio was tested on MTT assay.2 the enhancing effect of TanⅡA、SAAS on HUVEC HSV-tkGFP/GCV suicide gene therapy system(1) The inhibition was tested on MTT assay. The HUVEC and HUVEC HSV-tkGFP cells were inoculated into a 2:3 mix ratio of 96-well plates, mixed cells were divided into groups, Control group, ATRA positive group, GCV group, Tan ⅡA group, SAAS group, GCV combined Tan IIA and GCV combined SAAS group. Tan IIA was dosed in sequential. Absorbance was noted after 24h and 48h with drogs on MTT assay. The synergistic combined effect of TanIIA and SAAS was evaluated by "Q Value" method.(2) The apoptosis was tested on Annexin-V-FITC/PI assay cell. The HUVEC and HUVEC HSV-tkGFP cells were inoculated into a 2:3 mix ratio of 12-well plates, mixed cells were divided into groups, Control group, ATRA positive group, GCV group, Tan ⅡA group, SAAS group, GCV combined Tan ⅡA and GCV combined SAAS group. TanⅡA was dosed in sequential. Apoptosis of different drog groups was dyed by Annexin-V-FITC/PI and detected on Flow cytometry. Similarly, photoes were taken and analyzed.3 The effect of TanIIA and SAAS on gap junction function in Human Umbilical Vein Epithelial Cells(1) Inhibitive effect of TanIIA and SAAS on the growth of human umbilical vein epithelial cells was examined by MTT assay, and then the drug concentration was determined.(2) Flow cytometry with two kinds of fluorescent dyes is used to tested the strength of the gap junction intercellular communication from 0 μmol/L to 32 umol/L both TanIIA and SAAS. After added different concentrations of drugs and two kinds of fluorescent dyes, cells combined with Calcein-AM only is tested and outputed in the percentage of all. The stronger the value, the greater the representative function. Similarly, photoes were taken and analyzed. All the same, all-trans-retinoic acid (ATRA) served as the positive control drug.(3)The gene expression of Cx40 and Cx37 was detected by Quantitative real-time polymerase chain reaction and analyzed in relative quantitative method called the 2-delta delta from 0 to 32 μmol/L both TanⅡA and SAAS.(4) The protein expression of Cx40 and Cx37 was detected by Western Blotting from 0 to 32 umol/L both TanⅡA and SAAS and analyzed by Quantity One manual.Result1 construct HUVEC-tkGFP/GCV suicide gene therapy system On MTT assay:the proliferation of recombinant HUVECs were not effected by plasmid. Double time were 37.66 h and 42.78 h. HUVEC-tkGFP was inhibited by GCV from 2 to 32 μmol/L, while HUVEC from 32 μmol/L.16 μmol/L GCV is used on other assay. After 72h, different mixing ratio of HUVEC-tkGFP were obviously inhibited under 16 μ mol/L GCV. The HUVEC-tkGFP and HUVEC cells were inoculated into a 3:2 mix ratio to perform the experiment.2 the enhancing effect of TanⅡA、SAAS on HUVEC HSV-tkGFP/GCV suicide gene therapy system(1)MTT method assay the Inhibition rate of each group:Compared with GCV group and simplex Chinese medicine groups, the killing rate of 4,8,16μ mol/L Tan IIA combined GCV groups and 16,32 μmol/L SAAS combined GCV group were obviously improved. Their Q Value were more than 1.15 and indicated synergistic effect.(2) PI staining assay cell killing rate:The killing rate of TanⅡA and TanⅡA combined GCV groups were 2.1,14.8,1.7,14.3,20.4,18.7,32.9 and 40.1. The killing rate of SAAS and SAAS combined GCV groups were 3.4,17.0,2.1,5.6, 12.8,13.5,17.4,18.5,22.2,24.9,34.7 and 41.5. The killing rate of 4,8,16 μ mol/L Tan ⅡA combined GCV groups and 8,16,32 μ mol/L SAAS combined GCV groups were higher than GCV groups.Killing rate of 4,8,16μmol/L TanⅡA combined GCV groups was higher than relevant Tan ⅡA groups. Apoptosis rate of 8,16,32 μmol/L SAAS combined GCV groups were higher than relevant SAAS groups. Q Value of 4,8,16 μ mol/L Tan ⅡA combined GCV groups and 16,32 μ mol/L SAAS combined GCV groups were more than 1.15 and it indicated synergistic effect of Tan ⅡA and SAAS.(3)Annexin V-FITC/PI staining assay cell apoptosis:observation of fluores-cence microscope parallel to the result of MTT and PI assay, as well as Q value.(4)Annexin V-FITC/PI staining assay cell apoptosis:The killing rate of Tan II A and Tan ⅡA combined GCV groups were 2.0,7.2,5.0,6.1,9.8,6.0,12.7 and 37.7. The killing rate of SAAS and SAAS combined GCV groups were 2.8,4.4,3.4, 6.8,7.9,11.8,3.1,5.4,5.9,15.5,17.6 and 22.2. Compared with GCV group and simplex Chinese medicine groups, killing rate of 8,16μ mol/L Tan ⅡA combined GCV groups,4、8、16、32μ mol/L SAAS combined GCV groups get higher. Q Value of 16 μmol/L Tan IIA combined GCV groups and 8,32 μmol/L SAAS combined GCV groups were more than 1.15 and it indicated synergistic effect of TanⅡA and SAAS.3 The effect of TanⅡA and SAAS on gap junction function in Human Umbilical Vein Epithelial Cells(1)MTT assay cell multiplication:the proliferation of HUVECs were effected by 2-64μmol/L TanⅡA or SAAS after 48h.The toxicity of 64 μmol/L TanⅡA or SAAS was high, so 4-32 μmol/L were experimented.(2)Double immunofluorescent tracer method:The ratio of green cells in TanⅡA groups and positive control group were 8.4±0.6.9.8±0.4,12.2±0.5,12.8 ±1.1,10.8±1.3 and 10.5±0.4. Compared with the control group,8,16 and 32μmol/L TanⅡA distinctly improved the transmission function of the green fluorescent dye<P<0.01). The ratio of green cells in 8,16 μ mol/L Tan ⅡA groups was higher than ATRA group (P<0.05).The ratio of green cells in SAAS groups and positive control group were 9.7±0.4,10.0±0.6,14.1±0.9,16.2±0.9, 22.5±1.6 and 14.6±0.4. Compared with the control group,8,16 and 32 μmol/L SAAS improved the transmission function of the green fluorescent dye (P<0.05).The ratio of green cells in 16,32umol/L SAAS groups was higher than ATRA group (P<0.05).(3) qRT-PCR method:The expression of Cx40 and Cx37 mRNA was enhanced by Tan ⅡA And SAAS.The expression of Cx37 was distinctly enhanced by 8,16 and 32 μ mol/L TanⅡA or SAAS (P<0.05). The expression of Cx40 was distinctly enhanced by 8,16 and 32 μ mol/L TanⅡA or SAAS (P<0.05).The effect was concent-ration-dependent.(4) Western Blotting method:The results of Western blotting showed that Cx40 expression level were increased by 8,16μmol/L TanⅡA (P<0.05).while not 32 μ mol/L. The results of Western blotting showed that Cx40 expression level were increased by 16、32 μ mol/L SAAS (P<0.05) and The effect was concentration-dependent. The result was parallel to the result of MTT and PI assay. Expression of Cx37 were improved by Both TanⅡA and SAASConclusion1 The killing effects of GCV on HSV-tkGFP/GCV suicide gene therapy system was found out. Bystander effect of mixed HUVEC-tkGFP and HUVEC in certain ratio was tested out. Therefore, suicide gene therapy system was constructed success-fully.2 Both TanⅡA and Salvianic acid A sodium can enhance bystander effect of HSV-tk/GCV suicide gene therapy system. The mechanism may be partially due to TanⅡA or Salvianic acid A sodium can promote the expression of Cx proteins,thereby promoting GJIC function.3Gap junction mechanism of bystander effect, is playing an important mechanism for both TanⅡA and Salvianic acid A sodium with HUVEC HSV-tk/GCV suicide gene therapy system synergies.In the study, HSV-tkGFP/GCV suicide gene therapy system is successfully constructed in HUVEC cell lines. Gap junction function in Human Umbilical Vein Epithelial cells is Enhanced by TanⅡA and Salvianic acid A sodium in vitro and suicide gene therapy bystander effect is improved. Gene is further tested on Quantitative real-time polymerase chain reaction and protein on Western blot in order to demonstrate the gap junction mechanism of bystander effect. New ideas and methods of research suicide gene therapy system synergy mechanism from Traditional Chinese medicine were provided in tumor vessel.