| ObjectiveSuicide gene therapy has become a promising new approach that widely used in cancer treatment because of its bystander effect. Enhancing the effect of suicide gene therapy become a research hotspot. Our previous studies have shown that low concentration in vitro Dioscin can promote human kidney cancer786-0cells of gap junction intercellular communication function,but its mechanism is unclear. This study has identified the enhancing effect of dioscin on B16HSV-tk/GCV suicide gene therapy system in mice. By constructing the promotion or inhibition of gap junctional intercellular communication cell model to further study the gap junction mechanism, which may be an important mechanism of action in suicide gene therapy system.Methods1The Effect of dioscin on mouse melanoma cell gap junctional intercellular communication(1) Cells culture, passage, cryopreservation and recovery:We using adherent cell culture methods, the B16cells were cultured in the condition of RPMI-1640medium with10%FBS, at37℃5%CO2incubator,0.25%trypsinization was done when80%-90%cells were fused.The culture medium should be aspirated off when cell fusion rate reach more than90%, washed the cells1-2times with PBS and passaged with0.25%trypsinization (without EDTA) at37℃for2minutes. Add cell culture medium by gently pipetting (At this time gently shaking the cell plate or bottle can be a "quicksand" phenomenon) until the vast majority of adherent cells fall off and suspended. Then, After samples were put into centrifugal machine for5min at1000r/min, and then gently pipetting with the culture medium and form the cell suspension after supernatant was discarded.After trypsinization and centrifugation, beat upon the culture medium to obtain the cell suspension. Centrifugalte the sample for5min at1000r/min,and the supernatant were abandoned, inhalation vials at2×106/mL density. Put the vials into the frozen boxes which contain isopropanol, and remove the vials and moved into a container of liquid nitrogen after overnight in ice box at-80℃. Remove the vials containing the cells from the liquid nitrogen tank quickly, then water bath in the warm water at37℃-39℃. Remove the vials from the water bath and draw cryopreservation fluid containing cells, add3times the cells culture medium. Centrifugalte the sample for5min at1000r/min, the supernatant were abandoned, and pipetting with the culture medium to obtain the cell suspension. Added the cell culture or cell culture bottles and plates at1×104/cm2which calculated by flasks or plates bottom after counting under an inverted microscope. The medium was changed for every24hours.(2) MTT assay the effects of different concentrations of Dio on B16cell growth: The Mouse melanoma (B16) cells were4000/200μL/well were seeded into96-well plates. After24hours the cell culture medium was pipeted off, added diosgenin according to0,0.5,1,2,4,8μM final concentration(diluted in cell culture medium).48hours after aspirating the liquid measured the B16cell proliferation at different concentrations Dioscin by MTT assay, to determine the concentration used for research Dioscin gap junction intercellular communication functions.(3) Pre-labeled double-dye transfer flow cytometry analyze the effect of Dio on GJIC in B16cells:Set the B16cells which at a final concentration of diosgenin processing0,0.1,0.5,1,2,4μM as recipient cells, and set the untreated B16cells as donor cells.48hours after drug, incubated the donor cells for30min with5μM of DiI and5μM of Calcein-AM. Inoculate the donor cells by1:100in each group of receptor cells which treated by diosgenin. After cultured for six hours the culture medium was aspirated off, and the double anti-serum free medium RPMI-1640cells were washed twice; digested with0.25%trypsin for5minutes under room temperature, Gentle pipetting, the cell suspension after completely off, using flow cytometry analysis of gap junction intercellular function. Receptor double negative cells, the connection is passed through the slit into the Calcein (calcein, green fluorescent dye) to detect the number of green fluorescent cells with the fluorescent acceptor double negative cell ratio, as an evaluation index of gap junction function; greater the cell ratio between the formation of gap junctions between cells, the stronger the communication function.(4)qPCR analyze The effect of Dio on Cx43, Cx32, Cx26gene transcription in B16cells:B16cells respectively after0,0.1,0.5,1,2,4μM final concentration of Dio processing, extraction of total RNA, and reverse transcribed into cDNA; Primer5.0software design Cx43, Cx32, Cx26PCR primers, reference kit instructions, using the standard three-step method (95℃denaturation5seconds,60℃annealing for15seconds, extension72℃30seconds, and a total of40cycles) fluorescence quantitative PCR, the results with2-△△Ct method for relative quantitative analysis Cx43, Cx32, Cx26mRNA levels.(5) Western Blot detection the effect of Dio on Cx43, Cx32, Cx26protein expression in B16cells:B16were at a final concentration of Dio0,0.1,0.5,1,2,4μM after treatment, extracts from cell lysates by SDS-PAGE electrophoretic separation after transfer; successively added PVDF membrane after transfer is good corresponding to the closed a primary and secondary antibodies, membranes were incubated luminescent liquid discharge4000R after the image is scanned and analyzed using imaging workstation.2.The enhanceing effects of dioscin on B16HSV-tk/GCV suicide gene therapy system (1)MTT method assay the Inhibition rate of each group:The B16and B16HSV-tk cells were inoculated into a3:2mix ratio of96-well plates, mixed cells were divided into groups, Control group, GCV group, RA group, GCV combined RA group and GCV combined diosgenin group; The mixed cell culture24hours after dosing, the final concentration of GCV25μM, RA10μM, Dio4μM, the total volume of culture medium per well200μl, placed in37℃, the volume fraction of5%CO2incubator. Using MTT assay to the inhibition rate of each group after48hours.(2) Annexin-V-FITC assay cell apoptosis:The B16and B16HSV-tk cells were inoculated into a3:2mix ratio of96-well plates, mixed cells were divided into groups, Control group, GCV group, RA group, GCV combined RA group and GCV combined diosgenin group; The mixed cell culture24hours after dosing, the final concentration of GCV25μM, RA10μM, Dio4μM, the total volume of culture medium per well200μl, placed in37℃, the volume fraction of5%CO2incubator.Using Annexin-V-FITC flow cytometry assay cell apoptosis of each group.3. The effects of dioscin on B16HSV-tk/GCV suicide gene therapy system when blocking gap junction intercellular communication function (1) Construct the pLVmCherry-Cx43lentiviral vector plasmid:B16cells, HEK293cells,293T cells, our laboratory pre-frozen; pLVmCherry plasmid was given by Dr. Zhang Wenfeng, who is working in Guangdong College of Pharmacy. By Pubmed Cx43gene in human cDNA libraries provide full sequence design of the entire sequence primers. Instructions by TRIzol total RNA extracted HEK293cells, according to PrimeScript Double Strand cDNA Synthesis Kit kit instructions PCR reaction, amplification products were1%agarose gel electrophoresis, DNA gel extraction using Tiangen Gel Extraction Kit RT-PCR product.With restriction enzymes Cx43PCR amplification products and pLVmCherry plasmids were double digested digestion products after cutting plastic recycling ligase with T4DNA; The ligation product transformed into E. coli DH5a, including ampicillin-resistant coating flat, picked clones, shaking overnight bacteria, plasmid. Cut plasmid digested with restriction enzymes correctly after initial identification, sequencing further verification. The constructed plasmid was named pLVmCherry-Cx43.(2) Construct the pLVmCherry-Cx43G21R and pLVmCherry-Cx43G138R lentiviral vector plasmid by site-directed technique:Using the site-directed mutagenesis techniques, design contains a pair mutation primers, according Prime STAR Max DNA Polymerase manual operations to pre-constructed pLVmCherry-Cx43as a template, PCR amplification of plasmid full-length three-step method. PCR product was digested with DpnI and precipitated with ethanol, transformed into E. coli DH5a. Cloning and sequencing pick, point mutations resulting positive clones plasmid was named pLVmCherry-Cx43G21R, pLVmCherry-Cx43G138R.(3) Construct overexpression of wild-type Cx43, mutant Cx43G21R, mutant Cx43G138R B16cells:Extraction pLVmCherry-Cx43, pLVmCherry-Cx43G21R, pLVmCherry-Cx43G138R lentivirus recombinant plasmids,293T cells packaging virus, viral supernatant draw, then add Polybrene by membrane filtration, B16cells infected twice, observing mCherry expression after5days, amplification culture after the company FACSJazz type with BD FACS sorting red fluorescent cells, stable cell lines transfected with the recombinant plasmid. Western Blot assay strains obtained stable Cx43protein expression.(4) Fluorescence tracer method for the analysis of gap junction function:Donor cells with5μM Calcein-AM were incubated for30min. The donor cells at the ratio of1:100were inoculated into the receptor cells cultured for6hours, to form a stable, gap junction (gap junction, GJ), analysis of GJIC function were observed by fluorescence microscopy. Ratio of experimental group and control group, the average number of receptor cells of each donor cells around the green fluorescence of GJ, as the evaluation index of function.(5) MTT assay the effect of overexpression of wild-type Cx43, mutant Cx43G21R, mutant Cx43G138R in B16cells on B16HSV-tk/GCV suicide gene therapy system: The B16and B16HSV-tk cells according to the proportion of3:2mixture were inoculated into96well plate, hybrid cells were divided into control group, GCV group, GCV combined with dioscin group; B16-Cx43and B16HSV-tk according to the proportion of3:2mixture was inoculated into96well plate, hybrid cells were divided into control group, GCV group; B16-Cx43G21R and B16HSV-tk, B16-Cx43G138R and B16HSV-tk respectively by3:2the proportion of mixed inoculated into96well plate, hybrid cells were divided into control group, GCV group, GCV combined with dioscin group. The mixed cells were cultured for24hours after dosing, final concentration of GCV25μM, Dio4μM, each hole medium volume of200μL, incubator at37℃,5%volume fraction of CO2culture. The killing effect of MTT was detected in48hours.Result1. The Effect of dioscin on mouse melanoma cell gap junctional intercellular communication(1) MTT assay the effects of different concentrations of Dio on B16cells. The result reveals that B16-cells which has been treated by0-4μM Dio, had no significant effect on growth. Which means, within this concentration range of Dio, it has neither cytotoxicit nor proliferation effect on B16-cells. In subsequent experiments, I control the concentration of Dio within4μM, the reaction time was48hours.(2) Flow cytometry showed that, Compared with the control group, B16cells were treated with various concentration gradient Dio for48hours, the ratio of a single green fluorescent cells and cells with double vaginal dosing concentration increasing, showed Dio B16cells can effectively promote gap junction function, and there is a clear dose-response relationship.(3) Fluorescent quantitative PCR shows that after0-2μM Dio treated cells for48hours, no significant effect on Cx43and Cx32in RNA transcription, RNA transcripts for Cx26significant role in promoting;4μM Dio cells were treated for48hours have significantly promote Cx43and Cx32in RNA transcription of RNA transcription, but it significantly inhibited the RNA transcription of Cx26. (4) Western Blot showed that, compare with the control group, after each concentration gradient Dio48hours, the expression of Cx43and Cx26protein concentration increased with the dosage increased, the expression of Cx32decreases with increasing concentrations of dosing, indicating that Dio can effectively promote expression of cell gap junctional protein of B16Cx43and Cx26, and inhibit of the expression of Cx32.2. The enhanceing effects of dioscin on B16HSV-tk/GCV suicide gene therapy system(1) MTT method assay the Inhibition rate of each group:After preliminary tests, we found that when B16cells and B16HSV-tk cells mixed culture60%,40%to a final concentration of25μM GCV48hours, GCV can be substantially anti B16HSV-tk cells, but no cytotoxic effect on B16cells and no significant side killing effect on mixed cell. Therefore, in subsequent experiments, using a final concentration of GCV25μM,60%B16cells was mixed with40%B16HSV-tk ratio.MTT assay showed that the combined treatment group of60%B16cells were mixed with40%B16/tk inhibition was significantly higher than other groups,25μM GCV+2μM Dio set of actual inhibition rate49.2%,25μM GCV+4μM Dio set of actual inhibition rate56.5%, significantly higher than the theoretical inhibition rate (32.4%,35.3%), and there was a significant difference between25μM GCV group inhibition rate; Jinzheng Jun Q values were1.52,1.60, were greater than1.15,: indicating that Dio has a synergistic effect with GCV.(2) Annexin-V-FITC assay cell apoptosis:The results show that the apoptosis rates of Combination groups are higher than monodrug group.The apoptosis rate of25uM GCV+4μM Dio group is the highest, which is61.09%.3. The effects of dioscin on B16HSV-tk/GCV suicide gene therapy system when blocking gap junction intercellular communication function(1) After pLVmCherry-Cx43plasmid DNA was double digested and with1%agarose gel electrophoresis, showing approximately8kb (empty vector pLVmCherry size8.2kb) and lkb size of two bands, in line with the expected results; pLVmCherry-Cx43Cx43sequencing results in exactly the same sequence with NM000165, no accidental mutations(2)Plasmid pLV Cx43-mCherryG21R, pLVmCherry-Cx43G138R DNA was double digested by1%agarose gel electrophoresis, and were seen two bands about1kb and8kb; The recombinant plasmid pLVmCherry-Cx43-G21R sequencing showed that primers designed according to Section61of guanine to adenine mutation, the rest of the sequence exactly; pLVmCherry-Cx43-G138R sequencing showed that primers designed according to Section412of guanine mutation adenine, thymine mutation of the first414guanine, the rest of the sequence exactly.(3) The recombinant plasmids packaged virus were infected with recombinant lentivirus received B16cells,5d under a fluorescence microscope to see the cells expressing mCherry; expression of mCherry with time gradually increased, fluorescence enhancement. Western Blot Detection stable strains of connexin overexpression of Cx43protein expression of wild-type B16cells Cx43, mutant Cx43G21R, mutant Cx43G138R significantly higher.(4) Fluorescent tracer showed that the over-expression of wild-type Cx43between B16cells calcein transfer enhancement compared with the normal group, over-expression of mutant Cx43G21R, between B16cells Cx43G138R calcein transfer gap junction function than normal weakened.(5) Mixed cell culture24hours,48hours, without significant difference in growth;72hours, B16-Cx43Mixed cell growth than other mixing slowly. GCV group B16cells were mixed with B16HSV-tk inhibition rate of27.46%, B16-Cx43mixed with B16HSV-tk inhibition was46.32%(P<0.05vs B16cells were mixed with B16HSV-tk), B16-Cx43G21R and B16HSV-tk mixed cell inhibition rate of25.07%, B16-Cx43G138R mixed cell inhibition rate of26.44%; GCV+Dio combined group B16cells were mixed with B16HSV-tk inhibition rate of54.78%, B16-Cx43G21R mixed with B16HSV-tk cell inhibition rate of32.08%(P<0.05vs B16cells were mixed with B16HSV-tk), B16-Cx43G138R mixed cell inhibition rate33.21%(P <0.05vs B16cells were mixed with B16HSV-tk). GCV of hybrid cells containing Cx43over expression of wild-type cell lines B16inhibition rate higher than ordinary hybrid cells containing B16cells group; GCV over-expression of the hybrid cells containing mutant Cx43G21R or B16cell lines over expressing mutant Cx43G138R inhibition rate of B16cells containing less than ordinary group; Dio and GCV combined group, over-expression of the hybrid cells containing mutant Cx43G21R or over expressing mutant Cx43G138R inhibition rate of cell lines B16, B16cells group with lower than normal, and with the simple GCV was no significant difference.Conclusion(1)0-4μM Dio treated B16cells48hours had no significant effect on growth; Dio low concentrations in vitro can promote the GJIC function of B16cells and present a dose-effect relationship. The mechanism may be partially due to Dio can promote the expression of Cx proteins, thereby promoting GJIC function.(2) Dio do enhancing the effect of B16HSV-tk/GCV suicide gene therapy system.(3) Successfully constructed wild-type Cx43, mutant Cx43G21R, Cx43G138R recombinant fluorescent protein fusion lentiviral expression plasmid; stable over-expression of wild-type Cx43, and overexpression of mutant Cx43G21R, overexpressing mutant Cx43G138R of B16cells. B16cell lines overexpressing wild-type Cx43promote its GJIC function, over-expression of mutant Cx43G21R, Cx43G138R GJIC inhibit its function.(4) Gap junction mechanism of bystander effect, is playing an important mechanism for Dio with B16HSV-tk/GCV suicide gene therapy system synergies.This study medicine monomer in combination with suicide gene in vitro demonstrated lower concentrations Dioscin promote GJIC function of B16cells may enhance the efficacy of suicide gene system base on the overexpression of Cx43function properly constructed and functional defects in cell lines, by promote and blocking gap junctional communication way further study the suicide gene therapy side effects of anti-gap junction mechanism for prescription medicine provides new ideas and methods of research suicide gene therapy system synergy mechanism and so... |
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