| Primary hepatic carcinoma(PHC) is one of the highest incidences of malignant tumor in the worldwide,known as "cancer King".The number of the patient of PHC in China ranks first in the world.Every year about 23 million people die of the PHC because of its high degree of malignancy and poor prognosis.Using the gene therapy against tumors has become an important choice for scientists all over the world to choose.1989 -2003 years,the United States approved the registration of clinical trials of gene therapy for a total of 523,representing about 80%of the total of the world. The number of the patient who has been treated has achieved tol5,000,including 70%of the patient who was treated by the gene therapy.The Suicide gene therapy has a wide application prospects to become the new treatment of cancer because of the unique bystander effect.But the Suicide gene therapy alone can not completely cure the tumor,thereby enhancing the effectiveness of suicide gene therapy become a research hotspot.To improve the immune state of the tumor-bearing body is one of the measures of its efficiency,and the Chinese medicine has a definite role in the immune pharmacology.The study group makes it as a starting point,confirming the synergy of Liu Wei Di Huang bolus on hepatocarcinoma HSV-tk/GCV suicide gene therapy.The mechanism may be related with the fact that Liu Wei Di Huang bolus enhance the bystander effect of suicide gene by improving the inflammatory and immune micro-environment where the suicide gene faint against the tumor.The gap junction mechanism mediated by GJIC is considered the main mechanism of suicide gene's bystander effect.In view of Chinese herbs with multi-channel,multi-target characteristics,the synergy of Liu We Di Huang bolus on hepatocarcinoma suicide gene therapy may be related to the mechanism of gap junction.The subject of this study is to examine whether the synergy of Liu We Di Huang bolus on hepatocarcinoma suicide gene therapy is related to that it can improve the function of hepatocarcinoma cell's GJIC,and further clarify its mechanism.Objective:on the basis of preliminary work,we study the rat's hepatocarcinoma CBRH7919 cell to explore the gap junction mechanism of the synergy of Liu We Di Huang bolus on hepatocarcinoma HSV-tk/GCV suicide gene therapy,with a view to provide experimental data,scientific evidence and the basis of the work for the establishment of clinical suicide gene therapy combined with traditional Chinese medicine.Methods:1.Using the SABC immtmohistochemical assay to detect the expression of Cx26, Cx43,Cx32 in hepatocarcinoma tumor-bearing mice:We choose the paraffin-embedded tumor tissue of hepatocarcinoma tumor-bearing mice.The tumor tissue is divided into tumor model group,suicide gene therapy group,Liu We Di Huang bolus in treatment group,suicide gene therapy combined with Liu We Di Huang bolus in treatment group,a total of 4 groups.We selecte about 18 pieces of animal wax from each group,conventional biopsy DAB immunohistochemical staining,and then select 5 high power fields randomly under the microscope to count. According to the color intensity and the number of positive cells in the two indicators, then we determined positive rating.2 To examine the killing effect on rat's hepatoma cells CBRH7919 by using medicated serum of Liu Wei Di Huang bolus combined HSV-tk/GCV suicide gene therapy system.Selecte the pre-built HSV-tk/GCV CBRH7919 suicide gene therapy system,make sure that the GCV's working concentration is 39.2μmol/L;the mouse serum's adjunction is 10%(volume fraction);Medicated or non-medicated serums were obtained from SD rats which were treated with Liu Wei Di Huang Bolus(the dose was 32 g/kg.d).CBRH7919/tk+ cells and CBRH7919/tk - cells were mixed in proportion of 1:9 and were used as 10%tk+/GCV target cells.CBRH7919 cells were sorted into following experimental groups:non-medicated serum group(10% non-medicated serum,final concentration,v:v),GCV plus non-medicated serum group,non-medicated serum plus 10%tk+/GCV group,low-dose medicated serum group(2.5%medicated serum+ 7.5%non-medicated serum,final concentration,v:v), GCV plus low-dose medicated serum group,low-dose medicated serum+10% tk+/GCV group,mid-dose medicated serum group(5%medicated serum+ 5% non-medicated serum,final concentration,v:v),GCV plus mid-dose medicated serum group,mid-dose medicated serum plus 10%tk+/GCV group,high-dose medicated serum group(10%medicated serum,final concentration,v:v),GCV plus high-dose medicated serum group,high-dose medicated serum plus 10%tk+/GCV group. CBRH7919/tk- cells or 10%tk+ cells were seeded in quadruplicate in 96-wells plates at a density of 3×103/well,grown in complete medium for 24 hours,,treated with medicated or non-medicated serum for 12 hours and sequentially with GCV at 39.2μmol/L for another 60 hours.The viability of cells was determined by MTT assay. Q value analysis,the ratio of the actual effect of combined treatment to its theoretical effect,was used to value the synergistic effect of the medicated serums on the suicide gene system.The effect was classified into three categories:antagonistic effect(Q≤0.85),additive effect(0.85≤Q＜1.15),and synergistic effect(Q≥1.15).3.To examine the influence of the herbs serum of Liu Wei Di Huang Bolus on the GJIC function of CBRH7919 cell:CBRH7919 cells were sorted into following experimental groups:non-medicated serum group(10%non-medicated serum,final concentration,v:v),low-dose medicated serum group(2.5%medicated serum+ 7.5% non-medicated serum,final concentration,v:v),mid-dose medicated serum group(5% medicated serum+ 5%non-medicated serum,final concentration,v:v),high-dose medicated serum group(10%medicated serum,final concentration,v:v),treated with medicated serum of different concentration for 48h.The GJIC function is detected by the preloading and dye transfer and the fluorescence photo bleaching recover technique.4.To examine the influence of the herbs serum of Liu Wei Di Huang Bolus on the expression of Cx26,Cx43,Cx32 and mRNA on rats hepatoma cell line CBRH7919 by using Real-time fluorescent quantitative PCR assay:CBRH7919 cells were sorted into following experimental groups:non-cell group(without serum),non-medicated serum group(10%non-medicated serum,final concentration,v:v),low-dose medicated serum group(2.5%medicated serum+ 7.5%non-medicated serum,final concentration,v:v),mid-dose medicated serum group(5%medicated serum+ 5% non-medicated serum,final concentration,v:v),high-dose medicated serum group(10%medicated serum,final concentration,v:v),treated with medicated serum of different concentration for 48h.The expression of Cx26,Cx43,Cx32 and mRNA is detected by Real-time fluorescent quantitative PCR assay.Make the house-keeping gene GAPDH as the quod vide of the fluorescent quantitation RT-PCR reaction,result calculationΔct=ct's mean of Cx-ct's mean of GAPDH,then make 2-ΔCt to present the relative amount of primary cDNA.5 To examine the influence of the herbs serum of Liu Wei Di Huang Bolus on the the expression of Cx26,Cx43,Cx32 and mRNA on rats hepatoma cell line CBRH7919. The CBRH7919 cell is treated with medicated serum of different concentration for 48h.The expression of Cx26,Cx43,Cx32 and mRNA is detected by western blot, indirect immunofluorescence assay(FITC) and flow cytometry.Results:1.The expression of Cx26,Cx43,Cx32 in hepatocarcinoma tumor-bearing mice is detected by the SABC immunohistochemical assay,the positive expression were observed located in cytoplasm and cell membrane under the microscope.Cx43 in tumour adjacent tissues,especially the site which is infiltrated by fibrous tissue was strongly positive expression,but cancer cells in the tumor nodules are relatively weak expression.Cx26,Cx32 in tumor cell,especially near the tumour tissues which is infiltrated by adipose tissue show strong positive expression.The expression of Cx in GCV in treatment group is similar to that in tumor model group(P＞0.05),but The expression of Cx in Liu We Di Huang bolus in treatment group and suicide gene therapy combined with Liu We Di Huang bolus in treatment group is obviously stronger than that in tumor model group.The differences of expression of Cx between suicide gene therapy combined with Liu We Di Huang bolus in treatment group and tumor model group shows statistical significance.2.The cell of non-medicated serum group,medicated serum group,GCV plus non-medicated serum group,GCV plus medicated serum group shows no obvious cytotoxicity.Under the experimental conditions show that the serum and GCV on the growth of tumor cells had no significant effect;GCV plus low-dose medicated serum group,GCV plus different medicated serum groups both has obvious killing effect(P <0.05,compared with non-medicated serum group),Show that the suicide gene system has a biological function.The cell survival of mid-dose medicated serum plus 10%tk+/GCV group and high-dose medicated serum plus 10%tk+/GCV group is obvious lower than that of non-medicated serum plus 10%tk+/GCV group and corresponding medicated serum group;The actual inhibition rate of joint Group is significantly higher than the theoretical inhibition rate.Q values is 1.16,1.49,both larger than 1.15,shows that its interaction has the synergy,with the concentration of medicated serum increasing,Q values increases and synergies enhances.Non-medicated serum group and medicated serum group below 20%volume fraction do not show obvious cytotoxicity.When the volume fraction of the serum concentration is 5%and 7.5%,compared with seperate medicated serum group and GCV plus non-medicated serum group,GCV plus medicated serum group has a stronger killing effect on tumor cells,the cell survival rate were significantly different (P<0.01),and the effect of GCV combining with medicated serum has obvious synergies(Q>1.15).3.The influence of the herbs serum of Liu Wei Di Huang Bolus on the GJIC function of CBRH7919 cell:(1)The result of flow cytometry shows that,with treated with medicated serum for some time,compared with the control group,the proportion of the double positive cells gradually increases.It can go up to 32%after 48h.After saturated,with the time,the proportion of double-positive cells gradually decrease.After 48h,detect the proportion of double positive cells Of the three-dose group,the result is:the control group is 18.76±2.22%,low-dose medicated serum group is 27.93±3.55%,mid-dose medicated serum group is 35.23±4.15%,high-dose medicated serum group is 39.93±5.71%.With medicated serum concentration increased,the rate of double-positive cells gradually increase(low-dose group P<0.05;mid-dose group, high-dose group P<0.01),and shows a certain degree of dose-dependent effect.(2) Found from the image of fluorescence recovery after photobleaching (FRAP),before strong photobleaching,CBRH7919 cells in each group were inspired to produce a strong blue-green fluorescence.The fluorescence intensity of chooesn cell is significantly weaker after transient bleaching.Over time,the fluorescence gradually returns.After 4 min,the average fluorescence recovery rate of the non-medicated serum group is 5.93±0.72.But the mean fluorescence recovery rate of medicated serum group was significantly higher than that of non-medicated serum group,and showing a certain degree of concentration-dependent trend.From low to high dose group,fluorescence intensity is 15.71±3.81%,38.34±2.64%(P＜0.01),46.22±4.33%(P＜0.01).4.After being treated with medicated serum for 48h,with medicated serum concentration increased,the relative expression of Cx26,Cx32 and Cx43,mRNA in CBRH7919 cells gradually increase and show a dose-dependent effect.But the expression of Cx32 and Cx43,mRNA is most obvious in mid-dose medicated serum group.Make the expression of Cx mRNA as 1,from low to high dose group,the relative expression of Cx26mRNA is 6.63±0.78,19.29±3.62,29.04±5.35.Compared with the control group,differences is statistically significant(P＜0.01).The relative expression ofCx32 Mrna is 23.02±5.10,261.55±29.11,73.68±16.23,and the relative expression of Cx43 is 1.53±0.84,6.75±1.71,4.09±0.98.The different of the expression of Cx32 and Cx43 mRNA between mid,high-dose medicated serum group and the control group is statistically significant(P＜0.01,P＜0.05).5.Found from the result of laser confocal microscopy,each negative control group show no positive cells,CBRH7919 cells of no-cell group and non-medicated serum group expressed Cx26,Cx32 and Cx43 less,the fluorescence intensity of positive cells is less.The expression of fluorescent-positive cells is mainly located in the cytoplasm, some in the membrane,in particular parts of cell junction.CBRH7919 cells of medicated serum group expressed Cx26,Cx32 and Cx43 more,the fluorescence intensity of positive cells is stronger.The expressure of Cx 32 in CBRH7919 cell membrane is strongly positive,especially at the cell junction site.Fluorescence was granular or thread-like distributed along its cell membrane,partly cytoplasmic.The expression of Cx26 and Cx43 was mainly concentrated in the cytoplasm,cell junction, membrane and nuclear membrane surrounding,and the fluorescence was granular or thread-like distribution.Found from the result of flow cytometry,compared with no-cell group,the different of the expression of Cx in non-medicated serum group is no significant (P＞0.05);Compared with non-medicated serum group,The expression of Cx26, Cx32和Cx43 increase significantly,the difference was statistically significant(P＜0.05或P＜0.01),especially the proportion of the positive cells in Cx32 medicated serum group is up to 70%,and has a certain dose-response trend.Compared with low-medicated serum group,the difference of the proportion of Cx32-positive cells between mid-medicated serum group and high-medicated serum group is statistically significant(P＜0.05).The proportion of Cx43 and Cx32 positive cells is in the trend of increasing,but there is no statistical difference.Found from the result of Western-blot,medicated serum can significantly improve the expression ofCx26,Cx32,and shows a clear dose-effect relationship.Conclusion:1.Using the SABC immunohistochemical assay to detect the expression of Cx26, Cx43,Cx32 in hepatocarcinoma tumor-bearing mice.From the result,compared with tumor model group,suicide gene therapy combined with Liu We Di Huang bolus in treatment group can significantly improve the expression of Cx26,Cx43,Cx32, expecially Cx26.It is Prompte that the synergy of Liu Wei Di Huang bolus on Suicide gene bystander effect is related to gap junction.2.Liu Wei Di Huang bolus combined HSV-tk/GCV suicide gene therapy system has Synergies on killing tumour cells.3.Liu Wei Di Huang bolus medicated serum can improve the function of hepatocarcinoma cell's GJIC,and shows a certain degree of dose-response effect.4.Liu Wei Di Huang bolus medicated serum can increase the expression of Cx26, Cx43,Cx32 in CBRH7919 cell,and increase their localization in the cell membrane, expecially Cx32.It shows a certain degree of dose-response effect.5.Liu Wei Di Huang bolus medicated serum can increase the expression of Cx26, Cx32 and Cx43 mRNA.It increases the expression of Cx from the transcriptional level.In conclusion,Liu Wei Di Huang bolus may promote the expression,maturity, distribution and location of the Cx in CBRH7919 cell.Through this way,it can recover the the Cx-mediated GJIC function to a certain extent,re-establish the gap junctions between cells,thereby increase bystander effect of HSV-tk/GCV system achieve the synergies of suicide gene therapy system on killing the hepatocarcinoma cells. |
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