| The suicide-gene therapy is a promising project of treating tumor, but the problem in this remedy is the insufficient killing ability for the low efficiency of transfection and uncertain safety. It's reported by the last investigation that increasing the bystander effect was a good approach to resolve this problem. Considering the mechanism of bystander effect conducted by the Gap junction intercellular communication (GJIC) of cell, apoptosis of cell and immune factors, researchers today enhance the bystander effect by the methods of twin genes transfection and medical inducement to elevate GJIC, induce apoptosis and rise the function of immune system. The method of two genes transfection means that we should transfer the suicide gene with another effect-increasing gene at the same time, but in actual practice there is a low efficiency of transfection in vivo, so the difficulty of transfection practice of two genes simultaneously is far high, and the potential side-effect is much high either. In comparison, much merit such as simple practice, small expense, aiming much more target cells, etc. existed in the late method. So we adapted it.There were not many medicines especially natural medicine with this effect had been founded until today, but we knew some advantages and potentials have been illustrated by in apoptosis inducing, cell communication and adjustment of immune system, even some ingredients of TCM had been proved by the clinic or basic investigations on the effects of inducing apoptosis of tumor cells and increasing immunity of the patients. But few articles were released about the investigation on increasing bystander effect and cooperating the tumor gene therapy with TCM or their ingredients.Therefore, according to the development and trend of the studies in the mechanism of bystander effect and bystander effect-increasing induced by medicine, we presented the following hypothesis, there were some active ingredients in the Traditional Chinese Medicine could improve the GJIC, induce the apoptosis of tumor cells, improve the local inflammatory immune environment of tumor or enhance the function of immune system, and exert the effect of anti-tumor cooperated with other components by the manner of increasing bystander effect of suicide-gene which maybe increase GJIC, induce apoptosisof tumor cells and immunity so on. ObjectiveIn our research, firstly we intended to establish HSV-TK/GCV suicide-gene therapy system conducted by retrovirus aimed to liver cancer cells of rat and mice, then step up a technique and an effective and steady model of cells to screen high-scale medicines with effect of rising the bystander effect, all above served as the purpose to investigate the possibility improving the effect of suicide gene therapy cooperated with Traditional Chinese Medicine. After getting the several effective components of TCM on the cell level, we would validate them on the whole level repeatedly to find the real effective ingredients of TCM. And at the same time, we wanted to compose those ingredients acted on different mechanism a complex dose with known components, clarify pharmacology, stable quantity and maybe have a higher effect.During the research, we hoped to explore the possible mechanism of adjusting the tumor-cell cycle and inducing of cell apoptosis with TCM, then we could get the further comprehension about the mechanism of increasing bystander effect with TCM and constructed the foundation of investigating the pharmacology effect in signal transduction of tumor-cell, regulating the cell cycle, communication of cell and immunity of tumor cell etc. with the ingredients of TCM. Methods1. Constructing suicide-gene therapy of tumor, ?infecting the mice liver-cancer cell CBRH7919 with the active retrovirus particle excreted by the enveloped cell PT67/tk, then screening them three weeks with G418 to gain the recombinant CBRH7919/tk with high drug-fast ability and culture them on large scale. ?Abstracting DNA of cell CBRH7919 , CBRH7919/tkls, labeling the fragment of gene HSV-tk with Dig, dot hybridizing with the DNA of cell CBRH7919, CBRH7919/tk, those practice were for the purpose to examine whether the cell CBRH7919 > CBRH7919/tkls were integrated the gene HSV-tk or not. ?The killing effect of GCV to cell CBRH7919 , CBRH7919/tk in vitro, treating the cell CBRH7919 , CBRH7919/tkls with diverse dimension such as 0.1, 1,10, 50, lOOmg/L GCV, then examining the survive rate with MTT.2. Checking the bystander effect. Treating the composed cell of tk+ and tkon diverse ratio with GCV, checking the survive rate comparing the two bystander effect.3. Screening active compounds of TCM. The mice's liver cancer cell CBRH7919, the rat's liver cancer cell H22, and the mixed cell with 5%or 10% tk+ and tk" are treated separately by the tamshin, evodiamine, matrine, panax notoginseng saponins withdiverse concentration, then examining the survive rate with MTT, comparing one another and analyzing the difference of survive rate and the bystander effect.4. Investigating the effect of inducing the apoptosis of liver cancer cells with ingredients of tamshin, evodiamine matrine, panax notoginseng saponins, and the mechanism of inducing the apoptosis with tamshin, evodiamine. Apoptosis is examined by the flow-cell apparatus.5. The remedy of rat's transplanted liver tumor with combining suicide-gene therapy with active components of TCM. ?Observing the effect of tanshin that cured the rat's transplanted liver tumor with suicide-gene therapy cooperating. ?Observing the effect of lycium barbarum polysaccharides ( LBP) that cured the rat's transplanted liver tumor with suicide-gene therapy cooperating. Use the same methods as ?.Results1. Constructing suicide-gene therapy of tumor. ?The mice liver-cancer cells CBRH7919 were infected with the active reconstructive virus particle excreted by the enveloped cell PT67/tk, we obtained the recombinant CBRH7919/tk with high drug-fast ability.?After labeling the fragment of gene HSV-tk with Dig and then hybridizing with theDNAof cellCBRH7919 > CBRH7919/tkls, we found that the cell CBRH7919 > CBRH7919/tkls had integrated the gene HSV-tk while there was no gene HSV-tk in the cell CBRH7919. ?From the experiment of examining the killing effect of GCV to cell CBRH7919, CBRH7919/tkls in vitro we found there was obvious effect of poison to cell and inhibit the growth of CBRH7919 depending on the time and dimension while the cell CBRH7919 was not sensitive to the GCV. All above shown us the gene HSV-tk had expressed in the tumor-cell CBRH7919/tk and the expression product had a larger activity.2. Checking the bystander effect by treating the composed cell of tk+ and tk-on diverse ratio with GCV. We found that the bystander effect of suicide gene was lower in the cell lines CBRH7919/tk than many other tumor cells, the sensitivity was much higher when examining the bystander effected when the ratio of mixed tk+ with tk- is 5% to 10% than any other ratios. The results implied that we had constructed the tumor suicide gene therapy system affected to the mice's liver cancer successfully, found the model of cell to examine the effect of bystander effect, which founded the base to the further study to investigate the affect of active components in TCM to suicide-gene therapy and by-stand effects.3. The result of treating the mice's liver cancer cell CBRH7919or the rat's liver cancer cell H22 with the active components of TCM such as tamshin, evodiamine, matrine, panaxnotoginseng saponins in vitro combined with the tk/GCV system. The result showed that, when the concentration of GCV was lOOmg/L, the survive rate of tk/GCV declined 5.86% than the GCV group, while the survive rate of the low concentration tanshin with tk/GCV group decreased 36.76% than the low concentration tanshin with GCV group, the survive rate of concentration tanshin with tk/GCV group were less 46.51% than the tanshin with GCV group, there was the same result when the concentration of GCV is 50mg/L. the result of other components was achieved analogously with tanshin, there was not obvious decline of the survive rate in tk/GCV group than the GCV group, while the survive rate of evodiamine with GCV group downed 28.82% than the lmg/L evodiamine with tk/GCV group, the survive rate of 2mg/L evodiamine group descended 57.97% than the 2mg/L evodiamine with GCV group. All above illustrated that it's the components tanshin, evodiamine that could rise the killing effect on liver cancer cells cooperating with the tk/GCV system and the bystander effect of gene tk. matrine, panax notoginseng saponins had some ability to inhibit the growth of tumor cells while they could not cooperate with the tk/GCV system but only help the killing effect, no increasing effect with the bystander effect of gene tk has been found so far. 4. Presented by the experiment investigating the effect of inducing the apoptosis of liver cancer cells with some ingredients of TCM, tamshin, evodiamine both regulated the cell cycle, induced the apoptosis of liver cancer cells, excited the produce of apoptosis micro particles, they maybe exerted the killing tumor cells' effect cooperated by bystander effect of suicide gene conducted by the mechanism of apoptosis. In our experiment I also found that there was a higher activity of inducing the apoptosis of liver cancer cells with tanshin solution and evodiamine, the IC50 is 1.4g/L when affected by evodiamine 72 hours, the IC50 is 11.4mL/L when affected by tanshin injection solution 72 hours. The apoptosis index of cells affected by 5mL/L and lOmL/L tanshin injection solution for 24 hours is 2.2% and 11.6%, while the index of control group is 0.8%; and the apoptosis index of cells affected by the 1 mg/L and 2mg/L evodiamine for 24 hours is 6.4% and 9.1%, while the index of control group is 1.2%; Apoptosis index examined by the flow-cell apparatus.There were the similar effects of inducing the apoptosis of liver cancer cells with the concentration of 125mg/L matrine and lOOmg/L panax notoginseng saponins or higher in vivo, in such a concentration the survive rate is 76.18+10.31% and 69.74±8.54% respectively after affected three days.Then we deduce the IC50 is about 500 mg/L of matrine and 220 mg/Lof panax notoginseng saponins after affected 72 hours, which is not a much high survive rate comparing with tanshin and evodiamine.5. The remedy of rat's transplanted liver tumor with combining suicide-gene therapy with active components of TCM.?The tumor bump could be touched after inoculating for 7 days. With the treating of suicide gene therapy combined with tanshin, the growth speeds stepped down and implying the tumor was in a inhibit state, the average dimension of tumor was 1533.15±881.60mm3, decreased 52.01% than the control group's 3194.63±1763.93 mm3 (/M).020<0. 05), the average weight of tumor was 1.319±0.803g less 42.11% than the control group's 2.276+ 1.203g (/M).032<0.05), distinct difference in statistics, while there was no obvious difference in statistics of volume and weight in tanshin comedy group in contrast with the control group. In pathology check the number of tumor cells was smaller in combine therapy group than in the control group, the growth was not so agile either, some soft tissue proliferated around the tumor. And there were not highlight change in the simple suicide gene therapy group and tanshin therapy group and not distinction with the control group in statistics. All results alluded it's tanshin that capable to increase the killing liver cancer cell effects cooperating with the suicide gene system, and then improve the effect of suicide gene therapy.?With the treating of suicide gene therapy combined with lycium barbarum polysaccharides ( LBP), the growth speeds stepped down and implying the tumor was in a inhibit state, the average dimension of tumor was 2011.07+898.85mm3, decreased 40.91% than the control group's 3403.30 ± 1952.24 mm3 (P= 0.018<0. 05), but the average weight of tumor was 2.098 ±1.096g less 24.89% than the control group's 2.793 ±1.414g (/M).154>0. 05), not distinct difference in statistics. There was no obvious difference in statistics of volume and weight in comedy group and tk/GCV group in contrast with the control group. Whether LBP is capable of increasing the killing effects on liver cancer cells by cooperating with the suicide gene system or not, it needs more experiment evidence.In the single suicide gene therapy group, it presented the similar inhibition of tumor as the other group in every time while no distinct difference was found in statistic analysis. In our consideration the probable reason was there were much more tumor cells and it grew rapidly while the time was too short for the influence with GCV and not fully exerted on it. Adopting the tk+ cell on a low proportion for simulating the low transfer rate in the level of integrity. The impure tk+ cells maybe composed tk- cells. Etc.ConclusionEstablished successfully HSV-TK/GCV suicide-gene therapy system conducted by the...
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