【作者】 吴映雅；

【导师】 李杰芬；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2006， 博士

【摘要】 恶性肿瘤是严重危害人类健康和生命的重大疾病。基因治疗给恶性肿瘤患者带来了曙光,国外已把基因治疗作为恶性肿瘤常规治疗无效后的一种标准治疗尝试。但十多年来基因治疗的实验和临床研究结果表明,肿瘤基因治疗远未达到人们当初期望的那样有效。自杀基因治疗虽然存在旁杀伤机制,理论上能完全消除肿瘤细胞达到治愈的效果,但由于目前载体转染效率较低、靶向性不够等问题还没有解决,因而未能达到预期的效果,且因病毒载体的使用等带来了一定的毒副作用。如何有效地提高治疗效果,同时减少其毒副作用是目前仍须解决的关键问题。现有研究发现,几乎所有自杀基因系统都存在旁杀伤效应,增强旁杀伤效应能降低对高转染率的需求,减少载体及前体药物用量和毒性,增强基因治疗系统对肿瘤杀伤力。因此,增强旁杀伤效应目前已成为提高肿瘤基因治疗疗效的重要策略。产生旁杀伤效应的主要机制有:缝隙连接细胞通讯(Gap Junctional Intercellular Communication,GJIC)介导,细胞凋亡介导和免疫介导等。根据上述这几种机制,目前形成旁杀伤效应的增效策略主要有:促进GJIC、诱导癌细胞凋亡,提高患瘤机体免疫力等。常用的技术手段包括基因转染法及药物诱导法。中药是一个天然药物宝库,在促进肿瘤GJIC、诱导癌细胞凋亡或提高患瘤机体免疫功能等方面都有优势或潜力,且毒副作用相对较少,从中寻找自杀基因增效药物,建立中西医结合疗法具有可行性。 研究目的意义:本课题选择被报道有促细胞GJIC或诱导癌细胞凋亡作用的抑癌中药成分芹黄素、白藜芦醇、姜黄素,研究其对自杀基因系统是否有协同增效作用,获得有肯定作用的中药成分,探讨其通过GJIC或细胞凋亡机制增强自杀基因旁杀伤效应的药理靶点,阐明其增效的机理,为建立肿瘤基因治疗联合中医药疗法,推进肿瘤基因治疗的临床应用和抗癌中药新药开发打下基础。 1.实验方法: 1.1 中药活性成分对自杀基因旁观者效应影响的体外筛选:MTT法体外检测大鼠肝癌细胞CBRH7919对芹黄素、白藜芦醇、姜黄素的敏感程度,以确定用药浓度。选择对细胞抑制率低的浓度进行实验。上述三种中药活性成分各自以不同浓度与GCV分别或共同作用于大鼠肝癌CBRH7919的tk~-细胞,以及含10%tk~+细胞的tk~+、tk~-混合细胞,MTT检测各组存活率,两两比较分析各组存活率的差异和旁观者效应大小,并用金正钧Q值分析中药与自杀基因系统联合的相互作用是否具有协同性。Q值为联合用药时实测药效与理论药效的比值,Q≤0.85为拮抗作用,0.85≤Q<1.15为相加作用,q≥1.15为协同作用。 1.2 对有效中药成分对自杀基因治疗系统增效机制研究:分别用不同浓度的白藜芦醇、姜黄素与GCV分别或共同作用于大鼠肝癌CBRH7919的tk~-细胞以及含10%tk~+细胞的更多还原

【Abstract】 The suicide-gene therapy is a promising project of treating tumor, but the problem in this remedy is insufficient killing ability, low efficiency of transfection and uncertain safety. It’s reported that increasing the bystander effect is 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 methods of twin genes transfection and medical inducement to elevate GJIC, induce apoptosis and raise 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 later method. So we adapted it.There were not many medicines especially natural medicine with this effect had been found 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, 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 cell GJIC 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. Screening active compounds of TCM in vitro. The sensibilities of rat hepatocarcinoma cell CBRH7919 to apigenin, resveratrol and curcumin were detected by MTT assay. Then the cell CBRH7919, and the mixed cell with 5%or 10% tk+ and tk’ are treated separately by apigenin, resveratrol, curcumin in were treated with diverse concentrations of apigenin, resveratrol, curmin and GCV separately, or apigenin plus GCV , resveratrol plus GCV , curmin plus GCV (n = 3,6). Viability of cells was determined by MTT assay. The Q-value analysis was used to estimate the synergistic effects of the active components on the suicide gene system. The Q-value was equal to the ratio of the actual effect of combined treatment to its theoretical effect. 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).2. Exploring the mechanism of the synergistic effects of the active components: The cell CBRH7919, and the mixed cell with 5%or 10% tk+ and tk" are treated separately by apigenin, resveratrol, curcumin in were treated with diverse concentrations of apigenin, resveratrol, curmin and GCV separately, or apigenin plus GCV , resveratrol plus GCV , curmin plus GCV ( n = 3,).Then the cell cycle analysis and cell apoptotic index were performed using a FACScan cell analyzer. AGA was added in the combination therapeutic system in vitro and cell viability in every group was detected by MTT assay. The effects of resveratrol or curcumin on the GJIC of CBRH7919 were detected by SL/DT. The expression of Cx43 in cells were determined by FITC indirect immuno-fluorescent assay.3. Cloning of the tkgfp Fusion Gene and its expression: The cDNA encoding the thymidine kinase gene of HSV-1 was obtained by digestion of pLXSN-tk withEcoR I /BamHI. Thel.7-kb tk fragment was isolated and digested with Xma I. Xmal cuts 22 bp in front of the STOP codon. The resulting 1.3-kb tk fragment was isolated and ligated to the 4.7-kb DNA fragment obtained after plasmid pEGFP-Nl was digested sequentially with BamH I and Xmal. After ligation, the open reading frames of both tk and gfp are in-frame within the pTKGFP expression plasmid 6.0 kb, placing the tkgfp cDNA under control of the cytomegalo-virus CMV immediate early IE 1 promoter. Cells of B16, CBRH7919 and NIH3T3 were transfected with the plasmid pTKGFP or pEGFP-Nlor pDsRed2-Nl, with polyfect transfection reagent, according to the manufacturer’s protocol. For fluorescent detection of g#>-expressing cells or DsRed2-expressing cells, culture plates were examined with a standard fluorescence microscope 24 hours after transfection. For determination of the ganciclovir sensitivity, GCV-treatment was carried out at different final concentrations of medium for half of the dishes starting 24 hours after transfection. Three days later, surviving cells were determined as a percentage of non - GCV-treated transfected cells. 2. Result 2.1 The results of screening active compounds of TCM in vitro.Apigenin showed an inhibition effect on CBRH7919 when the final concentration was higher than 50uM but showed low inhibiting effect at 50uM.When combining with HSV-tk/GCV system, apigenin showed a synergistic effect at 50uM(Q value >1.15) but showed an additive effect on HSV-tk/GCV therapeutic system at 25uM(0.85<Q valueResvertrol showed a distinct inhibition effect on CBRH7919 in a range of concentration from 25uM to lOOuM. IC50 was 45uM And the effect showed in a dose-dependent manner. When combining with HSV-tk/GCV system, resveratrol showed a synergistic effect at lOuM and 25uM(Q values >1.15).Curcumin showed a distinct inhibition effect on CBRH7919 in a range of concentration from lOuM to 50uM. IC50 was 24uM .And the effect showed in a dose-dependent manner. When combining with HSV-tk/GCV system, curcumin showed a synergistic effect at 5uM and 10pM(Q values >1.15).2.2 The results of the mechanisms researchs of the synergistic effects of the active components:Results of FACS showed that when combining with HSV-tk/GCV system resveratrol showed an apoptotic rate-elevating effect on CBRH7919 but showed no effect on cells cell cycle. Curcumin showed an apoptotic rate-elevating effect on CBRH7919 when combiningwith HSV-tk/GCV system.Furthermore, it showed a S-phase lagging effect on CBRH7919 cell cycle. GJ inhibitor AGA showed a down-regulation effect on the killing effect in the combination group of resveratrol or curcum with tk/GCV system. These results indicated that mechanisms of the augmentation effect of resveratrol or curcum on tk/GCV system were related to GJIC. The result of SL/DT showed that ability of GJIC function of CBRH7919 was up-regulated by resveratrol or curcumin. The result of Cx43 expression detection by FTTC indirect-immuofluorescent assay showed a low expression of Cx43 in CBRH7919 control groups and a higher expression of Cx43 in resveratrol or curcumin treated groups.2.3 The levels of ^-expression were assessed days after transfection with a standard fluorescence mi croscope and were visually of similar intensity in tkgfp and native gfp transfected cells . The data indicate that tkgfp transduced cells can be readily identified by fluorescent light at least to the same extent as gfp transduced cells. The intracellular expression pattern of the TKGFP seems different than the native GFP. TKGFP is mostly concentrated in the nucleus, whereas the native GFP and DsRed displays a predominantly cytoplasmic distribution. GCV sensibility examination showed that the pTKGFP transfected cells were much more sensitive to GCV than the pEGFP-Nl transfected cells in a dose-dependent manner. Cone I us i onWe explore the effects of resveratrol, apigenin and curcumin on tumor HSV-TK /GCV suicide gene therapy system. Results show that all of them exibit synergistic killing effects on suicide gene system. Both of the effective concentration of resveratrol and curcumin are lower than apigenin. Mechanisms of the synergistic effect of resveratrol and curcum are related to the restoration of GJIC, or/and the apoptotic inducement or the regulation of cellular cycle.We construct a TKGFP fusion protein expression vector which is transfected into CBRH7919, B16, and NIH3T3 and expressed in these cells successively. The expressed fusion protein exibit the activity of thymidine kinase(TK) and green fluorescence of EGFP. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.更多还原