丹参酮IIA对自杀基因旁观者效应增效作用及机制研究
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摘要
恶性肿瘤是严重危害人类健康和生命的重大疾病。随着分子生物学的发展,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。自杀基因治疗是当前最有前景的肿瘤基因治疗方案,对肿瘤细胞具有特异性并能产生旁观者效应,理论上能完全消除肿瘤细胞达到治愈的效果,但由于尚存在载体转染效率低、靶向性不够等问题还没解决,因而未能达到预期的效果。如何有效的提高治疗效果,同时减少毒副作用是目前仍须解决的关键问题。增强旁观者效应能降低对高转染率的要求,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的杀伤力。因此,增强旁观者效应已成为提高肿瘤自杀基因治疗疗效的重要策略。目前主要采用基因转染法或药物诱导法以促进缝隙连接细胞通讯(GJIC)、诱导细胞凋亡或提高患瘤机体免疫功能等措施来提高自杀基因系统的旁观者效应。中药是一个天然药物宝库,中医研究肿瘤病学的历史源远流长,迄今为止,不少报道证实了中药在促进肿瘤GJIC、诱导癌细胞凋亡等方面都有独特的优势或潜力,加之价格低廉,给药方便,毒副作用较少,使其极有可能成为自杀基因疗法的有效增效成分,联合使用可发挥协同性抗肿瘤作用。
研究目的意义：
本研究选择被报道具有抗肿瘤作用的中药成分丹参酮ⅡA、丹参素钠和三七总皂甙,研究其对自杀基因系统是否有增效作用,在获得有明显作用的中药成分基础上进一步探讨其对肿瘤细胞生长、诱导细胞凋亡以及增强细胞缝隙连接通讯等方面的影响,以了解该有效中药成分增强旁观者效应的作用机制,探讨建立一种自杀基因疗法联合中药成分的中西医结合肿瘤基因治疗方案,为肿瘤治疗提供新的思路和模式。
实验方法：
1.中药活性成分对自杀基因旁观者效应影响的体外筛选：丹参酮ⅡA、丹参素钠和三七总皂甙成分各自以不同浓度单独或与GCV共同作用于大鼠肝癌细胞CBRH7919的10%tk+/tk-混合细胞,MTT检测各组抑制率,两两比较分析各组抑制率的差异和旁观者效应大小,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。筛选出的活性中药成分,用小鼠黑色素瘤细胞B16的10%tk+/tk-混合细胞对其联合自杀基因系统的增效作用进行验证。
2.丹参酮ⅡA联合自杀基因系统对小鼠移植性黑色素瘤细胞的治疗观察。实验分为模型对照组、丹参酮ⅡA组、GCV组和丹参酮IIA+GCV联合组;把tk+、tk-细胞按1：4比例混合接种C57BL／6J小鼠腋下皮下组织内,每只小鼠接种2×105个肿瘤细胞,次日雌雄均随机分组；接种后1-7天用中药治疗,7天后用自杀基因系统治疗7天,进行疗效观察。
3.丹参酮ⅡA对大鼠肝癌细胞和小鼠黑色素瘤细胞的影响：以大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤B16两种细胞株为对象,采用MTT法从浓度依赖和时间依赖两方面检测有效中药成分对两种细胞生长的影响；形态学观察药物作用后细胞的形态变化；彗星电泳检测药物对细胞DNA的影响。
4.采用流式细胞技术观察丹参酮ⅡA对HSV-tk/GCV系统的影响。检测丹参酮ⅡA联合10%tk+/GCV对大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤B16细胞周期和凋亡率的影响。PI单染法检测丹参酮ⅡA对10%CBRH7919/tk+和B16／tk+的影响：用终浓度为12.5μM、25、50μM的丹参酮ⅡA与终浓度为15.7μM的GCV分别和联合作用10% CBRH7919/tk+和B16／tk+细胞72h,各组细胞经收集后PBS洗2次,用预冷的70%乙醇固定后,PI染色,流式细胞仪进行凋亡率和细胞周期分析。
5.丹参酮ⅡA对大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤细胞B16缝隙连接的影响。采用降落伞法观察丹参酮ⅡA对细胞GJIC的影响：培养板内的细胞用绿色荧光染料钙黄绿素(Calecin-AM)孵育,该染料在细胞内代谢后所产生的代谢产物可通过GJ进行传递。把被绿色荧光染料标记的细胞制成细胞悬液,使绿色荧光细胞像降落伞一样降落到无标记的细胞组内,通过观察绿色荧光染料在细胞内的传输判断药物对细胞GJIC功能的影响；在丹参酮ⅡA和HSV-tk/GCV联合的系统中加入GJ长效抑制剂甘草次酸,MTT法检测甘草次酸对联合系统的影响：加入丹参酮ⅡA至终浓度为0μM、12.5μM、25μM、50μM四个不同浓度组,GCV终浓度分别为OgM和15.7μM,甘草次酸浓度分别0、15μM,药物和GCV两个浓度分别联合,药物和GCV与甘草次酸的两个浓度分别联合,药物作用72h后MTT法测定肿瘤细胞对不同组别的敏感程度;分别用终浓度为12.5μM、25μM和50μM的丹参酮ⅡA作用CBRH7919细胞和B16细胞72h,然后采用RT-PCR和Western-blot技术检测各组细胞Cx43的表达量。
实验结果：
1.中药活性成分对自杀基因旁观者效应影响的体外筛选：各浓度丹参酮ⅡA联合10%tk+/GCV组的细胞抑制率(%)分别为30.38±5.28、33.22±5.21、40.58±4.49和73.04±2.85,与单纯10%tk+/GCV组(21.04±3.96)和相应的各浓度丹参酮ⅡA组(3.65±2.84、4.74±4.48、17.34±5.08和52.81±5.95)的抑制率明显提高。从其两两比较的表2可知,6.25μM、12.5μM、25μM和50μM浓度下的丹参酮ⅡA联合10%tk+/GCV组的实际抑制率显著高于理论抑制率,金正钧Q值分别为1.270、1.341、1.168和1.164,均大于1.15。用小鼠恶性黑色素瘤细胞以相同浓度的丹参酮ⅡA联合自杀基因重做该实验,结果表明,6.25μM、12.5μM、25μM和50μM浓度下的丹参酮ⅡA联合10%tk+/GCV组的实际抑制率显著高于理论抑制率,金正钧Q值分别为2.276、1.892、1.150和1.211,均大于1.15。说明丹参酮ⅡA对自杀基因系统具有增效作用,且该增效作用为协同作用。25μM浓度的丹参素钠联合10%tk+/GCV组的实际抑制率大于理论抑制率,金正钧Q值大于1.15,说明该浓度的丹参素钠对自杀基因系统具有增效作用。但50μM、100gM和200μM浓度下的丹参素钠联合10%tk+/GCV组的实际抑制率均低于理论抑制率,Q值均低于1.15,说明在此浓度范围内丹参素钠对自杀基因系统没有协同增效作用。100mg／L、200mg/L、400mg/L和800 mg／L浓度下的三七总皂甙联合10%tk+/GCV组的实际抑制率均低于理论抑制率,金正钧Q值均低于1.15,说明在此浓度范围内三七总皂甙对自杀基因系统没有协同增效作用。
2.丹参酮ⅡA联合自杀基因系统对小鼠移植性黑色素瘤细胞的治疗观察。各组小鼠均在9-10日在右腋下触及肿块,自杀基因系统联合丹参酮ⅡA组治疗后,肿瘤生长速度减慢,肿瘤呈现生长抑制状态,平均肿瘤体积200.2 mm3,比模型对照组352.7 mm3减少43.24%(P<0.05),小鼠平均肿瘤质量0.278g,比模型对照组0.5628减少50.6%(P<0.05),统计学上差异有显著性意义。GCV组平均肿瘤体积比模型对照组减少34.76%(P<0.05)；小鼠的平均瘤块质量比模型对照组减少38.98%,但该结果没有统计学意义(P>0.05)。丹参酮ⅡA组平均肿瘤体积比模型对照组减少29.94%,小鼠的瘤块质量比模型对照组减少21.22%,但其肿瘤体积和质量与对照组相比都没有统计学意义(P>0.05)。以上结果提示：丹参酮ⅡA能提高自杀基因系统对小鼠移植瘤的杀伤力,协同自杀基因作用,达到提高自杀基因疗效的效果。
3.丹参酮ⅡA对大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤B16的影响：CBRH7919和B16经梯度浓度丹参酮ⅡA(12.5μM、25μM和50μM)作用后都出现了不同程度的生长抑制,而且相关分析显示,这一抑制作用表现出一定的浓度和时间依赖性,浓度增加,或药物作用时间延长,都会使抑制率增加。倒置相差显微镜观察到CBRH7919和B16的对照组细胞生长状况良好,贴壁很稳,密度高,细胞呈梭状、菱形、三角形或多边形,药物作用后细胞生长增殖明显受到抑制,增殖缓慢,几乎停滞,细胞逐渐由贴壁而脱落,药物浓度越高脱落细胞越多。Hoechst染色后,可见CBRH7919的细胞形态变圆,折光性差,体积减小,细胞核皱缩、染色质浓缩,胞内多见颗粒,高浓度组可见数个圆形凋亡小体围绕在细胞周围,但未观察到B16细胞的死亡或凋亡现象。彗星电泳结果显示,大鼠肝癌细胞CBRH7919细胞和小鼠恶性黑色素瘤细胞株B16经梯度丹参酮ⅡA(12.5μM、25μM和50μM)作用后,DNA发生损伤细胞后面形成长的拖尾,并呈典型凋亡彗星尾形态,平均光密度值较阴性对照均降低,差异具统计学意义,彗星尾距阴性对照均增加,差异具统计学意义,且二者的改变与作用浓度相关,即浓度越大,DNA受损伤的细胞越多。
4.采用流式细胞技术观察丹参酮ⅡA对HSV-tk/GCV系统的影响。丹参酮ⅡA联合10%tk+/GCV作用于大鼠肝癌细胞CBRH7919时,S期细胞较对照组和GCV单独作用组都有了显著的增加,说明丹参酮ⅡA联合10%tk+/GCV可有效地使大鼠肝癌细胞CBRH7919阻滞在S期,抑制DNA的合成。25μM联合组的凋亡率是23%,50μM联合组的凋亡率则达到35%,是相应浓度丹参酮ⅡA或GCV单独作用时凋亡率的5倍,说明丹参酮ⅡA能大大增加自杀基因系统的杀伤能力,抑止肝癌细胞CBRH7919的生长,促进它的凋亡。药物对B16细胞周期的影响较少,但25μM和50μM联合组的流式图上均观察到亚二倍体凋亡峰,50μM联合组的凋亡率达到34%,说明丹参酮ⅡA可促进B16 (10%tk+/GCV)细胞系统的细胞凋亡,对自杀基因系统具有增效作用。
5.丹参酮ⅡA对大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤细胞B16缝隙连接蛋白的影响。降落伞实验的结果显示,经过丹参酮ⅡA作用后,细胞之间出现了染料传输的现象,而且随着浓度的升高,含有绿色荧光的贴壁无标记细胞数量增多,提示丹参酮ⅡA极有可能具有增强细胞间GJIC的作用,而且浓度越高,效果越强；在丹参酮ⅡA和HSV-tk/GCV联合的系统中加入GJ长效抑制剂甘草次酸后,结果显示甘草次酸能显著下调丹参酮ⅡA与10%tk+/GCV的联合作用,使药物对细胞的抑制率降低,说明丹参酮ⅡA对自杀基因系统的增效作用机制很可能与改善细胞间缝隙连接细胞通讯有关；RT-PCR和Western-blot检测结果显示：高浓度(≥50μM)丹参酮ⅡA对CBRH7919细胞的Cx43mRNA和蛋白的表达有促进作用,而中、低浓度组则无明显上调作用；12.5μM、25μM和50μM的丹参酮ⅡA对B16细胞Cx43蛋白表达都有上调作用,但只有高浓度组对Cx43的mRNA表达有增强作用。
实验结论：
本文在细胞水平探讨了丹参酮ⅡA、丹参素钠和三七总皂甙三种中药活性成分对HSV-tk/GCV自杀基因疗法的旁观者效应,发现丹参酮ⅡA能有效增强自杀基因对细胞杀伤力,提高旁观者效应,而且有协同增效作用。丹参酮ⅡA还在小鼠移植瘤模型上得到了疗效验证：其联合自杀基因系统能明显抑制小鼠移植性黑色素瘤。通过体内实验对丹参酮ⅡA促进自杀基因旁观者效应的机制进行初步的研究,推测其增效机制可能是：丹参酮ⅡA作用于肿瘤细胞后在一定程度上恢复了Cx介导的GJIC功能,在一定程度上恢复或增强了细胞间的通讯,同时使肿瘤细胞阻滞于S期,令细胞毒素GCV-TP能更充分地起作用,进一步阻碍肿瘤细胞DNA的合成,使DNA发生损伤,并最终诱导细胞的凋亡。而细胞毒素则通过凋亡或细胞连接通讯的途径进入临近更多的细胞,从而使自杀基因的杀伤作用大大加强。
本研究为探讨中药增强自杀基因旁观者效应的可行性及其机制研究打下了基础,今后将在待选中药中继续筛选更多针对不同旁观者效应机制的强效中药成分,对有效中药成分的药理机制深入研究,从GJIC、细胞凋亡、免疫调节等及其与旁观者效应的关系这一新领域阐明中药的抗肿瘤机制。还可望进一步把针对多种增效机制的多种中药成分联合组成比单一成分更高效的复方,形成组分与药理药效明确、质量稳定的鸡尾酒式肿瘤基因治疗增效中药复方。研究有利于促进肿瘤基因治疗的临床推广应用,为建立新的肿瘤基因治疗联合疗法提供了实验依据。
Malignant tumor seriously threatens human health and life. With the development of molecular biology, gene therapy has brought the light of hope to malignant tumor patients. In foreign countries, gene therapy has been adopted as a standard therapeutic trial of malignant tumor after the failure of conventional treatment. Suicide gene therapy at present is the most promising therapeutically method for tumor gene treatment. It has specificness for tumor cells and produces bystander effect. Theoretically, it can completely eliminate the tumor cells and cure patients. The expected effects haven't been achieved due to problems like low transfection efficiency of carrier and weak targeting, ect. The key problems in urgent need to be solved are how to enhance curative effect and decrease the toxic side-effects. Enhancing the bystander effect can lower the requirement for high transfection efficiency, lower the dosage and toxicity of carrier and precursors and enhance lethality of gene therapy system on tumor. Therefore enhancing the bystander effect has become an important strategy to raise curative effect of the suicide gene therapy. The current methods adopted to raise bystander effect of the suicide gene are applying gene transfection methods or medicine revulsion methods to promote the GJIC, inducing apoptosis or improving the immune function of tumor mechanism. As a treasure house of natural medicine, Chinese medicine has a long history of phymatosis. Up to now, many reports have confirmed Chinese Herbal Medicine's unique advantages or potentials in promoting GJIC and inducing apoptosis, etc. It's economical, convenient and has little toxic side-effect. It has great potential of becoming the utility synergy element of suicide gene therapy together with which it can exert the cooperativity in tumor treatment.
Objective:
This research selects the Chinese Herbal Medicine elements with anti-tumor effect which are Tanshinone IIA, Salvianic acid A sodium and total saponins of Panax Notognseng to see whether they have the synergy effect on the suicide gene system. On the basis of acquiring the Chinese Herbal Medicine elements of significant effect, further exploration is made about their influence on tumor cell growth, the inducer cell apoptosis and the strengthening of GJIC, so as to understand the functional mechanism of enhancing the bystander effect by the effective element of Chinese Herbal Medicine and attempt to establish an Integrated Chinese and Western Medicine cancer gene therapy combining the suicide gene therapy with the Chinese Herbal Medicine which will offer a new thoughtway and model for tumor treatment.
Methods:
1. The vitro selection of suicide gene bystander effect by Chinese Medicine active components:Tanshinone IIA, Salvianic acid A sodium and total saponins of Panax Notognseng work on the 10% tk+/tk- mixed CBRH7919 rats'hepatoma carcinoma cells (HCC) at different density separately or together with GCV. Use MTT method to examine inhibition ratio of each group. Compare the difference of inhibition ratio and bystander effect of each group. Adopt JIN Zhengjun's Q Value formula to determine whether the Chinese Herbal Medicine and suicide gene system have cooperativity.
2. Observation of mice's transplanting melanoma cell treatment by Tanshinone IIA together with suicide gene system. The trial has 4 groups: Controlled Model Group, Tanshinone IIA Group, GCV Group and Tanshinone IIA+GCV Group. Mix the tk+ and tk- cells at the rate of 1:4 and inoculate at the armpit hypoderma of C57BL/6J mice. Each mouse has been inoculated 2×105 tumor cells and the next day the male and female mice are grouped at random. Traet with Chinese Herbal Medicine 1-7 days after inoculation. Treat with suicide gene system for 7 days and observe the curative effect.
3. Effect of Tanshinone IIA on rats'hepatoma carcinoma cells and mice's melanoma cells:Take the two kinds of cell lines which are the rats'hepatoma carcinoma cells CBRH7919 and mice's melanoma cells B16 as subjects. Adopt the MTT method to observe the Chinese Medicine active components'effect on the two kinds of cells'growth from density dependence and time dependence. Observe the cells'morphologic change after medication. Examine the effect of medicine on cell DNA by comet assay method.
4. Adopt the flow cytometry technique to observe the effect of Tanshinone on HSV-tk/GCV system. Observe the influence of cell cycle and apoptosis of rats'HCC and mice's carcinoma nigrum by Tanshinone IIA together with 10% tk+/GCV. Examine TanshinoneⅡA's effect on 10% CBRH7919/tk+ and B16/tk+ by PI simple staining method. Apply Tanshinone IIA with final concentration of 12.5μM,25μM and 50μM and GCV with final concentration of 15.7μM separately or together on the 10% CBRH7919/tk+ and B16/tk+ cell 72h. Wash twice the cells with PBS, fix with precooling 70% alcohol, dye with PI and use flow cytometry to make apoptosis rate and cell cycle analysis.
5. Observe the effect of Tanshinone IIA on the gap junction of rats' HCC CBRH 7919 cells and mice's carcinoma nigrum B 16 cells. Adopt parachute method to observe the effect of Tanshinone IIA on the cell GJIC. Incubate the cells within culture board by Calecin-AM. This green fluorescent dye metabolizes in cells and the metabolic products can be transmitted by GJ. Make cell suspension with the cells marked by the green fluorescent dye, cause the green fluorescent cells fall into the unmarked cell groups like parachute and determine the effect of medicine on cell GJIC by observing the intracellular transmission of the green fluorescent dye. Add the enoxolone, the prolonged-action inhibitor of GJ, to the integrated system of Tanshinone IIA and HSV-tk/GCV and observe the effect of enoxolone on the integrated system with MTT method:add Tanshinone IIA with the final concentration of 0μM,12.5μM,25μM, and 50μM to four groups of different density. The final concentrations of GCV are 0μM and 15.7μM, and the densities of enoxolone are 0 and 15μM. Integrate medicine with GCV at two densities separately. Integrate Medicine and GCV and enoxolone at two densities. After medication for 72 hours, use MTT method to determine the sensitivity of tumor cells to different groups. Apply Tanshinone IIA with the final concentration of 12.5μM,25μM, and 50μM to CBRH 7919 and B 16 for 72 hours. Adopt RT-PCR and Western-blot techniques to determine the expression value of Cx43 in each group.
Results:
1. The vitro selection of the effect of the active elements of Chinese Herbal Medicine on the bystander effect of the suicide gene:The cell inhibiting ratio (%) of Tanshinone IIA of different densities plus 10% tk+/GCV group are 30.38±5.28,33.22±5.21,40.58±4.49 and 73.04±2.85 which is obviously higher than that of the pure 10%tk+/GCV group (21.04±3.96) and the Tanshinone IIA of different densities group (3.65±2.84,4.74±4.48,17.34±5.08 and 52.81±5.95). Form 2 shows that the actual inhibiting ratio of the group of Tanshinone IIA at density of 6.25μM,12.5μM,25μM and 50μM plus 10%tk+/GCV is significantly higher than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are 1.270, 1.341,1.168 and 1.164 and all are above 1.15. Repeat the experiment with mice's carcinoma nigrum cells by Tanshinone IIA of the same density together with suicide gene. The result shows that the actual inhibiting ratio of the group of Tanshinone IIA at density of 6.25μM,12.5μM,25μM and 50μM plus 10%tk+/GCV is significantly higher than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are 2.276,1.892,1.150 and 1.211 and all are above 1.15. It proves that Tanshinone IIA has the synergy effect to the suicide gene system and the synergy effect is the cooperativity. Salvianic acid A sodium at density of 25μM plus 10%tk+/GCV is significantly higher than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are above 1.15. It proves that Salvianic acid A sodium has the synergy effect to the suicide gene system. However, Salvianic acid A sodium at density of 50μM, 100μM and 200PM plus 10%tk+/GCV is lower than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are lower than 1.15. It proves that Salvianic acid A sodium at these densities has not the synergy effect to the suicide gene system. Total saponins of Panax notognseng at density of 100 mg/L,200 mg/L,400 mg/L and 800 mg/L plus 10%tk+/GCV is lower, than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are lower than 1.15. It proves that the total saponins of Panax notognseng at these densities have not the synergy effect to the suicide gene system.
2. The observation of mice's transplanting melanoma cell treatment by Tanshinone IIA plus suicide gene system:On the ninth or the tenth day, tumors at right armpit can be touched in mice of each group. After being treated by suicide gene system plus Tanshinone IIA, the tumor growth slows down and is inhibited. The mean tumor volume is 200.2 mm3, and decreases 43.24%(P<0.05) compared with 352.7 mm3 of the Controlled Model Group. The mean tumor quality is 0.278g and decreases 50.6%(P＜0.05) compared with 0.5628 of the Controlled Model Group. There's statistical significance. In the GCV group, the mean tumor volume decreases 34.76% (P<0.05) compared with the Controlled Model Group. The mean tumor quality decreases 38.98%(P>0.05) compared with the Controlled Model Group and the result has no statistical significance. In the Tanshinone IIA group, the mean tumor volume decreases 29.94% compared with the Controlled Model Group. The mean tumor quality decreases 21.22% compared with the Controlled Model Group. There's no statistical significance (P >0.05)by comparing the tumor volume and quality with the Controlled Group. The above results show that Tanshinone IIA can raise the lethality of suicide gene system to mice's transplanted tumor and has synergy effect; therefore it can enhance the effect of suicide gene.
3. Tanshinone IIA's effect on rats'HCC CBRH7919 and mice's carcinoma nigrum B16:Growth inhibition at different degree has appeared in CBRH 7919 and B16 cells after being applied with TanshinoneⅡA at gradient densities (12.5μM、25μM and 50μM). The related analysis shows that this inhibition has certain density and time dependence. The increased density or the prolonged medication will increase the inhibition ratio. It can be found with inverted phase contrast microscope that the cells of the Controlled Group of CBRH 7919 and B16 grow well, has good adherence and high density. The cells have the shape of shuttle, lozenge, triangle or polygon. After medication, the growth is inhibited obviously and grows slowly or the growth even stops. Cells shed at different level with medication density. After Hoechst dying, CBRH 7919 cells turn circular and have poor refraction. Their volume decrease, the cellular nucleus crenate, and the chromatin condense. Granules can be seen in the cells. In the group of high density, several circular apoptosis body surrounds the cell, but there's no death or apoptosis of B16 cells. The comet assay results show that the after applying TanshinoneⅡA (12.5μM,25μM and 50μM) to rats'HCC CBRH7919 and mice's carcinoma nigrum B16 cell lines, DNA is injured and long trailing is formed behind the cells and takes the typical form of comet apoptosis. The average optical decreases compared with the negative control and the difference has statistical significance. The comet back range rises compared with the negative control and the difference has statistical significance. And the changes are related to the functional density that is the higher the density the more the cells with injured DNA.
4. Observe the Tanshinone IIA's effect on HSV-tk/GCV system by flow, cytometry. Apply Tanshinone IIA plus 10%tk+/GCV to HCC CBRH7919 cells of rats, S-phase cells increase significantly compared with the Controlled Group and the GCV group which indicates that Tanshinone IIA plus 10%tk+/GCV can effectively block rats'HCC CBRH7919 cells at the S-phase and inhibit the composition of DNA. The apoptosis rates of the integrated group of the density of 25μM and of 50μM are 23% and 35% which are 5 times of the apoptosis rate of the separate application of Tanshinone IIA or GCV of the same density. It indicates that Tanshinone IIA can greatly increase the lethality of suicide gene system, inhibit HCC CBRH7919's growth and promote apoptosis. Medicine has little effect on the B16 cell cycle, but from the flowing chart of the integrated group of density of 25μM and of 50μM, subdiploid apoptotic peak can be observed and the apoptosis rate of the integrated group of density of 50μM reaches 34%. It proves that Tanshinone IIA can promote the apoptosis of B16 (10%tk+/GCV) cell system and has synergy effect to the suicide gene system.
5. Tanshinone IIA's effect on the gap junction protein of rats'HCC CBRH7919 and mice's carcinoma nigrum cells. The parachute experiment shows that after applying Tanshinone IIA, there's dye transmission between cells and with the increase of density, the unmarked cells of green fluorescent adherence increase which indicates that Tanshinone IIA has high possibility of enhancing GJIC between cells and the effect rises with density. After adding GJ prolonged-action inhibitor of enoxolone to the integrated Tanshinone IIA and HSV-tk/GCV system, the result shows that enoxolone can significantly decrease the joint action of Tanshinone IIA and HSV-tk/GCV and lower the inhibition ratio of medicine which indicates that Tanshinone IIA's synergy effect to the suicide gene system is highly possible to be related with improving the GJIC. The RT-PCR and Western-blot examinations show:high-density (≥50μM) Tanshinone IIA promotes the expression of Cx43mRNA and protein, while the medium and low density groups don't have obvious promotion effect; Tanshinone IIA at density of 12.5μM,25μM and 50μM promotes the expression of Cx43 protein of B16 cells, while only the high density group has the enhancement property of mRNA of Cx43.
Conclusions:
This investigation explored the bystander effect of Tanshinone IIA, Salvianic acid A sodium and total saponins of Panax Notognseng, the active ingredient of Chinese Herbal Medicine on the suicide gene therapy and finds that Tanshinone IIA can effectively enhance the lethality of suicide gene, promote the bystander effect and has the synergy enhancement property. The curative effect of TanshinoneⅡA on the mice's transplanting tumor model proves that by integrating with the suicide gene system, it can significantly prohibit the mice's transplanting melanoma. By vivo experiment, the author makes preliminary research of the mechanism of Tanshinone IIA's promotion of bystander effect of suicide gene and infer that the synergy mechanism can be:Tanshinone IIA recovers Cx-mediated GJIC function of tumor cells, recovers or enhances the communication between cells, blocks the tumor cells at S-phase, exert the effect of cytotoxin GCV-TP, hinders the composition of DNA of tumor cells, injures DNA and finally induces the apoptosis of cells. Cytotoxin enters more cells by apoptosis or cell junction and communication; therefore it greatly improves the lethality of suicide gene.
This thesis lays foundation for researching on the feasibility and mechanism of bystander effect enhancement of suicide gene with Chinese Herbal Medicine. In the future, the author will select the effective ingredients for bystander effect mechanism research from the Chinese Herbal Medicine to be selected, make further study on the pharmacological mechanism of the active ingredients, and clarify the anti-tumor mechanism of Chinese Herbal Medicine from the new field of GJIC, cell apoptosis, immunological adjustment and the relationship with bystander effect, etc. Also, the author hopes to make a compound formula with multiple Chinese Herbal Medicine ingredients of synergy effect which are more effective than single ingredient and to form the cocktail-form synergy compound of clear pharmacology and pharmacodynamic action as well as stable quality. The research is beneficial for the clinical promotion of tumor gene therapy and provides trial basis for new integrated tumor gene therapy.
研究目的意义：
本研究选择被报道具有抗肿瘤作用的中药成分丹参酮ⅡA、丹参素钠和三七总皂甙,研究其对自杀基因系统是否有增效作用,在获得有明显作用的中药成分基础上进一步探讨其对肿瘤细胞生长、诱导细胞凋亡以及增强细胞缝隙连接通讯等方面的影响,以了解该有效中药成分增强旁观者效应的作用机制,探讨建立一种自杀基因疗法联合中药成分的中西医结合肿瘤基因治疗方案,为肿瘤治疗提供新的思路和模式。
实验方法：
1.中药活性成分对自杀基因旁观者效应影响的体外筛选：丹参酮ⅡA、丹参素钠和三七总皂甙成分各自以不同浓度单独或与GCV共同作用于大鼠肝癌细胞CBRH7919的10%tk+/tk-混合细胞,MTT检测各组抑制率,两两比较分析各组抑制率的差异和旁观者效应大小,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。筛选出的活性中药成分,用小鼠黑色素瘤细胞B16的10%tk+/tk-混合细胞对其联合自杀基因系统的增效作用进行验证。
2.丹参酮ⅡA联合自杀基因系统对小鼠移植性黑色素瘤细胞的治疗观察。实验分为模型对照组、丹参酮ⅡA组、GCV组和丹参酮IIA+GCV联合组;把tk+、tk-细胞按1：4比例混合接种C57BL／6J小鼠腋下皮下组织内,每只小鼠接种2×105个肿瘤细胞,次日雌雄均随机分组；接种后1-7天用中药治疗,7天后用自杀基因系统治疗7天,进行疗效观察。
3.丹参酮ⅡA对大鼠肝癌细胞和小鼠黑色素瘤细胞的影响：以大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤B16两种细胞株为对象,采用MTT法从浓度依赖和时间依赖两方面检测有效中药成分对两种细胞生长的影响；形态学观察药物作用后细胞的形态变化；彗星电泳检测药物对细胞DNA的影响。
4.采用流式细胞技术观察丹参酮ⅡA对HSV-tk/GCV系统的影响。检测丹参酮ⅡA联合10%tk+/GCV对大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤B16细胞周期和凋亡率的影响。PI单染法检测丹参酮ⅡA对10%CBRH7919/tk+和B16／tk+的影响：用终浓度为12.5μM、25、50μM的丹参酮ⅡA与终浓度为15.7μM的GCV分别和联合作用10% CBRH7919/tk+和B16／tk+细胞72h,各组细胞经收集后PBS洗2次,用预冷的70%乙醇固定后,PI染色,流式细胞仪进行凋亡率和细胞周期分析。
5.丹参酮ⅡA对大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤细胞B16缝隙连接的影响。采用降落伞法观察丹参酮ⅡA对细胞GJIC的影响：培养板内的细胞用绿色荧光染料钙黄绿素(Calecin-AM)孵育,该染料在细胞内代谢后所产生的代谢产物可通过GJ进行传递。把被绿色荧光染料标记的细胞制成细胞悬液,使绿色荧光细胞像降落伞一样降落到无标记的细胞组内,通过观察绿色荧光染料在细胞内的传输判断药物对细胞GJIC功能的影响；在丹参酮ⅡA和HSV-tk/GCV联合的系统中加入GJ长效抑制剂甘草次酸,MTT法检测甘草次酸对联合系统的影响：加入丹参酮ⅡA至终浓度为0μM、12.5μM、25μM、50μM四个不同浓度组,GCV终浓度分别为OgM和15.7μM,甘草次酸浓度分别0、15μM,药物和GCV两个浓度分别联合,药物和GCV与甘草次酸的两个浓度分别联合,药物作用72h后MTT法测定肿瘤细胞对不同组别的敏感程度;分别用终浓度为12.5μM、25μM和50μM的丹参酮ⅡA作用CBRH7919细胞和B16细胞72h,然后采用RT-PCR和Western-blot技术检测各组细胞Cx43的表达量。
实验结果：
1.中药活性成分对自杀基因旁观者效应影响的体外筛选：各浓度丹参酮ⅡA联合10%tk+/GCV组的细胞抑制率(%)分别为30.38±5.28、33.22±5.21、40.58±4.49和73.04±2.85,与单纯10%tk+/GCV组(21.04±3.96)和相应的各浓度丹参酮ⅡA组(3.65±2.84、4.74±4.48、17.34±5.08和52.81±5.95)的抑制率明显提高。从其两两比较的表2可知,6.25μM、12.5μM、25μM和50μM浓度下的丹参酮ⅡA联合10%tk+/GCV组的实际抑制率显著高于理论抑制率,金正钧Q值分别为1.270、1.341、1.168和1.164,均大于1.15。用小鼠恶性黑色素瘤细胞以相同浓度的丹参酮ⅡA联合自杀基因重做该实验,结果表明,6.25μM、12.5μM、25μM和50μM浓度下的丹参酮ⅡA联合10%tk+/GCV组的实际抑制率显著高于理论抑制率,金正钧Q值分别为2.276、1.892、1.150和1.211,均大于1.15。说明丹参酮ⅡA对自杀基因系统具有增效作用,且该增效作用为协同作用。25μM浓度的丹参素钠联合10%tk+/GCV组的实际抑制率大于理论抑制率,金正钧Q值大于1.15,说明该浓度的丹参素钠对自杀基因系统具有增效作用。但50μM、100gM和200μM浓度下的丹参素钠联合10%tk+/GCV组的实际抑制率均低于理论抑制率,Q值均低于1.15,说明在此浓度范围内丹参素钠对自杀基因系统没有协同增效作用。100mg／L、200mg/L、400mg/L和800 mg／L浓度下的三七总皂甙联合10%tk+/GCV组的实际抑制率均低于理论抑制率,金正钧Q值均低于1.15,说明在此浓度范围内三七总皂甙对自杀基因系统没有协同增效作用。
2.丹参酮ⅡA联合自杀基因系统对小鼠移植性黑色素瘤细胞的治疗观察。各组小鼠均在9-10日在右腋下触及肿块,自杀基因系统联合丹参酮ⅡA组治疗后,肿瘤生长速度减慢,肿瘤呈现生长抑制状态,平均肿瘤体积200.2 mm3,比模型对照组352.7 mm3减少43.24%(P<0.05),小鼠平均肿瘤质量0.278g,比模型对照组0.5628减少50.6%(P<0.05),统计学上差异有显著性意义。GCV组平均肿瘤体积比模型对照组减少34.76%(P<0.05)；小鼠的平均瘤块质量比模型对照组减少38.98%,但该结果没有统计学意义(P>0.05)。丹参酮ⅡA组平均肿瘤体积比模型对照组减少29.94%,小鼠的瘤块质量比模型对照组减少21.22%,但其肿瘤体积和质量与对照组相比都没有统计学意义(P>0.05)。以上结果提示：丹参酮ⅡA能提高自杀基因系统对小鼠移植瘤的杀伤力,协同自杀基因作用,达到提高自杀基因疗效的效果。
3.丹参酮ⅡA对大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤B16的影响：CBRH7919和B16经梯度浓度丹参酮ⅡA(12.5μM、25μM和50μM)作用后都出现了不同程度的生长抑制,而且相关分析显示,这一抑制作用表现出一定的浓度和时间依赖性,浓度增加,或药物作用时间延长,都会使抑制率增加。倒置相差显微镜观察到CBRH7919和B16的对照组细胞生长状况良好,贴壁很稳,密度高,细胞呈梭状、菱形、三角形或多边形,药物作用后细胞生长增殖明显受到抑制,增殖缓慢,几乎停滞,细胞逐渐由贴壁而脱落,药物浓度越高脱落细胞越多。Hoechst染色后,可见CBRH7919的细胞形态变圆,折光性差,体积减小,细胞核皱缩、染色质浓缩,胞内多见颗粒,高浓度组可见数个圆形凋亡小体围绕在细胞周围,但未观察到B16细胞的死亡或凋亡现象。彗星电泳结果显示,大鼠肝癌细胞CBRH7919细胞和小鼠恶性黑色素瘤细胞株B16经梯度丹参酮ⅡA(12.5μM、25μM和50μM)作用后,DNA发生损伤细胞后面形成长的拖尾,并呈典型凋亡彗星尾形态,平均光密度值较阴性对照均降低,差异具统计学意义,彗星尾距阴性对照均增加,差异具统计学意义,且二者的改变与作用浓度相关,即浓度越大,DNA受损伤的细胞越多。
4.采用流式细胞技术观察丹参酮ⅡA对HSV-tk/GCV系统的影响。丹参酮ⅡA联合10%tk+/GCV作用于大鼠肝癌细胞CBRH7919时,S期细胞较对照组和GCV单独作用组都有了显著的增加,说明丹参酮ⅡA联合10%tk+/GCV可有效地使大鼠肝癌细胞CBRH7919阻滞在S期,抑制DNA的合成。25μM联合组的凋亡率是23%,50μM联合组的凋亡率则达到35%,是相应浓度丹参酮ⅡA或GCV单独作用时凋亡率的5倍,说明丹参酮ⅡA能大大增加自杀基因系统的杀伤能力,抑止肝癌细胞CBRH7919的生长,促进它的凋亡。药物对B16细胞周期的影响较少,但25μM和50μM联合组的流式图上均观察到亚二倍体凋亡峰,50μM联合组的凋亡率达到34%,说明丹参酮ⅡA可促进B16 (10%tk+/GCV)细胞系统的细胞凋亡,对自杀基因系统具有增效作用。
5.丹参酮ⅡA对大鼠肝癌细胞CBRH7919和小鼠恶性黑色素瘤细胞B16缝隙连接蛋白的影响。降落伞实验的结果显示,经过丹参酮ⅡA作用后,细胞之间出现了染料传输的现象,而且随着浓度的升高,含有绿色荧光的贴壁无标记细胞数量增多,提示丹参酮ⅡA极有可能具有增强细胞间GJIC的作用,而且浓度越高,效果越强；在丹参酮ⅡA和HSV-tk/GCV联合的系统中加入GJ长效抑制剂甘草次酸后,结果显示甘草次酸能显著下调丹参酮ⅡA与10%tk+/GCV的联合作用,使药物对细胞的抑制率降低,说明丹参酮ⅡA对自杀基因系统的增效作用机制很可能与改善细胞间缝隙连接细胞通讯有关；RT-PCR和Western-blot检测结果显示：高浓度(≥50μM)丹参酮ⅡA对CBRH7919细胞的Cx43mRNA和蛋白的表达有促进作用,而中、低浓度组则无明显上调作用；12.5μM、25μM和50μM的丹参酮ⅡA对B16细胞Cx43蛋白表达都有上调作用,但只有高浓度组对Cx43的mRNA表达有增强作用。
实验结论：
本文在细胞水平探讨了丹参酮ⅡA、丹参素钠和三七总皂甙三种中药活性成分对HSV-tk/GCV自杀基因疗法的旁观者效应,发现丹参酮ⅡA能有效增强自杀基因对细胞杀伤力,提高旁观者效应,而且有协同增效作用。丹参酮ⅡA还在小鼠移植瘤模型上得到了疗效验证：其联合自杀基因系统能明显抑制小鼠移植性黑色素瘤。通过体内实验对丹参酮ⅡA促进自杀基因旁观者效应的机制进行初步的研究,推测其增效机制可能是：丹参酮ⅡA作用于肿瘤细胞后在一定程度上恢复了Cx介导的GJIC功能,在一定程度上恢复或增强了细胞间的通讯,同时使肿瘤细胞阻滞于S期,令细胞毒素GCV-TP能更充分地起作用,进一步阻碍肿瘤细胞DNA的合成,使DNA发生损伤,并最终诱导细胞的凋亡。而细胞毒素则通过凋亡或细胞连接通讯的途径进入临近更多的细胞,从而使自杀基因的杀伤作用大大加强。
本研究为探讨中药增强自杀基因旁观者效应的可行性及其机制研究打下了基础,今后将在待选中药中继续筛选更多针对不同旁观者效应机制的强效中药成分,对有效中药成分的药理机制深入研究,从GJIC、细胞凋亡、免疫调节等及其与旁观者效应的关系这一新领域阐明中药的抗肿瘤机制。还可望进一步把针对多种增效机制的多种中药成分联合组成比单一成分更高效的复方,形成组分与药理药效明确、质量稳定的鸡尾酒式肿瘤基因治疗增效中药复方。研究有利于促进肿瘤基因治疗的临床推广应用,为建立新的肿瘤基因治疗联合疗法提供了实验依据。
Malignant tumor seriously threatens human health and life. With the development of molecular biology, gene therapy has brought the light of hope to malignant tumor patients. In foreign countries, gene therapy has been adopted as a standard therapeutic trial of malignant tumor after the failure of conventional treatment. Suicide gene therapy at present is the most promising therapeutically method for tumor gene treatment. It has specificness for tumor cells and produces bystander effect. Theoretically, it can completely eliminate the tumor cells and cure patients. The expected effects haven't been achieved due to problems like low transfection efficiency of carrier and weak targeting, ect. The key problems in urgent need to be solved are how to enhance curative effect and decrease the toxic side-effects. Enhancing the bystander effect can lower the requirement for high transfection efficiency, lower the dosage and toxicity of carrier and precursors and enhance lethality of gene therapy system on tumor. Therefore enhancing the bystander effect has become an important strategy to raise curative effect of the suicide gene therapy. The current methods adopted to raise bystander effect of the suicide gene are applying gene transfection methods or medicine revulsion methods to promote the GJIC, inducing apoptosis or improving the immune function of tumor mechanism. As a treasure house of natural medicine, Chinese medicine has a long history of phymatosis. Up to now, many reports have confirmed Chinese Herbal Medicine's unique advantages or potentials in promoting GJIC and inducing apoptosis, etc. It's economical, convenient and has little toxic side-effect. It has great potential of becoming the utility synergy element of suicide gene therapy together with which it can exert the cooperativity in tumor treatment.
Objective:
This research selects the Chinese Herbal Medicine elements with anti-tumor effect which are Tanshinone IIA, Salvianic acid A sodium and total saponins of Panax Notognseng to see whether they have the synergy effect on the suicide gene system. On the basis of acquiring the Chinese Herbal Medicine elements of significant effect, further exploration is made about their influence on tumor cell growth, the inducer cell apoptosis and the strengthening of GJIC, so as to understand the functional mechanism of enhancing the bystander effect by the effective element of Chinese Herbal Medicine and attempt to establish an Integrated Chinese and Western Medicine cancer gene therapy combining the suicide gene therapy with the Chinese Herbal Medicine which will offer a new thoughtway and model for tumor treatment.
Methods:
1. The vitro selection of suicide gene bystander effect by Chinese Medicine active components:Tanshinone IIA, Salvianic acid A sodium and total saponins of Panax Notognseng work on the 10% tk+/tk- mixed CBRH7919 rats'hepatoma carcinoma cells (HCC) at different density separately or together with GCV. Use MTT method to examine inhibition ratio of each group. Compare the difference of inhibition ratio and bystander effect of each group. Adopt JIN Zhengjun's Q Value formula to determine whether the Chinese Herbal Medicine and suicide gene system have cooperativity.
2. Observation of mice's transplanting melanoma cell treatment by Tanshinone IIA together with suicide gene system. The trial has 4 groups: Controlled Model Group, Tanshinone IIA Group, GCV Group and Tanshinone IIA+GCV Group. Mix the tk+ and tk- cells at the rate of 1:4 and inoculate at the armpit hypoderma of C57BL/6J mice. Each mouse has been inoculated 2×105 tumor cells and the next day the male and female mice are grouped at random. Traet with Chinese Herbal Medicine 1-7 days after inoculation. Treat with suicide gene system for 7 days and observe the curative effect.
3. Effect of Tanshinone IIA on rats'hepatoma carcinoma cells and mice's melanoma cells:Take the two kinds of cell lines which are the rats'hepatoma carcinoma cells CBRH7919 and mice's melanoma cells B16 as subjects. Adopt the MTT method to observe the Chinese Medicine active components'effect on the two kinds of cells'growth from density dependence and time dependence. Observe the cells'morphologic change after medication. Examine the effect of medicine on cell DNA by comet assay method.
4. Adopt the flow cytometry technique to observe the effect of Tanshinone on HSV-tk/GCV system. Observe the influence of cell cycle and apoptosis of rats'HCC and mice's carcinoma nigrum by Tanshinone IIA together with 10% tk+/GCV. Examine TanshinoneⅡA's effect on 10% CBRH7919/tk+ and B16/tk+ by PI simple staining method. Apply Tanshinone IIA with final concentration of 12.5μM,25μM and 50μM and GCV with final concentration of 15.7μM separately or together on the 10% CBRH7919/tk+ and B16/tk+ cell 72h. Wash twice the cells with PBS, fix with precooling 70% alcohol, dye with PI and use flow cytometry to make apoptosis rate and cell cycle analysis.
5. Observe the effect of Tanshinone IIA on the gap junction of rats' HCC CBRH 7919 cells and mice's carcinoma nigrum B 16 cells. Adopt parachute method to observe the effect of Tanshinone IIA on the cell GJIC. Incubate the cells within culture board by Calecin-AM. This green fluorescent dye metabolizes in cells and the metabolic products can be transmitted by GJ. Make cell suspension with the cells marked by the green fluorescent dye, cause the green fluorescent cells fall into the unmarked cell groups like parachute and determine the effect of medicine on cell GJIC by observing the intracellular transmission of the green fluorescent dye. Add the enoxolone, the prolonged-action inhibitor of GJ, to the integrated system of Tanshinone IIA and HSV-tk/GCV and observe the effect of enoxolone on the integrated system with MTT method:add Tanshinone IIA with the final concentration of 0μM,12.5μM,25μM, and 50μM to four groups of different density. The final concentrations of GCV are 0μM and 15.7μM, and the densities of enoxolone are 0 and 15μM. Integrate medicine with GCV at two densities separately. Integrate Medicine and GCV and enoxolone at two densities. After medication for 72 hours, use MTT method to determine the sensitivity of tumor cells to different groups. Apply Tanshinone IIA with the final concentration of 12.5μM,25μM, and 50μM to CBRH 7919 and B 16 for 72 hours. Adopt RT-PCR and Western-blot techniques to determine the expression value of Cx43 in each group.
Results:
1. The vitro selection of the effect of the active elements of Chinese Herbal Medicine on the bystander effect of the suicide gene:The cell inhibiting ratio (%) of Tanshinone IIA of different densities plus 10% tk+/GCV group are 30.38±5.28,33.22±5.21,40.58±4.49 and 73.04±2.85 which is obviously higher than that of the pure 10%tk+/GCV group (21.04±3.96) and the Tanshinone IIA of different densities group (3.65±2.84,4.74±4.48,17.34±5.08 and 52.81±5.95). Form 2 shows that the actual inhibiting ratio of the group of Tanshinone IIA at density of 6.25μM,12.5μM,25μM and 50μM plus 10%tk+/GCV is significantly higher than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are 1.270, 1.341,1.168 and 1.164 and all are above 1.15. Repeat the experiment with mice's carcinoma nigrum cells by Tanshinone IIA of the same density together with suicide gene. The result shows that the actual inhibiting ratio of the group of Tanshinone IIA at density of 6.25μM,12.5μM,25μM and 50μM plus 10%tk+/GCV is significantly higher than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are 2.276,1.892,1.150 and 1.211 and all are above 1.15. It proves that Tanshinone IIA has the synergy effect to the suicide gene system and the synergy effect is the cooperativity. Salvianic acid A sodium at density of 25μM plus 10%tk+/GCV is significantly higher than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are above 1.15. It proves that Salvianic acid A sodium has the synergy effect to the suicide gene system. However, Salvianic acid A sodium at density of 50μM, 100μM and 200PM plus 10%tk+/GCV is lower than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are lower than 1.15. It proves that Salvianic acid A sodium at these densities has not the synergy effect to the suicide gene system. Total saponins of Panax notognseng at density of 100 mg/L,200 mg/L,400 mg/L and 800 mg/L plus 10%tk+/GCV is lower, than the theoretical inhibiting ratio. JIN Zhengjun's Q Values are lower than 1.15. It proves that the total saponins of Panax notognseng at these densities have not the synergy effect to the suicide gene system.
2. The observation of mice's transplanting melanoma cell treatment by Tanshinone IIA plus suicide gene system:On the ninth or the tenth day, tumors at right armpit can be touched in mice of each group. After being treated by suicide gene system plus Tanshinone IIA, the tumor growth slows down and is inhibited. The mean tumor volume is 200.2 mm3, and decreases 43.24%(P<0.05) compared with 352.7 mm3 of the Controlled Model Group. The mean tumor quality is 0.278g and decreases 50.6%(P＜0.05) compared with 0.5628 of the Controlled Model Group. There's statistical significance. In the GCV group, the mean tumor volume decreases 34.76% (P<0.05) compared with the Controlled Model Group. The mean tumor quality decreases 38.98%(P>0.05) compared with the Controlled Model Group and the result has no statistical significance. In the Tanshinone IIA group, the mean tumor volume decreases 29.94% compared with the Controlled Model Group. The mean tumor quality decreases 21.22% compared with the Controlled Model Group. There's no statistical significance (P >0.05)by comparing the tumor volume and quality with the Controlled Group. The above results show that Tanshinone IIA can raise the lethality of suicide gene system to mice's transplanted tumor and has synergy effect; therefore it can enhance the effect of suicide gene.
3. Tanshinone IIA's effect on rats'HCC CBRH7919 and mice's carcinoma nigrum B16:Growth inhibition at different degree has appeared in CBRH 7919 and B16 cells after being applied with TanshinoneⅡA at gradient densities (12.5μM、25μM and 50μM). The related analysis shows that this inhibition has certain density and time dependence. The increased density or the prolonged medication will increase the inhibition ratio. It can be found with inverted phase contrast microscope that the cells of the Controlled Group of CBRH 7919 and B16 grow well, has good adherence and high density. The cells have the shape of shuttle, lozenge, triangle or polygon. After medication, the growth is inhibited obviously and grows slowly or the growth even stops. Cells shed at different level with medication density. After Hoechst dying, CBRH 7919 cells turn circular and have poor refraction. Their volume decrease, the cellular nucleus crenate, and the chromatin condense. Granules can be seen in the cells. In the group of high density, several circular apoptosis body surrounds the cell, but there's no death or apoptosis of B16 cells. The comet assay results show that the after applying TanshinoneⅡA (12.5μM,25μM and 50μM) to rats'HCC CBRH7919 and mice's carcinoma nigrum B16 cell lines, DNA is injured and long trailing is formed behind the cells and takes the typical form of comet apoptosis. The average optical decreases compared with the negative control and the difference has statistical significance. The comet back range rises compared with the negative control and the difference has statistical significance. And the changes are related to the functional density that is the higher the density the more the cells with injured DNA.
4. Observe the Tanshinone IIA's effect on HSV-tk/GCV system by flow, cytometry. Apply Tanshinone IIA plus 10%tk+/GCV to HCC CBRH7919 cells of rats, S-phase cells increase significantly compared with the Controlled Group and the GCV group which indicates that Tanshinone IIA plus 10%tk+/GCV can effectively block rats'HCC CBRH7919 cells at the S-phase and inhibit the composition of DNA. The apoptosis rates of the integrated group of the density of 25μM and of 50μM are 23% and 35% which are 5 times of the apoptosis rate of the separate application of Tanshinone IIA or GCV of the same density. It indicates that Tanshinone IIA can greatly increase the lethality of suicide gene system, inhibit HCC CBRH7919's growth and promote apoptosis. Medicine has little effect on the B16 cell cycle, but from the flowing chart of the integrated group of density of 25μM and of 50μM, subdiploid apoptotic peak can be observed and the apoptosis rate of the integrated group of density of 50μM reaches 34%. It proves that Tanshinone IIA can promote the apoptosis of B16 (10%tk+/GCV) cell system and has synergy effect to the suicide gene system.
5. Tanshinone IIA's effect on the gap junction protein of rats'HCC CBRH7919 and mice's carcinoma nigrum cells. The parachute experiment shows that after applying Tanshinone IIA, there's dye transmission between cells and with the increase of density, the unmarked cells of green fluorescent adherence increase which indicates that Tanshinone IIA has high possibility of enhancing GJIC between cells and the effect rises with density. After adding GJ prolonged-action inhibitor of enoxolone to the integrated Tanshinone IIA and HSV-tk/GCV system, the result shows that enoxolone can significantly decrease the joint action of Tanshinone IIA and HSV-tk/GCV and lower the inhibition ratio of medicine which indicates that Tanshinone IIA's synergy effect to the suicide gene system is highly possible to be related with improving the GJIC. The RT-PCR and Western-blot examinations show:high-density (≥50μM) Tanshinone IIA promotes the expression of Cx43mRNA and protein, while the medium and low density groups don't have obvious promotion effect; Tanshinone IIA at density of 12.5μM,25μM and 50μM promotes the expression of Cx43 protein of B16 cells, while only the high density group has the enhancement property of mRNA of Cx43.
Conclusions:
This investigation explored the bystander effect of Tanshinone IIA, Salvianic acid A sodium and total saponins of Panax Notognseng, the active ingredient of Chinese Herbal Medicine on the suicide gene therapy and finds that Tanshinone IIA can effectively enhance the lethality of suicide gene, promote the bystander effect and has the synergy enhancement property. The curative effect of TanshinoneⅡA on the mice's transplanting tumor model proves that by integrating with the suicide gene system, it can significantly prohibit the mice's transplanting melanoma. By vivo experiment, the author makes preliminary research of the mechanism of Tanshinone IIA's promotion of bystander effect of suicide gene and infer that the synergy mechanism can be:Tanshinone IIA recovers Cx-mediated GJIC function of tumor cells, recovers or enhances the communication between cells, blocks the tumor cells at S-phase, exert the effect of cytotoxin GCV-TP, hinders the composition of DNA of tumor cells, injures DNA and finally induces the apoptosis of cells. Cytotoxin enters more cells by apoptosis or cell junction and communication; therefore it greatly improves the lethality of suicide gene.
This thesis lays foundation for researching on the feasibility and mechanism of bystander effect enhancement of suicide gene with Chinese Herbal Medicine. In the future, the author will select the effective ingredients for bystander effect mechanism research from the Chinese Herbal Medicine to be selected, make further study on the pharmacological mechanism of the active ingredients, and clarify the anti-tumor mechanism of Chinese Herbal Medicine from the new field of GJIC, cell apoptosis, immunological adjustment and the relationship with bystander effect, etc. Also, the author hopes to make a compound formula with multiple Chinese Herbal Medicine ingredients of synergy effect which are more effective than single ingredient and to form the cocktail-form synergy compound of clear pharmacology and pharmacodynamic action as well as stable quality. The research is beneficial for the clinical promotion of tumor gene therapy and provides trial basis for new integrated tumor gene therapy.
引文
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