【作者】 杨晓寰；

【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， 2007， 博士

【摘要】 研究目的迄今，恶性肿瘤仍然是严重威胁人类健康的疾病，尽管已有很多抗肿瘤药物应用于临床，但由于在很多情况下，效果仍然很不理想，再加上毒副作用大和耐药性的原因，因此人们正在努力寻找新的抗肿瘤药物。中医药累积了不少防治肿瘤的经验。研究与临床均证实，中医药以复方治疗为主，成分较复杂，具体到某种药或复方时，疗效尚难确切肯定，稳定性与重复性往往不高，药理机制及靶点尚不明确，这些均有待深入研究。因此，中药抗肿瘤的研究，无论是国外还是国内，仍然方兴未艾。研究表明，具有直接细胞生长抑制或杀伤作用的中药活性成分有姜黄素、β-榄香烯、苦参碱、大蒜素、紫杉醇、丹参酮、羟基喜树碱等。GJIC与肿瘤发生、发展密切相关，加强GJ能作为治疗肿瘤的一个手段。但有关中药与GJIC的研究尚少，已报道的有车前草的乙醇提取成分、丹参酮ⅡA等。本研究的创新点在于采用对肿瘤细胞的细胞生长抑制或杀伤作用进行筛选与GJIC的相关活性筛选相结合，初步找出具有直接细胞生长抑制或杀伤作用的中药活性成分，并探求能提高GJIC的活性成分，从而为中药的临床应用和作用新靶点的研究提供新的参考资料。实验方法1五种恶性肿瘤细胞株的生长时间和密度的测定实验采用活细胞增殖Alamar Blue试剂分别测定人胃癌细胞株SGC-7901、人肝癌细胞株Hep-G2、人急性旱幼粒白血病细胞株HL-60、小鼠肝癌细胞株H22和小鼠黑素瘤细胞株B16五种肿瘤细胞株在不同浓度各个时间段的吸光值，根据其还原率确定最佳的细胞生长条件。2中药活性成分抗肿瘤活性研究2．1所选药物黄芪甲苷、人参皂苷Rh2、人参皂苷Rg3、淫羊藿苷、川续断皂苷、阿魏酸、松果菊苷、葫芦巴碱、虫草素、灵芝多糖、灵芝三萜和参附注射液。2．2细胞增殖抑制或杀伤实验分别收获对数生长期的人胃癌细胞SGC-7901、人肝癌细胞Hep-G2和小鼠黑素瘤细胞B16，按照每孔4000个细胞接种入96孔板。过夜培养24h后加药。收获人白血病细胞HL-60和小鼠肝癌细胞H22，按照每孔4000个细胞接种入96孔板后加药。黄芪甲苷、人参皂苷Rh2、人参皂苷Rg3、淫羊藿苷、川续断皂苷、阿魏酸、松果菊苷、葫芦巴碱和虫草素设6个浓度，终浓度分别为0μM、5μM、10μ、20μM、40μM和80μM；灵芝多糖和灵芝三萜设6个浓度，终浓度分别为Omg／L、5mg／L、10mg／L、20mg／L、40mg／L和80mg／L；参附注射液设6个浓度，终浓度分别为OmL／L、5mL／L、10mL／L、20mL／L、40mL／L和80mL／L，每个浓度设5个复孔，补足180μl培养液于37℃、5％CO₂及饱和湿度条件下培养。每孔加入20μl Alamar Blue，用酶标仪分别测定0h、24h、48h和72h，570hm和630nm波长的吸收值，按照说明书的公式，计算每个浓度及时间段存活率。存活率=(34798×A₅₇₀-80586×A₆₃₀／34798×A₅₇₀⁰-80586×A₆₃₀⁰)×100其中，A₅₇₀和A₆₃₀分别是药物处理组细胞在570nm和630nm波长的吸收值，A₅₇₀⁰和A₆₃₀⁰分别是对照组细胞在570nm和630hm波长的吸收值。以细胞存活率为纵坐标，药物浓度为横坐标，绘制各组细胞的生存曲线，并计算药物对细胞的IC₅₀值。2．3流式细胞仪分析把人白血病细胞HL-60按每孔约2.5×10⁶个细胞接种到六孔板中，分为对照组，松果菊苷20μM、松果菊苷40μM、虫草素20μM、虫草素40μM、参附注射液20mL／L和参附注射液40mL／L共7组，每组3复孔。药物孵育48h后，各组细胞经胰酶消化收集后PBS洗3次，用预冷的70％乙醇固定后，PI染色，用流式细胞仪进行凋亡率和细胞周期分析检测。2．4琼脂糖凝胶电泳分别收集经过参附注射液不同浓度处理过的H22细胞，PBS洗涤后采用申能博彩生物科技有限公司试剂盒步骤提取细胞DNA，取7μL DNA提取液加溴芬兰3μL，上样于1.5％琼脂糖凝胶进行电泳（电压30V，2h），UV下观察结果，用凝胶成像系统拍照。3中药活性成分对GJIC和Cx43表达的影响对参附注射液和松果菊苷、虫草素等十一种补益中药活性成分中，分为正常对照组、芹黄素阳性对照组、18-α甘草酸阴性对照组、黄芪甲苷组、人参皂苷Rh2组、人参皂苷Rg3组、淫羊藿苷组、川续断皂苷组、阿魏酸组、松果菊苷组、葫芦巴碱组、虫草素组、灵芝多糖组、灵芝三萜组和参附注射液组共15组。药物孵育36h后进行荧光染料划痕实验，初步筛选出对GJIC有作用的药物，并对比这些中药活性成分对Cx43表达的影响。观察LY自伤沿列细胞向邻近细胞传递的距离进行半定量检测。FITC间接免疫荧光法通过FITC标记的Cx43抗体特异结合来检测中药活性成分对Hep-G2细胞株的Connexin 43表达的影响。结果1五种恶性肿瘤细胞株的生长时间和密度的测定结果显示：五种细胞株在24-72小时之间，20000个／ml（即4000个／孔，每孔总体积200μl）的细胞密度时，alamarBlue还原率与时间有线性关系。2中药活性成分对肿瘤细胞株的细胞生长抑制或杀伤作用①镜下观察有明显细胞生长抑制或杀伤作用的是：松果菊苷处理过的Hep-G2细胞株；虫草素处理过的HL-60细胞株；参附注射液处理过的HL-60和H22细胞株。②Alamar Blue法检测结果：松果菊苷在80μM浓度下对Hep-G2细胞株生长有抑制效应，但抑制率不高，80μM浓度下也只有30.91％。各组存活率为：92.69±3.35％、88.33±2.12％、80.97±2.50％、76.95±1.40％和69.09±1.11％。IC₅₀为467.8μM（368.3μg／mL）。虫草素在80μM浓度下对HL-60细胞株生长有较好的抑制效应，各组存活率为：100±3.73％、95.31±3.57％、90.66±2.30％、86.07±2.88％、70.64±1.56％、65.72±2.44％。其IC₅₀为155μM（46.5μg／mL）。参附注射液作用48h，Alamar Blue检测H22细胞株各组存活率分别为：100±4.93％、104.31±3.19％、97.31±4.53％、66.44±2.94％、18.01±3.93％、2.54±1.35％。IG₅₀为26.51mL／L。用MTT法检测HL-60存活率为87.5±8.02％、56.45±5.03％、38.38±7.48％，IC₅₀为68mL／L。其余各药物显微镜下观察未见明显杀伤作用。Alamar Blue法检测结果存活率很高。③PI单染流式细胞术检测结果显示，HL-60细胞株分别经虫草素40μM处理48h凋亡率为1.36％；参附注射液40mL几处理48h凋亡率为4.20％。其余均无明显诱导凋亡作用。细胞周期分析结果显示虫草素组S期细胞明显增多，G2／M期细胞比对照组明显减少。④参附注射液对H22作用，在20mL／L和40mL／L的浓度下均能出现典型的凋亡ladder条带。说明参附注射液能诱导H22细胞株产生凋亡。3中药活性成分对Hep-G2的GJIC和Cx43表达的影响①SL／DT实验结果显示：空白对照组细胞的荧光染料主要出现在划痕旁1-2列细胞中而且亮度低（±），细胞间染料传输功能差；Hep-G2细胞株分别经阳性对照芹黄素、松果菊苷、葫芦巴碱和参附注射液处理24小时后，荧光染料不仅出现在划痕的附近细胞内，而且在划痕以外的3-4列细胞内也出现了明显荧光（++++）。说明出现了细胞间染料传输的现象，说明细胞的间隙连接通讯功能获得增强。②间接免疫荧光检测Cx43，结果显示：空白对照组细胞得分为1.4±0.55，松果菊苷组得分为1.6±0.55，虫草素组得分为0.6±0.45，淫羊藿苷组得分为3.4±0.55，葫芦巴碱组得分为2.0±0.71，得分为2.2±0.84，参附注射液组得分为3.4±0.89。结果表明松果菊苷、葫芦巴碱、人参皂苷Rh2、淫羊藿苷组和参附注射液组的Cx43表达增强。结论①参附注射液对H22和HL-60细胞株有较明显的细胞毒作用：A．镜下观察发现药物作用组细胞有形态学改变，细胞发泡皱缩，细胞碎片增多，说明参附注射液对H22和HL-60有杀伤作用。B．MTT和Alamar Blue实验结果显示各组细胞存活率下降，且呈浓度依赖性。C．参附注射液作用HL-60细胞株48小时IC₅₀为26.51mL几，作用H22细胞株48小时IC₅₀为68mL／L。D．流式细胞术和琼脂糖凝胶电泳说明参附注射液的杀伤作用是通过诱导凋亡。②虫草素对HL-60细胞株生长呈现抑制增殖作用：A．镜下观察发现药物作用组细胞形态与对照组无差别，细胞碎片少，说明虫草素主要是抑制HL-60细胞的增殖，而不是促进杀伤。B．Alamar Blue实验结果显示各组细胞存活率下降，且呈浓度依赖性。C．虫草素作用HL-60细胞株24小时IC₅₀为155μM（46.5μg／mL）。D．流式细胞术分析说明虫草素作用HL-60细胞后，G2／M期细胞比对照组减少，且将细胞阻滞在S期。提示虫草素对靶肿瘤细胞的作用有周期特异性，其代谢产物可以掺入DNA，进而抑制DNA生成。③松果菊苷对Hep-G2细胞也有细胞生长抑制或杀伤作用，但活性很低，作用Hep-G2细胞24小时IC₅₀为46.．8μM（368.3μg／mL）。④淫羊藿苷等其余九种活性成分对上述的五种细胞株未显示细胞生长抑制或杀伤作用。⑤松果菊苷、葫芦巴碱和参附注射液作用Hep-G2细胞后出现明显细胞间荧光染料传输现象，提示这些成分可以促进GJIC功能。⑥松果菊苷、葫芦巴碱、人参皂苷Rh2、淫羊藿苷和参附注射液有促进Cx43表达的作用。更多还原

【Abstract】 ObjectiveCancer is a very severe threat to human lives. Although there are many anti-tumor drugs for cancer patients, the effect seems poor with the severe side-effects and drug resistance. Nowadays, it is necessary for us to find some new anti-tumor drugs.There are many treating experiences to cancer in Chinese medicine. Numerous researches proved that Chinese Medicine had an effect of anti-tumor with not only compound but also single herb or active ingredients. Researchers have made some Chinese medicines to treat cancer, but the effects are limited. Therefore, the field of anti-tumor Chinese medicine is in the ascendant to the whole world.Modern researches proved that there are some active ingredients with directed cytotoxic effect to cancer cells, such as curcumin, Elemene, Matrine, Garlicin, taxol, tanshinone, etc. There is a close relationship between Gap Junction Intercellular Communication（GJIC） and genesis and development of neoplasm, which has become a hot spot. But the reports of Chinese medicine and GJIC are still few, such as tanshinone IIA, etc.This study was to choose active ingredients with the inhibited or cytotoxic effect to five cancer cell lines, and choose the up-regulated active ingredients to GJIC. So we can find out some new data to Chinese medicine.Methods1 Measurement of growth time and density on five cancer cell line In this experiment, we measured the absorbent of five cancer cell line of SGC-7901、Hep-G2、H22、HL-60 and B16, with different densities and time. The reduction rate of Alamar Blue would be used to identify the most perfect factors to cell growth.2 Recover the active ingredients to tumor cell lines 2. 1 The active ingredientsAstragalosideⅣ, Ginsenoside Rh2, Ginsenoside Rg3, Icariin, xuduan, Ferulic Acid, Echinacoside, trigonelline, cordycepin, Ganoderma lucidum, ganoderic acid and Shenfu Injection.2. 2 The inhibited or cytotoxic experimentHavest SGC-7901, B16, Hep-G2 and culture overnight for 24h, and then plant in 96-well plate with 4000 per well. Havest HL-60 and H22, and plant in 96-well plate with 4000 per well. Add AstragalosideⅣ, Ginsenoside Rh2, Ginsenoside Rg3, Icariin, xuduan, Ferulic Acid, Echinacoside, trigonelline and cordycepin respectively with an eventual concentration of 0μM, 5μM, 10μM, 20μM, 40μM, 80μM, Ganoderma lucidum and ganoderic acid with Omg/L, 5mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, and Shenfu Injection with OmL/L, 5mL/L, 10mL/L, 20mL/L, 40mL/L, 80mL/L, for five wells. Culture in 37℃, 5％CO₂ with 180μl complete medium, then add 20μl Alamar Blue per well, and measure the absorbent of 570hm and 630hm for 0h、24h、48h and 72h. The survival rate was determined, with the curve of growth and IC₅₀ value.2.3 Flow cytometry analysisWe planted HL-60 in an 6-well plate with 2.5×10⁶ per well, with control group, ECH 20μM group, ECH 40μM group, cordycepin 20μM, cordycepin 40μM, Shenfu injection 25mL/L and Shenfu injection 50mL/L. After cultured with these drugs, we harvested the cells and measure with the flow cytometry.2.4 Agar electrophoresisHavest H22 cells with treatment of Shenfu injection（25mL/L and 50mL/L）, wash with PBS, and attract DNA to electrophoresis with 1.5％agar, 30V for 2 hours.Then observe with UV and take the picture.3 The influence of GJIC and Cx43 with active ingredients We developed 15 groups of control group, apigenin, AGA, AstragalosideⅣ, Ginsenoside Rh2, Ginsenoside Rg3, Icariin, xuduan, Ferulic Acid, Echinacoside, trigonelline, cordycepin, Ganoderma lucidum, ganoderic acid and Shenfu injection. After treated 36 hours with these ingredients, the effect on the GJIC of Hep-G2 were detected by SL/DT. The expression of Cx43 in cells were determined by FITC indirect immuno-fluorescent assay.Result1 The reduction rate of Alamar Blue showed 4000 cells per well with total 200μl have a linear relationship with time for 24 to 72 hours. 2 Inhibited or cytotoxic experiment showed①The severe cytotoxic effects were observed under microscope on ECH treated Hep-G2, cordycepin treated HL-60, and Shenfu injection treated HL-60 and H22.②Alamar Blue assay showed ECH had an inhibit effect to Hep-G2 in 80μM, IC₅₀ is 467.8μM（368.3μg/mL）, cordycepin to HL-60, IC₅₀ is 155μM（46.5μg/mL）. After treated with Shenfu injection, the survival rates of H22 were 100±4.93％、104.31±3.19％、97.31±4.53％、66.44±2.94％、18.01±3.93％、2.54±1.35％, respectly, with IC₅₀ value of 26.51mL/L. Other ingredients showed no cytotoxic effects. HL-60 cells treated with Shenfu Injection were detect by MTT assay. The survival rates of HL-60 were 87.5±8.02％, 56.45±5.03％and 38.38±7.48％, respectly, with IC₅₀ value of 68mL/L。③The apoptotic rate with HL-60 treated by cordycepin is 1.36％, Shenfu injection is 4.20％. The sub-G1 peak was observed in HL-60 treated with Shenfu Injection. The S phase was stuck in HL-60 treated cordycepin.④DNA ladder was observed on H22 treated by Shenfu injection.3 The effect of active ingredients to GJIC and Cx43①SL/DT showed there were an up-regulation of GJIC on Hep-G2 treated with Apigenin, ECH, cordycepin and Shenfu Injection.②The result of Cx43 detection is control group with 1.4±0.55 scores, ECH with 1.6±0.55, cordycepin with 0.6±0.45, ICA with 3.4±0.55, trigonelline with 2.0±0.71, Shenfu injection with 3.4±0.89. There were a up-regulation to ECH, trigonelline, G-Rh2, ICA and Shenfu injection.Conclusion①There are cytotoxic effects on H22 and HL-60 with treated Shenfu injection. A. We could observe the changes under microscope. B. MTT and Alamar Blue assay showed the survival rates decreased. C. The IC₅₀ of HL-60 was 26.51mL/L, and H22 was 68mL/L. D. The flow cytometry and agar electrophoresis had demonstrated that apoptotic cells were existed.②There are inhibited effects on HL-60 with treated cordycepin. A. We could observe no changes under microscope. B. Alamar Blue assay showed the survival rates decreased. C. The IC₅₀ of HL-60 was 155μM（46.5μg/mL）. D. The flow cytometry had demonstrated that the S phase was stagnation.③There are low effects on Hep-G2 treated with ECH. The IC₅₀ was 467.8μM （368.3μg/mL）.④The other nine active ingredients showed no inhibited and cytotoxic effects of the five cell lines.⑤ECH, trigonelline and Shenfu injection showed a up-regulation effect to GJIC.⑥Shenfu injection, trigonelline, ICA and G-Rh2 showed a up-regulation to Cx43.更多还原