蝙蝠葛活性成分抗胃癌细胞作用和机制的研究
|
摘要
研究背景与目的：
胃癌在我国的发病率和死亡率都非常之高,是消化道恶性肿瘤之首。早期胃癌的手术,治愈率还是很高的,但是胃癌本身缺乏特异性临床表现,近80%的患者就诊时就已经属于中晚期,失去了手术机会。此外胃癌患者在手术后很容易复发,尤其是有淋巴结转移者。对这些失去手术机会以及术后复发的患者,化疗是最主要的治疗手段之一。目前新辅助化疗,靶向药物治疗等取得了非常大地进步,但是胃癌的5年生存率还是没有得到明显地改善。因此,我们需要寻找一种新的更有效的抗肿瘤药物,增强胃癌的治疗疗效,改善患者的生活质量,延长患者的生存时间。
当前,对抗肿瘤药物的研究已经逐渐地转向了天然药物,故我国传统的中药越来越受到科学家们的关注,成为了肿瘤治疗的一大特色。大量的临床实践表明单独使用中药或者中西医结合治疗肿瘤,其疗效确切,并能减轻放疗、化疗的毒副作用。不仅如此,中草药可以相对减轻患者的经济负担,这也是中草药的独到之处。故对中草药治疗肿瘤的机制研究也越来越多,其中蝙蝠葛具有多种生物学活性,其根、茎叶、果实均有良好的药理效应,具有较好的应用前景和开发前景。现代研究认为蝙蝠葛活性成分除了具有抗菌、抗病毒和杀虫作用外,还具有较好的体内外抗肿瘤作用,而该作用也得到医学界越来越多的关注。蝙蝠葛活性成分对胃癌作用的报道比较少见,我们在多种细胞系中选择了胃癌SGC-7901细胞株,进一步探讨了其抗肿瘤作用及抑制转移侵袭作用。本实验所用的药物蝙蝠葛活性成分由延边大学天然药物研究所提供,由我们实验室用MTT等检测方法一步一步地筛选其有效成分,现已经接近于单体。其具体化学方程式正在进一步检验中。
本研究采用MTT法检测了蝙蝠葛活性成分体外对多种肿瘤细胞株的抗增殖作用,包括了人肺癌A-549细胞株,人肝癌HepG2细胞株,人肝癌Hep3B细胞株,人宫颈癌Hela细胞株,人乳腺癌MCF-7细胞株,人白血病K-562细胞株,人胃癌MGC-803细胞株,人胃癌SGC-7901细胞株和小鼠黑色素瘤B-16细胞株；研究了蝙蝠葛活性成分对人肺癌A-549细胞裸鼠皮下移植瘤生长的抑制作用；利用免疫细胞化学染色方法观察了蝙蝠葛活性成分对人胃癌SGC-7901细胞中Ki-67基因蛋白的表达变化；特殊染色后应用倒置显微镜、荧光显微镜、透射电镜观察了蝙蝠葛活性成分引起人胃癌SGC-7901细胞形态学方面的变化；应用了TUNEL检测法、DNAladder电泳法、Annexin-PI双染流式细胞仪检测法观察了蝙蝠葛活性成分诱导人胃癌SGC-7901细胞的凋亡作用；利用PI染色、流式细胞仪观察了对照组细胞和实验组细胞的细胞周期分布的变化；应用western blotting方法和流式细胞仪观察了细胞周期相关蛋白和凋亡通路相关蛋白表达的变化；通过划痕实验,transwell实验观察了蝙蝠葛活性成分对人胃癌SGC-7901细胞的运动能力,迁移能力以及侵袭能力的影响；用western blotting实验方法观察了蝙蝠葛活性成分降低人胃癌SGC-7901细胞转移侵袭作用的具体机制。通过这一系列的实验,较为深入地研究蝙蝠葛活性成分的抗肿瘤作用及其机制,有望能为将来采用蝙蝠葛活性成分进行抗癌治疗提供理论依据和实验基础。
本研究的主要方法和结果：
1、MTT比色法检测细胞活力
MTT结果显示蝙蝠葛活性成分对人肺癌A-549细胞株,人肝癌HepG2细胞株,人肝癌Hep3B细胞株,人宫颈癌Hela细胞株,人乳腺癌MCF-7细胞株,人白血病K-562细胞株,人胃癌MGC-803细胞株,人胃癌SGC-7901细胞株和小鼠黑色素瘤B-16细胞株均有明显抑制增殖作用,IC50值分别为4.58μg/ml,5.14/ig/ml,6.21/μg/ml,4.34μg/ml,3.00μg/ml,2.93μg/ml,3.92μg/ml,3.77μg/ml,6.22μg/ml。我们可以认为蝙蝠葛活性成分具有广谱的体外抗肿瘤作用,其中对人白血病K-562细胞作用最强。此外,对人胃癌SGC-7901细胞株也表现出较好的抗增殖作用。
2、蝙蝠葛活性成分对人肺癌A-549细胞裸鼠皮下移植瘤生长的抑制作用
将裸鼠随机分为4个组进行观察。阳性药MMC组的T/C值为6.0%,对裸小鼠移植瘤的生长具有显著的抑制作用。蝙蝠葛活性成分低剂量组T/C值为68.4%,对裸小鼠移植瘤的生长无明显的抑制作用。高剂量组的T/C值为58.8%,并P<0.05,故可以认为对裸小鼠移植瘤的生长具有抑制作用。各实验组裸鼠生长情况良好,整个实验过程均无死亡。阳性对照组,MMC组体重相对较轻,蝙蝠葛活性成分组体重均有增加,与阴性对照组相比无明显差异。
3、免疫细胞化学染色检测Ki-67蛋白的表达
本实验我们利用免疫细胞化学检测方法观察了蝙蝠葛活性成分对人胃癌SGC-7901细胞中的细胞增殖核抗原Ki-67表达的影响,发现它的表达水平发生了明显的改变。对照组细胞Ki-67主要表达在细胞核,为深棕色,并且细胞生长旺盛；实验组细胞变小变圆、成固缩状,几乎看不到棕色颗粒,细胞数目少。Ki-67蛋白阳性表达率,对照组为89.3%,实验组为10.1%,其差异具有统计学意义(P<0.05)。蝙蝠葛活性成分明显地降低了人胃癌SGC-7901细胞中Ki-67蛋白的表达,抑制了胃癌细胞的增殖能力。
4、形态学观察
我们应用倒置显微镜观察到了对照组细胞原本贴壁生长良好,经蝙蝠葛活性成分作用之后细胞变圆,贴壁不牢固,细胞生长明显受到抑制。HE染色、应用光学显微镜我们观察到了与倒置显微镜相同的改变,并可见染色质边集,细胞固缩,凋亡小体等典型的凋亡形态学改变。Hoechst33258染色发现蝙蝠葛活性成分作用之后的实验组细胞出现了染色质固缩,细胞核呈致密浓染和碎块状,颜色有些发白等典型的凋亡形态学改变。A0/EB荧光染色法是一种鉴别细胞坏死与凋亡的简便方法,实验结果发现蝙蝠葛活性成分引起了胃癌细胞的凋亡,很少看见坏死肿胀的细胞。透射电镜结果在微观水平上进一步证实了这一观点。上述实验运用多种特殊染色,通过多种显微镜均观察到了典型的凋亡形态学改变,从而我们能得出蝙蝠葛活性成分在体外引起了胃癌细胞凋亡的结论,可以看出它具有进一步研究的价值。
5、TUNEL检测法
通过TUNEL检测法我们发现对照组细胞在光学显微镜下,细胞生长旺盛,铺满整个盖玻片,但是在荧光显微镜下仅能见到少数的细胞；而实验组细胞在光学显微镜下观察到细胞数明显变少,但是在荧光显微镜下可以观察到更多的发出绿色荧光的细胞。可以看出蝙蝠葛活性成分作用之后人胃癌SGC-7901细胞发生了的凋亡的改变。
6、Annexin-PI双染法
Annexin V-Alexa Fluor和P工双染后利用流式细胞仪观察到对照组中正常细胞占大多数,平均凋亡率仅为5.12±0.87%；而实验组中正常细胞变少,凋亡细胞数变多,平均凋亡率为37.84±2.01%,并且其差异具有统计学意义(P<0.05)。
7、DNA ladder电泳检测
电泳结果发现对照组细胞DNA在胶顶部,呈现出一个高分子量条带；实验组细胞形成明显的DNA梯度,呈阶梯状排列。说明蝙蝠葛活性成分诱导了人胃癌SGC-7901细胞的凋亡,引起了DNA降解。
8、细胞周期分布的影响
我们用PI染色、酒精处理之后利用流式细胞仪观察到蝙蝠葛活性成分显著改变了人胃癌SGC-7901细胞的细胞周期分布。对照组G0/G1期细胞占55.17%,G。/M期细胞占13.07%,S期细胞占31.76%；而实验组G0/G1期细胞明显增多,为74.26%,G2/M期和S期细胞百分比均比对照组减少,分别为8.61%和17.13%。可以看出蝙蝠葛活性成分明显改变了胃癌细胞周期的分布,使G1期细胞的百分比明显增高,S和G2期细胞明显降低,使细胞阻滞在G1期,干预了细胞增殖。
9、细胞周期相关蛋白CyclinD1和p16
我们通过western blotting方法检测了CyclinD1和p16蛋白的表达变化。发现了蝙蝠葛活性成分明显下调了CyclinD1的表达水平,并上调了抑制因子p16基因蛋白表达水平,可以认为蝙蝠葛活性成分是通过调节上述基因蛋白的表达,启动了G1/S期监测点,干预了细胞周期的进行。
10、流式细胞仪检测线粒体膜电位的改变
我们用JC-1荧光染色细胞后利用流式细胞仪检测了线粒体膜电位的改变,发现蝙蝠葛活性成分作用之后线粒体膜电位下降,并且与对照组相比其差异具有统计学意义。线粒体跨膜电位的下降发生在细胞凋亡特征(染色质浓缩、DNA断裂)出现之前,它被认为是细胞凋亡级联反应过程中发生的最早事件。一旦线粒体膜电位崩溃,细胞凋亡就会不可逆转。
11、检测线粒体通路相关蛋白Bax和Bcl-2
我们用western blotting方法来检测胃癌SGC-7901细胞中的Bax和Bcl-2基因蛋白,发现蝙蝠葛活性成分能够上调Bax基因蛋白的含量,下调了Bcl-2基因蛋白的含量,Bcl-2/Bax比例明显减小,并且与对照组相比其差异均有统计学意义。这两种基因是线粒体通路中的关键蛋白酶,Bcl-2(抗凋亡因子)和Bax(促凋亡因子)与线粒体相互作用,改变跨膜电位,改变线粒体的通透性。
12、Caspase-3活性检测
在很多凋亡,不管是哪种因素,哪种途径,最终都要通过Caspase介导的信号途径导致细胞的凋亡,我们利用Caspase-3活性检测试剂盒,观察到蝙蝠葛活性成分明显提高了Caspase-3的活性,当终浓度分别为2.5、5、10μg/ml的蝙蝠葛活性成分作用之后Caspase-3的活性比对照组分别增高了1.37倍、2.5倍、2.7倍,并且与对照组相比具有统计学意义(P<0.05)。我们还观察到了Caspase-3的活性与细胞存活率成负相关,可以认为Caspase-3的活性增高使胃癌SGC-7901细胞的死亡增加,而Caspase-3是诱导凋亡的关键执行分子,Caspase-3的表达上调可以认为是细胞凋亡的特异性标志。
13、细胞划痕实验
细胞划痕实验,实验成本低,是一种简单易行地检测细胞运动能力的方法。本实验发现与对照组相比较,蝙蝠葛活性成分处理之后,细胞修复该划痕的能力减低。结果表明蝙蝠葛活性成分抑制了胃癌SGC-7901细胞的运动能力。
14、Transwell迁移实验
蝙蝠葛活性成分作用之后使人胃癌SGC-7901细胞对聚碳酸酯膜的迁移运动能力下降,其差异具有统计学意义(P<0.05),并且浓度越高其抑制作用就越强。
15、Transwell侵袭实验
Transwell侵袭实验方法与transwell迁移实验方法相似,不同的是transwell侵袭实验中在聚碳酸酯膜上涂抹matrigel胶,肿瘤细胞必须降解此成分才能穿过去,本实验发现蝙蝠葛活性成分不仅使迁移能力降低,还可使人胃癌SGC-7901细胞的侵袭能力下降,其差异具有统计学意义(P<0.05),并且浓度越高其抑制作用就越强。
16、检测侵袭转移相关蛋白MMP-2和Nm23
我们通过western blotting方法检测了侵袭转移相关蛋白MMP-2和Nm23的表达变化。发现了蝙蝠葛活性成分明显下调了MMP-2的表达水平,并上调了抑制因子Nm23的蛋白表达水平,可以认为蝙蝠葛活性成分通过调节上述基因蛋白的表达,来达到抑制胃癌SGC-7901细胞株转移侵袭作用的。
研究结论：
1.蝙蝠葛活性成分在体内和体外均表现出了较好的抗肿瘤作用。
2.蝙蝠葛活性成分通过线粒体途径启动和完成人胃癌SGC-7901细胞凋亡。
3.蝙蝠葛活性成分诱导凋亡作用可能与细胞周期发生G1期阻滞有关。
4.蝙蝠葛活性成分能使胃癌细胞的迁移运动能力,侵袭能力下降。这可能与下调MMP-2,上调Nm23蛋白表达有关。
5.蝙蝠葛活性成分有望开发成一种新的抗肿瘤药物。
Background and objective:
Gastric cancer has the high morbidity and mortality in China. In early stage of the gastric cancer, it could have clinical cure through the operation. Gastric cancer has lack of specific clinical manifestations. Nearly80%of the patients lost the cure chance of the operation, and on top of that, they were easy to replapse after operation. Chemotherapy is the most important treatment for them who lost the chance of the operation and replased after operation.5year survival rates of gastric cancer had not been improved obviously in despite of the neoadjuvant chemotherapy or targeted therapy. Therefore, we need to find a new drug which was effective and safe in the treatment of the gastric cancer to improve the quality of life and prolong the survival time of the patients.
Natural plants like Chinese herbal have been attached more and more consideration at present. A large number of study showed that the traditional Chinese medicine could improve the curative effect of the treatment, but also it could reduce the side effect of radiotherapy and chemotherapy. Additionaly, it has its own knack in economy of the patients. Menispermum dauricum has many biological activities. The root, stem and leaf of menispermum dauricum all have good pharmacological effects and well development prospect. More and more modern researches indicated that menispermum dauricum not only had anti-bacterial and anti-viral effects, but also had anti-tumor effect in vivo and vitro. The studies of anti-tumor effects on gastric cancer cells are still comparatively rare in our country. We discussed further research and possible application in human gastric cancer SGC-7901line treated with menispermum dauricum. Menispermum dauricum which used in this study were provided by Natural Medicin Research of Yanbian University. The selection and extraction of effective component of menispermum dauricum are doing at the same time. It is expected to select the final effective monomers or compound of drugs and analysis the chemical composition soon.
In this study, we used MTT assay to detect the anti-proliferation effect of menispermum dauricum on A-549, HepG2, Hep3B, Hela, MCF-7, K-562, MGC-803, SGC-7901and B-16cell lines; We also studied anti-tumor effect of menispermum dauricum in vivo, we used nude mice which subcutanelously transplanted human lung cancer A-549cells; We used ICC assay to detect the changes of expression of Ki-67protein; We used inverted microscope, fluorescence microscope and tranisssion electron microscope to observe morphology changes of human gastric cancer SGC-7901cell line; We used TUNEL assay, DNA ladder electrophoresis and Annexin-PI double staning to detect the inducing apoptosis effect of menispermum dauricum on grastric cancer SGC-7901cells; We used flow cytometry after PI staing and alchol treatment to detect the distribution of cell cycle; We used Western Blotting method to detect the proteins which did important rule in cell cycle; We did cell adhesion assay, wound healing assay, transwell migration assay and transwell invasion assay to detect the inhibiting metastasis and invasion effects on gastric cancer cells; We used Western Blotting method to detect the proteins which did important rule in tumor metastasis and invasion. Through above mentioned methods, we had further investigations of anti-tumor effects and mechanism of menispermum dauricum. These studies have layed the theoretical and experimental basis on using menispermum dauricum in the area of medicine. We expect that menispermum dauricum would be exploited a new anti-tumor drug soon.
Main methods and results of this research:
1. MTT assay:
The results of MTT assay showed that menispermum dauricum had anti-proliferation effects on A-549, HepG2, Hep3B, Hela, MCF-7, K-562, MGC-803, SGC-7901and B-16cell lines, and they showed concentration dependent.The IC50were4.58μg/ml,5.14μg/ml,6.21μg/ml,4.34μg/ml,3.00μg/ml,2.93μg/ml,3.92μg/ml,3.77μg/ml,6.22μg/ml, K562cell line showed best effect among of them. We could have a conclusion that menispermum dauricum had broad spectrum anti-tumor effect in vitro.
2. Anti-tumor effect in vivo:
We used nude mice which subcutanelously transplanted human lung cancer A-549cells to detect the anti-tumor effect of menispermum dauricum in vivo. The value of T/C in low dosage of menispermum dauricum group was68.4%. It was indicated that low dosage of menispermum dauricum did not have anti-tumor effect. The value of T/C in high dosage of menispermum dauricum group was58.8%, and P<0.05, it showed anti-tumor effect, and there were no obvious side effects. The value of T/C in MMC group was6.0%, it showed remarkable anti-tumor effect, but mice in this group experienced weight loss.
3. Immunocytochemistry:
We found the expression of the Ki-67protein which was detected by immunocytochemistry has been changed remarkble. Ki-67protein in control group was expressed in cell nucleus, the color of the protein showed dark brown. In experimental group, it was hardly see the expression of Ki-67. Besides, we use the formula to calculate the rate of positive expression of the Ki-67, we found it had significant difference with control group. Menispermum dauricum down-regulated the expression of Ki-67and inhibited the proliferation of gastric cancer SGC-7901cells.
4. Morphological observation:
We used inverted microscope to detecte the morphological changes in gastric cancer SGC-7901cells, we found that the cells in control group were growth well. After treated with menispermum dauricum, the growth of the cells was inhibited. HE staining showed similar changes with view which observed under the inverted microscope, it also showed chromatin margination, cell shrinkage and apoptotic cells. Hoechst staining showed morphological changes including chromatin margination, white color, fragmentation and nuclei dissolution and so on. AO/EB double staining is a simple method to identified aopotosis and necrosis cells. We found menispermum dauricum induced the apoptosis of gastric cancer cells, and rarely saw the necrosis of swollen cells. The results of the transmission electron microscopy further confirmed above mentioned results. Above all, we can have a conclusion that menispermum dauricum induced apoptosis of the gastric cancer SGC-7901cells. We can see that it has the value of further research and investigation.
5、TUNEL assay:
Through the TUNEL assay, we found that the cells of control group were growth well under the light microscope, but only few cells could be seen while they were detected under the fluorencence microscope; Under the light microscope, we found the number of experimental cells were got smaller than cells of control group, but the number of cells became more than the cells of control group while detected by the fluorescence microscope.
6. Annexin-PI double staining:
The results of Annexin-PI double staining showed that the majority of the cells in control group were normal cells. After they were treated with menispermum dauricum, the percentage of normal cells has become less, apoptosis cells became more, and it showed significant difference.
7. DNA ladder electrophoresis:
The results of DNA ladder electrophoresis showed that the DNA of the control group were located in rubber top, they were taken on a high molecular bands. After they were treated with menispermum dauricum, they were showed DNA ladder. We could have a conclusion that menispermum dauricum caused the degration of DNA of gastric cancer SGC-7901cells.
8. Distribution of cell cycle:
We used flow cytometry after PI staing and alchol treatment, found that menispermum dauricum significantly changed the distribution of gastric cancer SGC-7901cell cycle. In control group, the percentage of cells in Go/Gi phase was55.17%; percentage of cells in G2/M phase was13.07%; percentage of cells in S phase was31.76%. In experimental group, the percentage of cells in G0/G1phase was74.26%; percentage of cells in G2/M phase was8.61%; percentage of cells in S phase was17.13%. After treated with menispermum dauricum, the percentage of G0/G1phase was increased significantly, the percentage of G2/M phase and S phase were decreased. Menispermum dauricum blocked the human gastric cancer SGC-7901cells in Gi phase.
9. Cyclin D1and p16:
We examined the expression of CyclinD1and p16proteins by Western Blotting assay. We found that menispermum dauricum markedly down-regulated the expression of CyclinDl, and up-regulated the expression of p16. We could have a conclusion that the mechanism of blocking cells in G1phase may be related with that menispermum dauricum had regulated expression of these proteins which had important role in cell cycle.
10. Determination of mitochondria membrane potential
Reduction of mitochondria membrane potential is deemed to be the earliest event in the cascade reaction of the cell apoptosis. It occurs before the appearance of the karyon apoptosis characteristics like chromatin concentration and DNA-fracture.Once the mitochondria membrane potential collapses, the cell apoptosis would not be reversed. The experiment result showed that the mitochondria membrane potential rised after they were treated with menispermum dauricum. The ratio of FL2/FL1cell were decreased, and compared with control group, it showed significant difference.
11、Bax and Bcl-2
We detected the expression of Bax and Bcl-2proteins of gastric cancer SGC-7901cells by using Western Blonting method. We found menispermum dauricum up-regulated the expression of Bax and down-rugulated the expression of Bcl-2. The ratio of Bcl-2/Bax was decreased significantly in experimental group, and the difference was statistically significant. Bax and Bcl-2are keys of enzyme in the mitochondrial pathway, Bcl-2(anti-apoptotic factor) and Bax (pro-apoptotic factor) are interacted with each other, change mitochondrial transmembrane potential and mitochondrial permeability.
12. Detection of Caspase-3activity:
In many apoptosis, no matter what kind of factors or which signal pathways, they were ultimately passed the Caspase mediated apoptosis. We used Caspase-3assay kit, observed that menispermum dauricum significantly increased the activity of Caspase-3, when the concentration of Menispermum dauricum were2.5,5,10μg/ml, effects of Caspase-3were increased1.37times,2.5times and2.7times as much as control group. We also observed that the cell survival rates were negatively correlated with the activity of Caspase-3.
13. Wound healing assay:
Wound healing assay has been carried out for many years to estimate the migration and proliferation rates. A small area is disrupted and a group of cells destroyed or displaced by scratching a line through the layer. Then cells move in and fill the damaged area. This assay could detect the migration ability of the cancer cenlls. The results of this experiment showed that active components of menispermum menispermum dauricum inhibited the migration ability of gastric cancer cells.
14、Transwell migration assay:
The result of transwell migration assay showed menispermum dauricum could inhibit the migration ability of human gastric cancer SGC-7901cells. The higher concentration, the stronger inhibitory effect it did. The difference between control group and experimental group was statistical significance (P<0.05).
15、Transwell invasion assay:
Transwell invasion assay was similar with the transwell migration assay. The only difference between them was polycarbonate membrane in transwell invasion assay was covered with matrigel glue. Cancer cells were need to degradate this membrane to past. This study showed menispermum dauricum could not only inhibit the migration ability, but also inhibit the invasion ability of gastric cancer SGC-7901cells.
16、MMP-2and Nm23:
We detected the expression of MMP-2and Nm23protein by Western Blotting assay. We found menispermum dauricum markedly down-regulated the expression of MMP-2, and up-regulated the expression of Nm23. We could have a conclusion that the mechanism of inhibiting invasion and metastasis may be related with that menispermum dauricum regulated the expression of these proteins.
Conclusions of this research:
1. Menispermum dauricum had broad spectrum anti-tumor effect in vitro.
2. Menispermum dauricum had anti-tumor effect in vivo.
3. Menispermum dauricum could induce the apoptosis of human gastric cancer SGC-7901cells via mitochondrial pathway.
4. The mechanism of inducing apoptosis may be related with blocking cell cycle in Gi phase.
5. Menispermum dauricum could inhibit the motility ability, invasive ability of gastric cancer SGC-7901cells, these effects may be related with the down-regulation of MMP-2expression and up-regulation of Nm23expression.
6. We expect menispermum dauricum would be developed as a new anti-cancer drug soon.
胃癌在我国的发病率和死亡率都非常之高,是消化道恶性肿瘤之首。早期胃癌的手术,治愈率还是很高的,但是胃癌本身缺乏特异性临床表现,近80%的患者就诊时就已经属于中晚期,失去了手术机会。此外胃癌患者在手术后很容易复发,尤其是有淋巴结转移者。对这些失去手术机会以及术后复发的患者,化疗是最主要的治疗手段之一。目前新辅助化疗,靶向药物治疗等取得了非常大地进步,但是胃癌的5年生存率还是没有得到明显地改善。因此,我们需要寻找一种新的更有效的抗肿瘤药物,增强胃癌的治疗疗效,改善患者的生活质量,延长患者的生存时间。
当前,对抗肿瘤药物的研究已经逐渐地转向了天然药物,故我国传统的中药越来越受到科学家们的关注,成为了肿瘤治疗的一大特色。大量的临床实践表明单独使用中药或者中西医结合治疗肿瘤,其疗效确切,并能减轻放疗、化疗的毒副作用。不仅如此,中草药可以相对减轻患者的经济负担,这也是中草药的独到之处。故对中草药治疗肿瘤的机制研究也越来越多,其中蝙蝠葛具有多种生物学活性,其根、茎叶、果实均有良好的药理效应,具有较好的应用前景和开发前景。现代研究认为蝙蝠葛活性成分除了具有抗菌、抗病毒和杀虫作用外,还具有较好的体内外抗肿瘤作用,而该作用也得到医学界越来越多的关注。蝙蝠葛活性成分对胃癌作用的报道比较少见,我们在多种细胞系中选择了胃癌SGC-7901细胞株,进一步探讨了其抗肿瘤作用及抑制转移侵袭作用。本实验所用的药物蝙蝠葛活性成分由延边大学天然药物研究所提供,由我们实验室用MTT等检测方法一步一步地筛选其有效成分,现已经接近于单体。其具体化学方程式正在进一步检验中。
本研究采用MTT法检测了蝙蝠葛活性成分体外对多种肿瘤细胞株的抗增殖作用,包括了人肺癌A-549细胞株,人肝癌HepG2细胞株,人肝癌Hep3B细胞株,人宫颈癌Hela细胞株,人乳腺癌MCF-7细胞株,人白血病K-562细胞株,人胃癌MGC-803细胞株,人胃癌SGC-7901细胞株和小鼠黑色素瘤B-16细胞株；研究了蝙蝠葛活性成分对人肺癌A-549细胞裸鼠皮下移植瘤生长的抑制作用；利用免疫细胞化学染色方法观察了蝙蝠葛活性成分对人胃癌SGC-7901细胞中Ki-67基因蛋白的表达变化；特殊染色后应用倒置显微镜、荧光显微镜、透射电镜观察了蝙蝠葛活性成分引起人胃癌SGC-7901细胞形态学方面的变化；应用了TUNEL检测法、DNAladder电泳法、Annexin-PI双染流式细胞仪检测法观察了蝙蝠葛活性成分诱导人胃癌SGC-7901细胞的凋亡作用；利用PI染色、流式细胞仪观察了对照组细胞和实验组细胞的细胞周期分布的变化；应用western blotting方法和流式细胞仪观察了细胞周期相关蛋白和凋亡通路相关蛋白表达的变化；通过划痕实验,transwell实验观察了蝙蝠葛活性成分对人胃癌SGC-7901细胞的运动能力,迁移能力以及侵袭能力的影响；用western blotting实验方法观察了蝙蝠葛活性成分降低人胃癌SGC-7901细胞转移侵袭作用的具体机制。通过这一系列的实验,较为深入地研究蝙蝠葛活性成分的抗肿瘤作用及其机制,有望能为将来采用蝙蝠葛活性成分进行抗癌治疗提供理论依据和实验基础。
本研究的主要方法和结果：
1、MTT比色法检测细胞活力
MTT结果显示蝙蝠葛活性成分对人肺癌A-549细胞株,人肝癌HepG2细胞株,人肝癌Hep3B细胞株,人宫颈癌Hela细胞株,人乳腺癌MCF-7细胞株,人白血病K-562细胞株,人胃癌MGC-803细胞株,人胃癌SGC-7901细胞株和小鼠黑色素瘤B-16细胞株均有明显抑制增殖作用,IC50值分别为4.58μg/ml,5.14/ig/ml,6.21/μg/ml,4.34μg/ml,3.00μg/ml,2.93μg/ml,3.92μg/ml,3.77μg/ml,6.22μg/ml。我们可以认为蝙蝠葛活性成分具有广谱的体外抗肿瘤作用,其中对人白血病K-562细胞作用最强。此外,对人胃癌SGC-7901细胞株也表现出较好的抗增殖作用。
2、蝙蝠葛活性成分对人肺癌A-549细胞裸鼠皮下移植瘤生长的抑制作用
将裸鼠随机分为4个组进行观察。阳性药MMC组的T/C值为6.0%,对裸小鼠移植瘤的生长具有显著的抑制作用。蝙蝠葛活性成分低剂量组T/C值为68.4%,对裸小鼠移植瘤的生长无明显的抑制作用。高剂量组的T/C值为58.8%,并P<0.05,故可以认为对裸小鼠移植瘤的生长具有抑制作用。各实验组裸鼠生长情况良好,整个实验过程均无死亡。阳性对照组,MMC组体重相对较轻,蝙蝠葛活性成分组体重均有增加,与阴性对照组相比无明显差异。
3、免疫细胞化学染色检测Ki-67蛋白的表达
本实验我们利用免疫细胞化学检测方法观察了蝙蝠葛活性成分对人胃癌SGC-7901细胞中的细胞增殖核抗原Ki-67表达的影响,发现它的表达水平发生了明显的改变。对照组细胞Ki-67主要表达在细胞核,为深棕色,并且细胞生长旺盛；实验组细胞变小变圆、成固缩状,几乎看不到棕色颗粒,细胞数目少。Ki-67蛋白阳性表达率,对照组为89.3%,实验组为10.1%,其差异具有统计学意义(P<0.05)。蝙蝠葛活性成分明显地降低了人胃癌SGC-7901细胞中Ki-67蛋白的表达,抑制了胃癌细胞的增殖能力。
4、形态学观察
我们应用倒置显微镜观察到了对照组细胞原本贴壁生长良好,经蝙蝠葛活性成分作用之后细胞变圆,贴壁不牢固,细胞生长明显受到抑制。HE染色、应用光学显微镜我们观察到了与倒置显微镜相同的改变,并可见染色质边集,细胞固缩,凋亡小体等典型的凋亡形态学改变。Hoechst33258染色发现蝙蝠葛活性成分作用之后的实验组细胞出现了染色质固缩,细胞核呈致密浓染和碎块状,颜色有些发白等典型的凋亡形态学改变。A0/EB荧光染色法是一种鉴别细胞坏死与凋亡的简便方法,实验结果发现蝙蝠葛活性成分引起了胃癌细胞的凋亡,很少看见坏死肿胀的细胞。透射电镜结果在微观水平上进一步证实了这一观点。上述实验运用多种特殊染色,通过多种显微镜均观察到了典型的凋亡形态学改变,从而我们能得出蝙蝠葛活性成分在体外引起了胃癌细胞凋亡的结论,可以看出它具有进一步研究的价值。
5、TUNEL检测法
通过TUNEL检测法我们发现对照组细胞在光学显微镜下,细胞生长旺盛,铺满整个盖玻片,但是在荧光显微镜下仅能见到少数的细胞；而实验组细胞在光学显微镜下观察到细胞数明显变少,但是在荧光显微镜下可以观察到更多的发出绿色荧光的细胞。可以看出蝙蝠葛活性成分作用之后人胃癌SGC-7901细胞发生了的凋亡的改变。
6、Annexin-PI双染法
Annexin V-Alexa Fluor和P工双染后利用流式细胞仪观察到对照组中正常细胞占大多数,平均凋亡率仅为5.12±0.87%；而实验组中正常细胞变少,凋亡细胞数变多,平均凋亡率为37.84±2.01%,并且其差异具有统计学意义(P<0.05)。
7、DNA ladder电泳检测
电泳结果发现对照组细胞DNA在胶顶部,呈现出一个高分子量条带；实验组细胞形成明显的DNA梯度,呈阶梯状排列。说明蝙蝠葛活性成分诱导了人胃癌SGC-7901细胞的凋亡,引起了DNA降解。
8、细胞周期分布的影响
我们用PI染色、酒精处理之后利用流式细胞仪观察到蝙蝠葛活性成分显著改变了人胃癌SGC-7901细胞的细胞周期分布。对照组G0/G1期细胞占55.17%,G。/M期细胞占13.07%,S期细胞占31.76%；而实验组G0/G1期细胞明显增多,为74.26%,G2/M期和S期细胞百分比均比对照组减少,分别为8.61%和17.13%。可以看出蝙蝠葛活性成分明显改变了胃癌细胞周期的分布,使G1期细胞的百分比明显增高,S和G2期细胞明显降低,使细胞阻滞在G1期,干预了细胞增殖。
9、细胞周期相关蛋白CyclinD1和p16
我们通过western blotting方法检测了CyclinD1和p16蛋白的表达变化。发现了蝙蝠葛活性成分明显下调了CyclinD1的表达水平,并上调了抑制因子p16基因蛋白表达水平,可以认为蝙蝠葛活性成分是通过调节上述基因蛋白的表达,启动了G1/S期监测点,干预了细胞周期的进行。
10、流式细胞仪检测线粒体膜电位的改变
我们用JC-1荧光染色细胞后利用流式细胞仪检测了线粒体膜电位的改变,发现蝙蝠葛活性成分作用之后线粒体膜电位下降,并且与对照组相比其差异具有统计学意义。线粒体跨膜电位的下降发生在细胞凋亡特征(染色质浓缩、DNA断裂)出现之前,它被认为是细胞凋亡级联反应过程中发生的最早事件。一旦线粒体膜电位崩溃,细胞凋亡就会不可逆转。
11、检测线粒体通路相关蛋白Bax和Bcl-2
我们用western blotting方法来检测胃癌SGC-7901细胞中的Bax和Bcl-2基因蛋白,发现蝙蝠葛活性成分能够上调Bax基因蛋白的含量,下调了Bcl-2基因蛋白的含量,Bcl-2/Bax比例明显减小,并且与对照组相比其差异均有统计学意义。这两种基因是线粒体通路中的关键蛋白酶,Bcl-2(抗凋亡因子)和Bax(促凋亡因子)与线粒体相互作用,改变跨膜电位,改变线粒体的通透性。
12、Caspase-3活性检测
在很多凋亡,不管是哪种因素,哪种途径,最终都要通过Caspase介导的信号途径导致细胞的凋亡,我们利用Caspase-3活性检测试剂盒,观察到蝙蝠葛活性成分明显提高了Caspase-3的活性,当终浓度分别为2.5、5、10μg/ml的蝙蝠葛活性成分作用之后Caspase-3的活性比对照组分别增高了1.37倍、2.5倍、2.7倍,并且与对照组相比具有统计学意义(P<0.05)。我们还观察到了Caspase-3的活性与细胞存活率成负相关,可以认为Caspase-3的活性增高使胃癌SGC-7901细胞的死亡增加,而Caspase-3是诱导凋亡的关键执行分子,Caspase-3的表达上调可以认为是细胞凋亡的特异性标志。
13、细胞划痕实验
细胞划痕实验,实验成本低,是一种简单易行地检测细胞运动能力的方法。本实验发现与对照组相比较,蝙蝠葛活性成分处理之后,细胞修复该划痕的能力减低。结果表明蝙蝠葛活性成分抑制了胃癌SGC-7901细胞的运动能力。
14、Transwell迁移实验
蝙蝠葛活性成分作用之后使人胃癌SGC-7901细胞对聚碳酸酯膜的迁移运动能力下降,其差异具有统计学意义(P<0.05),并且浓度越高其抑制作用就越强。
15、Transwell侵袭实验
Transwell侵袭实验方法与transwell迁移实验方法相似,不同的是transwell侵袭实验中在聚碳酸酯膜上涂抹matrigel胶,肿瘤细胞必须降解此成分才能穿过去,本实验发现蝙蝠葛活性成分不仅使迁移能力降低,还可使人胃癌SGC-7901细胞的侵袭能力下降,其差异具有统计学意义(P<0.05),并且浓度越高其抑制作用就越强。
16、检测侵袭转移相关蛋白MMP-2和Nm23
我们通过western blotting方法检测了侵袭转移相关蛋白MMP-2和Nm23的表达变化。发现了蝙蝠葛活性成分明显下调了MMP-2的表达水平,并上调了抑制因子Nm23的蛋白表达水平,可以认为蝙蝠葛活性成分通过调节上述基因蛋白的表达,来达到抑制胃癌SGC-7901细胞株转移侵袭作用的。
研究结论：
1.蝙蝠葛活性成分在体内和体外均表现出了较好的抗肿瘤作用。
2.蝙蝠葛活性成分通过线粒体途径启动和完成人胃癌SGC-7901细胞凋亡。
3.蝙蝠葛活性成分诱导凋亡作用可能与细胞周期发生G1期阻滞有关。
4.蝙蝠葛活性成分能使胃癌细胞的迁移运动能力,侵袭能力下降。这可能与下调MMP-2,上调Nm23蛋白表达有关。
5.蝙蝠葛活性成分有望开发成一种新的抗肿瘤药物。
Background and objective:
Gastric cancer has the high morbidity and mortality in China. In early stage of the gastric cancer, it could have clinical cure through the operation. Gastric cancer has lack of specific clinical manifestations. Nearly80%of the patients lost the cure chance of the operation, and on top of that, they were easy to replapse after operation. Chemotherapy is the most important treatment for them who lost the chance of the operation and replased after operation.5year survival rates of gastric cancer had not been improved obviously in despite of the neoadjuvant chemotherapy or targeted therapy. Therefore, we need to find a new drug which was effective and safe in the treatment of the gastric cancer to improve the quality of life and prolong the survival time of the patients.
Natural plants like Chinese herbal have been attached more and more consideration at present. A large number of study showed that the traditional Chinese medicine could improve the curative effect of the treatment, but also it could reduce the side effect of radiotherapy and chemotherapy. Additionaly, it has its own knack in economy of the patients. Menispermum dauricum has many biological activities. The root, stem and leaf of menispermum dauricum all have good pharmacological effects and well development prospect. More and more modern researches indicated that menispermum dauricum not only had anti-bacterial and anti-viral effects, but also had anti-tumor effect in vivo and vitro. The studies of anti-tumor effects on gastric cancer cells are still comparatively rare in our country. We discussed further research and possible application in human gastric cancer SGC-7901line treated with menispermum dauricum. Menispermum dauricum which used in this study were provided by Natural Medicin Research of Yanbian University. The selection and extraction of effective component of menispermum dauricum are doing at the same time. It is expected to select the final effective monomers or compound of drugs and analysis the chemical composition soon.
In this study, we used MTT assay to detect the anti-proliferation effect of menispermum dauricum on A-549, HepG2, Hep3B, Hela, MCF-7, K-562, MGC-803, SGC-7901and B-16cell lines; We also studied anti-tumor effect of menispermum dauricum in vivo, we used nude mice which subcutanelously transplanted human lung cancer A-549cells; We used ICC assay to detect the changes of expression of Ki-67protein; We used inverted microscope, fluorescence microscope and tranisssion electron microscope to observe morphology changes of human gastric cancer SGC-7901cell line; We used TUNEL assay, DNA ladder electrophoresis and Annexin-PI double staning to detect the inducing apoptosis effect of menispermum dauricum on grastric cancer SGC-7901cells; We used flow cytometry after PI staing and alchol treatment to detect the distribution of cell cycle; We used Western Blotting method to detect the proteins which did important rule in cell cycle; We did cell adhesion assay, wound healing assay, transwell migration assay and transwell invasion assay to detect the inhibiting metastasis and invasion effects on gastric cancer cells; We used Western Blotting method to detect the proteins which did important rule in tumor metastasis and invasion. Through above mentioned methods, we had further investigations of anti-tumor effects and mechanism of menispermum dauricum. These studies have layed the theoretical and experimental basis on using menispermum dauricum in the area of medicine. We expect that menispermum dauricum would be exploited a new anti-tumor drug soon.
Main methods and results of this research:
1. MTT assay:
The results of MTT assay showed that menispermum dauricum had anti-proliferation effects on A-549, HepG2, Hep3B, Hela, MCF-7, K-562, MGC-803, SGC-7901and B-16cell lines, and they showed concentration dependent.The IC50were4.58μg/ml,5.14μg/ml,6.21μg/ml,4.34μg/ml,3.00μg/ml,2.93μg/ml,3.92μg/ml,3.77μg/ml,6.22μg/ml, K562cell line showed best effect among of them. We could have a conclusion that menispermum dauricum had broad spectrum anti-tumor effect in vitro.
2. Anti-tumor effect in vivo:
We used nude mice which subcutanelously transplanted human lung cancer A-549cells to detect the anti-tumor effect of menispermum dauricum in vivo. The value of T/C in low dosage of menispermum dauricum group was68.4%. It was indicated that low dosage of menispermum dauricum did not have anti-tumor effect. The value of T/C in high dosage of menispermum dauricum group was58.8%, and P<0.05, it showed anti-tumor effect, and there were no obvious side effects. The value of T/C in MMC group was6.0%, it showed remarkable anti-tumor effect, but mice in this group experienced weight loss.
3. Immunocytochemistry:
We found the expression of the Ki-67protein which was detected by immunocytochemistry has been changed remarkble. Ki-67protein in control group was expressed in cell nucleus, the color of the protein showed dark brown. In experimental group, it was hardly see the expression of Ki-67. Besides, we use the formula to calculate the rate of positive expression of the Ki-67, we found it had significant difference with control group. Menispermum dauricum down-regulated the expression of Ki-67and inhibited the proliferation of gastric cancer SGC-7901cells.
4. Morphological observation:
We used inverted microscope to detecte the morphological changes in gastric cancer SGC-7901cells, we found that the cells in control group were growth well. After treated with menispermum dauricum, the growth of the cells was inhibited. HE staining showed similar changes with view which observed under the inverted microscope, it also showed chromatin margination, cell shrinkage and apoptotic cells. Hoechst staining showed morphological changes including chromatin margination, white color, fragmentation and nuclei dissolution and so on. AO/EB double staining is a simple method to identified aopotosis and necrosis cells. We found menispermum dauricum induced the apoptosis of gastric cancer cells, and rarely saw the necrosis of swollen cells. The results of the transmission electron microscopy further confirmed above mentioned results. Above all, we can have a conclusion that menispermum dauricum induced apoptosis of the gastric cancer SGC-7901cells. We can see that it has the value of further research and investigation.
5、TUNEL assay:
Through the TUNEL assay, we found that the cells of control group were growth well under the light microscope, but only few cells could be seen while they were detected under the fluorencence microscope; Under the light microscope, we found the number of experimental cells were got smaller than cells of control group, but the number of cells became more than the cells of control group while detected by the fluorescence microscope.
6. Annexin-PI double staining:
The results of Annexin-PI double staining showed that the majority of the cells in control group were normal cells. After they were treated with menispermum dauricum, the percentage of normal cells has become less, apoptosis cells became more, and it showed significant difference.
7. DNA ladder electrophoresis:
The results of DNA ladder electrophoresis showed that the DNA of the control group were located in rubber top, they were taken on a high molecular bands. After they were treated with menispermum dauricum, they were showed DNA ladder. We could have a conclusion that menispermum dauricum caused the degration of DNA of gastric cancer SGC-7901cells.
8. Distribution of cell cycle:
We used flow cytometry after PI staing and alchol treatment, found that menispermum dauricum significantly changed the distribution of gastric cancer SGC-7901cell cycle. In control group, the percentage of cells in Go/Gi phase was55.17%; percentage of cells in G2/M phase was13.07%; percentage of cells in S phase was31.76%. In experimental group, the percentage of cells in G0/G1phase was74.26%; percentage of cells in G2/M phase was8.61%; percentage of cells in S phase was17.13%. After treated with menispermum dauricum, the percentage of G0/G1phase was increased significantly, the percentage of G2/M phase and S phase were decreased. Menispermum dauricum blocked the human gastric cancer SGC-7901cells in Gi phase.
9. Cyclin D1and p16:
We examined the expression of CyclinD1and p16proteins by Western Blotting assay. We found that menispermum dauricum markedly down-regulated the expression of CyclinDl, and up-regulated the expression of p16. We could have a conclusion that the mechanism of blocking cells in G1phase may be related with that menispermum dauricum had regulated expression of these proteins which had important role in cell cycle.
10. Determination of mitochondria membrane potential
Reduction of mitochondria membrane potential is deemed to be the earliest event in the cascade reaction of the cell apoptosis. It occurs before the appearance of the karyon apoptosis characteristics like chromatin concentration and DNA-fracture.Once the mitochondria membrane potential collapses, the cell apoptosis would not be reversed. The experiment result showed that the mitochondria membrane potential rised after they were treated with menispermum dauricum. The ratio of FL2/FL1cell were decreased, and compared with control group, it showed significant difference.
11、Bax and Bcl-2
We detected the expression of Bax and Bcl-2proteins of gastric cancer SGC-7901cells by using Western Blonting method. We found menispermum dauricum up-regulated the expression of Bax and down-rugulated the expression of Bcl-2. The ratio of Bcl-2/Bax was decreased significantly in experimental group, and the difference was statistically significant. Bax and Bcl-2are keys of enzyme in the mitochondrial pathway, Bcl-2(anti-apoptotic factor) and Bax (pro-apoptotic factor) are interacted with each other, change mitochondrial transmembrane potential and mitochondrial permeability.
12. Detection of Caspase-3activity:
In many apoptosis, no matter what kind of factors or which signal pathways, they were ultimately passed the Caspase mediated apoptosis. We used Caspase-3assay kit, observed that menispermum dauricum significantly increased the activity of Caspase-3, when the concentration of Menispermum dauricum were2.5,5,10μg/ml, effects of Caspase-3were increased1.37times,2.5times and2.7times as much as control group. We also observed that the cell survival rates were negatively correlated with the activity of Caspase-3.
13. Wound healing assay:
Wound healing assay has been carried out for many years to estimate the migration and proliferation rates. A small area is disrupted and a group of cells destroyed or displaced by scratching a line through the layer. Then cells move in and fill the damaged area. This assay could detect the migration ability of the cancer cenlls. The results of this experiment showed that active components of menispermum menispermum dauricum inhibited the migration ability of gastric cancer cells.
14、Transwell migration assay:
The result of transwell migration assay showed menispermum dauricum could inhibit the migration ability of human gastric cancer SGC-7901cells. The higher concentration, the stronger inhibitory effect it did. The difference between control group and experimental group was statistical significance (P<0.05).
15、Transwell invasion assay:
Transwell invasion assay was similar with the transwell migration assay. The only difference between them was polycarbonate membrane in transwell invasion assay was covered with matrigel glue. Cancer cells were need to degradate this membrane to past. This study showed menispermum dauricum could not only inhibit the migration ability, but also inhibit the invasion ability of gastric cancer SGC-7901cells.
16、MMP-2and Nm23:
We detected the expression of MMP-2and Nm23protein by Western Blotting assay. We found menispermum dauricum markedly down-regulated the expression of MMP-2, and up-regulated the expression of Nm23. We could have a conclusion that the mechanism of inhibiting invasion and metastasis may be related with that menispermum dauricum regulated the expression of these proteins.
Conclusions of this research:
1. Menispermum dauricum had broad spectrum anti-tumor effect in vitro.
2. Menispermum dauricum had anti-tumor effect in vivo.
3. Menispermum dauricum could induce the apoptosis of human gastric cancer SGC-7901cells via mitochondrial pathway.
4. The mechanism of inducing apoptosis may be related with blocking cell cycle in Gi phase.
5. Menispermum dauricum could inhibit the motility ability, invasive ability of gastric cancer SGC-7901cells, these effects may be related with the down-regulation of MMP-2expression and up-regulation of Nm23expression.
6. We expect menispermum dauricum would be developed as a new anti-cancer drug soon.
引文
[1]Li YJ, Sun LC, He Y, et al. The anti-tumor properties of two tumstatin peptide fragments in human gastric carcinoma[J]. Acta Pharmacol Sin, 2009,30(9):1307-1315.
[2]Zhengming Ge, Qiwen Ben,Junbo Qian, et al. Diabetes mellitus and risk of gastric cancer:a systematic review and meta-analysis of observational studies[J]. European Journal of Gastroenterology,2011,23(12):1127-130.
[3]朱金水.胃癌的治疗现状[J].中国临床医学,2004,11(2)：131-134.
[4]Lewis W G, Edwards P, Barry J D, et al. The gastrectomy question [J]. Gastric Cancer,2002,5(1):29-34.
[5]Lai, He X W, Jiang Y, et al. Preparative separation and determination of matrine from the Chinese Medicinal plant Sophora flavescents Ait by molecularly imprinted solid-phase extraction [J]. Anal Bioanal Chem, 2003,375(2):264-267.
[6]Guo DL, Zhou ZN, Zeng FD, et al. Dauricine inhibited L-type calcium current in single cardiomyocyte of guinea pig [J]. Acta Pharmacol Sin, 1997,18(5):418-421.
[7]Zeng GQ. Rui YC. Effect of anisodamine and daurieine on proliferation, DNA synthesis and calcium influx in bovine anterior cerebral arterial smooth muscle cells in culture [J]. Acta Phar Sic,1991,12(4):308-311.
[8]SHI Shao-jun, LI Zhong-fang, GU Shi-fen, et al. Pharmacokinetics and tissue of daurisoline in rabbits[J]. Chin J Clin Pharmacol Ther, 2005,10(6):622-626.
[9]SHI Shao-Jun, CHEN Hui, GU Shi-fen, et al. Pharmacokinetic pharmacodynamic modeling of daurisoline in beagle dogs[J]. Acta Pharmacol Sin,2003,24(10):1011-1013.
[10]杨万山,孙抒,吕贺.MTT法检测蝙蝠葛活性成分的抑制肿瘤增殖作用[J].延边大学医学学报,2010,33(4)：261-263.
[11]苏云明,李宜航,刘坤,等.蝙蝠葛酚性碱对BXPC-3肿瘤抑制作用及肿瘤组织中MMP-2含量的影响[J].2010,5(2)：113-115.
[12]SylvestreM, Legault J, Dufour D, et al. Chemical composition and anti-cancer activity of leaf essential oil of Myrica gale[J]. J Phytomedicine,2005,12(4):299-304.
[13]刘瑞,马养民,张弘弛.蝙蝠葛茎叶化学成分的初步研究[J].西北林学院学报,2010,25(5)：176-178.
[14]何志一,刘相辉,刚宏林.蝙蝠葛酚性碱诱导多药耐药细胞系K562/MDR1凋亡及逆转耐药性的研究[J].江苏大学学报,2010,20(2)：108-110.
[15]Park DJ, Lenz I-tJ. Determinants of chemosensitivity in gastric cancer [J]. Curr Opin Pharmaeol,2006,6:337-344.
[16]Moehler M, Galle PR, Gockel I, et al. The multidisciplinary management of gastrointestinal cancer. Multimodal treatment of gastric cancer [J]. Best Pratt Res Clin Gastroentrol,2007,21:965-981.
[17]周庚宴.组织病理学技术[M].北京：大学医学出版社,2006.149-152.
[18]孙宁.氧化苦参碱对人胃癌细胞裸鼠皮下移植瘤抑制作用的研究[D].郑州：河南中医学院.2007.
[19]王德娟,王栋.蝙蝠葛酚性碱药理作用研究进展[J].中医药学报,2008,36(5)：44-46.
[20]路艳艳,杨锦蓉,王菁,等.AO/EB染色法及流式细胞术检测DMF诱导人肝细胞凋亡[J].浙江预防医学,2007,19(6)：11-14.
[21]Wei X Y, Niu R F. The development history mechanism and application of Flow cytometer[J].Mod Instr,2006, (4):8-11.
[22]周庚宴.组织病理学技术[M].北京：大学医学出版社,2006.149-152.
[23]Mashimo K, Sato S, Adachi A, et al. Ethanol decreases cellular protein content and mitochondrial membrane potential of cultured neonatal rat cardiomyocytes:microassays with fluorometric and spectrometric plate readers[J]. Nihon Arukoru Yakubutsu Igakkai Zasshi,2010, 45(6):543-556.
[24]Pallis M, Grundy M, Turzan ski J, et al. Mitochondrial membrane sensitivity to depolarization in acute myeloblastic leukemia is associated with spontaneous in vitro apoptosis, wild type TP53 and vicinal thiol/disulfide status[J]. Blood,2001,98(2):405-413.
[25]Hu Y, Chen X, Duan H, et al. Chinese herbal medicinal ingredients inhibit secretion of IL-6, IL-8, E-selectin and TXB2 in LPS-induced rat intestinal micro vascular endothelial cells. Immunopharmacol Immunotoxicol,2009,31(4):550-555.
[26]Thangapasham RL, Singh AK, Sharma A, et al. Green tea poly-phenols and its constituent epigallocatechin gallate inhibits proliferation of human breast cancer cells in vitro and in vivo [J]. Cancer Letter, 2007,245(12):232-241.
[27]Hafez NH, Tahoun NS. Diagnostic value of p53 and ki67 immunostaining for distinguishing benign from malignant serous effusions[J]. J Egypt Natl Canc Inst,2011,23(4):155-162.
[28]Golmohammadi R, Pejhan A. The prognostic value of the P53 protein and the Ki67 marker in breast cancer patients[J]. J Pak Med Assoc, 2012,62(9):871-875.
[29]Jun Nian, ZHENG Yaf eng, SUN Dongsheng. Treatment with Vector-expressed Small Hairpin RNAs against Ki67 RNA-induced Cell Growth Inhibition and Apoptosis in Human Renal Carcinoma Cells[J]. Acta Biochimica et Biophysica Sinica,2006,38(4):254-261.
[30]Zhongchen, Liu Juan, Wang Ping. RGD-FasL Induces Apoptosis in hepatocellular Carcinoma [J]. Cellular and Molecular Immunology, 2009,6(4):285-293.
[31]Stephane Zalinski, Olivier Scatton, Bruto Randone, et al. Complete hepato cellular carcinoma necrosis following sequential porto-arterial embolization [J]. World Journal of Gastroenterology, 2008,14(44):6869-6872.
[32]Brooks C, Dong Z. Regulation of mitochondrial morphological dynamics during apoptosis by Bcl-2 family proteins:a key in Bak[J]. Cell Cycle, 2007,6(24):3043-3047.
[33]BhaUacharyya, Lahiry L, Mandal D, et al. Black tea induces rumor cell apoptosis by Bax translocation, loss in mitochondrial transmembrane potential cytochrome c release and caspase activation. Int J Cancer, 2005,117(2):308-315.
[34]Metrailler Ruchonnet I, Pagano A, Carnesecchi S, et al. Bcl-2 protects againsthy peroxia induced apoptosis through inhibition of themitochondria dependent pathway[J]. Free Radic Biol Med,2007,42(7):1062-7104.
[35]Chami M, Prandini A, Campanella M, et al. Bcl-2 and Bax exertopposing effects on Ca2+ signal, which do not depend on their putative poreforming region[J].J BiolChem,2004,279 (11): 54581-54589.
[36]Yasmine Nadler, Robert L Camp, Jennifer M Giltnane, et al. Expression patterns and prognostic value of Bag-1 and Bcl-2 in breast cancer [J]. Breast Cancer Research,2008,10(2):35-47.
[37]Jacquelyn M. Long, Charles W. Bell1 W, et al. Microarray and pathway analysis reveals decreased CDC25A and increased CDC42 associated with slow growth of Bcl-2 overexpressing immortalized breast cell line [J].Cell Cycle,2008,7(19):3062-3073.
[38]A Dhanasekaran, S Kotamraju, C Karunakaran, et al. Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis:role of mitochondrial superoxide[J]. Free Radic Biol Med,2005,39(5):567-583.
[39]C Du, M Fang, Y Li, et al. Smac a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition[J].Cell,2000,102(1):33-42.
[40]HK Lorenzo, SA Susin. Mitochondrial effectors in caspase-independent cell death[J]. FEBS Lett,2004,557(13):14-20.
[41]CH Ko, SC Shen, CS Hsu, et al. Mitochondrial-dependent, reactive oxygen species independent apoptosis by myricetin:roles of protein kinase C, cytochrome c, and caspase cascade [J]. Biochem Pharmacol, 2005,69(6):913-927.
[42]M van Gurp, N Festjens, Gvan Loo, et al. Mitochondrial intermembrane proteins in cell death[J]. Biochem Biophys Res,2003,304 (3):487-497.
[43]Larsen BD, Megeney LA. Parole terms for a killer:directing caspase3/CAD induced DNA strand breaks to coordinate changes in gene expression[J].Cell Cycle,2010,9(15):2940-2945.
[44]Padilla-Camberos E, Zaitseva G, Padilla C, et al. Anti-tumoral activity of allicin in murine lymphoma[J]. Asian Pac J Cancer Prev, 2010,11(5):1241-1244.
[45]Ming Sun, Yumei Zhao, Yi Gu, et al. Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke [J]. Neurochemistry International,2009, 54(11):339-346.
[46]Shihe Yang, Qiong Zhou, Xiaohe Yan. Caspase-3 status is a determinant of the differential responses to genistein between MDA-MB-231 and MCF-7 breast cancer cells[J]. Biochimical et Biophysica Acta,2007, (1773):903-911.
[47]Peek RM Jr, Wirth HP, Moss SF, et al. Helicobacter pylorialters Gastric epithelial cell cycle events and gastric secretion in m ongolian gerbils[J]. Gastroenterology,2000,118(1):48-59.
[48]Ke Xiu, Zhu Yali, Cao Bin. Changes of the cell cycle regulators and cell cycle arrest in cervical cancer cells after cisplatin therapy [J]. Academic Journal of Xian University,2009,21 (2):124-128.
[49]Zong Fang, Li Zhidong, Wang Yuan-yuan. Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract[J]. World Journal of Gastroenterology,2009, (36):4538-4546.
[50]Y. L. Hsu, P. L. Kuo, W. S. Tzeng, et al. Chalcone inhibits the proliferation of human breast cancer cell by blocking cell cycle progression and inducing apoptosis[J]. Food and Chemical Toxicology, 2006, (44):704-713.
[51]Kapral M, Strzalka-Mrozik B, Kowalczyk M, et al. Transcriptional activities of histone H3, cyclin D1 and claudin 7 encoding genes in laryngeal cancer[J]. Eur Arch Otorhinolaryngol,2011, 268(5):709-714.
[52]Musgrove EA, Caldon CE, Barraclough J, et al. Cyclin D as a therapeutic target in cancer [J]. Nat Rev Cancer,2011,11 (8):558-572.
[53]Wu L, Zhang W, Li H, et al. Inhibition of aortic intimal hyperplasia and cell cycle protein and extracellular matrix protein expressions by BuYang HuanWu Decoction[J]. J Ethnopharmacol,2009, 125(3):423-435
[54]Pietenpol JA, Stewart ZA. Cell cycle checkpoint signaling:cell cycle arrest versus apoptosis[J]. Toxicology,2002,181(182):475-481.
[55]Wang LM, Ren DM. Flavopiridol, the first cyclin-dependent kinase inhibitor:recent advances in combination chemotherapy [J]. Mini Rev Med Chem,2010,10(11):1058-1070.
[56]Gerard C, Goldbeter A. A skeleton model for the network of cyclin-dependent kinases driving the mammalian cell cycle[J]. Interface Focus,2011,1(1):24-35-
[57]Ilalina A, Artur P, Barbara MK, et al. Alterations in TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIPl expression associated with progression in B-CLL[J]. Folia Histochem Cytobiol,2010, 48(4):534-541.
[58]Tozawa-Ono A, Yoshida A, Yokomachi N, et al. Heat shock protein 27 and p16 immunohistochemistry in cervical intraepithelial neoplasia and squamous cell carcinoma[J]. Hum Cell,2012,25(1):24-28.
[59]Mahmut Yilmaz, Gerhard Christofori, Francois Lehembre. Distinct mechanism of tumor invasion and metastasis Trends [J].Molecular Medicine,2007,13(12):535-540.
[60]Lu XG, Zhan LB, FengBA, et al. Inhibition of growth and metastasis of human gastric cancer implanted in nudemice by delimonene[J]. World J Gastroenterol,2004,10(14):2140-2144.
[61]Bittoni A, Macearoni E, Scartozzi M, et al. Chemotherapy for locally advanced and metastatic gastric cancer:state of the art and future perpectives[J].Eur Rev Med Pharmacol Sci,2010,14(4):309-314.
[62]Gong J, Morishita A, Kurokohchi K, et al. Use of protein array to investigate receptor tyrosine kinases activated in gastric cancer [J].Int J Oncol.2010,36(1):101-106.
[63]Sahai E. Mechanisms of cancer cell invasion [J]. Curr Opin Genet Dev, 2005,15(1):87-96.
[64]Xu Gang, Wang Xing peng, Wu Kai, et al. Inhibition of invasive potential of pancreatic carcinoma cells by celecoxib in combination with gemcitabine[J]. Chin J Hepatobiliary Surg,2007,13(3):187-191.
[65]Waip Y, Kuo PC. Osteopontin:regulation in tumor metastasis [J]. Cancer Metastasis Rev,2008,27(1):103-118.
[66]Yingying X, Yong Z, Zhenning W, et al. Role of heparanase-1 in Gastric carcinoma invasion[J]. Asian Pac J Cancer Prey.2009,10(1): 151-154.
[67]Okayama H, Kumamoto K, Saitou K, et al. CD44v6, MMP-7 and nuclear Cdx2 are significant biomarkers for prediction of lymph node metastasis in primary gastric cancer[J]. Oncol Rep.2009,22 (4):745-755.
[68]Tian X, Cong M, Zhou W, et al. Relationship between protein expression of VEGF-C, MMP-2 and lymph node metastasis in papillary thyroid cancer[J].J Int Med Res,2008,36(4):699-703.
[69]Lee SJ, Bae SS, Kim KH, et al. High glucose enhances MMP-2 production in adventitial fibroblasts via Akt-dependent NF-kappaB pathway[J]. FEBS Lett,2007,581(22):4189-4194.
[70]Sampieri CL, Dela Pena S, Ochoa-Lara M, et al. Expression of matrix metalloproteinases 2 and 9 in human gastric cancer and superficial gastritis[J]. World J Gastroenterol,2010,16(12):1500-1505.
[71]Xu X, Wang Y, Lauer JL, et al. Contributions of the MMP-2 collagen binding domain to gelatin cleavage. Substrate binding via the collagen binding domain is required for hydrolysis of gelmin but not short peptides [J]. Matrix Biol,2004,23(3):171-181.
[72]Karadag A, Ogbureke KU, Fedarko NS, et al. Bone sialoprotein, matrix metallopro teinase 2 and alpha(v)beta3 integrin in osteotropic cancer cell invasion. [J]. Nail Cancer Inst,2004,96(12):956-965.
[73]Eren B, Sar M, Oz B, et al. MMP-2, TIMP-2 and CD44v6 expression in non-small cell lung carcinomas[J]. Ann Acad Med Singapore, 2008,37(1):32-39.
[74]Coussens LM, Tinkle CL, Hanahan D, et al. MMP supplied by bone marrow derived cells contributes to skin carcinogenesis[J]. Cell,2000, 103(3):481-490.
[75]Salerno M, Ouatas T, Palmieni D, et al. Inhibition of signal transduction by the Nm23 metastasis suppressor:possible mechanisms[J]. Clin Exp Metastasis,2003,20(1):3-10.
[76]Sema Biean, MD, Kedar V, et al. Nm23-H1 Expression in non-Hodgkin and Hodgkin Lymph-omas[J]. Appl Immunohistochem Mol Morphol,2008,16(3):207-210.
[77]Silvin B, Jean CM, Dana F, et al. Expression of Nm23-H1 in uveal melanoma[J]. Melanoma Research,2007,17(5):284-286.
[78]Gaszeri S, Brambilla E, Negoeseu, et al. Over expression of nueleoside diphos phate/kinase A/Nm23-H1 protein in human lung tumors:association with tumor progression in squamous careinoma[J]. Laboratory investigation,1996,74(1):158-167.
[79]Chen JQ, Zhan WH, He YL, et al. Expression of heparanase gene, CD44v6, MMP-7 and Nm23 protein and their relationship with the invasion and metastasis of gastric carcinomas[J]. World J Gastroenterol,2004,10(6):776-782.
[80]Huwer H, Kalweit G, Engel M, et al. Expression of the candidate tumor supression gene Nm23 in the bronchial system of patients with squamous cell lung cancer[J]. European journal of cardiothoracic surgery,1997,11(2):206-209.
[81]Liu YJ, Yah Ps, Li J, et al. Expreaaion and significance Of CD44s, CD44v6, and Nm23 mRNA in human cancer[J]. World J Gastroenterol, 2005,11(42):6601-6606.
[82]Katakura H, Tanaka F, Oyanagi H, et al. Clinical Significance of Nm23 expression in resected pathologic stage, rion small cell lung cancer[J]. The Annals of Thoracic Surgery,2002,73(4): 1060-1064.
[83]Wang LB, jiang ZN, Fan MY, et al. Changes ofhistology and pression of MMP-2 and Nm23-H1 in primary and metastatic gastric canccr[J]. WorldJ Gastroenterol,2008,14(10):1612-1616.
[84]Selfert M, Welle M, Mehraein Y, et al. Expression of the Nm23 homologues Nm23-H4, Nm23-H6, and Nm23-H7in human gastric and colon caneer[J]. J Pathol,2005,205(5):623-632.
[85]Marshall JC, Collins J, Marino N, et al. The Nm23-H1 metastasis supprossor as a translational target[J]. Eur J Cancer,2010,46(7):1278-1282.
[1]周珏.北豆根总碱的药学研究,提取工艺和指纹图谱的研究[D].哈尔滨：黑龙江中医药大学,2004.5
[2]李刚,岑东明.北豆根蝙蝠葛碱含量的测定方法[J].计算机光盘软件与应用,2010,1(2)：84-84.
[3]方立琼,郛济贤,郑立行.蝙蝠葛中药北豆根与山豆根质量评价[J].上海医科大学学报,1992,19(2)：124.
[4]刘瑞,马养民,张弘弛.蝙蝠葛茎叶化学成分的初步研究[J].西北林学院学报,2010,25(5)：176-178.
[5]龚宁,马养民,傅建熙.蝙蝠葛茎叶中化学成分的研究[J].陕西林业科技,2001,3(3)：8-10.
[6]龚宁,李玉平,刘拉平,等.蝙蝠葛果实脂肪酸和氨基酸成分分析[J].北基业学报,2006,15(4)：167-169.
[7]吴国新,李永兴,陈密玉,等.GC—MS法分析南瓜籽油脂肪酸组成[J].中草药,2003,34(12)：1079-1080.
[8]SHI Shao-jun, LI Zhong-fang, GU Shi-fen, et al. Pharmacokinetics and tissue of daurisoline in rabbits [J].Chin J Clin Pharmacol Ther,2005,10(6):622-626.
[9]SHI Shao-Jun, CHEN Hui, GU Shi-fen, et al. Pharmacokinetic-pharmacodynamic modeling of daurisoline in beagle dogs [J]. Acta Pharmacol Sin,2003,24(10):1011-1013.
[10]孔阳,马养民,余博,等.蝙蝠葛茎叶抗菌活性成分的研究[J].西北农林科技大学学报,2005,33(4)：151-153.
[11]刘瑞,马养民,张弘弛,等.蝙蝠葛茎叶正丁醇提取物抑菌作用研究[J].西北农业学报,2007,16(1)：77-81.
[12]Guo DL, Zhou ZN, Zeng FD, et al. Dauricine inhibited L-type calcium current in single cardiomyocyte of guinea pig. Acta Pharmacol Sin, 1997,18(5):418-421.
[13]Zeng GQ. Rui YC. Effect of anisodamine and daurieine on proliferation, DNA synthesis and calcium influx in bovine anterior cerebral arterial smooth muscle cells in culture. Acta Phar Sic,1991,12 (4): 308-311.
[14]杜佐华,曾晚成,龚培力,等.蝙蝠葛苏林碱抗实验性心律失常作用[J].中药药理与临床,1996(4)：2124.
[15]阎学斌,丁明,周利龙,等.蝙蝠葛碱抗离体家兔心房压增加所致机电反馈效应的研究[J].中国急救复苏与灾害医学杂志,2007,2(3)：142-144.
[16]冯克燕.蝙蝠葛碱治疗心律失常的临床研究[J].中华心血管病杂志.1984,12：265-267.
[17]龚培力,杜佐华.蝙蝠葛酚性碱的抗心律失常作用[J].中药新药与临床药理,1995,29(9)：647-651.
[18]苏云明,张树明,苏慧,等.蝙蝠葛酚性碱对垂体后叶素所致大鼠心肌缺血的保护作用[J].中医药学报,2004,32(2)：3-4.
[19]张长元,马建群,韩兆东,等.蝙蝠葛碱对兔心肌缺血再灌注时血清MDA浓度和SOD活性的影响[J].山东医药,2008,48(46)：25-27.
[20]黄晓东.蝙蝠葛碱对肾上腺素所致药物性高血压大鼠的影响研究[J].北华大学学报2007,8(1)：65-67.
[21]孙玉斌,关稍兰,金万宝.蝙蝠葛碱对麻醉犬心肌耗氧量的影响[J].中国医科大学学报,1995,24(3)：236-239.
[22]夏柳,柳强妮,龚培力.蝙蝠葛碱抗甲状腺素所致心肌肥厚的作用研究[J].医药导报,2006,25(11)：1109-1110.
[23]李艳红,黄向江,卢浩浩,等.蝙蝠葛碱对大鼠局灶性脑缺血及线粒体氧化损伤的保护作用[J].华中科技大学,2005,34(3)：270-272.
[24]张晓娟,罗宇芬,扬敏.蝙蝠葛酚性碱对大鼠局灶性脑缺血再灌注损伤的保护作用[J].中草药,2007,37(9)：1367-1369.
[25]孔祥英,龚培力.蝙蝠葛酚性碱对血栓形成和血小板聚集的影响[J].药学学报,2005,40(10)：916-919.
[26]孔祥英,杨晓燕,龚培力.蝙蝠葛酚性碱对血小板聚集的影响及其机制研 究[J].中国药学杂志,2006,41(12)：910-913.
[27]苏云明董培良,苏慧,等.蝙蝠葛酚性碱对“血瘀”模型大鼠凝血纤溶系统的影响[J].中医药学报,2004,(3)：3-3.
[28]田代华.实用中药辞典[M].第1版.北京：人民卫生出版社,2002.493,-.495.
[29]杨万山,孙抒,吕贺.MTT法检测蝙蝠葛活性成分的抑制肿瘤增殖作用[J].延边大学医学学报,2010,33(4)：261-263.
[30]苏云明,李宜航,刘坤,等.蝙蝠葛酚性碱对BXPC-3肿瘤抑制作用及肿瘤组织中MMP-2含量的影响[J].2010,5(2)：113-1 15.
[31]何志一,刘相辉,刚宏林.蝙蝠葛酚性碱诱导多药耐药细胞系K562/MDR1凋亡及逆转耐药性的研究[J].江苏大学学报,2010,20(2)：108-1 10.
[32]李铭,蔡文清,李颖,等.蝙蝠葛酚性碱抗突变作用的研究[J].第三军医大学学报,2006,28(7)：697-699.
[33]乔淑芬,顾美影,陈丹,等.蝙蝠葛不同提取液对三化螟的触杀活性[J].湖北农业科学,2009,48(1)：108-109.
[2]Zhengming Ge, Qiwen Ben,Junbo Qian, et al. Diabetes mellitus and risk of gastric cancer:a systematic review and meta-analysis of observational studies[J]. European Journal of Gastroenterology,2011,23(12):1127-130.
[3]朱金水.胃癌的治疗现状[J].中国临床医学,2004,11(2)：131-134.
[4]Lewis W G, Edwards P, Barry J D, et al. The gastrectomy question [J]. Gastric Cancer,2002,5(1):29-34.
[5]Lai, He X W, Jiang Y, et al. Preparative separation and determination of matrine from the Chinese Medicinal plant Sophora flavescents Ait by molecularly imprinted solid-phase extraction [J]. Anal Bioanal Chem, 2003,375(2):264-267.
[6]Guo DL, Zhou ZN, Zeng FD, et al. Dauricine inhibited L-type calcium current in single cardiomyocyte of guinea pig [J]. Acta Pharmacol Sin, 1997,18(5):418-421.
[7]Zeng GQ. Rui YC. Effect of anisodamine and daurieine on proliferation, DNA synthesis and calcium influx in bovine anterior cerebral arterial smooth muscle cells in culture [J]. Acta Phar Sic,1991,12(4):308-311.
[8]SHI Shao-jun, LI Zhong-fang, GU Shi-fen, et al. Pharmacokinetics and tissue of daurisoline in rabbits[J]. Chin J Clin Pharmacol Ther, 2005,10(6):622-626.
[9]SHI Shao-Jun, CHEN Hui, GU Shi-fen, et al. Pharmacokinetic pharmacodynamic modeling of daurisoline in beagle dogs[J]. Acta Pharmacol Sin,2003,24(10):1011-1013.
[10]杨万山,孙抒,吕贺.MTT法检测蝙蝠葛活性成分的抑制肿瘤增殖作用[J].延边大学医学学报,2010,33(4)：261-263.
[11]苏云明,李宜航,刘坤,等.蝙蝠葛酚性碱对BXPC-3肿瘤抑制作用及肿瘤组织中MMP-2含量的影响[J].2010,5(2)：113-115.
[12]SylvestreM, Legault J, Dufour D, et al. Chemical composition and anti-cancer activity of leaf essential oil of Myrica gale[J]. J Phytomedicine,2005,12(4):299-304.
[13]刘瑞,马养民,张弘弛.蝙蝠葛茎叶化学成分的初步研究[J].西北林学院学报,2010,25(5)：176-178.
[14]何志一,刘相辉,刚宏林.蝙蝠葛酚性碱诱导多药耐药细胞系K562/MDR1凋亡及逆转耐药性的研究[J].江苏大学学报,2010,20(2)：108-110.
[15]Park DJ, Lenz I-tJ. Determinants of chemosensitivity in gastric cancer [J]. Curr Opin Pharmaeol,2006,6:337-344.
[16]Moehler M, Galle PR, Gockel I, et al. The multidisciplinary management of gastrointestinal cancer. Multimodal treatment of gastric cancer [J]. Best Pratt Res Clin Gastroentrol,2007,21:965-981.
[17]周庚宴.组织病理学技术[M].北京：大学医学出版社,2006.149-152.
[18]孙宁.氧化苦参碱对人胃癌细胞裸鼠皮下移植瘤抑制作用的研究[D].郑州：河南中医学院.2007.
[19]王德娟,王栋.蝙蝠葛酚性碱药理作用研究进展[J].中医药学报,2008,36(5)：44-46.
[20]路艳艳,杨锦蓉,王菁,等.AO/EB染色法及流式细胞术检测DMF诱导人肝细胞凋亡[J].浙江预防医学,2007,19(6)：11-14.
[21]Wei X Y, Niu R F. The development history mechanism and application of Flow cytometer[J].Mod Instr,2006, (4):8-11.
[22]周庚宴.组织病理学技术[M].北京：大学医学出版社,2006.149-152.
[23]Mashimo K, Sato S, Adachi A, et al. Ethanol decreases cellular protein content and mitochondrial membrane potential of cultured neonatal rat cardiomyocytes:microassays with fluorometric and spectrometric plate readers[J]. Nihon Arukoru Yakubutsu Igakkai Zasshi,2010, 45(6):543-556.
[24]Pallis M, Grundy M, Turzan ski J, et al. Mitochondrial membrane sensitivity to depolarization in acute myeloblastic leukemia is associated with spontaneous in vitro apoptosis, wild type TP53 and vicinal thiol/disulfide status[J]. Blood,2001,98(2):405-413.
[25]Hu Y, Chen X, Duan H, et al. Chinese herbal medicinal ingredients inhibit secretion of IL-6, IL-8, E-selectin and TXB2 in LPS-induced rat intestinal micro vascular endothelial cells. Immunopharmacol Immunotoxicol,2009,31(4):550-555.
[26]Thangapasham RL, Singh AK, Sharma A, et al. Green tea poly-phenols and its constituent epigallocatechin gallate inhibits proliferation of human breast cancer cells in vitro and in vivo [J]. Cancer Letter, 2007,245(12):232-241.
[27]Hafez NH, Tahoun NS. Diagnostic value of p53 and ki67 immunostaining for distinguishing benign from malignant serous effusions[J]. J Egypt Natl Canc Inst,2011,23(4):155-162.
[28]Golmohammadi R, Pejhan A. The prognostic value of the P53 protein and the Ki67 marker in breast cancer patients[J]. J Pak Med Assoc, 2012,62(9):871-875.
[29]Jun Nian, ZHENG Yaf eng, SUN Dongsheng. Treatment with Vector-expressed Small Hairpin RNAs against Ki67 RNA-induced Cell Growth Inhibition and Apoptosis in Human Renal Carcinoma Cells[J]. Acta Biochimica et Biophysica Sinica,2006,38(4):254-261.
[30]Zhongchen, Liu Juan, Wang Ping. RGD-FasL Induces Apoptosis in hepatocellular Carcinoma [J]. Cellular and Molecular Immunology, 2009,6(4):285-293.
[31]Stephane Zalinski, Olivier Scatton, Bruto Randone, et al. Complete hepato cellular carcinoma necrosis following sequential porto-arterial embolization [J]. World Journal of Gastroenterology, 2008,14(44):6869-6872.
[32]Brooks C, Dong Z. Regulation of mitochondrial morphological dynamics during apoptosis by Bcl-2 family proteins:a key in Bak[J]. Cell Cycle, 2007,6(24):3043-3047.
[33]BhaUacharyya, Lahiry L, Mandal D, et al. Black tea induces rumor cell apoptosis by Bax translocation, loss in mitochondrial transmembrane potential cytochrome c release and caspase activation. Int J Cancer, 2005,117(2):308-315.
[34]Metrailler Ruchonnet I, Pagano A, Carnesecchi S, et al. Bcl-2 protects againsthy peroxia induced apoptosis through inhibition of themitochondria dependent pathway[J]. Free Radic Biol Med,2007,42(7):1062-7104.
[35]Chami M, Prandini A, Campanella M, et al. Bcl-2 and Bax exertopposing effects on Ca2+ signal, which do not depend on their putative poreforming region[J].J BiolChem,2004,279 (11): 54581-54589.
[36]Yasmine Nadler, Robert L Camp, Jennifer M Giltnane, et al. Expression patterns and prognostic value of Bag-1 and Bcl-2 in breast cancer [J]. Breast Cancer Research,2008,10(2):35-47.
[37]Jacquelyn M. Long, Charles W. Bell1 W, et al. Microarray and pathway analysis reveals decreased CDC25A and increased CDC42 associated with slow growth of Bcl-2 overexpressing immortalized breast cell line [J].Cell Cycle,2008,7(19):3062-3073.
[38]A Dhanasekaran, S Kotamraju, C Karunakaran, et al. Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis:role of mitochondrial superoxide[J]. Free Radic Biol Med,2005,39(5):567-583.
[39]C Du, M Fang, Y Li, et al. Smac a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition[J].Cell,2000,102(1):33-42.
[40]HK Lorenzo, SA Susin. Mitochondrial effectors in caspase-independent cell death[J]. FEBS Lett,2004,557(13):14-20.
[41]CH Ko, SC Shen, CS Hsu, et al. Mitochondrial-dependent, reactive oxygen species independent apoptosis by myricetin:roles of protein kinase C, cytochrome c, and caspase cascade [J]. Biochem Pharmacol, 2005,69(6):913-927.
[42]M van Gurp, N Festjens, Gvan Loo, et al. Mitochondrial intermembrane proteins in cell death[J]. Biochem Biophys Res,2003,304 (3):487-497.
[43]Larsen BD, Megeney LA. Parole terms for a killer:directing caspase3/CAD induced DNA strand breaks to coordinate changes in gene expression[J].Cell Cycle,2010,9(15):2940-2945.
[44]Padilla-Camberos E, Zaitseva G, Padilla C, et al. Anti-tumoral activity of allicin in murine lymphoma[J]. Asian Pac J Cancer Prev, 2010,11(5):1241-1244.
[45]Ming Sun, Yumei Zhao, Yi Gu, et al. Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke [J]. Neurochemistry International,2009, 54(11):339-346.
[46]Shihe Yang, Qiong Zhou, Xiaohe Yan. Caspase-3 status is a determinant of the differential responses to genistein between MDA-MB-231 and MCF-7 breast cancer cells[J]. Biochimical et Biophysica Acta,2007, (1773):903-911.
[47]Peek RM Jr, Wirth HP, Moss SF, et al. Helicobacter pylorialters Gastric epithelial cell cycle events and gastric secretion in m ongolian gerbils[J]. Gastroenterology,2000,118(1):48-59.
[48]Ke Xiu, Zhu Yali, Cao Bin. Changes of the cell cycle regulators and cell cycle arrest in cervical cancer cells after cisplatin therapy [J]. Academic Journal of Xian University,2009,21 (2):124-128.
[49]Zong Fang, Li Zhidong, Wang Yuan-yuan. Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract[J]. World Journal of Gastroenterology,2009, (36):4538-4546.
[50]Y. L. Hsu, P. L. Kuo, W. S. Tzeng, et al. Chalcone inhibits the proliferation of human breast cancer cell by blocking cell cycle progression and inducing apoptosis[J]. Food and Chemical Toxicology, 2006, (44):704-713.
[51]Kapral M, Strzalka-Mrozik B, Kowalczyk M, et al. Transcriptional activities of histone H3, cyclin D1 and claudin 7 encoding genes in laryngeal cancer[J]. Eur Arch Otorhinolaryngol,2011, 268(5):709-714.
[52]Musgrove EA, Caldon CE, Barraclough J, et al. Cyclin D as a therapeutic target in cancer [J]. Nat Rev Cancer,2011,11 (8):558-572.
[53]Wu L, Zhang W, Li H, et al. Inhibition of aortic intimal hyperplasia and cell cycle protein and extracellular matrix protein expressions by BuYang HuanWu Decoction[J]. J Ethnopharmacol,2009, 125(3):423-435
[54]Pietenpol JA, Stewart ZA. Cell cycle checkpoint signaling:cell cycle arrest versus apoptosis[J]. Toxicology,2002,181(182):475-481.
[55]Wang LM, Ren DM. Flavopiridol, the first cyclin-dependent kinase inhibitor:recent advances in combination chemotherapy [J]. Mini Rev Med Chem,2010,10(11):1058-1070.
[56]Gerard C, Goldbeter A. A skeleton model for the network of cyclin-dependent kinases driving the mammalian cell cycle[J]. Interface Focus,2011,1(1):24-35-
[57]Ilalina A, Artur P, Barbara MK, et al. Alterations in TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIPl expression associated with progression in B-CLL[J]. Folia Histochem Cytobiol,2010, 48(4):534-541.
[58]Tozawa-Ono A, Yoshida A, Yokomachi N, et al. Heat shock protein 27 and p16 immunohistochemistry in cervical intraepithelial neoplasia and squamous cell carcinoma[J]. Hum Cell,2012,25(1):24-28.
[59]Mahmut Yilmaz, Gerhard Christofori, Francois Lehembre. Distinct mechanism of tumor invasion and metastasis Trends [J].Molecular Medicine,2007,13(12):535-540.
[60]Lu XG, Zhan LB, FengBA, et al. Inhibition of growth and metastasis of human gastric cancer implanted in nudemice by delimonene[J]. World J Gastroenterol,2004,10(14):2140-2144.
[61]Bittoni A, Macearoni E, Scartozzi M, et al. Chemotherapy for locally advanced and metastatic gastric cancer:state of the art and future perpectives[J].Eur Rev Med Pharmacol Sci,2010,14(4):309-314.
[62]Gong J, Morishita A, Kurokohchi K, et al. Use of protein array to investigate receptor tyrosine kinases activated in gastric cancer [J].Int J Oncol.2010,36(1):101-106.
[63]Sahai E. Mechanisms of cancer cell invasion [J]. Curr Opin Genet Dev, 2005,15(1):87-96.
[64]Xu Gang, Wang Xing peng, Wu Kai, et al. Inhibition of invasive potential of pancreatic carcinoma cells by celecoxib in combination with gemcitabine[J]. Chin J Hepatobiliary Surg,2007,13(3):187-191.
[65]Waip Y, Kuo PC. Osteopontin:regulation in tumor metastasis [J]. Cancer Metastasis Rev,2008,27(1):103-118.
[66]Yingying X, Yong Z, Zhenning W, et al. Role of heparanase-1 in Gastric carcinoma invasion[J]. Asian Pac J Cancer Prey.2009,10(1): 151-154.
[67]Okayama H, Kumamoto K, Saitou K, et al. CD44v6, MMP-7 and nuclear Cdx2 are significant biomarkers for prediction of lymph node metastasis in primary gastric cancer[J]. Oncol Rep.2009,22 (4):745-755.
[68]Tian X, Cong M, Zhou W, et al. Relationship between protein expression of VEGF-C, MMP-2 and lymph node metastasis in papillary thyroid cancer[J].J Int Med Res,2008,36(4):699-703.
[69]Lee SJ, Bae SS, Kim KH, et al. High glucose enhances MMP-2 production in adventitial fibroblasts via Akt-dependent NF-kappaB pathway[J]. FEBS Lett,2007,581(22):4189-4194.
[70]Sampieri CL, Dela Pena S, Ochoa-Lara M, et al. Expression of matrix metalloproteinases 2 and 9 in human gastric cancer and superficial gastritis[J]. World J Gastroenterol,2010,16(12):1500-1505.
[71]Xu X, Wang Y, Lauer JL, et al. Contributions of the MMP-2 collagen binding domain to gelatin cleavage. Substrate binding via the collagen binding domain is required for hydrolysis of gelmin but not short peptides [J]. Matrix Biol,2004,23(3):171-181.
[72]Karadag A, Ogbureke KU, Fedarko NS, et al. Bone sialoprotein, matrix metallopro teinase 2 and alpha(v)beta3 integrin in osteotropic cancer cell invasion. [J]. Nail Cancer Inst,2004,96(12):956-965.
[73]Eren B, Sar M, Oz B, et al. MMP-2, TIMP-2 and CD44v6 expression in non-small cell lung carcinomas[J]. Ann Acad Med Singapore, 2008,37(1):32-39.
[74]Coussens LM, Tinkle CL, Hanahan D, et al. MMP supplied by bone marrow derived cells contributes to skin carcinogenesis[J]. Cell,2000, 103(3):481-490.
[75]Salerno M, Ouatas T, Palmieni D, et al. Inhibition of signal transduction by the Nm23 metastasis suppressor:possible mechanisms[J]. Clin Exp Metastasis,2003,20(1):3-10.
[76]Sema Biean, MD, Kedar V, et al. Nm23-H1 Expression in non-Hodgkin and Hodgkin Lymph-omas[J]. Appl Immunohistochem Mol Morphol,2008,16(3):207-210.
[77]Silvin B, Jean CM, Dana F, et al. Expression of Nm23-H1 in uveal melanoma[J]. Melanoma Research,2007,17(5):284-286.
[78]Gaszeri S, Brambilla E, Negoeseu, et al. Over expression of nueleoside diphos phate/kinase A/Nm23-H1 protein in human lung tumors:association with tumor progression in squamous careinoma[J]. Laboratory investigation,1996,74(1):158-167.
[79]Chen JQ, Zhan WH, He YL, et al. Expression of heparanase gene, CD44v6, MMP-7 and Nm23 protein and their relationship with the invasion and metastasis of gastric carcinomas[J]. World J Gastroenterol,2004,10(6):776-782.
[80]Huwer H, Kalweit G, Engel M, et al. Expression of the candidate tumor supression gene Nm23 in the bronchial system of patients with squamous cell lung cancer[J]. European journal of cardiothoracic surgery,1997,11(2):206-209.
[81]Liu YJ, Yah Ps, Li J, et al. Expreaaion and significance Of CD44s, CD44v6, and Nm23 mRNA in human cancer[J]. World J Gastroenterol, 2005,11(42):6601-6606.
[82]Katakura H, Tanaka F, Oyanagi H, et al. Clinical Significance of Nm23 expression in resected pathologic stage, rion small cell lung cancer[J]. The Annals of Thoracic Surgery,2002,73(4): 1060-1064.
[83]Wang LB, jiang ZN, Fan MY, et al. Changes ofhistology and pression of MMP-2 and Nm23-H1 in primary and metastatic gastric canccr[J]. WorldJ Gastroenterol,2008,14(10):1612-1616.
[84]Selfert M, Welle M, Mehraein Y, et al. Expression of the Nm23 homologues Nm23-H4, Nm23-H6, and Nm23-H7in human gastric and colon caneer[J]. J Pathol,2005,205(5):623-632.
[85]Marshall JC, Collins J, Marino N, et al. The Nm23-H1 metastasis supprossor as a translational target[J]. Eur J Cancer,2010,46(7):1278-1282.
[1]周珏.北豆根总碱的药学研究,提取工艺和指纹图谱的研究[D].哈尔滨：黑龙江中医药大学,2004.5
[2]李刚,岑东明.北豆根蝙蝠葛碱含量的测定方法[J].计算机光盘软件与应用,2010,1(2)：84-84.
[3]方立琼,郛济贤,郑立行.蝙蝠葛中药北豆根与山豆根质量评价[J].上海医科大学学报,1992,19(2)：124.
[4]刘瑞,马养民,张弘弛.蝙蝠葛茎叶化学成分的初步研究[J].西北林学院学报,2010,25(5)：176-178.
[5]龚宁,马养民,傅建熙.蝙蝠葛茎叶中化学成分的研究[J].陕西林业科技,2001,3(3)：8-10.
[6]龚宁,李玉平,刘拉平,等.蝙蝠葛果实脂肪酸和氨基酸成分分析[J].北基业学报,2006,15(4)：167-169.
[7]吴国新,李永兴,陈密玉,等.GC—MS法分析南瓜籽油脂肪酸组成[J].中草药,2003,34(12)：1079-1080.
[8]SHI Shao-jun, LI Zhong-fang, GU Shi-fen, et al. Pharmacokinetics and tissue of daurisoline in rabbits [J].Chin J Clin Pharmacol Ther,2005,10(6):622-626.
[9]SHI Shao-Jun, CHEN Hui, GU Shi-fen, et al. Pharmacokinetic-pharmacodynamic modeling of daurisoline in beagle dogs [J]. Acta Pharmacol Sin,2003,24(10):1011-1013.
[10]孔阳,马养民,余博,等.蝙蝠葛茎叶抗菌活性成分的研究[J].西北农林科技大学学报,2005,33(4)：151-153.
[11]刘瑞,马养民,张弘弛,等.蝙蝠葛茎叶正丁醇提取物抑菌作用研究[J].西北农业学报,2007,16(1)：77-81.
[12]Guo DL, Zhou ZN, Zeng FD, et al. Dauricine inhibited L-type calcium current in single cardiomyocyte of guinea pig. Acta Pharmacol Sin, 1997,18(5):418-421.
[13]Zeng GQ. Rui YC. Effect of anisodamine and daurieine on proliferation, DNA synthesis and calcium influx in bovine anterior cerebral arterial smooth muscle cells in culture. Acta Phar Sic,1991,12 (4): 308-311.
[14]杜佐华,曾晚成,龚培力,等.蝙蝠葛苏林碱抗实验性心律失常作用[J].中药药理与临床,1996(4)：2124.
[15]阎学斌,丁明,周利龙,等.蝙蝠葛碱抗离体家兔心房压增加所致机电反馈效应的研究[J].中国急救复苏与灾害医学杂志,2007,2(3)：142-144.
[16]冯克燕.蝙蝠葛碱治疗心律失常的临床研究[J].中华心血管病杂志.1984,12：265-267.
[17]龚培力,杜佐华.蝙蝠葛酚性碱的抗心律失常作用[J].中药新药与临床药理,1995,29(9)：647-651.
[18]苏云明,张树明,苏慧,等.蝙蝠葛酚性碱对垂体后叶素所致大鼠心肌缺血的保护作用[J].中医药学报,2004,32(2)：3-4.
[19]张长元,马建群,韩兆东,等.蝙蝠葛碱对兔心肌缺血再灌注时血清MDA浓度和SOD活性的影响[J].山东医药,2008,48(46)：25-27.
[20]黄晓东.蝙蝠葛碱对肾上腺素所致药物性高血压大鼠的影响研究[J].北华大学学报2007,8(1)：65-67.
[21]孙玉斌,关稍兰,金万宝.蝙蝠葛碱对麻醉犬心肌耗氧量的影响[J].中国医科大学学报,1995,24(3)：236-239.
[22]夏柳,柳强妮,龚培力.蝙蝠葛碱抗甲状腺素所致心肌肥厚的作用研究[J].医药导报,2006,25(11)：1109-1110.
[23]李艳红,黄向江,卢浩浩,等.蝙蝠葛碱对大鼠局灶性脑缺血及线粒体氧化损伤的保护作用[J].华中科技大学,2005,34(3)：270-272.
[24]张晓娟,罗宇芬,扬敏.蝙蝠葛酚性碱对大鼠局灶性脑缺血再灌注损伤的保护作用[J].中草药,2007,37(9)：1367-1369.
[25]孔祥英,龚培力.蝙蝠葛酚性碱对血栓形成和血小板聚集的影响[J].药学学报,2005,40(10)：916-919.
[26]孔祥英,杨晓燕,龚培力.蝙蝠葛酚性碱对血小板聚集的影响及其机制研 究[J].中国药学杂志,2006,41(12)：910-913.
[27]苏云明董培良,苏慧,等.蝙蝠葛酚性碱对“血瘀”模型大鼠凝血纤溶系统的影响[J].中医药学报,2004,(3)：3-3.
[28]田代华.实用中药辞典[M].第1版.北京：人民卫生出版社,2002.493,-.495.
[29]杨万山,孙抒,吕贺.MTT法检测蝙蝠葛活性成分的抑制肿瘤增殖作用[J].延边大学医学学报,2010,33(4)：261-263.
[30]苏云明,李宜航,刘坤,等.蝙蝠葛酚性碱对BXPC-3肿瘤抑制作用及肿瘤组织中MMP-2含量的影响[J].2010,5(2)：113-1 15.
[31]何志一,刘相辉,刚宏林.蝙蝠葛酚性碱诱导多药耐药细胞系K562/MDR1凋亡及逆转耐药性的研究[J].江苏大学学报,2010,20(2)：108-1 10.
[32]李铭,蔡文清,李颖,等.蝙蝠葛酚性碱抗突变作用的研究[J].第三军医大学学报,2006,28(7)：697-699.
[33]乔淑芬,顾美影,陈丹,等.蝙蝠葛不同提取液对三化螟的触杀活性[J].湖北农业科学,2009,48(1)：108-109.