九节龙皂苷-Ⅰ对胃癌生物学行为的影响及机制研究

发布时间：2018-06-03 00:56

本文选题：胃癌 + 九节龙皂苷-Ⅰ　；参考：《郑州大学》2016年博士论文

【摘要】：胃癌(gastric cancer)是最常见的恶性肿瘤之一,多种因素参与了该肿瘤的形成和发生,也涉及到了多种相关蛋白的表达和功能异常,具体的发病原因和机制目前还不清楚。虽然近年来胃癌的发病率在全世界范围内有所下降,但是在许多亚洲国家包括中国在内,胃癌的发病率仍居高不下。目前临床治疗胃癌的手段主要是手术、化疗及放疗等,尽管近年来基础和临床研究使胃癌的早期诊断及治疗效果取得很大进步,但提高胃癌患者的5年生存率仍是临床治疗的难题。由于胃癌早期症状不明显,患者被诊断时已是晚期或错过了最佳治疗机会。因此,寻找特异性、灵敏性高,毒副作用小的治疗药物尤为重要。天然药物具有副作用少、安全、廉价等优点,近年来在抗肿瘤研究中备受关注。九节龙皂苷(ardipusilloside,ADS)是从天然植物金牛科紫金牛属植物川产九节龙Ardisia pusilla中提取的一种天然化合物。大量研究证明,ADS具有抗肿瘤、降血压、抗病毒等多种生物学和药理学功能。越来越多的研究报道,九节龙皂苷-Ⅰ(ardipusilloside-Ⅰ,ADS-Ⅰ)对肺癌、肝癌、人脑胶质瘤、人子宫颈癌等多种肿瘤具有抑制作用,其抗肿瘤作用的主要机制有诱导细胞程序性死亡(PCD),诱导细胞自我吞噬,抑制肿瘤的侵袭和转移等。本研究首先观察了ADS-Ⅰ对人类胃癌TSGH、N87细胞增殖、侵袭、迁移及上皮间质转化(epithelial-mesenchymal transition,EMT)的影响,又进一步探索了ADS-Ⅰ对胃癌影响的分子机制,最后建立了胃癌N87细胞移植瘤,以进一步研究ADS-Ⅰ对胃癌移植瘤的影响及机制。本文最终确立了ADS-Ⅰ可能通过JAK/STAT3信号通路调控胃癌生长、侵袭、迁移及EMT,为胃癌的治疗提供了新的思路,为ADS-Ⅰ的抗肿瘤特性提供了新的依据。本研究共分以下三个部分。第一部分九节龙皂苷-Ⅰ对胃癌细胞增殖、侵袭、转移及上皮间质转化的影响方法1.利用MTT法检测不同浓度的ADS-I(10、20、40μM)处理胃癌TSGH、N87细胞24,48,72,96h后,对胃癌细胞增殖能力的影响,对照组为不含ADS-I的生理盐水。2.利用Transwell小室法检测不同浓度的ADS-I(10、20、40μM)处理胃癌TSGH、N87细胞24h后,对胃癌细胞侵袭能力的影响,主要是通过观察细胞穿透基质胶的数目判定胃癌细胞的侵袭能力。3.采用细胞划痕实验检测ADS-Ⅰ对胃癌TSGH、N87细胞迁移能力的影响,10、20及40μM的ADS-Ⅰ处理胃癌TSGH、N87细胞24h后,通过细胞的划痕距离的变化检测细胞的迁移能力。4.Western blot检测不同浓度ADS-Ⅰ(10、20、40μM)处理胃癌TSGH细胞24h后,通过EMT相关标记物E-钙粘蛋白和N-钙粘蛋白的表达来判断ADS-Ⅰ对胃癌细胞上皮间质转化的影响。结果1.MTT检测结果显示,不同浓度ADS-Ⅰ(10、20、40μM)处理,可以显著抑制胃癌TSGH、N87细胞的增殖。2.Transwell小室法检测细胞侵袭的结果显示,不同浓度ADS-Ⅰ(10、20、40μM)处理胃癌TSGH、N87细胞24h后,可以显著减少细胞穿透基质膜的数量。3.细胞划痕实验结果显示,不同浓度ADS-Ⅰ(10、20、40μM)处理胃癌TSGH、N87细胞24h后,可以显著抑制细胞的迁移能力。4.Western blot结果显示,不同浓度ADS-Ⅰ(10、20、40μM)处理胃癌TSGH细胞24h后,可以显著提高E-钙粘蛋白的表达,同时降低N-钙粘蛋白的表达,ADS-Ⅰ能够抑制胃癌的上皮细胞间质转化。第二部分九节龙皂苷-Ⅰ对胃癌细胞内JAK/STAT3信号通路的影响方法1.Western blot检测不同浓度的ADS-Ⅰ(10、20、40μM)处理胃癌TSGH细胞24h后,对细胞内p-JAK1、JAK1、p-JAK2、JAK2、p-STAT3及STAT3蛋白表达的影响。2.实时荧光定量PCR检测不同浓度的ADS-Ⅰ(10、20、40μM)处理胃癌TSGH细胞24h后,对细胞内Ki67、Bcl-2、MMP-9、Snail及Twist m RNA表达的影响。结果1.Western blot检测结果显示,ADS-Ⅰ可以抑制胃癌TSGH细胞内p-JAK1、p-JAK2及p-STAT3蛋白的表达,即ADS-Ⅰ可以抑制细胞内JAK/STAT3信号通路的活性。2.ADS-Ⅰ可以在m RNA水平上抑制Ki67、Bcl-2、MMP-9、Snail及Twist基因的表达。第三部分九节龙皂苷-Ⅰ对胃癌移植瘤的影响方法1.胃癌N87细胞裸鼠移植瘤模型的建立:采用BALB/c-nu裸鼠20只,每只裸鼠腋窝皮下注射200μL胃癌N87细胞(密度为3×106个/m L),待移植瘤体积大于100mm3时,将移植瘤模型分为对照组和处理组,对照组每日灌胃生理盐水200μL,处理组灌胃ADS-Ⅰ50mg/kg,每日1次,连续12天。2.每3日用游标卡尺测量移植瘤的最长径(a)和最短径(b)并观察肿瘤体积的变化,移植瘤体积的计算公式为:Tumor volume(mm3)=(the longest diameter)×(the shortest diameter)2/2。动物用药结束2天后,颈椎脱臼处死动物后剖取肿瘤块,称取每只裸鼠肿瘤块的重量,冷冻保存。3.Western blot检测对照组和ADS-Ⅰ处理组胃癌移植瘤中JAK/STAT3信号通路蛋白p-JAK1、JAK1、p-JAK2、JAK2、p-STAT3及STAT3蛋白的表达情况。4.Western blot检测对照组和ADS-Ⅰ处理组胃癌移植瘤中Ki67、Bcl-2、MMP-9、Snail及Twist蛋白表达情况。结果1.与对照组相比,使用50mg/kg的ADS-Ⅰ灌胃给药,显著抑制了胃癌N87细胞裸鼠移植瘤的生长。2.经过12天的ADS-Ⅰ灌胃给药,胃癌N87细胞裸鼠移植瘤的重量显著小于对照组。3.Western blot检测结果显示,ADS-Ⅰ显著抑制了胃癌N87细胞裸鼠移植瘤内p-JAK1、p-JAK2及p-STAT3蛋白的表达,阻断了JAK/STAT3信号通路。4.Western blot检测结果显示,ADS-Ⅰ显著抑制了胃癌N87细胞裸鼠移植瘤内Ki67、Bcl-2、MMP-9、Snail及Twist蛋白的表达。结论1、ADS-Ⅰ能够显著抑制胃癌细胞的增殖、侵袭、迁移及上皮间质转化。2、ADS-Ⅰ可能通过调控JAK/STAT3信号通路的活性来调控胃癌细胞的增殖、侵袭、迁移及细胞上皮间质转化等恶性生物学行为。3、ADS-Ⅰ能够抑制胃癌裸鼠移植瘤的生长,并阻断移植瘤中JAK/STAT3信号通路。
[Abstract]:Gastric cancer (gastric cancer) is one of the most common malignant tumors. A variety of factors have been involved in the formation and occurrence of the tumor, also involved in the expression and functional abnormalities of a variety of related proteins. The specific pathogenesis and mechanism are not yet clear. Although the incidence of gastric cancer has declined in the world in recent years, it is in many subtypes. The incidence of gastric cancer is still high in the state of China including China. The main means of clinical treatment for gastric cancer are surgery, chemotherapy and radiotherapy. Although basic and clinical studies have made great progress in the early diagnosis and treatment of gastric cancer, the 5 year survival rate of gastric cancer patients is still a difficult problem in clinical treatment. The early symptoms of cancer are not obvious, and the patients have been diagnosed at the advanced stage or missed the best treatment. Therefore, it is particularly important to find specific, high sensitivity, and small side effects. Natural drugs have the advantages of less side effects, safety and low cost. In recent years, nine ardipusilloside (ADS) has been paid much attention to. It is a natural compound extracted from the nine dragon Ardisia pusilla produced in the natural plant of the genus Taurus. A large number of studies have shown that ADS has many biological and pharmacological functions, such as anti-tumor, antihypertensive, antiviral, and so on. More and more studies have been reported that nine dragon saponins - I (ardipusilloside- I, ADS- I) are used for lung cancer, liver cancer, and human beings A variety of tumors, such as brain glioma and human cervical cancer, have inhibitory effects. The main mechanisms of its anti-tumor effect are inducing programmed cell death (PCD), inducing cell self phagocytosis, inhibiting tumor invasion and metastasis. This study first observed the effect of ADS- I on human gastric cancer TSGH, N87 cell proliferation, invasion, migration and epithelial transformation (Epithel The influence of ial-mesenchymal transition, EMT) further explored the molecular mechanism of ADS- I influence on gastric cancer, and finally established the N87 cell xenografts for gastric cancer in order to further study the effect and mechanism of ADS- I on the cancer of gastric cancer. This article finally established that ADS- I may regulate the growth, invasion, migration and E of gastric cancer through JAK /STAT3 signaling pathway. MT provides new ideas for the treatment of gastric cancer and provides a new basis for the anti-tumor properties of ADS- I. This study is divided into three parts. The first part of this study, part nine, the effects of saponins - I on the proliferation, invasion, metastasis and epithelial transformation of gastric cancer cells. 1. ADS-I (10,20,40 mu M) of different concentrations was used to detect TSGH in gastric cancer by MTT method. The effect of N87 cell 24,48,72,96h on the proliferation of gastric cancer cells, the control group was a saline.2. without ADS-I, using Transwell chamber method to detect the ADS-I (10,20,40 mu M) of different concentrations (10,20,40 mu M) to treat gastric cancer TSGH, and the effect of N87 cell 24h on the invasiveness of gastric cancer cells was mainly determined by the observation of the number of cells penetrating matrix glue to determine gastric cancer. Cell invasiveness.3. was used to detect the effect of ADS- I on the migration ability of TSGH and N87 cells in gastric cancer. 10,20 and ADS- I of 40 mu M were used to treat gastric cancer TSGH, N87 cell 24h, and the cell migration ability was detected by the change of scratch distance. After cell 24h, the expression of EMT related markers E- cadherin and N- cadherin was used to determine the effect of ADS- I on the epithelial mesenchymal transition of gastric cancer cells. Results 1.MTT detection results showed that different concentrations of ADS- I (10,20,40 u M) can significantly inhibit the gastric cancer TSGH, N87 cell proliferation.2.Transwell cell method detection of cell invasion results. The results showed that different concentrations of ADS- I (10,20,40 M) treatment of gastric cancer TSGH, N87 cells 24h, can significantly reduce the number of.3. cells in the cell penetrating matrix membrane, the results show that the different concentrations ADS- I (10,20,40 mu M) treatment of gastric cancer TSGH, N87 cells can significantly inhibit cell migration ability DS- I (10,20,40 M) treatment of gastric cancer TSGH cell 24h can significantly increase the expression of E- cadherin and decrease the expression of N- cadherin. ADS- I can inhibit the epithelial mesenchymal transition of gastric cancer. Second part nine the effect of saponin I on the JAK/STAT3 signaling pathway in gastric cancer cells 1.Western blot detection of different concentrations Effects of ADS- I (10,20,40 mu M) on the expression of p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3 and STAT3 protein in gastric cancer TSGH cells. The results show that ADS- I can inhibit the expression of p-JAK1, p-JAK2 and p-STAT3 protein in gastric cancer TSGH cells. That is, ADS- I can inhibit the activity of JAK/STAT3 signaling pathway in the cell, which can inhibit Ki67, Bcl-2, and the expression of the gene in M RNA level. The third part of the nine segment saponins - I on gastric cancer xenografts 1. nude mice transplantation tumor model of gastric cancer N87 cells was established: 20 nude mice were used in nude mice and 200 L gastric cancer N87 cells were injected subcutaneously in each nude mouse (3 x 106 /m L). The transplanted tumor model was divided into control group and treatment group when the volume of the transplanted tumor was greater than 100mm3. The control group was treated with gastric saline 200 u L daily, and the treatment group had gastric perfusion ADS-. 1 times a day, 1 times a day, 12 days for 12 days, the longest diameter (a) and the shortest path (b) of the transplanted tumor were measured with the vernier caliper every 3 days and the volume of the tumor was observed. The calculation formula of the volume of the transplanted tumor was Tumor volume (mm3) = (the longest diameter) * (the shortest) 2 days after the end of the animal drug use. The weight of tumor block in each nude mouse was called, and the expression of JAK/STAT3 signal pathway protein p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3 and STAT3 protein in the cancer transplanted tumor of the control group and the ADS- I treatment group were frozen and stored in the control group and the ADS- I treatment group. The expression of Twist protein. Results 1. compared with the control group, the use of 50mg/kg ADS- I intragastric administration significantly inhibited the growth of.2. in nude mice of gastric cancer after 12 days of ADS- I administration of ADS- I. The weight of nude mice transplanted tumor of gastric cancer was significantly less than that of the control group.3.Western blot detection results showed that ADS- I was significantly inhibited. The expression of p-JAK1, p-JAK2 and p-STAT3 protein in the transplanted tumor of gastric cancer N87 cells blocked the JAK/STAT3 signal pathway.4.Western blot detection results, and ADS- I significantly inhibited the Ki67, Bcl-2, MMP-9, and protein expression in the xenografts of gastric cancer N87 cells in nude mice. Conclusion 1, it can significantly inhibit the proliferation and invasion of gastric cancer cells. Migration and epithelial mesenchymal transformation.2, ADS- I may regulate the proliferation, invasion, migration and epithelial mesenchymal transition of gastric cancer cells by regulating the activity of JAK/STAT3 signaling pathway,.3. ADS- I can inhibit the growth of xenografts in nude mice and block the JAK/STAT3 signaling pathway in the transplanted tumor.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

【参考文献】