本文選題：胃癌 + 九節(jié)龍皂苷-Ⅰ�。� 參考：《鄭州大學》2016年博士論文

【摘要】：胃癌(gastric cancer)是最常見的惡性腫瘤之一,多種因素參與了該腫瘤的形成和發(fā)生,也涉及到了多種相關(guān)蛋白的表達和功能異常,具體的發(fā)病原因和機制目前還不清楚。雖然近年來胃癌的發(fā)病率在全世界范圍內(nèi)有所下降,但是在許多亞洲國家包括中國在內(nèi),胃癌的發(fā)病率仍居高不下。目前臨床治療胃癌的手段主要是手術(shù)、化療及放療等,盡管近年來基礎(chǔ)和臨床研究使胃癌的早期診斷及治療效果取得很大進步,但提高胃癌患者的5年生存率仍是臨床治療的難題。由于胃癌早期癥狀不明顯,患者被診斷時已是晚期或錯過了最佳治療機會。因此,尋找特異性、靈敏性高,毒副作用小的治療藥物尤為重要。天然藥物具有副作用少、安全、廉價等優(yōu)點,近年來在抗腫瘤研究中備受關(guān)注。九節(jié)龍皂苷(ardipusilloside,ADS)是從天然植物金牛科紫金牛屬植物川產(chǎn)九節(jié)龍Ardisia pusilla中提取的一種天然化合物。大量研究證明,ADS具有抗腫瘤、降血壓、抗病毒等多種生物學和藥理學功能。越來越多的研究報道,九節(jié)龍皂苷-Ⅰ(ardipusilloside-Ⅰ,ADS-Ⅰ)對肺癌、肝癌、人腦膠質(zhì)瘤、人子宮頸癌等多種腫瘤具有抑制作用,其抗腫瘤作用的主要機制有誘導細胞程序性死亡(PCD),誘導細胞自我吞噬,抑制腫瘤的侵襲和轉(zhuǎn)移等。本研究首先觀察了ADS-Ⅰ對人類胃癌TSGH、N87細胞增殖、侵襲、遷移及上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)的影響,又進一步探索了ADS-Ⅰ對胃癌影響的分子機制,最后建立了胃癌N87細胞移植瘤,以進一步研究ADS-Ⅰ對胃癌移植瘤的影響及機制。本文最終確立了ADS-Ⅰ可能通過JAK/STAT3信號通路調(diào)控胃癌生長、侵襲、遷移及EMT,為胃癌的治療提供了新的思路,為ADS-Ⅰ的抗腫瘤特性提供了新的依據(jù)。本研究共分以下三個部分。第一部分九節(jié)龍皂苷-Ⅰ對胃癌細胞增殖、侵襲、轉(zhuǎn)移及上皮間質(zhì)轉(zhuǎn)化的影響方法1.利用MTT法檢測不同濃度的ADS-I(10、20、40μM)處理胃癌TSGH、N87細胞24,48,72,96h后,對胃癌細胞增殖能力的影響,對照組為不含ADS-I的生理鹽水。2.利用Transwell小室法檢測不同濃度的ADS-I(10、20、40μM)處理胃癌TSGH、N87細胞24h后,對胃癌細胞侵襲能力的影響,主要是通過觀察細胞穿透基質(zhì)膠的數(shù)目判定胃癌細胞的侵襲能力。3.采用細胞劃痕實驗檢測ADS-Ⅰ對胃癌TSGH、N87細胞遷移能力的影響,10、20及40μM的ADS-Ⅰ處理胃癌TSGH、N87細胞24h后,通過細胞的劃痕距離的變化檢測細胞的遷移能力。4.Western blot檢測不同濃度ADS-Ⅰ(10、20、40μM)處理胃癌TSGH細胞24h后,通過EMT相關(guān)標記物E-鈣粘蛋白和N-鈣粘蛋白的表達來判斷ADS-Ⅰ對胃癌細胞上皮間質(zhì)轉(zhuǎn)化的影響。結(jié)果1.MTT檢測結(jié)果顯示,不同濃度ADS-Ⅰ(10、20、40μM)處理,可以顯著抑制胃癌TSGH、N87細胞的增殖。2.Transwell小室法檢測細胞侵襲的結(jié)果顯示,不同濃度ADS-Ⅰ(10、20、40μM)處理胃癌TSGH、N87細胞24h后,可以顯著減少細胞穿透基質(zhì)膜的數(shù)量。3.細胞劃痕實驗結(jié)果顯示,不同濃度ADS-Ⅰ(10、20、40μM)處理胃癌TSGH、N87細胞24h后,可以顯著抑制細胞的遷移能力。4.Western blot結(jié)果顯示,不同濃度ADS-Ⅰ(10、20、40μM)處理胃癌TSGH細胞24h后,可以顯著提高E-鈣粘蛋白的表達,同時降低N-鈣粘蛋白的表達,ADS-Ⅰ能夠抑制胃癌的上皮細胞間質(zhì)轉(zhuǎn)化。第二部分九節(jié)龍皂苷-Ⅰ對胃癌細胞內(nèi)JAK/STAT3信號通路的影響方法1.Western blot檢測不同濃度的ADS-Ⅰ(10、20、40μM)處理胃癌TSGH細胞24h后,對細胞內(nèi)p-JAK1、JAK1、p-JAK2、JAK2、p-STAT3及STAT3蛋白表達的影響。2.實時熒光定量PCR檢測不同濃度的ADS-Ⅰ(10、20、40μM)處理胃癌TSGH細胞24h后,對細胞內(nèi)Ki67、Bcl-2、MMP-9、Snail及Twist m RNA表達的影響。結(jié)果1.Western blot檢測結(jié)果顯示,ADS-Ⅰ可以抑制胃癌TSGH細胞內(nèi)p-JAK1、p-JAK2及p-STAT3蛋白的表達,即ADS-Ⅰ可以抑制細胞內(nèi)JAK/STAT3信號通路的活性。2.ADS-Ⅰ可以在m RNA水平上抑制Ki67、Bcl-2、MMP-9、Snail及Twist基因的表達。第三部分九節(jié)龍皂苷-Ⅰ對胃癌移植瘤的影響方法1.胃癌N87細胞裸鼠移植瘤模型的建立:采用BALB/c-nu裸鼠20只,每只裸鼠腋窩皮下注射200μL胃癌N87細胞(密度為3×106個/m L),待移植瘤體積大于100mm3時,將移植瘤模型分為對照組和處理組,對照組每日灌胃生理鹽水200μL,處理組灌胃ADS-Ⅰ50mg/kg,每日1次,連續(xù)12天。2.每3日用游標卡尺測量移植瘤的最長徑(a)和最短徑(b)并觀察腫瘤體積的變化,移植瘤體積的計算公式為:Tumor volume(mm3)=(the longest diameter)×(the shortest diameter)2/2。動物用藥結(jié)束2天后,頸椎脫臼處死動物后剖取腫瘤塊,稱取每只裸鼠腫瘤塊的重量,冷凍保存。3.Western blot檢測對照組和ADS-Ⅰ處理組胃癌移植瘤中JAK/STAT3信號通路蛋白p-JAK1、JAK1、p-JAK2、JAK2、p-STAT3及STAT3蛋白的表達情況。4.Western blot檢測對照組和ADS-Ⅰ處理組胃癌移植瘤中Ki67、Bcl-2、MMP-9、Snail及Twist蛋白表達情況。結(jié)果1.與對照組相比,使用50mg/kg的ADS-Ⅰ灌胃給藥,顯著抑制了胃癌N87細胞裸鼠移植瘤的生長。2.經(jīng)過12天的ADS-Ⅰ灌胃給藥,胃癌N87細胞裸鼠移植瘤的重量顯著小于對照組。3.Western blot檢測結(jié)果顯示,ADS-Ⅰ顯著抑制了胃癌N87細胞裸鼠移植瘤內(nèi)p-JAK1、p-JAK2及p-STAT3蛋白的表達,阻斷了JAK/STAT3信號通路。4.Western blot檢測結(jié)果顯示,ADS-Ⅰ顯著抑制了胃癌N87細胞裸鼠移植瘤內(nèi)Ki67、Bcl-2、MMP-9、Snail及Twist蛋白的表達。結(jié)論1、ADS-Ⅰ能夠顯著抑制胃癌細胞的增殖、侵襲、遷移及上皮間質(zhì)轉(zhuǎn)化。2、ADS-Ⅰ可能通過調(diào)控JAK/STAT3信號通路的活性來調(diào)控胃癌細胞的增殖、侵襲、遷移及細胞上皮間質(zhì)轉(zhuǎn)化等惡性生物學行為。3、ADS-Ⅰ能夠抑制胃癌裸鼠移植瘤的生長,并阻斷移植瘤中JAK/STAT3信號通路。
[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.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.2

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本文編號：1970843