本文選題：宮頸癌 + 藍(lán)萼乙素�。� 參考：《新鄉(xiāng)醫(yī)學(xué)院》2017年碩士論文

【摘要】：背景女性患者生殖系統(tǒng)惡性腫瘤的發(fā)病率逐年攀升,其中占第一位的是子宮頸癌[1]。研究表明人類乳頭狀瘤病毒(Human Papilloma Virus,HPV)感染是罹患子宮頸癌的高危因素,而中科院附屬腫瘤研究所流調(diào)顯示在我國城市和農(nóng)村地區(qū)HPV病毒攜帶率分別占其人口總數(shù)的15.2%和14.6%,這意味著我國對宮頸癌的防治應(yīng)引起足夠重視[2]。目前宮頸癌的治療方案主要依靠手術(shù)和放化療,而手術(shù)的創(chuàng)傷性、放射治療對機(jī)體造成的永久性損害、化療藥物的毒副反應(yīng)局限了其應(yīng)用范圍。近年來由于我國傳統(tǒng)醫(yī)學(xué)的不斷發(fā)展,中藥治療以療效高及毒副作用少等為特點(diǎn)日漸成為腫瘤研究的熱點(diǎn)之一[39];藍(lán)萼香茶菜屬于唇形科植物,在我國民間很常見,已有研究表明其對多種腫瘤細(xì)胞的生長均具有抑制作用[40];其活性成分主要是二萜類和黃酮類,藍(lán)萼甲素(Glaucocalyxin A,GLA)和藍(lán)萼乙素(Glaucocalyxin B,GLB)是從香茶菜中提取的二萜化合物。研究表明GLB對多種腫瘤細(xì)胞生長具有抑制作用[7],例如:對肺腺癌AGZY細(xì)胞、HL-60白血病細(xì)胞、人視網(wǎng)膜母細(xì)胞瘤Rb細(xì)胞的抑制作用等[15-17];本課題組前期研究表明GLB可以抑制宮頸癌HeLa和SiHa細(xì)胞的增殖,而動物實(shí)驗研究尚未有相關(guān)報道。目的在裸鼠皮下構(gòu)建子宮頸癌HeLa及SiHa細(xì)胞的體內(nèi)移植瘤,探討不同濃度的藍(lán)萼乙素對宮頸癌裸鼠體內(nèi)移植瘤的抑制作用及機(jī)理,為其應(yīng)用于臨床提供理論基礎(chǔ)。方法將體外培養(yǎng)的Hela、SiHa細(xì)胞分別移植至裸鼠右大腿根部背側(cè)皮下,建立兩種裸鼠移植瘤模型;待移植瘤體積約為0.1cm3時,于第10天將SiHa組隨機(jī)分為4組,于第14天將Hela組分為4組,分別是生理鹽水組、10%乙醇溶劑組、GLB低劑量組和高劑量組,每組5只;經(jīng)腹腔注射給藥,HeLa組給予生理鹽水0.4ml/只、10%乙醇0.4ml/只、GLB0.15umol/g 0.4ml/只和0.20umol/g 0.4ml/只,隔日1次,共20天;SiHa組給予生理鹽水0.4ml/只、10%乙醇0.4ml/只、GLB0.18umol/g 0.4ml/只和0.24umol/g 0.4ml/只,隔日1次,共20天。腹腔注射給藥的同時,隔天用標(biāo)準(zhǔn)體重秤稱量體重并做好記錄,每隔2天用游標(biāo)卡尺精確測量移植瘤的最大短徑和長徑,至少測量兩次取平均值。給藥結(jié)束后處死裸鼠前,眼球取血,測生化指標(biāo)和腫瘤標(biāo)志物,觀察各組模型鼠生化指標(biāo)和腫瘤標(biāo)志物的變化;進(jìn)行移植瘤的體積生長抑制試驗觀察各組移植瘤體積隨時間的變化,組織形態(tài)學(xué)上表現(xiàn)為組織病理切片后的HE染色的變化[13];免疫組化法移植瘤組織中張力蛋白同源在10號染色體有缺失的磷酸酶基因(Phosphatase and tensin ho-molog deleted from chromosome 10,PTEN)、自噬關(guān)鍵調(diào)控因子Beclin1,微管相關(guān)蛋白3(Microtubule associated protein I light chain 3,LC-3)的表達(dá)變化,Western-blot法進(jìn)一步檢驗相關(guān)蛋白變化。移植瘤生長曲線使用Graphpad軟件繪制,各組間瘤重、處理前后瘤體積變化、腫瘤標(biāo)記物等計量數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(sx±)表示,應(yīng)用SPSS19.0統(tǒng)計軟件分析,多組間比較采用單因素方差分析,差異有統(tǒng)計學(xué)意義的組間兩兩比較采用LSD法進(jìn)行比較,以P0.05為差異有統(tǒng)計學(xué)意義。結(jié)果1.裸鼠成瘤率為100%;裸鼠處死后,GLB處理組腫瘤標(biāo)志物較對照組(生理鹽水及10%乙醇組)降低,差異有統(tǒng)計學(xué)意義(P0.05);GLB處理組和對照組生化指標(biāo)比較差異無統(tǒng)計學(xué)意義(P0.05);GLB處理組的移植瘤體積均明顯小于對照組,差異有統(tǒng)計學(xué)意義(P0.05)。2.HE染色顯示:GLB處理組移植瘤組織出現(xiàn)凋亡、壞死改變;免疫組化法、Western-blot法結(jié)果示:GLB處理組的腫瘤組織PTEN、Beclin 1蛋白表達(dá)水平及LC3II/I比例較對照組明顯升高。結(jié)論1.藍(lán)萼乙素可以抑制人宮頸癌HeLa、SiHa細(xì)胞裸鼠體內(nèi)皮下移植瘤的生長,具有濃度依賴性;藍(lán)萼乙素可明顯降低實(shí)驗動物的腫瘤標(biāo)志物,具有濃度依賴性;藍(lán)萼乙素在一定劑量范圍內(nèi)可誘導(dǎo)裸鼠體內(nèi)移植瘤細(xì)胞發(fā)生自噬、凋亡。2.藍(lán)萼乙素抑制宮頸癌細(xì)胞生長的作用可能與上調(diào)PTEN和Beclin1及LC3蛋白的表達(dá)有關(guān)。
[Abstract]:The incidence of reproductive system malignant tumors in female patients is increasing year by year. The first of them is the [1]. study of cervical cancer. The Human Papilloma Virus (HPV) infection is a high risk factor for cervical cancer, and the Institute of cancer research affiliated to the Academy of Chinese Academy of Sciences shows the HPV virus in the urban and rural areas of China The carrying rate is 15.2% and 14.6% of the total number of the population, which means that the prevention and treatment of cervical cancer in China should pay enough attention to the treatment of [2]. cervical cancer mainly by surgery and radiotherapy and chemotherapy, and the trauma of the operation, the permanent damage to the body caused by radiation therapy, and the side effects of the chemotherapeutic drugs limited the scope of its application. Due to the continuous development of traditional medicine in China, traditional Chinese medicine has become one of the hotspots of cancer research with the characteristics of high curative effect and less toxic and side effects, which is one of the hot spots of cancer research [39]. Blue calyx incense tea is a lip plant. It is very common in the folk of our country. It has been studied that it has inhibitory effect on the growth of various tumor cells and its active components are [40]. Mainly two terpenoids and flavonoids, Glaucocalyxin A (GLA) and Glaucocalyxin B (GLB) are two terpenoids extracted from fragrant tea. Studies have shown that GLB inhibits the growth of various tumor cells, such as the inhibition of AGZY cells in lung adenocarcinoma, HL-60 leukemic cells, and Rb cells of human retinoblastoma. Effect of [15-17], and so on. The previous study showed that GLB could inhibit the proliferation of HeLa and SiHa cells in cervical cancer, but the animal experimental study had not been reported. Objective to construct the transplanted tumor of HeLa and SiHa cells of cervical cancer in nude mice and to explore the inhibitory effect of different concentrations of blue calyx on the transplanted tumor in nude mice of cervical cancer. The Hela and SiHa cells cultured in vitro were transplanted into the dorsal root of the right thigh of nude mice, and two nude mice transplanted tumor model was established. When the volume of the transplanted tumor was about 0.1cm3, the SiHa group was randomly divided into 4 groups on the tenth day, and the Hela group was divided into 4 groups at fourteenth days, respectively, the physiological saline. Group, 10% ethanol solvent group, GLB low dose group and high dose group, 5 rats in each group; HeLa group was given saline 0.4ml/ only, 10% ethanol 0.4ml/ only, GLB0.15umol/g 0.4ml/ and 0.20umol/g 0.4ml/ only, 1 times a day for 20 days, and SiHa group was given 0.4ml/ only, 10% ethanol 0.4ml/, GLB0.18umol/g 0.4ml/ only 1 times a day, a total of 20 days. At the same time of intraperitoneal injection, the body weight was weighed and recorded every other day. The maximum diameter and length of the transplanted tumor were measured with a vernier caliper every 2 days, at least two times to take the average. The changes in biochemical indexes and tumor markers of the model rats were observed. The volume growth inhibition test of the transplanted tumor was conducted to observe the changes of the volume of the transplanted tumor with time, and the morphological changes of the HE staining were [13] after the histopathological section. Enzyme gene (Phosphatase and tensin ho-molog deleted from chromosome 10, PTEN), the key regulator of autophagy, Beclin1, microtubule related protein 3 (Microtubule associated protein) expression changes. The tumor weight, the volume change of the tumor before and after treatment, the measurement data such as tumor markers were expressed with mean standard deviation (SX +), and the SPSS19.0 statistical software was used to analyze the data, and the single factor variance analysis was used in the multiple groups. The difference between the 22 groups was compared with the LSD method, and the difference was statistically significant with the difference of P0.05. The results were 1. nude mice. The rate of tumor formation was 100%. After the death of nude mice, the tumor markers in the GLB treatment group were lower than the control group (normal saline and 10% ethanol group), the difference was statistically significant (P0.05), and there was no statistical difference between the GLB treatment group and the control group (P0.05), and the volume of the transplanted tumor in the GLB treatment group was significantly smaller than that in the control group, and the difference was statistically significant (P0.05 .2.HE staining showed that the transplanted tumor tissue in the GLB treatment group appeared apoptosis and necrosis, and the immunohistochemical method and Western-blot method showed that the PTEN, Beclin 1 protein expression level and LC3II/I ratio in the GLB treatment group were significantly higher than those of the control group. Conclusion 1. blue calyx can inhibit the human cervical cancer HeLa, and the subcutaneous transplantation tumor of the SiHa cell nude mice The growth and concentration dependence; blue calyx can obviously reduce the tumor markers of experimental animals, and it has a concentration dependence. In a certain dose, blue calyx can induce autophagy in transplanted tumor cells in nude mice. The effect of apoptotic.2. blue calyx on the growth of cervical cancer cells may be associated with up regulation of PTEN and Beclin1 and LC3 protein. The expression is related.

【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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