蓝萼乙素对人宫颈癌裸鼠移植瘤抑制作用的研究

发布时间：2018-04-26 09:51

本文选题：宫颈癌 + 蓝萼乙素　；参考：《新乡医学院》2017年硕士论文

【摘要】：背景女性患者生殖系统恶性肿瘤的发病率逐年攀升,其中占第一位的是子宫颈癌[1]。研究表明人类乳头状瘤病毒(Human Papilloma Virus,HPV)感染是罹患子宫颈癌的高危因素,而中科院附属肿瘤研究所流调显示在我国城市和农村地区HPV病毒携带率分别占其人口总数的15.2%和14.6%,这意味着我国对宫颈癌的防治应引起足够重视[2]。目前宫颈癌的治疗方案主要依靠手术和放化疗,而手术的创伤性、放射治疗对机体造成的永久性损害、化疗药物的毒副反应局限了其应用范围。近年来由于我国传统医学的不断发展,中药治疗以疗效高及毒副作用少等为特点日渐成为肿瘤研究的热点之一[39];蓝萼香茶菜属于唇形科植物,在我国民间很常见,已有研究表明其对多种肿瘤细胞的生长均具有抑制作用[40];其活性成分主要是二萜类和黄酮类,蓝萼甲素(Glaucocalyxin A,GLA)和蓝萼乙素(Glaucocalyxin B,GLB)是从香茶菜中提取的二萜化合物。研究表明GLB对多种肿瘤细胞生长具有抑制作用[7],例如:对肺腺癌AGZY细胞、HL-60白血病细胞、人视网膜母细胞瘤Rb细胞的抑制作用等[15-17];本课题组前期研究表明GLB可以抑制宫颈癌HeLa和SiHa细胞的增殖,而动物实验研究尚未有相关报道。目的在裸鼠皮下构建子宫颈癌HeLa及SiHa细胞的体内移植瘤,探讨不同浓度的蓝萼乙素对宫颈癌裸鼠体内移植瘤的抑制作用及机理,为其应用于临床提供理论基础。方法将体外培养的Hela、SiHa细胞分别移植至裸鼠右大腿根部背侧皮下,建立两种裸鼠移植瘤模型;待移植瘤体积约为0.1cm3时,于第10天将SiHa组随机分为4组,于第14天将Hela组分为4组,分别是生理盐水组、10%乙醇溶剂组、GLB低剂量组和高剂量组,每组5只;经腹腔注射给药,HeLa组给予生理盐水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组给予生理盐水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。腹腔注射给药的同时,隔天用标准体重秤称量体重并做好记录,每隔2天用游标卡尺精确测量移植瘤的最大短径和长径,至少测量两次取平均值。给药结束后处死裸鼠前,眼球取血,测生化指标和肿瘤标志物,观察各组模型鼠生化指标和肿瘤标志物的变化;进行移植瘤的体积生长抑制试验观察各组移植瘤体积随时间的变化,组织形态学上表现为组织病理切片后的HE染色的变化[13];免疫组化法移植瘤组织中张力蛋白同源在10号染色体有缺失的磷酸酶基因(Phosphatase and tensin ho-molog deleted from chromosome 10,PTEN)、自噬关键调控因子Beclin1,微管相关蛋白3(Microtubule associated protein I light chain 3,LC-3)的表达变化,Western-blot法进一步检验相关蛋白变化。移植瘤生长曲线使用Graphpad软件绘制,各组间瘤重、处理前后瘤体积变化、肿瘤标记物等计量数据以均数±标准差(sx±)表示,应用SPSS19.0统计软件分析,多组间比较采用单因素方差分析,差异有统计学意义的组间两两比较采用LSD法进行比较,以P0.05为差异有统计学意义。结果1.裸鼠成瘤率为100%;裸鼠处死后,GLB处理组肿瘤标志物较对照组(生理盐水及10%乙醇组)降低,差异有统计学意义(P0.05);GLB处理组和对照组生化指标比较差异无统计学意义(P0.05);GLB处理组的移植瘤体积均明显小于对照组,差异有统计学意义(P0.05)。2.HE染色显示:GLB处理组移植瘤组织出现凋亡、坏死改变;免疫组化法、Western-blot法结果示:GLB处理组的肿瘤组织PTEN、Beclin 1蛋白表达水平及LC3II/I比例较对照组明显升高。结论1.蓝萼乙素可以抑制人宫颈癌HeLa、SiHa细胞裸鼠体内皮下移植瘤的生长,具有浓度依赖性;蓝萼乙素可明显降低实验动物的肿瘤标志物,具有浓度依赖性;蓝萼乙素在一定剂量范围内可诱导裸鼠体内移植瘤细胞发生自噬、凋亡。2.蓝萼乙素抑制宫颈癌细胞生长的作用可能与上调PTEN和Beclin1及LC3蛋白的表达有关。
[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.

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

【参考文献】