P物质在子宫内膜腺癌中的表达及其意义

发布时间：2018-05-18 12:24

本文选题：子宫内膜腺癌 + P物质　；参考：《河北医科大学》2016年硕士论文

【摘要】：目的:子宫内膜癌(Endometrial Carcinoma,EC)在全球女性中的发病率及病死率不断增加,是最为常见的女性生殖系统三大恶性肿瘤之一。据不完全统计,子宫内膜癌约占女性生殖系统恶性肿瘤的20%-30%,最常见的病理类型为腺癌,严重威胁着广大女性的身心健康。二十一世纪以来,子宫内膜癌在我国女性中的发病率显著升高,随着雌孕激素的非正规替代治疗及滥用的增加,内膜癌的发病人群逐渐呈现年轻趋势。子宫内膜癌的发生、发展是一个多因素、多环节的病理过程,其病因及发病机制尚未阐释清楚。随着分子生物学和免疫学的发展,越来越多的病因学研究集中在多种关键分子或信号通路上,其中P物质(Substance P,SP)因其分布广泛而备受关注。P物质是由11个氨基酸残基组成的小分子多肽,其分子量为1340,由TAC-1基因编码。P物质属于速激肽家族,广泛分布于中枢神经及外周组织中,具有多种生物学活性。P物质能够参与免疫功能的调节,增强创伤后的神经组织的修复,此外P物质对多种细胞有促增殖作用,当机体在受到创伤性刺激时,P物质可诱导血管内皮细胞和成纤维细胞的增殖。近年来,随着国内外学者们的深入研究P物质在肿瘤组织中的重要作用也逐渐被大家所熟知,在多种肿瘤如乳腺癌、胃癌、胰腺癌、喉癌、肺癌、黑色素瘤等中P物质的表达活性都有明显升高。迄今为止尚未见P物质与子宫内膜腺癌的研究报道,在子宫内膜腺癌中P物质的表达情况如何亦不清楚。因此,本实验利用实时荧光定量PCR法、Western Blotting法测定P物质在子宫内膜腺癌组织及细胞中的表达情况,采用MTT法及Transwell小室侵袭实验检测P物质对子宫内膜腺癌细胞增殖及侵袭的影响,旨在探讨P物质在子宫内膜腺癌发生、发展中的作用及意义。方法:1荧光定量PCR技术检测子宫内膜腺癌组织及Ishikawa细胞株中P物质mRNA的表达情况:采用子宫内膜腺癌组织及Ishikawa细胞株作为实验组,正常子宫内膜组织作为对照组,检测其中P物质的mRNA的表达情况。2 Western Blotting方法检测子宫内膜腺癌组织及Ishikawa细胞株中P物质表达的蛋白含量:采用子宫内膜腺癌组织及Ishikawa细胞株作为实验组,正常子宫内膜组织作为对照组,检测其中P物质蛋白的表达情况。3 MTT法检测不同浓度的P物质作用于Ishikawa细胞48h后的体外增殖情况:在处于对数生长期的Ishikawa细胞中分别加入不同浓度的P物质(10-9、10-8、10-7、10-6mol/l),置于5%的CO2、37℃的恒温培养箱中培养48h后,检测Ishikawa细胞的增殖情况。4 Transwell法检测经不同浓度P物质处理后Ishikawa细胞侵袭能力的变化:在Ishikawa细胞中分别加入不同浓度的P物质,Transwell小室用Matrigel胶包被后,逐滴加入各组细胞,置入5%的CO2、37℃恒温培养箱中培养24小时,固定染色后,计数落入小孔中的各组细胞数。5统计学方法:选用统计学软件SPSS 13.0进行分析,本实验数据满足正态性分布,计量资料以均数±标准差表示,用t检验或单因素方差分析比较各组之间差异,α=0.05为检验水准,P0.05有统计学意义。结果:1荧光定量PCR技术检测P物质mRNA在子宫内膜腺癌组织、Ishikawa细胞株和正常子宫内膜组织中的表达情况:子宫内膜腺癌组织中P物质表达量为2.62±0.13,Ishikawa细胞中为3.65±0.28,正常组织中为1.02±0.02,子宫内膜腺癌组织及Ishikawa细胞中P物质的表达明显高于正常组织,差异有统计学意义(P0.001)。2 Western Blotting方法检测子宫内膜腺癌组织、Ishikawa细胞株、正常子宫内膜组织中P物质表达的蛋白量:结果显示P物质蛋白在子宫内膜腺癌组织、Ishikawa细胞株及正常组织中的表达量分别为1.06±0.09、1.20±0.11及0.82±0.06;子宫内膜腺癌组织及Ishikawa细胞株中P物质表达明显高于正常组织(P0.001),结果有显著差异。3 MTT法检测不同浓度P物质(10-9、10-8、10-7、10-6mol/l)作用于子宫内膜腺癌Ishikawa细胞48h的增殖率分别为:8.933±0.615%、14.667±1.195%、26.523±3.374%、20.983±1.911%。结果显示,当P物质浓度在10-9mol/l-10-7mol/l时,与Ishikawa细胞共培养48小时后,随着P物质浓度的增加,细胞增殖能力逐渐增强,但当P物质增加到10-6mol/l时,细胞增殖能力有所下降。各组间差异明显(P0.01),有统计学意义。4 Transwell实验法检测Ishikawa细胞经不同浓度P物质处理后的迁移细胞数:在侵袭实验中P物质浓度选取10-9mol/l-10-7mol/l,与Ishikawa细胞共培养48小时后,肿瘤细胞穿过基底胶的数量分别为:26±5.612、37.6±7.603、49.6±9.737,随着P物质浓度增加迁移细胞数逐渐增多。各组间差异明显(P0.01),有统计学意义。结论:1 P物质在子宫内膜腺癌中的表达明显高于正常子宫内膜组织,提示P物质可能参与了子宫内膜腺癌的发生、发展过程,有希望为子宫内膜腺癌的诊断提供新的分子学标志物。2 P物质能显著增强子宫内膜腺癌细胞的增殖能力,说明P物质促进了子宫内膜腺癌的进展,有利于进一步阐释子宫内膜腺癌的发病机制。3 P物质能显著增强子宫内膜腺癌细胞的侵袭能力,并且随着P物质浓度升高其促侵袭能力越强,提示P物质可能在子宫内膜腺癌的恶变过程中起到重要作用,有助于对子宫内膜腺癌预后的判断。
[Abstract]:Objective: the incidence and mortality of Endometrial Carcinoma (EC) in the global women are increasing. It is one of the most common three malignant tumors in the female reproductive system. According to incomplete statistics, endometrial cancer accounts for about 20%-30% of the female reproductive system malignant tumor, and the most common pathological type is adenocarcinoma, which is a serious threat to the wide range of cancer. The incidence of endometrial cancer in women in China has increased significantly since twenty-first Century. With the increase of estrogen and progesterone, the incidence of endometrial cancer is becoming younger. The development of endometrial cancer is a multi factor, multi link pathological process, and the cause of the pathogenesis of endometrial cancer. And the pathogenesis is not clear. With the development of molecular biology and immunology, more and more etiological studies are focused on a variety of key molecules or signal pathways, in which substance P (Substance P (SP) is widely distributed,.P substance is a small molecular polypeptide composed of 11 amino acid residues, with a molecular weight of 1340, from TA C-1 gene encoding.P is a family of tachykinin, which is widely distributed in the central and peripheral tissues. A variety of biological activity.P can participate in the regulation of immune function and enhance the repair of nerve tissue after trauma. In addition, substance P can promote the proliferation of various cells. When the body is subjected to traumatic stimulation, substance P can be induced. The proliferation of vascular endothelial cells and fibroblasts has been well known in recent years with the in-depth study of the important role of substance P in tumor tissues at home and abroad. The expression of substance P in various tumors, such as breast cancer, gastric cancer, pancreatic cancer, laryngoma, lung cancer, melanoma and so on, has been significantly increased. No study of substance P and endometrial adenocarcinoma has been reported. The expression of substance P in endometrial adenocarcinoma is not clear. Therefore, the present experiment uses real-time fluorescence quantitative PCR and Western Blotting to determine the expression of substance P in endometrial adenocarcinoma tissue and cells. MTT method and Transwell chamber invasion test are used to detect P. The effect of substance on the proliferation and invasion of endometrial adenocarcinoma cells in order to explore the role and significance of substance P in the development of endometrial adenocarcinoma. Methods: 1 fluorescence quantitative PCR technique was used to detect the expression of substance mRNA in endometrial adenocarcinoma tissue and Ishikawa cell lines: endometrial adenocarcinoma tissue and Ishikawa cell lines were used as a method. In the experimental group, the normal endometrium tissue was used as the control group to detect the expression of mRNA in substance P..2 Western Blotting was used to detect the protein content of substance P in endometrial adenocarcinoma tissue and Ishikawa cell lines: endometrial adenocarcinoma tissue and Ishikawa cell lines were used as experimental group, and normal endometrium tissue was used as a pair. The expression of material protein of P was detected by.3 MTT method, and the proliferation of Ishikawa cell 48h was detected by different concentrations of P substance in Ishikawa cells: Ishikawa cells in the logarithmic growth period were added to different concentrations of P substance (10-9,10-8,10-7,10-6mol/l), and placed in the incubator of 5% CO2,37 C at the incubator for 48h. The proliferation of Ishikawa cells was measured by.4 Transwell method to detect the invasiveness of Ishikawa cells after the treatment of different concentrations of P substance: the P substance of different concentrations was added to Ishikawa cells, and the Transwell small room was filled with Matrigel glue package, and the cells were added to each group by drop by drop, and the cells were incubating in the incubator of 5% CO2,37 C for 24 hours and fixed. After dyeing, count the number of cells in each group in the small hole.5: statistical software SPSS 13 was selected for analysis. The experimental data met the normal distribution, the measurement data were expressed with mean standard deviation, and the difference between each group was compared with the t test or the single factor variance analysis. The alpha =0.05 was the test level, and the P0.05 had statistical significance. The results were statistically significant. 1 fluorescence quantitative PCR technique was used to detect the expression of substance P mRNA in endometrial adenocarcinoma, Ishikawa cell line and normal endometrium. The expression of substance P in endometrial adenocarcinoma was 2.62 + 0.13, 3.65 + 0.28 in Ishikawa cells, 1.02 + 0.02 in normal tissues, and P substance in endometrial adenocarcinoma and Ishikawa cells. The expression was significantly higher than that of normal tissue, and the difference was statistically significant (P0.001).2 Western Blotting method to detect the protein content of substance P in endometrial adenocarcinoma tissue, Ishikawa cell line and normal endometrium tissue. The results showed that the expression of substance P protein in endometrial adenocarcinoma group, Ishikawa cell line and normal tissue was respectively The expression of P substance in endometrial adenocarcinoma and Ishikawa cell lines was 1.06 + 0.09,1.20 + 0.11 and 0.82 + 0.06. The results showed significant difference between P substance (10-9,10-8,10-7,10-6mol/l) and.3 MTT assay in endometrial adenocarcinoma Ishikawa cell 48h (8.933 + 0.615%, 14.667), respectively. The results of 1.195%, 26.523 + 3.374%, 20.983 + 1.911%. showed that when the concentration of P was at 10-9mol/l-10-7mol/l, and after co culture with Ishikawa cells for 48 hours, the proliferation ability of the cells increased gradually with the increase of P substance concentration, but the proliferation ability of the cells decreased when the P substance increased to 10-6mol/l. The difference between each group was significant (P0.01), statistically significant. .4 Transwell test method was used to detect the number of migratory cells of Ishikawa cells treated with different concentrations of P substance: in the invasion experiment, the concentration of P substance was selected to 10-9mol/l-10-7mol/l, and after 48 hours co culture with Ishikawa cells, the number of tumor cells passing through basal gum were 26 + 5.612,37.6 + 7.603,49.6 + 9.737 respectively, with the increase of P substance concentration. The number of migratory cells increased gradually. There was significant difference between each group (P0.01). Conclusion: the expression of substance 1 P in endometrial adenocarcinoma is obviously higher than that of normal endometrium, suggesting that substance P may be involved in the occurrence of endometrial adenocarcinoma, the development process, and hope to provide a new molecular marker for the diagnosis of endometrial adenocarcinoma. Substance.2 P can significantly enhance the proliferation of endometrial adenocarcinoma cells, indicating that substance P promotes the progression of endometrial adenocarcinoma, which is beneficial to further explain the pathogenesis of endometrial adenocarcinoma..3 P can significantly enhance the invasion ability of endometrial adenocarcinoma cells, and the stronger the concentration of P substance, the stronger the invasion ability, the higher the increase of the invasion ability of the endometrial adenocarcinoma cells, and the increase of the invasion ability of endometrial adenocarcinoma cells. Substance P may play an important role in the malignant transformation of endometrial adenocarcinoma, and is helpful for the prognosis of endometrial adenocarcinoma.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.33

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