本文選題：子宮內(nèi)膜腺癌 + P物質(zhì)��； 參考：《河北醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:子宮內(nèi)膜癌(Endometrial Carcinoma,EC)在全球女性中的發(fā)病率及病死率不斷增加,是最為常見的女性生殖系統(tǒng)三大惡性腫瘤之一。據(jù)不完全統(tǒng)計(jì),子宮內(nèi)膜癌約占女性生殖系統(tǒng)惡性腫瘤的20%-30%,最常見的病理類型為腺癌,嚴(yán)重威脅著廣大女性的身心健康。二十一世紀(jì)以來,子宮內(nèi)膜癌在我國女性中的發(fā)病率顯著升高,隨著雌孕激素的非正規(guī)替代治療及濫用的增加,內(nèi)膜癌的發(fā)病人群逐漸呈現(xiàn)年輕趨勢(shì)。子宮內(nèi)膜癌的發(fā)生、發(fā)展是一個(gè)多因素、多環(huán)節(jié)的病理過程,其病因及發(fā)病機(jī)制尚未闡釋清楚。隨著分子生物學(xué)和免疫學(xué)的發(fā)展,越來越多的病因?qū)W研究集中在多種關(guān)鍵分子或信號(hào)通路上,其中P物質(zhì)(Substance P,SP)因其分布廣泛而備受關(guān)注。P物質(zhì)是由11個(gè)氨基酸殘基組成的小分子多肽,其分子量為1340,由TAC-1基因編碼。P物質(zhì)屬于速激肽家族,廣泛分布于中樞神經(jīng)及外周組織中,具有多種生物學(xué)活性。P物質(zhì)能夠參與免疫功能的調(diào)節(jié),增強(qiáng)創(chuàng)傷后的神經(jīng)組織的修復(fù),此外P物質(zhì)對(duì)多種細(xì)胞有促增殖作用,當(dāng)機(jī)體在受到創(chuàng)傷性刺激時(shí),P物質(zhì)可誘導(dǎo)血管內(nèi)皮細(xì)胞和成纖維細(xì)胞的增殖。近年來,隨著國內(nèi)外學(xué)者們的深入研究P物質(zhì)在腫瘤組織中的重要作用也逐漸被大家所熟知,在多種腫瘤如乳腺癌、胃癌、胰腺癌、喉癌、肺癌、黑色素瘤等中P物質(zhì)的表達(dá)活性都有明顯升高。迄今為止尚未見P物質(zhì)與子宮內(nèi)膜腺癌的研究報(bào)道,在子宮內(nèi)膜腺癌中P物質(zhì)的表達(dá)情況如何亦不清楚。因此,本實(shí)驗(yàn)利用實(shí)時(shí)熒光定量PCR法、Western Blotting法測(cè)定P物質(zhì)在子宮內(nèi)膜腺癌組織及細(xì)胞中的表達(dá)情況,采用MTT法及Transwell小室侵襲實(shí)驗(yàn)檢測(cè)P物質(zhì)對(duì)子宮內(nèi)膜腺癌細(xì)胞增殖及侵襲的影響,旨在探討P物質(zhì)在子宮內(nèi)膜腺癌發(fā)生、發(fā)展中的作用及意義。方法:1熒光定量PCR技術(shù)檢測(cè)子宮內(nèi)膜腺癌組織及Ishikawa細(xì)胞株中P物質(zhì)mRNA的表達(dá)情況:采用子宮內(nèi)膜腺癌組織及Ishikawa細(xì)胞株作為實(shí)驗(yàn)組,正常子宮內(nèi)膜組織作為對(duì)照組,檢測(cè)其中P物質(zhì)的mRNA的表達(dá)情況。2 Western Blotting方法檢測(cè)子宮內(nèi)膜腺癌組織及Ishikawa細(xì)胞株中P物質(zhì)表達(dá)的蛋白含量:采用子宮內(nèi)膜腺癌組織及Ishikawa細(xì)胞株作為實(shí)驗(yàn)組,正常子宮內(nèi)膜組織作為對(duì)照組,檢測(cè)其中P物質(zhì)蛋白的表達(dá)情況。3 MTT法檢測(cè)不同濃度的P物質(zhì)作用于Ishikawa細(xì)胞48h后的體外增殖情況:在處于對(duì)數(shù)生長期的Ishikawa細(xì)胞中分別加入不同濃度的P物質(zhì)(10-9、10-8、10-7、10-6mol/l),置于5%的CO2、37℃的恒溫培養(yǎng)箱中培養(yǎng)48h后,檢測(cè)Ishikawa細(xì)胞的增殖情況。4 Transwell法檢測(cè)經(jīng)不同濃度P物質(zhì)處理后Ishikawa細(xì)胞侵襲能力的變化:在Ishikawa細(xì)胞中分別加入不同濃度的P物質(zhì),Transwell小室用Matrigel膠包被后,逐滴加入各組細(xì)胞,置入5%的CO2、37℃恒溫培養(yǎng)箱中培養(yǎng)24小時(shí),固定染色后,計(jì)數(shù)落入小孔中的各組細(xì)胞數(shù)。5統(tǒng)計(jì)學(xué)方法:選用統(tǒng)計(jì)學(xué)軟件SPSS 13.0進(jìn)行分析,本實(shí)驗(yàn)數(shù)據(jù)滿足正態(tài)性分布,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示,用t檢驗(yàn)或單因素方差分析比較各組之間差異,α=0.05為檢驗(yàn)水準(zhǔn),P0.05有統(tǒng)計(jì)學(xué)意義。結(jié)果:1熒光定量PCR技術(shù)檢測(cè)P物質(zhì)mRNA在子宮內(nèi)膜腺癌組織、Ishikawa細(xì)胞株和正常子宮內(nèi)膜組織中的表達(dá)情況:子宮內(nèi)膜腺癌組織中P物質(zhì)表達(dá)量為2.62±0.13,Ishikawa細(xì)胞中為3.65±0.28,正常組織中為1.02±0.02,子宮內(nèi)膜腺癌組織及Ishikawa細(xì)胞中P物質(zhì)的表達(dá)明顯高于正常組織,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。2 Western Blotting方法檢測(cè)子宮內(nèi)膜腺癌組織、Ishikawa細(xì)胞株、正常子宮內(nèi)膜組織中P物質(zhì)表達(dá)的蛋白量:結(jié)果顯示P物質(zhì)蛋白在子宮內(nèi)膜腺癌組織、Ishikawa細(xì)胞株及正常組織中的表達(dá)量分別為1.06±0.09、1.20±0.11及0.82±0.06;子宮內(nèi)膜腺癌組織及Ishikawa細(xì)胞株中P物質(zhì)表達(dá)明顯高于正常組織(P0.001),結(jié)果有顯著差異。3 MTT法檢測(cè)不同濃度P物質(zhì)(10-9、10-8、10-7、10-6mol/l)作用于子宮內(nèi)膜腺癌Ishikawa細(xì)胞48h的增殖率分別為:8.933±0.615%、14.667±1.195%、26.523±3.374%、20.983±1.911%。結(jié)果顯示,當(dāng)P物質(zhì)濃度在10-9mol/l-10-7mol/l時(shí),與Ishikawa細(xì)胞共培養(yǎng)48小時(shí)后,隨著P物質(zhì)濃度的增加,細(xì)胞增殖能力逐漸增強(qiáng),但當(dāng)P物質(zhì)增加到10-6mol/l時(shí),細(xì)胞增殖能力有所下降。各組間差異明顯(P0.01),有統(tǒng)計(jì)學(xué)意義。4 Transwell實(shí)驗(yàn)法檢測(cè)Ishikawa細(xì)胞經(jīng)不同濃度P物質(zhì)處理后的遷移細(xì)胞數(shù):在侵襲實(shí)驗(yàn)中P物質(zhì)濃度選取10-9mol/l-10-7mol/l,與Ishikawa細(xì)胞共培養(yǎng)48小時(shí)后,腫瘤細(xì)胞穿過基底膠的數(shù)量分別為:26±5.612、37.6±7.603、49.6±9.737,隨著P物質(zhì)濃度增加遷移細(xì)胞數(shù)逐漸增多。各組間差異明顯(P0.01),有統(tǒng)計(jì)學(xué)意義。結(jié)論:1 P物質(zhì)在子宮內(nèi)膜腺癌中的表達(dá)明顯高于正常子宮內(nèi)膜組織,提示P物質(zhì)可能參與了子宮內(nèi)膜腺癌的發(fā)生、發(fā)展過程,有希望為子宮內(nèi)膜腺癌的診斷提供新的分子學(xué)標(biāo)志物。2 P物質(zhì)能顯著增強(qiáng)子宮內(nèi)膜腺癌細(xì)胞的增殖能力,說明P物質(zhì)促進(jìn)了子宮內(nèi)膜腺癌的進(jìn)展,有利于進(jìn)一步闡釋子宮內(nèi)膜腺癌的發(fā)病機(jī)制。3 P物質(zhì)能顯著增強(qiáng)子宮內(nèi)膜腺癌細(xì)胞的侵襲能力,并且隨著P物質(zhì)濃度升高其促侵襲能力越強(qiáng),提示P物質(zhì)可能在子宮內(nèi)膜腺癌的惡變過程中起到重要作用,有助于對(duì)子宮內(nèi)膜腺癌預(yù)后的判斷。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.33

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本文編號(hào)：1905825