本文選題：二氫楊梅素 + MicroRNA373　； 參考：《承德醫(yī)學(xué)院》2017年碩士論文

【摘要】：MicroRNA是一種進(jìn)化過(guò)程中高度保守,內(nèi)源性非編碼小RNA。它廣泛存在于真核生物,在線(xiàn)蟲(chóng),果蠅,小鼠和人等物種中作為參與調(diào)控基因表達(dá)的分子具有重要意義。近年來(lái)研究發(fā)現(xiàn),MicroRNA的異常表達(dá)能夠調(diào)控腫瘤的發(fā)展進(jìn)程。隨著精準(zhǔn)靶向治療的發(fā)展,多種MicroRNA被發(fā)現(xiàn)是腫瘤治療的靶點(diǎn),受到學(xué)者的關(guān)注。本課題組利用基因芯片技術(shù),檢測(cè)了絨毛膜上皮癌(簡(jiǎn)稱(chēng)絨癌)組織和正常絨毛組織中差異表達(dá)的MicroRNA。結(jié)果發(fā)現(xiàn)在絨癌組織中有63個(gè)MicroRNA上調(diào),74個(gè)MicroRNA下調(diào)。其中,MicroRNA373在絨癌組織中明顯下調(diào)。MicroRNA373作為原癌mi RNA或抑癌mi RNA以及細(xì)胞內(nèi)關(guān)鍵調(diào)節(jié)因子在多種腫瘤發(fā)生發(fā)展的過(guò)程中起著雙向調(diào)控作用。本研究探討了MicroRNA373在絨癌細(xì)胞中的表達(dá)和對(duì)絨癌細(xì)胞生長(zhǎng)的作用。應(yīng)用實(shí)時(shí)定量PCR技術(shù),檢測(cè)了MicroRNA373在絨癌細(xì)胞中的表達(dá),結(jié)果發(fā)現(xiàn)絨癌JAR和JEG-3細(xì)胞系中MicroRNA373表達(dá)量明顯低于正常滋養(yǎng)細(xì)胞HTR-8細(xì)胞系。在絨癌JAR細(xì)胞中瞬時(shí)轉(zhuǎn)染MicroRNA373 18h后,細(xì)胞增殖檢測(cè)法(CCK-8)檢測(cè)細(xì)胞增殖,結(jié)果發(fā)現(xiàn)絨癌JAR細(xì)胞的增殖顯著受到抑制,表明MicroRNA373能夠抑制絨癌JAR細(xì)胞的生長(zhǎng)。二氫楊梅素(Dihydromyacetin,DMY)主要活性成分是二氫黃酮醇,提取自我國(guó)特有草本植物藤茶莖葉,具有清除自由基、抗炎、抑菌等功效。近年來(lái)發(fā)現(xiàn),二氫楊梅素還具有抗腫瘤作用,能夠在體內(nèi)、外抑制多種腫瘤的生長(zhǎng)。本研究中發(fā)現(xiàn),二氫楊梅素作用于絨癌JAR細(xì)胞后促進(jìn)了MicroRNA373的表達(dá),而且隨著二氫楊梅素作用濃度的升高,MicroRNA373的表達(dá)水平也隨之升高。同時(shí),還發(fā)現(xiàn)二氫楊梅素作用后絨癌JAR細(xì)胞密度降低,細(xì)胞回縮,大小不一,細(xì)胞間隙變大,細(xì)胞透光性變差,提示二氫楊梅素可能影響了絨癌JAR細(xì)胞的生長(zhǎng)。進(jìn)一步通過(guò)CCK-8法檢測(cè)不同濃度二氫楊梅素作用下絨癌JAR細(xì)胞增殖的改變,結(jié)果發(fā)現(xiàn)隨著藥物濃度的升高,絨癌JAR細(xì)胞增殖抑制率逐步升高。以上研究表明,二氫楊梅素能夠促進(jìn)絨癌JAR細(xì)胞中MicroRNA373的表達(dá),并且抑制絨癌JAR細(xì)胞的增殖。本研究發(fā)現(xiàn)(1)MicroRNA373抑制了絨癌JAR細(xì)胞在體外的增殖;(2)二氫楊梅素能夠抑制JAR細(xì)胞的增殖,同時(shí)檢測(cè)MicroRNA373的表達(dá),發(fā)現(xiàn)其表達(dá)升高,與藥物濃度成正比。本研究對(duì)探索絨癌的發(fā)病機(jī)制和治療靶點(diǎn)具有重要意義,為二氫楊梅素應(yīng)用于絨癌臨床治療提供了理論依據(jù)。目的:⒈明確MicroRNA373對(duì)JAR細(xì)胞增殖的影響情況。⒉明確二氫楊梅素對(duì)JAR細(xì)胞增殖的影響情況。3.明確二氫楊梅素對(duì)JAR細(xì)胞增殖的影響是否通過(guò)MicroRNA373產(chǎn)生的。方法:1.實(shí)時(shí)定量PCR(Real-time PCR)技術(shù)檢測(cè)正常絨毛HTR-8細(xì)胞與絨癌JAR、JEG細(xì)胞間MicroRNA373的表達(dá)差異。2.應(yīng)用瞬時(shí)轉(zhuǎn)染技術(shù)(transient transfection)增強(qiáng)絨癌JAR細(xì)胞中MicroRNA373的表達(dá)。3.倒置熒光顯微鏡下觀察絨癌JAR細(xì)胞中MicroRNA373的轉(zhuǎn)染效率。4.實(shí)時(shí)定量PCR(Real-time PCR)技術(shù)驗(yàn)證轉(zhuǎn)染后MicroRNA373的表達(dá)改變。5.利用CCK8法檢測(cè)過(guò)表達(dá)MicroRNA373后的絨癌JAR細(xì)胞增殖情況。6.實(shí)時(shí)定量PCR(Real-time PCR)技術(shù)檢測(cè)二氫楊梅素作用后絨癌JAR細(xì)胞中MicroRNA373表達(dá)情況。7.倒置熒光顯微鏡下觀察二氫楊梅素作用后絨癌JAR細(xì)胞的細(xì)胞形態(tài)改變。8.通過(guò)CCK8法檢測(cè)不同濃度二氫楊梅素作用下絨癌JAR細(xì)胞的增殖情況。結(jié)果:1.絨癌細(xì)胞JAR和JEG中MicroRNA373的表達(dá)明顯低于正常絨毛滋養(yǎng)細(xì)胞HTR-8,具有統(tǒng)計(jì)學(xué)意義(P0.05)。2.轉(zhuǎn)染MicroRNA373 18h后,倒置顯微鏡下觀察發(fā)現(xiàn)轉(zhuǎn)染后的JAR細(xì)胞生長(zhǎng)狀況變差,部分細(xì)胞脫落死亡。倒置熒光顯微鏡熒光下觀察并記錄綠色熒光蛋白表達(dá)情況,觀察到轉(zhuǎn)染后的絨癌JAR細(xì)胞呈現(xiàn)綠色熒光,且細(xì)胞輪廓清晰可辨認(rèn)。3.實(shí)時(shí)定量PCR結(jié)果顯示,與陰性對(duì)照組相比,瞬時(shí)轉(zhuǎn)染后絨癌JAR細(xì)胞中MicroRNA373表達(dá)量顯著升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4.CCK8法檢測(cè)顯示,通過(guò)瞬時(shí)轉(zhuǎn)染后過(guò)表達(dá)MicroRNA373的絨癌JAR細(xì)胞吸光度較低,活細(xì)胞的數(shù)量減少,增殖抑制率顯著升高(P0.05),提示MicroRNA373有效降低了絨癌JAR細(xì)胞的增殖能力。5.實(shí)時(shí)定量PCR結(jié)果顯示,二氫楊梅素促進(jìn)絨癌JAR細(xì)胞中MicroRNA373的表達(dá),且隨著二氫楊梅素作用濃度的升高,MicroRNA373的表達(dá)逐步增強(qiáng),呈正相關(guān)的量效關(guān)系(P0.05)。6.倒置顯微鏡下觀察到:二氫楊梅素作用前,絨癌JAR細(xì)胞生長(zhǎng)緊湊、貼壁、輪廓清晰、大小形態(tài)一致。二氫楊梅素作用24h后,絨癌JAR細(xì)胞隨著作用濃度的增加,細(xì)胞密度降低,細(xì)胞回縮,大小不一,細(xì)胞間隙變大,細(xì)胞顏色加深,形成數(shù)量不等的細(xì)胞團(tuán)塊。7.CCK8法結(jié)果顯示,二氫楊梅素作用后絨癌JAR細(xì)胞活性減弱,且隨著作用濃度的升高其活性呈現(xiàn)遞減趨勢(shì),差異有統(tǒng)計(jì)學(xué)意義(P0.05),說(shuō)明二氫楊梅素抑制了絨癌JAR細(xì)胞的生長(zhǎng),且具有濃度依賴(lài)關(guān)系。結(jié)論:1.MicroRNA373是抑制絨癌細(xì)胞體外生長(zhǎng)的有效靶點(diǎn)。2.二氫楊梅素通過(guò)作用于靶點(diǎn)MicroRNA373抑制絨癌細(xì)胞的生長(zhǎng)。
[Abstract]:MicroRNA is a highly conservative, endogenous and non coding small RNA.. It is widely used in eukaryotes, online worms, Drosophila, mice and human beings as a molecule involved in regulating gene expression. In recent years, research has found that the abnormal expression of MicroRNA can regulate the development of tumor. The development of a variety of MicroRNA has been found to be the target of cancer treatment, which has attracted the attention of scholars. The research group used gene chip technology to detect the differential expression of MicroRNA. in chorionic epithelial carcinoma (choriocarcinoma) tissue and normal villi tissue, and found that there are 63 MicroRNA up-regulated and 74 MicroRNA down-regulation in choriocarcinoma tissue. Among them, M IcroRNA373 significantly downregulates.MicroRNA373 as the primary cancer mi RNA or MI RNA and the key regulatory factors in the cell. The expression of MicroRNA373 in choriocarcinoma cells and the effect on the cell growth of choriocarcinoma cells are discussed in this study. The real-time quantitative PCR technique is used. The expression of MicroRNA373 in choriocarcinoma cells was detected. The results showed that the expression of MicroRNA373 in choriocarcinoma JAR and JEG-3 cell lines was significantly lower than that of normal trophoblastic HTR-8 cell lines. After transient transfection of MicroRNA373 18h in choriocarcinoma JAR cells, the proliferation detection method (CCK-8) was used to detect the proliferation of cells. The results showed that the proliferation of JAR cells in choriocarcinoma was significantly affected by the proliferation of HTR-8 cells. Inhibition, indicating that MicroRNA373 can inhibit the growth of JAR cells in choriocarcinoma. Two the main active component of Dihydromyacetin (DMY) is the flavonol, which extracts the stem and leaves of the native herbaceous plant of the herbaceous plant, which has the functions of scavenging free radicals, anti-inflammatory and bacteriostasis. In recent years, two hydrogen myricetin has anti tumor effect and can be in vivo. In this study, it was found that two HP promoted the expression of MicroRNA373 in choriocarcinoma JAR cells, and the expression level of MicroRNA373 increased with the increase of the action concentration of two myricetin. At the same time, it was found that the density of JAR cells in choriocarcinoma cells decreased and the cell retracted after the action of two h myricetin. The cell gap became larger and the cell transmittance became worse. It suggested that two HP may affect the growth of JAR cells in choriocarcinoma. Further CCK-8 assay was used to detect the proliferation of JAR cells in choriocarcinoma cells under the action of two myricetin. The results showed that with the increase of drug concentration, the proliferation inhibition rate of choriocarcinoma JAR cells increased gradually. Studies have shown that two HP can promote the expression of MicroRNA373 in choriocarcinoma JAR cells and inhibit the proliferation of JAR cells in choriocarcinoma. This study found that (1) MicroRNA373 inhibited the proliferation of JAR cells in choriocarcinoma in vitro; (2) two HP could inhibit the proliferation of JAR cells and detected the expression of MicroRNA373, and found that the expression was elevated and the drug was used as a drug. This study is of great significance to the exploration of the pathogenesis and therapeutic targets of choriocarcinoma. It provides a theoretical basis for the clinical treatment of two choriocarcinoma. Objective: to clarify the effect of MicroRNA373 on the proliferation of JAR cells. Whether the effect of hormone on the proliferation of JAR cells was produced by MicroRNA373. Method: 1. real-time quantitative PCR (Real-time PCR) technique was used to detect the difference in the expression of MicroRNA373 between normal villi HTR-8 cells and choriocarcinoma JAR, JEG cells,.2. application transient transfection technique (transient transfection) expression inversion fluorescence in the strong choriocarcinoma cell The transfection efficiency of MicroRNA373 in choriocarcinoma JAR cells was observed under microscope..4. real-time quantitative PCR (Real-time PCR) technique was used to verify the expression of MicroRNA373 after transfection..5. using CCK8 method to detect the proliferation of JAR cells of choriocarcinoma after MicroRNA373 The expression of MicroRNA373 in the cell was observed by.7. inverted fluorescence microscope. The cell morphology changes of JAR cells after the action of two hydrogen myricetin.8. were detected by CCK8 method to detect the proliferation of JAR cells under the action of different concentrations of two myricetin. Results: the expression of MicroRNA373 in the JAR and JEG of 1. choriocarcinoma cells was significantly lower than that of normal villi. The cell HTR-8 was statistically significant (P0.05).2. transfected to MicroRNA373 18h. Under the inverted microscope, it was observed that the growth of JAR cells after transfection became poor and some cells fell off and died. The expression of green fluorescent protein was observed and recorded by fluorescence microscope, and the transfected choriocarcinoma JAR cells showed green fluorescence and fine. The.3. real-time quantitative PCR results showed that the MicroRNA373 expression in JAR cells after transient transfection was significantly higher than that in the negative control group, and the difference was statistically significant (P0.05).4.CCK8 assay showed that the number of living cells decreased and the number of living cells decreased by the transient transfection of MicroRNA373 in the choriocarcinoma JAR cells The proliferation inhibition rate was significantly increased (P0.05), suggesting that MicroRNA373 effectively reduced the proliferation ability of choriocarcinoma JAR cells.5. real-time quantitative PCR results showed that two h myricetin promoted the expression of MicroRNA373 in choriocarcinoma JAR cells, and the expression of MicroRNA373 was gradually enhanced with the increase of the action concentration of two myricetin (P0.), which was positively correlated with the dose effect relationship (P0.). 05) under the.6. inverted microscope, it was observed that before the action of two myricetin, the growth of choriocarcinoma JAR cells was compact, with a clear wall, a clear outline, and the same size and shape. After 24h, the cell density decreased, the cell density was reduced, the cell size was different, the cell gap became larger, the cell color deepened, and the cell color deepened. The results of cell mass.7.CCK8 showed that the activity of JAR cells in choriocarcinoma cells decreased after the action of two myricetin, and the activity decreased with the increase of action concentration, and the difference was statistically significant (P0.05), indicating that two myricetin inhibited the growth of choriocarcinoma JAR cells and had a concentration dependence. Conclusion: 1.MicroRNA373 is the inhibition of choriocarcinoma. Effective target for cell growth in vitro.2. two myricetin inhibited the growth of choriocarcinoma cells by targeting MicroRNA373.
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.33

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 曾曉聰;李醒三;文宏;;缺血性心肌病患者外周血MicroRNA-182、CITED2及HIF-1的表達(dá)及相關(guān)性研究[J];臨床心血管病雜志;2017年02期

2 丁小麗;胡蓉;;microRNA-25在腫瘤中的研究進(jìn)展[J];重慶醫(yī)學(xué);2017年04期

3 董海峰;趙亮;張學(xué)記;;癌癥精準(zhǔn)醫(yī)療的瞄準(zhǔn)鏡和雷達(dá)之“液體活檢”——微流控器件—核酸質(zhì)譜集成裝備研制及在腫瘤精準(zhǔn)醫(yī)學(xué)中的應(yīng)用解決方案[J];分析儀器;2017年01期

4 何艷玲;涂紅纓;;miRNA與非小細(xì)胞肺癌診治相關(guān)性的研究進(jìn)展[J];山東醫(yī)藥;2017年04期

5 張楚婕;程蕾蕾;;循環(huán)微小RNA診斷心肌病變的研究進(jìn)展[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版);2017年01期

6 王雪琴;何雪梅;周翔宇;何延政;;microRNA在動(dòng)脈硬化閉塞癥中調(diào)控血管平滑肌的作用機(jī)制[J];中國(guó)動(dòng)脈硬化雜志;2017年02期

7 董杰;韓輝;;MicroRNA-124作為缺血性腦卒中標(biāo)志物的研究進(jìn)展[J];醫(yī)學(xué)綜述;2017年02期

8 王濤;吳敬波;;血清或血漿中的miR-375在腫瘤診治和預(yù)后中的研究進(jìn)展[J];西南軍醫(yī);2017年01期

9 曹彬;郭仁維;;microRNA在早期診斷急性心肌梗死中的研究進(jìn)展[J];中西醫(yī)結(jié)合心腦血管病雜志;2017年01期

10 龔海燕;祖旭宇;申瑩瑩;劉江華;;microRNA調(diào)節(jié)血管鈣化[J];中國(guó)動(dòng)脈硬化雜志;2016年12期

相關(guān)碩士學(xué)位論文 前2條

1 平毅;Survivin、Fas及FasL在葡萄胎、侵蝕性葡萄胎、絨癌中的表達(dá)及意義[D];山西醫(yī)科大學(xué);2007年

2 簡(jiǎn)隆磊;中藥對(duì)腫瘤血管的影響[D];山東中醫(yī)藥大學(xué);2001年

，

本文編號(hào)：1903975