二氢杨梅素通过作用于MicroRNA373抑制绒癌JAR细胞生长的研究

发布时间：2018-05-18 02:26

本文选题：二氢杨梅素 + MicroRNA373　；参考：《承德医学院》2017年硕士论文

【摘要】：MicroRNA是一种进化过程中高度保守,内源性非编码小RNA。它广泛存在于真核生物,在线虫,果蝇,小鼠和人等物种中作为参与调控基因表达的分子具有重要意义。近年来研究发现,MicroRNA的异常表达能够调控肿瘤的发展进程。随着精准靶向治疗的发展,多种MicroRNA被发现是肿瘤治疗的靶点,受到学者的关注。本课题组利用基因芯片技术,检测了绒毛膜上皮癌(简称绒癌)组织和正常绒毛组织中差异表达的MicroRNA。结果发现在绒癌组织中有63个MicroRNA上调,74个MicroRNA下调。其中,MicroRNA373在绒癌组织中明显下调。MicroRNA373作为原癌mi RNA或抑癌mi RNA以及细胞内关键调节因子在多种肿瘤发生发展的过程中起着双向调控作用。本研究探讨了MicroRNA373在绒癌细胞中的表达和对绒癌细胞生长的作用。应用实时定量PCR技术,检测了MicroRNA373在绒癌细胞中的表达,结果发现绒癌JAR和JEG-3细胞系中MicroRNA373表达量明显低于正常滋养细胞HTR-8细胞系。在绒癌JAR细胞中瞬时转染MicroRNA373 18h后,细胞增殖检测法(CCK-8)检测细胞增殖,结果发现绒癌JAR细胞的增殖显著受到抑制,表明MicroRNA373能够抑制绒癌JAR细胞的生长。二氢杨梅素(Dihydromyacetin,DMY)主要活性成分是二氢黄酮醇,提取自我国特有草本植物藤茶茎叶,具有清除自由基、抗炎、抑菌等功效。近年来发现,二氢杨梅素还具有抗肿瘤作用,能够在体内、外抑制多种肿瘤的生长。本研究中发现,二氢杨梅素作用于绒癌JAR细胞后促进了MicroRNA373的表达,而且随着二氢杨梅素作用浓度的升高,MicroRNA373的表达水平也随之升高。同时,还发现二氢杨梅素作用后绒癌JAR细胞密度降低,细胞回缩,大小不一,细胞间隙变大,细胞透光性变差,提示二氢杨梅素可能影响了绒癌JAR细胞的生长。进一步通过CCK-8法检测不同浓度二氢杨梅素作用下绒癌JAR细胞增殖的改变,结果发现随着药物浓度的升高,绒癌JAR细胞增殖抑制率逐步升高。以上研究表明,二氢杨梅素能够促进绒癌JAR细胞中MicroRNA373的表达,并且抑制绒癌JAR细胞的增殖。本研究发现(1)MicroRNA373抑制了绒癌JAR细胞在体外的增殖;(2)二氢杨梅素能够抑制JAR细胞的增殖,同时检测MicroRNA373的表达,发现其表达升高,与药物浓度成正比。本研究对探索绒癌的发病机制和治疗靶点具有重要意义,为二氢杨梅素应用于绒癌临床治疗提供了理论依据。目的:⒈明确MicroRNA373对JAR细胞增殖的影响情况。⒉明确二氢杨梅素对JAR细胞增殖的影响情况。3.明确二氢杨梅素对JAR细胞增殖的影响是否通过MicroRNA373产生的。方法:1.实时定量PCR(Real-time PCR)技术检测正常绒毛HTR-8细胞与绒癌JAR、JEG细胞间MicroRNA373的表达差异。2.应用瞬时转染技术(transient transfection)增强绒癌JAR细胞中MicroRNA373的表达。3.倒置荧光显微镜下观察绒癌JAR细胞中MicroRNA373的转染效率。4.实时定量PCR(Real-time PCR)技术验证转染后MicroRNA373的表达改变。5.利用CCK8法检测过表达MicroRNA373后的绒癌JAR细胞增殖情况。6.实时定量PCR(Real-time PCR)技术检测二氢杨梅素作用后绒癌JAR细胞中MicroRNA373表达情况。7.倒置荧光显微镜下观察二氢杨梅素作用后绒癌JAR细胞的细胞形态改变。8.通过CCK8法检测不同浓度二氢杨梅素作用下绒癌JAR细胞的增殖情况。结果:1.绒癌细胞JAR和JEG中MicroRNA373的表达明显低于正常绒毛滋养细胞HTR-8,具有统计学意义(P0.05)。2.转染MicroRNA373 18h后,倒置显微镜下观察发现转染后的JAR细胞生长状况变差,部分细胞脱落死亡。倒置荧光显微镜荧光下观察并记录绿色荧光蛋白表达情况,观察到转染后的绒癌JAR细胞呈现绿色荧光,且细胞轮廓清晰可辨认。3.实时定量PCR结果显示,与阴性对照组相比,瞬时转染后绒癌JAR细胞中MicroRNA373表达量显著升高,差异有统计学意义(P0.05)。4.CCK8法检测显示,通过瞬时转染后过表达MicroRNA373的绒癌JAR细胞吸光度较低,活细胞的数量减少,增殖抑制率显著升高(P0.05),提示MicroRNA373有效降低了绒癌JAR细胞的增殖能力。5.实时定量PCR结果显示,二氢杨梅素促进绒癌JAR细胞中MicroRNA373的表达,且随着二氢杨梅素作用浓度的升高,MicroRNA373的表达逐步增强,呈正相关的量效关系(P0.05)。6.倒置显微镜下观察到:二氢杨梅素作用前,绒癌JAR细胞生长紧凑、贴壁、轮廓清晰、大小形态一致。二氢杨梅素作用24h后,绒癌JAR细胞随着作用浓度的增加,细胞密度降低,细胞回缩,大小不一,细胞间隙变大,细胞颜色加深,形成数量不等的细胞团块。7.CCK8法结果显示,二氢杨梅素作用后绒癌JAR细胞活性减弱,且随着作用浓度的升高其活性呈现递减趋势,差异有统计学意义(P0.05),说明二氢杨梅素抑制了绒癌JAR细胞的生长,且具有浓度依赖关系。结论:1.MicroRNA373是抑制绒癌细胞体外生长的有效靶点。2.二氢杨梅素通过作用于靶点MicroRNA373抑制绒癌细胞的生长。
[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.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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