本文選題：骨肉瘤 + miR-15a　； 參考：《第四軍醫(yī)大學(xué)》2015年博士論文

【摘要】：背景:骨肉瘤是最常見(jiàn)的原發(fā)性惡性骨腫瘤,臨床上由于該腫瘤發(fā)病及進(jìn)展迅速,早期易轉(zhuǎn)移,治療過(guò)程中容易產(chǎn)生耐藥,致使許多患者失去生命。而導(dǎo)致骨肉瘤發(fā)生和發(fā)展的機(jī)制仍未被研究透徹。已有的實(shí)驗(yàn)成果認(rèn)為,在癌細(xì)胞的基本代謝活動(dòng)過(guò)程中,mi R-15a和mi R-16-1起到了重要調(diào)控作用。研究它們?cè)诠侨饬黾?xì)胞內(nèi)的具體功能有望進(jìn)一步完善該腫瘤發(fā)生和發(fā)展的機(jī)制。目的:本實(shí)驗(yàn)旨在發(fā)現(xiàn)并驗(yàn)證mi R-15a和mi R-16-1在骨肉瘤細(xì)胞中的異常表達(dá)狀態(tài),研究它們對(duì)細(xì)胞生命程序的影響,揭示其在骨肉瘤細(xì)胞凋亡機(jī)制中的作用。方法:1.選取34例骨肉瘤病理蠟塊樣本進(jìn)行實(shí)時(shí)定量PCR分析,并與前期組織芯片結(jié)果對(duì)比,分析差異表達(dá)情況。2.采用瞬時(shí)轉(zhuǎn)染法構(gòu)建高表達(dá)mi R-15a和mi R-16-1的骨肉瘤細(xì)胞系,觀察轉(zhuǎn)染效果并計(jì)算轉(zhuǎn)染效率。3.搜集轉(zhuǎn)染48小時(shí)后的存活細(xì)胞,使用TUNEL、流式細(xì)胞儀及MTT法研究高表達(dá)mi R-15a和mi R-16-1的SOSP-9607細(xì)胞在細(xì)胞周期、凋亡及增殖過(guò)程中的變化。4.利用基因信息學(xué)軟件、文獻(xiàn)回顧及RT-PCR實(shí)驗(yàn)方法確定靶基因研究對(duì)象。5.使用免疫組織化學(xué)、Western blot、RT-PCR及熒光素酶報(bào)告基因依次從組織細(xì)胞、蛋白、m RNA及DNA水平驗(yàn)證靶基因。結(jié)果:1.34例骨肉瘤蠟塊標(biāo)本中mi R-15a和mi R-16-1的相對(duì)表達(dá)定量與瘤旁標(biāo)本相比有所降低,統(tǒng)計(jì)學(xué)分析有差異(P0.05)。2.轉(zhuǎn)染4小時(shí)后,經(jīng)過(guò)鏡下觀察并計(jì)算轉(zhuǎn)染效率,結(jié)果表明轉(zhuǎn)染成功,可用于后續(xù)實(shí)驗(yàn)。3.轉(zhuǎn)染48小時(shí)后,流式細(xì)胞儀和TUNEL法凋亡檢測(cè)結(jié)果表明高表達(dá)mi R-15a和mi R-16-1的SOSP-9607細(xì)胞凋亡率較抑制物組和對(duì)照組相比,顯著增加(P0.05)。流式細(xì)胞儀周期分析結(jié)果顯示轉(zhuǎn)染后處于G0/G1期的細(xì)胞明顯多于對(duì)照組,說(shuō)明細(xì)胞周期減緩(P0.05)。以MTT法繪制的細(xì)胞增殖曲線顯示轉(zhuǎn)染后的腫瘤細(xì)胞增殖速度較其他組有所減緩(P0.05)。上述實(shí)驗(yàn)中,抑制物組和對(duì)照組的組間和組內(nèi)結(jié)果對(duì)比均無(wú)差異(P0.05)。4.經(jīng)過(guò)基因信息學(xué)軟件篩選,文獻(xiàn)回顧分析以及RT-PCR的初步篩查,最終確定待研究靶基因?yàn)镃CND1。5.熒光素酶報(bào)告基因分析結(jié)果揭示出mi R-15a和mi R-16-1可以抑制CCND1-b組的熒光強(qiáng)度(P0.05),而CCND1-a組則不受影響(P0.05)。說(shuō)明mi R-15a和mi R-16-1通過(guò)與CCND1基因3’UTR區(qū)域的CCND1-b段結(jié)合抑制CCND1基因的表達(dá)。結(jié)論:mi R-15a和mi R-16-1可以通過(guò)抑制CCND1基因表達(dá)促進(jìn)SOSP-9607細(xì)胞凋亡、阻抑細(xì)胞周期,抑制細(xì)胞增殖。
[Abstract]:Background: osteosarcoma is the most common primary malignant bone tumor. Due to the rapid onset and progression of osteosarcoma early metastasis and drug resistance in the course of treatment many patients lost their lives. However, the mechanisms leading to the development of osteosarcoma have not been thoroughly studied. It has been suggested that mi R-15a and mi R-16-1 play an important role in the basic metabolic activity of cancer cells. The study of their specific function in osteosarcoma cells is expected to further improve the mechanism of tumorigenesis and development. Aim: to detect and verify the abnormal expression of mi R-15a and mi R-16-1 in osteosarcoma cells, to study their effects on cell life process and to reveal their role in the apoptosis mechanism of osteosarcoma cells. Method 1: 1. 34 cases of osteosarcoma were analyzed by real-time quantitative PCR and compared with the results of tissue microarray. Osteosarcoma cell lines with high expression of miR-15a and miR-16-1 were constructed by transient transfection method. The transfection effect was observed and the transfection efficiency was calculated. The survival cells were collected after 48 hours of transfection. Tunel, flow cytometry and MTT were used to study the changes in cell cycle, apoptosis and proliferation of SOSP-9607 cells with high expression of miR-15a and miR-16-1. Using gene informatics software, literature review and RT-PCR experiment method to determine the target gene research object. RT-PCR and luciferase reporter genes were used to identify target genes from tissue cells, protein RNA and DNA. Results the relative quantitative expression of miR-15a and miR-16-1 in paraffin specimens of 1. 34 cases of osteosarcoma was lower than that of the adjacent specimens, and there was a significant difference between them (P0.05. 2). After 4 hours of transfection, the transfection efficiency was observed and calculated under microscope. The results showed that the transfection was successful and could be used in the following experiment. 3. After 48 hours of transfection, the apoptotic rate of SOSP-9607 cells with high expression of miR-15a and miR-16-1 was significantly higher than that of the control group and the control group by flow cytometry and TUNEL assay, and the results showed that the apoptosis rate of SOSP-9607 cells with high expression of miR-15a and miR-16-1 was significantly higher than that of the control group. The results of flow cytometry showed that the number of cells in the G0/G1 phase after transfection was significantly higher than that in the control group, indicating that the cell cycle slowed down (P 0.05). The cell proliferation curve plotted by MTT method showed that the proliferation rate of tumor cells after transfection was slower than that of other groups. In the above experiments, there was no difference in the results between the inhibition group and the control group. After the screening of gene informatics software, literature review and preliminary screening of RT-PCR, the target gene was determined to be CCND 1.5. The results of luciferase reporter gene analysis showed that miR-15a and miR-16-1 could inhibit the fluorescence intensity of CCND1-b group (P0.05), but CCND1-a group was not affected. It is suggested that mi R-15a and mi R-16-1 inhibit the expression of CCND1 gene by binding to the CCND1-b segment of 3'UTR region of CCND1 gene. Conclusion the cell cycle and cell proliferation of SOSP-9607 cells can be inhibited by inhibiting the expression of CCND1 gene by inhibiting the apoptosis of SOSP-9607 cells.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R738.1
，

本文編號(hào)：1825844