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

【摘要】：背景:骨肉瘤是最常見的原發(fā)性惡性骨腫瘤,臨床上由于該腫瘤發(fā)病及進展迅速,早期易轉移,治療過程中容易產生耐藥,致使許多患者失去生命。而導致骨肉瘤發(fā)生和發(fā)展的機制仍未被研究透徹。已有的實驗成果認為,在癌細胞的基本代謝活動過程中,mi R-15a和mi R-16-1起到了重要調控作用。研究它們在骨肉瘤細胞內的具體功能有望進一步完善該腫瘤發(fā)生和發(fā)展的機制。目的:本實驗旨在發(fā)現(xiàn)并驗證mi R-15a和mi R-16-1在骨肉瘤細胞中的異常表達狀態(tài),研究它們對細胞生命程序的影響,揭示其在骨肉瘤細胞凋亡機制中的作用。方法:1.選取34例骨肉瘤病理蠟塊樣本進行實時定量PCR分析,并與前期組織芯片結果對比,分析差異表達情況。2.采用瞬時轉染法構建高表達mi R-15a和mi R-16-1的骨肉瘤細胞系,觀察轉染效果并計算轉染效率。3.搜集轉染48小時后的存活細胞,使用TUNEL、流式細胞儀及MTT法研究高表達mi R-15a和mi R-16-1的SOSP-9607細胞在細胞周期、凋亡及增殖過程中的變化。4.利用基因信息學軟件、文獻回顧及RT-PCR實驗方法確定靶基因研究對象。5.使用免疫組織化學、Western blot、RT-PCR及熒光素酶報告基因依次從組織細胞、蛋白、m RNA及DNA水平驗證靶基因。結果:1.34例骨肉瘤蠟塊標本中mi R-15a和mi R-16-1的相對表達定量與瘤旁標本相比有所降低,統(tǒng)計學分析有差異(P0.05)。2.轉染4小時后,經(jīng)過鏡下觀察并計算轉染效率,結果表明轉染成功,可用于后續(xù)實驗。3.轉染48小時后,流式細胞儀和TUNEL法凋亡檢測結果表明高表達mi R-15a和mi R-16-1的SOSP-9607細胞凋亡率較抑制物組和對照組相比,顯著增加(P0.05)。流式細胞儀周期分析結果顯示轉染后處于G0/G1期的細胞明顯多于對照組,說明細胞周期減緩(P0.05)。以MTT法繪制的細胞增殖曲線顯示轉染后的腫瘤細胞增殖速度較其他組有所減緩(P0.05)。上述實驗中,抑制物組和對照組的組間和組內結果對比均無差異(P0.05)。4.經(jīng)過基因信息學軟件篩選,文獻回顧分析以及RT-PCR的初步篩查,最終確定待研究靶基因為CCND1。5.熒光素酶報告基因分析結果揭示出mi R-15a和mi R-16-1可以抑制CCND1-b組的熒光強度(P0.05),而CCND1-a組則不受影響(P0.05)。說明mi R-15a和mi R-16-1通過與CCND1基因3’UTR區(qū)域的CCND1-b段結合抑制CCND1基因的表達。結論:mi R-15a和mi R-16-1可以通過抑制CCND1基因表達促進SOSP-9607細胞凋亡、阻抑細胞周期,抑制細胞增殖。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R738.1
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本文編號：1825844