【摘要】：目的:骨肉瘤是一種臨床常見的好發(fā)于青少年的成骨性惡性腫瘤,它惡性程度高,預(yù)后比較差。隨著化學(xué)療法、手術(shù)技術(shù)、骨重建等方法的進(jìn)展,5年總生存率明顯提高。阿霉素(adriamycin,ADM)、順鉑(diamminedichloroplantinum,DDP)是抗骨肉瘤的最有效藥物,但腫瘤細(xì)胞對化療藥物產(chǎn)生耐藥性是影響化療療效的主要原因。許多化療藥物通過誘導(dǎo)凋亡抗腫瘤生長。XIAP是凋亡調(diào)節(jié)因子家族中對凋亡調(diào)節(jié)作用最強的分子之一,同時是唯一的caspases抑制劑。研究顯示它在多種惡性腫瘤中上調(diào)表達(dá),并與腫瘤化療耐藥及預(yù)后差緊密相關(guān)。RNAi是近年來發(fā)展起來的新技術(shù),si RNA已經(jīng)成為繼反義核酸、核酶之后基因治療的又一新武器。為此,我們合成特異性干擾XIAP基因的si RNA,并在脂質(zhì)體介導(dǎo)下轉(zhuǎn)染骨肉瘤MG-63細(xì)胞,觀察其對骨肉瘤細(xì)胞生長抑制以及細(xì)胞侵襲性的影響,以及對骨肉瘤MG-63細(xì)胞ADM藥物敏感性及耐藥性的影響,從而為新的骨肉瘤的治療方法提供實驗依據(jù)。方法:1根據(jù)XIAP基因已知序列化學(xué)合成1條含21-23個堿基的si RNA(XIAP-si RNA)及陰性對照(si RNA-neg);用化學(xué)合成的XIAP-si RNA轉(zhuǎn)染骨肉瘤MG-63細(xì)胞,通過RT-PCR方法和流式細(xì)胞術(shù)檢測轉(zhuǎn)染前后MG-63細(xì)胞XIAP-m RNA和蛋白水平的表達(dá)情況,進(jìn)一步觀察轉(zhuǎn)染前后骨肉瘤MG-63細(xì)胞增殖抑制的改變,流式細(xì)胞術(shù)和MTT法進(jìn)一步檢測轉(zhuǎn)染前后MG-63細(xì)胞的凋亡及ADM對轉(zhuǎn)染前后骨肉瘤MG-63細(xì)胞的半數(shù)抑制濃度(IC50),觀察化學(xué)合成的特異性XIAP-si RNA對骨肉瘤細(xì)胞增殖抑制的影響以及對骨肉瘤MG-63細(xì)胞ADM藥物敏感性的影響。2統(tǒng)計學(xué)方法采用SPSS 13.0進(jìn)行分析處理,采用t檢驗和單因素方差分析,如多組間兩兩比較采用q檢驗,以P0.05認(rèn)為差異有統(tǒng)計學(xué)意義。1 XIAP-si RNA對骨肉瘤MG-63細(xì)胞增殖有抑制作用,使細(xì)胞周期阻滯發(fā)生在細(xì)胞周期的G0/1期,并能誘導(dǎo)凋亡。2 XIAP-si RNA降低了XIAP m RNA和蛋白的表達(dá)水平。3 XIAP-si RNA聯(lián)合ADM作用的細(xì)胞抑制率達(dá)(63.1±2.1)%,流式細(xì)胞儀分析顯示XIAP-si RNA組細(xì)胞的凋亡率明顯高于轉(zhuǎn)染非特異性si RNA組、空載組及未轉(zhuǎn)染組。結(jié)論:1特異性的化學(xué)合成XIAP-si RNA能夠下調(diào)骨肉瘤MG-63細(xì)胞XIAP基因和蛋白表達(dá),阻滯細(xì)胞周期于G0/1期和誘導(dǎo)凋亡。2特異性的化學(xué)合成XIAP-si RNA能增強骨肉瘤MG-63細(xì)胞對ADM的敏感性。RNAi技術(shù)有望成為腫瘤基因靶向治療的一種新的途徑。結(jié)果:
[Abstract]:Objective: osteosarcoma is a common osteogenic malignant tumor in adolescents, which has a high degree of malignancy and poor prognosis. With the progress of chemotherapy, surgical techniques and bone reconstruction, the 5-year overall survival rate improved significantly. Adriamycin (adriamycin,ADM) and cisplatin (diamminedichloroplantinum,DDP) are the most effective drugs against osteosarcoma. Many chemotherapeutic drugs are one of the most powerful apoptosis-regulating molecules in the family of apoptosis-regulating factors, and are the only caspases inhibitors. It has been shown that it is up-regulated in various malignant tumors and closely related to chemotherapy resistance and poor prognosis. RNAi, a new technique developed in recent years, has become a new weapon in gene therapy after antisense nucleic acid and ribozyme. Therefore, we synthesized si RNA, that specifically interfered with XIAP gene and transfected MG-63 cells into osteosarcoma MG-63 cells mediated by liposome, and observed its effects on the growth inhibition and cell invasion of osteosarcoma cells. The drug sensitivity and drug resistance of ADM in osteosarcoma MG-63 cells were also studied, so as to provide experimental evidence for the treatment of osteosarcoma. Methods one si RNA (XIAP-si RNA containing 21-23 bases and one negative control (si RNA-neg) were chemically synthesized according to the known sequence of XIAP gene, and then transfected into MG-63 cells of osteosarcoma by chemically synthesized XIAP-si RNA. The expression of XIAP-m RNA and protein in MG-63 cells before and after transfection was detected by RT-PCR and flow cytometry. The inhibition of proliferation of MG-63 cells in osteosarcoma was further observed before and after transfection. Flow cytometry and MTT were used to detect the apoptosis of MG-63 cells before and after transfection and the half inhibitory concentration (IC50) of ADM on osteosarcoma MG-63 cells before and after transfection. The effects of chemically synthesized specific XIAP-si RNA on the proliferation inhibition of osteosarcoma cells were observed. And the effect on the drug sensitivity of ADM in osteosarcoma MG-63 cells. 2 the SPSS 13.0 method was used to analyze the drug sensitivity of osteosarcoma cells. T test and single factor analysis of variance were used, such as Q test for comparison of multiple groups. The results showed that the difference was statistically significant (P0.05). 1 XIAP-si RNA could inhibit the proliferation of MG-63 cells of osteosarcoma, resulting in cell cycle arrest in G0 / 1 phase of cell cycle. Apoptosis induced by 2. 2 XIAP-si RNA decreased the expression level of XIAP m RNA and protein. 3. 3 XIAP-si RNA combined with ADM could inhibit the apoptosis of the cells (63.1 鹵2. 1). Flow cytometry analysis showed that the apoptosis rate of XIAP-si RNA group was significantly higher than that of non specific si RNA transfected group. No load group and no transfection group. Conclusion the specific chemical synthesis of XIAP-si RNA can down-regulate the expression of XIAP gene and protein in osteosarcoma MG-63 cells. Blocking cell cycle at G0 / 1 phase and inducing apoptosis. 2 specific chemical synthesis of XIAP-si RNA can enhance the sensitivity of osteosarcoma MG-63 cells to ADM. RNAi technique may be a new approach to target tumor gene therapy. Results:
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R738.1

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相關(guān)期刊論文 前2條

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本文編號：2222024