本文選題：膠質(zhì)瘤　切入點(diǎn)：增殖　出處：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的膠質(zhì)瘤呈浸潤(rùn)性生長(zhǎng),具有高度侵襲性,惡性膠質(zhì)瘤患者病死率高,并且缺乏有效的治療手段。新型CDK7抑制劑THZ1含有一個(gè)丙烯酰胺結(jié)構(gòu),能共價(jià)結(jié)合到傳統(tǒng)激酶區(qū)域外的半胱氨酸殘基上,是首個(gè)被報(bào)道的共價(jià)不可逆CDK抑制劑。THZ1在小細(xì)胞肺癌、三陰性乳腺癌、神經(jīng)母細(xì)胞瘤、T細(xì)胞急淋和其他造血系統(tǒng)腫瘤中顯示了較好療效,其能夠抑制多種癌基因的轉(zhuǎn)錄,阻滯細(xì)胞周期進(jìn)展,抑制增殖,誘發(fā)凋亡。本研究探討THZ1對(duì)人膠質(zhì)瘤細(xì)胞系U87及U251的增殖、凋亡、侵襲能力的影響及細(xì)胞周期的阻滯作用,進(jìn)一步探索THZ1的功能及其抗膠質(zhì)瘤的可能機(jī)制。方法將U87及U251細(xì)胞分成空白組、對(duì)照組及實(shí)驗(yàn)組,對(duì)于U87細(xì)胞,實(shí)驗(yàn)組分別采用終濃度為 25nmol/L、50 nmol/L、100 nmol/L、200 nmol/L、400 nmol/L的THZ1進(jìn)行處理,對(duì)于U251細(xì)胞實(shí)驗(yàn)組分別采用終濃度為5 nmol/L、10 nmol/L、20 nmol/L、40 nmol/L、80 nmol/L的THZ1進(jìn)行處理,空白組為加入等量的DMEM完全培養(yǎng)基,溶劑對(duì)照組(DMSO組)為含THZ1工作液中等量的DMS0的DMEM完全培養(yǎng)基。實(shí)驗(yàn)組加入不同濃度的THZ1處理后,以CCK-8法及平板克隆形成試驗(yàn)分析細(xì)胞增殖;Transwell侵襲試驗(yàn)觀測(cè)各組侵襲能力的變化;流式細(xì)胞儀檢測(cè)各組細(xì)胞周期分布及凋亡情況;Western blot實(shí)驗(yàn)檢測(cè)凋亡相關(guān)蛋白Caspase-3的表達(dá)量。結(jié)果 1.THZ1抑制U87及U251的增殖,72小時(shí)藥物IC50,U87細(xì)胞為83.1nmol/L、U251細(xì)胞為13.7nmol/L。平板克隆形成試驗(yàn)結(jié)果顯示對(duì)照組克隆形成率為(55.26±8.81)%,5nmol/L處理組的克隆形成率為(19.34±3.41)%,與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(t=6.586,P0.01),10nmol/L處理組未見明顯克隆形成,結(jié)果顯示低劑量THZ1即可明顯抑制U251細(xì)胞的單克隆形成率。2.膠質(zhì)瘤細(xì)胞U87具有較強(qiáng)的侵襲能力,通過體外transwell侵襲實(shí)驗(yàn)發(fā)現(xiàn),對(duì)照組細(xì)胞數(shù)為134±13個(gè),50nmol/L處理組細(xì)胞數(shù)為72±7個(gè),與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(t=7.141,P0.01),給予50nmol/L THZ1處理24小時(shí)后,其侵襲能力明顯減弱。所以,THZ1能明顯的降低膠質(zhì)瘤細(xì)胞的侵襲性。3.THZ1促進(jìn)U251細(xì)胞凋亡,Caspase-3表達(dá)增加。對(duì)照組凋亡率為(8.3±2.5)%,THZ1 處理 10nmol/L 組凋亡率為(15.6±3.7)%,20nmol/L 組凋亡率為(39.7±6.0)%,凋亡率隨著THZ1濃度的升高而增加。實(shí)驗(yàn)組與對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(t分別為2.832和8.367,P0.05)。Western blot結(jié)果顯示,凋亡相關(guān)蛋白Caspase-3表達(dá)隨著THZ1濃度升高而明顯增加。4.THZ1 阻滯U87及U251細(xì)胞周期進(jìn)展,G2期細(xì)胞顯著增加。流式細(xì)胞儀檢測(cè)結(jié)果顯示使用濃度為25、50、100nmol/L的THZ1處理U87細(xì)胞24h后,G2期隨著藥物濃度的增加,細(xì)胞比例逐漸增加,(分別為31.26± 1.97、34.09±2.09、41.22±2.40)與對(duì)照組(17.95±2.83)比較差異有統(tǒng)計(jì)學(xué)意義(P0.01)。同樣,使用濃度為5、10、20nmol/L的THZ1處理U251細(xì)胞24h后,G2期隨著藥物濃度的增加,細(xì)胞比例逐漸增加,(分別為9.27±2.01、41.35±0.73、56.92±0.69)與對(duì)照組(16.17±1.99)比較差異有統(tǒng)計(jì)學(xué)意義(P㩳0.01)。結(jié)論1.THZ1能夠抑制膠質(zhì)瘤細(xì)胞U87及U251的增殖,促進(jìn)凋亡,抑制侵襲,并阻滯細(xì)胞周期進(jìn)展。2.新型CDK抑制劑THZ1有望成為神經(jīng)膠質(zhì)瘤的靶向藥物
[Abstract]:Objective glioma infiltrative growth, highly invasive malignant glioma patients, mortality rate is high, and the lack of effective treatment. A novel CDK7 inhibitor THZ1 has a structure of acrylamide, covalently bound to cysteine residues to traditional kinase outside the region, is the first reported covalent irreversible CDK inhibitor.THZ1 in small cell lung cancer, three breast cancer, neuroblastoma, T cell acute lymphocytic leukemia and other hematopoietic tumors showed better curative effect, it can inhibit transcription of oncogenes, progress, cell cycle arrest and inhibit proliferation and induced apoptosis. This study investigated the effect of THZ1 on human glioma cell lines U87 and U251 the proliferation, apoptosis, invasion and inhibition effect of cell cycle, can further explore the mechanism of THZ1 function and anti glioma. Methods U87 and U251 cells were divided into blank group, control group and For the experimental group, U87 cells, experimental group respectively with the final concentration of 25nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L, 400 nmol/L THZ1 treatment for U251 cells in the experimental group were used at the concentration of 5 nmol/L, 10 nmol/L, 20 nmol/L, 40 nmol/L, 80 nmol/L THZ1 were treated as blank group adding the same amount of DMEM complete medium, solvent control group (group DMSO) an THZ1 containing working fluid in DMS0 DMEM complete medium. Different concentration in experimental group after THZ1 treatment, CCK-8 assay and colony formation test and analysis of changes of Transwell cell proliferation; invasion capability test observed the distribution of cells; each cycle and apoptosis was detected by flow cytometry; the expression of Western blot assay of apoptosis related protein Caspase-3. Results 1.THZ1 inhibited the proliferation of U251 and U87, 72 hours of drug IC50, U87 cell 83.1nmol/L, U251 cell 13.7nmol/L. colony formation test showed that the control group clone formation rate was (55.26 + 8.81)%, the treatment group 5nmol/L clone formation rate was (19.34 + 3.41)%, compared with the control group the difference was statistically significant (t=6.586, P0.01), 10nmol/L treatment group had no obvious colony formation, results showed that the monoclonal low dose of THZ1 can be inhibit U251 cell formation rate of glioma cells.2. U87 has strong ability of invasion in vitro by Transwell invasion assay showed that the cells in the control group were 134 + 13, in 50nmol/L treated cells was 72 + 7, compared with the control group the difference was statistically significant (t=7.141, P0.01), 50nmol/L THZ1 24 hours later, the invasion ability decreased. Therefore, THZ1 can significantly reduce the invasiveness of glioma cells.3.THZ1 promote U251 cell apoptosis, increase the expression of Caspase-3. The apoptosis rate of the control group (8.3 + 2.5)%, THZ1 The apoptosis rate of 10nmol/L group was (15.6 + 3.7)%, the apoptosis rate of 20nmol/L group was (39.7 + 6)%, the apoptosis rate increased with the increase of THZ1 concentration. Compared with the control group, the difference was statistically significant (t = 2.832 and 8.367, P0.05).Western blot results showed that the apoptosis related protein Caspase-3 the expression of THZ1 with the increased concentration of.4.THZ1 U87 increased significantly in U251 and block the cell cycle, a significant increase in G2 phase cells. Flow cytometry results showed that the concentration of THZ1 in U87 cells treated with 24h 25,50100nmol/L, G2 phase with the increase of drug concentration, the proportion of cells increased gradually, respectively (31.26 + 1.97,34.09 + 2.09,41.22 + 2.40) and control group (17.95 + 2.83) the difference was statistically significant (P0.01). Similarly, the use of concentration of THZ1 in U251 cells treated with 24h 5,10,20nmol/L, G2 phase with the increase of drug concentration, the proportion of cells increased gradually ( Were 9.27 + 2.01,41.35 + 0.73,56.92 + 0.69) and control group (16.17 + 1.99) and there was a statistically significant difference (P? 0.01). Conclusion: 1.THZ1 can inhibit U87 and U251 glioma cell proliferation, promote apoptosis, inhibit the invasion, and block the cell cycle progression of.2. CDK THZ1 is expected to become a new inhibitor of glioma the targeted drug

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 何潔;張大鵬;何靜;;替莫唑胺對(duì)比傳統(tǒng)化療藥治療高級(jí)別腦膠質(zhì)瘤的臨床療效[J];現(xiàn)代腫瘤醫(yī)學(xué);2015年17期

2 陳麗君;王建;范春芳;;CDK5逆轉(zhuǎn)Sirt1在宮頸癌細(xì)胞耐藥中的作用機(jī)制[J];現(xiàn)代腫瘤醫(yī)學(xué);2014年11期

3 張敬寧;賴年升;張帥;成宜軍;徐幸;藺玉昌;;不同起源膠質(zhì)瘤中microRNA-210表達(dá)差異及其與星形膠質(zhì)細(xì)胞腫瘤級(jí)別的相關(guān)性[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2014年07期

4 郝棟;胡世頡;董琛;;miRNA調(diào)控腦膠質(zhì)瘤癌基因表達(dá)研究進(jìn)展[J];中華神經(jīng)外科疾病研究雜志;2013年05期

，

本文編號(hào)：1657166