發(fā)布時(shí)間：2018-07-05 18:41

  本文選題：膠質(zhì)瘤 + HSP90��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：目的本研究對(duì)膠質(zhì)瘤組織及正常腦組織中HSP90蛋白的表達(dá)、細(xì)胞增殖和凋亡進(jìn)行測(cè)定,分析HSP90與膠質(zhì)瘤患者臨床病理特征的相關(guān)性及其對(duì)細(xì)胞增殖和凋亡的影響,以探討姜黃素靶向調(diào)控HSP90抑制膠質(zhì)瘤細(xì)胞增殖和侵襲的作用機(jī)制,旨在為膠質(zhì)瘤治療提供理論基礎(chǔ)及新的思路,同時(shí)也為尋找新的HSP90抑制劑治療膠質(zhì)瘤奠定基礎(chǔ)。方法(1)采集50例神經(jīng)膠質(zhì)瘤組織樣本,其中男性28例,女性22例。年齡在23-78歲之間,平均為37.55±11.2歲；所有膠質(zhì)瘤患者中,WHO I級(jí)6例,II級(jí)8例,III級(jí)17例,IV級(jí)19例；同時(shí)設(shè)立經(jīng)CT和MRI等影像學(xué)檢測(cè)證實(shí)無任何腫瘤性疾病的10例由于外傷或者腦出血而進(jìn)行顱內(nèi)減壓的正常腦組織樣本作為對(duì)照。運(yùn)用western blot法分別檢測(cè)上述樣本中HSP90蛋白的表達(dá)情況,分析HSP90的表達(dá)與臨床病理分級(jí)的相關(guān)性。(2)將HSP90 siRNA轉(zhuǎn)染膠質(zhì)瘤T98G細(xì)胞用以抑制其表達(dá),熒光定量PCR檢測(cè)轉(zhuǎn)染后細(xì)胞中HSP90 mRNA的表達(dá)情況；MTT法檢測(cè)轉(zhuǎn)染后細(xì)胞的增殖情況；流式細(xì)胞術(shù)檢測(cè)轉(zhuǎn)染后細(xì)胞的凋亡情況。(3)體外malachite green-molybdate顯色反應(yīng)以及ATP-瓊脂糖凝膠結(jié)合實(shí)驗(yàn)檢測(cè)姜黃素對(duì)HSP90的抑制活性,同時(shí)分別運(yùn)用MTT法、Transwell小室法檢測(cè)姜黃素對(duì)T98G細(xì)胞增殖和侵襲的影響,此外通過western blot法檢測(cè)姜黃素對(duì)基質(zhì)金屬蛋白9(MMP-9)以及HSP90表達(dá)的影響。結(jié)果(1)在膠質(zhì)瘤組織中HSP90蛋白表達(dá)量顯著高于其在正常腦組織中的表達(dá)(P0.01),且隨著病理級(jí)別的增高其表達(dá)量呈上升趨勢(shì)；運(yùn)用Spearman軟件分析膠質(zhì)瘤患者臨床病理分級(jí)與HSP90蛋白表達(dá)的相關(guān)性,結(jié)果為：HSP90蛋白表達(dá)的相關(guān)系數(shù)r=0.713(P0.001),表明膠質(zhì)瘤組織中HSP90蛋白表達(dá)與臨床病理分級(jí)呈正相關(guān),即在高級(jí)別病例組中高表達(dá),在低級(jí)別病例組中低表達(dá)。(2)隨著時(shí)間的延長(zhǎng),HSP90 siRNA作用后T98G細(xì)胞的增殖能力逐漸下降。當(dāng)作用第3、4、5、6天時(shí),siHSP90組T98G細(xì)胞的增殖活性較對(duì)照組均有下降,其中第4、5、6天時(shí)降低幅度顯著,差異具有顯著性統(tǒng)計(jì)學(xué)意義(P0.05)；對(duì)照組細(xì)胞的凋亡率為(8.35±1.27)%,siHSP90組細(xì)胞的凋亡率為(17.88±0.13)%,兩組相比差異具有高度顯著性統(tǒng)計(jì)學(xué)意義(P0.01)。(3)姜黃素能夠抑制HSP90重組蛋白的ATPase活性,其活性的IC50值為83.3μ/mol;姜黃素能夠抑制HSP90蛋白與ATP-瓊脂糖凝膠株結(jié)合,且這種結(jié)合抑制作用隨姜黃素濃度的增高而增強(qiáng)；T98G細(xì)胞增殖能力隨著姜黃素濃度的增高而逐漸降低,即姜黃素濃度越高其抑制T98G細(xì)胞增殖的作用越明顯,呈劑量依賴性；T98G細(xì)胞的侵襲能力隨著姜黃素濃度的增高而逐漸降低；MMP-9蛋白的表達(dá)量隨著姜黃素濃度的增高而逐漸降低,且呈劑量依賴性,而HSP90蛋白的表達(dá)則未發(fā)生明顯變化。結(jié)論姜黃素能夠顯著抑制膠質(zhì)瘤細(xì)胞增殖以及侵襲能力,其機(jī)制可能是通過抑制HSP90 ATPase活性致使其下游顧客蛋白MMP-9的表達(dá)量下降有關(guān)。
[Abstract]:Objective to investigate the expression of HSP90 protein, cell proliferation and apoptosis in glioma and normal brain tissues, and to analyze the relationship between HSP90 and clinicopathological features of glioma and its effect on cell proliferation and apoptosis. In order to explore the mechanism of curcumin targeting regulation of HSP90 on the proliferation and invasion of glioma cells, the aim is to provide a theoretical basis and new ideas for the treatment of glioma, and to find a new HSP90 inhibitor for the treatment of glioma. Methods (1) 50 cases of gliomas were collected, including 28 males and 22 females. The age ranged from 23 to 78 years old (mean 37.55 鹵11.2 years). Among all the glioma patients, there were 6 cases of WHO grade I, 8 cases of grade II, 17 cases of grade III and 19 cases of grade IV. At the same time, 10 cases of normal brain tissues with intracranial decompression due to trauma or intracerebral hemorrhage, which were confirmed by CT and MRI, were used as control group. The expression of HSP90 protein was detected by western blot method, and the correlation between HSP90 expression and clinicopathological grade was analyzed. (2) HSP90 siRNA was transfected into glioma T98G cells to inhibit the expression of HSP90 protein. The expression of HSP90 mRNA in transfected cells was detected by fluorescence quantitative PCR and the proliferation of transfected cells was detected by MTT assay. Flow cytometry was used to detect the apoptosis of transfected cells. (3) in vitro malachite green-molybdate reaction and ATP-agarose gel binding assay were used to detect the inhibitory activity of curcumin on HSP90. The effects of curcumin on the proliferation and invasion of T98G cells were detected by MTT assay and transwell chamber assay respectively. The effects of curcumin on the expression of matrix metalloprotein 9 (MMP-9) and HSP90 were detected by western blot assay. Results (1) the expression of HSP90 protein in glioma tissues was significantly higher than that in normal brain tissues (P0.01), and the expression of HSP90 protein increased with the increase of pathological grade. The correlation between the expression of HSP90 protein and the clinicopathological grade of glioma was analyzed by Spearman software. The results showed that the expression of HSP90 protein in glioma tissues was positively correlated with the clinicopathologic grade, and the correlation coefficient of the expression of HSP90 protein was rn0.713 (P0.001). That is, high expression in high grade cases, and low expression in low grade cases. (2) the proliferation ability of T98G cells decreased gradually with time prolonging after HSP90 siRNA treatment. The proliferation activity of T98G cells in HSP90 group was significantly lower than that in the control group on the 3rd day (P 0.05), and significantly decreased on the 4th day (P 0.05), while on the 6th day after treatment, the proliferation activity of T98G cells in HSP90 group was significantly lower than that in the control group (P 0.05). The apoptosis rate of the control group was (8.35 鹵1.27) and that of the control group was (17.88 鹵0.13). The difference between the two groups was statistically significant (P0.01). (3) curcumin could inhibit the ATPase activity of HSP90 recombinant protein. The IC50 of HSP90 protein was 83.3 渭 / mol. Curcumin could inhibit the binding of HSP90 protein to ATP-agarose gel strain, and the inhibitory effect increased with the increase of curcumin concentration, the proliferation ability of T98G cells decreased with the increase of curcumin concentration. The higher the curcumin concentration, the more obvious the inhibitory effect of curcumin on the proliferation of T98G cells. The invasion ability of T98G cells decreased with the increase of curcumin concentration, and the expression of MMP-9 protein decreased with the increase of curcumin concentration. The expression of HSP90 protein did not change in a dose-dependent manner. Conclusion curcumin can significantly inhibit the proliferation and invasion of glioma cells by inhibiting the activity of HSP90 ATPase resulting in the decrease of MMP-9 expression in the downstream of glioma cells.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.41

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