發(fā)布時間：2018-03-03 02:13

  本文選題：橫紋肌肉瘤　切入點：HDAC　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：橫紋肌肉瘤(rhabdomyosarcoma,RMS)是臨床上兒童常見的軟組織肉瘤,起源于骨骼肌細(xì)胞,約占小兒惡性實體瘤的10%左右,占兒童期所有軟組織肉瘤50%~70%。隨著在臨床上綜合治療的應(yīng)用,其預(yù)后已有了明顯提高,5年總生存率達(dá)到33%~50%,但仍有超過50%的患者死于腫瘤復(fù)發(fā)或遠(yuǎn)處轉(zhuǎn)移進(jìn)展。在我國,兒童橫紋肌肉瘤的患者人口基數(shù)大,總體治療效果雖然有所改善,但是其遠(yuǎn)期治愈率并不令人滿意。目前對于RMS的發(fā)病機(jī)制有了許多進(jìn)展,對于其綜合治療方法的改善有一定的幫助。但是對于復(fù)發(fā)的患者,目前主要依靠施行化療,但化療的毒副反應(yīng)則限制了其在兒童患者的使用。因此目前迫切需要治療橫紋肌肉瘤的新型藥物出現(xiàn)。研究報道腫瘤細(xì)胞中組蛋白往往呈低乙酰化水平,并且組蛋白去乙�；�(HDAC)活性異常造成的組蛋白乙酰化狀態(tài)失衡與腫瘤的發(fā)生發(fā)展有密切關(guān)系。HDAC抑制劑SAHA能可逆性地逆轉(zhuǎn)組蛋白乙酰化狀態(tài),從而調(diào)節(jié)基因表達(dá)、促進(jìn)凋亡、細(xì)胞周期的阻滯、抗血管生成、DNA的損傷和修復(fù)以及減弱腫瘤細(xì)胞的侵襲和遷移。因此SAHA有望成為治療橫紋肌肉瘤的一種新型靶向藥物。因此本課題的目的是研究SAHA對人橫紋肌肉瘤RD細(xì)胞增殖、細(xì)胞周期、凋亡、自噬和遷移能力的影響,并探索其誘導(dǎo)凋亡的機(jī)制。方法:不同濃度SAHA作用于RD細(xì)胞,MTT法檢測對細(xì)胞增殖作用的影響,western blot檢測ERK信號通路蛋白表達(dá);PI染色法分析細(xì)胞周期;Annexin V-FITC/PI雙染法檢測細(xì)胞凋亡,DAPI染色法驗證細(xì)胞凋亡,然后進(jìn)行以下實驗初步探討誘導(dǎo)凋亡的機(jī)制:用JC-1探針檢測線粒體膜電位的變化;western blot檢測凋亡相關(guān)蛋白、線粒體途徑相關(guān)蛋白的變化;MDC染色法檢測細(xì)胞自噬,western blot檢測自噬相關(guān)的蛋白的表達(dá);細(xì)胞劃痕檢測細(xì)胞遷移能力;western blot檢測細(xì)胞組蛋白H4的乙�；健＝Y(jié)果:1.saha抑制了rd細(xì)胞的增殖,并且具有時間和濃度依賴性。saha作用于rd細(xì)胞24h、48h、72h的半數(shù)抑制率ic50值分別為8.503μmol//l、3.965μmol/l和1.921μmol/l;與rd細(xì)胞增殖相關(guān)erk信號通路的變化是:erk蛋白磷酸化水平下降,而總erk蛋白水平?jīng)]有顯著的變化;2.流式細(xì)胞術(shù)結(jié)果顯示saha使rd細(xì)胞發(fā)生g2/m期阻滯;3.annexinv-fitc/pi雙染法結(jié)果顯示saha能夠誘導(dǎo)rd細(xì)胞凋亡;dapi染色法實驗表明,隨著saha濃度的增加,凋亡細(xì)胞的數(shù)量隨之增加,jc-1流式細(xì)胞術(shù)結(jié)果顯示,隨著saha濃度的增高,rd細(xì)胞線粒體膜電位水平逐漸降低;westernblot結(jié)果顯示:隨著saha濃度的增高,凋亡相關(guān)蛋白caspase-3和parp-1表達(dá)量增高,抗凋亡蛋白bcl-2表達(dá)量逐漸減低,促凋亡蛋白bax表達(dá)量逐漸增加,細(xì)胞色素c和caspase-9的表達(dá)也均有所增加;4.saha使rd細(xì)胞發(fā)生自噬:mdc染色結(jié)果顯示隨著saha濃度的增高,細(xì)胞內(nèi)的高亮點狀綠色熒光的斑點數(shù)量逐漸增多,表明細(xì)胞發(fā)生自噬。westernblot結(jié)果顯示:隨著saha濃度的增高,p62蛋白表達(dá)逐漸減少,beclin1蛋白表達(dá)水平逐漸增高,lc3-Ⅱ蛋白的表達(dá)也顯著增高。5.saha抑制rd細(xì)胞的遷移:細(xì)胞劃痕實驗結(jié)果顯示:隨著saha濃度的增高,劃痕愈合率逐漸降低。6.westernblot結(jié)果顯示:隨著saha濃度的增高,組蛋白h4的乙酰化水平逐漸增高。結(jié)論:1.hdac抑制劑saha抑制rd細(xì)胞的增殖,使rd細(xì)胞發(fā)生g2/m期阻滯,該抑制作用具有時間和濃度依賴性;2.hdac抑制劑saha誘導(dǎo)rd細(xì)胞的凋亡,介導(dǎo)凋亡的機(jī)制可能為依賴于線粒體途徑和具有caspase依賴性;3.hdac抑制劑saha誘導(dǎo)rd細(xì)胞自噬;4.hdac抑制劑saha抑制rd細(xì)胞的遷移能力。
[Abstract]:Rhabdomyosarcoma (rhabdomyosarcoma, RMS) is a clinically common soft tissue sarcoma, originated in skeletal muscle cells, accounting for about 10% of pediatric malignant solid tumor in childhood, accounting for all soft tissue sarcoma 50%~70%. with the application of comprehensive treatment in clinic, its prognosis has been improved obviously, 5 year survival rate of 33%~50%. But there are still more than 50% of the patients died of tumor recurrence or distant metastasis. In our country, children with rhabdomyosarcoma of the large population, the treatment effect was improved, but the long-term cure rate is not satisfactory. The pathogenesis of RMS has a lot of progress, for the comprehensive treatment can be improved help. But for patients who relapse, currently rely mainly on the implementation of chemotherapy, but the toxicity of chemotherapy is limited by its use in pediatric patients. So there is an urgent need for treatment The new drug research group reported rhabdomyosarcoma. Tumor cells often showed low protein acetylation, and histone deacetylase (HDAC) activity in the occurrence and development of abnormal histone acetylation imbalance and tumor is closely related to the.HDAC inhibitor SAHA can reversibly reverse histone acetylation, thereby regulating gene the expression of apoptosis, cell cycle arrest, anti angiogenesis, DNA damage and repair and weaken the invasion and migration of tumor cells. Therefore, SAHA is expected to become a new target for the treatment of rhabdomyosarcoma to drugs. So the purpose of this paper is to study the proliferation of SAHA on human rhabdomyosarcoma RD cells cell cycle, apoptosis, autophagy effect and migration, and to explore the mechanism of apoptosis. Methods: different concentrations of SAHA in RD cells. The effect of MTT method to detect cell proliferation, weste Expression of ERK signaling pathway protein RN blot; cell cycle analysis of PI staining method; Annexin V-FITC/PI method to detect cell apoptosis, apoptotic cells verified by DAPI staining, then the following experiments: preliminary study on the mechanism of apoptosis induced by changes in mitochondrial membrane potential detection JC-1 probe; Western blot detection of apoptosis related protein, mitochondrial pathway related changes protein; autophagy was detected by MDC staining, the expression of Western blot was used to detect the protein; cell scratch detection cell migration; acetylation level of histone H4 Western were detected by blot. Results: 1.saha inhibited the proliferation of RD cells, and has a time and concentration dependent effect of.Saha on RD 24h cells. 48h 72h, the half inhibition rate of IC50 was 8.503 mol//l, 3.965 mol/l and 1.921 mol/l; and RD cell proliferation and ERK signal pathway changes: ERK protein phosphorylation The level of decline, but no significant changes in the total protein level of ERK 2.; flow cytometry showed that RD Saha cells g2/m phase arrest; 3.annexinv-fitc/pi staining showed that Saha could induce Rd cell apoptosis; DAPI staining showed that the experiment, with the increase of SAHA concentration, the number of apoptotic cells increased JC-1 flow cytometry showed that with the increase of SAHA concentration, the level of RD cells mitochondrial membrane potential gradually decreased; Westernblot results showed that with the increase of SAHA concentration, apoptosis related protein caspase-3 and PARP-1 expression and anti apoptosis protein bcl-2 expression reduced gradually, the pro apoptotic protein Bax expression increased gradually, the increased expression of cytochrome c and caspase-9 are 4.saha; the autophagy in RD cells: MDC staining results show that with the increase of SAHA concentration, the number of spots of green fluorescent cells within the bright dot Gradually increased, indicating that cell autophagy.Westernblot results showed that with the increase of SAHA concentration, p62 protein expression decreased, Beclin1 protein expression gradually increased, the expression of lc3- protein also increased significantly inhibited Rd cell migration of.5.saha: the experimental results show that the node cell scratch with the increasing of SAHA concentration, the wound healing rate decreased.6.westernblot the results showed that with the increase of SAHA concentration, the acetylation level of histone H4 increased. Conclusion: 1.hdac inhibitor Saha inhibited the proliferation of RD cells, the g2/m phase arrest in RD cells, the inhibition is time and concentration dependent manner; the apoptosis of RD cells induced by 2.hdac inhibitor Saha mediated apoptosis, the possible mechanism for depending on the mitochondrial pathway and dependent on caspase; 3.hdac inhibitor Saha induces autophagy in RD cells; the migration ability of 4.hdac inhibitor Saha inhibited Rd cells.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R738.7
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本文編號：1559090