本文關(guān)鍵詞： 肝細(xì)胞癌 RBM8A 細(xì)胞增殖 細(xì)胞凋亡 細(xì)胞侵襲 細(xì)胞周期　出處：《廣西醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：一、RBM8A與人肝細(xì)胞癌相關(guān)性的臨床初步研究目的:有研究顯示RBM8A與惡性腫瘤的發(fā)生與發(fā)展具有密切相關(guān)性。然而,RBM8A與肝細(xì)胞癌的相關(guān)性尚未有明確報(bào)道。本研究旨在首先通過(guò)檢測(cè)RBM8A在肝細(xì)胞癌組織、相關(guān)癌旁組織、肝血管瘤組織中的表達(dá)水平的差異,以及檢測(cè)肝細(xì)胞癌患者與肝硬化患者、正常體檢人群血清樣本中RBM8A的表達(dá)差異,初步探討RBM8A與肝細(xì)胞癌發(fā)生相關(guān)性、以及RBM8A的表達(dá)水平與肝細(xì)胞癌患者腫瘤學(xué)特征及者預(yù)后的相關(guān)性。分析將RBM8A作為肝細(xì)胞癌輔助診斷指標(biāo)及預(yù)后預(yù)測(cè)指標(biāo)的潛在可能性。方法:收集廣西醫(yī)科大學(xué)附屬腫瘤醫(yī)院2012年3月至2014年12月間經(jīng)手術(shù)切除并經(jīng)術(shù)后病理證實(shí)為肝細(xì)胞癌的組織105例,并收集相應(yīng)距離腫瘤邊緣2cm處的癌旁組織。另收集經(jīng)手術(shù)切除并經(jīng)術(shù)后病理證實(shí)為肝血管瘤的組織67例。運(yùn)用qRT-PCR法、Western Blot法、免疫組織化學(xué)法檢測(cè)并分析rbm8a的mrna及蛋白在肝細(xì)胞癌組織、相應(yīng)癌旁組織、及肝血管瘤組織中的表達(dá)差異;根據(jù)rbm8a在肝細(xì)胞癌組織中的mrna含量及蛋白表達(dá)狀況,將肝細(xì)胞癌患者分成高表達(dá)組與低表達(dá)組,初步分析rbm8a的表達(dá)量和肝細(xì)胞癌患者腫瘤學(xué)及臨床特征的相關(guān)性;并根據(jù)免疫組織化學(xué)檢測(cè)結(jié)果,運(yùn)用kaplan-meier生存曲線法分析計(jì)算rbm8a低表達(dá)組與高表達(dá)組肝癌患者的無(wú)進(jìn)展生存(progression-freesurvival,pfs)和總生存(overallsurvival,os)時(shí)間,并用log-rank檢驗(yàn)兩組間是否存在統(tǒng)計(jì)學(xué)差異,初步分析rbm8a的表達(dá)量和肝細(xì)胞癌患者預(yù)后的關(guān)系。另收集肝細(xì)胞癌患者、肝硬化患者及正常體檢人群的血清標(biāo)本各110例,運(yùn)用elisa法聯(lián)合檢測(cè)上述人群血清中rbm8a、afp的表達(dá)差異,繪制roc曲線,根據(jù)cutoff值指示rbm8a+afp診斷hcc敏感度及特異性匹配最佳時(shí)的濃度值,分析聯(lián)合檢測(cè)rbm8a+afp對(duì)于提高診斷hcc敏感性及特異性的價(jià)值并將rbm8a作為肝癌輔助診斷指標(biāo)的可行性。結(jié)果:(1)qrt-pcr顯示:rbm8amrna在肝細(xì)胞癌組織中的平均表達(dá)量為△△ct=7.99595±5.49764,在癌旁組織中的平均表達(dá)量為△△ct=4.6667±3.78338,在肝血管瘤組織中的平均表達(dá)量為△△ct=1.6460±1.53852,結(jié)果顯示rbm8amrna的表達(dá)情況為肝細(xì)胞癌組織癌旁組織肝血管瘤組織(p0.05)。(2)免疫組化結(jié)果顯示:rbm8a蛋白在肝細(xì)胞癌組織、癌旁組織、肝血管瘤組織中陽(yáng)性率分別85%、61.67%、5.71%,結(jié)果顯示rbm8a蛋白的表達(dá)情況為肝細(xì)胞癌組織癌旁組織肝血管瘤組織(p0.0001);(3)westernblot結(jié)果顯示:rbm8a在肝細(xì)胞癌組織中平均灰度值為ia(rbm8a)/ia(β-actin)=1.86±0.36;癌旁組織中平均灰度值為ia(rbm8a)/ia(β-actin)=1.35±0.32;肝血管瘤組織中平均灰度值為ia(rbm8a)/ia(β-actin)=0.95±0.35(p0.05)。結(jié)果顯示rbm8a蛋白的表達(dá)情況為肝細(xì)胞癌組織癌旁組織肝血管瘤組織(p0.0001)。(4)將qrt-pcr與免疫組化檢測(cè)結(jié)果與肝癌患者臨床病特征進(jìn)行分析:(1)rbm8a的蛋白表達(dá)水平與腫瘤直徑、hbsag、tnm分期、edmondson病理分級(jí)呈正相關(guān)(p0.05)。(2)rbm8a低表達(dá)組患者的總體os和dfs均較高表達(dá)組延長(zhǎng)(p0.05)。(4)qrt-pcr檢測(cè)結(jié)果顯示,以rbm8amrna在肝細(xì)胞癌組織中表達(dá)量為7.99595為界,105例肝細(xì)胞癌患者中85例為高表達(dá)(80.95%)。rbm8amrna含量高表達(dá)與hbsag(p0.001)、edmondson病理分級(jí)(p=0.004)之間具有顯著相關(guān)性;免疫組化檢測(cè)結(jié)果顯示,105例肝細(xì)胞癌組織中rbm8a表達(dá)為陽(yáng)性的有92例(陽(yáng)性率87.62%)。rbm8a蛋白表達(dá)的陽(yáng)性率與hbsag(p0.001)、腫瘤直徑(p=0.013)、tnm分期(p0.001)、edmondson病理分級(jí)(p=0.003)之間具有顯著相關(guān)性。(5)免疫組化的結(jié)果顯示,肝細(xì)胞癌患者中為rbm8a高表達(dá)組(強(qiáng)陽(yáng)性及陽(yáng)性)的有64例(60.95%),低表達(dá)組(弱陽(yáng)性及陰性)的有41例(39.05%),低表達(dá)組患者和高表達(dá)組患者的rbm8a總體生存期(os)分別為229天和160天(p0.001),兩組患者的無(wú)進(jìn)展生存期(pfs)分別為114天和90天(p0.001)。結(jié)果表明rbm8a低表達(dá)組患者的總體生存期(os)和無(wú)進(jìn)展生存期(pfs)均長(zhǎng)于表達(dá)組患者。(6)elisa檢測(cè)結(jié)果顯示,肝細(xì)胞癌患者血清中rbm8a的蛋白表達(dá)水平明顯高于肝硬化組和正常肝組織患者血清中rbm8a的蛋白表達(dá)水平;并且,單純檢測(cè)血清afp時(shí),診斷肝癌的敏感性為70.0%,聯(lián)合檢測(cè)血清中rbm8a、afp時(shí),診斷肝癌的明顯提升為86.3%。提示聯(lián)合檢測(cè)血清中rbm8a及afp水平有助于提高診斷肝癌的敏感性,而對(duì)于提高診斷肝癌的特異性性則不明顯促進(jìn)作用。結(jié)論:rrbm8a的異常高表達(dá)可能與肝細(xì)胞癌的發(fā)生發(fā)展密切相關(guān),并且rrbm8a具有作為診斷、篩查肝細(xì)胞癌輔助指標(biāo)、預(yù)測(cè)肝細(xì)胞惡性程度及患者預(yù)后指標(biāo)的潛在可能性。二、rbm8a與肝癌細(xì)胞增殖及凋亡特性的基礎(chǔ)實(shí)驗(yàn)研究目的:在初步探討rbm8a的表達(dá)水平與肝細(xì)胞癌腫瘤學(xué)特征及肝細(xì)胞癌患者預(yù)后的相關(guān)性的基礎(chǔ)上,通過(guò)構(gòu)建rbm8a低表達(dá)及過(guò)表達(dá)的細(xì)胞株,檢測(cè)rbm8a低表達(dá)或過(guò)表達(dá)后肝細(xì)胞癌細(xì)胞的增殖能力、侵襲能力、凋亡能力及細(xì)胞周期的的變化,進(jìn)一步探討rbm8a基因?qū)τ诟渭?xì)胞癌形成的有關(guān)生物學(xué)活動(dòng)的影響。方法:(1)采用westernblot對(duì)一系列體外肝癌細(xì)胞株進(jìn)行檢測(cè),選取rbm8a表達(dá)水平最高的細(xì)胞株,設(shè)計(jì)并篩選rbm8a干擾序列,構(gòu)建敲低慢病毒和對(duì)照病毒,感染靶細(xì)胞;經(jīng)qrt-pcr驗(yàn)證rbm8a敲低效率(敲低效率70%以上),建立穩(wěn)定的轉(zhuǎn)染rbm8a-kd細(xì)胞株,并建立空載rbm8a-nc細(xì)胞株作為對(duì)照;(2)采用westernblot對(duì)一系列體外肝癌細(xì)胞株進(jìn)行檢測(cè),選取rbm8a表達(dá)水平最低的細(xì)胞株,調(diào)取rbm8a基因的編碼序列(codingsequence,cds)序列,構(gòu)建到慢病毒過(guò)表達(dá)載體上;采用包裝過(guò)表達(dá)慢病毒及對(duì)照慢病毒,感染靶細(xì)胞;經(jīng)westernblot驗(yàn)證rbm8a過(guò)表達(dá)效率;獲得穩(wěn)定rbm8a高表達(dá)的rbm8a-oe細(xì)胞株,并建立空載rbm8a-nc細(xì)胞株作為對(duì)照;(3)在以上構(gòu)建的細(xì)胞系)中采用cck-8檢測(cè)細(xì)胞的增殖能力變化;(4)facs(annexinⅤ-pe/7aad)檢測(cè)上述四株細(xì)胞凋亡能力變化;(5)facs檢測(cè)上述四株細(xì)胞周期變化;(6)劃痕實(shí)驗(yàn)檢測(cè)敲低及回補(bǔ)后rbm8a后細(xì)胞轉(zhuǎn)移能力的變化;(7)transwell實(shí)驗(yàn)檢測(cè)敲低及回補(bǔ)后rbm8a后細(xì)胞侵襲能力的變化;(8)細(xì)胞骨架染色檢測(cè)檢測(cè)敲低及回補(bǔ)后RBM8A后細(xì)胞骨架形態(tài)學(xué)的變化。結(jié)果:(1)Western blot篩選結(jié)果表明,Bel-7404細(xì)胞株的RBM8A表達(dá)水平最高,用于后續(xù)構(gòu)建Bel7404-RBM8A-KD細(xì)胞株;而HL-7702細(xì)胞株RBM8A表達(dá)水平最低,用于后續(xù)構(gòu)建HL7702-RBM8A-OE細(xì)胞株;(2)CCK8細(xì)胞增殖實(shí)驗(yàn)結(jié)果表明:Bel-7404細(xì)胞株在RBM8A被敲低后,細(xì)胞增值能力下降(P=0.00325);HL-7702細(xì)胞株在RBM8A過(guò)表達(dá)后,細(xì)胞增殖能力上升(P=0.00159);(3)流式細(xì)胞儀檢測(cè)結(jié)果表明:Bel-7404細(xì)胞株中RBM8A被敲低后,細(xì)胞凋亡明顯增加(P=0.0004);HL-7702細(xì)胞株中RBM8A過(guò)表達(dá)后,細(xì)胞凋亡明顯下降(P=0.000358);(4)流式細(xì)胞儀檢測(cè)結(jié)果表明:Bel-7404細(xì)胞株中RBM8A被敲低后,S期細(xì)胞量較NC組有所上升,G1期有所下降;(5)劃痕實(shí)驗(yàn)、transwell侵襲實(shí)驗(yàn)結(jié)果顯示:Bel-7404細(xì)胞株中RBM8A被敲低后,肝癌細(xì)胞遷移降低(P=0.04383)、侵襲能力下降(P=0.0089),而RBM8A回補(bǔ)后肝癌細(xì)胞侵襲再次明顯增強(qiáng)(P=0.020727)、轉(zhuǎn)移能力再次亦明顯增強(qiáng)(P=0.0016);(6)細(xì)胞骨架染色觀測(cè)結(jié)果顯示Bel-7404細(xì)胞株中RBM8A被敲低后,肝癌細(xì)胞形態(tài)變化明顯,呈現(xiàn)偏圓、少偽足形態(tài);而RBM8A回補(bǔ)后肝癌細(xì)胞形態(tài)再次變?yōu)榕c空載組細(xì)胞形態(tài)相似。結(jié)論:下調(diào)RBM8A基因后肝細(xì)胞癌細(xì)胞的分化增殖能力、轉(zhuǎn)移侵襲能力明顯下降,而凋亡能力明顯增強(qiáng),并且細(xì)胞周期有停滯在S期的趨勢(shì)。此研究表明RBM8A基因?qū)τ谡T導(dǎo)肝細(xì)胞癌細(xì)胞凋亡及細(xì)胞周期具有重要作用,RBM8A基因具有作為治療肝細(xì)胞癌的新的基因靶向位點(diǎn)的潛在可能性。
[Abstract]:First, objective to investigate the clinical relationship between RBM8A and hepatocellular carcinoma: studies have shown that closely associated with the occurrence and development of RBM8A and malignant tumor. However, the relationship between RBM8A and hepatocellular carcinoma has not been clearly reported. This study aims at first through the detection of RBM8A in hepatocellular carcinoma and related differences in paracancerous tissues. The expression level of liver hemangioma tissues, and patients with hepatocellular carcinoma and liver cirrhosis patients, the differential expression of RBM8A in serum samples of normal people, to study the correlation between RBM8A and the occurrence of hepatocellular carcinoma, and the correlation between patient characteristics and oncology expression level of RBM8A in hepatocellular carcinoma and prognosis analysis of RBM8A as a potential. The possibility of auxiliary diagnostic criteria and prognostic indicator for hepatocellular carcinoma. Methods: the tumor hospital affiliated to Guangxi Medical University from March 2012 to December 2014 were collected by surgical resection and The postoperative pathology confirmed hepatocellular carcinoma in 105 cases, and collect the corresponding 2cm away from the tumor margin of adjacent tissues. Other surgically resected and pathologically confirmed for hemangioma of liver tissue in 67 cases. Using qRT-PCR method, Western Blot method, immunohistochemistry and mRNA and protein the analysis of rbm8a in hepatocellular carcinoma tissues and corresponding noncancerous tissues, expression and hepatic hemangioma; according to the content of mRNA and the expression of rbm8a protein in hepatocellular carcinoma, hepatocellular carcinoma patients were divided into high expression group and low expression group. The correlation between the expression of rbm8a and preliminary analysis of oncology patients hepatocellular carcinoma and clinical features; and according to the test results by using Kaplan-Meier immunohistochemistry, the survival curve analysis calculation of rbm8a low expression group and high expression of progression free survival in patients with hepatocellular carcinoma group (progression-freesurvival, PFS) and The total survival time (overallsurvival, OS), and use log-rank to test whether the statistical difference exists between the two groups, preliminary analysis of the relationship between the expression of hepatocellular carcinoma and the prognosis of patients with rbm8a. Another collection of patients with hepatocellular carcinoma, liver cirrhosis and normal healthy population of serum samples from 110 patients with ELISA method, the combined detection of the serum in rbm8a, expression of AFP, ROC curve, according to the cutoff value indicates that the rbm8a+afp diagnosis of HCC sensitivity and specificity, the optimum concentration of the value analysis of the combined detection of rbm8a+afp in improving the diagnostic sensitivity and specificity of HCC value and rbm8a feasibility as auxiliary diagnostic index for hepatocellular carcinoma. Results: (1): qRT-PCR display the average expression of rbm8amrna in hepatocellular carcinoma was Delta ct=7.99595 + 5.49764, the average expression in the adjacent tissues as delta ct=4.6667 + 3.78338 in hepatic hemangiomas 鐨勫鉤鍧囪〃杈鵑噺涓衡柍鈻砪t=1.6460鹵1.53852,緇撴灉鏄劇ずrbm8amrna鐨勮〃杈炬儏鍐典負(fù)鑲濈粏鑳?yōu)鐧尵l勭粐鐧屾梺緇勭粐鑲濊綆＄槫緇勭粐(p0.05).(2)鍏嶇柅緇勫寲緇撴灉鏄劇ず:rbm8a铔嬬櫧鍦ㄨ倽緇嗚優(yōu)鐧岀粍緇，

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