【摘要】：腦膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)最常見(jiàn)的原發(fā)惡性腫瘤，約占成人顱內(nèi)腫瘤的35.26%-60.96%(平均44.69%)。由于其浸潤(rùn)性生長(zhǎng)，在腫瘤與正常組織之間常無(wú)明顯的邊界，因此手術(shù)治療難以徹底切除腫瘤組織。盡管我們努力嘗試各種治療方法，但是腦膠質(zhì)瘤仍然無(wú)法治愈。這就要求我們?yōu)槟z質(zhì)瘤的治療尋找新的策略。近年來(lái)，隨著基因工程技術(shù)的發(fā)展，人們開(kāi)始從分子生物學(xué)的角度探求腦膠質(zhì)瘤的發(fā)病機(jī)制，腦膠質(zhì)瘤的基因治療逐漸受到關(guān)注。CPEB4是新近發(fā)現(xiàn)的胞質(zhì)多聚腺苷酸化元件結(jié)合蛋白（CPEBs）家族中的一員，是一種具有序列特異性的RNA結(jié)合蛋白，含有一個(gè)RNA識(shí)別基序和一個(gè)鋅指結(jié)構(gòu)，位于人染色體5q21區(qū)域，定位于細(xì)胞質(zhì)，長(zhǎng)度為7769bp。胞質(zhì)多聚腺苷酸化元件結(jié)合蛋白（CPEBs）于大約二十年前被首次發(fā)現(xiàn)在卵母細(xì)胞成熟過(guò)程中，其在已確定的信使RNA的翻譯過(guò)程中起著重要的調(diào)控作用，人們?cè)谠缙谧隽撕芏嚓P(guān)于CPEB控制胞質(zhì)多聚腺苷酸化和翻譯的工作，發(fā)現(xiàn)CPEB通過(guò)調(diào)控mRNA這一活動(dòng)而影響諸多進(jìn)程如生殖細(xì)胞的發(fā)育、細(xì)胞分裂與細(xì)胞衰老、突觸可塑性以及學(xué)習(xí)與記憶等。然而，關(guān)于近年CPEBs的一些熱點(diǎn)研究表明它不僅在體細(xì)胞組織表達(dá)，而且在控制成年有機(jī)體時(shí)間和空間的基因表達(dá)方面具有重要的功能。這些新發(fā)現(xiàn)的功能包括調(diào)節(jié)衰老和增殖之間的平衡，及腫瘤的發(fā)生等。CPEBs在各種組織和腫瘤中高度表達(dá)，且有部分重疊。最近的一項(xiàng)新研究表明，CPEB4在胰腺癌和膠質(zhì)母細(xì)胞瘤中高度表達(dá)，這與腫瘤的生長(zhǎng)、惡性度的增加及血管生成等有關(guān)。然而，CPEB4在腦膠質(zhì)瘤中的表達(dá)沒(méi)有被詳細(xì)描述。 鑒于以上研究背景，我們通過(guò)施行免疫組織化學(xué)、蛋白免疫印跡以及實(shí)時(shí)熒光定量PCR等實(shí)驗(yàn)方法，檢測(cè)了CPEB4在正常腦組織和不同級(jí)別腦膠質(zhì)瘤的蛋白及RNA水平的表達(dá)情況。此外，應(yīng)用RNA干擾技術(shù)敲減膠質(zhì)瘤細(xì)胞系中CPEB4的表達(dá)，來(lái)探究其在膠質(zhì)瘤細(xì)胞增殖、凋亡和侵襲中的作用。本實(shí)驗(yàn)研究包括以下三個(gè)部分： 第一部分: CPEB4在人腦膠質(zhì)瘤組織中的表達(dá)及其臨床意義 目的：探討CPEB4在正常腦組織中和各級(jí)別腦膠質(zhì)瘤組織中的表達(dá)情況。 方法： 1收集樣本:收集2011年10月至2012年11月期間在河北醫(yī)科大學(xué)第二醫(yī)院住院，經(jīng)行手術(shù)切除的63例腦膠質(zhì)瘤組織及9例正常腦組織。腫瘤標(biāo)本均由神經(jīng)病理醫(yī)師確診，正常腦組織取自腦出血或腦外傷需行內(nèi)減壓術(shù)的患者。每例患者分兩部分留取標(biāo)本，一部分置于10%福爾馬林液中固定，另一部分保存于2ml無(wú)菌凍存管并立即投至液氮中速凍。 2應(yīng)用免疫組織化學(xué)法檢測(cè):9例正常腦組織及63例膠質(zhì)細(xì)胞瘤組織中CPEB4蛋白的表達(dá)情況，并比較其表達(dá)差異，分析其與腦膠質(zhì)瘤病理級(jí)別的相關(guān)性。 3應(yīng)用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)（Real-time qPCR）測(cè)定CPEB4mRNA的表達(dá)水平。 4應(yīng)用蛋白質(zhì)免疫印跡Western blot檢測(cè)正常腦組織及膠質(zhì)瘤組織中CPEB4蛋白的表達(dá)。 結(jié)果： 1免疫組織化學(xué)結(jié)果：CPEB4在膠質(zhì)細(xì)胞瘤組織中的總陽(yáng)性表達(dá)率為88.89%(56/63)，CPEB4在9例正常腦組織中表達(dá)呈陰性或很弱，腦膠質(zhì)瘤組織的CPEB4陽(yáng)性表達(dá)率明顯高于正常腦組織，具有顯著的統(tǒng)計(jì)學(xué)意義（P＜0.01）；低級(jí)別（I、II級(jí)）和高級(jí)別（III、IV級(jí)）膠質(zhì)瘤組織陽(yáng)性表達(dá)率分別為75%（15/20）和95.4%（41/43），差異具有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 2Western blot結(jié)果：CPEB4蛋白在腦膠質(zhì)瘤組織中的表達(dá)明顯高于正常腦組織。 CPEB4蛋白在正常腦組織及I、II、III、 IV級(jí)腦膠質(zhì)瘤中相對(duì)表達(dá)量分別為0.070±0.012,0.417±0.044,0.495±0.043,0.709±0.080,0.713±0.027。CPEB4蛋白在低級(jí)別（I、II級(jí)）腦膠質(zhì)瘤中的表達(dá)量低于高級(jí)別（III、IV級(jí)）（P＜0.05）。 3Real-time qPCR結(jié)果：CPEB4mRNA在正常腦組織、低級(jí)別及高級(jí)別中相對(duì)表達(dá)沒(méi)有統(tǒng)計(jì)學(xué)差異（P＞0.05）。 結(jié)論： 1免疫組化與Western blot檢測(cè)均得出CPEB4蛋白在人腦膠質(zhì)瘤組織中高度表達(dá)，并且其表達(dá)隨著膠質(zhì)瘤病理級(jí)別的增高而增加，III、IV級(jí)腦膠質(zhì)瘤中CPEB4蛋白表達(dá)要高于I、II級(jí)腦膠質(zhì)瘤，差異顯著；但是，在mRNA水平，CPEB4的表達(dá)卻無(wú)明顯差異。對(duì)于這種蛋白水平與RNA水平表達(dá)不一致的情況，我們分析可能是與此RNA的穩(wěn)定性差、易降解有關(guān)。 2CPEB4的高表達(dá)與膠質(zhì)瘤級(jí)別的升高呈顯著的正相關(guān)，可作為腦膠質(zhì)瘤臨床分級(jí)的重要指標(biāo)。 第二部分: CPEB4在人腦膠質(zhì)瘤細(xì)胞株U87、U251中的表達(dá) 目的：檢測(cè)CPEB4在人腦膠質(zhì)瘤細(xì)胞株U87和U251中的表達(dá)情況，選出CPEB4表達(dá)量較高的細(xì)胞株以進(jìn)行進(jìn)一步的生物學(xué)功能試驗(yàn)。 方法： 1應(yīng)用蛋白質(zhì)免疫印跡Western blot檢測(cè)U87和U251細(xì)胞中CPEB4蛋白表達(dá)情況。 2應(yīng)用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)（Real-time qPCR）測(cè)定U87和U251細(xì)胞中CPEB4mRNA的表達(dá)。 結(jié)果：人腦膠質(zhì)瘤細(xì)胞株U87的CPEB4蛋白和mRNA均高于U251（P＜0.05）。 結(jié)論：U87細(xì)胞株可被選擇作為CPEB4的優(yōu)勢(shì)表達(dá)株，進(jìn)一步行以CPEB4為研究靶點(diǎn)的生物學(xué)功能試驗(yàn)第三部分CPEB4慢病毒載體感染人腦膠質(zhì)瘤細(xì)胞株U87及其對(duì)U87細(xì)胞增殖、凋亡和侵襲力的影響研究 目的：構(gòu)建CPEB4干擾慢病毒載體，以構(gòu)建好的CPEB4siRNA干擾慢病毒載體感染U87細(xì)胞株，探討RNA干擾(RNAinierference，RNAi)對(duì)膠質(zhì)瘤細(xì)胞株U87中CPEB4表達(dá)的抑制作用及其對(duì)U87細(xì)胞增殖、凋亡和侵襲力的影響。 方法： 1構(gòu)建CPEB4干擾慢病毒載體：針對(duì)目的基因CPEB4的序列信息，嚴(yán)格按照RNA干擾序列設(shè)計(jì)原則，設(shè)計(jì)3個(gè)RNA干擾靶點(diǎn)序列，合成SiRNA并向U87細(xì)胞分別轉(zhuǎn)染3種si RNA，利用western blot篩選干擾效果最佳的siRNA，根據(jù)此siRNA序列，合成雙鏈DNA Oligo，，兩端帶有BamHI和ECORI酶切位點(diǎn)粘性末端，退火后得到的雙鏈產(chǎn)物無(wú)需酶切可直接連入BamHI和ECORI線(xiàn)性化的pSRL-SIH1-H1-GFP載體。將連接產(chǎn)物轉(zhuǎn)入細(xì)菌感受態(tài)細(xì)胞，提取細(xì)菌培養(yǎng)物中的質(zhì)粒，對(duì)長(zhǎng)出的克隆抽提質(zhì)粒并酶切鑒定，然后進(jìn)行測(cè)序比對(duì),鑒定陽(yáng)性克隆即為構(gòu)建成功的CPEB4RNA干擾慢病毒載體。 2在體外應(yīng)用RNA干擾技術(shù)以siCPEB4(CPEB4SiRNA)靶向感染膠質(zhì)瘤細(xì)胞株U87，分為轉(zhuǎn)染組、陰性對(duì)照組和空白對(duì)照組； 3應(yīng)用Western blot和Real time qPCR分別檢測(cè)CPEB4-siRNA慢病毒感染U87細(xì)胞后1天、2天、3天、4天、5天細(xì)胞中CPEB4蛋白和mRNA相對(duì)表達(dá)量變化； 4應(yīng)用流式細(xì)胞技術(shù)（FCM）檢測(cè)CPEB4SiRNA慢病毒感染U87細(xì)胞后的細(xì)胞周期變化，并檢測(cè)分析轉(zhuǎn)染前后細(xì)胞凋亡水平變化； 5應(yīng)用MTT比色實(shí)驗(yàn)檢測(cè)分析感染CPEB4-SiRNA慢病毒1天、2天、3天、4天、5天、6天及7天后U87細(xì)胞的增殖情況； 6應(yīng)用Transwell侵襲實(shí)驗(yàn)檢測(cè)分析轉(zhuǎn)染前后細(xì)胞體外侵襲能力的變化。 結(jié)果： 1成功構(gòu)建并包裝了攜帶CPEB4基因的慢病毒表達(dá)載體，經(jīng)過(guò)陽(yáng)性克隆測(cè)序證實(shí)插入序列完全正確； 2Western blot和Real time qPCR結(jié)果顯示，慢病毒感染組的CPEB4蛋白和mRNA相對(duì)表達(dá)量明顯低于空白對(duì)照組與陰性對(duì)照組，差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；空白對(duì)照組與陰性對(duì)照組間表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)； 3流式細(xì)胞儀檢測(cè)細(xì)胞周期結(jié)果顯示，相對(duì)于陰性對(duì)照組細(xì)胞以及空白對(duì)照組細(xì)胞，轉(zhuǎn)染CPEB4siRNA慢病毒后U87細(xì)胞阻滯于G1期，S期細(xì)胞數(shù)相對(duì)較少；陰性對(duì)照組和空白對(duì)照組的U87細(xì)胞的細(xì)胞狀態(tài)和細(xì)胞周期沒(méi)有變化；流式細(xì)胞儀分析各組細(xì)胞的凋亡水平顯示，相對(duì)于空白對(duì)照組細(xì)胞(0.13±0.08)%和陰性對(duì)照組細(xì)胞(0.47±0.12)%，U87細(xì)胞感染CPEB4si-RNA慢病毒后早期凋亡率增加，達(dá)(19.16±3.85)%，具有統(tǒng)計(jì)學(xué)差異(P0.01)； 4MTT檢測(cè)細(xì)胞活性顯示，從第三天起，與陰性對(duì)照組（0.476±0.048）和未轉(zhuǎn)染組（0.495±0.031）相比，慢病毒感染組（0.42±0.05）細(xì)胞生長(zhǎng)速度開(kāi)始減慢，并具有統(tǒng)計(jì)學(xué)差異(P＜0.05)，且隨感染時(shí)間的延長(zhǎng)，抑制程度越來(lái)越明顯。而未轉(zhuǎn)染組與陰性對(duì)照組組之間無(wú)明顯差異(P0.05)。說(shuō)明CPEB4siRNA對(duì)U87細(xì)胞生長(zhǎng)增殖具有顯著抑制作用； 5Transwell侵襲試驗(yàn)結(jié)果顯示，經(jīng)CPEB4siRNA慢病毒感染后，U87細(xì)胞穿膜數(shù)量明顯減少，低于陰性對(duì)照組及空白對(duì)照組，差異具有統(tǒng)計(jì)學(xué)意義。換句話(huà)說(shuō), CPEB4能夠促進(jìn)膠質(zhì)瘤細(xì)胞的體外侵襲力。 結(jié)論： CPEB4靶向轉(zhuǎn)染能夠有效敲減這個(gè)基因在人腦膠質(zhì)瘤U87細(xì)胞系內(nèi)的表達(dá)，使腫瘤細(xì)胞增殖和侵襲能力受到顯著抑制，阻滯細(xì)胞周期，并促進(jìn)其凋亡增加，為腦膠質(zhì)瘤基因治療提供了新的靶點(diǎn)。
[Abstract]:Human glioma is one of the most common primary malignant tumors in the central nervous system , accounting for 35.26 % - 60.96 % ( mean 44.69 % ) of intracranial tumors .


In view of the above background , we examined the expression of CPEB4 in normal brain tissue and brain glioma at different levels by performing immunohistochemistry , Western blotting and real - time fluorescence quantitative PCR .


The expression of CPEB4 in human glioma tissues and its clinical significance


Objective : To investigate the expression of CPEB4 in normal brain tissue and brain glioma at all levels .


Method :


Samples were collected : 63 cases of brain glioma and 9 normal brain tissues were collected from the Second Affiliated Hospital of Hebei Medical University between October 2011 and November 2012 . All the tumor specimens were confirmed by neurologists . Normal brain tissues were taken from patients with intracerebral hemorrhage or traumatic brain trauma . The samples were collected in two parts . One part was fixed in 10 % formalin solution , and the other part was stored in 2 ml of sterile frozen tubes and immediately frozen in liquid nitrogen .


The expression of CPEB4 protein in 9 normal brain tissues and 63 cases of gliocytoma were detected by immunohistochemical method . The correlation between the expression of CPEB4 and the pathological grade of glioma was analyzed .


3 . Real - time fluorescence quantitative polymerase chain reaction ( Real - time qPCR ) was used to determine the expression level of CPEB4mRNA .


4 . Western blot was used to detect the expression of CPEB4 protein in normal brain tissues and glioma tissues .


Results :


Results : The expression rate of CPEB4 was 88.89 % ( 56 / 63 ) . CPEB4 was negative or weak in 9 normal brain tissues , and the positive rate of CPEB4 was significantly higher than that of normal brain tissue ( P < 0.01 ) .
The positive expression rates of low - grade ( I , II ) and high - level ( III , IV ) glioma tissues were 75 % ( 15 / 20 ) and 95.4 % ( 41 / 43 ) , respectively . The difference was statistically significant ( P < 0.05 ) .


2 Western blot showed that the expression of CPEB4 protein in brain glioma was significantly higher than that of normal brain tissue . The relative expression levels of CPEB4 protein in normal brain tissue and I , II , III and IV gliomas were 0.070 鹵 0.012 , 0.417 鹵 0.044 , 0.495 鹵 0.043 , 0.709 鹵 0.080 , 0.713 鹵 0.027 . CPEB4 protein was lower in low grade gliomas ( grade I , II ) than in high level ( grade III , IV ) ( P < 0.05 ) .


3 Real - time qPCR results showed that the relative expression of CPEB4mRNA in normal brain tissue , low grade and high level was not significantly different ( P > 0.05 ) .


Conclusion :


1 Immunohistochemical and Western blot analysis showed that CPEB4 protein was highly expressed in human glioma tissues , and the expression of CPEB4 increased with the pathological grade of glioma .
However , in the case of mRNA levels , there was no significant difference in the expression of CPEB4 . In the case of non - uniform expression of this protein level with the level of RNA , we analyzed the possibility of poor stability and easy degradation of this RNA .


The high expression of 2CPEB4 is positively correlated with the increase of glioma grade , which can be used as an important index for clinical grading of glioma .


The second part : the expression of CPEB4 in human glioma cell line U87 and U251


Objective : To detect the expression of CPEB4 in human glioma cell lines U87 and U251 , and to select the cell line with high expression of CPEB4 for further biological function test .


Method :


1 . Western blot was used to detect the expression of CPEB4 protein in U87 and U251 cells .


2 . Real - time quantitative polymerase chain reaction ( Real - time qPCR ) was used to determine the expression of CPEB4mRNA in U87 and U251 cells .


Results : The CPEB4 protein and mRNA of human glioma cell line U87 were higher than U251 ( P < 0.05 ) .


Conclusion : U87 cell line can be selected as the dominant expression strain of CPEB4 , and further study the effect of CPEB4 on the biological function test of target site . The third part CPEB4 lentivirus vector is infected with human glioma cell line U87 and its effect on proliferation , apoptosis and invasion of U87 cell line .


Objective : To construct CPEB4 interfering slow virus vector , construct good CPEB4siRNA interfering slow virus vector to infect U87 cell line , investigate the inhibitory effect of RNA interference ( RNAi ) on the expression of CPEB4 in human glioma cell line U87 and its effect on proliferation , apoptosis and invasion of U87 cell line .


Method :


1 . Construction of CPEB4 interfering lentivirus vector : According to the sequence information of the target gene CPEB4 , three RNA interference target sequences were designed strictly according to the design principles of RNA interference sequence . Three kinds of siRNA were synthesized by Western blot . Two - stranded DNA oligo - stranded DNA Oligo was synthesized . Two - stranded DNA oligo - stranded DNA was synthesized and digested with BamHI and ECORI .


2 In vitro RNA interference technique was used to target cell line U87 with siCPEB4 ( CPEB4SiRNA ) , which was divided into transfection group , negative control group and blank control group .



3 . Western blot and Real time qPCR were used to detect the changes of CPEB4 protein and mRNA relative expression in the cells of CPEB4 - siRNA infected with CPEB4 - siRNA for 1 day , 2 days , 3 days , 4 days and 5 days respectively .



4 . Flow cytometry ( FCM ) was used to detect the changes of cell cycle after CPEB4SiRNA virus infection .



5 . MTT colorimetric assay was used to detect the proliferation of U87 cells infected with CPEB4 - SiRNA in 1 day , 2 days , 3 days , 4 days , 5 days , 6 days and 7 days .



6 . Transwell invasion assay was used to detect the changes of invasion ability of cells in vitro and after transfection .


Results :


1 . The lentivirus expression vector carrying CPEB4 gene was successfully constructed and packaged , and the insertion sequence was confirmed by positive clone sequencing .



2 Western blot and Real time qPCR showed that the relative expression of CPEB4 protein and mRNA in chronic viral infection group was significantly lower than that in control group and negative control group ( P0.05 ) .
There was no statistical difference between the blank control group and the negative control group ( P0.05 ) .



The results showed that the number of cells transfected with CPEB4siRNA was lower than that of negative control group and blank control group .
Cell status and cell cycle of U87 cells in the negative control group and the blank control group were not changed ;
Flow cytometry showed that the apoptosis rate of cells in each group was significantly higher than that of control group ( 0.13 鹵 0.08 ) % and negative control group ( 0.47 鹵 0.12 ) % , and the early apoptotic rate of U87 cells was increased by ( 19.16 鹵 3.85 ) % , which was statistically significant ( P0.01 ) .



Compared with the negative control group ( 0.476 鹵 0.048 ) and the untransfected group ( 0.495 鹵 0.031 ) , the growth rate of the slow virus infection group ( 0.42 鹵 0.05 ) was slower than that of the negative control group ( P < 0.05 ) .



5Transwell invasion assay showed that the number of U87 cells decreased significantly after CPEB4siRNA was infected with slow virus infection , which was lower than that of negative control group and blank control group . In other words , CPEB4 could promote the invasion of glioma cells in vitro .


Conclusion :


CPEB4 targeted transfection can effectively reduce the expression of the gene in human glioma U87 cell line , inhibit the proliferation and invasion ability of tumor cells , block the cell cycle , and promote the increase of apoptosis , thus providing a new target for gene therapy of glioma .
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R739.41

【引證文獻(xiàn)】

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