本文選題：脆性X智力低下一號(hào)基因(FMR1) + 雙熒光素酶報(bào)告基因系統(tǒng)　； 參考：《南方醫(yī)科大學(xué)》2012年碩士論文

【摘要】：脆性X綜合征[fragile X syndrome,Fra(X)]是最常見(jiàn)的遺傳性智力低下疾病之一。據(jù)保守估計(jì),我國(guó)至少有20萬(wàn)脆性X病人。目前認(rèn)為Fra(X)致病機(jī)制主要是由于脆性X智力低下一號(hào)基因(fragile X mental retardation-1gene,FMR1)啟動(dòng)子區(qū)內(nèi)(CGG)n三核苷酸重復(fù)序列的不穩(wěn)定擴(kuò)增及其上游的CPG島的異常甲基化,使FMR1基因轉(zhuǎn)錄及翻譯終止,其編碼產(chǎn)物-脆性X智力低下蛋白(fragile X mental retardation protein, FMRP)減少或缺如,最終表現(xiàn)為Fra(X)。目前對(duì)于Fra(X)的治療尚無(wú)有效的方法,基因治療仍困難重重,藥物重新活化FMR1的方法,也因其存在較強(qiáng)的毒副作用制約了臨床應(yīng)用,尋找有效、安全、低/無(wú)毒副作用的藥物用于治療Fra (X)仍是目前研究的熱點(diǎn)之一 已往很多研究提示智力低下與信號(hào)轉(zhuǎn)導(dǎo)通路之間有著某種相互聯(lián)系,其中,環(huán)磷酸腺苷(cyclic adenosine monophosphate,cAMP)信號(hào)通路與學(xué)習(xí)記憶和智力關(guān)系最為密切,故Fra(X)與cAMP通路之間的關(guān)系也引起了人們的密切關(guān)注。人們發(fā)現(xiàn)在Fra(X)患者細(xì)胞中、在FMR1基因敲除小鼠的腦組織及血小板內(nèi),以及在患有Fra(X)的果蠅腦組織內(nèi)cAMP水平均低于正常,而患有Fra(X)的果蠅可以通過(guò)重新植入FMR1基因來(lái)彌補(bǔ)這種缺陷,由此提示FMR1基因與cAMP之間可能存在相互調(diào)控關(guān)系。 Fra(X)患者細(xì)胞內(nèi)cAMP水平降低的原因是什么呢?cAMP的代謝主要與腺苷酸環(huán)化酶(adenylate cyclase,AC)和磷酸二酯酶(phosphodiesterase,PDE)的活性有關(guān)。前者作用于ATP可生成cAMP,而后者則降解cAMP,以保持胞內(nèi)cAMP水平的平衡。有臨床研究發(fā)現(xiàn)一部分Fra(X)患者存在腺苷酸環(huán)化酶催化亞單位的功能或者調(diào)節(jié)的異常。我們前期研究發(fā)現(xiàn)FMR1基因封閉后細(xì)胞內(nèi)cAMP水平下降,腺苷酸環(huán)化酶活性也明顯降低,而磷酸二酯酶的活性則無(wú)顯著變化。提示FMR1基因的功能缺陷可影響腺苷酸環(huán)化酶的活性,推測(cè)腺苷酸環(huán)化酶活性抑制可能是影響Fra(X)患者細(xì)胞內(nèi)cAMP水平降低的主要原因。 如果FMR1基因轉(zhuǎn)錄及翻譯終止與腺苷酸環(huán)化酶活性降低有關(guān),反之,用藥物提高腺苷酸環(huán)化酶的活性能否重啟被封閉的FMR1基因呢?為了證明我們的猜想,前期在封閉的FMR1基因細(xì)胞模型上,我們采用特異性腺苷酸環(huán)化酶(AC)激活劑藥物弗司可林(forskolin,FSK)改善腺苷酸環(huán)化酶的低活性狀態(tài),結(jié)果發(fā)現(xiàn)它的確可有效地重新誘導(dǎo)沉默F(xiàn)MR1基因的轉(zhuǎn)錄和蛋白表達(dá),但forskolin對(duì)FMR1基因調(diào)節(jié)的具體機(jī)制尚不清楚,是否通過(guò)提高cAMP水平這一途徑誘導(dǎo)的有待進(jìn)一步證實(shí)。 已知FMRI基因啟動(dòng)子區(qū)與FMR1基因的轉(zhuǎn)錄密切相關(guān),該區(qū)內(nèi)存在一個(gè)具有增強(qiáng)子活性的甲基化敏感區(qū)(1methylation sensitive element,MSE),值得注意的是該區(qū)增強(qiáng)子內(nèi)又同時(shí)包含一個(gè)cAMP反應(yīng)單位(cAMP-responsive element,CRE)堿基序列,兩者結(jié)構(gòu)重疊,關(guān)系緊密。而CRE序列是環(huán)磷酸腺苷反應(yīng)單元結(jié)合蛋白(CREB)的結(jié)合位點(diǎn)。CREB是一種重要的核轉(zhuǎn)錄因子,具有調(diào)節(jié)包括記憶功能在內(nèi)的廣泛的生物學(xué)功能,且研究發(fā)現(xiàn)CREB可以與FMR1啟動(dòng)子結(jié)合并可增強(qiáng)FMR1啟動(dòng)子的活性,因此推測(cè)其機(jī)制可能是cAMP誘導(dǎo)的。AC激動(dòng)劑forskolin是否經(jīng)cAMP通路,通過(guò)FMR1啟動(dòng)子區(qū)內(nèi)的MSE/CRE重疊位點(diǎn)實(shí)現(xiàn)對(duì)FMR1基因調(diào)節(jié)的尚有待證實(shí)。 為了證實(shí)以上推測(cè),本研究1.擬直接采用外源性cAMP藥物-二丁酰環(huán)磷酸腺苷(db-cAMP)誘導(dǎo)沉默F(xiàn)MR1基因的轉(zhuǎn)錄和蛋白表達(dá),試圖證實(shí)forskolin是通過(guò)提高細(xì)胞內(nèi)cAMP水平這一途徑而達(dá)到對(duì)FMR1基因再激活作用的；2.以MSE/CRE位點(diǎn)為研究靶點(diǎn),構(gòu)建FMR1啟動(dòng)子區(qū)MSE/CRE位點(diǎn)雙熒光素酶報(bào)告基因系統(tǒng),在不影響MSE整體啟動(dòng)子活性的情況下,分別突變CRE序列,形成突變啟動(dòng)子M1、M2的雙熒光素酶報(bào)告基因,試圖驗(yàn)證forskolin是經(jīng)FMR1啟動(dòng)子區(qū)MSE/CRE位點(diǎn)實(shí)現(xiàn)了對(duì)FMR1基因的調(diào)節(jié),為進(jìn)一步研究AC激動(dòng)劑forskolin重啟FMR1基因的機(jī)制奠定基礎(chǔ),試圖為Fra(X)患者的治療提供新的思路和治療前景。 研究方法： 1.制作pc12細(xì)胞FMR1基因封閉的細(xì)胞模型 采用硝普鈉(sodium nitroprusside,SNP)封閉FMR1基因。硝普鈉為經(jīng)典的一氧化氮(nitric oxide,NO)供體,可通過(guò)產(chǎn)生的NO活化DNA甲基轉(zhuǎn)移酶(DNA MeTase),從而使FMR1啟動(dòng)子區(qū)CpG島的MSE中的胞嘧啶發(fā)生高度甲基化,阻斷了核酸因子與啟動(dòng)了的結(jié)合,抑制了FMRl mRNA的轉(zhuǎn)錄。最后通過(guò)real-time PCR觀察FMR1基因的封閉效果。 2.觀察二丁酰環(huán)磷酸腺苷(db-cAMP)對(duì)pc12細(xì)胞FMRl mRNA的影響 1000umol/LSNP作用pc12細(xì)胞24h后,實(shí)驗(yàn)組分別加入濃度為0.25mmo1/L0.5mmo1/L、1mmol/L的db-cAMP,分別于db-cAMP作用6h、12h、24h、48h后收集細(xì)胞,提取總RNA。以正常的pc12細(xì)胞為正常對(duì)照組,以僅加SNP作用的pc12細(xì)胞為封閉抑制對(duì)照組,用熒光定量PCR(染料法)檢測(cè)FMR1基因的表達(dá)情況。 3.觀察二丁酰環(huán)磷酸腺苷(db-cAMP)對(duì)FMR1蛋白水平(FMRP)的影響 硝普鈉封閉pc12細(xì)胞FMR124h后,加入0.5mmol/Ldb-cAMP,分別于加藥24h、48h、72h、96h后收集細(xì)胞(12小時(shí)補(bǔ)充失效的db-cAMP,48小時(shí)補(bǔ)充失效的SNP),提取蛋白質(zhì),進(jìn)行Western Blot,對(duì)FMRP進(jìn)行半定量分析。 4.FMR1啟動(dòng)子MSE/CER重疊序列及突變CRE雙熒光素酶報(bào)告基因的構(gòu)建 采用PCR的方法從K562細(xì)胞DNA中釣取MSE/CRE重疊序列,然后采用PCR點(diǎn)突變的方法分別突變CRE序列,形成突變了M1、M2,分別獲得MSE、M1、M2片段,然后連接到PGL3載體上,進(jìn)而轉(zhuǎn)化到DH5a細(xì)胞中,提取質(zhì)粒。MSE/PGL3、M1/PGL3、M2/PGL3分別與海腎質(zhì)粒共轉(zhuǎn)染pc12細(xì)胞后,對(duì)所測(cè)熒光素酶活性強(qiáng)度值進(jìn)行分析。 5.驗(yàn)證CRE位點(diǎn)是腺苷酸環(huán)化酶激活劑forskolin重啟FMR1基因的關(guān)鍵位點(diǎn) 從DH50細(xì)菌中1提取MSE/PGL3、M1/PGL3、M2/PGL3質(zhì)粒,分別轉(zhuǎn)染pc12細(xì)胞后進(jìn)行分組,加1000umol/L SNP和(或)50umo1/Lforskolin,檢測(cè)熒光素酶活性,比較在正常CRE與突變后CRE狀態(tài)下,forskolin對(duì)FMRl基因開(kāi)啟的影響,通過(guò)對(duì)熒光素酶值的比較,判斷啟動(dòng)子活性(熒光素酶值高,啟動(dòng)子活性則高；反之,則低),以此驗(yàn)證CRE位點(diǎn)是否是腺苷酸環(huán)化酶激活劑forskolin重啟FMR1基因的關(guān)鍵位點(diǎn)。 6.統(tǒng)計(jì) 實(shí)驗(yàn)所得數(shù)據(jù)經(jīng)SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)處理,數(shù)據(jù)用均數(shù)士標(biāo)準(zhǔn)差(χ-±s)表示。熒光定量PCR結(jié)果采用2-△△cT值分析,采用單因素方差分析；因方差不齊,組間兩兩比較采用Dunnett T3;與正常組的比較采用單樣本t檢驗(yàn)。WesternBlot數(shù)據(jù)采用Bia-Rad Gel-Pro Analyzer軟件分析所得A值之比,因方差齊性,組間兩兩比較采用LSD；相對(duì)熒光素酶活性檢測(cè)數(shù)據(jù)應(yīng)用相對(duì)熒光素酶活性強(qiáng)度值分析,采用析因設(shè)計(jì)資料方差分析,因方差齊性,組間兩兩比較采用LSD。p0.05表示有統(tǒng)計(jì)學(xué)差異。 研究結(jié)果 1.不同濃度及不同時(shí)間點(diǎn)db-cAMP對(duì)封閉的FMR1轉(zhuǎn)錄水平的影響 db-cAMP在0.5mmo1/L作用12h時(shí)FMR1mRNA激活效果最強(qiáng),FMR1mRNA的表達(dá)量約為封閉組的9.7倍(P0.05),約為正常組的0.86倍(P=0.135)。0.5mmol/Ldb-cAMP的激活效果維持時(shí)間小于18小時(shí)。 2.db-cAMP對(duì)FMRP表達(dá)的影響 用SNP1000μmol/L封閉FMR1基因,作用72h時(shí)FMRP表達(dá)量最低,約為正常組的0.56倍；在SNP封閉的基礎(chǔ)上,加入0.5mmol/Ldb-cAMP可提高FMRP表達(dá)量,作用72h時(shí)激活效果最好,為正常組的0.98倍(P0.05),是封閉組的1.25倍(P0.05)。 3.熒光素酶載體構(gòu)建 PCR、雙酶切驗(yàn)證后送測(cè)序,測(cè)序結(jié)果均符合設(shè)計(jì)。pGL-3/MSE位、pGL-3/M1位、pGL-3/M2位質(zhì)粒、pGL3basic與海腎質(zhì)粒共轉(zhuǎn)染pc12細(xì)胞,質(zhì)粒均成功轉(zhuǎn)入pc12細(xì)胞,并且具有啟動(dòng)子活性,顯示脆性X智力低下一號(hào)基因啟動(dòng)子雙熒光素酶報(bào)告基因載體構(gòu)建成功。 4、腺苷酸環(huán)化酶激活劑forskolin重啟FMR1基因CRE位點(diǎn)驗(yàn)證 (1)經(jīng)SNP封閉作用后,MSE位、M1位、M2位三組與未加SNP的各自正常對(duì)照組比較均有統(tǒng)計(jì)學(xué)意義(P0.05),提示經(jīng)SNP作用,三組FMR1均被SNP封閉成功。 (2)未經(jīng)SNP封閉,單經(jīng)forskolin處理后,MSE位、M1位、M2位三組分別與各自的對(duì)照組相比較均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),提示forskolin對(duì)正�；钚缘腇MR1啟動(dòng)子,無(wú)論CRE序列結(jié)構(gòu)正常或突變,均無(wú)增強(qiáng)作用。 (3)已被SNP封閉后的FMR1,再添加forskolin處理,當(dāng)CRE位點(diǎn)正常時(shí),MSE組的熒光素酶活性顯著高于未添加forskolin的封閉對(duì)照組(P0.05)；在突變了CRE結(jié)合位點(diǎn)的M1組和M2組中,添加forskolin組與未添加forskolin封閉對(duì)照組均無(wú)顯著性差異(P0.05)。由此提示,當(dāng)FMR1啟動(dòng)子活性低下時(shí),在CRE結(jié)構(gòu)正常情況下,forskolin可開(kāi)啟封閉的FMR1基因,而當(dāng)突變CRE序列后,forskolin對(duì)FMR1封閉基因的開(kāi)啟作用則消失。 以上結(jié)果顯示當(dāng)CRE位點(diǎn)正常時(shí),forskolin對(duì)活性低下的FMR1啟動(dòng)了有顯著地激活作用,而對(duì)正�；钚缘腇MR1啟動(dòng)子則無(wú)增強(qiáng)作用；當(dāng)CRE位點(diǎn)突變之后,forskolin對(duì)正�；钚浴⒌拖禄钚缘腇MR1啟動(dòng)子均無(wú)開(kāi)啟作用。 結(jié)論 1.發(fā)現(xiàn)直接采用外源性環(huán)磷酸腺苷(db-cAMP)也可有效地重新誘導(dǎo)被封閉的FMR1基因和蛋白表達(dá),這有望為Fra(X)發(fā)病機(jī)制的研究和臨床治療提供新的思路和治療前景。 2.證實(shí)了cAMP激活劑forskolin開(kāi)啟FMR1封閉基因的機(jī)制之一是通過(guò)提高細(xì)胞內(nèi)cAMP水平而實(shí)現(xiàn)的。 3.成功構(gòu)建FMR1啟動(dòng)子區(qū)MSE/CRE位點(diǎn)雙熒光素酶報(bào)告基因系統(tǒng),結(jié)果顯示當(dāng)CRE位點(diǎn)正常時(shí),forskolin對(duì)活性低下的FMR1啟動(dòng)子有顯著地激活作用,而當(dāng)CRE位點(diǎn)突變之后,forskolin則無(wú)此作用,由此證實(shí)CRE位點(diǎn)是forskolin重啟FMR1基因的關(guān)鍵位點(diǎn)。推測(cè)AC激動(dòng)劑forskolin可能主要是通過(guò)FMR1啟動(dòng)子區(qū)內(nèi)的MSE/CRE重疊位點(diǎn),經(jīng)cAMP通路實(shí)現(xiàn)了對(duì)FMR1基因的調(diào)控。
[Abstract]:Fra ( X ) is one of the most common hereditary mental retardation diseases . It is estimated that there are at least 200,000 fragile X patients in our country . At present , it is considered that Fra ( X ) pathogenesis is mainly due to the unstable amplification of ( CGG ) n trino - nucleotide repeat sequence in the promoter region ( CGG ) of fragile X mental retardation - 1 gene ( FMR1 ) and the abnormal methylation of CpG islands upstream .

There is a certain correlation between the mental retardation and the signal transduction pathways in many researches . The relationship between the signal pathway of cyclic adenosine monophosphate ( cAMP ) and learning and memory and intelligence is closely related . It is found that in Fra ( X ) patient cells , the level of cAMP in brain tissue and platelets of mice bearing Fra ( X ) is lower than that of normal , and that of Fra ( X ) can compensate for this deficiency by re - implanting the FMR1 gene , which suggests that there may be mutual regulation between FMR1 gene and cAMP .

In this study , we found that the activity of adenylyl cyclase ( cAMP ) decreased and the activity of adenylyl cyclase ( cAMP ) decreased and the activity of adenylyl cyclase ( cAMP ) decreased .

If FMR1 gene transcription and translation termination are related to the decrease of adenylyl cyclase activity , whether the activity of adenylyl cyclase can be restarted by drugs ? In order to prove our guess , we use the specific adenylyl cyclase ( AC ) activator , forskolin ( FSK ) , to improve the low activity state of adenylyl cyclase . The results show that it can effectively re - induce the transcription and protein expression of the silencing FMR1 gene , but the specific mechanism forskolin ' s regulation of FMR1 gene is not clear .

It is known that the promoter region of the FMRI gene is closely related to the transcription of FMR1 gene . In this region , there is a methylation sensitive element ( MSE ) with enhancer activity , which is a binding site of cAMP - responsive element ( CRE ) .

In order to confirm the above assumption , this study is intended to investigate the transcription and protein expression of FMR1 gene induced by exogenous cAMP - induced silence FMR1 gene . It is attempted to demonstrate that forskolin is activated by increasing cAMP level in cells .
2 . Using the MSE / CRE site as the research target , the MR1 promoter region MSE / CRE site dual luciferase reporter gene system was constructed . In the absence of the overall promoter activity of MSE , the CRE sequence was mutated respectively to form the dual luciferase reporter gene of the mutant promoters M1 and M2 . The attempt to verify that forskolin was the mechanism of the FMR1 promoter region MSE / CRE site was used to establish the basis for further study the mechanism of the AC agonist forskolin to restart the FMR1 gene , and to provide a new thought and treatment prospect for the treatment of Fra ( X ) patients .

Study method :

1 . Production of pc12 cell FMR1 gene - enclosed cell model

The FMR1 gene was blocked by sodium nitrogenase ( SNP ) . The classical nitric oxide ( NO ) donor could be activated by NO to activate DNA methyltransferase ( DNA MeTase ) , so that cytosine in the MSE of CpG island of FMR1 promoter was highly methylated , blocking the transcription of FMRl mRNA . Finally , the closing effect of FMR1 gene was observed by real - time PCR .

2 . To observe the effect of dibutyrylamide ( db - cAMP ) on the expression of FMRl mRNA in pc12 cells

After 24 h of pc12 cells , the cells were harvested at 6h , 12h , 24h and 48h at 6h , 12h , 24h and 48h respectively , and the total RNA was extracted . The normal pc12 cells were used as control groups , and the expression of FMR1 gene was detected by fluorescence quantitative PCR ( dye method ) .

3 . Observe the effect of dibutyrylamide ( db - cAMP ) on FMR1 protein level ( FMRP )

After blocking pc12 cell FMR124h , 0.5 mmol / L db - cAMP was added . After 24 h , 48 h , 72 h , 96 h after drug administration , the cells were collected ( 12 hours supplemented with failed db - cAMP , 48 hour supplemented failed SNP ) , protein was extracted , Western Blot was performed , and semi - quantitative analysis was performed on FMRP .

4 . Construction of the FMR1 promoter MSE / CER overlapping sequence and mutant CRE dual luciferase reporter gene

The MSE / CRE overlapping sequences were isolated from K562 cell DNA by PCR , and then the CRE sequences were mutated by PCR - point mutation . The MSE , M1 , M2 were amplified by PCR and then ligated to the PGL3 vector , and then transformed into the DH5a cells . Then , the plasmids were extracted . The MSE / PGL3 , M1 / PGL3 , M2 / PGL3 were co - transfected with the marine renal plasmid pc12 cells , and the intensity values of luciferase activity were analyzed .

5 . Verify that the CRE site is the key site of the Adenylate Cyclase activator forskolin to restart the FMR1 gene

The effect of forskolin on the opening of FMRl gene was determined from DH50 bacteria by extracting MSE / PGL3 , M1 / PGL3 , and M2 / PGL3 plasmids , respectively .
Conversely , low ) , thereby verifying whether the CRE site is a key site for the adenylyl cyclase activator forskolin to restart the FMR1 gene .

6 . Statistics

The data of the experiment was statistically processed by SPSS 13.0 , and the data was expressed by mean standard deviation ( 蠂 ~ 鹵 s ) . The results of fluorescence quantitative PCR were analyzed by 2 - 鈻，

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