本文選題：錯(cuò)配修復(fù)基因 + hMLH1�。� 參考：《第三軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：研究背景及目的 人DNA錯(cuò)配修復(fù)（mismatch repair, MMR）系統(tǒng)在維持基因組穩(wěn)定性中起著關(guān)鍵作用。MMR功能障礙導(dǎo)致的微衛(wèi)星不穩(wěn)定（microsatellite instability,MSI）與許多惡性腫瘤的發(fā)生有密切關(guān)系。大部分遺傳性非息肉病性結(jié)直腸癌（hereditary nonpolyposis colorectal cancer, HNPCC）由MMR基因種系突變引起。此外，一部分散發(fā)性結(jié)直腸癌也與MMR功能障礙相關(guān)。hMLH1是體內(nèi)最重要的MMR基因之一。前期研究發(fā)現(xiàn)，正常人血清雌激素（Estradiol，E2）水平與結(jié)腸上皮細(xì)胞中hMLH1基因表達(dá)水平呈正相關(guān)。細(xì)胞實(shí)驗(yàn)證實(shí)，E2能上調(diào)結(jié)腸癌細(xì)胞株COLO205的hMLH1基因表達(dá)量，并且這種上調(diào)作用很可能發(fā)生在轉(zhuǎn)錄水平。然而，E2如何調(diào)節(jié)錯(cuò)配修復(fù)基因hMLH1表達(dá)的具體機(jī)制尚不清楚。本研究旨在構(gòu)建含hMLH1啟動(dòng)子片段的熒光素酶報(bào)告基因載體，檢測(cè)其在HEK293和LoVo細(xì)胞株中E2誘導(dǎo)下的轉(zhuǎn)錄活性。 研究方法 1、根據(jù)UCSC（www.geome.ucsc.edu）數(shù)據(jù)庫(kù)確定hMLH1基因轉(zhuǎn)錄起始位點(diǎn)，用PCR方法克隆hMLH1啟動(dòng)子序列（-1953/+53），，定向插入到雙熒光報(bào)告基因載體pGL3-Basic，抽提質(zhì)粒并經(jīng)雙酶切和測(cè)序鑒定。構(gòu)建好的含hMLH1啟動(dòng)子的真核表達(dá)質(zhì)粒命名為pGL3-Promoter-luc。 2、用瞬時(shí)轉(zhuǎn)染的方法將pGL3-Promoter-luc、陰性對(duì)照（pGL3-Basic）和陽(yáng)性對(duì)照（pGL3-Control）分別與內(nèi)參質(zhì)粒（pRL-SV40）共轉(zhuǎn)入HEK293和LoVo細(xì)胞。檢測(cè)不同作用時(shí)間和不同劑量的E2對(duì)報(bào)告基因熒光素酶活性值的影響，以及E2-BSA和ICI182.780對(duì)hMLH1啟動(dòng)子轉(zhuǎn)錄活性的影響。 3、運(yùn)用Western Blot法檢測(cè)HEK293和LoVo細(xì)胞中hMLH1的相對(duì)表達(dá)量。 結(jié)果 1、構(gòu)建的含hMLH1啟動(dòng)子的重組質(zhì)粒經(jīng)酶切和測(cè)序鑒定,插入的核苷酸序列與UCSC數(shù)據(jù)庫(kù)中hMLH1啟動(dòng)子區(qū)序列完全吻合，說(shuō)明報(bào)告基因重組質(zhì)粒構(gòu)建成功。 2、報(bào)告基因熒光素酶活性對(duì)E2存在一定的劑量依賴和時(shí)間依賴關(guān)系。10~(-9)mol/L的E2處理24小時(shí)后，報(bào)告基因熒光素酶活性值增強(qiáng)最明顯（n=3，P0.01），而這種效應(yīng)能被雌激素受體拮抗劑ICI182.780抑制。E2-BSA對(duì)目的基因的上調(diào)作用不如E2顯著。 3、運(yùn)用Western Blot檢測(cè)HEK293和LoVo細(xì)胞株內(nèi)源性hMLH1基因蛋白表達(dá)量，轉(zhuǎn)染pRST7-ERβ組高于對(duì)照組（n=3，P0.01）。結(jié)論 E2能上調(diào)HEK293和LoVo細(xì)胞錯(cuò)配修復(fù)基因MLH1的表達(dá)，并能顯著增強(qiáng)由hMLH1啟動(dòng)子序列引導(dǎo)的熒光素酶表達(dá)活性，說(shuō)明hMLH1啟動(dòng)子序列中存在與E2相關(guān)的調(diào)控序列，且ERβ在此過(guò)程中起到重要作用。該報(bào)告基因載體為進(jìn)一步明確參與調(diào)控的順式作用元件和轉(zhuǎn)錄因子奠定了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Background and objective Human DNA mismatch repair (mismatch repair,) system plays a key role in maintaining genomic stability. The microsatellite instability (microsatellite instability) caused by MMR dysfunction is closely related to the occurrence of many malignant tumors. Most hereditary nonpolyposis colorectal cancer (hereditary nonpolyposis colorectal cancer, HNPCC) is caused by mutation of MMR gene. In addition, some sporadic colorectal cancer is also associated with MMR dysfunction. HMLH1 is one of the most important MMR genes in vivo. Previous studies showed that the level of serum estradiol E _ 2 (E _ 2) was positively correlated with the expression of hMLH1 gene in colonic epithelial cells. Cell experiments confirmed that E _ 2 could up-regulate the expression of hMLH1 gene in colon cancer cell line COLO205, and this up-regulation may occur at the transcription level. However, it is unclear how E2 regulates the expression of mismatch repair gene hMLH1. The aim of this study was to construct a luciferase reporter gene vector containing hMLH1 promoter fragment and to detect its transcriptional activity induced by E2 in HEK293 and LoVo cell lines. Methods 1.According to the UCSC (www.geome.ucsc.edu) database, hMLH1 promoter sequence (-1953 / 53) was cloned and inserted into the double fluorescent reporter gene vector pGL3-Basic. the plasmid was extracted and identified by double enzyme digestion and sequencing. The constructed eukaryotic expression plasmids containing hMLH1 promoter were named pGL3-Promoter-Luc.2. PGL3-Promoter-luc, pGL3-basic and pGL3-Control were co-transfected into HEK293 and LoVo cells with pRL-SV40, respectively. The effects of different time and dose of E2 on luciferase activity of reporter gene and the transcriptional activity of E2-BSA and ICI182.780 on hMLH1 promoter were detected. 3 the relative expression of hMLH1 in HEK293 and LoVo cells was detected by Western Blot. Results 1. The recombinant plasmid containing hMLH1 promoter was identified by restriction endonuclease digestion and sequencing. The inserted nucleotide sequence was consistent with the sequence of hMLH1 promoter in UCSC database. The results showed that the reporter gene recombinant plasmid was successfully constructed. 2. The luciferase activity of the reporter gene had a dose-dependent and time-dependent relationship with E2 for 24 hours after treatment with E2 of 10 ~ (-9) mol / L. The luciferase activity of the reporter gene was increased most significantly (nng3, P0.01), and this effect was inhibited by estrogen receptor antagonist ICI182.780. E2-BSA was not as up-regulated as E2. 3. Western Blot was used to detect the effect in HEK293 and LoVo cell lines. Protein expression of hMLH1 gene, The transfection of pRST7-ER 尾 group was higher than that of control group (nrST7-ER 尾 group). Conclusion E2 can up-regulate the expression of mismatch repair gene MLH1 in HEK293 and LoVo cells, and enhance the luciferase expression activity guided by hMLH1 promoter, indicating that there are E2 related regulatory sequences in hMLH1 promoter sequence. And ER 尾 plays an important role in this process. The report gene vector lays the experimental foundation for further clarifying the cis-acting elements and transcription factors involved in regulation.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R346

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相關(guān)期刊論文 前2條

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