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

【摘要】：研究背景及目的 人DNA錯配修復（mismatch repair, MMR）系統(tǒng)在維持基因組穩(wěn)定性中起著關鍵作用。MMR功能障礙導致的微衛(wèi)星不穩(wěn)定（microsatellite instability,MSI）與許多惡性腫瘤的發(fā)生有密切關系。大部分遺傳性非息肉病性結直腸癌（hereditary nonpolyposis colorectal cancer, HNPCC）由MMR基因種系突變引起。此外，一部分散發(fā)性結直腸癌也與MMR功能障礙相關。hMLH1是體內最重要的MMR基因之一。前期研究發(fā)現(xiàn)，正常人血清雌激素（Estradiol，E2）水平與結腸上皮細胞中hMLH1基因表達水平呈正相關。細胞實驗證實，E2能上調結腸癌細胞株COLO205的hMLH1基因表達量，并且這種上調作用很可能發(fā)生在轉錄水平。然而，E2如何調節(jié)錯配修復基因hMLH1表達的具體機制尚不清楚。本研究旨在構建含hMLH1啟動子片段的熒光素酶報告基因載體，檢測其在HEK293和LoVo細胞株中E2誘導下的轉錄活性。 研究方法 1、根據(jù)UCSC（www.geome.ucsc.edu）數(shù)據(jù)庫確定hMLH1基因轉錄起始位點，用PCR方法克隆hMLH1啟動子序列（-1953/+53），，定向插入到雙熒光報告基因載體pGL3-Basic，抽提質粒并經雙酶切和測序鑒定。構建好的含hMLH1啟動子的真核表達質粒命名為pGL3-Promoter-luc。 2、用瞬時轉染的方法將pGL3-Promoter-luc、陰性對照（pGL3-Basic）和陽性對照（pGL3-Control）分別與內參質粒（pRL-SV40）共轉入HEK293和LoVo細胞。檢測不同作用時間和不同劑量的E2對報告基因熒光素酶活性值的影響，以及E2-BSA和ICI182.780對hMLH1啟動子轉錄活性的影響。 3、運用Western Blot法檢測HEK293和LoVo細胞中hMLH1的相對表達量。 結果 1、構建的含hMLH1啟動子的重組質粒經酶切和測序鑒定,插入的核苷酸序列與UCSC數(shù)據(jù)庫中hMLH1啟動子區(qū)序列完全吻合，說明報告基因重組質粒構建成功。 2、報告基因熒光素酶活性對E2存在一定的劑量依賴和時間依賴關系。10~(-9)mol/L的E2處理24小時后，報告基因熒光素酶活性值增強最明顯（n=3，P0.01），而這種效應能被雌激素受體拮抗劑ICI182.780抑制。E2-BSA對目的基因的上調作用不如E2顯著。 3、運用Western Blot檢測HEK293和LoVo細胞株內源性hMLH1基因蛋白表達量，轉染pRST7-ERβ組高于對照組（n=3，P0.01）。結論 E2能上調HEK293和LoVo細胞錯配修復基因MLH1的表達，并能顯著增強由hMLH1啟動子序列引導的熒光素酶表達活性，說明hMLH1啟動子序列中存在與E2相關的調控序列，且ERβ在此過程中起到重要作用。該報告基因載體為進一步明確參與調控的順式作用元件和轉錄因子奠定了實驗基礎。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R346

【參考文獻】

相關期刊論文 前2條

1 陸曉娟;余東亮;王佳鋅;潘笑露;金鵬;李世榮;盛劍秋;;雌激素對結腸癌細胞株COLO205錯配修復基因表達的影響[J];細胞與分子免疫學雜志;2011年07期

2 牟韶嬌;盛劍秋;謝惠;金鵬;陸曉娟;高巍;;雌激素對結腸細胞錯配修復活性的影響[J];胃腸病學和肝病學雜志;2011年05期

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