發(fā)布時間：2018-03-24 22:20

  本文選題：人乳頭瘤病毒　切入點：11　出處：《重慶醫(yī)科大學》2005年碩士論文

【摘要】：人乳頭瘤病毒(human papillomavirus,HPV)是一種無包膜的環(huán)狀閉合雙鏈 DNA 病毒,以特異感染人類皮膚和粘膜上皮為特征。迄今,經鑒定和測序的 HPV 基因型已超過 100 多種。根據 HPV 所致疾病的惡性程度,可將其分為高危型和低危型。高危型 HPV(如 HPV16、18、31)與生殖道、皮膚及喉部鱗狀細胞癌密切相關,其基因組整合入宿主細胞染色體中,引起細胞惡性轉化,致病機制已基本闡明;而低危型 HPV(如 HPV6、11)引起尖銳濕疣等良性增生,很少發(fā)展成為惡性,所以長期以來不被重視。 由于缺乏對尖銳濕疣發(fā)病機制的了解,至今對尖銳濕疣只能進行對癥治療。近年來,隨著尖銳濕疣的發(fā)病率大幅度上升,人們逐漸認識到“低危并不等于不重要”,從病因學入手,研究低危型 HPV,特別是最常見的 HPV11 的作用機制顯得尤為重要與迫切。 已知高危型 HPV E6 蛋白可以通過泛素蛋白水解途徑特異性的降解細胞 p53 蛋白。由于 p53 能夠通過 p21WAF1/CIP1間接導致 G1 期阻滯,使細胞贏得時間,在進入 S 期前修復損傷的 DNA。E6 引起的 p53 的降解,解除了 p53 對生長的負向調節(jié)功能。這就允許遺傳改變的細胞繼續(xù)增殖,導致基因組不穩(wěn)定進而引發(fā)腫瘤。由于低危型 HPV 和高危型HPV 的基因組結構和核酸序列具有高度一致性,因此可以推斷低危型HPV E6 蛋白也有相似的作用機制。 本研究選用低危型 HPV 中有代表性的 HPV11 為研究對象,主要目的是通過構建帶有增強型綠色熒光蛋白(EGFP)的 HPV11E6 基因 重慶醫(yī)科大學碩士研究生學位論文 4 的真核表達質粒,應用體外(in vitro)基因轉染技術將該質粒轉染入 人真皮成纖維細胞,進而研究 HPV11E6 對該細胞 p53 蛋白穩(wěn)定性的影 響,借以了解 HPV11E6 的功能,為進一步全面闡明 HPV11 引起尖銳 濕疣的機制奠定一定的基礎。 本研究主要由三部分組成: 一、HPV11E6 基因綠色熒光蛋白真核表達載體的構建和鑒定 根據已知的 HPV11E6 序列,設計在目的片段兩端分別帶 BamHI 和 EcoRI 酶切位點的引物。用 PCR 方法以質粒 pBR322/HPV11 為模板 擴增該基因片段,經 BamHI 和 EcoRI 雙酶切后回收純化,定向克隆至 含有以增強型綠色熒光蛋白為報告基因的真核表達質粒 pIRES2-EGFP 中。重組質粒 pIRES2-HPV11E6-EGFP 經限制性酶切和 DNA 序列測定 證明構建正確,為研究 HPV11E6 在細胞內的功能提供了一個方便而重 要的工具。 二、重組質粒 pIRES2-HPV11E6-EGFP 在人真皮成纖維細胞中的表 達 取臨床包皮組織,用含氨芐青霉素和硫酸鏈霉素的 PBS 反復漂洗 后,剪棄脂肪和結締組織。將皮膚剪成 1mm3左右的小塊,膠原酶和透 明質酸酶消化,離心細胞加入 DMEM,在 37℃、5%CO2 培養(yǎng)箱中培 養(yǎng),形態(tài)學觀察鑒定。收獲處于指數生長期的成纖維細胞約 1×105個, 將細胞接種于 6 孔細胞培養(yǎng)板中培養(yǎng) 24h,使細胞達到 50-80%融合。 用轉染試劑 DOTAP,按照說明書將 pIRES2-HPV11E6-EGFP 質粒轉染 入成纖維細胞。轉染后 12h,在熒光顯微鏡下可以觀察到熒光蛋白的表 達3,6-48h為熒光蛋白表達的高峰期;收集細胞提取RNA后,用RT-PCR 方法檢測細胞總 RNA 中目的基因 mRNA 的表達,結果為陽性,說明 在轉錄水平有目的基因的表達。 三、HPV11E6 在人真皮成纖維細胞內功能的初步研究
[Abstract]:Human papilloma virus (human papillomavirus HPV) is a non enveloped closed circular double stranded DNA virus, with specific infection of human skin and mucosal epithelial features. So far, the HPV genotyping and sequencing has been more than 100. According to the degree of malignancy of HPV disease, which can be divided into high-risk and the low-risk type. The high-risk HPV (such as HPV16,18,31) and the reproductive tract, skin and laryngeal squamous cell carcinoma is closely related to the genome integration into the chromosome of the host cell, cause cell malignant transformation, the pathogenic mechanism has been clarified; and the low risk HPV (such as HPV6,11) caused by benign hyperplasia of condyloma acuminatum, rarely become for a long time not to be malignant, so seriously.
Due to the lack of understanding of the pathogenesis of condyloma acuminatum, since the symptomatic treatment of condyloma acuminatum. Only in recent years, with the incidence of condyloma acuminatum rate increased to a great extent, people gradually realize that "low risk is important, starting from etiology, of low risk HPV, especially the most common mechanism of HPV11 it is particularly important and urgent.
Known high-risk HPV E6 protein through the ubiquitin proteolytic pathway specific degradation of cell p53 protein. Because p53 can cause G1 arrest by indirect p21WAF1/CIP1, the cell gain time, degradation caused by in the S period before the repair of DNA.E6 p53 p53, released on the growth of the negative regulation of this function. Allow the genetically altered cells continue to proliferate, leading to genomic instability and tumor initiation. The nucleic acid sequence of genome structure and low risk HPV and high-risk HPV has a high degree of consistency, it can be concluded that the low risk HPV E6 protein has a similar mechanism.
In this study, the representative HPV11 in low risk HPV was selected as the research object, and the main purpose was to construct HPV11E6 gene with enhanced green fluorescent protein (EGFP).
Master's degree thesis of Medical University Of Chongqing
Four
The eukaryotic expression plasmid was transfected into the plasmid by in vitro gene transfection technique.
Human dermal fibroblasts, and then study the effect of HPV11E6 on the stability of p53 protein in this cell
Ringing to understand the function of HPV11E6 and to sharpen the HPV11 for further clarifying
The mechanism of condyloma acuminata lays a certain foundation.
This study mainly consists of three parts:
Construction and identification of the eukaryotic expression vector of HPV11E6 gene green fluorescent protein
According to the known HPV11E6 sequence, BamHI is designed at both ends of the target segment.
Primers at the site of the EcoRI enzyme cutting site. Plasmid pBR322/HPV11 is used as a template by PCR
The gene fragment was amplified and purified by BamHI and EcoRI double enzyme digestion.
Eukaryotic expression plasmid pIRES2-EGFP containing enhanced green fluorescent protein as the reporter gene
Determination of recombinant plasmid pIRES2-HPV11E6-EGFP by restriction enzyme digestion and DNA sequencing
Proving that the construction is correct, it provides a convenient and heavy study for the function of HPV11E6 in cell.
The tools you want.
Two, the table of recombinant plasmid pIRES2-HPV11E6-EGFP in human dermal fibroblasts
reach
The clinical prepuce tissue, repeated rinse with ampicillin and streptomycin sulfate PBS
After that, cut off the fat and connective tissue. Cut the skin into small pieces around 1mm3, collagenase and dialysis
After digestion, the centrifugation cells were added to DMEM and were cultured at 37 C, 5%CO2 culture box.
1 * 105 fibroblasts were harvested at an exponential growth period.
The cells were inoculated into the 6 cell culture plate and cultured 24h to achieve 50-80% fusion.
Transfection of pIRES2-HPV11E6-EGFP plasmid with the transfection reagent DOTAP according to the instructions
Into fibroblasts. After transfection of 12h, a fluorescent protein table can be observed under the fluorescence microscope.
3,6-48h is the peak of the expression of the fluorescent protein; after the collection of RNA, RT-PCR
Methods the expression of the target gene mRNA in the total cell RNA was detected, and the results were positive, indicating that the expression of the target gene was positive.
The expression of the target gene at the transcriptional level.
A preliminary study of the function of three, HPV11E6 in human dermal fibroblasts

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

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1 左國偉;人乳頭瘤病毒11型E6基因的克隆、真核表達及其功能的初步研究[D];重慶醫(yī)科大學;2005年

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本文編號：1660271