本文關(guān)鍵詞： HPV16E6蛋白 HPV18E7蛋白 宮頸癌 早期診斷 免疫組化　出處：《重慶理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：宮頸癌是由人乳頭瘤病毒(Human papllomavirus,HPV)引起的一種常見的高發(fā)于女性的惡性腫瘤,其死亡率高居女性腫瘤第二位。近年來統(tǒng)計發(fā)現(xiàn),我國農(nóng)村地區(qū)宮頸癌篩查范圍覆蓋低,農(nóng)村地區(qū)發(fā)病率和致死率均高于城市,并呈逐年增長趨勢且發(fā)病年齡趨向年輕化,嚴重威脅女性健康。高危型HPV呈持續(xù)感染,尤其是HPV16和HPV18兩個型,是導(dǎo)致宮頸癌的根本原因。此外,E6和E7蛋白的過表達在細胞惡性轉(zhuǎn)化中起到至關(guān)重要的作用,為潛在的宮頸癌早期診斷標(biāo)志物。目的:目前我國尚無商業(yè)化的基于HPV16E6和HPV18E7蛋白的免疫組化試劑盒,用于檢測臨床樣本中的HPV16和HPV18。用于臨床HPV16和HPV18檢測的多為HPV DNA檢測,不利于資源匱乏地區(qū)推廣。為此,建立擁有自主知識產(chǎn)權(quán)的,基于HPV16E6和HPV18E7蛋白的免疫組化試劑盒更具優(yōu)勢。我們的目的是,以HPV16E6和HPV18E7蛋白為診斷標(biāo)志物,建立特異性好、成本低的HPV16E6和HPV18E7蛋白免疫組化試劑盒,用于宮頸癌的早期診斷。方法:本課題采用經(jīng)鑒定的HPV16E6和HPV18E7重組蛋白為抗原,分別用于抗HPV16E6和HPV18E7單克隆抗體的制備。并基于抗HPV16E6和抗HPV18E7單抗,分別建立宮頸癌細胞系的HPV16E6和HPV18E7蛋白檢測免疫組化試劑盒。在此基礎(chǔ)上,將建立的免疫組化試劑盒應(yīng)用于臨床樣本的檢測。結(jié)果:(1)以純化的HPV16E6和HPV18E7蛋白為抗原免疫小鼠,經(jīng)過亞克隆篩選,分別得到了7株和8株可穩(wěn)定分泌特異性高、親和性好的抗HPV16E6單抗和抗HPV18E7單抗的雜交瘤細胞株,并分別選擇效價最高的屬Ig G亞類單抗的HPV16E6-4D4、HPV18E7-E11細胞株進行單抗的制備與鑒定;(2)基于HPV16E6-4D4、HPV18E7-E11單克隆抗體,分別初步建立了宮頸癌細胞系的HPV16E6和HPV18E7蛋白免疫組化檢測試劑盒;(3)用所建立的HPV16和HPV18蛋白免疫組化檢測試劑盒分別檢測臨床樣本,都成功檢測出HPV16E6和HPV18E7蛋白表達陽性,與宮頸癌病理檢查結(jié)果的符合率分別達95.23%和100%,均顯示了良好的診斷性能與潛力。結(jié)論:本實驗初步建立了HPV16E6和HPV18E7蛋白免疫組化檢測試劑盒,并用于不同時期的宮頸癌或癌前病變組織臨床樣本檢測,其具有成本低、特異性好的特點,可用于宮頸癌的早期診斷。
[Abstract]:Cervical cancer is a common malignant tumor in women caused by human papillomavirus (HPV). In recent years, it is found that the scope of cervical cancer screening in rural areas in China is low, the morbidity and mortality in rural areas are higher than those in urban areas. And the disease age tends to be younger, which is a serious threat to the health of women. The high-risk type of HPV is persistent infection, especially HPV16 and HPV18. In addition, the overexpression of E6 and E7 proteins plays a crucial role in the malignant transformation of cells. Objective: there is no commercial immunohistochemical kit based on HPV16E6 and HPV18E7 protein in China. For the detection of HPV16 and HPV18 in clinical samples. Most of the clinical HPV16 and HPV18 detection is HPV DNA detection, which is not conducive to the promotion of resource-poor areas. The establishment of HPV16E6 and HPV18E7 protein based immunohistochemical kits with independent intellectual property rights is more advantageous. Our aim is. Using HPV16E6 and HPV18E7 proteins as diagnostic markers, an immunohistochemical kit of HPV16E6 and HPV18E7 proteins with good specificity and low cost was established. Methods: the recombinant proteins of HPV16E6 and HPV18E7 were used as antigen. It was used in the preparation of monoclonal antibodies against HPV16E6 and HPV18E7, respectively, and was based on anti HPV16E6 and anti HPV18E7 monoclonal antibodies. The HPV16E6 and HPV18E7 protein detection kits of cervical cancer cell lines were established respectively. Results the purified HPV16E6 and HPV18E7 proteins were used as antigen to immunize mice. Seven and eight hybridoma cell lines with stable secretion specificity and good affinity were obtained, respectively. The HPV16E6-4D4D4 HPV18E7-E11 cell line with the highest titer of Ig G subclass McAb was selected to prepare and identify the McAb. (2) based on monoclonal antibody HPV16E6-4D4, HPV18E7-E11. HPV16E6 and HPV18E7 protein immunohistochemical detection kits of cervical cancer cell lines were established. The positive expression of HPV16E6 and HPV18E7 protein was detected successfully by using the established HPV16 and HPV18 protein immunohistochemical detection kit to detect the clinical samples. The coincidence rate with pathological examination of cervical cancer was 95.23% and 100% respectively. Conclusion: the HPV16E6 and HPV18E7 protein immunohistochemical detection kit was established in this experiment. It can be used in the early diagnosis of cervical cancer because of its low cost and good specificity.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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