發(fā)布時間：2018-03-19 01:14

  本文選題：鼻咽癌　切入點：腺病毒-胸苷激酶　出處：《瀘州醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：本實驗利用進入Ⅱ期臨床試驗的重組腺病毒-胸苷激酶(Adenovirus - thymidine kinase, ADV-TK)轉(zhuǎn)染鼻咽癌CNE-2細胞進行體外實驗,觀察ADV-TK/GCV(Adenovirus - thymidine kinase/ ganciclovir)自殺基因前藥系統(tǒng)對鼻咽癌CNE-2細胞的殺傷作用以及放射增敏作用。方法：分別用不同感染復(fù)數(shù)(Multiplicity of Infection,MOI)的ADV-TK轉(zhuǎn)染鼻咽癌CNE-2細胞,3h后更換新鮮培養(yǎng)基,繼續(xù)培養(yǎng)72h,倒置顯微鏡下觀察轉(zhuǎn)染前后細胞形態(tài)變化,WST-1檢測不同MOI轉(zhuǎn)染后細胞存活率。不同濃度GCV作用于鼻咽癌CNE-2細胞,繼續(xù)培養(yǎng)72h,倒置顯微鏡下觀察細胞存活情況,WST-1檢測不同濃度GCV作用下的細胞存活率。選取對鼻咽癌CNE-2細胞無明顯毒性作用的MOI為100作為下一步實驗研究,轉(zhuǎn)染鼻咽癌CNE-2細胞,3h后更換新鮮培養(yǎng)基,繼續(xù)培養(yǎng)24h,用不同濃度GCV作用,繼續(xù)培養(yǎng)72h,倒置顯微鏡下觀察細胞存活情況,WST-1檢測細胞存活率,計算GCV的IC10、IC50。選取MOI為100及IC10進行放射增敏實驗,實驗分單純放療組、ADV-TK+放療組、GCV+放療組、ADV-TK/GCV+放療組；MOI為100的ADV-TK轉(zhuǎn)染細胞3h,更換新鮮培養(yǎng)基,繼續(xù)培養(yǎng)24h,加入GCVIC10,繼續(xù)培養(yǎng)48h,四組分別給予0、0.5、1、2、3、4、6、8、10Gy Y射線照射,每個照射劑量組設(shè)三個培養(yǎng)皿,照射后立即更換新鮮培養(yǎng)基,繼續(xù)培養(yǎng)14d后,觀察各不同處理組對CNE-2細胞的殺傷作用,計算克隆形成率、細胞存活分?jǐn)?shù),用單擊多靶數(shù)學(xué)模型進行曲線擬合作圖,計算放射增敏比。結(jié)果：倒置顯微鏡下觀察ADV-TK基因轉(zhuǎn)染后的鼻咽癌CNE-2細胞體積較對照組增大,輪廓較模糊,邊界欠清,細胞內(nèi)出現(xiàn)較多粗大的黑色顆粒樣物質(zhì),主要集中于細胞核內(nèi),而未轉(zhuǎn)染ADV-TK基因的細胞內(nèi)無明顯顆粒狀物質(zhì)出現(xiàn)。MOI100時存活率為98.21%,MOI為1000時存活率為85.97%,MOI≥1000時對鼻咽癌CNE-2細胞有明顯毒性作用。GCV濃度為100ug/ml及1000ug/ml時,細胞存活率分別為98.38%和84.63%,GCV濃度為≥1000ug/ml時對細胞具有明顯的抑制作用。選取MOI為100轉(zhuǎn)染鼻咽癌CNE-2細胞,不同濃度GCV作用于細胞,計算IC10和IC50分別為7.1968ug/ml和196.0358ug/ml, GCV對鼻咽癌CNE-2細胞的殺傷作用具有濃度依賴性。放射增敏實驗,單純照射組：DO值為1.238 Gy,Dq值為2.838Gy,N值為3.284；ADV-TK+放射組：DO值為1.237Gy,Dq值為2.607 Gy,N值為3.108,放射增敏比SERD0為1.001,SERDq為1.089,SERSF2為1.036; GCV+放射組：DO值為1.232Gy, Dq值為2.800Gy, N值為3.274,放射增敏比SERD0為1.005, SERDq為1.014, SERSF2為1.005; AD V-TK/GCV+放射組：DO值為0.880Gy, Dq值為1.561Gy,N值為2.775,放射增敏比SERD0為1.407, SERDq為1.818, SERSF2為2.141。結(jié)論：(1)ADV-TK成功轉(zhuǎn)染CNE-2細胞。(2)ADV-TK(MOI)≥1000對CNE-2細胞具有明顯的毒性作用。(3) GCV≥1000ug/ml對鼻咽癌CNE-2細胞具有明顯抑制作用。(4) ADV-TK/GCV自殺基因前藥系統(tǒng)對鼻咽癌CEN-2細胞有明顯的殺傷作用,并具有GCV濃度依賴性。(5) ADV-TK/GCV自殺基因系統(tǒng)對鼻咽癌CEN-2細胞有明顯放射增敏作用。
[Abstract]:Objective: to investigate the effect of recombinant adenovirus-thymidine kinase thymidine kinase (ADV-TK) on nasopharyngeal carcinoma (NPC) CNE-2 cells in vitro. To observe the killing effect and radiosensitization of ADV-TK/GCV(Adenovirus thymidine kinase/ ganciclovirus (ADV-TK/GCV(Adenovirus thymidine kinase/ ganciclovirl) suicide gene prodrug system on CNE-2 cells of nasopharyngeal carcinoma (NPC). After 72 hours of culture, the morphological changes of nasopharyngeal carcinoma CNE-2 cells were observed under inverted microscope. WST-1 was used to detect the survival rate of different MOI transfected cells. Different concentrations of GCV acted on nasopharyngeal carcinoma CNE-2 cells. After 72 hours of culture, cell survival was observed under inverted microscope. The survival rate of CNE-2 cells treated with different concentrations of GCV was determined by WST-1. The MOI of 100 was chosen as the next step of the experimental study, which had no obvious toxicity to CNE-2 cells of nasopharyngeal carcinoma. After transfection of nasopharyngeal carcinoma (NPC) CNE-2 cells, fresh culture medium was changed for 3 h, cultured for 24 h, and then cultured for 72 h with different concentrations of GCV. The survival rate of nasopharyngeal carcinoma CNE-2 cells was observed under inverted microscope and WST-1 was used to detect the survival rate of the cells. The IC10 / IC50 of GCV was calculated. The radiosensitization test was carried out with 100 MOI and IC10. The experiment was divided into two groups: single radiotherapy group (ADV-TK radiotherapy group), GCV radiotherapy group (ADV-TK / GCV radiotherapy group) and ADV-TK transfection cell with MOI of 100 (ADV-TK / GCV radiation group) for 3 h, and the fresh culture medium was replaced. After 24 hours of culture, GCVIC10 was added to GCVIC10, and 48 hours after culture, the four groups were irradiated with 0 ~ (0.5) ~ (0.5) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (3) C ~ (-1) ~ (-) ~ (-) ~ (8) ~ (10) Gy Y radiation respectively. Three petri dishes were set up in each irradiation dose group. The killing effect of different treatment groups on CNE-2 cells was observed. The clone formation rate and cell survival fraction were calculated. Results: the volume of nasopharyngeal carcinoma CNE-2 cells transfected with ADV-TK gene was larger than that of the control group, the contour was blurred, the boundary was not clear, and a lot of coarse black particles appeared in the cells under inverted microscope. The survival rate of the cells without transfection of ADV-TK gene was 98.21 and the survival rate of MOI 1000 was 85.97 moi 鈮，

本文編號：1632163