重组腺病毒—胸苷激酶自杀基因前药系统对鼻咽癌CNE-2细胞放射增敏作用的体外实验研究

发布时间：2018-03-19 01:14

  本文选题：鼻咽癌　切入点：腺病毒-胸苷激酶　出处：《泸州医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的：本实验利用进入Ⅱ期临床试验的重组腺病毒-胸苷激酶(Adenovirus - thymidine kinase, ADV-TK)转染鼻咽癌CNE-2细胞进行体外实验,观察ADV-TK/GCV(Adenovirus - thymidine kinase/ ganciclovir)自杀基因前药系统对鼻咽癌CNE-2细胞的杀伤作用以及放射增敏作用。方法：分别用不同感染复数(Multiplicity of Infection,MOI)的ADV-TK转染鼻咽癌CNE-2细胞,3h后更换新鲜培养基,继续培养72h,倒置显微镜下观察转染前后细胞形态变化,WST-1检测不同MOI转染后细胞存活率。不同浓度GCV作用于鼻咽癌CNE-2细胞,继续培养72h,倒置显微镜下观察细胞存活情况,WST-1检测不同浓度GCV作用下的细胞存活率。选取对鼻咽癌CNE-2细胞无明显毒性作用的MOI为100作为下一步实验研究,转染鼻咽癌CNE-2细胞,3h后更换新鲜培养基,继续培养24h,用不同浓度GCV作用,继续培养72h,倒置显微镜下观察细胞存活情况,WST-1检测细胞存活率,计算GCV的IC10、IC50。选取MOI为100及IC10进行放射增敏实验,实验分单纯放疗组、ADV-TK+放疗组、GCV+放疗组、ADV-TK/GCV+放疗组；MOI为100的ADV-TK转染细胞3h,更换新鲜培养基,继续培养24h,加入GCVIC10,继续培养48h,四组分别给予0、0.5、1、2、3、4、6、8、10Gy Y射线照射,每个照射剂量组设三个培养皿,照射后立即更换新鲜培养基,继续培养14d后,观察各不同处理组对CNE-2细胞的杀伤作用,计算克隆形成率、细胞存活分数,用单击多靶数学模型进行曲线拟合作图,计算放射增敏比。结果：倒置显微镜下观察ADV-TK基因转染后的鼻咽癌CNE-2细胞体积较对照组增大,轮廓较模糊,边界欠清,细胞内出现较多粗大的黑色颗粒样物质,主要集中于细胞核内,而未转染ADV-TK基因的细胞内无明显颗粒状物质出现。MOI100时存活率为98.21%,MOI为1000时存活率为85.97%,MOI≥1000时对鼻咽癌CNE-2细胞有明显毒性作用。GCV浓度为100ug/ml及1000ug/ml时,细胞存活率分别为98.38%和84.63%,GCV浓度为≥1000ug/ml时对细胞具有明显的抑制作用。选取MOI为100转染鼻咽癌CNE-2细胞,不同浓度GCV作用于细胞,计算IC10和IC50分别为7.1968ug/ml和196.0358ug/ml, GCV对鼻咽癌CNE-2细胞的杀伤作用具有浓度依赖性。放射增敏实验,单纯照射组：DO值为1.238 Gy,Dq值为2.838Gy,N值为3.284；ADV-TK+放射组：DO值为1.237Gy,Dq值为2.607 Gy,N值为3.108,放射增敏比SERD0为1.001,SERDq为1.089,SERSF2为1.036; GCV+放射组：DO值为1.232Gy, Dq值为2.800Gy, N值为3.274,放射增敏比SERD0为1.005, SERDq为1.014, SERSF2为1.005; AD V-TK/GCV+放射组：DO值为0.880Gy, Dq值为1.561Gy,N值为2.775,放射增敏比SERD0为1.407, SERDq为1.818, SERSF2为2.141。结论：(1)ADV-TK成功转染CNE-2细胞。(2)ADV-TK(MOI)≥1000对CNE-2细胞具有明显的毒性作用。(3) GCV≥1000ug/ml对鼻咽癌CNE-2细胞具有明显抑制作用。(4) ADV-TK/GCV自杀基因前药系统对鼻咽癌CEN-2细胞有明显的杀伤作用,并具有GCV浓度依赖性。(5) ADV-TK/GCV自杀基因系统对鼻咽癌CEN-2细胞有明显放射增敏作用。
[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，

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