本文選題：4-1BBL　切入點：CD28單克隆抗體　出處：《汕頭大學》2010年碩士論文

【摘要】：目的在細胞水平,研究口腔癌瘤苗TCV-4-1BBL聯(lián)合CD28單克隆抗體(CD28 mAb)誘導T淋巴細胞抗腫瘤活性的能力。 方法1.建立穩(wěn)定表達4-1BBL克隆細胞株：通過脂質體法將真核載體pEGFP-4-1BBL轉染人口腔鱗癌細胞Tca8113,經G418(400μg/ml)篩選及有限稀釋后獲得穩(wěn)定高表達4-1BBL克隆細胞株Tca8113-4-1BBL,分別用RT-PCR和Western blot檢測轉染細胞中4-1BBL mRNA和蛋白的表達。2.制備瘤苗：將轉染與未轉染4-1BBL基因的Tca8113細胞用絲裂霉素C (MMC)處理后,制成腫瘤細胞瘤苗,分別命名TCV-4-1BBL、TCV-Tca8113。3. TCV-4-1BBL瘤苗聯(lián)合CD28 mAb誘導抗腫瘤活性的能力：TCV-4-1BBL聯(lián)合CD28 mAb與經體外CD3單克隆抗體(CD3 mAb)誘導的人外周血T淋巴細胞共同培養(yǎng),實驗分四組,①Tca8113組：T淋巴細胞+TCV-Tca8113;②Tca8113+CD3 mAb組：T淋巴細胞+CD3 mAb+ TCV-Tca8113;③Tca8113-4-1BBL+CD3 mAb組：T淋巴細胞+CD3 mAb+TCV-4-1BBL;④Tca8113-4-1BBL +CD3 mAb組+CD28 mAb:T淋巴細胞+CD3 mAb+TCV-4-1BBL+CD28 mAb。各實驗組培養(yǎng)72小時后,臺盼藍染色計數T細胞、用Cell Counting Kit-8 (CCK-8)測細胞毒性T細胞(CTL)殺傷活性,ELISA法檢測細胞因子IL-2、IFN-γ的分泌。 結果1.熒光顯微鏡下觀測到轉染了pEGFP-4-1BBL的Tca8113細胞(Tca8113-4-1BBL)綠色熒光蛋白的表達,RT-PCR、western blot在mRNA和蛋白水平檢測到4-1BBL表達。2.72h后臺盼藍染色計數上述混合培養(yǎng)的T細胞,④組1.78±0.16,高于①組0.917±0.12,②組1.01±0.14及③組1.38±0.15,P0.05,促進T細胞的增殖。3.72h后測CTL的殺傷活性：④組59.2±5.1,高于②組25.7±3.5及③組38.9±3.4,P0.05,促進T細胞的活化。4.72h后各組IL-2的分泌：④組881.2131±78,顯著高于①組56.1619±13,②組176.4235±33,③組526.1235±67,P0.05,促進IL-2分泌。5.72h后各組IFN-γ的分泌：④組402.81±34高于其他各組(②組(69.83±13),③組246.87±24,①組37.85±11),促進IFN-γ的分泌。結論pEGFP/neo-4-1BBL真核表達載體在Tca8113細胞中獲得穩(wěn)定表達,口腔癌瘤苗Tca8113-4-lBBL構建成功。在細胞水平顯示,TCV-4-1BBL瘤苗聯(lián)合CD28 mAb,能增強T細胞抗腫瘤免疫功能,促進T細胞增殖、活化,促進IL-2、IFN-γ分泌可能是其機制之一。
[Abstract]:Objective to study the antitumor activity of T lymphocytes induced by oral cancer vaccine (TCV-4-1BBL) combined with CD28 monoclonal antibody (CD28 mAb) at the cell level.Method 1.Expression of 4-1BBL mRNA and protein.Preparation of tumor vaccine: Tca8113 cells transfected and untransfected with 4-1BBL gene were treated with mitomycin C (MMC), and TCV-4-1BBLTCV-TCV-Tca8113.3 respectively.The ability of TCV-4-1BBL tumor Vaccine combined with CD28 mAb to induce Antitumor activity: 1 TCV-4-1BBL combined with CD28 mAb was co-cultured with human peripheral blood T lymphocytes induced by CD3 monoclonal antibody CD3 mAb. the experiment was divided into four groups.T lymphocytes in 1Tca8113 group: TCV-Tca8113Tca8113 CD3 mAb: CD3 mAb TCV-Tca8113TCV-Tca8113-4-1BBL CD3 mAb: CD3 mAb TCV-4-1BBL 4Tca8113-4-1BBL CD3 mAb CD28 mAb:T CD3 mAb TCV-4-1BBL CD28 mAb.After 72 hours of culture, the T cells were counted by trypan blue staining. The cytotoxic T cells were detected by Cell Counting Kit-8 CCK-8 and the secretion of cytokine IL-2 IFN- 緯 was detected by Elisa.Result 1.The expression of green fluorescent protein (RT-PCRwestern blot) was observed in Tca8113 cells transfected with pEGFP-4-1BBL under fluorescence microscope. 4-1BBL expression was detected at the level of mRNA and protein. The expression of 4-1BBL was detected by backstage trypan blue staining for 2.72 hours.Conclusion the eukaryotic expression vector of pEGFP/neo-4-1BBL was stably expressed in Tca8113 cells, and Tca8113-4-lBBL of oral cancer vaccine was successfully constructed.At the cell level, TCV-4-1BBL vaccine combined with CD28 mAbcould enhance the anti-tumor immune function of T cells, promote the proliferation and activation of T cells, and promote the secretion of IL-2 IFN- 緯, which may be one of the mechanisms of TCV-4-1BBL combined with CD28 mAb.
【學位授予單位】：汕頭大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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1 雷靜,錢e，

本文編號：1729145