CAJ | 학술논문

目的：研究肝癌特异性TCR基因TCRVα12畅2-Vβ7畅1转染T细胞后，促进T细胞识别肿瘤抗原，活化抗肿瘤免疫反应的效果。方法：构建重组腺病毒Ad5F35-TRAV-TRBV，以之感染T细胞并优化最佳感染条件。钙黄绿素染色法检测T细胞对不同肿瘤靶细胞株的杀伤活性。 Annexin V-PI双染法检测靶细胞凋亡比例。流式细胞术检测T细胞表面FasL表达比例。 ELISA法检测T细胞作用于靶细胞后IFN-γ与IL-2分泌水平。结果：成功将TCRVα12畅2-Vβ7畅1基因片段构建入嵌合型腺病毒载体，获得重组腺病毒 Ad5 F35-TRAV-TRBV。感染复数为100时，重组腺病毒有最高转染效率。感染3天后， TCRVα12 Vβ7阳性细胞比例超过25％。特异性TCR基因转染有效促进T细胞杀伤AFP阳性肝癌细胞，特异性裂解率显著提高（ P＜0畅001）。特异性TCR基因转染有效促进T细胞诱导靶细胞凋亡（ P＜0畅001）。同时，T细胞表面FasL表达比例上升（ P＜0畅001）。 TCR基因转染T细胞作用于AFP抗原阳性靶细胞后IFN-γ分泌显著提高（P＜0畅001）。结论：重组腺病毒Ad5F35-TRAV-TRBV成功介导肿瘤特异性TCR基因转染T细胞。 TCR基因转染可有效促进T细胞识别抗原阳性肿瘤细胞，并发挥抗肿瘤免疫效应。
목적：연구간암특이성TCR기인TCRVα12창2-Vβ7창1전염T세포후，촉진T세포식별종류항원，활화항종류면역반응적효과。방법：구건중조선병독Ad5F35-TRAV-TRBV，이지감염T세포병우화최가감염조건。개황록소염색법검측T세포대불동종류파세포주적살상활성。 Annexin V-PI쌍염법검측파세포조망비례。류식세포술검측T세포표면FasL표체비례。 ELISA법검측T세포작용우파세포후IFN-γ여IL-2분비수평。결과：성공장TCRVα12창2-Vβ7창1기인편단구건입감합형선병독재체，획득중조선병독 Ad5 F35-TRAV-TRBV。감염복수위100시，중조선병독유최고전염효솔。감염3천후， TCRVα12 Vβ7양성세포비례초과25％。특이성TCR기인전염유효촉진T세포살상AFP양성간암세포，특이성렬해솔현저제고（ P＜0창001）。특이성TCR기인전염유효촉진T세포유도파세포조망（ P＜0창001）。동시，T세포표면FasL표체비례상승（ P＜0창001）。 TCR기인전염T세포작용우AFP항원양성파세포후IFN-γ분비현저제고（P＜0창001）。결론：중조선병독Ad5F35-TRAV-TRBV성공개도종류특이성TCR기인전염T세포。 TCR기인전염가유효촉진T세포식별항원양성종류세포，병발휘항종류면역효응。
Objective:To investigate effects of tumor specific TCR gene Vα12.2-Vβ7.1 modification on recognition of tumor antigen and activation of anti-tumor reactivity of T cells.Methods: T cells were transduced using recombinant Ad 5F35-TRAV-TRBV adenovirus ,and multiplicity of infection was optimized.Specific lysis of T cells was evaluated by calcein release assay.The frequency of apoptotic cells in target cells was detected by Annexin V /PI double-labeled FACS.The expression of FasL on T cells was analyzed by FACS.The secretion of cytokine IFN-γand IL-2 of T cells was determined by ELISA assays.Results: The highest tranduce efficiency was obtained at MOI 100 by recombinant Ad5F35-TRAV-TRBV adenovirus.The frequency of TCRVα12+Vβ7+cells reached above 25%3 days after transduction.TCR gene modification enhanced the ability of T cells to lyse HLA-A2+AFP+target cells(P<0.001), the ability of T cells to induce HepG-2 apoptosis(P<0.001),and expression of FasL on T cells(P<0.001).TCR gene modification also enhanced T cells to secret IFN-γafter coculture with antigen positive tumor cells ( P<0.001 ).Conclusion: Specific TCR gene modification by recombinant adeno virus effectively promotes T cells to recognize antigen positive tumor cell and exert anti -tumor reac-tivity.