中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2011年
1期
52-57
,共6页
赵军%路静%刘雅琴%杨洪艳%黄幼田%赵继敏%李珊%翟景明%赵明耀%张曦%董子明
趙軍%路靜%劉雅琴%楊洪豔%黃幼田%趙繼敏%李珊%翟景明%趙明耀%張晞%董子明
조군%로정%류아금%양홍염%황유전%조계민%리산%적경명%조명요%장희%동자명
宫颈肿瘤%细胞系,肿瘤%树突细胞%血管内皮生长因子受体2%整合素αV
宮頸腫瘤%細胞繫,腫瘤%樹突細胞%血管內皮生長因子受體2%整閤素αV
궁경종류%세포계,종류%수돌세포%혈관내피생장인자수체2%정합소αV
Uterine cervical neoplasms%Cell line,tumor%Dendritic cells%Vascular endothelial growth factor receptor-2%Integrin alphaV
目的 探讨负载小鼠脑微血管内皮细胞bEnd.3抗原的树突状细胞(DC)诱导小鼠抗同种宫颈癌U14细胞的体液和细胞免疫反应.方法 培养小鼠脑微血管内皮细胞bEnd.3,逆转录(RT)-PCR技术检测血管内皮细胞增殖相关抗原血管内皮生长因子受体2(VEGF-R2)和整合素αV的表达,并制备bEnd.3细胞的冻融抗原,用抗原负载DC(即bEnd.3-DC).采用bEnd.3-DC免疫小鼠(bEnd.3-DC组),共4次,每周1次,设DC组和磷酸盐缓冲液(PBS)组作为对照;免疫结束后1周皮下注射小鼠宫颈癌U14细胞,观察荷瘤小鼠的肿瘤生长情况,检测免疫后小鼠脾T淋巴细胞体外靶向血管内皮细胞的细胞毒T淋巴细胞(CTL)效应、脾CD+3 CD+8T淋巴细胞百分率和免疫后小鼠血清的抗体滴度,通过免疫细胞化学方法和蛋白印迹法进行小鼠抗血清的特异性免疫反应分析.结果 经RT-PCR技术证实,bEnd.3细胞在mRNA水平表达VEGF-R2和整合素αV.PBS组小鼠的肿瘤生长最快,DC组和bEnd.3-DC组小鼠肿瘤也有生长,但生长较慢;在免疫小鼠2周后,bEnd.3-DC组小鼠肿瘤消失,PBS组小鼠肿瘤体积为(3.38 ±0.34)cm3,DC组小鼠肿瘤体积为(0.11±0.13)cm3.小鼠脾T淋巴细胞体外CTL实验结果显示,bEnd.3-DC组有明显的特异性靶向杀伤bEnd.3细胞的作用,其杀伤率超过60%.脾CD+3 CD+8 T淋巴细胞的百分率,PBS组为(30.0±0.9)%,DC组为(32.8±0.4)%,bEnd.3-DC组为(38.6 ±0.7)%,3组间比较,差异有统计学意义(P＜0.05).bEnd.3-DC组免疫小鼠血清的抗体滴度为1∶3200,DC组为1∶800,PBS组则为0.免疫细胞化学检测显示,bEnd.3-DC组的抗血清与bEnd.3细胞有特异性的抗原抗体反应;蛋白印迹法结果显示,在相对分子质量为220 000处有特异性的VEGF-R2蛋白条带.结论 负载小鼠bEnd.3细胞抗原的DC对小鼠宫颈癌Ui4细胞的生长有抑制作用,其原因是诱导小鼠产生了特异性的靶向血管内皮细胞的CTL和针对bEnd.3细胞中的某些血管内皮细胞增殖相关抗原的细胞和体液免疫应答.
目的 探討負載小鼠腦微血管內皮細胞bEnd.3抗原的樹突狀細胞(DC)誘導小鼠抗同種宮頸癌U14細胞的體液和細胞免疫反應.方法 培養小鼠腦微血管內皮細胞bEnd.3,逆轉錄(RT)-PCR技術檢測血管內皮細胞增殖相關抗原血管內皮生長因子受體2(VEGF-R2)和整閤素αV的錶達,併製備bEnd.3細胞的凍融抗原,用抗原負載DC(即bEnd.3-DC).採用bEnd.3-DC免疫小鼠(bEnd.3-DC組),共4次,每週1次,設DC組和燐痠鹽緩遲液(PBS)組作為對照;免疫結束後1週皮下註射小鼠宮頸癌U14細胞,觀察荷瘤小鼠的腫瘤生長情況,檢測免疫後小鼠脾T淋巴細胞體外靶嚮血管內皮細胞的細胞毒T淋巴細胞(CTL)效應、脾CD+3 CD+8T淋巴細胞百分率和免疫後小鼠血清的抗體滴度,通過免疫細胞化學方法和蛋白印跡法進行小鼠抗血清的特異性免疫反應分析.結果 經RT-PCR技術證實,bEnd.3細胞在mRNA水平錶達VEGF-R2和整閤素αV.PBS組小鼠的腫瘤生長最快,DC組和bEnd.3-DC組小鼠腫瘤也有生長,但生長較慢;在免疫小鼠2週後,bEnd.3-DC組小鼠腫瘤消失,PBS組小鼠腫瘤體積為(3.38 ±0.34)cm3,DC組小鼠腫瘤體積為(0.11±0.13)cm3.小鼠脾T淋巴細胞體外CTL實驗結果顯示,bEnd.3-DC組有明顯的特異性靶嚮殺傷bEnd.3細胞的作用,其殺傷率超過60%.脾CD+3 CD+8 T淋巴細胞的百分率,PBS組為(30.0±0.9)%,DC組為(32.8±0.4)%,bEnd.3-DC組為(38.6 ±0.7)%,3組間比較,差異有統計學意義(P＜0.05).bEnd.3-DC組免疫小鼠血清的抗體滴度為1∶3200,DC組為1∶800,PBS組則為0.免疫細胞化學檢測顯示,bEnd.3-DC組的抗血清與bEnd.3細胞有特異性的抗原抗體反應;蛋白印跡法結果顯示,在相對分子質量為220 000處有特異性的VEGF-R2蛋白條帶.結論 負載小鼠bEnd.3細胞抗原的DC對小鼠宮頸癌Ui4細胞的生長有抑製作用,其原因是誘導小鼠產生瞭特異性的靶嚮血管內皮細胞的CTL和針對bEnd.3細胞中的某些血管內皮細胞增殖相關抗原的細胞和體液免疫應答.
목적 탐토부재소서뇌미혈관내피세포bEnd.3항원적수돌상세포(DC)유도소서항동충궁경암U14세포적체액화세포면역반응.방법 배양소서뇌미혈관내피세포bEnd.3,역전록(RT)-PCR기술검측혈관내피세포증식상관항원혈관내피생장인자수체2(VEGF-R2)화정합소αV적표체,병제비bEnd.3세포적동융항원,용항원부재DC(즉bEnd.3-DC).채용bEnd.3-DC면역소서(bEnd.3-DC조),공4차,매주1차,설DC조화린산염완충액(PBS)조작위대조;면역결속후1주피하주사소서궁경암U14세포,관찰하류소서적종류생장정황,검측면역후소서비T림파세포체외파향혈관내피세포적세포독T림파세포(CTL)효응、비CD+3 CD+8T림파세포백분솔화면역후소서혈청적항체적도,통과면역세포화학방법화단백인적법진행소서항혈청적특이성면역반응분석.결과 경RT-PCR기술증실,bEnd.3세포재mRNA수평표체VEGF-R2화정합소αV.PBS조소서적종류생장최쾌,DC조화bEnd.3-DC조소서종류야유생장,단생장교만;재면역소서2주후,bEnd.3-DC조소서종류소실,PBS조소서종류체적위(3.38 ±0.34)cm3,DC조소서종류체적위(0.11±0.13)cm3.소서비T림파세포체외CTL실험결과현시,bEnd.3-DC조유명현적특이성파향살상bEnd.3세포적작용,기살상솔초과60%.비CD+3 CD+8 T림파세포적백분솔,PBS조위(30.0±0.9)%,DC조위(32.8±0.4)%,bEnd.3-DC조위(38.6 ±0.7)%,3조간비교,차이유통계학의의(P＜0.05).bEnd.3-DC조면역소서혈청적항체적도위1∶3200,DC조위1∶800,PBS조칙위0.면역세포화학검측현시,bEnd.3-DC조적항혈청여bEnd.3세포유특이성적항원항체반응;단백인적법결과현시,재상대분자질량위220 000처유특이성적VEGF-R2단백조대.결론 부재소서bEnd.3세포항원적DC대소서궁경암Ui4세포적생장유억제작용,기원인시유도소서산생료특이성적파향혈관내피세포적CTL화침대bEnd.3세포중적모사혈관내피세포증식상관항원적세포화체액면역응답.
Objective To explore the specific cellular and humoral immunity induced by dendritic cells (DC) vaccine loading allogenic microvascular endothelial cell bEnd. 3 antigen against U14 cervical cancer cell of mice. Methods Mouse brain microvascular endothelial cell bEnd. 3 was cultured and identified for preparation endothelial cell bEnd. 3 antigen. The level of mRNA expression of vascular endothelial growth factor receptor 2 (VEGF-R2) and integrin αV was detected by reverse transcription (RT)-PCR. The BALB/c mice were immuned with DC loading bEnd. 3 antigen 4 times in 4 weeks (bEnd. 3-DC group), while the mice only were immuned with DC or injected with phosphate buffer saline (PBS group) as control group. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, cytotoxic T lymphocyte (CTL) response of spleen lymphocytes in vitro, the percentage of CD3+ CD+8 surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and western blot. Results The expression of VEGF-R2 and integrin αV gene in bEnd. 3 cells were expressed highly.After the vaccine was injected, the tumors of mice in PBS group grew faster than those in other groups, while the tumors in bEnd. 3-DC group grew slowly and disappeared after 2 weeks. The volume of tumors in DC group grew slower than those in PBS group [(0.11± 0.13) cm3 versus (3.38 ±0.34) cm3]. The CTL response of spleen iymphocytes in vitro showed that bEnd. 3-DC cells could kill bEnd. 3 cells, the special lysis rate was more than 60% . The percentage of CD+3 CD+8 spleen lymphocytes in bEnd. 3-DC group[(38.6 ± 0.7) %] was higher than those in other groups (P ＜ 0.05). The titer of serum antibody of Immunocytochemistry analysis indicated there were specific antigen-antibody reaction to bEnd. 3 cell in bEnd. 3-DC group. Western blot analysis revealed that there were specific bands at 220 000 (VEGF-R2).Conclusions bEnd. 3-DC vaccine can inhibit the tumor growth of U14 cervical cancer cell of mice, which indicates that the special cellular and humorai immunity are induced by bEnd. 3-DC antigen which maybe have some antigens in bEnd. 3 cells that reacts with endothelial cell proliferation-related antigens.
