中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2008年
12期
902-907
,共6页
刘红菊%辛建保%李卓亚%熊先智%陶晓南%胡豫
劉紅菊%辛建保%李卓亞%熊先智%陶曉南%鬍豫
류홍국%신건보%리탁아%웅선지%도효남%호예
树突细胞%基因,p53%细胞毒性,免疫
樹突細胞%基因,p53%細胞毒性,免疫
수돌세포%기인,p53%세포독성,면역
Dendritic cell%Genes,p53%Cytotoxicity immunologic
目的 观察加载了野生型p53基因的树突细胞(DC)对不同位点p53基因突变肿瘤得庖咧瘟谱饔?方法通过锥虫蓝染色、同种异体混合白细胞反应及流式细胞仪检测DC细胞表面分子,评估腺病毒(Ad)-p53感染DC是否影响DC的免疫功能.以Ad或转导了野生型p53基因的Ad分别感染骨髓De(Ad-DC和Ad-p53-DC)后,静脉注射免疫C57BL/6小鼠各5只,分离脾细胞,采用标准6 h51 Cr释放试验测定其诱导不同肿瘤细胞系(MethA、D459和P815)细胞毒性T淋巴细胞(CTL)的杀伤活性;效应细胞(Ad-p53-DC免疫后的小鼠脾细胞)和靶细胞(Ad-p53-P815和D459)孵育时分别加入抗CD4抗体或抗CD8抗体,观察CTL的活性变化.使用MethA和D459肿瘤细胞系得到不同的荷瘤鼠,于肿瘤形成前后分别使用Ad-p53-DC免疫,Ad-DC对照,当实体瘤三维直径之和＞20 cm时处死小鼠,用生存曲线评估Ad-p53-DC免疫的预防或治疗作用.结果 (1)Ad-p53-DC免疫诱导的抗Ad-p53-P815、D459和MethA的CTL反应(效应细胞:靶细胞=50:1)分别为(27.8±3.4)%、(23.5±2.7)%及(58.3±9.2)%,与Ad-DC免疫诱导的反应[(9.3±1.8)%、(4.6±1.0)%及(23.5 ±3.7)%]相比,差异有统计学意义(td值分别为5.79、3.68、5.02,均P＜0.05).Ad-p53-DC免疫小鼠T淋巴细胞与靶细胞Ad-p53-P815或D459的CTL活性,抗CD4组[(59.8 ±4.6)%、(18.9±2.4)%]与无抗体组[(64.4±6.3)%、(22.2±3.0)%]相比,差异无统计学意义(td值分别为0.84、0.91,均P＞0.05),而抗CD8组[(26.7±2.8)%、(6.1±1.2)%]差异有统计学意义(td值分别为9.03、7.67,均P＜0.05).抗CD8组与抗CD4组比较,差异有统计学意义(td值分别为8.79、9.18,均P＜0.05).(2)Ad-p53-DC和Ad-DC静脉注射2次免疫小鼠后,分别以D459细胞或MethA肉瘤细胞荷瘤20只小鼠.在Ad-p53-DC免疫组分别有14只和16只小鼠肿瘤的生长得到完全抑制,与Ad-DC免疫组比较差异有统计学意义(x2值分别为6.72、5.86,P＜0.05).皮下接种小鼠D459后,Ad-p53-DC免疫治疗组的小鼠肿瘤生长速度比Ad-DC组延缓2周左右,二组比较差异有统计学意义(x2 值为9.48,P＜0.05).结论 Ad-p53-DC可诱导抗MethA、P815和D459靶细胞的由CD8+T淋巴细胞介导的CTL反应,并抑制鼠体内肿瘤细胞的形成和生长.
目的 觀察加載瞭野生型p53基因的樹突細胞(DC)對不同位點p53基因突變腫瘤得庖咧瘟譜饔?方法通過錐蟲藍染色、同種異體混閤白細胞反應及流式細胞儀檢測DC細胞錶麵分子,評估腺病毒(Ad)-p53感染DC是否影響DC的免疫功能.以Ad或轉導瞭野生型p53基因的Ad分彆感染骨髓De(Ad-DC和Ad-p53-DC)後,靜脈註射免疫C57BL/6小鼠各5隻,分離脾細胞,採用標準6 h51 Cr釋放試驗測定其誘導不同腫瘤細胞繫(MethA、D459和P815)細胞毒性T淋巴細胞(CTL)的殺傷活性;效應細胞(Ad-p53-DC免疫後的小鼠脾細胞)和靶細胞(Ad-p53-P815和D459)孵育時分彆加入抗CD4抗體或抗CD8抗體,觀察CTL的活性變化.使用MethA和D459腫瘤細胞繫得到不同的荷瘤鼠,于腫瘤形成前後分彆使用Ad-p53-DC免疫,Ad-DC對照,噹實體瘤三維直徑之和＞20 cm時處死小鼠,用生存麯線評估Ad-p53-DC免疫的預防或治療作用.結果 (1)Ad-p53-DC免疫誘導的抗Ad-p53-P815、D459和MethA的CTL反應(效應細胞:靶細胞=50:1)分彆為(27.8±3.4)%、(23.5±2.7)%及(58.3±9.2)%,與Ad-DC免疫誘導的反應[(9.3±1.8)%、(4.6±1.0)%及(23.5 ±3.7)%]相比,差異有統計學意義(td值分彆為5.79、3.68、5.02,均P＜0.05).Ad-p53-DC免疫小鼠T淋巴細胞與靶細胞Ad-p53-P815或D459的CTL活性,抗CD4組[(59.8 ±4.6)%、(18.9±2.4)%]與無抗體組[(64.4±6.3)%、(22.2±3.0)%]相比,差異無統計學意義(td值分彆為0.84、0.91,均P＞0.05),而抗CD8組[(26.7±2.8)%、(6.1±1.2)%]差異有統計學意義(td值分彆為9.03、7.67,均P＜0.05).抗CD8組與抗CD4組比較,差異有統計學意義(td值分彆為8.79、9.18,均P＜0.05).(2)Ad-p53-DC和Ad-DC靜脈註射2次免疫小鼠後,分彆以D459細胞或MethA肉瘤細胞荷瘤20隻小鼠.在Ad-p53-DC免疫組分彆有14隻和16隻小鼠腫瘤的生長得到完全抑製,與Ad-DC免疫組比較差異有統計學意義(x2值分彆為6.72、5.86,P＜0.05).皮下接種小鼠D459後,Ad-p53-DC免疫治療組的小鼠腫瘤生長速度比Ad-DC組延緩2週左右,二組比較差異有統計學意義(x2 值為9.48,P＜0.05).結論 Ad-p53-DC可誘導抗MethA、P815和D459靶細胞的由CD8+T淋巴細胞介導的CTL反應,併抑製鼠體內腫瘤細胞的形成和生長.
목적 관찰가재료야생형p53기인적수돌세포(DC)대불동위점p53기인돌변종류득포렬온보옹?방법통과추충람염색、동충이체혼합백세포반응급류식세포의검측DC세포표면분자,평고선병독(Ad)-p53감염DC시부영향DC적면역공능.이Ad혹전도료야생형p53기인적Ad분별감염골수De(Ad-DC화Ad-p53-DC)후,정맥주사면역C57BL/6소서각5지,분리비세포,채용표준6 h51 Cr석방시험측정기유도불동종류세포계(MethA、D459화P815)세포독성T림파세포(CTL)적살상활성;효응세포(Ad-p53-DC면역후적소서비세포)화파세포(Ad-p53-P815화D459)부육시분별가입항CD4항체혹항CD8항체,관찰CTL적활성변화.사용MethA화D459종류세포계득도불동적하류서,우종류형성전후분별사용Ad-p53-DC면역,Ad-DC대조,당실체류삼유직경지화＞20 cm시처사소서,용생존곡선평고Ad-p53-DC면역적예방혹치료작용.결과 (1)Ad-p53-DC면역유도적항Ad-p53-P815、D459화MethA적CTL반응(효응세포:파세포=50:1)분별위(27.8±3.4)%、(23.5±2.7)%급(58.3±9.2)%,여Ad-DC면역유도적반응[(9.3±1.8)%、(4.6±1.0)%급(23.5 ±3.7)%]상비,차이유통계학의의(td치분별위5.79、3.68、5.02,균P＜0.05).Ad-p53-DC면역소서T림파세포여파세포Ad-p53-P815혹D459적CTL활성,항CD4조[(59.8 ±4.6)%、(18.9±2.4)%]여무항체조[(64.4±6.3)%、(22.2±3.0)%]상비,차이무통계학의의(td치분별위0.84、0.91,균P＞0.05),이항CD8조[(26.7±2.8)%、(6.1±1.2)%]차이유통계학의의(td치분별위9.03、7.67,균P＜0.05).항CD8조여항CD4조비교,차이유통계학의의(td치분별위8.79、9.18,균P＜0.05).(2)Ad-p53-DC화Ad-DC정맥주사2차면역소서후,분별이D459세포혹MethA육류세포하류20지소서.재Ad-p53-DC면역조분별유14지화16지소서종류적생장득도완전억제,여Ad-DC면역조비교차이유통계학의의(x2치분별위6.72、5.86,P＜0.05).피하접충소서D459후,Ad-p53-DC면역치료조적소서종류생장속도비Ad-DC조연완2주좌우,이조비교차이유통계학의의(x2 치위9.48,P＜0.05).결론 Ad-p53-DC가유도항MethA、P815화D459파세포적유CD8+T림파세포개도적CTL반응,병억제서체내종류세포적형성화생장.
Objective To explore the anti-tumor immune responses of dendritic cells (DCs) loaded with intact wild-type p53 to mice challenged with tumor cells expressing p53 genes with mutations at different sites.Methods Ad-p53-DC immunization function was assessed by the expression of surface molecules and allogeneic MLR.DCs derived from bone marrow were transduced with adenovims or a human wild-type p53 containing recombinant adenovirus (Ad-DC and Ad-p53-DC)and immunized C57BL/6 mice.Splenocytes were separated and cell cytotoxicity was measured against tumor cells expressing mutant 003 ( MethA,D459 and PS15)in a standard 6-h5t Cr-release assay.Effector and target cells were incubated in the presence of anti-CD4 or anti-CD8 antibody.Ad-p53-DC was immunized in control Ad-DC before or after mice were challenged with either D459 tumor or with MethA sarcoma cells to observe whether immune response would provide tumor protection.Results Immunization with Ad-p53-DC developed significantly higher substantial CTL responses against Ad-p53-P815,D459 and MethA cells (effectors:target cells = 50:1 ),(27.8±3.4)%,(23.5±2.7)%,(58.3±9.2)% than with Ad-DC(9.3±1.8)%,(4.6±1.0)%,(23.5±3.7) % (td = 5.79,3.68,5.02,all P ＜ 0.05 ).In Ad-p53-DC immunized mice,anfi-CD8 antibody blocked the cytotoxicity against Ad-p53-PS15 (26.7±2.8)% or D459(6.1 ± 1.2)%,but not anti-CD4 antibody [(59.8± 4.6) %,( 18.9±2.4 ) %,td = 8.79 or 9.18,all P ＜ 0.05].Ad-p53-DC immunization provided complete tumor protection in 80% of mice challenged with D459 and in 70% of mice challenged with MethA,while none protected in Ad-DC immunization group(x2 = 6.72,5.86,all P ＜0.05).Treated with Ad-p53-DC after D459 inoculation subcutaneously,mice were killed due to the bulky tumor more than 2 weeks later than the mice in the Ad-DC treatment group during 7 week observation ( X2 = 9.48,P ＜ 0.05 ).Conclusion DCs tranfected with 100 MOI Ad-p53 induced intense CTL responses against P815,D459 and MethA.This CTL response is mediated by CDs+ T ceils.Treatment with Ad-p53-DC significantly developed tumor immunology and slowed the growth of established tumors.
