MHC五聚体方法检测基因疫苗体外诱导的抗原特异性细胞毒T淋巴细胞
MHC오취체방법검측기인역묘체외유도적항원특이성세포독T림파세포
Determination of antigen-specific CTL induced by the gene vaccine in vitro with MHC pentamer
  • 万方数据
    中华检验医学杂志 중화검험의학잡지
    CHINESE JOURNAL OF LABORATORY MEDICINE
    2010年 7期 686-690 ,共5页

    李绵洋%朱平%王成彬%王红霞%潘玉玲%丛玉隆%达万明

    리면양%주평%왕성빈%왕홍하%반옥령%총옥륭%체만명

    疫苗,DNA%T淋巴细胞,细胞毒性%组织相容性抗原I类 疫苗,DNA%T淋巴細胞,細胞毒性%組織相容性抗原I類
    역묘,DNA%T림파세포,세포독성%조직상용성항원I류
    Vaccines,DNA%T-lymphocytes,cytotoxic%Histocompatibility antigens class I
    目的 探讨MHC五聚体方法检测在体外用抗B细胞淋巴瘤基因疫苗(简称基因疫苗)刺激PBMC生成抗原特异性CTL的效率.方法 用于细胞培养的4份血液标本来自2例B淋巴细胞肿瘤患者(1例为滤泡性淋巴瘤患者,另1例为毛细胞白血病患者)和2名健康对照者.分别检测上述标本中PBMC受基因疫苗刺激后CTL生成情况.PBMC贴壁培养获得DC前体,在重组细胞因子GM-CSF和IL4的支持下诱导培养DC,采用基因枪方法将基因疫苗质粒导入DC,RT-PCB方法检测转染后细胞IgHV1-GM-CSF mRNA的转录,ELISA方法检测细胞因子GM-CSF的表达,并验证转染是否获得有效表达.转染后的DC再与从PBMC中获得的淋巴细胞共培养,诱导抗原特异性的CTL;共进行2次DC刺激,共计培养24 d,分别在培养前、培养第7天、第17天及第24天收获培养细胞,培养分为转染基因疫苗组和转染对照质粒组,流式细胞术分析培养细胞中CD3+ CD8+细胞亚群的水平;用针对基因疫苗抗原肽抗原表位的MHC五聚体荧光抗体检测CTL产生水平.结果 基因枪颗粒轰击方法转染DC后,RT-PCR结果显示转染得到了表达;转染基因疫苗组的DC中GM-CSF含量为(28±6)ng/106个细胞,而转染对照质粒组的DC中GM-CSF含量为(10±3)ng/106个细胞,差异有统计学意义(t=5.191,P<0.01).分析培养后的T淋巴细胞亚群,显示随着培养时间的延长,CD3+CD8+细胞亚群比例明显增加,转染对照质粒组在培养前、培养第7天、第17天和第24天的比例分别为(34.24±2.72)%,(46.06±3.08)%,(65.34±4.26)%,(73.86±4.85)%,而在转染基因疫苗组,其比例分别为(32.28±2.08)%,(45.32±3.81)%,(63.37±4.21)%,(75.01±3.20)%.两因素方差分析显示培养不同时间点的CD3+CD8+细胞亚群比例差异有统计学意义(F培养时间=176.966,P<0.01),但转染因素本身对其产生的影响差异无统计学意义(F转染因素=0.657,P>0.05).MHC五聚体检测结果显示转染基因疫苗组的DC能够诱导针对该表位的抗原特异性CTL产生,CTL水平随着DC的刺激逐渐增高;在培养第24天,健康对照者最高获得2.03%的CTL,滤泡性淋巴瘤患者的CTL达4.36%,而毛细胞白血病患者为3.89%.结论 MHC五聚体方法能够有效检测基因疫苗诱导的抗原特异性CTL;该方法对于抗肿瘤细胞免疫的检测、肿瘤患者的细胞免疫状态及早期预后判定可能是一项有前景的临床检验方法.
    목적 탐토MHC오취체방법검측재체외용항B세포림파류기인역묘(간칭기인역묘)자격PBMC생성항원특이성CTL적효솔.방법 용우세포배양적4빈혈액표본래자2례B림파세포종류환자(1례위려포성림파류환자,령1례위모세포백혈병환자)화2명건강대조자.분별검측상술표본중PBMC수기인역묘자격후CTL생성정황.PBMC첩벽배양획득DC전체,재중조세포인자GM-CSF화IL4적지지하유도배양DC,채용기인창방법장기인역묘질립도입DC,RT-PCB방법검측전염후세포IgHV1-GM-CSF mRNA적전록,ELISA방법검측세포인자GM-CSF적표체,병험증전염시부획득유효표체.전염후적DC재여종PBMC중획득적림파세포공배양,유도항원특이성적CTL;공진행2차DC자격,공계배양24 d,분별재배양전、배양제7천、제17천급제24천수획배양세포,배양분위전염기인역묘조화전염대조질립조,류식세포술분석배양세포중CD3+ CD8+세포아군적수평;용침대기인역묘항원태항원표위적MHC오취체형광항체검측CTL산생수평.결과 기인창과립굉격방법전염DC후,RT-PCR결과현시전염득도료표체;전염기인역묘조적DC중GM-CSF함량위(28±6)ng/106개세포,이전염대조질립조적DC중GM-CSF함량위(10±3)ng/106개세포,차이유통계학의의(t=5.191,P<0.01).분석배양후적T림파세포아군,현시수착배양시간적연장,CD3+CD8+세포아군비례명현증가,전염대조질립조재배양전、배양제7천、제17천화제24천적비례분별위(34.24±2.72)%,(46.06±3.08)%,(65.34±4.26)%,(73.86±4.85)%,이재전염기인역묘조,기비례분별위(32.28±2.08)%,(45.32±3.81)%,(63.37±4.21)%,(75.01±3.20)%.량인소방차분석현시배양불동시간점적CD3+CD8+세포아군비례차이유통계학의의(F배양시간=176.966,P<0.01),단전염인소본신대기산생적영향차이무통계학의의(F전염인소=0.657,P>0.05).MHC오취체검측결과현시전염기인역묘조적DC능구유도침대해표위적항원특이성CTL산생,CTL수평수착DC적자격축점증고;재배양제24천,건강대조자최고획득2.03%적CTL,려포성림파류환자적CTL체4.36%,이모세포백혈병환자위3.89%.결론 MHC오취체방법능구유효검측기인역묘유도적항원특이성CTL;해방법대우항종류세포면역적검측、종류환자적세포면역상태급조기예후판정가능시일항유전경적림상검험방법.
    Objective To determine the antigen-specific CTL in PBMC induced by a fusional family-gene vaccine of the immunoglobulin heavy chain variable gene framework region combined with the sequence of cytokine CM-CSF in vitro with MHC pentamer. Methods Peripheral blood samples were collected from two healthy donors and two patients. One was follicular lymphoma and another was hair cell leukemia. PBMC were isolated by density gradient centrifugalization with Ficoll and then subsequently differentiated into immature DCs (imDCs) induced by recombinant human GM-CSF and recombinant human IL-4. Gene gun was used to deliver the plasmids of the gene vaccine or the control plasmids into the imDCs. RT-PCR and ELISA assay were used to detect IgHVl-GM-CSF mRNA and GM-CSF in order to validate the transfection of the vaccine. After adding the cytokine cocktail, the imDCs became mature DCs. Then the mature DCs were co-cultured with lymphocytes from the blood samples for the induction of the antigen-specific CTL. The cultured cells were classified into vaccine group and control group and harvested at different time points of 0 d,7d, 17 d and 24 d after transfection. The subset of CD3+CD8+ T cells was analyzed by FCM assay. Finally, the CTL levels were detected with fluorescently labeled MHC pentamer antibody targeting vaccine epitopes. Results With the induction of cytokines, the imDCs with typical morphology were generated in PBMC. After delivering, the efficient expressions of the vaccine in the imDCs were determined by RT-PCR. And ELISA results also confirmed that GM-CSF was produced at a level of (28 ±6) ng/106 cells of the imDCs loaded with the vaccine, which was significantly different from that of control group (10 ± 3) ng/106 cells (t = 5. 191, P <0.01). FCM assay result showed that the CD3+ CD8+ T cells increased in a stepwise pattern during the culture. For control group, the levels at 0 d,7d, 17d and 24 d were ( 34. 24 ± 2. 72 )% , (46.06 ± 3.08)%, ( 65. 34 ± 4. 26 )% and (73.86 ±4.85 )% , respectively. For vaccine group, the results were (32. 28 ± 2. 08 ) % , (45. 32 ± 3. 81)% , ( 63. 37 ± 4. 21)% and (75. 01 ±3. 20)%. The differences between each time point had statistical significance (F = 176. 966 ,P <0.01) ,but there was no statistical differences between vaccine group and control group ( F = 0.657,P>0.05). The MHC pentamer analysis showed that the DCs loaded with IgHV1-GM-CSF fusional vaccine could efficiently induce the antigen-specific CTL response and the CTL levels increased gradually with the culture time, with the highest level of 4. 36% in the lymphoma blood and 3. 89% in the hair cell leukemia blood. Conclusions MHC pentamer assay could efficiently determine the antigen-specific CTLs response induced by the gene vaccine of family IgHV frame region in vitro. It could be a useful method for monitoring of anti-tumor cell immunity and evaluating of diagnosis and prognosis of the tumors in clinical application.
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