摘要
实验目的在体外探讨不同浓度IFN-α对人脐血来源DC分化成熟及功能的影响,对DC表面标志和功能进行检测；同时进行肝癌细胞冻融抗原负载DC介导CTL对肝癌细胞杀伤实验；评价该实验方法培养DC的可行性及应用肝癌细胞冻融抗原负载DC介导的抗肝癌效应。
实验方法从脐血中分离单个核细胞(CBMNC),在完全培养液(RPMI-1640+5%胎牛血清)中添加GM-CSF(1000U/ml)、IL-4(500U/ml)及不同浓度INF-a(A至E五组浓度为0、100、500、700、1000U/ml)联合诱导CBMNC分化为DC,第7天收获DC。倒置显微镜下观察细胞形态,流式细胞仪测定DC表面标志CD14、CD83、CD86、CD40、CDla和HLA-DR的表达,MTT法测定DC对同种异体淋巴细胞的增殖能力和DC介导CTL对肝癌细胞的杀伤率,ELISA测定DC上清液中IL-12、IFN-y和IL-10细胞因子的含量。
实验结果1.CBMNC在细胞因子GM-CSF+IL-4及不同浓度INF-a的共同作用下,培养3天大部分细胞仍贴壁生长,并可见部分细胞形成小的集落,未见明显树突状样突起；第7天可见细胞表面有明显伪足样突起,呈现典型的树突状形态。2.流式细胞仪分析表明第7天收获的细胞高表达成熟DC的表面标志,其中本实验第1部分的INF-a浓度为500 U/ml时的表达率(%)分别为CD86(95.10±1.53)、CD83(75.11±1.63)、CD1a(38.75±1.93)、HLA-DR(88.75±1.65)。3.在一定浓度范围内(0、100、500、700U/ml), INF-a诱导培养的DC刺激异体淋巴细胞增殖反应呈递增趋势(P<0.05),其中以INF-a浓度为700U/ml且T：DC=10：1时刺激异体淋巴细胞增殖的OD值最高(0.485±0.100),与其余组存在明显统计学差异(P<0.05)。4.本实验中,INF-a浓度为700U/ml时,负载SMMC-7721抗原的DC诱导异体CTL对肝癌细胞的杀伤率(%)最高(30.1±1.6)；与其余组存在统计学差异(P<0.05)。5. ELISA方法检测A、B、C、D、E组第3天DC上清液细胞因子含量各组无明显统计学差异(P>0.05)；第7天时各组的细胞因子含量均有升高,其中D组的IL-12(37.69±2.67 pg/ml)、IFN-y(49.94±1.35 pg/ml)含量最高,与其余各组存在统计学差异(P<0.05)；而D组的IL-10(32.34±2.49 pg/ml)含量最低,与其余各组存在统计学差异(P<0.05)。
实验结论脐血分离的CBMNC在细胞因子GM-CSF, IL-4及不同浓度INF-a(0、100、500、700、1000U/ml)共同作用下在体外均可诱导分化成熟DC；不同浓度INF-a均能提高DC分泌的免疫刺激细胞因子IL-12、IFN-γ的能力,均能促进负载抗原的DC诱导的异体淋巴细胞增殖及提高DC介导CTL对肝癌的杀伤率。在本实验中,INF-a浓度为700U/ml是诱导培养DC的最适浓度。本实验将为DC应用于临床提供一定的实验基础。
Objectives To investigate the influence of different concentrations of IFN-a on differentiation, maturation and immune function of human cord blood derived dendritic cells in vitro. To evaluate DC with hepatoma cell lysates induced antit-hepatoma effect and the feasibility of experimental methods of cultivation DC and antitumor efficiency.
Methods Isolated from umbilical cord blood mononuclear cells (CBMNC), in complete culture medium (RPMI-1640 +5% fetal calf serum) added GM-CSF (1000U/ml), IL-4 (500U/ml) and different concentrations of INF-a (A to E five concentration 0,100,500,700,1000 U/ml) to induced DC through CBMNC, on 7th day, DC Cell morphology was observed in microscope, flow cytometry detected DC surface markers CD14, CD83, CD86, CD40, CDla and HLA-DR expression, The methed of MTT determination the proliferation of allogeneic lymphocytes and the rate of cell killing about DC-mediated CTL, The methed of ELISA determination of DC supernatants of cytokine levels of IL-12, IFN-y and IL-10.
Results 1.cell factor GM-CSF+IL-4 and INF-a with different concentrations to cultivate CBMNC, CBMNC cultured for 3 days, most of the cells still adhered to the wall and some cells forming small colonies, no dendritic-like processes,On the 7th day, the surface of cells significantly pseudopod-like protrusions, showing a typical dendritic morphology; 2.Cultured cells detectd surface markers by flow cytometry,we found that D groups cell surface expression of CD83, CD1a, HLA-DR, the expression rate (%) were CD86 (95.10±1.53), CD83 (75.11±1.63), CDla (38.75±1.93), HLA-DR (88.75±1.65);3.Within a certain range (0,100,500,700 U/ml), INF-a induced DC to stimulate allogeneic lymphocyte proliferation increased gradually (P <0.05),When the concentration of INF-a reach 700U/ml and the T: DC= 10:1, stimulating the proliferation of allogeneic lymphocytes about OD value (0.485±0.100)is highest, and obvious significant difference with other groups (P<0.05);4.In this study,when INF-a concentration reach 700U/ml, the load of the DC antigen SMMC-7721 liver cancer cells induce allogeneic CTL killing is the highest percentage (%), (59.0±2.0); and had significantly difference with other goups (P <0.05); 5.the methed of ELISA to detect A, B, C, D, E groups cytokines in DC supernatants, on 3th day cytokines in DC supernatants had no significant difference with other groups (P> 0.05); 7th day cytokines levels of each group were high, in which D group IL-12 (37.69±2.67 pg/ml), IFN-y (49.94±1.35 pg/ml) was highest, and had significant difference with other groups (P<0.05); and D group of IL-10 (32.34±2.49 pg/ml) were the lowest, and had significant difference with other groups (P<0.05).
Conclusion CBMNC isolated from cord blood, GM-CSF, IL-4 and different concentrations of INF-a (0,100,500,700,1000 U/ml) can promote differentiation and maturation of DC in vitro; different concentrations of INF- a can enhance contention of cytokine IL-12 which DC secretion, IFN-y, DC loaded with tumor antigen can promote the induction of allogeneic lymphocyte proliferation and enhance DC-mediated CTL killing rate of hepatocellular carcinoma. In this experiment, INF-a concentration reaching 700U/ml is the optimal concentration. this experiment provide some experimental basis for clinical application.
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