新型诱导剂钙离子载体联合GM-CSF体外诱导人外周血单核细胞生成树突状细胞的研究

发布时间：2018-06-08 06:10

本文选题：树突状细胞 + 钙离子载体　；参考：《广州医学院》2010年硕士论文

【摘要】：目的: 1.用组合细胞因子诱导人外周血单核细胞(human peripheral blood mononuclear cell,PBMC)生成树突状细胞(Dendritic cell,DC)。 2.用新型诱导剂钙离子载体(Calcium Ionophore,CI)A23187联合重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)体外诱导人外周血单核细胞生成DC。 3.观察DC刺激细胞毒性T淋巴细胞(CTL)对K562细胞(慢性髓性白血病细胞)产生的特异性杀伤作用。方法: 试验一: 1.采用密度梯度离心法及黏附法分离出PBMC,加入rhGM-CSF+重组人白介素- 4(rhIL-4)诱导,第5天加入重组人肿瘤坏死因子-α(rhTNF-α),继续培养2天,刺激DC成熟。 2.倒置显微镜观察细胞形态、数量的变化。 3.流式细胞仪检测DC的表面标志。 4.通过四甲基偶氮唑盐比色法(methyl thiazolyl tetrazolium,MTT)检测DC刺激同种异体外周血T淋巴细胞增殖的能力。试验二: 1.采用密度梯度离心法及黏附法分离出PBMC,分组培养: A:传统方法组,rhGM- CSF+ rhIL- 4,诱导72h后,加入rhTNF-α,继续培养24h; B:新型诱导剂组,加入rhGM- CSF+A23187,诱导96h。 2. K562细胞抗原的制备:收集对数生长期K562细胞,反复冻融(-80℃/37℃),低温离心,取上清用微孔滤膜过滤,-20℃保存备用。 3.培养开始时分别予K562细胞冻融抗原致敏,培养96小时后分别收集负载抗原的DC。 4.倒置显微镜观察细胞形态、数量的变化。 5.流式细胞仪检测DC的表面标志。 6.将负载K562细胞抗原的DC刺激T淋巴细胞,通过MTT比色法检测各组DC刺激同种异体外周血T淋巴细胞增殖的能力。 7.通过MTT比色法检测各组DC对K562细胞的抑制作用。 8.通过MTT比色法检测各组DC激活的CTL对K562细胞的杀伤作用。结果: 试验一: 1. PBMC经rhGM-CSF及rhIL-4诱导培养5天后,多数细胞呈集落生长,加入rhTNF-α继续培养2天后,可见细胞有典型的树枝状突起并呈散在分布。 2.培养第5天时,DC细胞表型CD83、CD1a、CD86、CD40及CD14分别是(14.3±2.2)%,(12.8±2.1)%,(20.1±1.8)%,(19.9±3.8)%及(16.2±3.3)%。加入TNF-α诱导后,即培养第7天,细胞表型CD83、CD1a、CD86、CD40及CD14分别是(29.8±4.4)%,(18.2±4.5)%,(33.6±4.2)%,(28.1±4.3)%及(8.0±2.8)%(与第5天比较,P值均0.05)。 3.成熟后的DC具备较强刺激T淋巴细胞增殖的能力,且增殖效应随效靶比的增加而增强。试验二: 1. A23187联合rhGM- CSF诱导培养的DC具有典型的树突形态。 2.与传统组比较,新型诱导剂组诱导的DC表面分子表达水平明显升高,分别为CD83(45.2±1.8)%vs(16.9±1.3)%;CD1a(31.5±3.9)% vs(20.4±3.4)%;CD86(40.1±7.8)% vs(26.5±2.2)%;CD40(36.4±6.3)% vs(22.3±3.0)%,P值均0.05;而CD14表达明显下降,分别为(5.7±0.8)% vs(19.0±1.6)%,P0.05。 3.采用MTT比色法检测各组方法培养的细胞对同种异体T淋巴细胞的刺激作用,发现两组DC在体外均能有效地激发同种异体外周血T淋巴细胞的增殖,与传统方法相比,新型诱导剂组除效靶比为1:40的增值效应与前者相似,P0.05,其它效靶比均明显强,P0.05,且增殖效应随效靶比的增加而增强。 4.与传统组比较,新型诱导剂组负载K562冻融抗原后的DC对K562细胞抑制作用更明显,效靶比为1:1及10:1时,抑瘤率分别为(25.33±3.76)% vs(15.55±2.39)%、(35.57±5.23)% vs(22.91±3.21)%,P值均0.05。 5.与传统组比较,新型诱导剂组负载K562冻融抗原后的DC激活的CTL对K562细胞有明显的杀伤作用,效靶比为10:1及40:1时,杀瘤率分别为(43.33±6.20)% vs(29.93±2.75)%、(60.97±5.18)% vs(43.14±4.75)%,P值均0.05。结论: 1.组合细胞因子rhGM-CSF+rhIL-4+rhTNF-α能诱导出成熟的DC,但培养时间长、容易污染及费用昂贵。 2.新型诱导剂CI A23187联合rhGM-CSF能更有效地诱导PBMC生成成熟DC,培养时间短且费用低廉。 3. K562冻融抗原冲击DC激活的CTL能够获得较强的杀伤K562细胞的能力。
[Abstract]:Objective:
1. the use of combined cytokines to induce human peripheral blood mononuclear cell (PBMC) to generate dendritic cells (Dendritic cell, DC).
2. a new inducer calcium carrier (Calcium Ionophore, CI) A23187 combined with recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) to induce human peripheral blood mononuclear cells to produce DC. in vitro
3. to observe the specific killing effect of DC stimulated cytotoxic T lymphocytes (CTL) on K562 cells (chronic myeloid leukemia cells).
Method:
Test 1:
1. the density gradient centrifugation and adhesion method were used to isolate PBMC, rhGM-CSF+ recombinant human interleukin-4 (rhIL-4) was added, and recombinant human tumor necrosis factor - alpha (rhTNF- alpha) was added for fifth days, and it continued to be cultured for 2 days, stimulating the maturation of DC.
2. inverted microscope was used to observe cell morphology and quantity changes.
3. flow cytometry was used to detect the surface markers of DC.
4. the ability of DC to stimulate the proliferation of T lymphocytes in peripheral blood was detected by methyl thiazolyl tetrazolium (MTT). Four
Test two:
1. the density gradient centrifugation and adhesion method were used to separate PBMC and group culture: A: traditional method group, rhGM- CSF+ rhIL- 4. After inducing 72h, rhTNF- a was added to 24h, B: new inducer group, rhGM- CSF+A23187, inducement 96h..
2. K562 cell antigen preparation: collecting K562 cells in the logarithmic growth period, freezing and thawing repeatedly (-80 C /37 C), centrifugation at low temperature, filtering the supernatant with microporous filter membrane, and storing it at -20.
3. at the beginning of culture, K562 cells were sensitized by freeze thawed antigen, and DC. was collected after 96 hours of culture.
4. inverted microscope was used to observe cell morphology and quantity changes.
5. flow cytometry was used to detect the surface markers of DC.
6. the DC cells loaded with K562 cell antigen stimulated T lymphocytes, and the ability of DC to stimulate the proliferation of T lymphocytes in peripheral blood was detected by MTT colorimetric method.
7. the inhibitory effect of DC on K562 cells was detected by MTT colorimetric assay.
8. the cytotoxicity of DC activated CTL on K562 cells was detected by MTT colorimetric assay.
Result:
Test 1:
1. PBMC was induced by rhGM-CSF and rhIL-4 for 5 days. Most of the cells were colonies, and rhTNF- a continued to be cultured for 2 days.
2. after fifth days of culture, the phenotype of DC cells, CD1a, CD86, CD40 and CD14 were (14.3 + 2.2)%, (12.8 + 2.1)%, (20.1 + 1.8)%, (19.9 + 3.8)% and (16.2 + 3.3)% respectively. After the induction of TNF- a, the cell phenotype CD83, CD1a, CD86, CD40 and CD14 were%, (2.1%)%,% +%,% and% In comparison, the P value is 0.05).
3. the mature DC has stronger ability to stimulate T lymphocyte proliferation, and the proliferation effect increases with the increase of target target ratio.
Test two:
1. A23187 combined with rhGM- CSF induced DC exhibited typical dendrite morphology.
2. compared with the traditional group, the expression level of DC surface molecules induced by the new inducer group was significantly increased, which were CD83 (45.2 + 1.8)%vs (16.9 + 1.3)%, CD1a (31.5 + 3.9)% vs (20.4 + 3.4)%, CD86 (40.1 + 7.8)% vs (26.5 + 2.2)%, CD40 (40.1%)% vs (20.4)%, P values, and CD14 expression, respectively. .05.
3. MTT colorimetric assay was used to detect the stimulating effect of cells cultured in each group on allogeneic T lymphocytes. It was found that the two groups of DC could effectively stimulate the proliferation of T lymphocytes of the allogenic peripheral blood in vitro. Compared with the traditional methods, the value added effect of the new inducer group at 1:40 was similar to the former, P0.05 and the other target ratio were both. Obviously stronger, P0.05, and the proliferation effect increased with the increase of target target ratio.
4. compared with the traditional group, the inhibitory effect of DC on K562 cells after the new inducer group loaded with K562 freeze-thaw antigen was more obvious. When the target ratio was 1:1 and 10:1, the tumor suppressor rate was (25.33 + 3.76)% vs (15.55 + 2.39)%, (35.57 + 5.23)% vs (22.91 + 3.21)% and P value 0.05.
5. compared with the traditional group, the DC activated CTL after the new inducer group loaded with K562 freeze-thaw antigen had an obvious killing effect on K562 cells. When the target ratio was 10:1 and 40:1, the tumor killing rate was (43.33 + 6.20)% vs (29.93 + 2.75)%, (60.97 + 5.18)% vs (43.14 + 4.75)% and P value of 0.05..
Conclusion:
1. combined cytokine rhGM-CSF+rhIL-4+rhTNF- alpha can induce mature DC, but it can be cultured for a long time, which is easy to pollute and expensive.
2. the new inducer CI A23187 combined with rhGM-CSF can induce PBMC to produce mature DC more effectively, and the incubation time is short and the cost is low.
3. K562 freeze-thaw antigen impacted on DC activated CTL can obtain a strong ability to kill K562 cells.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】