本文選題：樹突狀細胞 + 鈣離子載體　； 參考：《廣州醫(yī)學院》2010年碩士論文

【摘要】：目的: 1.用組合細胞因子誘導人外周血單核細胞(human peripheral blood mononuclear cell,PBMC)生成樹突狀細胞(Dendritic cell,DC)。 2.用新型誘導劑鈣離子載體(Calcium Ionophore,CI)A23187聯(lián)合重組人粒細胞-巨噬細胞集落刺激因子(rhGM-CSF)體外誘導人外周血單核細胞生成DC。 3.觀察DC刺激細胞毒性T淋巴細胞(CTL)對K562細胞(慢性髓性白血病細胞)產(chǎn)生的特異性殺傷作用。 方法: 試驗一: 1.采用密度梯度離心法及黏附法分離出PBMC,加入rhGM-CSF+重組人白介素- 4(rhIL-4)誘導,第5天加入重組人腫瘤壞死因子-α(rhTNF-α),繼續(xù)培養(yǎng)2天,刺激DC成熟。 2.倒置顯微鏡觀察細胞形態(tài)、數(shù)量的變化。 3.流式細胞儀檢測DC的表面標志。 4.通過四甲基偶氮唑鹽比色法(methyl thiazolyl tetrazolium,MTT)檢測DC刺激同種異體外周血T淋巴細胞增殖的能力。 試驗二: 1.采用密度梯度離心法及黏附法分離出PBMC,分組培養(yǎng): A:傳統(tǒng)方法組,rhGM- CSF+ rhIL- 4,誘導72h后,加入rhTNF-α,繼續(xù)培養(yǎng)24h; B:新型誘導劑組,加入rhGM- CSF+A23187,誘導96h。 2. K562細胞抗原的制備:收集對數(shù)生長期K562細胞,反復凍融(-80℃/37℃),低溫離心,取上清用微孔濾膜過濾,-20℃保存?zhèn)溆谩?3.培養(yǎng)開始時分別予K562細胞凍融抗原致敏,培養(yǎng)96小時后分別收集負載抗原的DC。 4.倒置顯微鏡觀察細胞形態(tài)、數(shù)量的變化。 5.流式細胞儀檢測DC的表面標志。 6.將負載K562細胞抗原的DC刺激T淋巴細胞,通過MTT比色法檢測各組DC刺激同種異體外周血T淋巴細胞增殖的能力。 7.通過MTT比色法檢測各組DC對K562細胞的抑制作用。 8.通過MTT比色法檢測各組DC激活的CTL對K562細胞的殺傷作用。 結(jié)果: 試驗一: 1. PBMC經(jīng)rhGM-CSF及rhIL-4誘導培養(yǎng)5天后,多數(shù)細胞呈集落生長,加入rhTNF-α繼續(xù)培養(yǎng)2天后,可見細胞有典型的樹枝狀突起并呈散在分布。 2.培養(yǎng)第5天時,DC細胞表型CD83、CD1a、CD86、CD40及CD14分別是(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誘導后,即培養(yǎng)第7天,細胞表型CD83、CD1a、CD86、CD40及CD14分別是(29.8±4.4)%,(18.2±4.5)%,(33.6±4.2)%,(28.1±4.3)%及(8.0±2.8)%(與第5天比較,P值均0.05)。 3.成熟后的DC具備較強刺激T淋巴細胞增殖的能力,且增殖效應隨效靶比的增加而增強。 試驗二: 1. A23187聯(lián)合rhGM- CSF誘導培養(yǎng)的DC具有典型的樹突形態(tài)。 2.與傳統(tǒng)組比較,新型誘導劑組誘導的DC表面分子表達水平明顯升高,分別為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表達明顯下降,分別為(5.7±0.8)% vs(19.0±1.6)%,P0.05。 3.采用MTT比色法檢測各組方法培養(yǎng)的細胞對同種異體T淋巴細胞的刺激作用,發(fā)現(xiàn)兩組DC在體外均能有效地激發(fā)同種異體外周血T淋巴細胞的增殖,與傳統(tǒng)方法相比,新型誘導劑組除效靶比為1:40的增值效應與前者相似,P0.05,其它效靶比均明顯強,P0.05,且增殖效應隨效靶比的增加而增強。 4.與傳統(tǒng)組比較,新型誘導劑組負載K562凍融抗原后的DC對K562細胞抑制作用更明顯,效靶比為1:1及10:1時,抑瘤率分別為(25.33±3.76)% vs(15.55±2.39)%、(35.57±5.23)% vs(22.91±3.21)%,P值均0.05。 5.與傳統(tǒng)組比較,新型誘導劑組負載K562凍融抗原后的DC激活的CTL對K562細胞有明顯的殺傷作用,效靶比為10:1及40:1時,殺瘤率分別為(43.33±6.20)% vs(29.93±2.75)%、(60.97±5.18)% vs(43.14±4.75)%,P值均0.05。 結(jié)論: 1.組合細胞因子rhGM-CSF+rhIL-4+rhTNF-α能誘導出成熟的DC,但培養(yǎng)時間長、容易污染及費用昂貴。 2.新型誘導劑CI A23187聯(lián)合rhGM-CSF能更有效地誘導PBMC生成成熟DC,培養(yǎng)時間短且費用低廉。 3. K562凍融抗原沖擊DC激活的CTL能夠獲得較強的殺傷K562細胞的能力。
[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.
【學位授予單位】：廣州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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