本文選題：明膠 + 單核細胞��； 參考：《山西醫(yī)科大學》2012年碩士論文

【摘要】：目的：觀察明膠法分離外周血單核細胞的效率以及將分離出的單核細胞刺激成熟為樹突狀細胞(dendritic cell, DC),觀察其形態(tài)及表型特征,并與普通塑料粘附法對比。 方法：使用人淋巴細胞分離液密度梯度離心法分離人外周血得到外周血單個核細胞(peripheral blood mononuclear cell, PBMC),根據(jù)培養(yǎng)瓶是否進行明膠包被分為明膠包被組(實驗組)和普通塑料組(對照組)。均分外周血單個核細胞,按不同分組分離獲得單核細胞。在重組人白細胞介素4(recombinant human interleukin-4, rhIL-4)和粒-巨噬細胞集落刺激因子(recombinant human granulocyte-macrophage colony-stimulating factor, rhGM-CSF)作用下刺激為非成熟樹突狀細胞,在促成熟雞尾酒組合重組人白細胞介素1β (recombinant human interleukin-1β, rhIL-1β)、重組人白細胞介素6(recombinant human interleukin-6,rhIL-6)、重組人腫瘤壞死因子a (recombinant human tumor necrosis factor-a, rhTNF-a)和前列腺素E2(prostaglandin E2, PGE2)作用下誘導(dǎo)刺激為成熟樹突狀細胞。計數(shù)各組分離獲得的單核細胞數(shù)。鏡下觀察對比兩組血小板污染情況。流式細胞儀檢測兩組單核細胞的CD14、CD3、CD19陽性率,分別代表所得單核細胞的純度、T、B淋巴細胞污染率。錐蟲藍拒染法計算單核細胞活率。單核細胞分化為樹突狀細胞后鏡下觀察對比兩組所得樹突狀細胞的形態(tài)。使用流式細胞儀檢測樹突狀細胞非成熟期和成熟期CDla,CD83的表達情況。 結(jié)果：平均每組30ml外周血,普通塑料組和明膠包被組分別獲得(12.3±3.56)和(15.8±3.05)×106個單核細胞,兩組相比P0.05,差異有顯著性意義,表明明膠法分離獲得的單核細胞多于普通塑料粘附法。鏡下觀察到普通塑料組的血小板污染率明顯高于明膠包被組。普通塑料組和明膠包被組所得單核細胞的CD14、CD3、CD19陽性率分別為(67.25±5.77)%和(81.56±5.83)%、(4.68±1.01)%和(2.89±0.81)%、(10.89±1.45)%和(7.68±1.54)%,各組相比P0.05,差異有顯著性意義,表明明膠法獲得的單核細胞的純度大于普通塑料粘附法,T、B淋巴細胞污染率低于普通塑料粘附法。兩組細胞活率分別為(94.6±1.58)%和(95.2±1.64)%,相比P0.05,差異無顯著性意義,表明明膠法對細胞活力無明顯不利影響。兩組細胞刺激為樹突狀細胞后,均具有典型樹突狀細胞的形態(tài)學特征,非成熟期和成熟期表型CDla、CD83相比差異無顯著性意義,認為明膠法對單核細胞刺激成熟為樹突狀細胞的功能無不利影響。 結(jié)論：明膠法可以簡單高效分離出外周血中單核細胞并成功刺激其分化為具有典型形態(tài)學及成熟表型的樹突狀細胞。
[Abstract]:Objective: to observe the efficiency of isolation of peripheral blood monocytes by gelatin method, and to observe the morphological and phenotypic characteristics of dendritic cells (DCN), which were stimulated by the isolated monocytes and matured into dendritic cells (DCN). Methods: human peripheral blood mononuclear cells (PBMC) were isolated from human peripheral blood by density gradient centrifugation method, and divided into gelatin according to whether the culture bottle was coated with gelatin. Coated group (experimental group) and ordinary plastic group (control group). Mononuclear cells were isolated from peripheral blood mononuclear cells in different groups. 4(recombinant human interleukin-4 (rhIL-4) and granulocyte-macrophage colony stimulating factor recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) stimulated immature dendritic cells. Recombinant human interleukin-1 尾, rhIL-1 尾, recombinant 6(recombinant human interleukin-6hIL-6, recombinant human tumor necrosis factor-a (rhTNF-a) and prostaglandin E2prostaglandin E2 (PGE2) were used to induce mature dendritic cells (dendritic cells) in the presence of a cocktail of prostaglandin 2 (PGE2), recombinant human interleukin-1 尾 (rhIL-1 尾), recombinant human interleukin (6(recombinant human) interleukin-6 (rhIL-6), recombinant human tumor necrosis factor-a (rhTNF-a) and prostaglandin E2 (PGE2). The number of monocytes isolated from each group was counted. The platelet contamination in the two groups was observed under microscope. The positive rate of CD14, CD3, CD19 in monocytes was detected by flow cytometry. The viability of monocytes was calculated by trypanosome blue exclusion method. The morphology of dendritic cells in the two groups was observed and compared with the differentiation of monocytes into dendritic cells. Flow cytometry was used to detect the CD83 expression of dendritic cells in immature and mature stages. Results: on average, 12.3 鹵3.56) and 15.8 鹵3.05) 脳 106 monocytes were obtained in each group of 30ml peripheral blood, ordinary plastic group and gelatin coated group, respectively. Compared with P0.05, the difference between the two groups was significant, indicating that the monocytes isolated by gelatin method were more than those by ordinary plastic adhesion method. The rate of platelet contamination in ordinary plastic group was significantly higher than that in gelatin coated group. The positive rates of CD14 and CD3 + CD19 were 67.25 鹵5.77 and 81.56 鹵5.83% and 2.89 鹵0.81% and 10.89 鹵1.45% and 7.68 鹵1.54 of monocytes in the plastic group and gelatin coated group, respectively, and there was significant difference between the two groups (P0.05, P 0.05), and the positive rates of CD14 + CD3 + CD19 in the normal plastic group and gelatin coated group were 67.25 鹵5.77% and 81.56 鹵5.83%, respectively, and 2.89 鹵0.81% and 7.68 鹵1.54% in the normal plastic group and gelatin coated group, respectively. The results showed that the purity of monocytes obtained by gelatin method was higher than that by ordinary plastic adhesion method. The cell viability of the two groups was 94.6 鹵1.58% and 95.2 鹵1.64%, respectively. Compared with P0.05, there was no significant difference between the two groups, indicating that the gelatin method had no significant adverse effect on cell viability. After stimulated with dendritic cells, the two groups had the morphological characteristics of typical dendritic cells, and there was no significant difference in the phenotypes of CDla-CD83 between immature and mature stages. It is concluded that gelatin method has no adverse effect on the function of monocytes in stimulating maturation to dendritic cells. Conclusion: gelatin method can be used to isolate monocytes from peripheral blood and successfully stimulate their differentiation into typical morphology and formation. Mature phenotypic dendritic cells.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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