本文選題：樹突狀細(xì)胞 + 細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞�。� 參考：《新鄉(xiāng)醫(yī)學(xué)院》2013年碩士論文

【摘要】：目的： 本實(shí)驗(yàn)通過分離健康人外周血單個(gè)核細(xì)胞,應(yīng)用不同的細(xì)胞因子體外誘導(dǎo)分化出樹突狀細(xì)胞(Dendritic Cells, DC)和細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞(Cytokine-induced killer cells, CIK)細(xì)胞,將凍融抗原(Antigen, Ag)致敏DC并與CIK細(xì)胞共培養(yǎng)。 (1)觀察視網(wǎng)膜母細(xì)胞瘤(Retinoblasotma, RB)細(xì)胞株RB-Y79細(xì)胞凍融抗原致敏的DC與CIK細(xì)胞共培養(yǎng)后,DC與CIK細(xì)胞的形態(tài)學(xué)改變、細(xì)胞表型以及細(xì)胞增殖情況的變化； (2)探討凍融抗原致敏的DC-CIK細(xì)胞(DC-Ag-CIK細(xì)胞)對(duì)RB-Y79細(xì)胞的殺傷作用以及聯(lián)合應(yīng)用卡鉑后對(duì)RB-Y79細(xì)胞的協(xié)同殺傷作用。 (3)探討DC-Ag-CIK細(xì)胞聯(lián)合卡鉑對(duì)RB-Y79細(xì)胞可能的殺傷機(jī)制。 方法： (1)觀察凍融抗原致敏的DC與CIK細(xì)胞的體外增殖與生物學(xué)活性。取健康人外周血單個(gè)核細(xì)胞,加入不同細(xì)胞因子培養(yǎng)DC及CIK細(xì)胞,第3天用RB-Y79細(xì)胞凍融抗原致敏DC并于第7天將DC與CIK共培養(yǎng),分為CIK組, DC-CIK組和DC-Ag-CIK組。第7天倒置顯微鏡下觀察DC及CIK細(xì)胞形態(tài),以及3組細(xì)胞在共培養(yǎng)后的增殖情況。流式細(xì)胞儀檢測(cè)DC第3、7天及CIK細(xì)胞第7、15天的細(xì)胞表型變化。CBA細(xì)胞因子試劑盒檢測(cè)共培養(yǎng)至第15天時(shí)3組細(xì)胞上清液中IL-6及IL-10的含量。 (2)檢測(cè)殺瘤活性。PI法檢測(cè)卡鉑對(duì)RB-Y79及hTERT-RPE1細(xì)胞的殺傷活性；CFSE/PI法檢測(cè)效應(yīng)細(xì)胞(CIK、DC-CIK及DC-Ag-CIK細(xì)胞)在不同效靶比下對(duì)RB-Y79細(xì)胞、hTERT-RPE1細(xì)胞(正常視網(wǎng)膜色素上皮細(xì)胞)、RB耐藥細(xì)胞(RB-resistant cells, RB-R)的殺傷作用,以及效應(yīng)細(xì)胞聯(lián)合卡鉑對(duì)RB-Y79細(xì)胞的殺傷作用和CIK細(xì)胞在不同培養(yǎng)液條件下對(duì)RB-Y79細(xì)胞的殺傷活性; CFSE/PI法檢測(cè)卡鉑與DC-Ag-CIK細(xì)胞在不同應(yīng)用順序下對(duì)RB-Y79細(xì)胞的殺傷作用。 (3)在RB-Y79細(xì)胞上轉(zhuǎn)染GFP綠色熒光蛋白并篩選出RB-GFP細(xì)胞,免疫熒光顯微鏡拍攝卡鉑和／或DC-Ag-CIK細(xì)胞對(duì)RB-GFP細(xì)胞的動(dòng)態(tài)殺瘤過程；免疫熒光染色法及流式細(xì)胞術(shù)檢測(cè)卡鉑和／或DC-Ag-CIK細(xì)胞作用RB細(xì)胞后,RB細(xì)胞膜外Annexin V蛋白的表達(dá)情況。 結(jié)果： (1)經(jīng)過培養(yǎng)的DC呈現(xiàn)出典型的“樹突狀”結(jié)構(gòu),CIK細(xì)胞則聚集成團(tuán)。培養(yǎng)至第15天時(shí),DC-Ag-CIK和DC-CIK組的細(xì)胞數(shù)絕對(duì)值明顯高于CIK組(P0.01),DC-CIK組與DC-Ag-CIK組的IL-6含量明顯高于CIK組,而DC-Ag-CIK組IL-10含量明顯低于CIK組與DC-CIK組(P0.01)。經(jīng)過7天的培養(yǎng),DC細(xì)胞表型CD80、CD83、CD86的表達(dá)率均明顯高于第3天時(shí)的DC細(xì)胞(P0.01)。第15天的各組CIK細(xì)胞的細(xì)胞表型均明顯高于第7天未與DC共培養(yǎng)時(shí)的CIK細(xì)胞(P0.01),而在第15天的CIK組、DC-CIK組及Ag-DC-CIK組中,Ag-DC-CIK組的CD3+CD56+及CD3+CD8+表達(dá)率均明顯高于CIK組及DC-CIK組(P0.01)。 (2)效應(yīng)細(xì)胞隨著效靶比的增高對(duì)RB-Y79細(xì)胞的殺傷活性也隨之增高,在相同效靶比下,Ag-DC-CIK細(xì)胞對(duì)RB-Y79細(xì)胞的殺傷活性明顯高于CIK組與DC-CIK組(P0.05)；效應(yīng)細(xì)胞對(duì)正常視網(wǎng)膜色素上皮細(xì)胞hTERT-RPE1細(xì)胞幾乎沒有殺傷作用,且各組效應(yīng)細(xì)胞之間沒有明顯差異(P0.05)；CIK細(xì)胞在三種不同培養(yǎng)液條件下對(duì)RB-Y79細(xì)胞的殺傷活性無(wú)明顯差異(P0.05)；在效應(yīng)細(xì)胞與卡鉑聯(lián)合作用后,在相同效靶比的條件下對(duì)RB-Y79細(xì)胞的殺傷活性明顯高于單獨(dú)應(yīng)用效應(yīng)細(xì)胞和單獨(dú)應(yīng)用卡鉑(P0.01),同時(shí)對(duì)RB耐藥細(xì)胞的殺傷活性也明顯高于單獨(dú)應(yīng)用效應(yīng)細(xì)胞(P0.01)；在兩次卡鉑中加入應(yīng)用DC-Ag-CIK細(xì)胞后,其殺瘤率高(71.16±4.65%),與其他各組相比具有非常顯著的統(tǒng)計(jì)學(xué)差異(P0.01)。 (3)當(dāng)RB-GFP細(xì)胞死亡時(shí),RB細(xì)胞的綠色熒光消失,同時(shí)變被PI染成紅色。RB細(xì)胞預(yù)先與卡鉑共培養(yǎng)12小時(shí)后再加入DC-Ag-CIK細(xì)胞,其出現(xiàn)RB凋亡細(xì)胞比單獨(dú)應(yīng)用卡鉑及單獨(dú)應(yīng)用DC-Ag-CIK細(xì)胞需較短的時(shí)間；DC-Ag-CIK組Annexin V陽(yáng)性細(xì)胞表達(dá)率明顯高于CIK組及DC-CIK組(P0.01),與CIK細(xì)胞組相比,DC-Ag-CIK細(xì)胞能引發(fā)更多的RB凋亡細(xì)胞(P0.01),卡鉑聯(lián)合DC-Ag-CIK細(xì)胞后,其引發(fā)RB凋亡細(xì)胞數(shù)明顯多于DC-Ag-CIK組及單獨(dú)卡鉑組(P0.01)。 結(jié)論： (1)RB凍融抗原致敏的DC與CIK細(xì)胞共培養(yǎng)后,可促進(jìn)DC及CIK細(xì)胞的成熟與分化,使CIK細(xì)胞具有更強(qiáng)的增殖活性,并與IL-6的分泌上調(diào)以及IL-10的分泌下調(diào)有關(guān)。 (2)RB凍融抗原致敏的DC可增強(qiáng)CIK細(xì)胞對(duì)RB-Y79細(xì)胞的特異性殺傷作用,可能由于DC-Ag與CIK細(xì)胞共培養(yǎng)后可使CIK細(xì)胞大量增殖并誘導(dǎo)出大量CD3+CD56+的T細(xì)胞,從而提高了殺瘤活性。而DC-Ag-CIK細(xì)胞對(duì)正常視網(wǎng)膜色素上皮細(xì)胞(hTERT-RPE1細(xì)胞)無(wú)明顯殺傷作用。 (3)效應(yīng)細(xì)胞聯(lián)合卡鉑后對(duì)RB-Y79細(xì)胞及RB-R細(xì)胞均可產(chǎn)生明顯的協(xié)同殺傷作用,DC-Ag-CIK細(xì)胞聯(lián)合低劑量的卡鉑對(duì)RB-Y79細(xì)胞仍可產(chǎn)生明顯的殺瘤效果。 (4)卡鉑聯(lián)合DC-Ag-CIK細(xì)胞后能縮短殺瘤時(shí)間,其殺瘤機(jī)制可能是通過增加誘導(dǎo)RB細(xì)胞出現(xiàn)早期凋亡。
[Abstract]:Objective:
In this experiment, the human peripheral blood mononuclear cells were isolated from healthy human peripheral blood, and different cytokines were used to induce Dendritic Cells (DC) and cytokine induced killer cells (Cytokine-induced killer cells, CIK) cells, and to sensitized DC with freeze-thaw antigen (Antigen, Ag) and co culture with CIK cells.
(1) to observe the morphological changes of DC and CIK cells, the changes of cell phenotype and cell proliferation after the co culture of DC and CIK cells sensitized by the freeze-thaw antigen of Retinoblasotma (RB) cell line RB-Y79 cells.
(2) to investigate the killing effect of DC-CIK cells (DC-Ag-CIK cells) sensitized by freeze-thaw antigen on RB-Y79 cells and the synergistic killing effect of combined application of carboplatin on RB-Y79 cells.
(3) to explore the possible killing mechanism of DC-Ag-CIK cells combined with carboplatin on RB-Y79 cells.
Method錛，

本文編號(hào)：2018915