目的 建立一种稳定的人外周血树突状细胞(DCs)体外培养的方法,并与磁珠分选法进行比较.方法 通过密度梯度离心法分离出志愿者的外周血单个核细胞(PBMC),再分别应用磁珠分选法、贴壁法对PBMC进行培养,应用重组人集落刺激因子(rhGM-CSF)、重组人白细胞介素-4(rhIL-4)诱导获得DCs.倒置显微镜观察细胞形态变化,并分别在第3、5、6天用台盼蓝染色法进行细胞活力检测;经过1、2、5 h的贴壁培养后,应用流式细胞仪检测单核细胞表面CD14、CD1a、HLA-DR的表达以确定最佳贴壁时间;经人重组细胞因子诱导培养后,对所获得的细胞检测CD14、CD1a、CD86、CD83、HLA-DR的表达.采用同种混合淋巴细胞反应,评价DCs刺激T淋巴细胞增殖的能力.结果 经贴壁2 h后诱导培养的DCs形态较典型.磁珠分选法获得的DCs第5、6天细胞活力[(53.333±5.774)%、(38.333±7.638)%]明显低于第3天[(68.667±3.215)%,P均＜0.05];贴壁培养法获得的DCs第3、5、6天的细胞活力[(92.667±3.055)%、(94.000±1.000)%和(94.667±1.528)%]比较,差异无统计学意义(F=0.737,P＞0.05);贴壁培养法获得的DCs第3、5、6天细胞活力均高于磁珠分选法(t值分别为9.374、12.021、12.527,P均＜0.05).PBMC经磁珠分选前后CD14的阳性表达率分别为(32.457±12.351)%、(41.914±14.858)%,二者比较差异无统计学意义(t=1.295,P＞0.05).单核细胞表面CD14的阳性表达率在培养2 h时[(35.267±4.658)%]高于培养1、5 h时[(15.033±6.189)%、(21.233±4.895)%,P均＜0.05].培养第6天,DCs表面CD14的阳性表达率[(2.200±1.356)%]较第1天[(32.328±14.517)%]明显下降(t=5.467,P＜0.05),CD1a的阳性表达率[(43.371±16.250)%]较第1天[(12.300±6.223)%]显著升高(t=2.545,P＜0.05);而CD86、CD83、HLA-DR的阳性表达率[(16.857±5.686)%、(9.343±5.230)%、(72.800±17.881)%]与第1天[(12.550±16.758)%、(6.250±1.323)%、(64.671±15.588)%]比较,差异无统计学意义(t值分别为0.652、1.137、0.907,P均＞0.05).同种混合淋巴细胞反应,随着淋巴细胞的增多,增殖能力下降.磁珠分选法中,DCs与淋巴细胞的比例为1:50、1:100时,细胞增殖能力(1.502±0.055、1.507±0.029)较1:10时(1.859±0.049)降低(P均＜0.05);贴壁培养法中,DCs与淋巴细胞的比例为1:100时,细胞增殖能力(1.545±0.066)较1:10时(2.015±0.301)降低(P＜0.05).在DCs与淋巴细胞的比例相同时,两种方法得到的DCs在刺激T淋巴细胞方面的能力相近(P＞0.05).结论 与磁珠分选法比较,贴壁培养2 h后的人外周血PBMC再行诱导可获得形态与功能较优的DCs,且此法稳定、简便、经济,是一种适于基础、临床研究的DCs体外培养方法.
目的 建立一種穩定的人外週血樹突狀細胞(DCs)體外培養的方法,併與磁珠分選法進行比較.方法 通過密度梯度離心法分離齣誌願者的外週血單箇覈細胞(PBMC),再分彆應用磁珠分選法、貼壁法對PBMC進行培養,應用重組人集落刺激因子(rhGM-CSF)、重組人白細胞介素-4(rhIL-4)誘導穫得DCs.倒置顯微鏡觀察細胞形態變化,併分彆在第3、5、6天用檯盼藍染色法進行細胞活力檢測;經過1、2、5 h的貼壁培養後,應用流式細胞儀檢測單覈細胞錶麵CD14、CD1a、HLA-DR的錶達以確定最佳貼壁時間;經人重組細胞因子誘導培養後,對所穫得的細胞檢測CD14、CD1a、CD86、CD83、HLA-DR的錶達.採用同種混閤淋巴細胞反應,評價DCs刺激T淋巴細胞增殖的能力.結果 經貼壁2 h後誘導培養的DCs形態較典型.磁珠分選法穫得的DCs第5、6天細胞活力[(53.333±5.774)%、(38.333±7.638)%]明顯低于第3天[(68.667±3.215)%,P均＜0.05];貼壁培養法穫得的DCs第3、5、6天的細胞活力[(92.667±3.055)%、(94.000±1.000)%和(94.667±1.528)%]比較,差異無統計學意義(F=0.737,P＞0.05);貼壁培養法穫得的DCs第3、5、6天細胞活力均高于磁珠分選法(t值分彆為9.374、12.021、12.527,P均＜0.05).PBMC經磁珠分選前後CD14的暘性錶達率分彆為(32.457±12.351)%、(41.914±14.858)%,二者比較差異無統計學意義(t=1.295,P＞0.05).單覈細胞錶麵CD14的暘性錶達率在培養2 h時[(35.267±4.658)%]高于培養1、5 h時[(15.033±6.189)%、(21.233±4.895)%,P均＜0.05].培養第6天,DCs錶麵CD14的暘性錶達率[(2.200±1.356)%]較第1天[(32.328±14.517)%]明顯下降(t=5.467,P＜0.05),CD1a的暘性錶達率[(43.371±16.250)%]較第1天[(12.300±6.223)%]顯著升高(t=2.545,P＜0.05);而CD86、CD83、HLA-DR的暘性錶達率[(16.857±5.686)%、(9.343±5.230)%、(72.800±17.881)%]與第1天[(12.550±16.758)%、(6.250±1.323)%、(64.671±15.588)%]比較,差異無統計學意義(t值分彆為0.652、1.137、0.907,P均＞0.05).同種混閤淋巴細胞反應,隨著淋巴細胞的增多,增殖能力下降.磁珠分選法中,DCs與淋巴細胞的比例為1:50、1:100時,細胞增殖能力(1.502±0.055、1.507±0.029)較1:10時(1.859±0.049)降低(P均＜0.05);貼壁培養法中,DCs與淋巴細胞的比例為1:100時,細胞增殖能力(1.545±0.066)較1:10時(2.015±0.301)降低(P＜0.05).在DCs與淋巴細胞的比例相同時,兩種方法得到的DCs在刺激T淋巴細胞方麵的能力相近(P＞0.05).結論 與磁珠分選法比較,貼壁培養2 h後的人外週血PBMC再行誘導可穫得形態與功能較優的DCs,且此法穩定、簡便、經濟,是一種適于基礎、臨床研究的DCs體外培養方法.
목적 건립일충은정적인외주혈수돌상세포(DCs)체외배양적방법,병여자주분선법진행비교.방법 통과밀도제도리심법분리출지원자적외주혈단개핵세포(PBMC),재분별응용자주분선법、첩벽법대PBMC진행배양,응용중조인집락자격인자(rhGM-CSF)、중조인백세포개소-4(rhIL-4)유도획득DCs.도치현미경관찰세포형태변화,병분별재제3、5、6천용태반람염색법진행세포활력검측;경과1、2、5 h적첩벽배양후,응용류식세포의검측단핵세포표면CD14、CD1a、HLA-DR적표체이학정최가첩벽시간;경인중조세포인자유도배양후,대소획득적세포검측CD14、CD1a、CD86、CD83、HLA-DR적표체.채용동충혼합림파세포반응,평개DCs자격T림파세포증식적능력.결과 경첩벽2 h후유도배양적DCs형태교전형.자주분선법획득적DCs제5、6천세포활력[(53.333±5.774)%、(38.333±7.638)%]명현저우제3천[(68.667±3.215)%,P균＜0.05];첩벽배양법획득적DCs제3、5、6천적세포활력[(92.667±3.055)%、(94.000±1.000)%화(94.667±1.528)%]비교,차이무통계학의의(F=0.737,P＞0.05);첩벽배양법획득적DCs제3、5、6천세포활력균고우자주분선법(t치분별위9.374、12.021、12.527,P균＜0.05).PBMC경자주분선전후CD14적양성표체솔분별위(32.457±12.351)%、(41.914±14.858)%,이자비교차이무통계학의의(t=1.295,P＞0.05).단핵세포표면CD14적양성표체솔재배양2 h시[(35.267±4.658)%]고우배양1、5 h시[(15.033±6.189)%、(21.233±4.895)%,P균＜0.05].배양제6천,DCs표면CD14적양성표체솔[(2.200±1.356)%]교제1천[(32.328±14.517)%]명현하강(t=5.467,P＜0.05),CD1a적양성표체솔[(43.371±16.250)%]교제1천[(12.300±6.223)%]현저승고(t=2.545,P＜0.05);이CD86、CD83、HLA-DR적양성표체솔[(16.857±5.686)%、(9.343±5.230)%、(72.800±17.881)%]여제1천[(12.550±16.758)%、(6.250±1.323)%、(64.671±15.588)%]비교,차이무통계학의의(t치분별위0.652、1.137、0.907,P균＞0.05).동충혼합림파세포반응,수착림파세포적증다,증식능력하강.자주분선법중,DCs여림파세포적비례위1:50、1:100시,세포증식능력(1.502±0.055、1.507±0.029)교1:10시(1.859±0.049)강저(P균＜0.05);첩벽배양법중,DCs여림파세포적비례위1:100시,세포증식능력(1.545±0.066)교1:10시(2.015±0.301)강저(P＜0.05).재DCs여림파세포적비례상동시,량충방법득도적DCs재자격T림파세포방면적능력상근(P＞0.05).결론 여자주분선법비교,첩벽배양2 h후적인외주혈PBMC재행유도가획득형태여공능교우적DCs,차차법은정、간편、경제,시일충괄우기출、림상연구적DCs체외배양방법.
Objective To establish a economic and stable method to induce and culture dendritic cells (DCs) from peripheral blood of human being, and compare with the magnetic activated cell sorting. Methods Monocytes were isolated from health donors peripheral blood mononuclear cells(PBMC) by density gradient separation,cultured and compared with that of cells isolated by the magnetic activated cell sorting or adherent culture,respectively. PBMC were cultured with recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4(rhIL-4) for 6 days to induce the growth of DCs. Morphological changes was observed under inverted microscope. Meanwhile, cell viability was tested at the 3rd, 5th, 6th day,respectively. The phenotypes, like CD14, CDla, HLA-DR were analyzed with flow cytometry after PBMC were adherent cultured for 1, 2, 5 h. After adding human recombinant cytokines, the phenotypes of acquired cells surface markers, CD14, CD1a, CD86, CD83 and HLA-DR would be detected and compared with flow cytometry. T cells proliferating activity was determined by allogeneic mixed lymphocyte reaction in vitro. Results After adherent culture for 2 h, the acquired DCs showed typical morphology. Cell viability was decreased at days 5th, 6th[(53.333 ±5.774)%,(38.333 ± 7.638)%] than that at day of 3rd[(68.667 ± 3.215)%, all P ＜ 0.05] with the magnetic activated cell sorting, but with adherent culture method, the difference was not statistically significant (F = 0.737,P＞ 0.05) at days of 3rd, 5th, 6th[(92.667 ± 3.055)%,(94.000 ± 1.000)%,(94.667 ± 1.528)%]. Moreover,compared with the magnetic activated cell sorting, there were differences in cell viability of adherent culture method at days of 3rd, 5th, 6th(t = 9.374, 12.021,12.527, all P ＜ 0.05). Before and after using the magnetic activated cell sorting, the expression of CD14 were (32.457 ± 12.351) %, (41.914 ± 14.858)%, respectively. The difference was not statistically significant(t = 1.295, P ＞ 0.05). After culturing for 2 h, the expression of CD14[(35.267 ± 4.658)%]was higher than those of culturing for 1, 5 h[(15.033 ± 6.189)%, (21.233 ± 4.895)%, all P ＜ 0.05]. Compared with the 1st day[(32.328 ± 14.517)%], the CD14 expression level[(2.200 ± 1.356)%] on surface of DCs was significantly reduced(t = 5.467, P ＜ 0.05) at the 6th day of culturing, the CD1a expression level[(43.371 ±16.250)%] was remarkablely increased than that of the 1st day[(12.300 ± 6.223)%, t = 2.545, P ＜ 0.05];while the expressions of CD86, CD83, HLA-DR[(16.857 ± 5.686)%,(9.343 ± 5.230)%,(72.800 ± 17.881)%] were similar(t = 0.652,1.137,0.907, all P ＞ 0.05) compared with that of the 1st day[(12.550 ± 16.758)%, (6.250 ±1.323)%, (64.671 ± 15.588)%]. In mixed lymphocytes reactions, with increasing of lymphocytes, T lymphocytes proliferating activities were reduced. In the magnetic activated cell sorting, when the ratio of DCs and lymphocytes were 1: 50, 1: 100, cells proliferation ability(1.502 ± 0.055,1.507 ± 0.029) were lower than that of ratio of 1: 10(1.859 ± 0.049, all P ＜ 0.05);in adherent culture method, the ratio of DCs and lymphocytes was 1: 100, the cells proliferation ability(1.545 ± 0.066) was decreased than that of ratio 1: 10(2.015 ± 0.301, P ＜ 0.05). When the proportion of DCs and lymphocytes remained the same, the capacity to stimulate T lymphocyte was similar of the two methods(P ＞ 0.05). Conclusions Comparied with the magnetic activated cell sorting, after culture of PBMC for 2 h the induction of DCs can produce better formed and functional cells, and this method is stable, simple,economic, and is a suitable method for basic and clinical research of DCs in vitro.
