CAJ | 학술논문

背景：来自外周血单核细胞的自体DC疫苗作为一种很有前途的抗肿瘤免疫疗法，其面临的局限性包括细胞数量不足和功能缺陷。CD34+造血干细胞则是另一个理想的DC来源，已经有多种扩增CD34-DC的方法报道，都可以得到大量的细胞数和各种功能不同的DC。目的：本实验旨在探讨脐带血CD34+造血干细胞来源的树突状细胞(CD34-DC)与外周血单核细胞来源的树突状细胞(Mo-DC)在细胞形态、细胞表型、诱导生成的CTL细胞毒性方面的差异，并探讨不同来源的DC超微结构的变化与其抗原处理与抗原提呈功能之间的相关性，以期制备功能更加强大的DC疫苗。方法：取健康成人外周血，采用密度梯度离心法获取单核细胞后诱导生成DC(Mo-DC)；取健康足月产孕妇脐带血，采用磁珠分选法分离纯化CD34+造血干细胞，并培养于含粒细胞-巨噬细胞刺激因子(GM-CSF)/干细胞因子(SCF)的培养基诱导CD34+造血干细胞扩增及DC前体细胞的生成，GM-CSF/白细胞介素-4(IL-4)培养基进一步诱导前体细胞向DC细胞的分化(CD34-DC)。电镜下观察CD34-DC及Mo-DC的形态，比较其细胞突起数量、细胞核大小及内体小泡数量；流式细胞术检测不同培养时间(d1、d3、d5、d7)的CD34-DC及Mo-DC表面分子表达，并给予FITC-OVA257–264冲击，培养24 h后检测其抗原提呈能力；取培养至d5的CD34-DC及Mo-DC均负载CEA蛋白并加入肿瘤坏死因子(TNF-α)制备成CEA特异性的CD34-mDC、Mo-mDC(即DC疫苗)，分别体外诱导细胞毒性T淋巴细胞(CTL)生成，并利用MTS法检测其各自对CEA高表达的肿瘤细胞系Ls174-T的细胞毒性，以检测其激活T细胞的功能。结果：1)经过细胞因子和肿瘤抗原刺激之后的DC，悬浮生长而失去树突状外观，表现为类圆形，细胞膜清晰且光滑。2)与抗原提呈能力密切相关的内体小泡数量在CD34-DC中多于Mo-DC(P<0.05)。3)Mo-DC与CD34-DC经抗原及细胞因子刺激生成的Mo-mDC与CD34-mDC均可成熟(P>0.05)。4)CD34-DC比Mo-DC具有更强的抗原提呈能力(P<0.05)。5)CD34-DC与Mo-DC在刺激同种异体T淋巴细胞的增殖能力方面无差异(P>0.05)。6)CC-CTLs及CM-CTLs都可以杀伤CEA高表达的肿瘤细胞Ls174-T，但CC-CTLs比CM-CTLs有更高的杀伤率(P<0.05)。结论：1)经GM-CSF/SCF培养基可诱导脐带血CD34+造血干细胞扩增及生成DC前体细胞，并在培养d8开始贴壁生长，每3 d可收获一批DC前体细胞，用GM-CSF/IL-4培养基可进一步诱导生成CD34-DC，其表型与Mo-DC无差异。2)脐血造血干细胞来源的CD34-DC具有更强的抗原提呈、促淋巴细胞增殖及诱导激活CTL的能力。3)电镜下树突状突起数量、细胞核大小及内体小泡数量的变化是评估DCs功能差异的微观机制。4)脐带血来源的CD34-DC有望成为制备DC疫苗新的、理想的原料细胞。揹景：來自外週血單覈細胞的自體DC疫苗作為一種很有前途的抗腫瘤免疫療法，其麵臨的跼限性包括細胞數量不足和功能缺陷。CD34+造血榦細胞則是另一箇理想的DC來源，已經有多種擴增CD34-DC的方法報道，都可以得到大量的細胞數和各種功能不同的DC。目的：本實驗旨在探討臍帶血CD34+造血榦細胞來源的樹突狀細胞(CD34-DC)與外週血單覈細胞來源的樹突狀細胞(Mo-DC)在細胞形態、細胞錶型、誘導生成的CTL細胞毒性方麵的差異，併探討不同來源的DC超微結構的變化與其抗原處理與抗原提呈功能之間的相關性，以期製備功能更加彊大的DC疫苗。方法：取健康成人外週血，採用密度梯度離心法穫取單覈細胞後誘導生成DC(Mo-DC)；取健康足月產孕婦臍帶血，採用磁珠分選法分離純化CD34+造血榦細胞，併培養于含粒細胞-巨噬細胞刺激因子(GM-CSF)/榦細胞因子(SCF)的培養基誘導CD34+造血榦細胞擴增及DC前體細胞的生成，GM-CSF/白細胞介素-4(IL-4)培養基進一步誘導前體細胞嚮DC細胞的分化(CD34-DC)。電鏡下觀察CD34-DC及Mo-DC的形態，比較其細胞突起數量、細胞覈大小及內體小泡數量；流式細胞術檢測不同培養時間(d1、d3、d5、d7)的CD34-DC及Mo-DC錶麵分子錶達，併給予FITC-OVA257–264遲擊，培養24 h後檢測其抗原提呈能力；取培養至d5的CD34-DC及Mo-DC均負載CEA蛋白併加入腫瘤壞死因子(TNF-α)製備成CEA特異性的CD34-mDC、Mo-mDC(即DC疫苗)，分彆體外誘導細胞毒性T淋巴細胞(CTL)生成，併利用MTS法檢測其各自對CEA高錶達的腫瘤細胞繫Ls174-T的細胞毒性，以檢測其激活T細胞的功能。結果：1)經過細胞因子和腫瘤抗原刺激之後的DC，懸浮生長而失去樹突狀外觀，錶現為類圓形，細胞膜清晰且光滑。2)與抗原提呈能力密切相關的內體小泡數量在CD34-DC中多于Mo-DC(P<0.05)。3)Mo-DC與CD34-DC經抗原及細胞因子刺激生成的Mo-mDC與CD34-mDC均可成熟(P>0.05)。4)CD34-DC比Mo-DC具有更彊的抗原提呈能力(P<0.05)。5)CD34-DC與Mo-DC在刺激同種異體T淋巴細胞的增殖能力方麵無差異(P>0.05)。6)CC-CTLs及CM-CTLs都可以殺傷CEA高錶達的腫瘤細胞Ls174-T，但CC-CTLs比CM-CTLs有更高的殺傷率(P<0.05)。結論：1)經GM-CSF/SCF培養基可誘導臍帶血CD34+造血榦細胞擴增及生成DC前體細胞，併在培養d8開始貼壁生長，每3 d可收穫一批DC前體細胞，用GM-CSF/IL-4培養基可進一步誘導生成CD34-DC，其錶型與Mo-DC無差異。2)臍血造血榦細胞來源的CD34-DC具有更彊的抗原提呈、促淋巴細胞增殖及誘導激活CTL的能力。3)電鏡下樹突狀突起數量、細胞覈大小及內體小泡數量的變化是評估DCs功能差異的微觀機製。4)臍帶血來源的CD34-DC有望成為製備DC疫苗新的、理想的原料細胞。
배경：래자외주혈단핵세포적자체DC역묘작위일충흔유전도적항종류면역요법，기면림적국한성포괄세포수량불족화공능결함。CD34+조혈간세포칙시령일개이상적DC래원，이경유다충확증CD34-DC적방법보도，도가이득도대량적세포수화각충공능불동적DC。목적：본실험지재탐토제대혈CD34+조혈간세포래원적수돌상세포(CD34-DC)여외주혈단핵세포래원적수돌상세포(Mo-DC)재세포형태、세포표형、유도생성적CTL세포독성방면적차이，병탐토불동래원적DC초미결구적변화여기항원처리여항원제정공능지간적상관성，이기제비공능경가강대적DC역묘。방법：취건강성인외주혈，채용밀도제도리심법획취단핵세포후유도생성DC(Mo-DC)；취건강족월산잉부제대혈，채용자주분선법분리순화CD34+조혈간세포，병배양우함립세포-거서세포자격인자(GM-CSF)/간세포인자(SCF)적배양기유도CD34+조혈간세포확증급DC전체세포적생성，GM-CSF/백세포개소-4(IL-4)배양기진일보유도전체세포향DC세포적분화(CD34-DC)。전경하관찰CD34-DC급Mo-DC적형태，비교기세포돌기수량、세포핵대소급내체소포수량；류식세포술검측불동배양시간(d1、d3、d5、d7)적CD34-DC급Mo-DC표면분자표체，병급여FITC-OVA257–264충격，배양24 h후검측기항원제정능력；취배양지d5적CD34-DC급Mo-DC균부재CEA단백병가입종류배사인자(TNF-α)제비성CEA특이성적CD34-mDC、Mo-mDC(즉DC역묘)，분별체외유도세포독성T림파세포(CTL)생성，병이용MTS법검측기각자대CEA고표체적종류세포계Ls174-T적세포독성，이검측기격활T세포적공능。결과：1)경과세포인자화종류항원자격지후적DC，현부생장이실거수돌상외관，표현위류원형，세포막청석차광활。2)여항원제정능력밀절상관적내체소포수량재CD34-DC중다우Mo-DC(P<0.05)。3)Mo-DC여CD34-DC경항원급세포인자자격생성적Mo-mDC여CD34-mDC균가성숙(P>0.05)。4)CD34-DC비Mo-DC구유경강적항원제정능력(P<0.05)。5)CD34-DC여Mo-DC재자격동충이체T림파세포적증식능력방면무차이(P>0.05)。6)CC-CTLs급CM-CTLs도가이살상CEA고표체적종류세포Ls174-T，단CC-CTLs비CM-CTLs유경고적살상솔(P<0.05)。결론：1)경GM-CSF/SCF배양기가유도제대혈CD34+조혈간세포확증급생성DC전체세포，병재배양d8개시첩벽생장，매3 d가수획일비DC전체세포，용GM-CSF/IL-4배양기가진일보유도생성CD34-DC，기표형여Mo-DC무차이。2)제혈조혈간세포래원적CD34-DC구유경강적항원제정、촉림파세포증식급유도격활CTL적능력。3)전경하수돌상돌기수량、세포핵대소급내체소포수량적변화시평고DCs공능차이적미관궤제。4)제대혈래원적CD34-DC유망성위제비DC역묘신적、이상적원료세포。
Background:As a kind of promising anti-tumor immunotherapy, autologous dendritic cells (DCs) vaccine derived from peripheral blood monocytes faced limitations including deifciency of function and shortage of cell number. CD34+ progenitors are another ideal source of DC generation and different methods were explored and resulted in different cell number, various function of DC.Objective: Present study aimed to explore the differences between DCs derived from CD34+ hematopoietic stem cells (CD34-DC) of the cord blood and those derived from monocytes (Mo-DC) of the peripheral blood, in their cell morphologies, cell phenotypes, and the function of inducing antigen-specific CTLs, which could specifically induce tumor lyses, and explore different sources DC ultrastructural changes related to its antigen processing and antigen-presenting function of differences , in order to explore more powerful therapeutic DC vaccines. Methods: Monocytes were obtained from peripheral blood of healthy volunteers by using density gradient centrifugation, and the dendritic cells derived from monocytes (Mo-DC) were harvested by adherent assay. CD34+ hematopoietic stem cells were harvested from the cord blood of healthy full-term pregnant women, and puriifed by using magnetic bead separation, and expanded in GM-CSF/SCF medium for 8-10 days. DC progenitors were differentiated continuously in the GM-CSF/IL-4 medium, and CD34+ stem cell derived DCs (CD34-DC) were harvested every 3 days. Take d3, d5, d7 days immature DC (CD34-imDC, Mo-imDC) and d7 harvested CD34-mDC, Mo-mDC (DC vaccine) in the TEM compare their cell processes, nuclear size and endosomal vesicles quantity. Taken after different incubation time (d1, d3, d5, d7) CD34-DC and Mo-DC surface molecules detected by flow cytometry expression, some cells pulsed with FITC-OVA257-264 and continued to culture 24 hours, respectively, detecting the antigen-presenting ability by lfow cytometry. Take culture iftfh day DCs load CEA protein and tumor necrosis factor (TNF-α) was added. Preparation of the mature CD34-DC, Mo-DC (DC vaccine), in vitro induced cytotoxic T lymphocyte (CTL) generation, both vaccines induced the CTL were of high CEA-expressing tumor cell lines Ls174-T do killing assay, using MTS cytotoxicity assay. Results: 1) Atfer stimulated by cytokine and tumor antigen, they displayed the relative consistency in cell appearance. Antigen loaded DCs were lfoated with no dendrite, round-like shape, and the membrane was clear and smooth. 2) Number of endosomal vesicles was determined and results showed CD34-DC had more endosomal vesicles than Mo-DC at each time points (P<0.05). 3) CD34-mDC and Mo-mDC generated from CD34-DC and Mo-DC stimulated by antigen and cytokine can become mature (P<0.05). 4) CD34-DC has more antigen presenting ability than Mo-DC. 5) CD34-DC induced higher amount of additional lymphocytes over Mo-DC, but there are no significant differences (P>0.05). 6) CD34-DC and Mo-DC displayed an increased oncolytic efficacy and CC-CTLs showed stronger cytotoxicity than CM-CTLs (P<0.05).Conclusion: 1) With GM-CSF/SCF medium can induce cord blood CD34+ hematopoietic stem cell expansion and generate DC precursor cells and cultured adherent growth d8 beginning, a group can be harvested every three days DC precursor cells with GM-CSF/IL-4 -induced medium can further generate CD34-DC, no phenotypic differences between CD34-DC and Mo-DC. 2) Umbilical cord blood -derived CD34-DC has a stronger antigen-presenting, promoting lymphocyte proliferation and the ability to induce activation of CTLs. 3) By electron microscope, changes of the number of dendritic protrusions, nuclear size and the number of endosomal vesicles are to assess in DCs’ micro-mechanism. 4) Umbilical cord blood -derived CD34-DC is expected to become the new ideal raw cells for DC vaccine.