CAJ | 학술논문

背景:造血干细胞是构筑免疫系统的最早的细胞,能分化为多种细胞,其中具有包括免疫应答调控树突状细胞.树突状细胞的诱导培养因前体细胞来源不同,所采用的细胞因子,及最佳的细胞因子配伍、应用顺序、实验室培养条件亦不相同,树突状细胞的发育、各种表犁的表达及成熟度也不尽相同.目的:观察肿瘤坏死因子α和白细胞介素4对脐血CD34+造血干细胞来源的树突状细胞诱导培养体系的影响,探寻该培养体系优化方法.设计、时间及地点:观察性实验,于2005-03/11在南京医科大学微生物与免疫学实验室完成.材料:健康新生儿脐血为南京市八一医院产妇同意捐赠.CD34单克隆抗体一磁珠分离系统为德国Miltenyi Biotec公司产品:重组人粒细胞巨噬细胞集落刺激因子(GM-CSF)、重组人白细胞介素4和重组人肿瘤坏死因子α为美国PeproTech公司产品.方法:淋巴细胞分离液分离获得脐血单个核细胞,免疫磁珠阳性分选CD34+造血干细胞,并用流式细胞术鉴定CD34+造血干细胞纯度:比较GT(GM-CSF+肿瘤坏死因子α)方案和GTI(GM-CSF+肿瘤坏死因子α+自细胞介素4)方案及GTI方案中肿瘤坏死因子α和白细胞介素4不同时段加入对诱导培养产生的树突状细胞成熟的影响;通过激光共聚焦显微镜观察细胞形态,流式细胞仪分析细胞表型及3H-TdR检测树突状细胞激发异体T细胞增殖能力.结果:免疫磁珠阳性分选CD34+造血干细胞纯度町达90%以上.将CD34+造血干细胞按GT方案和GTI方案进行培养.均可诱导产生树突状细胞,CD34的阳性表达率逐渐下降,HLA-DR的表达下降(P＜0.05),树突状细胞的相关分化抗原CD80,CD86,CD83和CDla的表达均相应增加,培养13～15 d的细胞各表型表达较7～9 d,10～12 d充分.但经GT方案诱导的树突状细胞CD14表达较高,cD80,CDB6,CD83,CD1a表达不如经GTI方案诱导的高;而GTI方案中,以肿瘤坏死因子aoh、白细胞介素448 h加入诱导培养的树突状细胞各表型表达相对较佳,其细胞表达CD80,CD86均较其他组高,尤以CD86表达为著,并具有激发异体T细胞增殖能力.结论:CD34+造血干细胞经过合适的培养体系能够诱导分化为功能性树突状细胞,以GM-CSF与肿瘤坏死因子α O h加入、白细胞介素4 48 h加入的GM-CSF+肿瘤坏死因子α+白细胞介素4方案更为可取. 揹景:造血榦細胞是構築免疫繫統的最早的細胞,能分化為多種細胞,其中具有包括免疫應答調控樹突狀細胞.樹突狀細胞的誘導培養因前體細胞來源不同,所採用的細胞因子,及最佳的細胞因子配伍、應用順序、實驗室培養條件亦不相同,樹突狀細胞的髮育、各種錶犛的錶達及成熟度也不儘相同.目的:觀察腫瘤壞死因子α和白細胞介素4對臍血CD34+造血榦細胞來源的樹突狀細胞誘導培養體繫的影響,探尋該培養體繫優化方法.設計、時間及地點:觀察性實驗,于2005-03/11在南京醫科大學微生物與免疫學實驗室完成.材料:健康新生兒臍血為南京市八一醫院產婦同意捐贈.CD34單剋隆抗體一磁珠分離繫統為德國Miltenyi Biotec公司產品:重組人粒細胞巨噬細胞集落刺激因子(GM-CSF)、重組人白細胞介素4和重組人腫瘤壞死因子α為美國PeproTech公司產品.方法:淋巴細胞分離液分離穫得臍血單箇覈細胞,免疫磁珠暘性分選CD34+造血榦細胞,併用流式細胞術鑒定CD34+造血榦細胞純度:比較GT(GM-CSF+腫瘤壞死因子α)方案和GTI(GM-CSF+腫瘤壞死因子α+自細胞介素4)方案及GTI方案中腫瘤壞死因子α和白細胞介素4不同時段加入對誘導培養產生的樹突狀細胞成熟的影響;通過激光共聚焦顯微鏡觀察細胞形態,流式細胞儀分析細胞錶型及3H-TdR檢測樹突狀細胞激髮異體T細胞增殖能力.結果:免疫磁珠暘性分選CD34+造血榦細胞純度町達90%以上.將CD34+造血榦細胞按GT方案和GTI方案進行培養.均可誘導產生樹突狀細胞,CD34的暘性錶達率逐漸下降,HLA-DR的錶達下降(P＜0.05),樹突狀細胞的相關分化抗原CD80,CD86,CD83和CDla的錶達均相應增加,培養13～15 d的細胞各錶型錶達較7～9 d,10～12 d充分.但經GT方案誘導的樹突狀細胞CD14錶達較高,cD80,CDB6,CD83,CD1a錶達不如經GTI方案誘導的高;而GTI方案中,以腫瘤壞死因子aoh、白細胞介素448 h加入誘導培養的樹突狀細胞各錶型錶達相對較佳,其細胞錶達CD80,CD86均較其他組高,尤以CD86錶達為著,併具有激髮異體T細胞增殖能力.結論:CD34+造血榦細胞經過閤適的培養體繫能夠誘導分化為功能性樹突狀細胞,以GM-CSF與腫瘤壞死因子α O h加入、白細胞介素4 48 h加入的GM-CSF+腫瘤壞死因子α+白細胞介素4方案更為可取.
배경:조혈간세포시구축면역계통적최조적세포,능분화위다충세포,기중구유포괄면역응답조공수돌상세포.수돌상세포적유도배양인전체세포래원불동,소채용적세포인자,급최가적세포인자배오、응용순서、실험실배양조건역불상동,수돌상세포적발육、각충표리적표체급성숙도야불진상동.목적:관찰종류배사인자α화백세포개소4대제혈CD34+조혈간세포래원적수돌상세포유도배양체계적영향,탐심해배양체계우화방법.설계、시간급지점:관찰성실험,우2005-03/11재남경의과대학미생물여면역학실험실완성.재료:건강신생인제혈위남경시팔일의원산부동의연증.CD34단극륭항체일자주분리계통위덕국Miltenyi Biotec공사산품:중조인립세포거서세포집락자격인자(GM-CSF)、중조인백세포개소4화중조인종류배사인자α위미국PeproTech공사산품.방법:림파세포분리액분리획득제혈단개핵세포,면역자주양성분선CD34+조혈간세포,병용류식세포술감정CD34+조혈간세포순도:비교GT(GM-CSF+종류배사인자α)방안화GTI(GM-CSF+종류배사인자α+자세포개소4)방안급GTI방안중종류배사인자α화백세포개소4불동시단가입대유도배양산생적수돌상세포성숙적영향;통과격광공취초현미경관찰세포형태,류식세포의분석세포표형급3H-TdR검측수돌상세포격발이체T세포증식능력.결과:면역자주양성분선CD34+조혈간세포순도정체90%이상.장CD34+조혈간세포안GT방안화GTI방안진행배양.균가유도산생수돌상세포,CD34적양성표체솔축점하강,HLA-DR적표체하강(P＜0.05),수돌상세포적상관분화항원CD80,CD86,CD83화CDla적표체균상응증가,배양13～15 d적세포각표형표체교7～9 d,10～12 d충분.단경GT방안유도적수돌상세포CD14표체교고,cD80,CDB6,CD83,CD1a표체불여경GTI방안유도적고;이GTI방안중,이종류배사인자aoh、백세포개소448 h가입유도배양적수돌상세포각표형표체상대교가,기세포표체CD80,CD86균교기타조고,우이CD86표체위저,병구유격발이체T세포증식능력.결론:CD34+조혈간세포경과합괄적배양체계능구유도분화위공능성수돌상세포,이GM-CSF여종류배사인자α O h가입、백세포개소4 48 h가입적GM-CSF+종류배사인자α+백세포개소4방안경위가취.
BACKGROUND: Blood stem cells (BSCs) are the most primitive cells in the immune system and can be differentiated into many kinds of cells. As the regulatory cells of immune response, dendritic cells (DCs) have been attracted more and more attention in the field of autoimmune diseases. Due to different resources of precursor cells, DCs have different cytokines, ideal cytokine matching,applying orders, and experimental cultured conditions. Furthermore, development, phenotypic expression, and mature degree are still different.OBJECTIVE: To investigate the influence of tumor necrosis factor- α (TNF- a ) and interleukin-4 (IL-4) on the culture system and provide improved method for inducing functional DCs in vitro, derived from cord blood CD34+ hematopoietic precursor cells (HPC).DESIGN, TIME AND SETTING: Observational study, which was performed in the Department of Microbiology and Immunology, Nanjing Medical University from March 2005 to November 2005.MATERIALS: The cord blood was collected from neonatal umbilical cord in the 81 Hospital of Nanjing City. CD34 monoclonal antibody coated magnetic bead system (MACS) was provided by Miltenyi Biotec Company, Germany; human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF), human recombinant interleukin-4 (rhlL-4), and human recombinant tumor necrosis factor-α (rhTNF-α) by Pepro Tech Company, USA.METHODS: CD34+HPC were isolated and purified from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Then the cells were cultured with different culture medium which contained different combinations of cytokines:GM-CSF and TNF-α (GT) or GM-CSF, TNF-α and IL-α (GTI) in order to be induced to differentiate into dendritic cells (DCs).The DCs derived from CD34+HPC were identified for their morphology and phcnotype by FACS and laser scanning con_focal fluorescence microscopy, and also for their abilities of inducing proliferation of allogenic T cells by 3H-TdR incorporation assay.RESULTS: The purity of selected CD34+ cells with MACS was more than 90%. DCs could be obtained from CD34+HPC by the culture in presence of GM-CSF and TNF-α or GM-CSF, TNF- o and IL-4. With the time of culture lasting, the cells expressed lower level surface antigen of CD34 and HLA-DR (P ＜ 0.05), and possessed the phenotypes of DCs characterized by higher expression of CD80, CD86, CD83 and CDIa. At 13-15 days, the cells possessed higher level of phenotypes of DCs compared to 7-9 days and 10-12 days. DCs induced with GTI culture system expressed higher levels of surface antigen CD80, CD86, CD83,CD 1 a and a lower level of CD 14 than those induced with GT culture system. DCs displayed typical morphology and property and expressed higher level surface antigen of CD86 and CD80, especially expressed CD86 when they were induced with proper cytokines of GM-CSF and TNF- α added at 0 hour, and of IL-4 added at 48 hours.CONCLUSION: DCs can be generated from CD34+HPC by proper culture system, the design of GM-CSF + TNF- α + IL-4(GM-CSF and TNF- α were added at 0 hour, IL-4 was added at 48 hours) is preferred.