人脐带基质间充质干细胞免疫调节机制及其相关应用的研究进展

间充质干细胞(mesenchymal stem cells,MSC)是成体干细胞的一种,因其具有自我更新、多向分化潜能、高可塑性、调节免疫应答、易于遗传修饰的特性[1],在细胞治疗及组织工程等领域显示出极大的应用价值.目前,MSC的常见来源有骨髓、胚胎、脂肪、脐带等.一般认为骨髓是MSC的经典来源,而人脐带来源的MSC因其易于获得,获取方法具有非侵袭性及无伦理学问题,故其相关研究进展迅速[ 2].人脐带来源MSC简单可分为脐血来源MSC (human umbilical cord blood MSCs,hUCB- MSC)和脐带MSC (human umbilical cord MSCs,hUC-MSC).hUC-MSC主要来自脐带胶质,也称华尔通胶或沃顿胶(Wharton's jelly),因此hUC-MSC亦常称为hWJ-MSC.

流式细胞仪与免疫磁珠分选法纯化脐血嗜碱粒细胞的比较

近年来研究结果显示,嗜碱粒细胞在过敏反应炎症中担当重要角色,是炎症的发起者和传播者~[1].嗜碱粒细胞占外周血白细胞的小部分(<1%),提纯一定数量的嗜碱粒细胞就需要较多的血,这限制了对它的深入研究.产妇脐带血血量较多,可得到50-150 ml的量,而且脐带血尚未直接受到外界抗原刺激,是研究过敏性疾病发病机制的理想标本.本研究比较流式细胞仪分选和免疫磁珠分选两种方法提纯产妇脐带血中嗜碱粒细胞的分选效率.

Objective:In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transduced cell clones (HFCL/EF) had been selected by the addition of G418. The cells were exposed to γ-radiation by 60 Co source for 0.5-20Gy. The expressions of transduced cells were detected with FACS, Northern blot ELISA and CFU assay. The HFCL/EF and CD34+ cells from human umbilical cord blood were one after the other transplanted i.v. into sublethally irradiated severe combined immunodeficient (SCID) mice. The white blood cell amount in peripheral blood and human cell engrafted in recipent mice were detected by flow cytometry and CFU-GM etc. Results:The activity of EGFP in transduced cells increased by 3.1 fold as compared to non-transduced cells at 18h after exposure to 2.5Gy. The amounts of secreted FL in serum-free supernatants of Egr-EF increased by 605.46±107.21pg/ml, which were significantly higher than the control group (214.45±35.61pg/ml). The effects of FL in HFCL/EF cultural supernatants on expansion of CD34+ cells derived from cord blood in the presence of SCF, IL-6 and IL-3 were also studied. The results showed that at day 10 of culture the number of CD34+ cells increased by 173. 09±11.58×103/ml, which was significantly higher than that of non-radiation group(68. 04± 13. 73 × 103/ml). It showed that radiation can enhance the ability of the supernatants containing FL of HFCL/EF to expand early hematopoietic progenitor cells and protect hematopoietic cells from radiation-injury effects. The HFCL/EF and CD34+cells from human umbilical cord blood were one after the other transplanted i. v. into sublethally irradiated severe combined immunodeficient (SCID) mice. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr-1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, while no significant differences were found for CD45+ 、CD34+ cells in bone marrow cells. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, without significant differences being found for CD45+、CD34+ 、CFU-GM and marrow nucleared cells in bone marrow cells. Conclusions:The results suggested both in vivo and in vitro use of the gene therapy of FL gene regulated by Egr-1 promoter could protect hematopoiesis from irradiation-induced damage.

人脐血干细胞的特性及其神经分化的研究进展

寻找一种合适的细胞来源进行移植以代替受损的神经元、星形胶质细胞和少突胶质细胞来修复神经损伤已成为人们的研究热点.早在20年以前,Ogawa等人就发现在人脐带血(human umbilical cord blood,HUCB)中存在原始的造血干细胞,随着近年来研究的深入,发现脐带血中不但含有丰富的造血干细胞,而且还含有大量的多潜能干细胞,可以向内皮、血管以及神经等组织分化,由于其具有来源丰富、采集方便且无伦理学等问题,人脐血干细胞(HUCBC)的研究逐渐引起研究者的广泛兴趣.

对脐带血临床采集问题分析

干细胞是一种未分化的细胞,具有自我更新、分化、发育再生各种组织器官和人体的潜在功能.1989年,法国的鲁克罗曼教授用HLA相合的同胞脐带血干细胞进行移植,成功地治疗了1例遗传性疾病-范可尼贫血症,之后短短的15年的时间内,医学界利用脐带血干细胞已成功治愈和改善了多种疾病,包括血液系统疾病、免疫系统疾病、神经和血管系统疾病、脑部疾病、肿瘤、糖尿病等.

特异性脐血DC疫苗对Beige裸鼠人结肠癌动物模型荷瘤免疫治疗作用的实验研究

背景：肿瘤免疫治疗目前在临床治疗中已广泛开展，但是临床上经过反复输注后，由于机体内肿瘤免疫抑制因素的增多出现了治疗效果下降的情况，严重影响了长期治疗效果，本实验通过构建二次成瘤模型尽可能的模拟人体的肿瘤发生及术后复发转移的肿瘤微环境，通过采用外源性脐血特异性Dc疫苗，起到改善体内免疫细胞功能，减少体内免疫抑制因素，从而达到改善肿瘤患者体内免疫微环境，发挥增强肿瘤免疫治疗效果的临床治疗目的。目的：通过成功构建SCID/Beige裸鼠人结肠癌动物模型，研究脐血来源特异性Dc疫苗对人源化免疫重建荷瘤裸鼠成瘤的免疫抑制作用。方法：分别选取健康志愿者外周血及正常分娩足月产孕妇脐带血，利用密度梯度离心法贴壁培养不同来源Dc细胞制备负载人结肠腺癌SW-1116抗原的特异性抗原提呈细胞，促其成熟后收获；选用健康雄性SCID/Beige裸鼠构建动物模型，将人SW-1116细胞接种于裸鼠腋窝皮下，观察并确定成瘤。空白对照组无任何预先处理，实验组、对照组及阴性对照组确定成瘤后自鼠尾静脉注入健康志愿者外周血PBMC行人免疫细胞微环境重建，在行二次荷瘤前24 h实验组给予脐血Dc疫苗尾静脉注射，实验对照组给予健康志愿者Dc疫苗尾静脉注射，阴性对照组给予健康志愿者无负载抗原的Dc疫苗尾静脉注射；经上述处理后在荷瘤裸鼠对侧腋窝皮下接种人SW-1116细胞，分别观察不同荷瘤组裸鼠二次荷瘤后的一般生活状态、成瘤时间、成瘤大小及瘤重。结果：空白组裸鼠双侧肿瘤生长快，裸鼠短时间内出现肿瘤破溃，部分出现活动差及死亡；经免疫预防处理后荷瘤裸鼠一般生活情况良好，肿瘤生长缓慢，肿瘤体积较小，无破溃、坏死及裸鼠死亡；脐血来源Dc细胞能够负载人结肠癌细胞株SW-1116抗原，并进一步活化成熟发挥其专职抗原提成能力；脐血Dc实验组裸鼠瘤重及瘤终体积平均值明显小于对照组及阴性对照组，比较统计学差异显著。结论：脐血特异性DC疫苗能够改善外周血免疫细胞的免疫功能，促进增强其自身肿瘤免疫杀伤能力，对瘤细胞生长有明显抑制，延缓其肿瘤进展及程度，有积极的肿瘤免疫治疗作用。

人脐血干细胞的特性及其肝细胞分化的研究进展

干细胞一般是指那些来自胚胎、胎儿或成体未分化的,具有无限或长期自我维持和自我更新能力的细胞.在人体内,分化的细胞大多是具有一定的形态特征,并具有特殊的功能,如心脏、肌肉、表皮、红细胞等.但干细胞是非定型的,并一直保持这种非定型.当机体受损时,干细胞会收到特殊信号,细胞在形态和功能上都发生改变并开始进入终末分化.早在20年前,Ogaw[1]等人就发现在人脐带血(human umbilical cord blood HUCB)中存在原始的造血干细胞,由于来源丰富,采集方便,近年来人脐血干细胞(HUCB)的研究已经成为热门话题.

体外培养的脐血间充质干细胞的生物学特性及影响因素

脐血间充质干细胞是-类从脐带血中分离和培养的成体干细胞,具有高度自我更新和多向分化的潜能.脐血间充质干细胞的这些特性,吸引了众多国内外学者的目光,目前,脐血间充质干细胞移植的临床治疗取得了一定进展.本文就脐血间充质干细胞的采集分离、纯化及生物学特性及研究前景作一简要综述.

Wnt/β-catenin调控骨形成分子机制的研究进展

OP的病理机制主要与成骨分化能力减弱、成脂分化能力增强，骨组织微循环血供减少有关[1-2]。BMSCs（Bone mesenchymal stem cel s，BMSCs）是成骨细胞的起源。在老龄OP患者中，BMSCs的含量不仅显著减少，分化能力明显减弱，且增殖缓慢，移植过程病毒感染风险大，免疫原性与成本也较高。人脐血间充质干细胞（Human umbilical cord blood mesenchymal stem cel s，hUCB-MSCs）在体外诱导条件下具有向成骨细胞定向分化的巨大潜能[3]，来源更丰富,临床取材方便，分离纯度更高，具有强大的增殖与自我更新能力，免疫原性较低，能耐受更大程度的HLA配型不符，蕴藏着比BMSCs更加优越的临床应用价值[4]。因此，通过持续激活Wnt/β-catenin信号通路，启动与增强hUCB-MSCs的自身成骨分化能力，为临床OP的干细胞治疗提供新的策略。

Background:Hematopoietic stem cell (HSC) transplantation can be used to treat blood and immune system disorders. Fresh umbilical cord blood (UCB), a major source of HSC for potential clinical applications, contains a limited number of HSCs. Stem cell factor (SCF) activates HSC self-renewal and is being used to stimulate ex vivo expansion of HSCs for treating various hematologic diseases in clinic. Yet, the mechanism by which SCF stimulates and supports HSCs expansion remains poorly understood. Thus, the purpose of the study is to obtain novel monoclonal antibodies for structural and functional SCF characterizations, as well as for the optimization of HSCs ex vivo expansion. Methods:Recombinant human stem cell factor (rhSCF) was used for producing monoclonal antibody (mAb). High-titer mAb speciifc to rhSCF was selected by following immunochemical screening to various mAb cell lines. HSCs with CD34+ epitope were isolated from UCB using affinity chromatography. SCF activity was tested in an ex vivo HSC expansion assay, with use of flow cytometry for detection of CD34+ cell and total mononuclear cells. Part of rhSCF that contained the antibody-binding site was identified via immunoblot analysis of rhSCF tryptic peptides, rhSCF-speciifc mAb, and subsequent NH2-terminal amino acid sequence analysis of the detected peptides. Results: The mAb cell line 23C8 with a high titer was found to be speciifc for rhSCF. In ex vivo cord blood expansion assays, the ability of rhSCF to stimulate the expansion of CD34+ cells was significantly inhibited by 23C8 in a dose-dependet fashion(?). Through peptide mapping, the binding site of 23C8 on rhSCF was mapped to the ifrst 104 amino acids.. Conclusion: The mAb cell line 23C8 produces speciifc and inhibitory anti-rhSCF mAb. The mAb appears to bind directly to a part of rhSCF that is critical for biological activity. This functionally active site of rhSCF is located in the ifrst 104 amino acids from the NH2-terminus. The novel anti-rhSCF mAb will be valuable for further dissection of SCF functional domains and optimization of HSCs ex vivo expansion.