Effects of bone marrow mesenchymal stem cell combined with platelet-rich plasma treatment of bone nonunion after long bone fracture surgery on bone metabolism and cytokines

Objective: To study the effects of bone marrow mesenchymal stem cell combined with platelet-rich plasma treatment of bone nonunion after long bone fracture surgery on bone metabolism and cytokines. Methods: Patients who were treated in our hospital due to bone nonunion after long bone fracture surgery between March 2011 and October 2017 were selected and randomly divided into two groups, combined group received bone marrow mesenchymal stem cell combined with platelet-rich plasma therapy, and control group received bone marrow mesenchymal stem cell therapy. The levels of bone metabolism markers and growth cytokines in serum as well as the expression of bone metabolism-related signal molecules in peripheral blood were determined before treatment and 1 month after treatment. Results: Compared with those of same group before treatment, serum PINP, OPG, BALP, VEGF, TGF-β1, IGF-I, IGF-II and bFGF levels as well as peripheral blood Runx2, Wnt1, Wnt3a and β-catenin expression intensity of both groups of patients significantly increased whereas serum β-CTX and RANKL levels as well as peripheral blood NOX4 and NF-κB expression intensity significantly decreased after treatment, and serum PINP, OPG, BALP, VEGF, TGF-β1, IGF-I, IGF-II and bFGF levels as well as peripheral blood Runx2, Wnt1, Wnt3a and β-catenin expression intensity of combined group after treatment were higher than those of control group whereas serum β-CTX and RANKL levels as well as peripheral blood NOX4 and NF-κB expression intensity were lower than those of control group. Conclusion: Bone marrow mesenchymal stem cell combined with platelet-rich plasma treatment of bone nonunion after long bone fracture surgery can be more effective than bone marrow mesenchymal stem cell monotherapy to improve the bone metabolism and increase the cytokines.

How mesenchymal stem cell cotransplantation with hematopoietic stem cells can improve engraftment in animal models

BACKGROUND Bone marrow transplantation(BMT)can be applied to both hematopoietic and nonhematopoietic diseases;nonetheless,it still comes with a number of challenges and limitations that contribute to treatment failure.Bearing this in mind,a possible way to increase the success rate of BMT would be cotransplantation of mesenchymal stem cells(MSCs)and hematopoietic stem cells(HSCs)to improve the bone marrow niche and secrete molecules that enhance the hematopoietic engraftment.AIM To analyze HSC and MSC characteristics and their interactions through cotransplantation in murine models.METHODS We searched for original articles indexed in PubMed and Scopus during the last decade that used HSC and MSC cotransplantation and in vivo BMT in animal models while evaluating cell engraftment.We excluded in vitro studies or studies that involved graft versus host disease or other hematological diseases and publications in languages other than English.In PubMed,we initially identified 555 articles and after selection,only 12 were chosen.In Scopus,2010 were identified,and six were left after the screening and eligibility process.RESULTS Of the 2565 articles found in the databases,only 18 original studies met the eligibility criteria.HSC distribution by source showed similar ratios,with human umbilical cord blood or animal bone marrow being administered mainly with a dose of 1×10^(7) cells by intravenous or intrabone routes.However,MSCs had a high prevalence of human donors with a variety of sources(umbilical cord blood,bone marrow,tonsil,adipose tissue or fetal lung),using a lower dose,mainly 106 cells and ranging 104 to 1.5×107 cells,utilizing the same routes.MSCs were characterized prior to administration in almost every experiment.The recipient used was mostly immunodeficient mice submitted to low-dose irradiation or chemotherapy.The main technique of engraftment for HSC and MSC cotransplantation evaluation was chimerism,followed by hematopoietic reconstitution and survival analysis.Besides the engraftment,homing and cellularity were also evaluated in some studies.CONCLUSION The preclinical findings validate the potential of MSCs to enable HSC engraftment in vivo in both xenogeneic and allogeneic hematopoietic cell transplantation animal models,in the absence of toxicity.

Similarities and differences between mesenchymal stem/progenitor cells derived from various human tissues

BACKGROUND Mesenchymal stromal/stem cells (MSCs) constitute a promising tool in regenerative medicine and can be isolated from different human tissues. However, their biological properties are still not fully characterized. Whereas MSCs from different tissue exhibit many common characteristics, their biological activity and some markers are different and depend on their tissue of origin. Understanding the factors that underlie MSC biology should constitute important points for consideration for researchers interested in clinical MSC application. AIM To characterize the biological activity of MSCs during longterm culture isolated from: bone marrow (BM-MSCs), adipose tissue (AT-MSCs), skeletal muscles (SMMSCs), and skin (SK-MSCs). METHODS MSCs were isolated from the tissues, cultured for 10 passages, and assessed for: phenotype with immunofluorescence and flow cytometry, multipotency with differentiation capacity for osteo-, chondro-, and adipogenesis, stemness markers with qPCR for mRNA for Sox2 and Oct4, and genetic stability for p53 and c-Myc;27 bioactive factors were screened using the multiplex ELISA array, and spontaneous fusion involving a co-culture of SM-MSCs with BM-MSCs or AT-MSCs stained with PKH26 (red) or PKH67 (green) was performed. RESULTS All MSCs showed the basic MSC phenotype;however, their expression decreased during the follow-up period, as confirmed by fluorescence intensity. The examined MSCs express CD146 marker associated with proangiogenic properties;however their expression decreased in AT-MSCs and SM-MSCs, but was maintained in BM-MSCs. In contrast, in SK-MSCs CD146 expression increased in late passages. All MSCs, except BM-MSCs, expressed PW1, a marker associated with differentiation capacity and apoptosis. BM-MSCs and AT-MSCs expressed stemness markers Sox2 and Oct4 in long-term culture. All MSCs showed a stable p53 and c-Myc expression. BM-MSCs and AT-MSCs maintained their differentiation capacity during the follow-up period. In contrast, SK-MSCs and SM-MSCs had a limited ability to differentiate into adipocytes. BM-MSCs and AT-MSCs revealed similarities in phenotype maintenance, capacity for multilineage differentiation, and secretion of bioactive factors. Because AT-MSCs fused with SM-MSCs as effectively as BM-MSCs, AT-MSCs may constitute an alternative source for BM-MSCs. CONCLUSION Long-term culture affects the biological activity of MSCs obtained from various tissues. The source of MSCs and number of passages are important considerations in regenerative medicine.

Local signals in stem cell-based bone marrow regeneration

The cellular basis of bone marrow （BM） tissue development and regeneration is mediated through hematopoietic stem cells （HSCs） and mesenchymal stem cells （MSCs）. Local interplays between hematopoietic cells and BM stromal cells （BMSCs） determine the reconstitution of hematopoiesis after myelosuppression. Here we review the BM local signals in control of BM regeneration after insults. Hematopoietic growth factors （HGFs） and cytokines produced by BMSCs are primary factors in regulation ofBM hematopoiesis. Morphogens which are critical to early embryo development in multiple species have been added to the family of HSCs regulators, including families of Wnt proteins, Notch ligands, BMPs, and Hedgehogs. Global gene expression analysis of HSCs and BMSCs has begun to reveal signature groups of genes for both cell types. More importantly, analysis of global gene expression coupled with biochemical and biological studies of local signals during BM regeneration have strongly suggested that HGFs and cytokines may not be the primary local regulators for BM recovery, rather chemokines （SDF- 1, FGF-4） and angiogenic growth factors （VEGF-A, Ang- 1） play instructive roles in BM reconstitution after myelosuppression. A new direction of management of BM toxicity is emerging from the identification of BM regenerative regulators.

Comparison of mesenchymal stem cells from human placenta and bone marrow

Background Nowadays bone marrow represents the main source of mesenchymal stem cells (MSCs). We identified a new population of MSCs derived from human placenta and compared its biological characterization with bone marrow derived MSCs.Methods Mononucleated cells (MNC) were isolated from the human placenta tissue perfusate by density gradient fractionation. Individual colonies were selected and cultured in tissue dishes. At the same time,human bone marrow derived MSCs were identified. Culture-expanded cells were characterized by immune phenotyping and cultured under conditions promoting differetiation to osteoblasts or adipocytes. The hematopoietic cytokines were assayed using reverse transcriptase polymerase chain reaction (RT-PCR). Results Human placental MSCs exhibited fibroblastoid morphology. Flow cytometric analyses showed that the placental MSC were CD29,CD44,CD73,CD105,CD166,HLA-ABC positive; but were negative for CD34,CD45,and HLA-DR. Functionally,they could be induced into adipocytes or osteocytes. Moreover,several hematopoietic cytokine mRNA was found in placenta-derived MSCs by RT-PCR analysis,including IL-6,M-CSF,Flt3-ligand and SCF. These results were consistent with the properties of bone marrow derived MSCs.Conclusion These observations implicate the postpartum human placenta as an important and novel source of multipotent stem cells that could potentially be used for investigating mesenchymal differentiation and regulation of hematopoiesis.

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (selfrenewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous,muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancerstemcells(CSCs) viaG-protein coupled receptorsS1Pn(n=1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptoractivated downstream effectors influenced the rate of selfrenewal and should be further explored as regeneration related targets. Considering malignant transformation,it is essential to control the level of self-renewal capacity.Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged ordead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations exploredpharmacologicaltoolsthattargetsphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.

Mechanisms of action of neuropeptide Y on stem cells and its potential applications in orthopaedic disorders

Musculoskeletal disorders are the leading causes of disability and result in reduced quality of life.The neuro-osteogenic network is one of the most promising fields in orthopaedic research.Neuropeptide Y(NPY)system has been reported to be involved in the regulations of bone metabolism and homeostasis,which also provide feedback to the central NPY system via NPY receptors.Currently,potential roles of peripheral NPY in bone metabolism remain unclear.Growing evidence suggests that NPY can regulate biological actions of bone marrow mesenchymal stem cells,hematopoietic stem cells,endothelial cells,and chondrocytes via a local autocrine or paracrine manner by different NPY receptors.The regulative activities of NPY may be achieved through the plasticity of NPY receptors,and interactions among the targeted cells as well.In general,NPY can influence proliferation,apoptosis,differentiation,migration,mobilization,and cytokine secretion of different types of cells,and play crucial roles in the development of bone delayed/non-union,osteoporosis,and osteoarthritis.Further basic research should clarify detailed mechanisms of action of NPY on stem cells,and clinical investigations are also necessary to comprehensively evaluate potential applications of NPY and its receptor-targeted drugs in management of musculoskeletal disorders.

Stem cells of the reproductive tract of women

Research in stem cells is one of the most rapidly evolving fields of investigation in medicine today. Stem cells are defined as cells that have the capacity to both generate daughter cells identical to the cell of origin (self-renewal) and to produce progeny with more restricted, specialized potential (differentiated cells). This dual ability to self-renew and differentiate offers great promise for expanding our understanding of organ systems, elucidating disease pathophysiology, and creating therapeutic approaches to difficult diseases. The goal of this review is to offer an overview of the different types of stem cells and to provide an introduction to the applications of stem cells to the field of obstetrics and gynecology.

Identification of cytokines involved in hepatic differentiation of mBM-MSCs under liver-injury conditions

AIM: To identify the key cytokines involved in hepatic differentiation of mouse bone marrow mesenchymal stem cells (mBM-MSCs) under liver-injury conditions. METHODS: Abdominal injection of CCl4 was adopted to duplicate a mouse acute liver injury model. Global gene expression analysis was performed to evaluate the potential genes involved in hepatic commitment under liver-injury conditions. The cytokines involved in hepatic differentiation of mBM-MSCs was function-ally examined by depletion experiment using specifi c antibodies, followed by rescue experiment and direct inducing assay. The hepatic differentiation was characterized by the expression of hepatic lineage genes and proteins, as well as functional features. RESULTS: Cytokines potentially participating in hepatic fate commitment under liver-injury conditions were initially measured by microarray. Among the up-regulated genes determined, 18 cytokines known to closely relate to liver growth, repair and development, were selected for further identif ication. The f ibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF) and oncostatin M (OSM) were fi nally found to be involved in hepatic differentiation of mBM-MSCs under liver-injury conditions. Hepatic differentiation could be dramatically decreased after removing FGF-4, HGF and OSM from the liver-injury conditioned medium, and could be rescued by supplementing these cytokines. The FGF-4, HGF and OSM play different roles in the hepatic differentiation of mBM-MSCs, in which FGF-4 and HGF are essential for the initiation of hepatic differentiation, while OSM is critical for the maturation of hepatocytes. CONCLUSION: FGF-4, HGF and OSM are the key cytokines involved in the liver-injury conditioned medium for the hepatic differentiation of mBM-MSCs.

Recombinant human bone morphogenetic protein-2 promotes the proliferation of mesenchymal stem cells in vivo and in vitro

Background Bone morphogenetic protein （BMP） is a member of the superfamily of transforming growth factor-13. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect of recombinant human BMP （rhBMP）-2 in hematopoiesis, we set out to determine whether rhBMP-2 could promote the proliferation of mesenchymal stem cells （MSCs） and increase the levels of hematopoietic cytokines in MSCs. Methods 2,3-bis （2-methoxy-4-nitro-5-sulfophenyl）-5-（（phenylamino） carbonyl）-2H-tetrazolium hydroxide （XTT）, real-time polymerase chain reaction （PCR） and enzyme-linked immunosorbent assay （ELISA） were used to detect the effects of rhBMP-2 on the proliferation and hematopoietic cytokine levels of MSCs. In addition, MSCs marked with Hoechst33342 were transplanted into BALB/c mice by the intravenous route or intra-bone marrow transplantation, and cluster numbers were counted. Results The XTT test revealed that rhBMP-2 significantly induced proliferation of MSCs in doses ranging from 10 ng/ml to 0.1 mg/ml in a dose-dependent manner. The experiments in vivo showed that there were more clusters of donor cells in bone marrow, spleen, liver and lung of the BMP group than those in the control group after both intra-bone marrow transplantation （P 〈0.001, P 〈0.001, P 〈0.001, and P=0.001, respectively） and intravenous transplantation （P 〈0.001, P 〈0.001, and P 〈0.001 respectively）. The results of real-time PCR and ELISA revealed that rhBMP-2 significantly increased mRNA expressions and protein levels of IL-6, IL-7, IL-11, G-CSF, M-CSF and SCF. Conclusions The treatment with rhBMP-2 promotes the proliferation of MSCs in vivo and in vitro and increases the levels of hematopoietic cytokines in MSCs, which may contribute to the improvement of hematopoietic function.

Injectable bone marrow microniches by co-culture of HSPCs with MSCs in 3D microscaffolds promote hematopoietic reconstitution from acute lethal radiation

Hematopoietic syndrome of acute radiation syndrome(h-ARS)is an acute illness resulted from the damage of bone marrow(BM)microenvironment after exposure to radiation.Currently,the clinical management of h-ARS is limited to medication-assisted treatment,while there is still no specific therapy for the hematopoietic injury from high-dose radiation exposure.Our study aimed to assemble biomimetic three-dimensional(3D)BM microniches by co-culture of hematopoietic stem and progenitor cells(HSPCs)and mesenchymal stem cells(MSCs)in porous,injectable and viscoelastic microscaffolds in vitro.The biodegradable BM microniches were then transplanted in vivo into the BM cavity for the treatment of h-ARS.We demonstrated that the maintenance of HSPCs was prolonged by co-culture with MSCs in the porous 3D microscaffolds with 84μm in pore diameter and 11.2 kPa in Young’s modulus compared with 2D co-culture system.Besides,the minimal effective dose and therapeutic effects of the BM microniches were investigated on a murine model of h-ARS,which showed that the intramedullary cavity-injected BM microniches could adequately promote hematopoietic reconstitution and mitigate death from acute lethal radiation with a dose as low as 1000 HSPCs.Furthermore,the mRNA expression of Notch1 and its downstream target gene Hes1 of HSPCs were increased when co-cultured with MSCs,while the Jagged1 expression of the co-cultured MSCs was upregulated,indicating the significance of Notch signaling pathway in maintenance of HSPCs.Collectively,our findings provide evidence that biomimetic and injectable 3D BM microniches could maintain HSPCs,promote hematopoiesis regeneration and alleviate post-radiation injury,which provides a promising approach to renovate conventional HSPCs transplantation for clinical treatment of blood and immune disorders.

基于间充质干细胞的小鼠急性移植物抗宿主病骨髓微环境损伤体外细胞模型的建立与评价策略研究

目的:建立小鼠急性移植物抗宿主病(aGVHD)骨髓微环境损伤的体外细胞模型并进行评价。方法:以6-8周龄雌性C57BL/6N小鼠作为骨髓和淋巴细胞的供体,6-8周龄雌性BALB/c小鼠作为aGVHD模型的受鼠。受鼠接受致死剂量(8.0 Gy,72.76 cGy/min)γ射线全身照射后6-8 h内,尾静脉输注供鼠来源骨髓细胞(1×10^(7)/只),建立骨髓移植(BMT)组小鼠模型(n=20);尾静脉输注供鼠来源骨髓细胞(1×10^(7)/只)及脾脏淋巴细胞(2×10^(6)/只),建立小鼠aGVHD模型(n=20)。造模后d 7麻醉小鼠,摘取眼球取血,静置离心后吸取血清。分离小鼠间充质干细胞(MSC),分别用添加了2%、5%和10%的BMT组血清和相同浓度的aGVHD组血清的培养基进行培养。通过成纤维细胞集落形成实验(CFU-F)评价两组血清对MSC集落形成能力的影响;通过CD29和CD105免疫荧光染色评价两组MSC表面分子表达的差异;通过实时荧光定量PCR(RT-qPCR)检测MSC自我更新相关基因Oct-4、Sox-2和Nanog的表达情况。结果:成功建立起可以模拟小鼠aGVHD骨髓微环境损伤的体外细胞模型。CFU-F实验表明,在培养后d 7,与BMT组相比,aGVHD血清浓度为2%和5%时,MSC集落形成能力显著降低(P<0.05);在培养后d 14,与BMT组相比,不同aGVHD血清浓度组MSC集落形成能力均显著降低(P<0.05)。免疫荧光染色实验表明,MSC表面分子CD29^(+)和CD105^(+)细胞百分比在不同aGVHD血清浓度组较BMT组均降低,在aGVHD血清浓度为10%时差异最为显著(P<0.001,P<0.01)。RT-qPCR检测结果显示,aGVHD血清浓度组MSC自我更新相关基因Oct-4、Sox-2和Nanog表达下降,在aGVHD血清浓度为10%时差异最为显著(P<0.01,P<0.001,P<0.001)。结论:不同浓度的小鼠aGVHD血清和小鼠MSC共同培养,发现添加不同浓度的小鼠aGVHD血清MSC的自我更新能力受损程度不同,为开展aGVHD骨髓微环境损伤领域研究提供了新的工具。

异位高表达OCT4的人骨髓间充质干细胞对T淋巴细胞功能的影响

目的:探讨异位高表达OCT4的人骨髓间充质干细胞(MSC)在体外对T细胞增殖、活化及分泌功能的影响。方法:分离健康儿童外周血单个核细胞,使用Anti-CD3/CD28单克隆抗体体外激活T淋巴细胞,白介素(IL)2体外刺激培养的T细胞1周。建立异位高表达OCT4的MSC(MSC-OCT4)与活化T细胞的体外共培养体系,收集共培养1周后的上清液,流式细胞仪测定Th1/Th2细胞因子(IL-2、IL-4、IL-6、IL-10、肿瘤坏死因子-α、γ干扰素)水平。收集共培养1周后的淋巴细胞,通过Countstar软件进行计数,用流式细胞仪测定T细胞及活化T细胞亚群的比例后,计算出绝对值,以均数±标准差表示。结果:与对照单独T细胞培养组相比,MSC及MSC-OCT4均能显著抑制CD3^(+)T细胞、CD3^(+)CD4^(+)T细胞和CD3^(+)CD8^(+)T细胞增殖。与MSC相比,MSC-OCT4能更好地抑制CD3^(+)CD8^(+)T细胞增殖(P=0.049),且主要抑制早期T细胞活化。与对照单独T细胞培养组相比,MSC及MSC-OCT4均能显著下调细胞因子IL-2和γ干扰素水平。与T细胞共培养1周后,MSC与MSC-OCT4组细胞因子IL-6水平显著增加。与对照MSC组相比,MSC-OCT4组在共培养1周后活细胞数更高(P=0.019),且能耐受更高浓度丝裂霉素C的抑制增殖作用。结论:MSC及MSC-OCT4在体外均能抑制IL-2刺激的T细胞增殖及活化,过表达OCT4后MSC能更强地抑制CD3^(+)CD8^(+)T细胞增殖,且具有更好的体外增殖能力,可能具有更好更持久地调节Th1/Th2造血因子平衡的作用。

多发性骨髓瘤患者来源间充质干细胞的最新研究进展

多发性骨髓瘤(MM)是一种浆细胞恶性增殖性疾病,骨髓间充质干细胞(MSC)在MM的疾病进展过程中发挥着重要作用。与正常供者来源MSC(ND-MSC)相比,MM来源MSC(MM-MSC)表现出基因、信号通路、蛋白质表达水平和自身分泌细胞因子的异常,且其外泌体与骨髓微环境相互影响,以上原因可导致MM细胞增殖、化疗耐药、MM-MSC成骨分化障碍,并影响MM患者的免疫调节能力。为进一步了解MM的发病机理及相关影响因素,本文针对MM-MSC的最新研究进展作一综述。

补肾生血药对顺铂损伤的小鼠骨髓间充质干细胞的影响

目的:观察补肾生血药对顺铂损伤的骨髓间充质干细胞(BMSC)的影响,探讨补肾生血改善化疗后骨髓抑制的机制。方法:将50只SD雄性大鼠随机分为空白对照组、补肾生血药组,每组25只,分别制备补肾生血血清和空白血清,原代培养BALB/c小鼠BMSC,随机分组,CCK-8法确定顺铂和补肾生血血清干预浓度。干预48 h后,采用流式细胞仪检测细胞凋亡率和细胞周期,酶联免疫吸附试验法检测培养液中造血调控因子含量。结果:干预48 h后,骨髓细胞凋亡率明显增高(P<0.01),补肾生血血清组G_(1)期细胞比例显著降低(P<0.05),S期细胞比例显著增加(P<0.05),增殖期细胞比例明显增加(P<0.05)。补肾生血血清组骨髓细胞凋亡率明显降低(P<0.001),G_(1)期细胞比例显著降低(P<0.05),增殖期细胞比例显著增加(P<0.05),IL-3含量明显升高(P<0.01),TNF-α、TGF-β、IFN-γ含量显著降低(均P<0.05)。结论:补肾生血药能减轻顺铂造成的BMSC凋亡,促进细胞进入增殖期,促进造血生长因子分泌,减少造血抑制因子分泌,提示该药对化疗后骨髓抑制具有一定的改善作用。

人骨髓间充质干细胞表达多种造血细胞因子

为了研究培养的人骨髓间充质干细胞在造血中的生物作用 ,采用RT PCR方法在mRNA水平上分析体外培养的人骨髓间充质干细胞 (BM MSC)造血因子的表达 ,以及氢化考的松对BM MSC表达造血细胞因子的影响。结果显示 ,体外传代培养的正常人、白血病和淋巴瘤等血液病人BM MSC均能表达SCF ,Flt3 ligandTPO ,LIF ,IL 6和IL 11等重要的造血细胞因子 ,但不表达G CSF ,GM CSF和IL 3。不同代数的BM MSC细胞 ,造血细胞因子的表达相同。BM MSC经氢化考的松作用 7- 2 1天后 ,可诱导G CSFmRNA表达 ,但不诱导GM CSF的表达 ,细胞在形态学上也未发生明显改变。结论 :体外培养的正常人和本组血液病病人BM

人骨髓间充质干细胞支持体外造血(英文)

体内的稳态造血依赖于复杂而完整的骨髓造血微环境系统 ,其中的细胞成分是该系统的关键。存在于骨髓中的间充质干细胞 (MSCs)是成纤维细胞、内皮细胞、成骨细胞、脂肪细胞等多种骨髓基质细胞的前体细胞 ,在造血调控中可能具有一定的作用。本研究首先建立了成人骨髓MSCs的分离及体外培养的方法 ,并应用长期骨髓细胞培养体系 ,观察了MSCs滋养层体外维持长期培养启动细胞 (LTC IC)的能力及其促进造血细胞分化的功能。结果显示 :①脐带血来源的CD34+细胞粘附于滋养层上形成造血灶 ,表明MSCs可形成与骨髓基质细胞相似的体外造血微环境 ;②共培养 5周后造血细胞仍具有体外集落形成能力 ,说明MSCs具有维系LTC IC的能力 ;③流式细胞术分析显示 ,体外培养 5周后约 1%悬浮细胞表达CD34,15 %细胞CD4 1a阳性 ,提示MSCs促进造血细胞向巨核系细胞分化。

骨髓间质干细胞降低大鼠移植物抗宿主反应的实验研究

目的探讨MSCs对GVHD的作用及其机制。方法建立大鼠同种异体骨髓移植模型,同时输入供者的T淋巴细胞诱导出移植物抗宿主反应,联合或不联合移植供体来源的MSCs,观察受鼠的生存时间,同时利用RT-PCR法研究Th1/Th2淋巴细胞亚群的比例,用ELISA法检测移植后体内IL-4细胞因子的浓度。结果GVHD组的平均生存时间为(17.30±2.33)天,实验组的平均生存时间为(24.10±2.36)天,与单独移植HSCs相比,MSCs与HSCs共移植明显延长的受鼠的生存时间。同时,GVHD组Th1/Th2细胞比值为1.29±0.06,IL-4因子的浓度平均为(14.84±2.59)pg/mL,实验组Th1/Th2细胞比值为(0.77±0.14),IL-4因子的浓度平均为(40.09±13.99)pg/mL。MSCs与HSCs共移植降低了体内Th1/Th2淋巴细胞亚群的比例,提高了体内IL-4细胞因子的浓度。结论MSCs与HSCs共移植能有效抑制HSCs移植后致死性GVHD的发生,延长生存时间,同时MSCs可能通过作用于体内Th1/Th2淋巴细胞亚群的比例,促进体内IL-4细胞因子的分泌从而间接发挥了抑制GVHD的作用。

骨髓间充质干细胞在CD4^+CD25^+调节性T细胞减轻大鼠移植物抗宿主反应中的作用

本研究探讨间充质干细胞(MSC)对GVHD的作用及其机制。建立大鼠同种异体骨髓移植模型,同时输入供者的T淋巴细胞诱导出移植物抗宿主反应,联合或不联合移植供体来源的MSC,观察受鼠的GVHD的发生情况;利用双荧光标记抗体标记受鼠脾脏和胸腺单个淋巴细胞,通过流式细胞术分析CD4+CD25+调节性T细胞亚群比例的变化,分析MSC的作用机制。结果显示实验组的GVHD的发生程度减轻,存活率提高,而CD4/CD8比值在GVHD组出现不同程度的减少,CD4+CD25+调节性T细胞在实验组中的脾淋巴细胞和胸腺淋巴细胞的比例分别为31.55±7.58%、93.20±2.69%,在GVHD组中的比例分别为20.90±1.90%、57.17±6.79%,实验组中CD4+CD25+调节性T细胞比例比GVHD组中增多,具有显著性差异。结论MSC能有效抑制HSC移植后致死性GVHD的发生,提高生存率,同时MSC可能通过作用于体内调节性T淋巴细胞而间接发挥了抑制GVHD的作用。

骨髓间充质干细胞体外对T淋巴细胞分泌IL-2、IL-4功能的影响

目的:探讨间充质干细胞(Mesenchymal stem cells,MSCs)对T淋巴细胞分泌功能的调节作用。方法:体外分离培养、扩增人骨髓MSCs,并通过形态学特征及流式细胞术检测其表面标志加以鉴定。将不同数量的MSCs(5×103、1×104、5×104个细胞/孔)分别与PHA激活的T细胞和混合淋巴细胞反应(MLR)体系共培养,并将不同浓度(25%、50%、75%)的MSCs培养上清和MLR体系共培养。应用ELISA分别检测各培养上清液中T细胞分泌IL-2、IL-4的水平。结果:骨髓MSCs能抑制PHA作用下的T细胞分泌IL-2、IL-4(P<0.05),并呈MSCs数量相关性(P<0.05),且对IL-2的抑制作用更显著;也可抑制MLC体系中T细胞分泌IL-2、IL-4(P<0.05),但各数量组的MSCs的抑制作用无显著性差异(P>0.05)。不同浓度的MSCs培养上清均可抑制MLC体系中T细胞分泌IL-2、IL-4(P<0.05),但不同浓度的MSCs培养上清组对IL-4抑制作用无显著性差异(P>0.05)。结论:骨髓MSCs及其培养上清均可抑制PHA或异体抗原作用下的T细胞分泌IL-2、IL-4,提示MSCs可能是直接作用于T细胞或通过分泌可溶性因子调节Th1/Th2反应平衡而发挥免疫调节作用的。