小鼠骨髓间充质干细胞和内皮祖细胞的分离?培养及鉴定

目的:建立一种高效,稳定的从小鼠骨髓中同时分离培养间充质干细胞(MSC)和内皮祖细胞(EPC)的方法.方法:从小鼠骨髓分离单个核细胞,经差速贴壁结合专用血清或特殊培养基分别扩增MSC和EPC.以成骨、成脂诱导分化鉴定MSC,并以流式细胞术(FCM)检测MSC纯度;以Dil-ac-LDL、FITC-UEA-1荧光双标,结合vWF、CD31免疫组化染色鉴定EPC,并计算其纯度.结果:早期贴壁细胞48 h换液时即可见明显集落形成,1周后即达80%融合,传至第3代后经诱导能够向成骨细胞和脂肪细胞分化,FCM检测CD29、CD34、CD45、CD90阳性率分别为(93.86±1.12)%,(0.48±0.38)%,(1.89±1.49)%,(94.11±3.32)%;2次贴壁细胞经EGM-2 MV专用培养基培养后,第3天开始伸展,第5天可见集落形成,约2周左右可融合近80%,传代后Dil-ac-LDL、FITC-UEA-1双荧光染色阳性率(75.2±4.5)%,vWF、CD31免疫组化染色阳性率分别为(55.7±4.7)%和(52.5±3.6)%.结论:采用该方法可以同时培养扩增骨髓间充质干细胞和内皮祖细胞,效率高,稳定性和重复性好.     

Endothelial progenitor cell down-regulation in a mouse model of Kawasaki disease

Background  Cardiovascular complications of Kawasaki disease (KD) are a common cause of heart disease in pediatric populations. Previous studies have suggested a role for endothelial progenitor cells (EPCs) in coronary artery lesions associated with KD. However, long-term observations of EPCs during the natural progression of this disorder are lacking. Using an experimental model of KD, we aimed to determine whether the coronary artery lesions are associated with down-regulation of EPCs.

Methods  To induce KD, C57BL/6 mice were administered an intraperitoneal injection of Lactobacillus casei cell wall extract (LCWE; phosphate buffered saline used as control vehicle). Study groups included: group A (14 days following LCWE injection), group B (56 days following LCWE injection) and group C (controls). Numbers of circulating EPCs (positively staining for both CD34 and Flk-1 while staining negative for CD45) were evaluated using flow cytometry. Bone marrow mononuclear cells were cultured in vitro to expand EPCs for functional analysis. In vitro EPC proliferation, adhesion and migration were assessed.

Results  The model was shown to exhibit similar coronary artery lesions to KD patients with coronary aneurysms. Numbers of circulating EPCs decreased significantly in the KD models (groups A and B) compared to controls ((0.017±0.008)% vs. (0.028±0.007)%, P <0.05 and (0.016±0.007)% vs. (0.028±0.007)%, P <0.05). Proliferative, adhesive and migratory properties of EPCs were markedly impaired in groups A and B.

Conclusion  Coronary artery lesions in KD occur as a consequence of impaired vascular injury repair, resulting from excess consumption of EPCs together with a functional impairment of bone marrow EPCs and their precursors.

     

兔全骨髓培养诱导分化获取内皮祖细胞

目的探讨采用将兔全骨髓直接体外培养诱导分化的方法获取内皮祖细胞(EPCs),同时观察EPCs的扩增能力。方法新西兰兔10只,对每只兔穿刺并抽取骨髓3 ml,用EGM-2培养基对全骨髓进行培养,观察细胞生长及形态变化,绘制细胞生长曲线并评估其扩增能力。对培养12 d后的贴壁细胞行CD133、CD34、VEGFR-2三抗原免疫组化鉴定及吞噬乙酰化低密度脂蛋白(ac-LDL)和结合荆豆凝集素(UEA-1 lectin)的内皮细胞功能鉴定;同时对其行CD133免疫磁珠分选及流式细胞术检测,比较分选前后的CD34+/VEGFR-2+和CD133+/CD34+/VEGFR-2+细胞比例的差异。结果全骨髓直接培养48 h后可见细胞呈丛状或集落样生长,细胞呈梭形、三角形、多边形,细胞生长曲线呈"S"型。经过12 d的培养,每3 ml骨髓可以获得(1.51±0.29)×106个贴壁生长的EPCs,细胞呈铺路石样外观;检测发现CD133、CD34、VEGFR-2三抗原阳性表达,并具有吞噬ac-LDL和结合荆豆凝集素(UEA-1 lectin)的内皮细胞功能。经CD133免疫磁珠分选后CD34+/VEGFR-2+和CD133+/CD34+/VEGFR-2+的细胞比例数分别为分选前的3.38倍和6.14倍。结论通过全骨髓直接培养诱导分化获取兔EPCs的方法简单、可行。     

小鼠骨髓内皮祖细胞的培养与鉴定

目的建立分离小鼠骨髓内皮祖细胞(endothelial progenitor cell,EPC)的方法。方法通过密度梯度分离法从小鼠骨髓中分离单个核细胞,行流式细胞检测,培养7d后,行DIL-acLDL和FITC-UEA-1双荧光染色、细胞移行实验和血管内皮网络掺入实验。结果流式细胞技术检测结果显示,骨髓来源的CD34/CD133/VEGFR2三阳细胞为0.029%±0.008%;细胞培养7d后,可见梭形细胞呈优势生长;DIL-acLDL与FITC-UEA-1荧光标记的双阳性细胞占细胞总数的比例为86.085%±5.622%;细胞移行实验结果可见平均每视野移行细胞数为(19.458±2.251)个;血管内皮网络掺入实验结果为,每视野掺入细胞数平均为(67.750±8.823)个。结论用流式细胞技术检测分离小鼠骨髓单个核细胞不同表面标志物的表达,用选择性内皮生长体系培养EPC,再通过细胞免疫及细胞功能检测所分离的细胞,是一种较为理想的分离与鉴定EPC的方法。     

Endothelial progenitor cell transplantation ameliorates elastin breakdown in a Kawasaki disease mouse model

Background  Coronary artery damage from Kawasaki disease (KD) is closely linked to the dysfunction of endothelial progenitor cells (EPCs). The aim of the present study was to evaluate the therapeutic effect of EPCs transplantation in KD model.

Methods  Lactobacillus casei cell wall extract (LCWE)-induced KD model in C57BL/6 mice was established. The model mice were injected intravenously with bone marrow-derived in vitro expanded EPCs. Histological evaluation, number of circulating EPCs and the function of bone marrow EPCs were examined at day 56.

Results  Inflammation was found around the coronary artery of the model mice after 14 days, Elastin breakdown was observed after 56 days. CM-Dil labeled EPCs incorporated into vessel repairing foci was found. At day 56, the number of peripheral EPCs in the KD model group was lower than in EPCs transplanted and control group. The functional index of bone marrow EPCs from the KD model group decreased in proliferation, adhesion and migration. Increased number of circulating EPCs and improved function were observed on the EPCs transplanted group compared with model group.

Conclusion  Exogenously administered EPCs, which represent a novel strategy could prevent the dysfunction of EPCs, accelerate the repair of coronary artery endothelium lesion and decrease the occurrence of aneurysm.

     

Angiogenesis effect of Astragalus polysaccharide combined with endothelial progenitor cells therapy in diabetic male rat following experimental hind limb ischemia

Background Diabetes mellitus （DM） is a common disease accompanied with a high incidence of hind limb ischemia （HLI）.In recent years,numerous studies demonstrated that endothelial progenitor cells （EPCs） are involved in angiogenesis and maintenance of vascular integrity following HLI.On the other side,it has been proved that Astragalus polysaccharide （APS） could promote angiogenesis.In the present study,we aimed to evaluate the effect of APS and EPCs on enhancing angiogenesis after experimental HLI caused by femoral artery ligation in rats with streptozotocin （STZ）-induced diabetes.Methods Rats （n=110） were randomly assigned to the following groups：sham group,ischemia group,APS group,EPCs group and APS＋EPCs group.APS,EPCs or an equal volume of vehicle was administered intramuscularly after HLI induction,and 6 rats were assessed by angiography at 28 days after induction of HLI,6 rats were sacrificed at the same time point to take histological studies,biochemical tests were also performed at that point in the rest rats.Results APS or EPCs treatment induced an increase,respectively,in the protein expression of vascular endothelial growth factor （VEGF） （36.61%,61.59%）,VEGF receptor-1 （VEGFR-1） （35.50%,57.33%）,VEGFR-2 （31.75%,41.89%）,Angiopoietin-1 （Ang-1） （37.57%,64.66%） and Tie-2 （42.55%,76.94%） （P 〈0.05）,after HLI injury.And combined therapy of APS and EPCs enhanced the effort of angiogenesis after HLI induction in diabetic rats,through elevating protein expression of VEGF （99.67%）,VEGFR-1 （105.33%）,VEGFR2 （72.05%）,Ang-1 （114.30%） and Tie-2 （111.87%） （P〈0.05）.Similarly,mRNA expression of VEGF,VEGFR-1,VEGFR2,Ang-1,Tie-2 also show similar trends as well as protein expression （P〈0.05）.Conclusion APS or EPCs could enhance angiogenesis,and the combined treatment leads to better effort,at least,partially via VEGFNEGFR and Ang-1/Tie-2 signaling pathway.     

大鼠骨髓源性内皮祖细胞的分离培养及鉴定

目的: 探讨内皮祖细胞(endothelial progenitor cells,EPCs)分离培养鉴定方法及其生物学特性。方法： 采用梯密度离心法结合差速贴壁法分离获取大鼠骨髓单核细胞,培养于EGM 2 MV SingleQuots内皮细胞培养液中,分别取48 h内贴壁细胞(早期EPCs)和48 h后贴壁细胞(晚期EPCs),在倒置显微镜下观察细胞形态,应用流式细胞术鉴定EPCs表面抗原标志CD34,CD133和血管内皮生长因子受体 2（VEGFR 2）,激光共聚焦检测EPCs吞噬功能,透射电镜观察EPCs内部超微结构。结果： 单核细胞接种后呈小圆形,早期EPCs呈长梭形,晚期以纺锤形为主,培养7 d后有明显干细胞集落;增殖至第6代以后,形态开始逐渐接近于内皮细胞,并出现管腔样结构。内皮祖细胞表面标志CD34,CD133和VEGFR 2均呈阳性表达,能吞噬Dil标记的乙酰化低密度脂蛋白并结合FITC标记的凝集素 1。透射电镜可观察到内皮细胞特征性细胞器Weible Palade小体。结论： 梯密度离心法结合差速贴壁法能够成功地从骨髓中分离、纯化、培养出内皮祖细胞,其增殖能力强,数量充足,生物学特性稳定。     

小鼠骨髓来源内皮祖细胞的分离、培养及鉴定

目的：探讨自小鼠骨髓分离培养内皮祖细胞（endothelial progenitor cells,EPC）的方法及其鉴定和生物学特性。方法：密度梯度离心法分离获取小鼠骨髓单核细胞,培养于EGM-2 SingleQuots培养液中,每天于倒置显微镜下观察EPC的形态。应用细胞免疫荧光法、流式细胞技术鉴定EPC表面抗原标志CD34、血管内皮生长因子受体-2（VEGFR-2）,荧光显微镜检测EPC吞噬DiI标记的乙酰化低密度脂蛋白（DiI-AC-LDL）及结合FITC标记的荆豆凝集素（nTC-UEA-lectin）功能。结果：早期EPC呈长梭形,细胞群落可呈现线状、管状、网状生长状态,培养7d后出现干细胞集落,并逐渐增多;培养3周后,细胞集落逐渐消失,细胞呈现铺路石样生长状态。培养获得的EPC绝大部分可同时表达细胞表面标记物CD34及VEGFR2,并具有吞噬DiI-AC-LDL及结合FITC-UEA-lectin功能。结论：自小鼠骨髓中分离、培养、纯化EPC是一种方便、经济、高效的培养方法。通过该方法培养获得的EPC增殖能力强,数量充足,生物学特性稳定。该方法对今后利用EPC进行细胞移植治疗的相关实验有着重要意义。     

Monitoring the source of mesenchymal stem cells in patients after transplantation of mismatched-sex hematopoietic stem cells plus third-party cells

Background In bone marrow transplant patients, the microenvironment in bone marrow is damaged after chemotherapy or radiotherapy. Subsequent to allogenic hematopoietic stem cell transplantation in patients with clinically successful engraftments, the source of mesenchymal stem cells （MSCs） remains controversial. To further verify the stimulatory effect of the simultaneous transplantation of cells from second donors on engraftment success for hematopoietic stem cell transplantation in support of donor MSCs engraftments, the aim of this study is to monitor the dynamics of the engraftment of bone marrow-derived MSCs in patients after transplantation with mismatched-sex hematopoietic stem and third-party cells. Methods In this study, the hematopoietic stem cells from 32 clinical donors of different sexes that resulted in successful engraftments were selected for transplantation and were classified into three groups for research purposes： group A consisted of 14 cases of transplantation with bone marrow and recruited peripheral hematopoietic stem cell transplantation, group B contained 8 cases of simultaneous re-transfusion of MSCs from the second donor, and group C contained 10 cases of simultaneous re-transfusion of umbilical blood from the second donor. The bone marrow from 32 patients with successful engraftments of hematopoietic transplantation were selected and sub-cultured with MSCs. Flow cytometry （FCM） was used to measure the expression of surface antigens on MSCs. Denaturing high-performance liquid chromatography （DHPLC） in combination with polymerase chain reaction amplification of short tandem repeats （STR- PCR） was used to measure the engraftment status of fifth-generation MSCs in patients. Fluorescence in situ hybridization （FISH） revealed the sex origin of the fifth-generation MSCs in 32 patients. Dynamic examinations were performed on patients receiving donor transplantations. Results The progenies of fifth-generation MSCs were successfully cultured in 32 cases. The results of FCM demonstrated that the expression levels of CD14＋ and CD45＋ cells were lower than 0.04% in the fifth-generation MSCs. The analysis using DHPLC and FISH showed similar results. One patient from group B also received a temporary transplantation of MSCs from the donor. The MSCs in the remaining 31 patients all originated from the patients themselves. Conclusions After transplantation, the MSCs present in patients originated from the host. In patients transplanted with MSCs from a second donor, the phenomenon of temporary chimerization of MSCs was observed.     

Early changes of CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation

Background  Hemorrhagic shock induces immune dysfunction. Regulatory T cells (Tregs), T-helper (Th) cells, and cytotoxic T-lymphocytes (CTLs) can execute many crucial actions in immune and inflammatory responses. This study was conducted to investigate the early pathophysiological changes of CD4⁺CD25⁺Foxp3⁺ Treg and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation.

Methods  A rat model of controlled hemorrhagic shock with no fluid resuscitation was established. Peripheral blood samples were taken before and four hours after hemorrhagic shock with no fluid resuscitation. Three color flow cytometry was used to detect Tregs, Th1, Th2, Tc1 and Tc2 cells in the samples.

Results  In the peripheral blood of rats, the percentage of Tregs four hours after hemorrhagic shock was significantly lower than before hemorrhagic shock (P=0.001). The ratios of Th1/Th2 and Tc1/Tc2 were changed from (23.08±8.98)% to (23.91±15.36)%, and from (40.40±21.56)% to (65.48±23.88)%, respectively.

Conclusions  At an early stage, the advent of hemorrhagic shock is related to an early decrease of Tregs, and a mild shift in the Th1/Th2, Tc1/Tc2 balance toward Th1 and Tc1 dominance. These changes are part of a hyper-inflammatory state of the host, and will deteriorate the maintenance of immune balance. Further influences and detailed mechanisms need to be investigated.     

冻存骨髓来源单个核细胞分化成内皮祖细胞的功能特性

目的:对新鲜和超低温保存的骨髓来源单个核细胞(MNC)体外扩增的内皮祖细胞(EPC)进行功能比较。方法:从猪髂骨抽取骨髓,对分离后的MNC进行培养或-80℃冻存3个月后再培养;冻存后培养的P1代细胞利用免疫组化及流式细胞技术进行EPC表面标志抗原鉴定。同时分别对新鲜和冻存培养的EPC获得率、细胞迁徙、黏附和增殖功能进行比较。结果:冻存组细胞免疫组化鉴定:CD133(+)、CD34(+)、CD31()、KDR(),流式细胞技术鉴定:CD133的阳性率(17.24±3.12)%,CD34的阳性率(37.21±10.85)%,CD31的阳性率(72.07±13.34)%,KDR的阳性率(89.09±16.40)%。新鲜和冻存的MNC经诱导培养后EPC获得率分别为(1.1±0.078)%、(1.03±0.061)%,P=0.054;细胞迁徙率分别为(15±0.71)%、(14.2±0.63)%,P=0.17;贴壁率分别为(42.7±2.1)%、(39.5±1.7)%,P=0.11;增殖功能分别为(25.06±2.82)×104、(21.64±2.34)×104,P=0.089。结论:超低温保存骨髓来源的MNC经诱导培...     

大鼠骨髓来源内皮祖细胞的分离培养及鉴定

目的建立体外分离培养及鉴定大鼠骨髓来源内皮祖细胞（endothelialprogenitorcells,EPCs）的方法。方法采用密度梯度离心法从大鼠骨髓中分离得到骨髓单个核细胞,并结合差时贴壁法,收集24h后未贴壁细胞,然后用添加有多种细胞因子的内皮细胞基础培养基（endothelial basal medium,EBM-2）诱导培养获取EPCs。通过形态学观察、CD133和VEGFR-2抗体免疫荧光染色并结合FITC-UEA-1和Dil—ae—LDL的摄取功能来对EPCs进行鉴定分析,同时通过体外管样结构形成能力对EPCs进行功能性评价。结果在EBM-2培养基中诱导培养7d后,出现类似于血岛样的细胞集落,集落中央的细胞呈圆形,周边的细胞呈放射状;80％以上的细胞都是CD133＋／VEGFR＋2＋细胞,并且这些细胞同时能够摄取Dil-ac—LDL和结合FITC-UEA-1;EPCs在基质胶表面形成管腔样结构。结论密度梯度离心法结合差时贴壁法从骨髓中分离获得的单个核细胞,然后经EBM-2条件培养基诱导培养可获得较高纯度的EPCs。     

猪骨髓来源内皮祖细胞体外培养条件的优化

目的 优化选择猪骨髓来源内皮祖细胞(endothelial progenitor cell, EPC)体外培养条件,为后续研究奠定基础。方法 从猪髂骨抽取骨髓,利用密度梯度离心法分离得到单个核细胞(MNC),体外培养分化为EPC。在其他培养条件相同的前提下,分别比较不同的细胞接种密度(2×103/cm2、5×103/cm2、1×104/cm2、2×104/cm2),不同基础培养液(EGM、M199、DMEM),不同FBS浓度(5%、10%、20%、30%),以及血管内皮细胞生长因子(VEGF)与不同细胞因子\[碱性成纤维细胞生长因子(bFGF)、基质细胞衍生因子（SDF）、胰岛素样生长因子（IGF）和表皮生长因子（EGF）\]组合(VEGF+bFGF、VEGF+SDF、VEGF+bFGF+SDF、VEGF+bFGF+IGF+EGF、VEGF+bFGF+SDF+IGF)对EPC细胞增殖及迁徙功能的影响;采用细胞形态观察、双荧光染色法及免疫细胞化学染色方法对培养的EPC进行鉴定。结果 猪骨髓EPC以1×104/cm2密度接种在盛有M199,并添加10% FBS和VEGF+bFGF+SDF+IGF细胞因子的培养液时,细胞的增殖能力和迁徙率最高。每组细胞经结合Dil标记的乙酰化低密度脂蛋白(Dil-ac-LDL)和FITC标记的荆豆凝集素(FITC-UEA-1)双色荧光染色鉴定双阳性率>76%,免疫细胞化学检测CD133、CD34、KDR均为阳性。结论 通过优化猪骨髓来源EPC的体外培养条件,可使细胞数量增多及功能增强,为后续研究奠定了基础。     

Correlation of Thl7 cells and CD4＋CD25＋ regulatory T cells with clinical parameters in patients with systemic sclerosis

Background Systemic sclerosis （SSc） is an autoimmune disease that has three major components： inflammation, fibrosis, and vasculopathy. T-helper 17 cell （Th17） and regulatory T cell （Treg） are considered to be critical for autoimmune disease pathogenesis. The role of Th17 and Treg in SSc is still unclear. The aim of this study was to detect the presence of Th17s and CD4＊CD25~ Tregs in peripheral blood samples from SSc patients and to investigate the possible roles of these two T cell subsets in SSc pathogenesis. Methods Th17s （CD4 and IL-17 positive） and CD4＊CD25~ Tregs （CD4, CD25 and Foxp3 positive） in the peripheral blood mononuclear cells of 53 SSc patients and 27 healthy controls were counted by flow cytometry. The differences between SSc and control patients were analyzed. Clinical parameters, including disease duration, duration of the second symptoms, Modified Rodnan Skin Score （MRSS）, anti-topoisomerase I antibody, anti-U1 ribonucleoprotein （RNP） antibody, systemic involvements, pulmonary function test （PFT） and high resolution computed tomography （HRCT） score were prospectively collected following EUSTAR （EULAR scleroderma trial and research group） protocols. The correlations between the experimental and clinical data were investigated. Results The ratio of Th17 in SSc patients was significantly elevated compared to healthy controls （8.74% vs. 4.41%, P 〈0.001）. The amount of Th17 was positively correlated with disease duration （R=-0.531, P=-0.013） and duration of the second symptoms （R=-0.505, P=0.023）. The ratio of CD4＊CD25＊ Treg in SSc patients also significantly differed from the healthy controls （3.04% vs. 2.24%, P=0.018）. Elevated Tregs were more frequently observed in patients with a high interstitial lung disease （ILD） score on computed tomography （24/36） compared with patients with normal ILD scores （4/12, ,P=-0.043）. Elevated Tregs were also more often observed in patients with low carbon monoxide diffusing capacity     

人脐血内皮祖细胞体外诱导分化的研究

目的研究从人脐血中诱导分化出内皮祖细胞（endothelial progenitor cells,EPCs）,并对此类细胞进行鉴定。方法术中取健康产妇的新鲜脐静脉血,采用淋巴细胞分离液梯度离心法分离出单个核细胞（mononuclear cells,MNCs）,应用添加诱导因子的培养基于体外诱导分化,观察细胞生长状态。培养7d后将贴壁细胞与含Dil-ac-LDL和FITC-UEA-1的培养基进行孵育并于荧光显微镜下观察,流式细胞仪检测培养细胞表面分子CD34及CD133的阳性率,RT-PCR检测培养细胞表达VEGFR-2mRNA。结果体外诱导7d后90%以上贴壁细胞摄取Dil-ac-LDL呈红色荧光和FITC-UEA-1呈绿色荧光双阳性。流式细胞仪检测CD34及CD133阳性细胞分别为（50.48±5.17）%和（19.12±4.37）%。RT-PCR检验培养细胞VEGFR-2mRNA呈阳性。结论采用密度梯度离心法从人脐血中提取MNCs在体外经诱导分化可以形成EPCs。     

Ectopic osteogenesis and scaffold biodegradation of tissue engineering bone composed of chitosan and osteo-induced bone marrow mesenchymal stem cells in vivo

Background Chitosan （CS） scaffolds combined with osteogenically induced bone marrow mesenchymal stem cells （BMSCs） have been proved to be promising substitutes for repairing bone defects.Nevertheless,the bone-forming and scaffold-biodegrading processes are seldom studied.This study aimed to determine the osteogenic ability of CS/osteoinduced BMSC composites by observing the bone-forming process and explore the relationship between bone formation and scaffold biodegradation.Methods The CS/osteo-induced BMSC composites （CS＋cells group） and the CS scaffolds （CS group） were,respectively,implanted into SD rat thigh muscles.At 2,4,6,8,and 12 weeks postoperatively,the rat femurs were scanned by CT,and the CT values of the implants were measured and comparatively analyzed.Subsequently,the implants were harvested and stained with hematoxylin and eosin and Masson trichrome,and the percentages of bone area,scaffold area,and collagen area were calculated and compared between the two groups.Results The imaging results showed that the densities of implants of the two groups gradually increased along with time,but the CT values of implants in the CS＋cells group were much higher than in the CS group at the same time point （P ＜0.05）.The histological results showed that the de novo bone and collagen formed in the pores of the scaffolds and gradually increased since 2 weeks postoperation in both groups,and the scaffold gradually degraded along with the boneforming process.However,the comparative analysis results showed that the CS＋cells group gained more de novo bone and collagen formation and had less scaffold than the CS group at the same time point （P ＜0.05）.Conclusion The CS/osteo-induced BMSC composites are excellent bone tissue engineering substitutes,and the scaffold biodegradation is accordant with the bone formation.     

Immature CD4+ dendritic cells conditioned with donor kidney antigen prolong renal allograft survival in rats

Background  Allogeneic transplant rejection is currently a major problem encountered during organ transplantation. The dendritic cell (DC) is the most effective powerful known professional antigen-presenting cell, and recent studies have found that DCs can also induce immune tolerance, and avoid or reduce the degree of transplant rejection. The aim of this study was to evaluate the effect of transfused immature CD4⁺ DCs on renal allografts in the rat model.

Methods  In this study, we induced CD4⁺ immature DCs from rat bone marrow cells by a cytokine cocktail. The immature CD4⁺ DCs were identified by morphological analysis and then the suppressive activity of these cells conditioned with donor kidney antigen was evaluated in vitro and in vivo.

Results  Immature CD4⁺ DCs conditioned with donor kidney antigen possessed immunosuppressive activity in vitro and they were able to prolong renal transplant survival in an allograft rat model in vivo.

Conclusions  Our study provides new information on efficacious renal transplantation, which might be useful for understanding the function of immature CD4⁺ DCs in modulating renal transplant rejection and improving clinical outcome in future studies.

     

Transduction of mesenchymal stem cells with multidrug resistance gene provides protection for bone marrow toxicity after being transplanted into a nude mice model

Background  Myelosuppression is the main dose-related toxicity of many chemotherapeutic drugs. The human multidrug resistance (mdr1) gene is well-known for its ability to confering drug resistance. In this study, we meant to transplant the placenta mesenchymal stem cells (P-MSCs) moderated by mdr1 gene into a nude mice model radiated by g-Co⁶⁰ and to explore the chemoprotection for bone marrow (BM) toxicity.

Methods  Human P-MSCs were isolated from trypsin-digested term placentas and then transduced by with reconstructed retroviral vector containing mdr1 gene and green fluorescent protein (GFP) reporter gene. The integration and expression of mdr1 gene was observed indirectedly by the expression of GFP. A nude mice model was constructed after irradiation with a sublethal dosage of g-Co⁶⁰. These irradiated mice were transplanted with mdr1-MSCs through the caudal vein and then received paclitaxel (PAC) intraperitoneal chemotherapy. The Peripheral peripheral blood (PB) of the nude mice was collected, and the PB cells counts and values were determined using an automatic analyzer.

Results  After PAC treatment, mdr1-MSCs transplanted mice showed markedly improved survival upon compared to MSCs transplanted mice (85.7% vs. 57.1%). White blood cell (WBC) and red blood cell (RBC) counts as well as the hemoglobin (Hb) values were significantly increased in PAC treated mdr1-MSCs mice compared to PAC treated control mice when PAC chemotherapy had been finished (all P <0.05), but the difference was not found in the plateltes (PLT) count (P >0.05).

Conclusion  Human P-MSCs moderated by mdr1 gene when transplanted into nude mice may provide chemoprotection for hematopoietic toxicity.

     

ProCOUNT与单平台ISHAGE方法检测外周血及采集物中CD34+细胞绝对计数的比较

目的评估ProCOUNT方法和单平台ISHAGE方法检测外周血及其采集物中CD34+细胞绝对计数的优缺点。方法采用流式细胞仪ProCOUNT方法和单平台ISHAGE方法,对23份动员外周血及50份采集物行CD34+细胞绝对计数,并对2种方法所得的结果进行Spearman相关分析和配对t检验。结果Spearman相关分析结果:ProCOUNT方法和单平台ISHAGE方法所得的CD34+细胞绝对数、CD34+细胞百分比高度相关(r均>0.9,P均<0.001)。配对t检验分析结果:ProCOUNT方法和单平台ISHAGE方法检测动员外周血的CD34+细胞绝对数分别为(42.7±7.5)μL-1和(52.1±7.2)μL-1(P<0.01),CD34+细胞百分比分别为(0.26±0.07)%和(0.13±0.05)%(P<0.01);检测采集物中CD34+细胞绝对数分别为(3 515.2±684.7)μL-1和(3 923.8±711.1)μL-1(P<0.01),CD34+细胞百分比分别为(0.79±0.18)%和(0.83±0.17)%(P=0.056)。ProCOUNT方法所得的结果均低于单平台ISHAGE方法所得的结果。结论ProCOUNT方法和单平台ISHAGE方法检测CD34+细胞绝对计数和百分比所得结果具有高度相关性,但前者所得结果要低于后者。