Transplantation of bone marrow-derived endothelial progenitor cells and hepatocyte stem cells from liver fibrosis rats ameliorates liver fibrosis

AIM To explore the effectiveness for treating liver fibrosisby combined transplantation of bone marrow-derived endothelial progenitor cells(BM-EPCs) and bone marrow-derived hepatocyte stem cells(BDHSCs) from the liver fibrosis environment.METHODS The liver fibrosis rat models were induced with carbon tetrachloride injections for 6 wk. BM-EPCs from rats with liver fibrosis were obtained by different rates of adherence and culture induction. BDHSCs from rats with liver fibrosis were isolated by magnetic bead cell sorting. Tracing analysis was conducted by labeling EPCs with PKH26 in vitro to show EPC location in the liver. Finally, BM-EPCs and/or BDHSCs transplantation into rats with liver fibrosis were performed to evaluate the effectiveness of BM-EPCs and/or BDHSCs on liver fibrosis.RESULTS Normal functional BM-EPCs from liver fibrosis rats were successfully obtained. The co-expression level of CD133 and VEGFR2 was 63.9% ± 2.15%. Transplanted BM-EPCs were located primarily in/near hepatic sinusoids. The combined transplantation of BM-EPCs and BDHSCs promoted hepatic neovascularization, liver regeneration and liver function, and decreased collagen formation and liver fibrosis degree. The VEGF levels were increased in the BM-EPCs(707.10 ± 54.32) and BM-EPCs/BDHSCs group(615.42 ± 42.96), compared with those in the model group and BDHSCs group(P < 0.05). Combination of BM-EPCs/BDHSCs transplantation induced maximal up-regulation of PCNA protein and HGF m RNA levels. The levels of alanine aminotransferase(AST), aspartate aminotransferase, total bilirubin(TBIL), prothrombin time(PT) and activated partial thromboplastin time in the BMEPCs/BDHSCs group were significantly improved, to be equivalent to normal levels(P > 0.05) compared with those in the BDHSC(AST, TBIL and PT, P < 0.05) and BM-EPCs(TBIL and PT, P < 0.05) groups. Transplantation of BM-EPCs/BDHSCs combination significantly reduced the degree of liver fibrosis(staging score of 1.75 ± 0.25 vs BDHSCs 2.88 ± 0.23 or BMEPCs 2.75 ± 0.16, P < 0.05).CONCLUSION The combined transplantation exhibited maximal therapeutic effect compared to that of transplantation of BM-EPCs or BDHSCs alone. Combined transplantation of autogenous BM-EPCs and BDHSCs may represent a promising strategy for the treatment of liver fibrosis, which would eventually prevent cirrhosis and liver cancer.

Effect of Intracoronary Infusion of Bone Marrow Mononuclear Cells or Peripheral Endothelial Progenitor Cells on Myocardial Ischemia-reperfusion Injury in Mini-swine

Objective To simulate and assess the clinical effect of intracoronary infusion of bone marrow mononuclear cells or peripheral endothelial progenitor cells on myocardial reperfusion injury in mini-swine model.Methods Twenty-three mini-swine with myocardial reperfusion injury were used as designed in the study protocol.About(3.54±0.90)×108 bone marrow mononuclear cells(MNC group,n=9) or(1.16± 1.07)×107 endothelial progenitor cells(EPC group,n=7) was infused into the affected coronary segment of the swine.The other mini-swine were infused with phosphate buffered saline as control(n=7).Echocardio-graphy and hemodynamic studies were performed before and 4 weeks after cell infusion.Myocardium infarction size was calculated.Stem cell differentiation was analyzed under a transmission electromicroscope.Results Left ventricular ejection fraction dropped by 0% in EPC group,2% in MNC group,and 10% in the control group 4 weeks after cell infusion,respectively(P<0.05).The systolic parameters increased in MNC and EPC groups but decreased in the control group.However,the diastolic parameters demonstrated no significant change in the three groups(P>0.05).EPC decreased total infarction size more than MNC did(1.60±0.26 cm2 vs.3.71±1.38 cm2,P<0.05).Undermature endothelial cells and myocytes were found under transmission electromicroscope.Conclusions Transplantation of either MNC or EPC may be beneficial to cardiac systolic function,but might not has obvious effect on diastolic function.Intracoronary infusion of EPC might be better than MNC in controlling infarction size.Both MNC and EPC may stimulate angiogenesis,inhibit fibrogenesis,and differentiate into myocardial cells.

Bone marrow progenitor cells do not contribute to liver fibrogenic cells

AIM:To investigate the contribution of bone marrow(BM) cells to hepatic fibrosis.METHODS:To establish a model of chimerism,C57Bl/6 female mice were subjected to full-body irradiation(7 Gy) resulting in BM myeloablation.BM mononuclear cells obtained from male transgenic mice expressing enhanced green fluorescent protein(GFP) were used for reconstitution.Engraftment was confirmed by flow cytometry.To induce liver injury,chimeric animals received carbon tetrachloride(CCl4) 0.5 mL/kg intraperitoneally twice a week for 30 d(CCl4 30 d) and age-matched controls received saline(Saline 30 d).At the end of this period,animals were sacrificed for post mortem analysis.Liver samples were stained with hematoxylin and eosin to observe liver architectural changes and with Sirius red for collagen quantification by morphometric analysis.α-smooth muscle actin(α-SMA) was analyzed by confocal microscopy to identify GFP+ cells with myofibroblast(MF) characteristics.Liver tissue,BM and peripheral blood were collected and prepared for flow cytometric analysis using specific markers for detection of hepatic stellate cells(HSCs) and precursors from the BM.RESULTS:Injury to the liver induced changes in the hepatic parenchymal architecture,as reflected by the presence of inflammatory infiltrate and an increase in collagen deposition(Saline 30 d = 11.10% ± 1.12% vs CCl4 30 d = 12.60% ± 0.73%,P = 0.0329).Confocal microscopy revealed increased reactivity against α-SMA in CCl4 30 d compared to Saline 30 d,but there was no co-localization with GFP+ cells,suggesting that cells from BM do not differentiate to MFs.Liver flow cytometric analysis showed a significant increase of CD45+/GFP+ cells in liver tissue(Saline 30 d = 3.2% ± 2.2% vs CCl4 30 d = 5.8% ± 1.3%,P = 0.0458),suggesting that this increase was due to inflammatory cell infiltration(neutrophils and monocytes).There was also a significant increase of common myeloid progenitor cells(CD117+/CD45+) in the livers of CCl4-treated animals(Saline 30 d = 2.16% ± 1.80% vs CCl4 30 d = 5.60% ± 1.30%,P = 0.0142).In addition the GFP-/CD38+/CD45-subpopulation was significantly increased in the CCl4 30 d group compared to the Saline 30 d group(17.5% ± 3.9% vs 9.3% ± 2.4%,P = 0.004),indicating that the increase in the activated HSC subpopulation was not of BM origin.CONCLUSION:BM progenitor cells do not contribute to fibrosis,but there is a high recruitment of inflammatory cells that stimulates HSCs and MFs of liver origin.? 2012 Baishideng.All rights reserved.

Endothelial progenitor cells as factors in neovascularization and endothelial repair

Endothelial progenitor cells(EPCs)are a heterogeneous population of cells that are provided by the bone marrow and other adult tissue in both animals and humans.They express both hematopoietic and endothelial surface markers,which challenge the classic dogma that the presumed differentiation of cells into angioblasts and subsequent endothelial and vascular differentiation occurred exclusively in embryonic development.This breakthrough stimulated research to understand the mechanism(s)underlying their physiologic function to allow development of new therapeutic options.One focus has been on their ability to form new vessels in injured tissues,and another has been on their ability to repair endothelial damage and restore both monolayer integrity and endothelial function in denuded vessels.Moreover,measures of their density have been shown to be a better predictor of cardiovascular events,both in healthy and coronary artery disease populations than the classical tools used in the clinic to evaluate the risk stratification.In the present paper we review the effects of EPCs on revascularization and endothelial repair in animal models and human studies,in an attempt to better understand their function,which may lead to potential advancement in clinical management.

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma

AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein(GFP) + bone marrow cells.The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting.Serum and tissue levels of vascular endothelial growth factor(VEGF) and colony-stimulating factor(CSF) were quantified by enzyme-linked immunosorbent assay.The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction.The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry.The proportion of EPCs in vessels was then calculated.RESULTS:The HCC model was successful established.The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively.These values are much higher than in the sham-operation group(0.11% ± 0.13%,0.05% ± 0.11%,n = 9) at 14 d after modeling.At 21 d,the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%,0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%,0.12% ± 0.11% in control.Compared to the transient increase observed in controls,the higher level of circulating EPCs were induced by HCC.In addition,the level of serum VEGF and CSF increased gradually in HCC,reaching its peak 14 d after modeling,then slowly decreased.Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels.Under fluorescence microscopy,the bone-marrow(BM)-derived cells labeled with GFP were concentrated in the same area.The relative levels of CD133 and CD34 gene expression were elevated in tumors,around 5.0 and 3.8 times that of the tumor free area.In frozen liver sections from HCC mice,cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies.In tumor tissue,the double-positive cells were incorporated into vessel walls.In immunofluorescent staining.These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells(VECs) come partly from BM-derived EPCs.The proportion of GFP CD31 double positive VECs(out of all VECs) on day 21 was around 35.3% ± 21.2%.This is much higher than the value recorded on day 7 group(17.1% ± 8.9%).The expression of intercellular adhesion molecule 1,vascular adhesion molecule 1,and VEGF was higher in tumor areas than in tumor-free tissues.CONCLUSION:Mobilized EPCs were found to participate in tumor vasculogenesis of HCC.Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.

A novel and feasible way to cultivate and purify endothelial progenitor cells from bone marrow of children with congenital heart diseases

Background Endothelial progenitor cells （EPCs） are used in vascular tissue engineering and clinic therapy. Some investigators get EPCs from the peripheral blood for clinic treatment, but the number of EPCs is seldom enough. We have developed the cultivation and purification of EPCs from the bone marrow of children with congenital heart disease, to provide enough seed cells for a small calibre vascular tissue engineering study. Methods The 0.5-ml of bone marrow was separated from the sternum bone, and 5-ml of peripheral blood was collected from children with congenital heart diseases who had undergone open thoracic surgery. CD34＋ and CD34＋NEGFR＋ cells in the bone marrow and peripheral blood were quantified by flow cytometry. CD34＋/VEGFR＋ cells were defined as EPCs. Mononuclear cells in the bone marrow were isolated by Ficoll density gradient centrifugation and cultured by the EndoCult Liquid Medium KitTM. Colony forming endothelial cells was detected. Immunohistochemistry staining for Dil-ac-LDL and FITC-UEA-1 confirmed the endothelial lineage of these cells. Results CD34＋ and CD34＋NEGFR＋ cells in peripheral blood were （0.07±0.05）% and （0.05±0.02）%, respectively. The number of CD34＋ and CD34＋/VEGFR＋ cells in bone marrow were significantly higher than in blood, （4.41±1.47）% and （0.98±0.65）%, respectively （P 〈0.0001）. Many colony forming units formed in the culture. These cells also expressed high levels of Dil-ac-LDL and FITC-UEA-I. Conclusion This is a novel and feasible approach that can cultivate and purify EPCs from the bone marrow of children with congenital heart disease, and provide seed cells for small calibre vascular tissue engineering.

Stem cell therapy for erectile dysfunction

Erectile dysfunction(ED)is an important health problem that has commonly been clinically treated using phosphodiesterase type 5 inhibitors(PDE5Is).However,PDE5Is are less effective when the structure of the cavernous body has been severely injured,and thus regeneration is required.Stem cell therapy has been investigated as a possible means for regenerating the injured cavernous body.Stem cells are classified into embryonic stem cells and adult stem cells(ASCs),and the intracavernous injection of ASCs has been explored as a therapy in animal ED models.Bone marrowderived mesenchymal stem cells and adipose tissuederived stem cells are major sources of ASCs used for the treatment of ED,and accumulated evidence now suggests that ASCs are useful in the restoration of erectile function and the regeneration of the cavernous body.However,the mechanisms by which ASCs recover erectile function remain controversial.Some studies indicated that ASCs were differentiated into the vascular endothelial cells,vascular smooth muscle cells,and nerve cells that originally resided in the cavernous body,whereas other studies have suggested that ASCs improved erectile function via the secretion of anti-apoptotic and/or proangiogenic cytokines ratherthan differentiation into other cell types.In this paper,we reviewed the characteristics of stem cells used for the treatment of ED,and the possible mechanisms by which these cells exert their effects.We also discussed the problems to be solved before implementation in the clinical setting.

附子理中汤对变应性鼻炎大鼠骨髓干细胞鼻腔归巢的影响

目的研究附子理中汤对变应性鼻炎大鼠骨髓间充质干细胞(MSCs)和内皮祖细胞(EPCs)归巢的影响及意义。方法复制变应性鼻炎的疾病模型,将12只模型大鼠分为治疗组(附子理中汤干预)和空白组,雌雄各半,每组6只。在模型建立后的7 d分别评价两组的症状积分及鼻腔黏膜取材免疫荧光检测鼻腔黏膜MSCs和EPCs的阳性率。结果空白组0、7天的积分比较差异无统计学意义(P>0.05)。治疗组在治疗后积分减少(P<0.05)。两组间同期比较,第0天时积分比较差异无统计学意义(P>0.05)。第7天的积分差异有统计学意义(P<0.05)。变应性鼻炎造模成功后,骨髓MSCs和EPCs都能够从循环系统向鼻腔黏膜归巢,在鼻腔黏膜都有阳性表达。经χ2检验,治疗组在治疗后鼻腔黏膜的MSCs和EPCs阳性率均较空白组提高(P<0.05)。说明治疗组附子理中汤能有效改善变应性鼻炎大鼠的症状,且能促进变应性鼻炎大鼠的骨髓MSCs和EPCs向鼻腔归巢。结论骨髓MSCs和EPCs能够向变应性鼻炎的鼻腔黏膜归巢,而附子理中汤能够促进骨髓MSCs和EPCs的鼻腔归巢。

Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture

Background Macrophage stimulating protein （MSP） is produced by human bone marrow endothelial cells. In this study we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor （SCF）, interleukin 3 （IL-3）, interleukin 6 （IL-6）, granulocyte macrophage-colony stimulating factor （GM-CSF）, erythropoietin （EPO） （Cys） and MSP or of Cys and bone marrow endothelial cell conditioned medium （EC-CM）. Methods Human bone marrow CD34^＋ cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit （CFU-GM） and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte （CFU-GEMM） were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium （NBT） reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively. Results MSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow （BM） CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys＋EC-CM, Cys＋MSP or Cys compared with 0 hour control in liquid culture system after 6 days. Conclusion MSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

兔骨髓源性血管内皮祖细胞的分离、培养及鉴定

本研究探讨兔骨髓源性血管内皮祖细胞(endothelial progenitor cells,EPC)的分离、培养及鉴定方法。抽取兔骨髓细胞,用梯度密度离心法获得单个核细胞,以内皮细胞培养液培养,通过细胞形态观察、免疫组织化学试验、流式细胞术以及内皮祖细胞吞噬功能进行鉴定。结果表明,新分离的骨髓单个核细胞呈圆形,培养48小时后可见贴壁细胞呈集落样生长,细胞呈圆形或不规则形,核分裂相明显,至培养第7天成片生长的细胞集落相互连接呈梭形的内皮样细胞。内皮祖细胞免疫组织化学检测结果显示CD133(+),CD34(+),Ⅷ因子(++),KDR(++);流式细胞术鉴定结果显示CD133的阳性率为(18.23±7.12)%,CD34的阳性率为(47.71±14.85)%,CD31的阳性率为(71.61±13.51)%,KDR的阳性率为(87.24±11.40)%。细胞吞噬功能鉴定说明超过80%的贴壁细胞都特异性地摄取了Dil-acLDL和FITC-UEA-1。结论:密度梯度离心法体外分离兔骨髓源的单个核细胞,在一定的诱导培养条件下能分化成为血管内皮祖细胞。

黄芪、三七促进骨髓干细胞体外转化并扩增血管内皮前体细胞(EPC)作用的研究

目的:探讨黄芪、三七对骨髓干细胞体外转化并扩增EPC的促进作用及其可能的机制。方法:常规采集下肢缺血患者骨髓血,密度梯度离心法分离骨髓单个核细胞,不同条件下贴壁扩增细胞。镜下细胞的形态学观察,流式细胞仪检测CD3+4细胞的百分比。结果:细胞呈梭形,束状排列,间杂有少量圆形细胞。与对照组相比,黄芪中、低剂量组,三七中、高剂量组CD3+4细胞的百分比均显著增加。结论:黄芪、三七能促进EPC的转化和增殖。

微孔法分离大鼠骨髓内皮祖细胞

用一种杂交瘤皿,根据内皮祖细胞集落形成单位(endothelial progenitor cells colony-forming units,EPCs-CFUs)的形态特征和EPCs表面特异性标记物分离EPCs.取大鼠股骨、胫骨骨髓,将全骨髓接种在聚苯乙烯制作的杂交瘤皿上,培养4～7天后出现CFUs,将这些集落分别挑选出来后,取单个集落的部分细胞免疫荧光鉴定EPCs表面特异性标记物CD133/VEGFR-2.CD133/VEGFR-2双阳性即为EPCs-CFUs.与此对应的余下一部分继续传代增殖,流式细胞术鉴定CD133/VEGFR-2/CD34,并把此方法命名为微孔法.发现接种后第4天,显微镜下可见明显的CFUs.免疫荧光鉴定大约7%的CFUs为CD133+/VEGFR-2+,进一步传代培养,流式细胞术鉴定CD133+/VEGFR-2+/CD34+细胞纯度达70%以上.传代细胞可在体外形成血管样结构,并表达内皮细胞特异性标记物vWF.结果表明通过微孔法能成功地从大鼠骨髓分离到EPCs.

差速贴壁法分离兔骨髓源性间充质干细胞和内皮祖细胞及其生物学特性的研究

本研究探讨一种高效、稳定的从兔骨髓中同时分离培养间充质干细胞(Mesenchymal Stem Cells,MSC)和内皮祖细胞(Endothelial Progenitor Cells,EPC)的方法并观察其生物学特性。抽取兔骨髓,应用密度梯度分离单个核细胞,采用差速贴壁经纤连蛋白包被并结合EGM-2MV培养基分别扩增MSC和EPC。用台盼蓝法测定细胞传代成活率,通过绘制生长曲线法、MTT法、DNA周期检测MSC和EPC的增殖能力及诱导分化成骨细胞、成脂肪细胞能力,并结合流式细胞术(FCM)检测MSC免疫原型以鉴定MSC;细胞吞噬功能特异性地摄取Dil-ac-LDL及FITC-UEA-1,并结合CD133、VEGFR2/KDR、CD34免疫荧光鉴定EPC,并计算其纯度。结果表明,经密度梯度分离单个核细胞,在早期贴壁细胞24 h换液时即可见明显集落形成,8 d后达80%融合,细胞呈均匀一致的长梭形排列;2次贴壁细胞经EGM-2MV培养基培养,第3天开始伸展,约8 d可融合近80%,细胞呈多角形,出现条索状结构;2种细胞台盼蓝法测定细胞传代成活率均在90%以上,传至第2代后,生长曲线均近似"S"形;MTT法检测显示,细胞生长d 3至d 5时光密度值变化较明显;MSC G0-G1期为(93.32±1.65)%、EPC G0-G1为(93.05±1.95)%,2种细胞DNA周期无明显差异;早期贴壁细胞FCM检测CD90、CD44阳性率为(99.7±1.12)%、(99.1±2.33)%;CD14、CD45、CD79a阳性率分别为(4.8±0.38)%、(6.8±0.49)%及(0.4±0.08)%,经体外诱导能够向成骨细胞及脂肪细胞分化,鉴定为MSC;2次贴壁细胞传至第2代经Dil-ac-LDL、FITC-UEA-1双荧光染色阳性率(82.1±3.4)%,CD133、VEGFR2/KDR、CD34免疫荧光染色阳性率分别为(74.2±3.2)%、(64.7±4.3)%及(43.5±1.5)%,鉴定为EPC。结论:应用密度梯度分离法结合差速贴壁筛选法可培养出高纯度的MSC,2次贴壁细胞经纤连蛋白预包被并结合EGM-2MV培养基体外诱导可培养出增殖能力较强的EPC,为组织工程学研究提供种子细胞。

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48h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-2MV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。

丹参酮ⅡA增效大鼠骨髓源性内皮祖细胞VEGF、SDF-1表达

目的:研究在丹参酮ⅡA的辅助下,骨髓源性内皮祖细胞(EPCs)VEGF与SDF-1表达是否能增强,从而促进细胞归巢。方法:Percoll密度梯度离心法分离SD大鼠骨髓单个核细胞,血管内皮生长因子(VEGF)、表皮生长因子((EGF)、碱性成纤维细胞生长因子(bFGF)诱导培养,并进行形态学、免疫学(免疫细胞化学染色)、功能学(Dil-acLDL与FITC-UEA-1双荧光染色)鉴定。将鉴定为EPCs的细胞分为单纯EPCs组(对照组)、EPCs+丹参酮ⅡA组(加药组)。细胞培养第9d,Western blot与实时荧光定量PCR检测各组VEGF、SDF-1基因与蛋白表达。结果:EPCs+丹参酮IIA组VEGF、SDF-1基因与蛋白表达均高于EPCs组(p均<0.05)。结论:丹参酮ⅡA可上调大鼠骨髓源性内皮祖细胞VEGF及SDF-1表达,从而可能在缺血环境中更好促进EPCs归巢,更有效促进血管修复与再生。

滋肾调冲法对大鼠外周血内皮祖细胞影响的实验研究

[目的]观察滋肾调冲法代表方功血宁Ⅱ号方对骨髓单个核细胞移植大鼠外周血内皮祖细胞(EPCs)数量的影响,探讨该法对骨髓EPCs的动员作用。[方法]受鼠雌性大鼠经137Cs全身照射后,从尾静脉输注雄性大鼠标记4′,6-二脒基-2-苯基吲哚(DAPI)荧光的骨髓单个核细胞。受鼠随机分为移植对照组、功血宁Ⅱ号组和乌鸡白凤丸组,另取未照射大鼠尾静脉输注等量培养液为假孕对照组。骨髓移植24 h后各组给予相应药物或等容积的生理盐水。受鼠造血重建后,各组均制备假孕模型。在黄体早中晚期各组处理1/3数的大鼠,眼内眦取血,流式细胞术检测外周血EPCs的数量。血液涂片,荧光显微镜观察DAPI标记细胞的分布。[结果]荧光显微镜下显示,移植各组黄体各期血液涂片可见DAPI标记细胞,且早期数量为多,功血宁Ⅱ号组尤为明显。各组黄体早期外周血EPCs数目最多,中期略有下降,晚期明显减少(P<0.05或P<0.01)。功血宁Ⅱ号组、乌鸡白凤丸组黄体各期EPCs数目较假孕对照组显著增多(P<0.05或P<0.01);功血宁Ⅱ号组早期较移植对照组和乌鸡白凤丸组显著增多(P<0.05)。[结论]滋肾调冲法能够动员骨髓EPCs,提高外周血中EPCs数量,对EPCs进一步归巢至卵巢黄体,促进黄体血管新生具有重要作用。

小鼠骨髓源性内皮祖细胞的培养和表面标志物鉴定

目的建立一种从小鼠骨髓中分离、培养内皮祖细胞(EPC)的方法,并对EPC多种表面标志物进行鉴定。方法密度梯度离心法获取小鼠骨髓源性单个核细胞,应用专门培养基EGM-2培养。观察不同时期细胞增殖情况;在培养7 d后行DIL-AC-LDL和FITC-UEA-1双荧光染色鉴定及流式细胞检测;应用RT-PCR分析0,4,7 d时CD31、CD34、CD45、CD117、CD133、FLK-1、VE-Cad等表面标志物的表达情况。结果诱导培养的骨髓单个核细胞72 h后基本完成贴壁,8～9 d时贴壁70%～80%可进行传代,2～3周后呈典型的鹅卵石样外观。细胞培养7 d后DIL-AC-LDL和FITC-UEA-1双染色阳性细胞比例为(90.16±6.77)%,流式细胞检测PE-Flk-1、APC-CD133、FITC-CD34均阳性概率为(1.73±0.27)%。RT-PCR结果显示随时间推移CD34、CD117、CD133的表达量逐渐下降,而CD31、FLK-1、VE-Cad等内皮系标志物的表达逐渐增加。结论密度梯度离心法结合定向诱导培养可获得较多数量的EPC,以流式细胞检测技术为主,结合细胞形态学和功能学对EPC进行鉴定是一种较为理想的方法。

干细胞因子联合粒细胞集落刺激因子对单侧输尿管梗阻大鼠骨髓干细胞及内皮祖细胞的动员作用

目的探讨干细胞因子(SCF)联合粒细胞集落刺激因子(G-CSF)对单侧输尿管梗阻(UUO)大鼠骨髓干细胞及内皮祖细胞的动员作用。方法56只Wistar大鼠随机分为7组:正常对照组、SCF组、G-CSF组、SCF联合G-CSF组(SCF-G组)、假手术组、UUO组、SCF联合G-CSF用于UUO组(UUO+SCF-G组)。实验第5天采集血标本后:①流式细胞仪检测静脉血单个核细胞中CD34+、CD34+/CD133+细胞;②检测血清谷丙转氨酶、谷草转氨酶、尿素氮、肌酐水平。结果①对照组大鼠静脉血单个核细胞中CD34+细胞为(0.13±0.01)%,假手术组CD34+细胞为(0.24±0.06)%与对照组比较差异无显著性(P>0.05);其余各组与对照组比较CD34+细胞百分率均明显增高(P<0.05),以UUO+SCF-G组(3.04±0.42)%及SCF-G组(2.10±0.28)%增高最为明显;②对照组大鼠静脉血单个核细胞中CD34+/CD133+细胞为(0.02±0.01)%,假手术组CD34+/CD133+细胞(0.05±0.02)%与对照组比较差异无显著性(P>0.05);其余各组与对照组比较CD34+/CD133+细胞百分率均明显增高(P<0.05),以UUO+SCF-G组(0.73±0.17)%增高最为明显;③7组血清尿素氮,肌酐、谷丙转氨酶水平无明显增高,UUO组谷草转氨酶水平较其他6组增高,差异有显著性。结论SCF和G-CSF对干细胞和内皮祖细胞的动员效果并非完全呈平行关系,联合使用可提高内皮祖细胞和干细胞的动员率,短期内未见肝、肾毒副作用。

大鼠骨髓与外周血来源内皮祖细胞生物学特性比较

背景:内皮祖细胞不仅参与胚胎血管生成,也参与出生后血管发生和血管内膜损伤后修复,对治疗缺血性疾病意义重大,但目前对内皮祖细胞的分离、培养、鉴定还存在争议。目的:体外分离、培养大鼠骨髓与外周血来源的内皮祖细胞,并比较其生物学特性。方法:密度梯度离心法分离SD大鼠骨髓和外周血单个核细胞,接种于纤维连接蛋白铺被的培养瓶中贴壁培养,用加入血管内皮生长因子、碱性成纤维细胞生长因子及表皮生长因子的完全培养基诱导培养,对获得的贴壁细胞进行细胞形态学,免疫细胞化学染色,流式细胞仪,透射电镜,以及Dil-acLDL、FITC-UEA-1双荧光染色法检测。结果与结论:骨髓来源的内皮祖细胞数量多,集落状生长,增殖能力强;外周血来源的内皮祖细胞数量较少,散在生长,消化后能贴壁但不能传代。两种不同来源的内皮祖细胞免疫细胞化学检测贴壁细胞CD133、CD34、Flk-1、Ⅷ因子在不同时段呈阳性表达;激光共聚焦显微镜观察,Dil-acLDL、FITC-UEA-1均为双染。透射电镜检查外周血来源的内皮祖细胞发现W-P小体。提示大鼠骨髓和外周血均能分离培养出内皮祖细胞,但前者是早期内皮祖细胞,后者为晚期内皮祖细胞,两者生物学特性各不相同。

丹酚酸B对内皮祖细胞与骨髓间充质干细胞联合心脏移植后增殖状况的影响

目的通过丹酚酸B预处理的内皮祖细胞(endothelial progenitor cells,EPCs)与骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)混合后对心肌梗死大鼠进行心脏移植,观察移植细胞的增殖情况。方法密度梯度离心法和贴壁筛选法培养、纯化EPCs与BMSCs;免疫细胞化学法(CD34、CD133、CD44)分别鉴定2种细胞。采用左冠状动脉结扎法制作大鼠急性心肌梗死模型。丹酚酸B最佳药物浓度(8μg/mL)预处理EPCs,并以不同比例与BMSCs混合(EPCs/BMSCs分别为1∶1、2∶1、4∶1和8∶1),手术移植至心肌梗死区。HE和N-BT染色法检测心肌梗死面积;免疫组化法检测Ki-67的表达情况。结果与模型组(19.60%±3.23%)比较,各细胞移植组心肌梗死面积均明显下降(P<0.05),其中4∶1组(11.37%±2.18%)和8∶1组(9.23%±2.35%)效果最明显(P<0.05)。与模型组[(5.17±2.31)个/高倍视野]比较,各细胞移植组Ki-67阳性细胞数均显著增多(P<0.05),其中8∶1组阳性细胞数[(15.00±3.16)个/高倍视野]明显高于其他细胞移植组(P<0.05)。结论丹酚酸B预处理的EPCs联合BMSCs进行心脏移植,能够减少大鼠心肌梗死面积,提高BMSCs在梗死周边和缺血局部的增殖。并且随着EPCs移植比例的增加,梗死面积逐渐减少,增殖表达逐渐增强。