表皮生长因子刺激对脂肪干细胞促血管生成作用的影响

目的 探讨表皮生长因子（epidermal growth factor,EGF）对脂肪干细胞（adipose-derived stem cells,ADSCs）旁分泌促血管生成因子及促进人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVEC）小管形成的影响.方法 体外原代培养ADSCs,流式细胞术分析细胞表面标志物表达,酶联免疫吸附法（ELISA）检测不同浓度EGF刺激前后ADSCs-CM中血管内皮生长因子（vascular endothelial growth factor,VEGF）、肝细胞生长因子（hepatoeyte growth factor,HGF）、间质源性因子-1 （stromal-cell derived factor-1,SDF-1）含量变化;HUVEC小管形成实验检测EGF预处理对ADSCs促进HUVEC形成管样毛细血管的影响.结果 利用脂肪抽吸液成功培养出ADSCs,ADSCs具有其特异的表面标记物表达;ADSCs可分泌各种促血管生成因子,且EGF刺激可增加其分泌量;ADSCs可促进共培养的HUVEC形成管样毛细血管,用EGF预处理ADSCs后该作用显著提高.结论 体外环境下ADSCs可分泌多种促血管生成因子,并可促进共培养的HUVEC形成管样毛细血管;EGF刺激可增强该作用,15 mg/L EGF即可达到最佳效果.     

SDF-1/CXCR4调控Tip内皮细胞成血管效应的研究进展

基质细胞衍生因子－1 (stromal cell derived factor,SDF-1)是新近发现的一种趋化因子,SDF-1和趋化因子受体4 (CXC chemokine receptor 4,CXCR4)共同构成了特异性的SDF-1/CXCR4.血管内皮尖端细胞(endothelial tip cell)在新生血管的数量、分支及导向中发挥着重要作用.研究表明,SDF- 1/CXCR4轴对tip内皮细胞的行为和促血管形成起一定作用.本文就SDF- 1/CXCR4轴与Tip内皮细胞的关系及其促血管生成的机制做一综述.     

人脂肪来源间充质干细胞促进Matrigel培养内皮细胞血管形成

目的:通过体外细胞学研究(共培养体系建立)直观表现脂肪干细胞(Adipose derived stem cells,ADSCs)体外进血管形成能力,阐述脂肪干细胞作为辅助细胞通过促进早期血供建立而提高移植脂肪存活率的可能机制。方法:临床通过Coleman法抽吸成人大腿或腹部浅层脂肪,并通过经典方法培养获取ADSCs。对ADSCs进行形态学、表面分子标记物及多向分化等生物学特性鉴定,并通过Matrigel培养脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)管腔样结构形成实验探讨ADSCs体外促血管形成能力,说明ADSCs促进血管新生及移植物早期血供建立的潜力。结果:ADSCs/HUVECs共培养体系可于体外促进内皮细胞形成血管腔样结构,提高早期血管新生。结论:通过共培养体系模拟提示ADSCs体外具备促血管形成的能力,具有促进早期血供建立而提高移植物存活的潜力,为临床细胞辅助脂肪移植提高存活率的可能机制。     

基质细胞衍生因子-1对神经干细胞的趋化作用

目的 观察基质细胞衍生因子-1(SDF-1)对神经干细胞(NSCs)迁移的影响.方法 由GFP转基因SD大鼠胚胎脑组织获取NSCs并进行传代培养,免疫细胞化学染色法检测SDF-1特异性受体CXCR4的表达,利用Blind-Well小室体外迁移体系观察不同浓度的SDF-1(0、1、10、50、100、500、1000μg/L)对NSCs定向迁移数量的影响,随后分别使用CXCR4激动剂和阻断剂处理NSCs,再次利用上述方法观察最适浓度SDF-1时NSCs的迁移.结果 成功分离培养得到能够稳定表达GFP的NSCs,且CXCR4在该种NSCs上有表达.体外趋化实验结果表明,SDF-1对NSCs有较强的趋化作用,随着SDF-1浓度的升高,发生迁移的细胞数量也随之增加,并于SDF-1浓度为500μg/L时达到最高峰;CXCR4特异性激动剂和阻断剂分别能够增强和减弱SDF-1对NSCs定向迁移的趋化作用.结论 SDF-1与其特异性受体CXCR4相互作用,能够对NSCs的定向迁移产生靶向性作用.     

基质细胞衍生因子-1及其受体CXCR4对结肠癌肝转移潜能的影响

目的 探讨趋化因子基质细胞衍生因子-1(SDF-1)及其受体CXCR4对结肠癌肝转移潜能的影响.方法 采用Western-blot法检测不同结肠癌细胞株中CXCR4蛋白及不同组织中SDF-1蛋白的表达,MTT法检测SDF-1及抗CXCR4单抗对结肠癌细胞HT-29增殖能力的影响,体外趋化实验检测HT-29细胞定向迁移能力的变化.建立裸鼠结肠癌肝转移瘤模型,观察CXCR4特异性拮抗剂AMD3100对裸鼠肝转移率和转移瘤数目的 影响.结果 HT-29细胞表达较高强度的CXCR4蛋白,而肝组织表达高强度的SDF-1蛋白.与对照组相比,SDF-1可以诱导HT-29细胞增殖(0.76±0.11 vs0.38±0.06,P<0.05),抗CXCR4单抗对SDF-1的诱导增殖具有显著的抑制作用(0.42±0.08 vs0.76±0.11,P<0.05);SDF-1可促进HT-29细胞的趋化迁移,抗CXCR4单抗可显著抑制SDF-1诱导下HT-29细胞的迁移能力(104.6±18.3 vs 148.8±26.2,P<0.05).AMD3100治疗组裸鼠结肠癌肝转移率显著低于对照组(40% vs 100%,P<0.05),平均瘤结节数目显著低于对照组(7.8±2.6 vs 22.4±8.6,P<0.05).结论 SDF-1/CXCR4生物轴参与了结肠癌肝转移过程,拮抗CXCR4功能可抑制裸鼠结肠癌肝转移,其机制与抑制CXCR4能够有效阻断结肠癌细胞在SDF-1诱导下的细胞增殖和定向迁移有关.     

SDF-1/CXCR4介导体外冲击波促进大鼠骨髓间充质干细胞的黏附、迁移作用

目的探讨体外冲击波(extracorporeal shock wave,ESW)对大鼠骨间充质干细胞(marrow mesenchymal stem cells,MSCs)黏附、迁移及成骨分化的影响及SDF-1/CXCR4通路在其中的作用。方法体外试验分四组:空白对照组(Ctrl组)、冲击波组(ESW组)及CXCR4特异性抑制剂AMD3100对照组(AMD组),采用最适宜能力ESW(500脉冲次数、10 kV)处理MSCs,通过黏附率、划痕试验、Transwell试验对比MSCs黏附及迁移能力的差别,通过ELISA检测SDF-1分泌表达及蛋白免疫印迹法检测CXCR4的表达,研究SDF-1/CXCR4通路的作用;体内试验则选取12周龄SD大鼠48只,随机分成Ctrl、ESW及AMD 3组各16只,采用右侧股骨中段骨缺损模型,各组骨缺损处植入载有该组细胞的PLGA支架,AMD组术后接受AMD3100注射(1 mg/kg/day),于术后第4、8周取材,通过HE染色评估骨缺损区域的骨愈合及新生骨形成情况。结果 ESW明显增加了MSCs的SDF-1分泌量及CXCR4受体表达,促进了MSCs的黏附、迁移及骨缺损的愈合,AMD3100可部分抑制ESW此促进作用。结论 ESW可促进MSCs的黏附、迁移能力,并促进骨缺损区域的骨愈合,其分子机制与分泌型蛋白SDF-1及其受体CXCR4的表达相关,此结果为ESW在促进骨愈合治疗中的临床应用提供了理论基础。     

SDF-1α/CXCR4信号通路在轴向应力刺激促进骨再生中的作用研究

目的通过轴向应力刺激促进骨再生,观察基质细胞衍生因子1α/趋化因子CXC亚族受体4(stromal cell-derived factor 1α/cysteine X cysteine receptor 4,SDF-1α/CXCR4)信号通路变化,探讨轴向应力刺激促进骨再生的机制。方法取72只雄性新西兰大白兔,于右后肢胫骨近端内侧制备直径8 mm圆形皮质骨缺损并脱蛋白松质骨支架修复模型,随机分为3组(n=24)。A组腹腔注射PBS,B组术肢给予应力刺激治疗+腹腔注射PBS,C组术肢给予应力刺激治疗+腹腔注射CXCR4拮抗剂(AMD3100)。术后2、4、8、12周,摄X线片并采用Lane-Sandhu X线评分标准评价骨愈合情况,取标本行HE染色观察新生骨组织及支架降解,免疫组织化学染色观察VEGF、CXCR4表达水平;4、8周取标本Western blot检测SDF-1α及CXCR4蛋白表达水平;12周行Micro-CT检查,计算新生骨体积及新生骨密度。结果 X线片检查示,除术后2周各组骨缺损区及支架无明显变化外,4、8及12周时B组骨愈合评分均高于A、C组(P0.05)。12周时Micro-CT扫描可见B组骨缺损修复、髓腔再通,新生骨体积及骨密度均高于A、C组(P0.05)。HE染色显示,术后4周开始B组骨再生及支架降解均明显快于A、C组。免疫组织化学染色示,各组VEGF及CXCR4阳性表达均在4周达峰值;各时间点B组VEGF及CXCR4表达量均显著高于A、C组(P0.05)。Western blot检测显示,4、8周时B组SDF-1α与CXCR4表达量均显著高于A、C组(P0.05)。结论轴向应力刺激促进骨再生可能与其促进骨缺损区组织高表达SDF-1α,激活与其下游调控BMSCs募集的CXCR4信号有关。     

低氧预处理促进大鼠脂肪来源干细胞体外促血管化的实验研究

目的为了观察不同低氧预处理条件对大鼠脂肪来源干细胞(rat adipose-derived stem cells, r ASCs)旁分泌功能及其体外血管化的影响,研究其相关促血管化机制。方法 1、将r ASCs分别放置于常氧培养箱(21%O_2),低氧培养箱1(2%O_2),低氧培养箱2(0.1%O_2),体外预置培养6、12、24和48小时。利用RT-PCR技术检测,细胞培养液中的VEGF-A,SDF-1a,bFGF,HFG基因在m RNA水平的表达情况。探索低氧预处理r ASCs促血管化的优选条件。2、在上述体外预置培养条件下,测定常氧与低氧组r ASCs旁分泌VEGF-A,SDF-1a蛋白水平情况以及体外血管化能力。结果研究结果发现,氧含量0.1%O_2和体外培养24小时促进血管再生能力更强,可以作为低氧预置优选条件。ASCs经过低氧预置后,条件培养基中VEGF-A和SDF-1a的含量明显高于常氧预置组(VEGF:2.2±0.3倍,P0.05;SDF-1a:3.2±0.5倍,P0.05)。与常氧组相比,低氧组的条件培养基显著促进了其体外管状形成能力(成管数量,常氧组:5.8±1.1,低氧组:11.9±2.6,P0.05)结论本研究通过优化体外低氧预处理条件,激发r ASCs的旁分泌功能,通过分泌促血管再生细胞因子,增加组织血管化再生能力,提示其具有改善勃起功能的潜能。     

SDF-1、CXCR4及ERK1／2在病理性瘢痕中的表达及临床意义

目的：检测病理性瘢痕组织中SDF-1,CXCR4及ERK1／2的表达并探讨其在病理性瘢痕中的相互作用机制。方法：采用免疫组织化学SABC法检测66例病理性瘢痕,25例非病理性瘢痕及27例正常皮肤中SDF-1,CXCR4及ERK1／2的表达。结果：与正常皮肤及非病理性瘢痕相比,SDF-1、CXCR4及ERK1／2三者在病理性瘢痕中均高表达（P〈0．05）;在病理性瘢痕中,SDF—1与ERK1／2的表达呈正相关关系（r=0．293,P〈0．05）,CXCR4和ERK1／2的表达呈正相关关系（r=0．284,P〈O．05）结论：SDF-1／CXCR4生物轴在病理性瘢痕形成过程中可能通过调节ERK1／2途径从而在病理性瘢痕的增生中发挥重要作用。     

肛周脓肿局部组织中SDF-1和CXCR4的表达及其临床意义

目的探讨肛周脓肿局部组织中基质细胞衍生因子1(SDF-1)和趋化因子受体4(CXCR4)的表达情况及其与患者临床病理学特征及预后的关系。方法回顾性收集2017年1月至2018年2月期间在北京市第一中西医结合医院进行手术治疗的47例肛周脓肿患者(肛周脓肿组)及58例混合痔患者(混合痔组)的临床资料。术中采集组织,采用实时定量逆转录聚合酶链式反应(qRT-PCR)法检测2组局部组织中SDF-1 mRNA和CXCR4mRNA的表达水平,采用免疫组织化学染色法检测局部组织中SDF-1蛋白和CXCR4蛋白的表达,分析SDF-1蛋白和CXCR4蛋白的表达与肛周脓肿患者临床病理学特征以及复发的关系。结果肛周脓肿组组织中SDF-1mRNA和CXCR4 mRNA的表达水平均相应高于混合痔组,且肛周脓肿组组织中SDF-1蛋白和CXCR4蛋白的表达阳性率也均相应高于混合痔组,差异均有统计学意义(P0.05)。肛周脓肿患者组织中SDF-1蛋白和CXCR4蛋白的表达与患者的性别、年龄、脓肿位置及病程均无关(P0.05),但均与脓肿直径、愈合时间及肛瘘有关(P0.05)。SDF-1蛋白阴性组和CXCR4蛋白阴性组的术后未复发率均相应低于SDF-1蛋白阳性组及CXCR4蛋白阳性组,差异均有统计学意义(P0.05)。结论 SDF-1和CXCR4分子在肛周脓肿局部组织中的表达上调,且SDF-1蛋白和CXCR4蛋白的表达与脓肿直径、愈合时间、肛瘘形成以及复发有关。     

Skeletal localization and neutralization of the SDF-1(CXCL12)/CXCR4 axis blocks prostate cancer metastasis and growth in osseous sites in vivo.

To delineate the role of SDF-1 and CXCR4 in metastatic prostate cancer (CaP), positive correlations were established between SDF-1 levels and tumor metastasis. Neutralization of CXCR4 limited the number and the growth of intraosseous metastasis in vivo. Together, these in vivo metastasis data provide critical support that SDF-1/CXCR4 plays a role in skeletal metastasis. INTRODUCTION: Previously we determined that the stromal-derived factor-1 (SDF-1)/CXCR4 chemokine axis is activated in prostate cancer (CaP) metastasis to bone. To delineate the role of SDF-1/CXCR4 in CaP, we evaluated SDF-1 levels in a variety of tissues and whether neutralization of SDF-1 prevented metastasis and/or intraosseous growth of CaPs. MATERIALS AND METHODS: SDF-1 levels were established in various mouse tissues by ELISA, immunohistochemistry, and in situ hybridization. To assess the role of SDF-1/CXCR4 in metastasis, bone metastases were established by administering CaP cells into the left cardiac ventricle of nude animals in the presence or absence of neutralizing CXCR4 antibody. The effect of SDF-1 on intraosseous growth of CaP cells was determined using intratibial injections and anti-CXCR4 antibodies and peptides. RESULTS: There was a positive correlation between the levels of SDF-1 and tissues in which metastatic CaP lesions were observed. SDF-1 levels were highest in the pelvis, tibia, femur, liver, and adrenal/kidneys compared with the lungs, tongue, and eye, suggesting a selective effect. SDF-1 staining was generally low or undetectable in the center of the marrow and in the diaphysis. SDF-1 mRNA was localized to the metaphysis of the long bones nearest to the growth plate where intense expression was observed near the endosteal surfaces covered by osteoblastic and lining cells. Antibody to CXCR4 significantly reduced the total metastatic load compared with IgG control-treated animals. Direct intratibial injection of tumor cells followed by neutralizing CXCR4 antibody or a specific peptide that blocks CXCR4 also decreased the size of the tumors compared with controls. CONCLUSIONS: These data provide critical support for a role of SDF-1/CXCR4 in skeletal metastasis. Importantly, these data show that SDF-1/CXCR4 participate in localizing tumors to the bone marrow for prostate cancer.     

胰岛素对脂肪干细胞生长因子分泌功能的影响

Objective To study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs). Methods ADSCs were isolated from human ab-dominal adipose tissue and cultured. The immunophenotype and adipose induced-differenciation were identi-fied, and the third generation cells were collected. The collected cells were assigned to 1 × 10-8, 1 × 10-7, 1 × 10-6 mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry. Results The secretion amounts of VEGF and HGF of ADSCs in 1 × 10-8 and 1 × 10-7 mol/L insulin groups [ (471±41, 762±66 ng/L), (643±64, 930±67 ng/L) , respectively ] were significantly higher as compared with those in control group (286±47, 577±84 ng/L) ( P <0.05 orP <0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1±10-6 mol/L insulin group ( P >0.05 ). The supernatant fluid of ADSCs' nutrient medium of 1 ± 10-8 ,1 ± 10-7 mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 ± 10-7 mol/L group ( P < 0.05 or P < 0. 01 ). Conclusions Insulin in the concentrations of 1 ± 10-8 and 1 ± 10-7 mol/L can notably promote ADSCs' function of secreting VEGF and HGF.     

CXCR4 is highly expressed at the tumor front but not in the center of prostate cancers

Objective

To evaluate the expression of CXCR4, its ligand SDF-1, β-catenin and E-cadherin throughout the local tumor microenvironment of prostate cancer.

Patients and methods

A total of 64 prostate cancer specimens, 24 frozen and 40 paraffin-embedded sections, were obtained from patients treated with radical prostatectomy for clinically localized cancer. Real-time RT-PCR was used for mRNA quantification of CXCR4 and SDF-1 in the tumor center (T), tumor front (F) and distant peritumoral tissue (D). Immunohistochemical analysis was used to investigate the expression patterns of CXCR4, E-cadherin and β-catenin. Clinical records of these patients were studied for follow-up data, and the prognostic value of these molecules’ expression was statistically assessed.

Results

CXCR4 mRNA and protein were significantly increased at the tumor front as compared to distant tissue or tumor center. In comparison, SDF-1 mRNA level gradually increased from the tumor center to the distant peritumoral tissue. High CXCR4 at the tumor front was associated with high Gleason score. Low SDF-1 at the tumor front was associated with locally advanced cancer and disease recurrence. Moreover, high CXCR4 staining at the tumor front and increased cytosolic E-cadherin expression in the same location was associated with locally advanced disease.

Conclusions

CXCR4 seems overexpressed at the tumor front of prostate tumors, where it potentially promotes cell migration toward the SDF-1 centrifugal attracting gradient, as well as epithelial–mesenchymal transition. High CXCR4 and low SDF-1 levels at tumor front were both associated with adverse histological features.

     

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 in the formation of postburn hypertrophic scar (HTS)

Recent data support the involvement of stromal cell-derived factor 1 (SDF-1) in the homing of bone marrow-derived stem cells to wound sites during skeletal, myocardial, vascular, lung, and skin wound repair as well as some fibrotic disorders via its receptor CXCR4. In this study, the role of SDF-1/CXCR4 signaling in the formation of hypertrophic scar (HTS) following burn injury and after treatment with systemic interferon α2b (IFNα2b) is investigated. Studies show SDF-1/CXCR4 signaling was up-regulated in burn patients, including SDF-1 level in HTS tissue and serum as well as CD14+ CXCR4+ cells in the peripheral blood mononuclear cells. In vitro, dermal fibroblasts constitutively expressed SDF-1 and deep dermal fibroblasts expressed more SDF-1 than superficial fibroblasts. Lipopolysaccharide increased SDF-1 gene expression in fibroblasts. Also, recombinant SDF-1 and lipopolysaccharide stimulated fibroblast-conditioned medium up-regulated peripheral blood mononuclear cell mobility. In the burn patients with HTS who received subcutaneous IFNα2b treatment, increased SDF-1/CXCR4 signaling was found prior to treatment which was down-regulated after IFNα2b administration, coincident with enhanced remodeling of their HTS. Our results suggest that SDF-1/CXCR4 signaling is involved in the development of HTS by promoting migration of activated CD14+ CXCR4+ cells from the bloodstream to wound sites, where they may differentiate into fibrocyte and myofibroblasts and contribute to the development of HTS.     

SDF-1/CXCR4生物轴对胆囊癌细胞增殖及迁移的影响

目的 研究胆囊腺癌中趋化因子SDF-1及其受体CXCR4的表达情况,探讨其与胆囊腺癌临床病理特点及淋巴转移的关系.方法 采用免疫组化SP法检测41例胆囊腺癌中SDF-1及其受体CXCR4蛋白阳性表达情况,并分析其与临床病理参数的关系.结果 SDF-1在胆囊癌、胆囊炎、胆囊结石组和正常对照组胆囊黏膜中的表达率分别为68.3%(28/41)、6.7%(6/90)和5.0%(1/20),CXCR4的表达率分别为51.2%(21/41)、5.6%(5/90)和5.0%(1/20),SDF-1和CXCR4在胆囊癌与慢性胆囊炎、胆囊结石组胆囊黏膜、正常对照组胆囊黏膜中的阳性率比较,差异均有统计学意义(SDF-1:χ2=64.33,P＜0.001;CXCR4:χ2=42.52,P＜0.001),胆囊癌不同病理组织学分级、Nevin不同分期、组织学不同分化程度、有无淋巴结或远处转移组间的SDF-1和CXCR4的阳性率表达差异均有统计学意义(P均＜0.05),而不同性别、年龄、有无伴发胆囊结石组、肿瘤大小间SDF-1和CXCR4的阳性率表达差异均无统计学意义(P均＞0.05),胆囊癌组织中SDF-1阳性表达率(68.3%)与CX-CR4阳性表达率(51.2%)之间存在显著正相关(r=0.68,P＜0.01).结论 本研究表明,SDF-1/CXCR4生物轴与胆囊癌关系密切,提示可以通过干预SDF-1/CXCR4生物轴来治疗胆囊癌.     

SDF-1/CXCR4在肝移植急性排斥反应中的表达

目的 通过检测基质细胞衍生因子-1(stromal-dirived factors-1,SDF-1)的受体CXCR4在移植肝脏急性排斥反应中的表达,探讨其在肝移植急性排斥反应中的作用机制.方法 根据Banff病理分级将原位肝移植术后肝穿刺活检标本分成4组,应用RT-PCR方法 半定量分析各组标本中CXCR4的mRNA水平.结果 对照组和未排斥组肝穿标本大多呈现CXCR4的轻、中度表达;排斥组肝穿标本CXCR4高度表达的例数较多,而且随着排斥程度的加重,肝穿标本CXCR4表达的相对R值呈现递增趋势.结论 CXCR4在人类肝移植急性排斥反应中起着重要作用,通过各种生物学手段抑制CXCR4在移植肝脏中的表达有可能成为防治急性排斥反应有效途径.