CXCR4在肺癌转移中的作用及其机制研究

目的 探讨CXCR4在肺癌转移中的作用及其机制。方法 将CXCR4不同表达水平的肺癌细胞(95C、95C-pc、95C-X4、95D、95D-pC、95D-ASX4)接种于裸鼠皮下并分析其转移能力。分别通过趋化侵袭实验、明胶酶谱法、黏附实验、RT-PCR法检测CXCR4／SDF-1对肺癌细胞迁移、基质金属蛋白酶(MMP-2)活性、黏附能力、生长相关癌基因-α(GRO-α)表达的调控;通过流式细胞术和共聚焦显微镜观察肺癌细胞内纤维肌动蛋白的合成和聚合情况;Western印迹分析CXCR4／SDF-1对ERK1／2磷酸化的影响。结果 CXCR4不同表达水平的肺癌细胞在裸鼠体内具有不同的转移能力,95D-ASX4组有2／5的小鼠发生了转移,其肺转移结节的数目明显少于95D、95D-pC组(P=0．044)。CXCR4特异配体SDF-1α可以诱导肺癌细胞的迁移和细胞骨架蛋白纤维肌动蛋白的合成和聚合;SDF-1α促进肺癌细胞MMP-2活性、黏附能力和GRO-α表达的增加;CXCR4中和抗体可以在一定程度上抑制这些作用。SDF-1α可以诱导ERK1／2的磷酸化。结论 肺癌转移在一定程度上依赖于CXCR4／SDF-1的相互作用,他们通过调控肺癌细胞的运动性、MMP活性、黏附能力及GRO-α的表达参与肺癌的转移。     

LRRC4通过SDF-1α/CXCR4生物学轴抑制脑胶质瘤细胞侵袭迁移能力

目的:探讨LRRC4(leucine-rich repeats containing 4)通过SDF-1 α/CXCR4(stromal cell-derived factor-1 α/CXC receptor 4)对脑胶质瘤细胞侵袭迁移能力的影响.方法:将LRRC4基因转入脑胶质母细胞瘤U251细胞,分别通过RT-PCR实验、黏附实验、侵袭实验、细胞运动试验、划痕标记荧光染料示踪实验,研究SDF-1α干预前后LRRC4对U251细胞迁移能力的影响.结果:将LRRC4基因转入脑胶质瘤细胞U251,通过RT-PCR实验发现CXCR4的表达下调.用CXCR4的特异配体SDF-1α干预后,肿瘤黏附内皮实验、侵袭实验、细胞迁移实验、划痕标记染料示踪实验表明脑胶质瘤细胞迁移能力增强,LRRC4可以抑制细胞的这种侵袭迁移能力.SDF-1 α能够改善细胞间的通讯能力,LRRC4可以进一步增强这种通讯能力.结论:SDF-1 α/CXCR4的相互作用可以增强脑胶质瘤细胞U251的运动迁移能力,LRRC4基因可以通过调控SDF-1α/CXCR4生物学轴有效地抑制肿瘤细胞的侵袭迁移能力.     

CXCR4在VEGF-C介导的胃癌淋巴道转移中的作用

目的 探索趋化因子受体CXCR4和VEGF-C与胃癌淋巴道转移的关系,为研究干预胃癌淋巴转移的新方法提供理论依据.方法 采用RT-PCR方法检测86例胃癌组织中趋化因子受体CXCR4 mRNA的表达及VEGF-C mRNA的表达,探索其与胃癌淋巴结转移的关系;Boyden趋化小室法检测CXCR4不同表达状态胃癌细胞的趋化活性和趋化抑制性.结果 86例胃癌组织均有不同程度的CXCR4 mRNA和VEGF-C mRNA的表达.53例CXCR4 mRNA呈高表达,表达率为61.63%,VEGF-C mRNA高表达率为56.98%(49/86);VEGF-C mRNA高表达组中的CXCR4 mRNA的表达率也较高;CXCR4 mRNA的表达与胃癌淋巴结转移呈正相关(P＜0.05);CXCR4的配体SDF-1对胃癌细胞的平均趋化率为81%,经抗CXCR4单克隆抗体处理后癌细胞的平均趋化率下降至30%,CXCR4被特异性抗体封闭前后的癌细胞趋化活性有显著性差异(P＜0.05).结论 胃癌CXCR4的表达和VEGF-C的表达与胃癌淋巴结转移呈正相关;CXCR4的功能状态影响胃癌细胞的迁移活性.通过干预趋化因子受体CXCR4和VEGF-C的活性可能成为阻断胃癌淋巴转移的新靶点.     

CXCR7在胃癌细胞中的表达及对SGC-7901细胞迁移及侵袭的影响

目的 探讨趋化因子受体7(CXCR7)在不同分化程度的胃癌细胞系中的表达以及siRNA沉默CXCR7后对胃癌SGC-7901细胞迁移及侵袭能力的影响。方法 体外培养5种人胃癌细胞系:未分化腺癌HGC-27、低分化黏液腺癌MGC-803、低分化腺癌BGC-823、中分化腺癌SGC-7901和高分化腺癌MKN-28。采用Western blot及RT-PCR法分别检测CXCR7的蛋白及mRNA表达。应用脂质体转染siRNA的方法沉默SGC-7901细胞CXCR7的表达,并采用其配体基质细胞衍生因子-1（SDF-1）干预,实验分为4组:阴性对照siRNA(NC siRNA组), NC siRNA+SDF-1组,CXCR7 siRNA组和CXCR7 siRNA+SDF-1组。应用Transwell小室检测细胞迁移及侵袭能力。结果 CXCR7在不同人胃癌细胞系中表达程度不一,其中高分化MKN-28几乎不表达,中分化SGC-7901细胞表达程度最高。与 NC siRNA组相比,NC siRNA＋SDF-1组的迁移细胞数和侵袭细胞数增加（P CXCR7 siRNA组的迁移细胞数和侵袭细胞数减少（P CXCR7 siRNA＋SDF-1组的迁移细胞数和侵袭细胞数减少（P CXCR7 siRNA抑制。     

过表达趋化因子受体4的内皮祖细胞影响血管平滑肌细胞迁移、增殖

目的 研究过表达趋化因子受体4(chemokine receptor 4,CXCR4)的内皮祖细胞(endothelial progenitor cells,EPCs)对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)迁移、增殖的影响.方法 分离培养并鉴定大鼠骨髓来源EPCs.CXCR4的重组腺病毒感染EPCs后,流式细胞术、实时荧光定量PCR(qRT-PCR)分别检测细胞膜CXCR4受体及mRNA表达,CCK-8法、Transwell法分别检测EPCs增殖活性、迁移能力.建立过表达CXCR4的EPCs与VSMCs非接触共培养模型,Transwell法、CCK-8法检测EPCs对VSMCs的迁移、细胞存活的影响.结果 CXCR4重组腺病毒感染EPCs 48 h后,EPCs细胞膜CXCR4受体和mRNA表达明显增高(P＜0.01),过表达CXCR4对EPCs增殖活性无明显影响,但可增强EPCs定向迁移能力(P＜0.01).共培养模型中,SDF-1α诱导下,CXCR4组VSMCs迁移、细胞存活率明显降低(P＜0.05),无SDF-1α诱导下空白对照组、GFP组及CXCR4组VSMCs的迁移、细胞存活率整体高于SDF-1α诱导下各组(P＜0.05).结论 过表达CXCR4可增强EPCs的迁移能力,在SDF-1/CXCR4调控轴中加强EPCs对VSMCs迁移和增殖的抑制作用,可用于防治血管再狭窄的细胞治疗.     

CXCR4/SDF-1对鼻咽癌细胞增殖与迁移能力的影响

目的 观察鼻咽癌不同恶性程度的细胞中趋化因子受体CXCR4的表达,检测CXCR4/SDF-1反应轴对鼻咽癌细胞的增殖作用,SDF-1对CXCR4阳性肿瘤细胞的趋化作用,探讨CXCR4/SDF-1生物学轴对人鼻咽癌细胞增殖与迁移能力的影响.方法 以鼻咽癌成瘤高转移细胞株5-8F及成瘤不转移细胞株6-10B为研究对象,采用Western blot免疫印迹法检测鼻咽癌细胞株CXCR4蛋白的表达情况,SDF-1及CXCR4阻断剂AMD3100作用于两种细胞后,MTT法检测细胞的增殖能力,体外迂徙实验检测CXCR4/SDF-1反应轴对鼻咽癌细胞的趋化作用.结果 5-8F细胞株CXCR4蛋白的表达明显高于6-10B细胞株;SDF-1能明显增强5-8F细胞增殖与迁移能力,CXCR4阻断剂AMD3100作用后,5-8F细胞增殖与迁移能力明显降低;SDF-1对6-10B细胞的迁移及增殖能力无明显影响.结论 CXCR4/SDF-1生物学轴与鼻咽癌细胞株的增殖与迁移有一定的关系,AMD3100可明显抑制鼻咽癌细胞的增殖与迁移能力.     

趋化因子受体CXCR4小干扰RNA对人大肠癌细胞株迁移的抑制作用

目的:探讨趋化因子受体CXCR4小干扰RNA(siRNA)对人大肠癌细胞株迁移的抑制作用.方法:采用RT-PCR、Western印迹和体外迁移实验检测趋化因子受体CXCR4在大肠癌细胞株SW480、SW620、LoVo 细胞的表达及迁移能力.将与CXCR4 mRNA互补的 siRNA及非抑制性双链RNA(Non-silencing dsRNA),在脂质体介导下以150 nmol/L终浓度转染大肠癌细胞SW480,以RT-PCR和Western印迹法检测CXCR4表达的变化,Transwell迁移实验检测大肠癌细胞SW480迁移的变化.结果:大肠癌细胞SW480高表达CXCR4,大肠癌细胞LoVo低表达CXCR4,SW620的CXCR4表达水平介于二者之间.与LoVo 细胞相比,SW480和SW620迁移细胞明显增多(P＜0.01).与对照组、脂质体组和Non-silencing dsRNA组相比,siRNA组SW480细胞内CXCR4 mRNA和蛋白均明显降低,细胞迁移被明显抑制 (P＜0.01).结论:CXCR4 siRNA可通过下调CXCR4的表达抑制人大肠癌细胞SW480的迁移.     

SDF-1/CXCR4在恶性胶质瘤细胞体外增殖、迁移及侵袭中的作用

目的探讨趋化因子SDF-1及受体CXCR4在恶性胶质瘤细胞中的增殖、迁移及侵袭的作用,为临床治疗提供依据.方法免疫组化法检测CXCR4在恶性胶质瘤C6细胞中的表达;用MTT法分别检测不同浓度的趋化因子SDF-1促恶性胶质瘤C6细胞体外增殖,并应用CXCR4受体抑制剂AMD3100与SDF-1共培养细胞观察增殖情况及抗CXCR4单克隆抗体在恶性胶质瘤中上述指标的变化.通过细胞迁移实验和细胞侵袭实验评价不同处理组C6细胞的迁移和侵袭能力.结果 CXCR4在恶性胶质瘤C6细胞系表达呈阳性.SDF-1可以促进恶性胶质瘤C6细胞的体外增殖、迁移和侵袭,而抗CXCR4单克隆抗体可以有效抑制其增殖、迁移和侵袭,并呈浓度依赖性.AMD3100可以抑制SDF-1的诱导作用.结论 SDF-1/CXCR4轴在恶性胶质瘤细胞株的体外增殖、迁移及侵袭中的作用,可能与恶性胶质瘤的侵袭和转移有关,为治疗恶性胶质瘤提供新的治疗策略.     

体外合成CXCR4 mRNA对人脐带间充质干细胞迁移的影响

目的:探讨体外合成的趋化因子受体4(CXCR4)mRNA对人脐带血间充质干细胞迁移的影响。方法:构建合成CXCR4 mRNA的质粒,通过PCR和测序鉴定。体外合成CXCR4 mRNA转染人脐带间充质干细胞(h UCMSCs)免疫荧光检测CXCR4蛋白的表达。Transwell检测转染CXCR4 mRNA的h UC-MSCs向SDF-1迁移的能力。结果:成功构建了CXCR4 mRNA载体,免疫荧光证实体外合成的CXCR4 mRNA可高效转染h UC-MSCs并增加其向SDF-1迁移的能力。结论:CXCR4 mRNA可高效转染h UC-MSCs并增强其向SDF-1迁移的能力,为h UC-MSCs靶向治疗的临床运用奠定基础。     

基质细胞衍生因子1及其受体趋化因子受体4生物学轴与头颈肿瘤

许多恶性肿瘤细胞均能分泌趋化因子,其与肿瘤细胞相互作用。关于趋化因子的研究已成为肿瘤研究的热点领域之一。基质细胞衍生因子1(SDF-1)及其受体CXC趋化因子受体4(CXCR4)广泛参与肿瘤的发生、增殖、侵袭、转移等过程,并可能与肿瘤的治疗、TNM分期及预后相关。但SDF-1/CXCR4生物学轴与头颈肿瘤的关系尤其是作用和机制的研究较少。近年来,SDF-1/CXCR4生物学轴与头颈肿瘤的研究逐渐成为热门,为头颈肿瘤的诊疗指出新思路。     

Role of CXCL12 in metastasis of human ovarian cancer

Background In a previous study, we have verified that CXCR4 expression is correlated with tumor aggressive progression and poor prognosis in patients with epithelial ovarian cancer. The aim of this study was to explore the effect of CXCL12-CXCR4 axis on the metastasis of human ovarian cancer. Methods The expressions of CXCR4 and CXCL12 mRNA and protein in human ovarian cancer cell line CAOV-3 was detected by RT-PCR and immunocytochemistry. Methythiazolyltetrazolium (MTT) was used to analyze the effect of different concentrations of CXCL12 on the proliferation of CAOV-3 cells. Transwell invasion chamber and matrigel were used to evaluate the effect of various concentrations of CXCL12 and ascites on the migration and invasion of CAOV-3 cells. The expressions of integrin β(1) and vascular endothelial growth factor-C (VEGF-C) mRNA were detected by RT-PCR. Data were analyzed using ANOVA by SAS 6.12.Results Under serum-free suboptimal culture conditions, CXCL12 (100 ng/ml) significantly enhanced the proliferation of CAOV-3 cells compared with the control and 10 ng/ml CXCL12 groups (0.428 ± 0.051 vs. 0.325 ± 0.045 and 0.328±0.039, P<0.05). This enhancing effect of CXCL12 was significantly inhibited by 10 μg/ml neutralizing CXCR4 antibody or 1 μg/ml CXCR4 antagonist AMD3100. However, 10 μg/ml neutralizing CXCR4 antibody could not inhibit cell proliferation without CXCL12. The levels of migration and invasion of the CAOV-3 cells treated with 100 ng/ml CXCL12 were significantly higher than those in the control (migration: 523.3 ± 25.2 vs 108.0 ± 7.2; invasion: 39.3 ± 4.0 vs. 4.0 ± 1.0). The enhancing effect of CXCL12 on cell migration and invasion increased with the concentration of CXCL12 (100 ng/ml vs10 ng/ml: migration, 523.3 ± 25.2 vs 211.7 ± 24.7; invasion, 39.3 ± 4.0 vs 15.7 ± 3.1, P<0.05), and was strongly inhibited by 10 μg/ml neutralizing CXCR4 antibody or 1 μg/ml AMD3100. The number of migrated and invading cells in the CAOV-3 added with ascites was significantly higher than those in the 100 ng/ml CXCL12 group (migration: 706.6 ± 30.6 vs 523.3 ± 25.2, invasion: 61.7 ± 7.6 vs 39.3 ± 4.0, P<0.05). The level of integrin β(1) mRNA was greatly increased at 3 hours after being treated with CXCL12 (0.53±0.10 vs. 1.53±0.16, P<0.05), and VEGF-C mRNA displayed significant augment at 24 hours after being treated with CXCL12 (0.52 ± 0.09 vs 1.11 ± 0.15, P<0.05).Conclusions CXCL12 and its receptor CXCR4 can promote the proliferation, migration, invasion of ovarian cancer cell line CAOV-3 and enhance its secretion of integrin β(1) and VEGF-C. These effects can be inhibited by neutralizing CXCR4 antibody or AMD3100. CXCL12-CXCR4 axis plays an important role in ovarian cancer growth and metastasis.     

趋化因子受体4在神经胶质瘤中的表达及血管生成中的作用

目的:探讨趋化因子受体4(chemokine receptor 4,CXCR4)在神经胶质瘤中的表达及在血管生成中的作用?方法:通过免疫组化法检测正常或神经胶质瘤患者的瘤体组织标本CXCR4?血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,其结果做方差分析及相关性分析;采用ELISA观察CXCR4的配体基质细胞衍生因子-1(stromal derived factor-1,SDF-1)对U87胶质瘤细胞系VEGF分泌的影响;同时利用多种处理方式(对照组?SDF-1处理组?CXCR4拮抗剂AMD3100处理组?CXCR4 RNA干扰处理组)作用于U87后,取其条件培养上清与人脐静脉内皮细胞系(human umbilical vein endothelial cells,HUVECs)共培养,比较小管样结构(tubule-like structure,TLS)生成数量的变化?结果:神经胶质瘤中CXCR4?VEGF的表达量随着肿瘤恶性度的升高而增加,且二者呈线性正相关(P < 0.01)?ELISA实验表明SDF-1可通过CXCR4促进VEGF的分泌(P < 0.01)?成管实验提示CXCR4与胶质瘤血管生成相关?结论:CXCR4与神经胶质瘤恶性度相关,且其配体SDF-1可通过CXCR4影响VEGF的表达,将CXCR4拮抗或干扰明显影响胶质瘤血管增生,提示CXCR4可能为神经胶质瘤的治疗提供相应靶点和思路?     

基质细胞衍生因子-1对人脐静脉血管内皮细胞迁移的影响

目的:利用Boyden chamber体外迁移体系初步探索基质细胞衍生因子-1α(SDF-1α)在人脐静脉血管内皮细胞(HUVEC)迁移中的作用及其信号转导机制。方法:以HUVEC为实验材料,首先利用免疫荧光技术及蛋白印迹方法检测HUVEC表达SDF-1α受体CXCR4,其次利用Boyden chamber体外迁移体系,先观察不同浓度的SDF-1α对HUVEC迁移的影响,最后以磷脂酰肌醇-3激酶(PI-3K)阻断剂wortmannin(50 M)或LY294002(10μM)、丝裂酶原活化蛋白激酶(MAPK)阻断剂PD98059(50μM)、磷脂酰肌醇特异性磷脂酶C(PI-PLC)阻断剂U73122(10μM)、CXCR4特异性阻断剂AMD3100(0.1 mg/ml)等不同预处理HUVEC 30 min,观察分析SDF-1α对HUVEC迁移影响的信号转导通路。结果:培养的HUVEC表达SDF-1α受体CXCR4,HUVEC体外迁移能力随着SDF-1α浓度的递增而逐渐增强,并且SDF-1α浓度在100 ng/ml时,HUVEC迁移到滤膜上的细胞数最多;Wort-mannin、LY294002、PD98059、U73122、AMD3100对HUVEC迁移均有影响,其中U73122、AMD3100对HUVEC迁移阻断的效应最显著。结论:SDF-1α/CXCR4所介导的HUVEC迁移与MAPK)、PI-PLC和蛋白激酶C(PKC)等信号途径有关,且PKC途径可能处于中心环节。     

基质细胞衍生因子-1通过促进C-kit表达提高小鼠心肌干细胞增殖和迁移能力的研究

目的：探讨基质细胞衍生因子-1（ SDF-1）提高小鼠心肌干细胞（ CSCs ）增殖和迁移能力的机制。方法：从小鼠心肌中分离、培养出CSCs ,并用免疫磁珠法筛选出c-kit （＋） CSCs ,用流式细胞仪检测CSCs表面标志：干细胞因子受体c-kit、干细胞抗原Sca-1。用SDF-1及SDF-1特异性拮抗剂AMD3100干预c-kit（＋） CSCs,qPCR及Western blotting检测c-kit的mRNA和蛋白表达,用CCK-8试剂盒及transwell趋化实验检测细胞增殖和迁移能力。结果：分离、培养出的细胞经免疫磁珠筛选后,流式细胞仪检测c-kit和Sca-1表达均大于80％。 SDF-1干预后, qPCR和Western blotting 结果显示c-kit mRNA 和蛋白表达均增高, CCK-8及transwell趋化实验结果显示SDF-1干预后细胞的增殖及迁移能力明显提高,并且可以被AMD3100拮抗。结论：SDF-1通过促进c-kit表达提高c-kit（＋） CSCs的增殖及迁移能力。     

Suppression of breast cancer proliferation and induction of apoptosis via AKT and ERK1/2 signal transduction pathways by synthetic polypeptide derived from viral macrophage inflammatory protein II

SDF-1α,a ligand for the chemokine receptor CXCR4,is well known for mediating the migration of breast cancer cells.In a previous study we demonstrated that a synthetic 21-mer peptide antagonist of CXCR4(NT21MP) derived from the viral macrophage inflammatory protein Ⅱ could antagonize tumor growth in vivo by inhibiting cellular proliferation and inducing apoptosis in breast cancer cells.However,the role of SDF-1α in the signaling pathways underlying the proliferation of human breast cancer cells and associated signaling pathways and inhibiting signal pathways of NT21MP remained unclear.The present study investigated the mechanism of NT21MP on anti-tumor in breast cancer in vitro.The effect of NT21MP on the viability of cells was determined by the MTT assay.Annexin V-FITC and PI staining was performed to detect early stage apoptosisin SKBR3 cells treated with SDF-1α and AMD3100 or NT21MP.Western blotting techniques were used to assay the composition of phosphoproteomics and total proteins present in the SKBR3 breast cancer cells.RT-PCR and Western blotting technique were used to detect the effect of NT21MP and AMD3100 on Bcl-2 and Bax expression.The results indicated that SDF-1α prevented apoptosis and promoted the proliferation of SKBR3 human breast cancer cells.As compared with untreated SKBR3 cells,Treatment with SDF-1α significantly increased cell viability,and NT21MP abolished the protective effects of SDF-1α dose-dependently(P<0.05).There was a significant decrease in the percentage of apoptotic cells after SDF-1α treatment as compared with control group(2.7%±0.2% vs.5.7%±0.4%,P<0.05).But pretreatment of SKBR3 cells with NT21MP significantly attenuated the antiapoptotic effects of SDF-1α as compared with SKBR3 cells without NT21MP pretreatment.The proliferative and anti-apoptotic effects of SDF-1α in SKBR3 cells were associated with an increase in AKT and ERK1/2 phosphorylation as well as a decrease in Bax expression and an increase in Bcl-2 expression.These changes in intracellular processes were blocked by NT21MP in a dose-dependent manner(P<0.05).In conclusion,NT21MP efficiently inhibits SDF-1α-induced proliferation and antiapoptosis in SKBR3 cells by reducing the levels of phosphorylated AKT and ERK1/2,as well as decreasing the ratio of expression of Bcl-2 relative to Bax.     

蜕膜自然杀伤细胞趋化因子受体的表达及募集机制的研究

目的　研究早孕期人蜕膜自然杀伤 (NK)细胞趋化因子受体谱的表达及其特异性募集的机制。方法　收集早孕期蜕膜组织 ,免疫磁珠分选CD5 6 brightCD16 -NK细胞 ;逆转录 聚合酶链反应(RT PCR)检测CD5 6 brightCD16 -NK细胞中 18种趋化因子受体的转录水平 ,筛选出高表达的趋化因子受体 (即CXCR4及CXCR3)。原位杂交及免疫组织化学分析CXCR4特异性配体基质细胞衍生因子 (SDF 1)在早孕胎盘的定位表达 ;ELISA检测早孕滋养细胞培养上清液中SDF 1α的浓度水平。趋化试验研究重组人SDF 1α(rhSDF 1α)及滋养细胞培养上清液对蜕膜CD5 6 brightCD16 -NK细胞的趋化作用。结果　人蜕膜CD5 6 brightCD16 -NK细胞可转录多种趋化因子受体 ,其中CXCR4及CXCR3mRNA呈高水平表达。人早孕期滋养细胞表达趋化因子SDF 1;原代培养的滋养细胞可自发分泌SDF 1α,培养 6 0h后上清液中SDF 1α的浓度达 385ng/L± 91ng/L。rhSDF 1α及滋养细胞培养上清液对蜕膜CD5 6 brightCD16 -NK细胞具有显著趋化作用。当rhSDF 1α为 10 μg/L时趋化至Transwell下室中的细胞可达总细胞数的2 2 9%± 4 3%。结论　人早孕期蜕膜CD5 6 brightCD16 -NK细胞高水平转录CXCR4 ,而滋养细胞则表达并分泌CXCR4的配体SDF 1。SDF 1趋化、募集CD5 6 br     

基质细胞衍生因子-1对骨髓源间充质干细胞迁移的影响

目的:利用Boyden chamber体外迁移体系初步探索基质细胞衍生因子-1(SDF-1)在骨髓源间充质干细胞(MSC)迁移中的作用及其信号转导机制。方法:采用经典的全骨髓贴壁法培养MSC,通过成骨、成脂肪等多向诱导分化以及流式细胞分析其表面标记(CD133、CD34、CD90、CD105)等鉴定MSC特征;以P3-MSC为实验材料,利用Boyden chamber体外迁移体系,先观察不同浓度的hSDF-1α对MSC迁移的影响,随后以50 M wortman-nin、10 uM LY294002、50 uM PD98059、10 uM U73122、0.1 mg/ml AMD3100等不同处理P3-MSC,观察最适宜浓度的hSDF-1α对MSC迁移影响的信号转导机制。结果:培养的MSC呈现出CD90、CD105强阳性,具有成骨、成脂肪等多向分化能力;MSC体外迁移能力随着hSDF-1α浓度(1 ng/ml、10 ng/ml、100 ng/ml、500 ng/ml)的递增而逐渐增强,并且hSDF-1α浓度在100 ng/ml时,MSC迁移到滤膜上的细胞数接近于锋值;Wortmannin、LY294002、PD98059、U70312、AMD3100对MSC迁移均有影响,其中U73122、AMD3100对MSC迁移阻断的效应最显著。结论:SDF-1/CXCR4所介导的MSC迁移与丝裂酶原活化蛋白激酶(MAPK)、磷脂酰肌醇特异性磷脂酶C(PI-PLC)和蛋白激酶C(PKC)等信号途径有关,且PKC途径可能处于中心环节。