摘要
脑梗死是中老年人致残的主要疾病之一,目前对脑梗死的治疗尚缺乏理想的方法。在脑缺血损伤模型中,通过动、静脉注射的骨髓间质干细胞(MSCs)都能够迁移、聚集到损伤部位表明局部缺血微环境可能表达某些信号以促使循环中的MSCs迁移到该区域。目前,大多数学者倾向于用基质细胞衍生因子-1( SDF-1)及其唯一特异性受体CXCR4的相互作用来解释这一现象。
SDF-1/CXCR4在骨髓干细胞的动员和归巢、内皮细胞的迁移、血管的发生、粘附分子表达、移植细胞增殖和存活方面起到重要作用。研究表明组织损伤后,损伤部位分泌的SDF-1与血循环中MSCs表达的CXCR4相互作用对MSCs产生向损伤组织的趋化聚集,在移植细胞向损伤部位迁移中发挥重要作用。由于CXCR4在MSCs的表达率比较低,可能影响其向损伤部位迁移。因此我们设想通过转基因技术将外源性CXCR4基因导入MSCs,并推测提高CXCR4在MSCs的表达率能增强MSCs向脑梗死区组织的趋化聚集。
因此本实验构建表达CXCR4的慢病毒载体,并转导rMSCs,采用体内、体外实验观察过表达CXCR4基因的rMSCs是否具有较高的细胞迁移率,及过表达CXCR4基因的rMSCs移植治疗脑梗死是否有更好效果。本课题分四部分。
第一部分:CXCR4重组慢病毒载体(pNL-CXCR4-IRES2-EGFP)的构建及鉴定
1、材料和方法
1)根据Genbank大鼠CXCR4编码区序列全长及两端酶切位点需要设计上下游引物;
2)用RT-PCR方法从大鼠肝脏获取CXCR4的cDNA;
3)双酶切将pNL- IRES2-EGFP慢病毒载体的线性化与含有CXCR4基因片段连接,转化,小量抽提质粒DNA,进行双酶切、PCR及测序鉴定。
2、结果
1)酶切鉴定和测序都证实pNL-CXCR4-IRES2-EGFP质粒构建正确。
3、结论
1)成功构建大鼠CXCR4基因重组慢病毒载体(pNL-CXCR4-IRES2- EGFP)。
第二部分:建立过表达CXCR4骨髓间充质干细胞系(CXCR4-rMSCs)
1、材料和方法
1)将三质粒pNL-CXCR4-IRES2- EGFP、pHELPER和pVSVG共转染293T细胞,包装生产慢病毒;
2)经超速离心收集病毒颗粒,转导293T细胞,用流式细胞术测定EGFP蛋白表达率来测定慢病毒功能滴度;
3)用相同滴度的转CXCR4基因慢病毒、空载体慢病毒、CXCR4基因沉默慢病毒(由本课题组的前期研究所构建)转导rMSCs,建立稳定过表达CXCR4基因细胞系CXCR4-rMSCs;
4)用RT-PCR、Western Blot、细胞免疫荧光组织化学和流式细胞术检测转CXCR4基因rMSCs组(CXCR4-rMSCs组)、转空载体rMSCs组(null-rMSCs组)及CXCR4基因沉默rMSCs组(siRNA-rMSCs组中CXCR4表达情况。
2、结果
1)三质粒pNL-CXCR4-IRES2-EGFP、pHELPER和pVSVG共转染293T细胞,包装产生慢病毒。
2)转CXCR4基因、空载体及CXCR4基因沉默rMSCs强烈表达EGFP。
3) RT-PCR、Western Blot、细胞免疫荧光组织化学和流式细胞术一致证实CXCR4-rMSCs组强烈表达CXCR4,siRNA-rMSCs组的CXCR4表达明显被抑制。
3、结论
1)成功建立稳定过表达CXCR4骨髓间充质细胞系(CXCR4-rMSCs)。
第三部分:SDF-1α对rMSCs迁移作用的体外研究
1、材料和方法
1)体外实验在内置8μm孔径聚碳酸酯膜的48孔微迁移板中进行,趋化因子SDF-1α以不同浓度加入到迁移板下室,CXCR4-rMSCs、null-rMSCs及siRNA-rMSCs加入到上室,观察细胞迁移情况,计算细胞迁移指数;
2)抗CXCR4多抗阻断后各组rMSCs,观察细胞迁移情况,计算细胞迁移指数。
2、结果
1) SDF-1α可明显促进rMSCs的跨膜迁移,而且在一定浓度范围随着SDF-1α浓度梯度增高而增强。
2)相同浓度SDF-1α作用下CXCR4-rMSCs跨膜细胞迁移指数明显高于null-rMSCs及siRNA-rMSCs。
3) CXCR4-rMSCs及null-rMSCs抗体阻断后迁移指数明显下降,与抗体阻断前差别有显著性意义;siRNA-rMSCs抗体阻断前与抗体阻断后迁移指数无显著改变。
3、结论
1)体外微迁移板实验表明,表达于rMSCs表面的CXCR4在SDF-1α介导rMSCs跨膜迁移中起很重要作用。
第四部分:rMSCs静脉移植后的迁移、分化及对脑梗死保护作用的研究
1、材料和方法
1)取健康成年雄性SD大鼠分为四组①CXCR4-rMSCs移植组;②null-rMSCs移植组;③siRNA-rMSCs移植组;④PBS组,在MCAO后24h将各组rMSCs或PBS经股静脉注入大鼠体内,在MCAO后7天观察以下各指标;
2)体视荧光显微镜下观察EGFP+细胞的分布情况;
3) TTC染色测定脑梗死体积;
4)检测各组大鼠神经功能(mNSS)评分的变化;
5)免疫荧光组化观察CXCR4表达、神经元、神经胶质细胞及内皮细胞表面标志观察;
6)激光扫描共聚焦显微镜观察梗死灶周边区脑血流灌注量。
2、结果
1)移植EGFP+ rMSC主要存在于大鼠的脑梗死半球大脑皮质、皮质下和海马等处,对侧半球只可见少量EGFP+细胞。CXCR4-rMSCs移植组脑梗死半球特别在缺血半暗带区EGFP+ rMSC明显多于其他各组,siRNA-rMSCs移植组脑梗死半球缺血区EGFP+ rMSC明显少于null-rMSCs移植组,PBS组大鼠大脑两侧未发现EGFP+细胞聚集。
2) MCAO后7天CXCR4-rMSCs和null-rMSCs两移植组脑梗死体积均低于PBS组及siRNA-rMSCs移植组,差异有显著性意义,且CXCR4-rMSCs移植组脑梗死体积明显低于null-rMSCs移植组,两移植组间差别亦有显著性意义。
3) MCAO后7天CXCR4-rMSCs和null-rMSCs移植组mNSS评分均低于PBS组及siRNA-rMSCs移植组,差异有显著性意义,且CXCR4-rMSCs和null-rMSCs两移植组间mNSS评分差别亦有显著性意义,CXCR4-rMSCs移植组功能恢复明显优于null-rMSCs移植组。
4)大部分移植EGFP阳性细胞表达CXCR4蛋白,小部分移植EGFP阳性细胞表达神经元标记物NSE、星形胶质细胞标记物GFAP或血管内皮细胞标记物vWF。
5) MCAO后7天CXCR4-rMSCs和null-rMSCs两移植组梗死灶周边区脑血流灌注量均高于PBS组及siRNA-rMSCs移植组,差异有显著性意义,CXCR4-rMSCs移植组梗死灶周边区脑血流灌注量明显高于null-rMSCs移植组,两移植组间差别亦有显著性意义。
3、结论
1)脑梗死后脑组织局部产生的SDF-1α和表达于rMSCs表面的CXCR4在介导rMSCs向损伤组织迁移中起很重要作用。
2)过表达CXCR4基因rMSCs能明显增强rMSCs向损伤组织迁移,增加梗死灶周边区脑血流灌注量,从而增进脑梗死后神经功能的恢复。
Ischemic stroke remains a leading cause of adult disability and no pharmaco- logical treatment is presently available to protect brain tissue from the injury that are arosed by ischemia and reperfusion. Accumulated studies suggest that bone marrow mesenchymal stem cells (MSCs) participate in neuroprotection following Stroke. Increasing evidences suggest that systemically transplanted MSCs can survive, migrate toward injured tissue, and promote recovery of neurological function following cerebral infarction. It seems that the injured tissues can attract MSCs and mediate their migration behavior. However, the mechanisms regulating MSCs migration and accumulation in the injured brain remain to be revealed.
Stromal cell-derived factor-1(SDF-1) and its unique receptor, CXCR4 play an important role in stem cell migration, chemotaxis, expression of adhesion molecules, engraftment, proliferation, and survival. Recent studies have shown that SDF-1 is expressed in the ischemic boundary zone of the brain and plays an important role in the migration of the transplanted cells. Due to low native levels of CXCR4 expression in MSCs, they migrate sluggishly toward injured tissue. We hypothesized that CXCR4 gene-modified MSCs will promote stem cell recruitment and injured brain tissue regeneration.
In this study, we constructed the lentiviral vectors (LV)carrying the CXCR4 and genetically engineered rMSCs overexpressing CXCR4, and focused on that over-expression of CXCR4 in rMSCs will enhance their engraftment and protect brain subjected to MCAO. This study consisted of four parts.
Part1:Construction and identification of the lentiviral vector carrying CXCR4
Materials and Methods
1) The total RNA was isolated from SD rat's liver with Trizol.
2) The CXCR4 gene was amplified by RT-PCR.
3) CXCR4 was inserted into the transfer vector of lentivirus after being digested with restriction endonuclease.
4) Then the product pNL-CXCR4-IRES2-EGFP was confirmed by sequencing and being digested with restriction endonuclease.
Results
1) The result of restriction enzyme digestion and sequencing showed that the full-length fragment of CXCR4 gene was successfully cloned into the transfer vector of lentivirus.
Conclusions
1) The lentiviral vectors carrying the CXCR4 gene were successfully constructed.
Part2:Construction of rMSCs over-expressing CXCR4 Materials and Methods
1) pNL-CXCR4-IRES2-EGFP was cotransfected along with pHELPER and pVSVG into 293T to package lentivirus particles.
2) According to the enhanced green fluorescent protein (EGFP) expression,the functional titer was determined by flow cytometry after transduction into 293T cells.
3) Lentiviral transduction was carried out to over-express either CXCR4/EGFP (CXCR4-rMSCs group), siRNA targeting CXCR4/EGFP (siRNA-rMSCs group) or EGFP alone (null-rMSCs group) in rMSCs. The rMSCs were selected for stable integrants by using EGFP reporter gene.
4) The expressing of CXCR4 gene in rMSCs was evaluated with RT-PCR, Western blotting, cellular immunofluorescence and flow cytometry.
Results
1) Lentiviral vector can be packaged in 293T cells by cotransfection.
2) The rMSCs from CXCR4-rMSCs group, siRNA-rMSCs group or null-rMSCs group strongly expressed EGFP.
3) The result of FCM showed that CXCR4 expression was significantly higher in CXCR4-rMSCs group as compared with that of null-rMSCs group, and was lowest in siRNA-rMSCs group. The result was confirmed by RT-PCR, Western blotting and cellular immunofluorescence.
Conclusions
1) The rMSCs overexpressing the CXCR4 gene were successfully constructed. Part3:In vitro migration assay of rMCSs induced by SDF-1
Materials and Methods
1) In vitro migration of rMSCs in response to SDF-1αwas assessed in a 48-well microchemotaxis chamber using polycarbonate membranes with 8μm pore size. The rMSCs were added to the upper chambers. SDF-1αwas added to the lower wells in different concentrations. The migrated cells were counted and the migration index was calculated to show the difference of migration.
2) The rMSCs were incubated by anti CXCR4 monoclonal antibody then the chemotaxis assay was performed..
Results
1) The result showed that exposure of rMSCs to SDF-1αcaused a robust cell migration in a concentration dependent manner.
2) The number of migrated rMSCs was significantly higher in CXCR4 -rMSCs group than that in null-rMSCs group. However, the migration of the rMSCs from the siRNA-rMSCs group in response to SDF-1αwas blocked.
3) The migration of the rMSCs incubated by anti CXCR4 monoclonal antibody in response to SDF-1αwas blocked
Conclusions
1) Over expression of CXCR4 enhances the ability of rMSCs to respond to SDF-1 induced chemotaxis.
Part4:Migration, differentiation, neuroprotection and angiogenesis of donor rMSCs in stroke rats
Materials and Methods
1) The healthy adult male SD rats were divided into four groups: CXCR4-rMSCs group, siRNA-rMSCs group, null-rMSCs group and PBS group.Rats were received rMSCs or PBS transplantation via femoral vein injection 24 hours after the left middle cerebral artery occlusion (MCAO).
2) 7 days after MCAO, the distribution of EGFP+ cells was observed under the fluorescent microscope.
3) The total infarct volumes were calculated by 2,3,5- triph-enyltetrazolium chloride (TTC) stain.
4) Behavioral tests ( modified Neurological Severity Score [mNSS]) was performed 1 and 7 days after MCAO.
5) The expressing of CXCR4, neuronal marker NSE, astrocytic marker GFAP, and vascular phenotypes (vWF) in EGFP+ rMSCs was evaluated with immunohistochemical fluorescence.
6) The volumes of the microvessels were analysed with a laser scanning confocal imaging system .
Results
1) The EGFP-positive cells were found in multiple areas of the ipsilateral hemisphere including cortex, striatum, and few cells were observed in the contralateral hemisphere. The number of migrating rMSCs to damaged brain area especially in ischemic boundary zone was significantly higher in CXCR4-rMSCs group as compared with null-rMSCs group. However, the number of EGFP-positive cells was significantly reduced in siRNA-rMSCs group as compared with null-rMSCs group.
2) The average infarct volumes were significantly reduced in the CXCR4-rMSCs and null-rMSCs groups compared with those of the PBS and siRNA -rMSCs groups. The infarct volume was significantly decreased in the CXCR4 -rMSCs group as compared with the null-rMSCs group. There was no significant difference found between the infarct volumes of the siRNA -rMSCs and the PBS group..
3) On 7th day, the mNSS scores of the null-rMSCs and CXCR4-rMSCs groups were significantly decreased compared with those of the siRNA-rMSCs and PBS groups. In addition, the score of the CXCR4-rMSCs group was significant decrease compared with that of the null-rMSCs group. There was no significant difference between the sores of the siRNA-rMSCs and PBS group.
4) Double-label fluorescence immunohistochemistry of brain sections of CXCR4-rMSCs group revealed that a number of EGFP-positive cells in the cerebral cortex, were neuronal marker NSE and astrocytic marker GFAP positive, and several EGFP positive cells showed vascular phenotypes (vWF) positive. Most of EGFP positive cells were CXCR4 positive.
5) The result of three-dimensional image acquisition of brain slices indicated that the capillary vascular volume ratios were significantly higher in both null-rMSCs and CXCR4-rMSCs groups as compared with those in the PBS and siRNA-rMSCs groups, and the ratio in the CXCR4-rMSCs group was significantly higher than that in the null-rMSCs group. There was no significant difference found between the siRNA-rMSCs and the PBS group.
Conclusions
1)The interaction of locally produced SDF-1αand CXCR4 expressing on the rMSCs surface plays an important role in the migration of transplanted cells to infarcted brain.
2)Overexpressing of CXCR4 in rMSCs was extremely effective in their engraftment in the infarcted brain for post-infarction recovery of neural function.
引文
1 Kraitchman DL, Tatsumi M, Gilson WD, et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction[J]. Circulation, 2005, 112: 1451–1461.
2 Donald G, Phinney, Iryna I. Plasticity and therapeutic potential of mesenchymal stem cells in the nervous system[J]. Cur Pharma Design, 2005, 11:1255-1265
3 Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors [J]. Science, 1998, 279: 1528-1530.
4 Chen J, Li Y, Wang L, et a1.Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats[J]. J Neurol Sci, 2001, 189: 49-57.
5 Shen LH, Li Y, Chen J, et al. Therapeutic benefit of bone marrow stromal cells administered 1 month after stroke [J]. J Cereb Blood Flow Metab. 2007, 27: 6-13.
6 Masaaki H, Satoshi K, Hideo S, et al. Bone marrow stromal cells protect and repair damaged neurons through multiple mechanisms[J]. J Neuro Res, 2008,86: 1024-1035.
7 Phinney DG, Building a consensus regarding the nature and origin of mesen- chymal stem cells [J]. J Cell Biochem Suppl, 2002, 38: 7–12.
8 Shyu WC, Lee YJ, Liu DD, et al. Homing genes, cell therapy and stroke [J]. Front Biosci, 2006, 11: 899-907.
9 Picinich SC, Mishra PJ, Mishra PJ, et al. The therapeutic potential of mesenchymal stem cells: Cell- & tissue-based therapy [J]. Expert Opin Biol Ther, 2007, 7: 965-973.
10 Fox JM, Chamberlain G, Ashton BA, et al. Recent advances into the understanding of mesenchymal stem cell trafficking[J]. Br J Haematol, 2007, 137: 491-502.
11 Chen J, Li Y, Wang L, et a1. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats[J]. Stroke, 2001, 32: 1005-1011.
12 Mahmood, A, Lu, D, Wang, L, et al. Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells[J]. Neurosurgery, 2001, 49: 1196-1204.
13 Abbott JD, Huang Y, Liu D, et a1. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence ofinjury [J]. Circulation, 2004, 110(21): 3300-3305.
14 Kollet O, Shivtiel S, Chen YQ, et a1. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver[J]. J Clin Inest, 2003, 112(2): 160-169.
15 Hattori K, Heissig B, Rafii S. The regulation of hematopoietic stem cell and progenitor mobilization by chemokine SDF-1[J]. Leuk Lymphoma, 2003, 44: 575-582.
16 Salvucci O, Yao L, Villalba S, et al. Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal-derived factor-1[J]. Blood, 2002, 99: 2703-2711.
17 Yamaguchi J, Kusano KF, Masuo O, et al. Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization[J]. Circulation, 2003, 107: 1322-1328.
18 Egawa T, Kawabata K, Kawamoto H, et al. The earliest stages of B cell development require a chemokine stromal cell - derived factor pre - B cell growth- stimulating factor[J]. Immunity, 2001, 15 (2): 323-334
19 Zhang XF, Wang JF, Matczak, et a1. Janus kinase 2 is involved in stromal cell-derived factor-l alpha-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoietic progenitor cells [J]. Blood, 2001, 97(11): 3342-3348.
20 Ding Z, Issekutz TB, Downey GP, et al L-selectin stimulation enhances functional expression of surface CXCR4 in lymphocytes: implications for cellular activation during adhesion and migration. [J] . Blood, 2003, 10l(11): 4245-4252.
21 Bhakta S, Hong P, Koc O. The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromalcell-derived factor-1 in vitro butdoes not decrease apoptosis under serum deprivation[J]. Cardiovasc Revasc Med, 2006, 7: 19–24.
22 Wojakowski W, Tendera M, Michalowska A, et al. Mobilization of CD34/ CXCR4+, CD34/CD117+, c-met+ stemcells, and mononuclear cells expressing early cardiac, muscle, and endothelial markers into peripheral blood in patients with acute myocardial infarction [J]. Circulation, 2004,110(20): 3213-3220.
23 Askari AT, Unzek S, Popovic ZB, et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy [J]. Lancet, 2003, 362: 697-703.
24 Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a condition packaging system [J]. Viology, 1998, 72(11): 8463-8471.
25 Federico M. Lentiviruses as gene delivery vectors[J]. Curr Opin Biotechnol, 1999, 10(5): 448-453.
26 Kafri T, van Praag H. Lentiviral vectors: regulated gene expression[J]. Mol Ther, 2000, 6: 516-521.
27 Bleul C, Farzan M, Choe H, et a1. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature, 1996a, 382: 829-833.
28 Oberlin E, Amara A, Bachelerie F, et a1. The CXC chemokine SDF-1 is the ligand for LESTR / fusin and prevents infection by T-cell-line-adapted HIV-1[J]. Nature, 1996, 382: 833-835.
29 Horuk R. Chemokines beyond inflamation[J]. Nature, 1998, 393(6685): 524- 525.
30 Vaday GG, Lidey O. Extracellular matrix motictis, Cytokines, and enzymes, dynamic effects on immune cell behavior and inflammation [J]. J Leukoe Biol, 2000, 67: 149-159.
31 Taichman RS, Cooper C, Keller ET, et al. Use of the stromal cell-derived factor-1/ CXCR4 pathway in prostate cancer metastasis to bone[J]. Cancer Res, 2002, 62: 1832-1837.
32 Kucia M, Reca R, Miekus K, et a1. Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1/CXCR4 axis[J]. Stem Cells, 2005, 23 (7): 879-894.
33 Zhang D, Fan GC, Zhou X , et al. Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium[J]. J Mol and Cell Cardio, 2008, 44: 281–292.
34陈东平,张志坚,吴秀丽,等. SDF-1/CXCR4增强骨髓间质干细胞的造血支持作用[J].基础医学与临床, 2008, 28(2): 123-127.
35 Lataillade JJ, Clay D, Dupuy C, et al. Chemokine SDF- 1 enhances circulating CD34+ cell proliferation in synergy with cytokines possible role in rogenitor survival [J]. Blood, 2000, 95: 756-768.
36 Neuhaus T, Stier S, Totzke G, et al. Stromal cell-derived factor1 (SDF-1) induces gene-expression of early growth response-1 (Egr-1) and VEGF in human arterial endothelial cells and enhances VEGF induced cell proliferation [J]. Cell Prolif , 2003, 36: 75–86.
37屈伸,刘志国等.分子生物学实验技术.北京:化学工业出版社, 1999, 137-160.
38 Inder MV, Nikaj S. Gene therapy - promises, problems and prospects[J]. Nature, 1997, 389: 239-241
39 Miller AD. Cell-surface receptors for retroviruses and implications for gene transfer[J]. Proc Natl Acad Sci, 1996, 93 (11): 407-412.
40 Xu K, Ma H, McCown TJ, et al.Generation of a stable cell line producing high-titer self- inactivating lentiviral vectors[J]. Mol Ther, 2001, 3(1): 97-104.
41 Buchschacher GL, Wong-Staal F. Development of lentiviral vectors for gene therapy for human diseases [J]. Blood, 2000, 95(8): 2499-2504.
42 Zufferey R, Nagy D, Mandel RJ, et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo [J]. Nat Biotedmol, 1997, 15(9): 871-875.
43 Friedenstein A J. Precursor cells of mechanocytes[J]. Int Revcytol, 1976, 47: 327-355.
44 Haynesworth SE, Goshima J, Goldberg VM, et a1. Characterization of cells with osteogenic potential from human bone marrow[J]. Bone, 1992, l3: 8l-88
45 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284: 143-147.
46 Lou S, Gu P, Chen F, et al. The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague-Dawley rats [J]. Brain Res, 2003, 968: 114-121.
47 Migliaccio AR, Quarto R, Piacibello W. Cell therapy: filling the gap between basic science and clinical trials[J]. Stem Cells, 2003, 21: 348-356.
48 Shichinohe H, Kuroda S, Lee JB, et al. In vivo tracking of bone marrow stromal cells transplanted into mice cerebral infarct by fluorescence optical imaging[J]. Brain Res Protoc, 2004, 13: 166-175.
49 Ratajczak MZ, Kuczynski WI, Sokol DL, et al. Expression and physiologic significance of Kit ligand and stem cell tyrosine kinase-1 receptor ligand in normal human CD34+, c-Kit+ marrow cells[J]. Blood, 1995, 86: 2161-2167.
50 Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow: Analysis of precursor cells for osteogenic and hematopoietic tissues[J]. Transplantation, 1968, 6: 230-247.
51 Zohar R, Sodek J, McCulloch CA, et al. Characterization of stromal progenitor cells enriched by flow cytometry[J]. Blood, 1997, 90: 3471-3481.
52 Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells [J]. J Cell Physiol, 1998, 176: 57-66.
53. Majumdar MK, Keane-Moore M, Buyaner D, et al. Characterization and functionality of cell surface molecules on human mesenchymal stem cells [J]. J Biomed Sci, 2003, 10: 228-241.
54 Ruster B, Gottig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells [J]. Blood, 2006, 108: 3938-3944.
55 Beyer Nardi N, da Silva Meirelles L. Mesenchymal stem cells: isolation, in vitro expansion and characterization [J]. Handb Exp Pharmacol, 2006: 249-282.
56 Dominici M, Le Blanc K, Mueller I, et a1. Minimal cntena for defitning multipotent mesenchymal stromal cells[J]. Cyto Ther, 2006, 8(4): 315-317.
57 Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets[J]. Blood, 2005, 106: 419-427.
58 Ji JF, He BP, Dheen ST, et al. Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury[J]. Stem Cells, 2004, 22: 415-427.
59 Honczarenko M, Le Y, Swierkowski M, et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors[J]. Stem Cells, 2006, 24: 1030-1041.
60 Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymalstem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow[J]. Blood, 2004, 104: 2643-2645.
61 Wang Y, Deng Y, Zhou GQ, et al. SDF-1α/CXCR4-mediated migration of systemically transplanted bone marrow stromal cells towards ischemic brain lesion in a rat model[J]. Brain Res, 2008, 1195: 104-112.
62 Wang L, Li Y, Chen X, et al. MCP-1, MIP-1, IL-8 and ischemic cerebral tissue enhance human bone marrow stromal cell migration in interface culture[J]. Hematology, 2002, 7: 113-117.
63 Cui X, Chen J, Zacharek A, et al. Nitric oxide donor upregulation of stromal cell-derived factor-1/chemokine (CXC motif) receptor 4 enhances bone marrow stromal cell migration into ischemic brain after stroke[J]. Stem Cells, 2007, 25: 2777-2785.
64 Swedlow JR, Goldberg L, Brauner E, et al. Informatics and quantitative analysis in biological imaging [J]. Science, 2003, 300(56l6): 100-102.
65 Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression [J]. Science, 1994, 263: 802-805.
66 Riede CL, Khodjakov A. Mitosis throught the microscope: advances in seeing inside live dividing cells [J]. Science, 2003, 300(56l6): 91-96.
67 Olson TS, Ley K. Chemokines and chemokine rceptors in leukocyte trafficking[J]. AM J Physiol, 2002, 283 (1): 7-28.
68 Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity [J]. Immunity, 2000, 12: 121-127.
69 Nagasawa T, Kikutani H, Kishimoto T. Molecular cloning and structure of a pre-B-cell growth-stimulating factor[J]. Proc Natl Acad Sci, 1994, 91(6): 2305-2309.
70 Shirozu M, Nakano T, Inazawa J, et al. Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene[J]. Genomics, 1995, 28(3): 495-500.
71 Gear AR, Suttitanamongkol S, Viisoreanu D, et al. Adenosine diphosphate strongly potentiates the ability of the chemokines MDC, TARC, and SDF-1 to stimulate platelet function[J]. Blood, 2001, 97(4): 937-945.
72 Bleul CC, Fuhlbrigge RC, Casasnovas JM, et al. A highly efficacious lymphocyte chemoattractant,stromal cell-derived factor 1(SDF-1)[J]. J Exp Med, 1996, 184(3): 1101-1109.
73 Bleul CC, Farzan M, Choe H, et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry[J]. Nature, 1996, 382(6594): 829-833.
74 Loetseher P, Gong J, Dewald B, et al. N-terminal peptides of stromal cell-derived factor-1 with CXC chemokin receptor 4 agonist and antagonist activities [J]. J Biol Chem, 1998, 27(35): 22279-22283.
75 Sallusto F, Baggiolini M. Chemokines and leukocyte traffic [J]. Nat Immunol, 2008, 9: 949-952.
76 Lavi E, Strizki JM, Ulrich AM, et al. CXCR-4 (Fusin), a co-receptor for the type 1 human immunodeficiency virus (HIV-1), is expressed in the human brain in a variety of cell types, including microglia and neurons[J]. Am J Pathol, 1997, 151(4): 1035-1042.
77 Tanabe S, Heesen M, Yoshizawa I, et al. Functional expression of the CXC-chemokine receptor-4/fusin on mouse microglial cells and astrocytes[J]. J Immunol, 1997, 159(2): 905-911.
78 Roland J, Mushy BJ, Ahr B, et a1. Role of the intraeellular domains of CXCR4 in SDF-1 mediated signaling[J]. Blood, 2003, l0(2): 399-406.
79 Liles WC, Broxmeyer HE, Rodger E, et al. Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD a CXCR4 antagonist [J]. Blood, 2003, 102: 2728-2730.
80 Peled A, Kollet O, Ponomaryov T, et a1. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice [J]. Blood, 2000, 95 (11): 3289-3296.
81 Odemis V, Moepps B, Gierschik P, et al. Interleukin-6 and cAMP induce stromal cell-derived factor-1 chemotaxis in astroglia by up-regulating CXCR4 cell surface expression: Implications for brain inflammation[J]. J Biol Chem, 2002, 277(42): 39801-39808.
82 Hideo S, Satoshi K, Shunsuke Y, et a1. Role of SDF-1/CXCR4 system in survival and migration of bone marrow stromal cells after transplantation into mice cerebral infarct[J]. Brain Res, 2007, 1183: 138-147.
83 Bhakta S, Hong P, Koc O. The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro butdoes not decrease apoptosis under serum deprivation [J]. Cardiovasc Revasc Med, 2006, 7: 19-24.
84陈东平,张志坚,吴秀丽,等.大鼠CXCR4基因RNAi慢病毒载体的构建及其在骨髓间质干细胞中的表达[J].生物工程学报. 2009, 25(2): 299-305.
85 Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20: 84-91.
86 Zhang ZG, Bower L, Zhang RL, et al. Three-dimensional measurement of cerebral microvascular plasma perfusion, glial fibrillary acidic protein and microtubule associated protein-2 immunoreactivity after embolic stroke in rats: a double fluorescent labeled laser-scanning confocal microscopic study[J]. Brain Res, 1999, 844: 55-66.
87 Ruster B, Gottig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells [J]. Blood, 2006, 108: 3938-3944.
88 Zhao MZ, Nonoguchi N, Ikeda N, et al. Novel therapeutic strategy for stroke in rats by bone marrow stromal cells and ex vivo HGF gene transfer with HSV-1 vector [J]. J Cereb Blood Flow Metab, 2006, 26: 1176-1188.
89 Horita Y, Honmou O, Harada K, et a1. Intravenous administration of GDNF gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat[J]. J Neurosci Res, 2006, 84(7): 1495-1504.
90 Liu H, Honmou O, Harada K, et a1. Neuroprotection by PIGF gene modifled human mesenchymal stem cells after cerebral ischemia[J]. Brain, 2006, 129(10): 2734-2745.
91 Zhao LX, Zhang J, Cao F, et a1. Modification of the brain-derived neurotrephic factor gene: a portal to transform mesenchymal stem cells into advantageous engineering cells for neuroregeneration and neuroprotection [J]. Exp Neural, 2004, 190(2): 396-406.
92 Wei L, Erinjeri JP, Rovainen CM, et a1. Collateral growth and angiogenesis around cortical stroke[J]. Stroke, 2001, 32: 2179-2184.
93 Hess DC, Hill WD, Martin-Studdard A, et a1. Bone marrow as a source of endothelial cells and neunoexpressing cells after stroke[J]. Stroke, 2002, 33: 1362-1368.
94 Li Y, Chen J, Chen XG, et a1. Human marrow stremal cell therapy for stroke in rat: neuretrophins and functional recovery[J]. Neurology, 2002, 59(4): 514-523.
95 Chen J, Sanberg PR, Li Y, et al. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats[J]. Stroke, 2001, 32: 2682-2688.
96 Stumm RK, Rummel J, Junker V, et al. A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia[J]. J Neurosci, 2002, 22: 5865-5878.
97 Hill WD, Hess DC, Martin-Studdard A, et al. SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury[J]. J Neuropathol Exp Neurol, 2004, 63: 84-96.
98 Wang Y, Deng Y, Zhou GQ. SDF-1alpha/CXCR4-mediated migration of systemically transplanted bone marrow stromal cells towards ischemic brain lesion in a rat model[J]. Brain Res, 2008, 1195: 104-112.
99 Ceradini DJ, Kulkarni AR, Callaghan MJ, et a1. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1[J]. Nat Med, 2004, 10(8): 858-864.
100 Miller JT, Bartley JH, Wimborne HJ, et a1. The neuroblast and angioblast chemotaxic factor SDF-1 (CXCL12) expression is briefly up regulated by reactive astrocytes in brain following neonatal hypoxic–ischemic injury [J]. BMC Neurosci, 2005, 6: 63-72.
101 Shyu WC, Lin SZ, Yen PS, et al. Stromal cel1 derived factor-1 alpha promotes neuropotection, angiogenesis,and mobilization/homeing of bone marrow- derived cells in stroke rats [J]. J Pharmacol Exp Ther. 2008, 324(2): 834-849.
102 Kollet O, Shivtiel S, Chen YQ, et a1. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver[J]. J Clin Inest, 2003, 112(2): 160-169.
103 Liu H, Honmou O, Harada K, et a1. Neuroprotection by PIGF gene-modifled human mesenchymal stem cells after cerebral ischemia[J]. Brain, 2006, 129(10): 2734-2745
104 Ryo Ukai, Osamu Honmou, Kuniaki Harada, et a1. Mesenchymal stem cellsderived from peripheral blood protects against ischemia[J]. J Neurotra, 2007, 24(3): 508-520.
105 Kanda S, Mochizuki Y, Kanetake H. Stromal cell derived factor-1 alpha induces tube-like structure formation of endothelial cells through phosphoinositide 3-kinase[J]. J Boil Chem, 2003, 278(1): 257-262.
1 Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic transplantsof bone marrow: analysisof precursor cells for osteogenic and hematopoietic tissues[J]. Trans Plantation, 1968, 6: 230-247
2 Li WJ,Tuli R,Okafor C,et a1.A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells[J]. Biomaterials, 2005, 26: 599-609.
3 Olson TS, Ley K. Chemokines and chemokine rceptors in leukocyte trafficking. J Physiol Regul Integr Comp, 2002, 283 (1): 7-28.
4 Nagasawa T, Kikutani H, Kishimoto T. Molecular cloning and structure of a pre-B-cell growth-stimulating factor[J]. Proc Natl Acad Sci, 1994, 91(6): 2305-2309.
5 Shirozu M, Nakano T, In,azawa J, et al. Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene[J]. Genomics, 1995, 28(3): 495-500.
6 Gleichmann M, Gillen C, Czardybon M, et al. Cloning and characterization of SDF-1 gamma, a novel SDF-1 chemokine transcript with developmentally regulated expression in the nervous system[J]. Eur J Neurosci, 2000, 12(6): 1857-1866.
7 Gear AR, Suttitanamongkol S, Viisoreanu D, et al. Adenosine diphosphate strongly potentiates the ability of the chemokines MDC, TARC, and SDF-1 to stimulate platelet function[J]. Blood, 2001, 97(4): 937-945.
8 Bleul CC, Fuhlbrigge RC, Casasnovas JM, et al. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor-1[J]. J Exp Med, 1996, 184(3): 1101-1109.
9 Sallusto F, Baggiolini M. Chemokines and leukocyte traffic. Nat Immunol, 2008, 9: 949-952.
10 Bleul CC, Farzan M, Choe H, et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry[J]. Nature, 1996, 382(6594):829-833.
11 Loetseher P, Gong J, Dewald B, et al. N-terminal peptides of stromal cell- derived factor-1 with CXC chemokin receptor 4 agonist and antagonist activities [J]. J Biol Chem, 1998, 27(35): 22279-22283.
12 Horuk R. Chemokines beyond inflammation[J]. Nature, 1998, 393: 524-525.
13 Peled A, Petit I, Kollet O, et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4[J]. Science, 1999, 283: 845-848.
14 Hesselgesser J, Halks-Miller M, DelVecchio V, et al. CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons[J]. Curr Biol, 1997, 7: 112-121.
15 Taichman RS, Cooper C, Keller ET, et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone[J]. Cancer Res, 2002, 62: 1832-1837.
16 Roland J, Mushy BJ, Ahr B, et a1. Role of the intraeellular domains of CXCR4 in SDF-1 mediated signaling[J]. Blood, 2003, l0(2): 399-406.
17 Peled A, Kollet O, Ponomaryov T,et a1.The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood, 2000, 95 (11): 3289-3296.
18 Baggiolini M. Chemokines and leukocyte traffic. Nature, 1998, 392: 565-568.
19 Odemis V, Moepps B, Gierschik P, et al. Interleukin-6 and cAMP induce stromal cell-derived factor-1 chemotaxis in astroglia by up-regulating CXCR4 cell surface expression: Implications for brain inflammation[J]. J Biol Chem, 2002, 277(42): 39801-39808.
20 Zhang D, Fan GC, Zhou X , et al.Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium [J]. J Mol and Cell Cardio, 2008, 44: 281-292.
21 Hideo S, Satoshi K, Shunsuke Y, et a1. Role of SDF-1/CXCR4 system in survival and migration of bone marrow stromal cells after transplantation into mice cerebral infarct [J].. Brain Res, 2007, 1183: 138-147.
22 Bhakta S, Hong P, Koc O. The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation. Cardiovasc Revasc Med,2006, 7: 19-24.
23 Lataillade JJ, Clay D, Dupuy C, et al. Chemokine SDF- 1 enhances circulating CD34+ cell proliferation in synergy with cytokines possible role in rogenitor survival[J]. Blood, 2000, 95: 756-768.
24 Kuang Y, Wu Y, Jiang H, et al. Selective G protein coupling by C-C chemokine receptors [J]. J Biol Chem, 1996, 271: 3975–8.
25 Gillard SE, Lu M, Mastracci RM, et al. Expression of functional chemokine receptors by rat cerebellar neurons [J]. J Neuroimmunol, 2002, 124: 16–28.
26 Florio T, Casagrande S, Diana F, et al. Chemokine stromal cell-derived factor 1alpha induces proliferation and growth hormone release in GH4C1 rat pituitary adenoma cell line through multiple intracellular signals [J]. Mol Pharmacol, 2006, 69: 539–546.
27 Alvarez S, Serramia MJ, Fresno M, et al. Human immuno deficiency virus type-1 envelope glycoprotein 120 induces cyclooxygenase-2 expression in neuroblastoma cells through a nuclear factor-kappaB and activating protein-1 mediated mechanism [J]. J Neurochem, 2005, 94: 850–861.
28 Soldevila G, Licona I, Salgado A, et al. Impaired chemokine-induced migration during T-cell development in the absence of Jak 3[J]. Immunology 2004, 112: 191–200.
29 Petit I, Goichberg P, Spiegel A, et a1. A typical PKC zeta regulates SDF-1 mediated migration and development of human CD34 progenitor cells[J]. J Clin Invest, 2005, 115(1): 168-l76.
30 Kollet O, Shivtiel S, Chen YQ, et a1. HGF, SDF-1, and MMP-9 are involved in stress induced human CD34 stem cell recruitment to the liver[J]. J Clin Invest, 2003, 112(2): l60-169.
31 Stumm RK, Rummel J, Junker V, et al. A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia [J]. J Neurosci, 2002, 22: 5865-5878.
32 Hill WD, Hess DC, Martin-Studdard A, et al. SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury [J]. J Neuropathol Exp Neurol, 2004, 63:84-96.
33 Wang Y, Deng Y, Zhou GQ, et al. SDF-1alpha/CXCR4-mediated migration ofsystemically transplanted bone marrow stromal cells towards ischemic brain lesion in a rat model[J]. Brain Res, 2008, 1195: 104-112.
34 Miller, JT, Bartley, JH, Wimborne, HJ, et al. The neuroblast and angioblast chemotaxic factor SDF-1 (CXCL12) expression is briefly up regulated by reactive astrocytes in brain following neonatal hypoxic–ischemic injury [J]. BMC Neurosci, 2005, 6, 63.
35 Ceradini DJ, Kulkarni AR, Callaghan MJ, et a1. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1[J]. Nat Med, 2004, 10(8): 858-864.
36 Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow [J].. Blood, 2004, 104: 2643-2645.
37 Shyu WC, Lin SZ, Rang HI, et a1. Functional recovery of stroke rats induced by granulocyte colony-stimulating factor-stimulated stem cells[J]. Circulation, 2004, 110: 1847-1854.
38 Shyu WC, Lin SZ, Yen PS, et al. Stromal cel1 derived factor-1 alpha promotes neuropotection, angiogenesis, and mobilization/homeing of bone marrow- derived cells in stroke rats [J]. J Pharmacol Exp Ther, 2008 , 324(2): 834-849.
39 Abbott JD, Huang Y, Liu D, et al. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury [J]. Circulation, 2004, 110: 3300-3305.
40 Ye Wang , Yubin Deng, Guang-Qian Zhou, et al. SDF-1α/CXCR4-mediated migration of systemically transplanted bone marrow stromal cells towards ischemic brain lesion in a rat model [J]. Brain Res, 2008, 1195: 104-112
41 Dongsheng Zhang , Guo-Chang Fan , Xiaoyang Zhou, et al.Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium [J].. J Molecular and Cellular Cardiology ,44 (2008): 281-292
42 Ruster B, Gottig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells [J]. Blood, 2006, 108: 3938-3944.
43 Peled A, Kollet O, Ponomaryov T, et al. The chemokine SDF-1 activates the integrins LFA-l, VLA-4, and VLA-5 on immature human CD34(+)cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice[J].Blood, 2000, 95(11): 3289-3296.
44 Ding Z, Issekutz TB, Downey GP, et al. L-selectin stimulation enhances functional expression of surface CXCR4 in lymphocytes: implications for cellular activation during adhesion and migration [J]. Blood, 2003, 10l(11): 4245-4252.
45 Carmeliet P. Mechanisms of angiogenesis and arteriogenesis[J]. Nature Medicine, 2000, 6(3): 389-395.
46 Salvucci O, Yao L, Villalba S, et al. Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal -derived factor-1 [J]. Blood, 2002, 99: 2703-2711.
47 Kanda S, Mochizuki Y, Kanetake H, et al. Stromal cel1 derived factor-1 alpha induces tube-like structure formation of endothelial cells through phosphoinosi- tide 3-kinase[J]. J Boil Chem, 2003, 278(1): 257-262.
