蛋白芯片的应用

1、Islet-1 Overexpression in Human Mesenchymal Stem Cells Promotes Vascularization Through Monocyte Chemoattractant Protein-3Major： Biochemistry and Molecular BiologyDate：2014.12.19Contents Conclusion 5Introduction 2Materials and methods3Results 4Thinking6Abstract 1ISL1： islet-1胰岛素基因增强结合蛋白-1，由人类胚胎干细胞发育

2、形成，对心肌细胞、内皮细胞、平滑肌细胞的生成发展有重要作用hMSC： human mesenchymal stem cells来源于早期中胚层和外胚层，属多能干细胞，主要存在于结缔组织和器官间质中，骨髓中含量最丰富，有分化潜能、造血支持、免疫调控、自我复制等特点HUVEC：human umbilical vein endothelialb cells用于血管内皮细胞实验，理论上能无限次传代MCP3： monocyte chem oattractant protein-3趋化性细胞因子，主要由白细胞和造血微环境中的基质细胞分泌，对单核细胞、淋巴细胞细胞等有趋化和激活活性GlossaryA. 不影

3、响不影响hMSC的存活、增殖、迁移和分化成成骨细胞、脂肪细胞、心肌细胞、内皮细胞的能力C. 体外体外实验，ISL1-hMSC条件培养基有高MCP3水平，增强HUVECs的存活、迁移、管形成能力D. 体内体内实验， ISL1-hMSC与HUVECs共移植增强了血管生成能力B. 影响影响hMSC有效分化成平滑肌细胞Abstract慢病毒载体慢病毒载体EF1-ISL1转染转染hMSC，hMSC的分化能力、血管生成特性？的分化能力、血管生成特性？Introduction血液循环不畅可引发心肌缺血、中风、伤口愈合延迟等缺血性血管疾病。因此，血管形成对于组织修复以及血管疾病治疗有重要意义。目前临床研究集中

4、在基因治疗和小分子治疗促进血管再生，其中深入研究的是hMSC，它能分化成平滑肌细胞、心肌细胞、内皮细胞等细胞，分泌促进血管形成的可溶性因子。干细胞治疗血管疾病的障碍是缺血组织中细胞存活率太低。有报告指出：MSC与AKT共同超表达通过旁泌机制增加细胞存活，促进血管形成。ISL1对心血管细胞的发展很重要，先前有文献报道，ISL1敲除后，血管和间质发育受损导致小鼠胚胎成长阻滞，最终坏死。后续有研究表明ISL1在体内可以促进形成不同类型的心血管细胞，分化成心肌细胞，内皮细胞、平滑肌细胞等。然而，然而，ISL1是否影响是否影响hMSC的生物学特性仍然是未知的。的生物学特性仍然是未知的。1、Cell Cu

5、lturehMSC： DMEM，用于初代病毒宿主细胞培养及单一细胞培养的培养基HUVEC：EGM-2，内皮细胞生长培养基2、Lentiviral Vector Construction-GatewayBP反应利用attB DNA片段和attP供载体之间的重组反应，创建入门克隆LR反应是attL入门克隆和attR目的载体之间的重组反应，产生表达克隆Materials and methods3、Chracterization of ISL1-hMSC -Fluorescence activated cell sorting利用荧光标记的蛋白抗体，进行利用荧光标记的蛋白抗体，进行FACS实验实验 -

6、Cell proliferation assaydirect cell counting (trypan blue exclusion) / fluorescence-based Cell Counting Kit-8 (CCK8)-Osteogenic differentiationAlizarin Red S staining for calcium；成骨相关基因；成骨相关基因RUNX2、OPN、COL1A1的表达的表达-Adipogenesisoil red O staining；脂肪形成相关基因；脂肪形成相关基因PPARc、LPL、FABP4的表达的表达- Neural differe

7、ntiation神经相关基因神经相关基因PAX6、ISL2、LHX3、LHX4、HB9、CHAT、TUBB3、MAP2、NFL、GFAP的表达的表达4、Cell Survival and Migration AssayAlexa Fluor 488 Annexin V/PI Kit；Transwell，Hoechst 33342 staining5、Cardiac，Endothelial，Smooth Muscle Cell Differentiation of ISL1-hMSC6、In Vitro Tube-Formation Assay Growth factor-reduced Mat

8、rigel and Cytodex-3 Beads用于体外细胞的培养和分化以及对细胞形态、生化功能、迁移、基因表达的研究8、Angiogenesis Antibody Arraysandwich immunoassay检测血管生成相关细胞因子的表达9、Neutralization Assay病毒或毒素与相应抗体结合，失去对动物致病力的试验方法（MCP3）7、In Vivo Angiogenesis Assayvascular density was performed by immunostaining for CD31 and counterstaining with hematoxylin

9、（1）Generation of an ISL1-hMSC Cell LineResults 纺锤形细胞，呈现出成纤维细胞的形态ISL1超表达与对照组没有显著的形态学差异(B) Immunofluorescence staining of hMSCs and ISL1-hMSCs with anti-ISL1 antibody(C)qRT-PCR analysis of ISL1 in ISL1-hMSCs and ohMSCs1) 转入ISL1的hMSC大部分细胞，约98%都能表达ISL12) 进一步证实ISL1-hMSCs中ISL1的成功表达（2）ISL1-hMSCs Retained t

10、he Characteristics of hMSCUsed FACS to analyze hMSCs surface markersCD29, CD34, CD44, CD45, CD73, CD90, CD105, CD166 expression in hMSCsand ISL1-hMSCs were analyzed by fluorescence activated cell sortingISL1-hMSCs维持了原始hMSCs的表型Cell-proliferation ability was analyzed by CCK8 assayISL1-hMSCs没有改变hMSCs的增

11、殖特性population doubling time(PDT)ISL1-hMSCs和对照组在第5代细胞、第10代细胞的PDT没有显著差异Alizarin Red S staining of osteogenic-differentiated cells茜素红S染色分化为成骨细胞的ISL1-hMSC以及对照组，没有显著差异ISL1-hMSCs和对照组中，骨形成相关蛋白表达没有显著差异qRT-PCR and WB assay were used to analyze expression of osteogenic related genesOsteocalcin secretion by di

12、fferentiated cells was also determined using ELISA kitISL1-hMSCs和对照组中骨钙蛋白分泌没有显著差异Oil red O staining of adipogenic-differentiated cells油红O染色分化为脂肪细胞的ISL1-hMSCs以及对照组，没有显著差异qRT-PCR and WB assay were used to analyze expression of adipogenesis markerISL1-hMSCs和对照组中，脂肪形成标记蛋白表达没有显著差异The expression of neural

13、 lineage related genes were analyzed by immunostaining 免疫染色分化为神经细胞的ISL1-hMSC以及对照组，发现TUBB3、NF-L没有显著差异，但是都没有检测到SOX2、HB9qRT-PCR was used to analyze expression neural lineage related genes ISL1-hMSCs以及对照组中，发现PXP6、TUBB3、MAP2的表达没有显著差异，但是都没有检测到ISL2、LHX3、LHX4、HB9、CHAT的表达Tested whether ISL1 overexpression al

14、tered cell survival or migration capacity ？Using a LIVE/DEAD viability/cytotoxicity kit ( Alexa Fluor 488 Annexin V/PI Kit)ISL1-hMSCs以及对照组培养48小时后均没有显著增加细胞死亡Migration assays were performed in transwell dishes with 8-lm pore filters. Migrated cells were identified by Hoechst and Calcein AM staining培养6

15、小时后，ISL1-hMSCs以及对照组的细胞迁移率均没有显著差异（3）ISL1 Enhanced the Smooth Muscle Differentiation Capacity of hMSC先前有文献报道，ISL1阳性细胞是心血管发展的祖细胞。ISL超表达是否增强hMSC的血管分化潜能？Cardiac differentiation was assessed by immunofluorescence using anti MHC and anti-cTnT antibodies.ISL1-hMSC和对照组结果一样，像心肌细胞一样具有阳性MHC 和cTnT的细胞较少Endothelia

16、l cell differentiation was assessed by CD31 immunostainingFluorescence activated cell sorting analyses indicatedthat about 0.1% of cells in both groups were CD34-positiveISL1-hMSC和对照组结果一样，像内皮细胞一样具有阳性CD31和CD34的细胞较少Smooth muscle differentiation was evaluated by immunofluorescence detection of SMA expr

17、ession. qRT-PCR detection of SMA expressionISL1-hMSC增加了阳性平滑肌细胞标记SMA的细胞数量（4）ISL1 Improved the Angiogenic Properties of hMSCs In VitroISL1-hMSC的条件培养基是否影响HUVEC的存活？HUVECs were cultured in CM from ISL1-hMSCs or control hMSCsISL1-hMSC细胞存活率达到了90%以上，而对照组细胞存活率只有50%左右ISL1影响hMSC的旁泌活性，增加了细胞存活推测：旁泌因子影响HUVEC的迁移Mi

18、gration assays were performed in transwell dishes with 8-lm pore filters Migrated cells were identified by Hoechst and Calcein AM staining条件培养基作用下，ISL1-hMSC的细胞迁移率远远大于对照组旁泌因子影响HUVEC的管形成能力The tube-formation ability of HUVECs ISL1-hMSC的条件培养基增强了HUVEC的管形成能力The Cytodex-3 Beads-based tube formation assayIS

19、L1-hMSC的条件培养基培养细胞48小时后，增强了HUVEC的芽和腔形成能力（5）ISL1 Enhanced the In Vivo Angiogenesis Properties of hMSCH&E staining of Matrigel plugs from mice共移植hMSCs与HUVECs增加了具功能小管的形成H&E staining and fluorescence detection of HUVECs单独移植hMSCs, ISL1-hMSCs, HUVECs几乎不能形成有功能的小管H&E staining and Immunostaining for CD31 共移植I

20、SL1-hMSCs与HUVECs比对照组增加了血管密度共移植ISL1-hMSCs与HUVECs比增加了阳性CD31细胞和阳性SMA细胞比率Immunostaining assays（6）ISL1 Expression Resulted in Elevated Expression of MCP3 in hMSCsUsing human angiogenesis antibody arrsay to establish the involvement of cytokines secreted from ISL1-hMSCs in enhancing the angiogenic capacit

21、y of HUVECqRT-PCR analysis of ISL1-hMSCs and control hMSCsISL1-hMSCs条件培养基显著增加了MCP3的表达qRT-PCR analysis of ISL1-hMSCs and control hMSCsISL1-hMSCs条件培养基还显著增加了细胞外粘附因子ICAM1的表达In Matrigel plug assays, the tube formation ability of human umbilical vein endothelial cells (HUVECs) grown in CM from ISL-hMSCs c

22、ultures was significantly impaired by addition of an anti-MCP3 antibody中和试验添加MCP3抗体，降低了ISL1-hMSC条件培养基中HUVEC小管形成Test the impact of secreted MCP3 on the biological properties of HUVECs using a neutralizing antibody against MCP3中和试验添加MCP3抗体，降低了ISL1-hMSC条件培养基中细胞存活HUVECs survival in CM from ISL1-hMSCs cultures, with and without the addition of anti-MCP3 neutralizing antibodyIn transwell assays, the percentage of migrated HUVECs in CM from I