血管内皮祖细胞的基因修饰与体外三维培养的研究
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摘要
血管内皮祖细胞是血管内皮细胞的前体细胞具有定向迁移分化为成熟血管内皮细胞的能力。参与出生后的血管生成和血管发生,近年来血管内皮祖细胞被广泛用于治疗缺血性心血管疾病和组织工程血管生成的研究中。为了提高血管内皮祖细胞的移植效率,克服传统干细胞移植治疗方法中的不足。将血管内皮祖细胞种植于模拟体内细胞外基质结构的三维载体支架上,进行体外培养和体内移植,是近年来研究的热点。
本研究采用从大鼠骨髓分离单个核细胞,用特定的诱导培养基在体外培养、扩增血管内皮祖细胞。并将构建的表达组织因子途径抑制因子(TFPI)基因的重组逆转录病毒载体包装成逆转录病毒,感染血管内皮祖细胞,获得稳定表达TFPI和GFP基因的血管内皮祖细胞,用复乳有机溶剂挥发法添加蔗糖做为致孔剂,制备了聚乳酸(poly (D, L-lactide), PDLLA)多孔微球,考察了血管内皮祖细胞在RGD修饰的聚乳酸多孔微球载体上的粘附、增殖、分化情况。具体研究内容如下：
为获得一种方便、有效的血管内皮祖细胞分离、培养和扩增方法,本研究选用密度梯度离心法分离大鼠骨髓单个核细胞,结合贴壁培养法,在添加特定生长因子的诱导体系培养血管内皮祖细胞。通过观察其生长过程中的形态改变、检测EPCs的功能和表面标志来进行鉴定。倒置显微镜观察,在贴壁培养的第4d出现EPCs集落,细胞呈纺锤形,增长速度快,于培养的第14天集落相互融合,细胞分化成铺路石样细胞。荧光显微镜观察可见大部分EPCs能同时摄取Dil-acLDL和结合FITC-UEA-1。荧光显微镜观察到培养14d的EPCs CD31、v-WF免疫荧光染色呈阳性。流式细胞仪检测结果显示CD133、Flk.1在诱导培养的3.7d呈升高趋势,至培养的第14d两者的表达量明显降低。在培养的第3、7d,CD31几乎没有表达,至培养的第14d,EPCs的CD31表达量明显升高。以上试验结果证实此方法分离培养后得到的细胞是相对纯度较高的血管内皮祖细胞。
为获得稳定表达TFPI和GFP基因的血管内皮祖细胞,本研究将人全长TFPI的cDNA亚克隆到逆转录病毒载体pMSCV-IRES-GFP中,获得定向插入重组子pMSCV-TFPI-IRES-GFP,酶切图谱证实重组病毒中含有人TFPI的cDNA片段,基因测序结果与Genebank中TFPI cDNA序列相符。用构建的重组质粒转染293T细胞,成功包装出含有TFPI和GFP基因的逆转录病毒,将重组的逆转录病毒感染EPCs,荧光显微镜观察及流式细胞仪检测显示,90%以上的感染细胞中存在GFP表达。RT-PCR分析显示,重组的逆转录病毒感染的EPCs中TFPI mRNA水平明显增高,ELISA法检测显示,感染重组病毒的EPCs培养上清中有TFPI蛋白表达。以上研究结果为TFPI基因结合血管内皮祖细胞进行血管再狭窄防治的研究奠定了良好的实验基础。
用复乳有机溶剂挥发法,同时选用蔗糖做为致孔剂,制备了聚乳酸多孔微球,粒径为100-300μm左右,微球支架表面孔径为16±3.78μm,球形态好,粒径、孔径均匀分布,通过共价交联的方法用RGD修饰PDLLA多孔微球,修饰后的微球形态无改变,通过红外、XPS等检测技术证实RGD成功修饰多孔微球表面。本阶段的研究为用PDLLA多孔微球作为血管内皮祖细胞的微载体研究奠定了基础。
为考察RGD-PDLLA多孔微球支架的生物相容性,将血管内皮祖细胞种植于RGD-PDLLA多孔微球支架,以未经RGD修饰的PDLLA微球为对照,通过MTT、扫描电镜、荧光染色等生物学性能检测后,对比研究RGD修饰的PDLLA微球对EPCs增殖和分化的影响。MTT结果显示EPCs种植于多孔微球支架后的第1d、3d、7d,细胞数量逐渐增多,这说明EPCs能够在PDLLA多孔微球支架上生长、增殖。而且RGD-PDLLA多孔微球上细胞数量明显高于未修饰的PDLLA多孔微球支架。扫描电镜和荧光显微镜观察结果与MTT结果相一致。EPCs种植于多孔微球支架后的7d,保持着摄取Dil-acLDL和结合FITC-UEA-1的功能。
Endothelial Progenitor Cells (EPCs) are endothelial precursors that have an enhanced potential for differentiation into the endothelial cell lineage. EPCs are recruited into injured tissues and contribute to vasculogenesis and angiogenesis by directly incorporating into vessel walls or by secreting a variety of angiogenic growth factors. Subsequently, EPCs transplantation has been widely used in treatment of non-curable ischemic diseases and construction of tissue engineered vessels. To overcome the drawbacks of conventional cell transplantation methods and improve the quality of EPCs grafted into the injured sites. A variety of biosynthetic scaffolds have been developed as EPCs carrier for cells in vitro expension and in vivo tansplantation.
In our study, EPCs obtained from rat bone marrow mononuclear cells(BMMNCs) were cultured in special medium. The constructed recombinant retroviral vector capable of expressing human tissue factor pathway inhibitor(TFPI) and GFP was packed into TFPI retrovirals. and infected EPCs successfully. Biological compatibility of the poly (D, L-lactide) (PDLLA) and the RGD modified PDLLA porous microspheres scaffolds were assessed by in vitro culture of EPCs and cervical cancer cells. The main content of this research are shown as follows. To establish an efficient and stable method for obtaining endothelial progenitor cells from rat bone marrow. Bone marorw mononuclear cells were isolated from rat bone marrow of femurs and tibias by Histopaque-1083 density gradient centrifugation. Then were cultured in endothelial basal medium(EGM-2) supplemented with vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, ascorbic acid. The form of colonis of EPCs was observed under inverted microscope. The function of EPCs were identified through uptaking DiL-acLDL and binding FITC-UEA-1. The expressions of surface markers (CD133、flk-1,CD31,v-WF) were analyzed by immunofluorescence and Fluorescence-activated cell sorting.The adhesive cells formed clusters after 4 days plating. These colonies proliferated rapidly and exhibited spindle-shaped morphology. Twelve days after plating most colonies had reached near confluence and differentiated into cobblestone-shaped cells. The majority of the cells expressed endothelial markers of CD31 and v-WF After 14 days culture by immunofluorescence detection. Furthermore, These cells were positive for Dil-acLDL incorporation and UEA-1 lectin binding after 14 days culture. Fluorescence-activated cell sorting (FACS) detection demonstrated that the expression of the early markers of EPCs (CD133、Flk-1) increased gradually after 3d、7d culture.Then decreased after 14 days culture. The expression of the late markers of EPCs (CD31) was detected highly increased after 14d culture.
To construct the recombinant retroviral vector capable of expressing human tissue factor pathway inhibitor and GFP in rat endothelial progenitor cells (EPCs). Full length TFPI cDNA obtained from pIRES-TFPI by PCR amplification was digested with EcoRI and Xhol restriction enzymes and subsequently inserted into pMSCV-IRES-GFP expression vector to create the recombinant bicistronic retroviral vector pMSCV- TFPI- IRES -GFP encoding both TFPI and GFP. The recombinant plasmid was identified with restrictive endonuclease digestion and DNA sequencing. The recombinant plasmid was transfected into 293T and the supernatant containing packaged recombinant retroviral particles was collected and used to infect the EPCs isolated from rat bone marrow. TFPI mRNA was measured by RT-PCR and the amount of TFPI protein secreted from the transfected cells was determined by ELISA. GFP expression in the infected cells was analyzed by fluorescent microscopy and fluorescence activated cell sorting (FACS). Restriction endonuclease mapping and DNA sequencing confirmed the in-frame insertion of TFPI cDNA into the constructed vector-pMSCV-TFPI-IRES-GFP. Both RT-PCR and ELISA analysis demonstrated increased TFPI expression in the EPCs infected with TFPI retroviral. FACS analysis demonstrated that the transduction efficiency of EPCs with TFPI retroviral in vitro was over 90%. pMSCV-TFPI-IRES-GFP could be effectively expressed in cultured EPCs and may provide a useful tool for further study on the application of TFPI in the prevention of restenosis.
Solvent-evaporation method was modified using sucrose as an additive to form large porous microspheres of PDLLA polymers.Then porous PDLLA microspheres were modified by RGD via a method of hydrolysis and grafting-coating to improve their compatibility to cells. Surface morphology of PDLLA microspheres before and after hydrolysis was visualized by scanning electron microscopy. The SEM images of PDLLA microspheres revealed that there was a highly open porous structure between the surface and the internal.There are more porous on the hydrolysis microspheres than untreated microspheres.Modified microspheres were characterized for surface chemistry by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR). FT-IR spectra exhibited characteristic peaks of amide at 3430cm-1 and 1630cm-1, XPS spectra shows the N 1s (atomic orbital 1s of nitrogen) region of the surface of the microspheres.
Biological Compatibility of the control PDLLA and the RGD modified porous PDLLA microspheres were assessed by in vitro culture of endothelial progenitor cells. The morphology and distribution of cells on the microspheres were observed under SEM、inverted fluorescence microscopy after cultured for 1d、7 d. Cell viability was measured by MTT assay.RGD modified PDLLA microspheres exhibited stronger ability to promote EPCs attachment and proliferation after 24 h、3d、7 d culture compared to unmodified microspheres. Morever, EPCs on RGD modified microspheres maintain the function of uptaking DiL-acLDL and binding FITC-UEA-1. Scanning electron microscopy also revealed copious amount of extra-cellular matrix production by EPCs.Therefore, the RGD-coated PDLLA microspheres can be potentially used as the injectable cell microcarriers for EPCs in the treatment of Ischemic disease and tissue engineering vessels.
本研究采用从大鼠骨髓分离单个核细胞,用特定的诱导培养基在体外培养、扩增血管内皮祖细胞。并将构建的表达组织因子途径抑制因子(TFPI)基因的重组逆转录病毒载体包装成逆转录病毒,感染血管内皮祖细胞,获得稳定表达TFPI和GFP基因的血管内皮祖细胞,用复乳有机溶剂挥发法添加蔗糖做为致孔剂,制备了聚乳酸(poly (D, L-lactide), PDLLA)多孔微球,考察了血管内皮祖细胞在RGD修饰的聚乳酸多孔微球载体上的粘附、增殖、分化情况。具体研究内容如下：
为获得一种方便、有效的血管内皮祖细胞分离、培养和扩增方法,本研究选用密度梯度离心法分离大鼠骨髓单个核细胞,结合贴壁培养法,在添加特定生长因子的诱导体系培养血管内皮祖细胞。通过观察其生长过程中的形态改变、检测EPCs的功能和表面标志来进行鉴定。倒置显微镜观察,在贴壁培养的第4d出现EPCs集落,细胞呈纺锤形,增长速度快,于培养的第14天集落相互融合,细胞分化成铺路石样细胞。荧光显微镜观察可见大部分EPCs能同时摄取Dil-acLDL和结合FITC-UEA-1。荧光显微镜观察到培养14d的EPCs CD31、v-WF免疫荧光染色呈阳性。流式细胞仪检测结果显示CD133、Flk.1在诱导培养的3.7d呈升高趋势,至培养的第14d两者的表达量明显降低。在培养的第3、7d,CD31几乎没有表达,至培养的第14d,EPCs的CD31表达量明显升高。以上试验结果证实此方法分离培养后得到的细胞是相对纯度较高的血管内皮祖细胞。
为获得稳定表达TFPI和GFP基因的血管内皮祖细胞,本研究将人全长TFPI的cDNA亚克隆到逆转录病毒载体pMSCV-IRES-GFP中,获得定向插入重组子pMSCV-TFPI-IRES-GFP,酶切图谱证实重组病毒中含有人TFPI的cDNA片段,基因测序结果与Genebank中TFPI cDNA序列相符。用构建的重组质粒转染293T细胞,成功包装出含有TFPI和GFP基因的逆转录病毒,将重组的逆转录病毒感染EPCs,荧光显微镜观察及流式细胞仪检测显示,90%以上的感染细胞中存在GFP表达。RT-PCR分析显示,重组的逆转录病毒感染的EPCs中TFPI mRNA水平明显增高,ELISA法检测显示,感染重组病毒的EPCs培养上清中有TFPI蛋白表达。以上研究结果为TFPI基因结合血管内皮祖细胞进行血管再狭窄防治的研究奠定了良好的实验基础。
用复乳有机溶剂挥发法,同时选用蔗糖做为致孔剂,制备了聚乳酸多孔微球,粒径为100-300μm左右,微球支架表面孔径为16±3.78μm,球形态好,粒径、孔径均匀分布,通过共价交联的方法用RGD修饰PDLLA多孔微球,修饰后的微球形态无改变,通过红外、XPS等检测技术证实RGD成功修饰多孔微球表面。本阶段的研究为用PDLLA多孔微球作为血管内皮祖细胞的微载体研究奠定了基础。
为考察RGD-PDLLA多孔微球支架的生物相容性,将血管内皮祖细胞种植于RGD-PDLLA多孔微球支架,以未经RGD修饰的PDLLA微球为对照,通过MTT、扫描电镜、荧光染色等生物学性能检测后,对比研究RGD修饰的PDLLA微球对EPCs增殖和分化的影响。MTT结果显示EPCs种植于多孔微球支架后的第1d、3d、7d,细胞数量逐渐增多,这说明EPCs能够在PDLLA多孔微球支架上生长、增殖。而且RGD-PDLLA多孔微球上细胞数量明显高于未修饰的PDLLA多孔微球支架。扫描电镜和荧光显微镜观察结果与MTT结果相一致。EPCs种植于多孔微球支架后的7d,保持着摄取Dil-acLDL和结合FITC-UEA-1的功能。
Endothelial Progenitor Cells (EPCs) are endothelial precursors that have an enhanced potential for differentiation into the endothelial cell lineage. EPCs are recruited into injured tissues and contribute to vasculogenesis and angiogenesis by directly incorporating into vessel walls or by secreting a variety of angiogenic growth factors. Subsequently, EPCs transplantation has been widely used in treatment of non-curable ischemic diseases and construction of tissue engineered vessels. To overcome the drawbacks of conventional cell transplantation methods and improve the quality of EPCs grafted into the injured sites. A variety of biosynthetic scaffolds have been developed as EPCs carrier for cells in vitro expension and in vivo tansplantation.
In our study, EPCs obtained from rat bone marrow mononuclear cells(BMMNCs) were cultured in special medium. The constructed recombinant retroviral vector capable of expressing human tissue factor pathway inhibitor(TFPI) and GFP was packed into TFPI retrovirals. and infected EPCs successfully. Biological compatibility of the poly (D, L-lactide) (PDLLA) and the RGD modified PDLLA porous microspheres scaffolds were assessed by in vitro culture of EPCs and cervical cancer cells. The main content of this research are shown as follows. To establish an efficient and stable method for obtaining endothelial progenitor cells from rat bone marrow. Bone marorw mononuclear cells were isolated from rat bone marrow of femurs and tibias by Histopaque-1083 density gradient centrifugation. Then were cultured in endothelial basal medium(EGM-2) supplemented with vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, ascorbic acid. The form of colonis of EPCs was observed under inverted microscope. The function of EPCs were identified through uptaking DiL-acLDL and binding FITC-UEA-1. The expressions of surface markers (CD133、flk-1,CD31,v-WF) were analyzed by immunofluorescence and Fluorescence-activated cell sorting.The adhesive cells formed clusters after 4 days plating. These colonies proliferated rapidly and exhibited spindle-shaped morphology. Twelve days after plating most colonies had reached near confluence and differentiated into cobblestone-shaped cells. The majority of the cells expressed endothelial markers of CD31 and v-WF After 14 days culture by immunofluorescence detection. Furthermore, These cells were positive for Dil-acLDL incorporation and UEA-1 lectin binding after 14 days culture. Fluorescence-activated cell sorting (FACS) detection demonstrated that the expression of the early markers of EPCs (CD133、Flk-1) increased gradually after 3d、7d culture.Then decreased after 14 days culture. The expression of the late markers of EPCs (CD31) was detected highly increased after 14d culture.
To construct the recombinant retroviral vector capable of expressing human tissue factor pathway inhibitor and GFP in rat endothelial progenitor cells (EPCs). Full length TFPI cDNA obtained from pIRES-TFPI by PCR amplification was digested with EcoRI and Xhol restriction enzymes and subsequently inserted into pMSCV-IRES-GFP expression vector to create the recombinant bicistronic retroviral vector pMSCV- TFPI- IRES -GFP encoding both TFPI and GFP. The recombinant plasmid was identified with restrictive endonuclease digestion and DNA sequencing. The recombinant plasmid was transfected into 293T and the supernatant containing packaged recombinant retroviral particles was collected and used to infect the EPCs isolated from rat bone marrow. TFPI mRNA was measured by RT-PCR and the amount of TFPI protein secreted from the transfected cells was determined by ELISA. GFP expression in the infected cells was analyzed by fluorescent microscopy and fluorescence activated cell sorting (FACS). Restriction endonuclease mapping and DNA sequencing confirmed the in-frame insertion of TFPI cDNA into the constructed vector-pMSCV-TFPI-IRES-GFP. Both RT-PCR and ELISA analysis demonstrated increased TFPI expression in the EPCs infected with TFPI retroviral. FACS analysis demonstrated that the transduction efficiency of EPCs with TFPI retroviral in vitro was over 90%. pMSCV-TFPI-IRES-GFP could be effectively expressed in cultured EPCs and may provide a useful tool for further study on the application of TFPI in the prevention of restenosis.
Solvent-evaporation method was modified using sucrose as an additive to form large porous microspheres of PDLLA polymers.Then porous PDLLA microspheres were modified by RGD via a method of hydrolysis and grafting-coating to improve their compatibility to cells. Surface morphology of PDLLA microspheres before and after hydrolysis was visualized by scanning electron microscopy. The SEM images of PDLLA microspheres revealed that there was a highly open porous structure between the surface and the internal.There are more porous on the hydrolysis microspheres than untreated microspheres.Modified microspheres were characterized for surface chemistry by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR). FT-IR spectra exhibited characteristic peaks of amide at 3430cm-1 and 1630cm-1, XPS spectra shows the N 1s (atomic orbital 1s of nitrogen) region of the surface of the microspheres.
Biological Compatibility of the control PDLLA and the RGD modified porous PDLLA microspheres were assessed by in vitro culture of endothelial progenitor cells. The morphology and distribution of cells on the microspheres were observed under SEM、inverted fluorescence microscopy after cultured for 1d、7 d. Cell viability was measured by MTT assay.RGD modified PDLLA microspheres exhibited stronger ability to promote EPCs attachment and proliferation after 24 h、3d、7 d culture compared to unmodified microspheres. Morever, EPCs on RGD modified microspheres maintain the function of uptaking DiL-acLDL and binding FITC-UEA-1. Scanning electron microscopy also revealed copious amount of extra-cellular matrix production by EPCs.Therefore, the RGD-coated PDLLA microspheres can be potentially used as the injectable cell microcarriers for EPCs in the treatment of Ischemic disease and tissue engineering vessels.
引文
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