VEGF抑制肝硬化大鼠肝窦毛细血管化的研究纤维血管镜技术在动脉闭塞性疾病中的应用研究
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摘要
研究背景:
肝窦毛细血管化包括内皮细胞的失窗孔和基底膜形成,是肝硬化过程中的重要病理改变。正常肝窦内皮细胞拥有跨肝窦内皮的窗孔,并且缺乏基底膜。窗孔、吞饮小泡及跨膜通道共同控制组织与血液之间的物质交换。有窗孔的肝窦内皮的结构完整性是维持肝窦和肝细胞间体液、溶质、大分子颗粒、和代谢产物交换的基本保证。尽管导致肝硬化的原因多样,但共有的一点是内皮的失窗孔和基底膜形成。正常滤过屏障的消失将导致肝细胞和肝窦血液问的物质交换障碍。
血管内皮生长因子(VEGF)可诱导血管发生、内皮细胞增殖及提高血管通透性,在调节血管生成方面起重要作用。近来,VEGF已被证实可促进内皮细胞的窗孔形成,增加内皮细胞通透性。通过点注射VEGF,在正常或者肝硬化的大鼠肝脏中促进了肝窦周围新生血管的形成。
既然内皮细胞的失窗孔和基底膜形成导致了肝窦血液和肝细胞间的物质代谢紊乱。因此改善肝窦内皮细胞功能可以减轻肝硬化。同时,VEGF具有促进内皮细胞窗孔形成和提高渗透性的作用。据此,我们进行了VEGF转基因治疗肝硬化大鼠肝窦毛细血管化的初步研究。
目的:
1、分离培养大鼠肝脏细胞,以EGFP/VEGF质粒转染原代肝细胞,通过观察EGFP(绿色荧光蛋白)和VEGF(血管内皮生长因子)的表达,证实细胞转染的有效性。
2、分离培养大鼠肝窦内皮细胞,以EGFP/VEGF质粒转染肝窦内皮细胞,观察VEGF对内皮细胞窗孔的影响。
3、以硫代乙酰胺复制肝硬化大鼠模型,电镜观察肝窦毛细血管化在肝硬化发生过程中的作用。
4、EGFP/VEGF质粒通过门静脉转染肝硬化大鼠活体肝脏,研究VEGF对肝窦内皮细胞化的影响。通过血管生成基因芯片探讨VEGF抑制肝窦内皮细胞化的机制。
方法:
1、胶原酶消化法获取大鼠肝脏细胞,EGFP/VEGF质粒转染原代肝细胞,免疫荧光显微镜观测EGFP的表达,VEGF免疫组织化学染色观察VEGF的表达。
2、胶原酶消化法获取大鼠肝窦内皮细胞,EGFP/VEGF质粒转染原代肝窦内皮细胞,扫描电镜及透射电镜观察转染前后内皮细胞窗孔数量的变化。
3、40只大鼠参与造模,采取前5周使用0.03%的硫代乙酰胺,后5周使用0.04%硫代乙酰胺作为其饮用水,共诱导10周成肝硬化模型。对成模大鼠测定门静脉压力,取硬化后肝脏标本,并门静脉内注入EGFP/VEGF质粒150μg。饲养三周后重复测定门静脉压力,处死全部大鼠,取肝组织标本。行透射电子显微镜观察转基因前后肝脏超微结构变化,统计肝窦内皮细胞窗孔数量及门静脉压力在转基因前后的变化。取肝脏组织行基因芯片实验,比较转基因前后血管生成相关基因的变化。
结果:
1、原代肝细胞转基因后,免疫荧光显微镜下可观测到绿色荧光蛋白的表达,免疫组织化学染色可观测到VEGF的表达。
2、肝窦内皮细胞转基因后,扫描电镜及透射电镜可见窗孔数量和直径增加。
3、在硫代乙酰胺诱导的肝硬化大鼠中,透射电镜下可见肝窦内皮细胞窗孔数量明显减少,内皮细胞下基底膜形成,肝细胞微绒毛脱落,肝细胞坏死,胆红素外溢,纤维细胞、胶原增生。硬化肝脏PCHO&VEGF质粒经门静脉转基因后,伴随肝窦内皮细胞窗孔的增加,门静脉压力显著降低,同时肝细胞微绒毛再生,肝细胞再生,胆红素外溢消失。
4、基因芯片结果显示,肝硬化时,VEGF家族转录明显减少,而其受体家族转录增加;转基因后,VEGF家族转录增加,而其受体家族转录减少。
结论:
1、EGFP/VEGF质粒细胞转染,可使肝脏细胞分泌VEGF增多,并可使肝窦内皮细胞窗孔直径增大、数量增多。
2、肝窦毛细血管化在肝硬化门脉高压发生中起重要作用,肝窦内皮细胞窗孔直径减小并数量减少,内皮细胞下基底膜形成使肝细胞与外界物质交换减少,导致细胞缺氧、代谢产物聚集,肝细胞坏死,是门脉高压发生过程中基本的病理生理改变。
3、EGFP/VEGF质粒转染,可通过VEGF的表达促进肝窦内皮细胞窗孔增加,基底膜降解,因此肝细胞与外界物质的交换恢复,肝细胞再生,门脉压力降低。VEGF导致的内皮细胞的窗孔化和通透性的增加在抑制肝窦毛细血管化方面起重要作用。
4、VEGF转基因,是通过众多生长因子的交互作用,促进肝窦内皮细胞生长,基底膜降解的,其中包括:血管内皮细胞生长因子Figf(VEGFD)、Pgf、Vegf、Vegfb、Vegfc;Ephrin家族Efna2(EphrinA2)、Efnb2(Ephrin B2)、Ephb4(Ephrin B4);成纤维细胞生长因子Fgf1(aFGF)、Fgf6和其受体Fgfr3;血小板源性生长因子和受体:Pdgfa、Pdgfb和其受体Pdgfrb,转化生长因子和受体Tgfb1、Tgfb2、Tgfb3和其受体Tgfbr2;其他生长因子:组织生长因子Ctgf、表皮生长因子Ege胰岛素样生长激素1Igf1、Gro1。
Capillarization of the sinusoidal endothelium includes a defenestrated sinusoidal endothelium and the presence of a subendothelial basement membrane,which is an important contributor to the hepatic cirrhosis.Liver sinusoidal endothelial cells (LSECs)possess open fenestrae that perforate the hepatic endothelial lining,but lack a basal lamina.Fenestrae,vesicles and channels together control the bulk of trans-endothelial transport between blood and tissues.Structural integrity of the fenestrated sinusoidal liver endothelium is believed to be essential for the maintenance of the normal exchange of fluids,solutes,particles and metabolites between the sinusoidal blood and hepatocytes.Although there are various causes and morphologies of hepatic cirrhosis,all forms of cirrhosis are characterized by a defenestrated sinusoidal endothelium and the presence of a subendothelial basement membrane.It has been demonstrated that the disappearance of the normal filtration barrier in cirrhotic livers results in an impaired bidirectional exchange between the sinusoidal blood and parenchymal cells.
Vascular endothelial growth factor(VEGF),which plays a role in regulating vasculogenesis,induces angiogenesis and endothelial cell proliferation.In recent years,it has been proven that VEGF is relevant to the increased number of fenestrae and endothelial permeability in endothelial cells.Injection of VEGF-D plasmid into both normal and ischemic rat liver resulted in an increased number of new capillaries around hepatic sinuses.
Because defenestration and basement membrane formation result in a disordered exchange between the sinusoidal blood and hepatocytes,it is necessary to restore the function of liver sinusoidal endothelial cells in order to reverse cirrhosis.VEGF provides the perfect means to achieve this,because of it promotes fenestration and permeability.On account of these ideas,we studied the effects of VEGF transfection in cirrhotic rat livers.
Objective:To investigate the efficacy of EGFP/VEGF plasmid transfection and the effect of VEGF on fenestrae of LECs by primary hepatocytes and LECs being transfected by EGFP/VEGF plasmid.
Cirrhotic rats were generated by thioacetamide(TAA)administration. EGFP/VEGF plasmid were injected into the portal vein to observe the super microstructural change of the liver.Gne array was carried out to clarify the mechanism of the super microstructural change.
Method:
Primary hepatocytes and LECs were isolated from the livers of male Wistar rats by collagenase perfusion.EGFP/VEGF transfection was confirmed by immunofluorescence microscopy and immunohistochemistry both in primary hepatocytes and in normal liver.Scanning electron microscope was used to observe the number of fenestrae in LECs.
40 rats accepted 0.03%TAA administration,after 5 weeks,the concentration of TAA was changed to 0.04%.After 10 weeks the portal hypertension models were built.Determined the pressure of superior mesenteric vein and obtain cirrhosis liver example,then infused VEGF/EGFP plasmid 150μg through superior mesenteric vein.After 3 weeks,determined the pressure of superior mesenteric vein again,then sacrificed all rats,took the liver organization specimen.The expression of VEGF in protein level were identified.The change of the liver super microstructural after gene transfection was observed by electronic microscope.Taking the liver organization to go the gene chip experiment,detected the change of Angiogenesis Gene after gene transfection.
Result:
Green fluorescent protein was observed in the cytoplasms of liver cells under immunofluorescence microscopy 24 h after transfection with EGFP/VEGF plasmid in vitro.Staining with polyclonal antibodies against VEGF illustrated that hepatocytes expressed immunodetectable VEGF both in vitro and in vivo.Much fenestrae in LECs was found under SEM after gene transfection.
In TAA induced cirrotic rats,the transmission electronic microscope showed a reduced number of fenestrae accompanied with the increased portal vein pressure.A basement membrane appeared.Cell conjunction between hepatocytes was destroyed and particles of bilirubin overflowed into the cytoplasm of hepatocytes and LECs, even into the hepatic sinusoid.The microvilli of hepatocytes in the space of Disse and the cholangiole were ablated.After VEGF plasmid transfection,accompanied with the increased number of fenestrae the portal vein pressure decreased.Furthermore, morphology changed after transfection with the EGFP/VEGF plasmid.The fenestrae, cell conjunctions,and microvilli of hepatocytes were restored,the basement membrane disappeared and cell apoptosis decreased.Newborn capillaries formed by a single liver endothelial cell emerged Gene-array analysis revealed that the relative abundance of transcripts of VEGF family members decreased in the cirrhotic state and increased after transfection.
Conclusion:
Hepatic sinusoid capillarization performed an important actor in the happen of portal hypertension.The formation of basement membrane impaired the substance exchange between the sinusoidal blood and hepatic cells.Oxygen deficiency, metabolic product accumulation and cell necrosis were the basic pathologic physiology change.
The VEGF plasmid transfection increased the number of fenestra and impaired basement membrane.The exchange between the sinusoidal blood and hepatic cells restored,so the liver cell regenerated and the portal vein pressure decreased.The VEGF transfection promoted endothelial cell in hepatic sinusoid growth and the basement membrane degradation through many growth factors interaction which mainly include:Vascular Endothelial Growth Factors & Receptors:Figf(VEGFD), Pgf,Vegf,Vegfb,Vegfc;Ephrin Family Members:Efna2(the Ephrin A2), Efnb2(the Ephrin B2),Ephb4(the Ephrin B4);Fibroblast Growth Factors & Receptors:Fgf1(aFGF),Fgf6 /Fgfr3;Platelet-derived Growth Factors & Receptors:The Pdgfa,Pdgfb/Pdgfrb,Transforming Growth Factors & Receptors: Tgfo1,Tgfb2,Tgfb3/Tgfbr2;Other Growth Factors:Ctgf,Egf,Igf1,Gro1. Chemokines:Nrp(neuropilin).
肝窦毛细血管化包括内皮细胞的失窗孔和基底膜形成,是肝硬化过程中的重要病理改变。正常肝窦内皮细胞拥有跨肝窦内皮的窗孔,并且缺乏基底膜。窗孔、吞饮小泡及跨膜通道共同控制组织与血液之间的物质交换。有窗孔的肝窦内皮的结构完整性是维持肝窦和肝细胞间体液、溶质、大分子颗粒、和代谢产物交换的基本保证。尽管导致肝硬化的原因多样,但共有的一点是内皮的失窗孔和基底膜形成。正常滤过屏障的消失将导致肝细胞和肝窦血液问的物质交换障碍。
血管内皮生长因子(VEGF)可诱导血管发生、内皮细胞增殖及提高血管通透性,在调节血管生成方面起重要作用。近来,VEGF已被证实可促进内皮细胞的窗孔形成,增加内皮细胞通透性。通过点注射VEGF,在正常或者肝硬化的大鼠肝脏中促进了肝窦周围新生血管的形成。
既然内皮细胞的失窗孔和基底膜形成导致了肝窦血液和肝细胞间的物质代谢紊乱。因此改善肝窦内皮细胞功能可以减轻肝硬化。同时,VEGF具有促进内皮细胞窗孔形成和提高渗透性的作用。据此,我们进行了VEGF转基因治疗肝硬化大鼠肝窦毛细血管化的初步研究。
目的:
1、分离培养大鼠肝脏细胞,以EGFP/VEGF质粒转染原代肝细胞,通过观察EGFP(绿色荧光蛋白)和VEGF(血管内皮生长因子)的表达,证实细胞转染的有效性。
2、分离培养大鼠肝窦内皮细胞,以EGFP/VEGF质粒转染肝窦内皮细胞,观察VEGF对内皮细胞窗孔的影响。
3、以硫代乙酰胺复制肝硬化大鼠模型,电镜观察肝窦毛细血管化在肝硬化发生过程中的作用。
4、EGFP/VEGF质粒通过门静脉转染肝硬化大鼠活体肝脏,研究VEGF对肝窦内皮细胞化的影响。通过血管生成基因芯片探讨VEGF抑制肝窦内皮细胞化的机制。
方法:
1、胶原酶消化法获取大鼠肝脏细胞,EGFP/VEGF质粒转染原代肝细胞,免疫荧光显微镜观测EGFP的表达,VEGF免疫组织化学染色观察VEGF的表达。
2、胶原酶消化法获取大鼠肝窦内皮细胞,EGFP/VEGF质粒转染原代肝窦内皮细胞,扫描电镜及透射电镜观察转染前后内皮细胞窗孔数量的变化。
3、40只大鼠参与造模,采取前5周使用0.03%的硫代乙酰胺,后5周使用0.04%硫代乙酰胺作为其饮用水,共诱导10周成肝硬化模型。对成模大鼠测定门静脉压力,取硬化后肝脏标本,并门静脉内注入EGFP/VEGF质粒150μg。饲养三周后重复测定门静脉压力,处死全部大鼠,取肝组织标本。行透射电子显微镜观察转基因前后肝脏超微结构变化,统计肝窦内皮细胞窗孔数量及门静脉压力在转基因前后的变化。取肝脏组织行基因芯片实验,比较转基因前后血管生成相关基因的变化。
结果:
1、原代肝细胞转基因后,免疫荧光显微镜下可观测到绿色荧光蛋白的表达,免疫组织化学染色可观测到VEGF的表达。
2、肝窦内皮细胞转基因后,扫描电镜及透射电镜可见窗孔数量和直径增加。
3、在硫代乙酰胺诱导的肝硬化大鼠中,透射电镜下可见肝窦内皮细胞窗孔数量明显减少,内皮细胞下基底膜形成,肝细胞微绒毛脱落,肝细胞坏死,胆红素外溢,纤维细胞、胶原增生。硬化肝脏PCHO&VEGF质粒经门静脉转基因后,伴随肝窦内皮细胞窗孔的增加,门静脉压力显著降低,同时肝细胞微绒毛再生,肝细胞再生,胆红素外溢消失。
4、基因芯片结果显示,肝硬化时,VEGF家族转录明显减少,而其受体家族转录增加;转基因后,VEGF家族转录增加,而其受体家族转录减少。
结论:
1、EGFP/VEGF质粒细胞转染,可使肝脏细胞分泌VEGF增多,并可使肝窦内皮细胞窗孔直径增大、数量增多。
2、肝窦毛细血管化在肝硬化门脉高压发生中起重要作用,肝窦内皮细胞窗孔直径减小并数量减少,内皮细胞下基底膜形成使肝细胞与外界物质交换减少,导致细胞缺氧、代谢产物聚集,肝细胞坏死,是门脉高压发生过程中基本的病理生理改变。
3、EGFP/VEGF质粒转染,可通过VEGF的表达促进肝窦内皮细胞窗孔增加,基底膜降解,因此肝细胞与外界物质的交换恢复,肝细胞再生,门脉压力降低。VEGF导致的内皮细胞的窗孔化和通透性的增加在抑制肝窦毛细血管化方面起重要作用。
4、VEGF转基因,是通过众多生长因子的交互作用,促进肝窦内皮细胞生长,基底膜降解的,其中包括:血管内皮细胞生长因子Figf(VEGFD)、Pgf、Vegf、Vegfb、Vegfc;Ephrin家族Efna2(EphrinA2)、Efnb2(Ephrin B2)、Ephb4(Ephrin B4);成纤维细胞生长因子Fgf1(aFGF)、Fgf6和其受体Fgfr3;血小板源性生长因子和受体:Pdgfa、Pdgfb和其受体Pdgfrb,转化生长因子和受体Tgfb1、Tgfb2、Tgfb3和其受体Tgfbr2;其他生长因子:组织生长因子Ctgf、表皮生长因子Ege胰岛素样生长激素1Igf1、Gro1。
Capillarization of the sinusoidal endothelium includes a defenestrated sinusoidal endothelium and the presence of a subendothelial basement membrane,which is an important contributor to the hepatic cirrhosis.Liver sinusoidal endothelial cells (LSECs)possess open fenestrae that perforate the hepatic endothelial lining,but lack a basal lamina.Fenestrae,vesicles and channels together control the bulk of trans-endothelial transport between blood and tissues.Structural integrity of the fenestrated sinusoidal liver endothelium is believed to be essential for the maintenance of the normal exchange of fluids,solutes,particles and metabolites between the sinusoidal blood and hepatocytes.Although there are various causes and morphologies of hepatic cirrhosis,all forms of cirrhosis are characterized by a defenestrated sinusoidal endothelium and the presence of a subendothelial basement membrane.It has been demonstrated that the disappearance of the normal filtration barrier in cirrhotic livers results in an impaired bidirectional exchange between the sinusoidal blood and parenchymal cells.
Vascular endothelial growth factor(VEGF),which plays a role in regulating vasculogenesis,induces angiogenesis and endothelial cell proliferation.In recent years,it has been proven that VEGF is relevant to the increased number of fenestrae and endothelial permeability in endothelial cells.Injection of VEGF-D plasmid into both normal and ischemic rat liver resulted in an increased number of new capillaries around hepatic sinuses.
Because defenestration and basement membrane formation result in a disordered exchange between the sinusoidal blood and hepatocytes,it is necessary to restore the function of liver sinusoidal endothelial cells in order to reverse cirrhosis.VEGF provides the perfect means to achieve this,because of it promotes fenestration and permeability.On account of these ideas,we studied the effects of VEGF transfection in cirrhotic rat livers.
Objective:To investigate the efficacy of EGFP/VEGF plasmid transfection and the effect of VEGF on fenestrae of LECs by primary hepatocytes and LECs being transfected by EGFP/VEGF plasmid.
Cirrhotic rats were generated by thioacetamide(TAA)administration. EGFP/VEGF plasmid were injected into the portal vein to observe the super microstructural change of the liver.Gne array was carried out to clarify the mechanism of the super microstructural change.
Method:
Primary hepatocytes and LECs were isolated from the livers of male Wistar rats by collagenase perfusion.EGFP/VEGF transfection was confirmed by immunofluorescence microscopy and immunohistochemistry both in primary hepatocytes and in normal liver.Scanning electron microscope was used to observe the number of fenestrae in LECs.
40 rats accepted 0.03%TAA administration,after 5 weeks,the concentration of TAA was changed to 0.04%.After 10 weeks the portal hypertension models were built.Determined the pressure of superior mesenteric vein and obtain cirrhosis liver example,then infused VEGF/EGFP plasmid 150μg through superior mesenteric vein.After 3 weeks,determined the pressure of superior mesenteric vein again,then sacrificed all rats,took the liver organization specimen.The expression of VEGF in protein level were identified.The change of the liver super microstructural after gene transfection was observed by electronic microscope.Taking the liver organization to go the gene chip experiment,detected the change of Angiogenesis Gene after gene transfection.
Result:
Green fluorescent protein was observed in the cytoplasms of liver cells under immunofluorescence microscopy 24 h after transfection with EGFP/VEGF plasmid in vitro.Staining with polyclonal antibodies against VEGF illustrated that hepatocytes expressed immunodetectable VEGF both in vitro and in vivo.Much fenestrae in LECs was found under SEM after gene transfection.
In TAA induced cirrotic rats,the transmission electronic microscope showed a reduced number of fenestrae accompanied with the increased portal vein pressure.A basement membrane appeared.Cell conjunction between hepatocytes was destroyed and particles of bilirubin overflowed into the cytoplasm of hepatocytes and LECs, even into the hepatic sinusoid.The microvilli of hepatocytes in the space of Disse and the cholangiole were ablated.After VEGF plasmid transfection,accompanied with the increased number of fenestrae the portal vein pressure decreased.Furthermore, morphology changed after transfection with the EGFP/VEGF plasmid.The fenestrae, cell conjunctions,and microvilli of hepatocytes were restored,the basement membrane disappeared and cell apoptosis decreased.Newborn capillaries formed by a single liver endothelial cell emerged Gene-array analysis revealed that the relative abundance of transcripts of VEGF family members decreased in the cirrhotic state and increased after transfection.
Conclusion:
Hepatic sinusoid capillarization performed an important actor in the happen of portal hypertension.The formation of basement membrane impaired the substance exchange between the sinusoidal blood and hepatic cells.Oxygen deficiency, metabolic product accumulation and cell necrosis were the basic pathologic physiology change.
The VEGF plasmid transfection increased the number of fenestra and impaired basement membrane.The exchange between the sinusoidal blood and hepatic cells restored,so the liver cell regenerated and the portal vein pressure decreased.The VEGF transfection promoted endothelial cell in hepatic sinusoid growth and the basement membrane degradation through many growth factors interaction which mainly include:Vascular Endothelial Growth Factors & Receptors:Figf(VEGFD), Pgf,Vegf,Vegfb,Vegfc;Ephrin Family Members:Efna2(the Ephrin A2), Efnb2(the Ephrin B2),Ephb4(the Ephrin B4);Fibroblast Growth Factors & Receptors:Fgf1(aFGF),Fgf6 /Fgfr3;Platelet-derived Growth Factors & Receptors:The Pdgfa,Pdgfb/Pdgfrb,Transforming Growth Factors & Receptors: Tgfo1,Tgfb2,Tgfb3/Tgfbr2;Other Growth Factors:Ctgf,Egf,Igf1,Gro1. Chemokines:Nrp(neuropilin).
引文
1.Senger DR,et al.Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.Science 1983;219:983-985.
2.Leung DW,et al.Vascular endothelial growth factor is a secreted angiogenic mitogen.Science 1989;246:1306-1309.
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