门脉高压症脾血管病变的研究

Splenic angiopathy in portal hypertension

目的:通过观察门脉高压症患者脾动脉和脾静脉的病理学改变和门静脉高压时其血管内皮细胞eNOS,ET-1,PKC, NF-κB的表达以及Ⅰ,Ⅲ型前胶原mRNA在门脉高压症时脾静脉壁的表达,从而探讨门静脉局部内皮源性血管活性物质异常与内皮细胞应力信号转导通路激活的关系和细胞外基质在门脉高压性血管病变发生中的作用以及门脉高压症、内脏高动力循环和门脉高压性内脏血管病变三者之间的相互关系. 方法:采用光镜和电镜方法观察20例门脉高压症患者和10 例正常对照组的脾动脉和脾静脉的形态学改变;用免疫细化和免疫荧光激光扫描共聚焦显微镜技术检测20例门静脉高压症患者脾静脉和15只门静脉高压大鼠模型门静脉内皮细胞eNOS,ET-1,PKC,NF-κB的表达,探讨门静脉高压时其血管内皮细胞eNOS,ET-1表达改变与信息分子PKC, NF-κB的关系;用逆转录聚合酶链反应检测Ⅰ、Ⅲ型前胶原mRNA在20例门静脉高压症患者脾静脉壁的表达. 结果:光镜下可见脾动脉内膜破损,内弹性膜和弹力纤维断裂和变性;脾静脉内膜局灶性增厚,内皮细胞不完整,附壁血栓形成,中膜平滑肌显著增厚.纤维结缔组织明显增多.电镜下可见脾动脉平滑肌细胞变性,坏死,表型从收缩型向合成型转化,红细胞、血小板聚集在破损内皮周围;脾静脉血管平滑肌细胞以合成型为主,其胞质中含有丰富的粗面内质网和高尔基体,整个管壁沉积有大量胶原纤维,并排列紊乱,内膜损伤,血液有形成分聚集,内皮表面形态不规则,凹凸不平.PKC的阳性表达见于细胞的胞质、胞膜. 门静脉高压患者脾静脉和实验组大鼠模型门静脉内皮细胞PKC表达呈阳性或强阳性,其血管平滑肌细胞也可见阳性信号.对照组则呈阴性或弱阳性表达.eNOS,ET-1与NF- κB双重免疫荧光标记的激光扫描共聚焦显微镜检测显示荧光定位均主要在血管内皮,三者在门静脉高压症患者脾静脉和实验组大鼠模型门静脉内皮的荧光信号强度均显著高于对照组.门静脉高压症患者脾静脉壁Ⅰ型前胶原mRNA表达强度与非门脉高压症患者无显著性差异(P>0.05);门静脉高压症患者脾静脉壁Ⅲ型前胶原mRNA表达强度明显高于非门脉高压症患者(P<0.01). 结论:门静脉高压时其血管内皮细胞ET-1和eNOS表达上调与该通路激活有关.Ⅲ型前胶原及胶原可能是门静脉高压症时导致血管构型改建的重要细胞外基质,门脉高压症时合并有脾动脉和脾静脉病变,门脉高压症和内脏高动力循环以及内脏血管病变之间互为因果关系.

AIM: To investigate the relationship among portal hypertension, splanchnic hyperdynamic disturbances and splanchnic angiopathy by observing splenic arterial and venous pathological changes, to discuss the relationship between the abnormal local endothelium-derived vasoactive substances in portal veins and the activation of the pathway of mechanical force signal transduction in vascular endothelial cells by investigating the expression of eNOS ET-1 PKC NF-κB in vascular endothelial cells of portal hypertension, and to explore the role of extracellular matrix in the pathogen-esis of portal hypertensive angiopathy by detecting the expression of type Ⅰ and type Ⅱ procollagen mRNA in splenic vein of portal hypertensive patients. METHODS: Splenic arteries, veins from portal hypertensive patients (n =20) and normal people (n=10) were removed and observed under optical and electron microscopes. Immunohistochemistry and double labeling immunofluo-rescence combined with laser scanning confocal microscope were used to investigate the expression of eNOS ET-1 NF-κB and PKC protein in endothelial cells of splenic veins from portal hypertensive patients (n =20) and portal veins from Wistar rats (n =15). Total RNA was extracted and type Ⅰ and type Ⅱ procollagen mRNAs in splenic vein of portal hypertensive patients (n =20) were examined by using the method of reverse transcriptional polymerase chain reaction with semiquantitative method. RESULTS: The endothelium of splenic arteries was damaged and the internal elastic membrane and medial elastic fibers of the splenic artery wall were broken and degenerated. The endothelium of splenic veins was remarkably thickened and endothelial cells integrated with the formation of mural thrombus. The tunica media thickened significantly because of hypertrophy of smooth muscle. Fiber and connective tissues increased in amount. Under the electron microscope, atrophy, apoptosis and phenotypic changes were seen in smooth muscle cells of splenic arteries. There were some red blood cells and pathelets congregation around the damaged endothelium. Synthesis type of smooth muscle cells accounted for a large part of the total cells in splenic veins. There were plentiful rough endoplasmic reticulum and Golgi complex within the cytoplasm of smooth muscle cells. The endothelium of splenic veins was damaged, indicating that a lot of collagen fibers and some blood components accumulating around the damaged endotheliun. The positive signal of PKC was observed in cytoplasm and cell membrane.The PKC expression in endothelial cells in the splenic/portal vein of portal hypertensive patients/rats showed positive or strong positive signal, and positive signal were also observed in some smooth muscle cells in these specimens. But the PKC expression in endothelial cells in the control groups was negative or mild positive. The result of eNOS, ET-1 and NF-κB expression examined by double labeling immunofluorescence combined with laser scanning confocal microscope showed that the fluorescence were mostly localized in the endothelium of vessel. The intensity of fluorescence in the portal hypertensive patients/rats were significantly higher than that in control group. Human I procollagen mRNA expression of portal hypertensive patients in splenic vein showed a insignificant pattern with control group (P>0.05), however, human Ⅲ procollagen mRNA expression in portal hypertensive patients were much higher than that of control group (P<0.01). CONCLUSION: In this study, the mechanical signal pathway of endothelial cell is activated in portal hypertension and the upregulation of ET-1 and eNOS are related with the activation of this pathway. Type Ⅲ procollagen and collagen may be one of the major extracellular matrix which deposits and results in neointimal formation and vascular remodeling in the pathogenesis of portal hypertensive vasopathy. Our research also shows that pathological changes of splenic arteries and veins are accompanied with portal hypertension. There may be an interactive relationship among portal hypertension, splanchnic hyper-dynamic disturbances and splanchnic angiopathy.