摘要
研究背景
舌诊是中医独具特色的诊法之一,为临床诊断不可或缺的依据。舌下络脉作为中医舌诊的重要组成部分,因其显露易见,成为观察人体气血、津液盈亏和瘀血的重要指征。近年来在心血管病、消化系统疾病、糖尿病及恶性肿瘤等的中医诊治中得到广泛应用。课题组在长期原发性肝癌的中西医结合临床中发现,肝癌患者多伴有舌下络脉的扩张、迂曲等形态及青紫、绛紫等颜色变化,这种变化非常灵敏,且多与患者的病情进展和治疗预后明显相关。根据传统中医理论,肝通过其经络直接与舌相连,故肝的异常可在舌上出现相应的改变。但对于异常舌下络脉形成的分子机制,目前尚鲜有报道,对肝癌异常舌象的研究就更少之又少。既往研究认为,异常舌下络脉形成可能与舌的静脉压力升高有关,但我们工作中注意到肝癌患者伴有门静脉压增高者更易出现舌下络脉异常,通过对肝癌患者的舌下络脉变化与肝门脉血管及其局部血液动力学指标的检测也证实了二者存在密切的关系。另有学者通过临床研究证实,门脉高压症患者的舌下络脉曲张率高达100%。在此基础上,我们提出了“舌下络脉和肝门静脉作为人体静脉血管系统的组成部分,二者在病理上必然存在密切的内在联系”这一假说。而门脉高压的形成存在肝外阻塞、肝内阻塞和肝外肝内复合阻塞三种类型,不同的门脉高压形成机制是否会导致舌下络脉的的不同的变化。继续的临床观察注意到肝癌伴有的肝硬化病理可以引起舌下络脉的颜色及性状异常,而如果患者门脉高压系继发于门脉血管受到肿瘤侵犯之后(门脉癌栓),其舌下络脉更具有根底部粗张迂曲、络脉周围瘀血细络增多,出现新的小血管等自身特点,这很难简单地用门静脉压力增高引起舌下络脉被动扩张来完整解释,进一步提示不同门脉高压的形成机制,同样引起了舌下络脉构型上的多样性。而且,引起二者舌下络脉构型差异的背后必然存在着并不完全一致的分子机制。
成体中血管的形成主要依赖于生理性和病理性的血管生成机制,近年来许多研究也已证实,在门静脉高压症过程中,肺脏和脾脏中均存在血管生成现象。舌下络脉作为全身静脉的一部分,其异常改变极可能与机体的血管生成现象存在内在联系。因此我们设想根据“血管生成”理论,重点从血管生成的角度,探讨不同门脉高压模型异常舌下络脉形成的可能分子机制。许多分子在血管生成中起着正向调节作用,血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)是近年来发现的参与血管生成的最主要的分子。缺氧反应、机械应力等多种机制均可通过激活VEGF/VEGFR信号通路调节血管生成。新近研究发现,脂肪炎症因子及其受体Apelin/APJ通路的调节与血管生成也有密切联系,可能与VEGF/VEGFR信号通路存在“串话”并起到协同作用,可以看作是治疗门脉高压新的靶点。
研究目的
本研究通过复制肝前型、肝内型、复合型三种门脉高压犬模型,观察模型犬舌下络脉的整体和微观变化,并从血管生成的角度,重点探寻VEGF/ VEGF2型受体(vascular endothelial growth factor receptor 2, VEGFR2)及Apelin/APJ信号通路与异常舌下络脉形成之间的关系,为揭示异常舌下络脉形成的分子机制提供数据支持。
研究方法
(1)三种门脉高压犬模型的制备
将Beagle犬随机分为正常组、假手术组和模型组。以门静脉窄缩术复制肝前型门脉高压模型;以二甲基亚硝胺(dimethylnitrosamine,DMN)连续给药诱导肝硬化制备肝内型门脉高压犬模型;以上两种方法结合复制复合型门脉高压模型。经皮下留置针以生物电记录系统连续监测其门脉压力通过血清肝功能测定和肝脏组织病理学观察进行成模检测。
(2)三种门脉高压模型犬舌下络脉的特征观察
检测成模后,肉眼观察舌下络脉形态和颜色变化;游标卡尺测量舌底根部络脉宽度;动物处死后,取舌底组织,免疫组织化学显色法检测舌底组织毛细血管密度;透射电镜观察舌下络脉的内皮细胞形态学变化。
(3)三种门脉高压模型犬异常舌下络脉的形成机制研究
硝酸还原酶法检测血清一氧化氮(nitric oxide, NO)浓度;Elisa法检测血清VEGF及其VEGFR2、缺氧诱导因子1α(Hypoxia-induciable factor 1-α, HIF-1α)浓度;免疫组织化学显色法和Western blot法检测犬舌下VEGF、VEGFR2、HIF-1α、Apelin、APJ蛋白表达;Real-time PCR法检测VEGF、VEGFR2、HIF-1αmRNA的表达量。
结果
(1)三种门脉高压犬模型的制备
与假手术组比较,肝内型组和复合型组各实验犬在DMN诱导2周门静脉压力即开始升高(P<0.05),随实验进展模型组门静脉压力在4周、6周、8周呈阶梯状上升(P<0.01),8周后肝内型组门脉压力持续稳定,复合型组在进一步行门脉窄缩术后压力进一步升高(P<0.01);肝前型组在行门脉窄缩术后,门脉压力迅速升高达峰值后逐渐下降后,处死前仍然处于较高水平(P<0.01)。另外,与正常组和假手术组比较,三个模型组实验犬的的肝脏组织学和(或)肝功能出现不同程度的异常改变,显示模型复制成功。
(2)三种门脉高压模型犬舌下络脉的特征观察
肉眼观察发现,肝内型门脉高压组和复合型组模型犬舌下络脉形态较正常组和假手术组迂曲粗大,血管周围出现瘀点和细络,颜色变暗变紫,舌根部络脉直径明显增粗(P<0.01),肝前型门脉高压组犬仅络脉颜色变暗,舌根部络脉直径增粗不明显;与正常组和假手术组比较,免疫组化结果显示三个模型组实验犬舌底CD31表达均显著增加(P<0.01),以复合型组增加最为明显;透射电镜显示,三个模型组实验犬血管壁全部或局部增厚,管腔变小,出现新的毛细血管,内皮细胞膜界限不清,细胞核损失甚或消失,复合型组改变程度最重,肝内组次之,肝前型组改变较小。
(3)三种门脉高压模型犬异常舌下络脉的形成机制研究
与正常组和假手术组比较,三种模型组实验犬血清NO浓度明显升高(P<0.01);肝内型组和复合型组血清VEGF、HIF-1α浓度明显升高(P<0.01),肝前型组升高不明显;三种模型组实验犬舌底组织VEGF、VEGFR2、HIF-1α蛋白表达均显著升高(P<0.05),趋势为复合型组>肝内型组>肝前型组;舌底组织VEGF、VEGFR2、HIF-1α的mRNA表达量也较正常组和假手术组明显升高,趋势与蛋白表达一致;Apelin、APJ蛋白表达也较正常组和假手术组明显升高(P<0.05),以复合型组表达增多最为明显,肝内型次之,肝前型升高较小。
结论
肝前型、肝内型、复合型三种门脉高压模型犬舌下络脉均从宏观和微观方面出现相应的改变,肝内阻塞因素所致的门静脉压力升高可能是导致其异常改变形成的一个重要因素,而肝外阻塞又往往成为诱发络脉变化加重的因素;VEGF及Apelin诱导的血管生成参与了模型犬异常舌下络脉的形成,分别与HIF-1α/ VEGF/ VEGFR2/ NO及Apelin/APJ通路诱导的血管生成密切相关,这可能是其舌下络脉血管重建的重要机制之一。
Background
Sublingual venae diagnosis is an important component of traditional tongue diagnosis. Because it is easy to check and reflects fast, it is also an important indications of human qi-blood, fluid-humor and static blood in clinical diagnosis. Recently, sublingual venae diagnosis became an important method to diagnose the cardiovascular disease, the digestive system disease and diabetes and other diseases. With long time clinical observation of patients with primary liver cancer and cirrhosis, it is found that a large percent of those patients are suffering sublingual venae expansion, morphology circle change and the color change to blue, purple and crimson. These changes are sensitive and significantly related to the progress of treatment and the prognosis of the patients. In Traditional Chinese Medicine, liver and tongue are deep related. Abnormalities of liver may reflect on the tongues with corresponding changes. Many researches suggest that abnormal formation of sublingual collateral vessels of these patients may be related to the increased stress of portal vein. However, the mechanism of abnormal sublingual venae was rarely reported. Previous researches reported that the increasing pressure of sublingual venae resulted in the abnormal sublingual venae. However, we found that primary hepatic carcinoma patients with portal vein pressure are more likely to have the sublingual collaterals abnormalities and the width of Sublingual collaterals vessels was related to portal vascular and certain local hemodynamic indicators directly. Meanwhile, the result that the pathological changes of sublingual collateral vessels and determined the stress of portal vein of 118 portal hypertension patients at surgery also confirm that sublingual collateral vessels varicose rate is 100% in the portal hypertension patients. Based on this, we put forward one hypothesis that sublingual collateral is a component of human vein system as well as portal vein, and there must be some relationship in pathology between them. However, the formation of portal hypertension has three types, and there are extrahepatic obstruction, intrahepatic blockages and extrahepatic intrahepatic compound jam. Different formation mechanism of portal hypertension would lead to the different change of sublingual collaterals. The following days, we discovered that there were different changes between patients with cirrhosis and patients with portal hypertension caused by tumor obstruction. Therefore, it is difficult to explain by passive expansion of sublingual venae. This suggests us that abnormal sublingual collateral vessels which are caused by liver portal hypertension have deeper material basis.
With the developing research of modern cancer and molecular biology, molecular mechanism of angiogenesis has been gradually revealed. For the adults, physiological and pathological mechanisms of angiogenesis are the main blood vessel formation. Lots of elements are playing positive regulatory roles in angiogenesis. In factors that known in angiogenesis, the vascular endothelial growth factor (Vascular Endothelial Growth Factor, VEGF) are the most important. Hypoxic response, mechanical stress and a variety of mechanisms are involved in the VEGF gene induction. VEGF adjust angiogenesis through VEGF/VEGFR signal passage. Recent studies found that fat inflammation factors and its receptor, Apelin/APJ path, also have close relations with the angiogenesis, and are looked as new targets of treatment to the portal hypertension. In recent years research has been confirmed that the lungs and spleen also exists angiogenesis in the process of portal hypertension. Therefore, envisaging under the "angiogenesis" theory, pay the focus on the perspective of angiogenesis to explore the possible molecular mechanism of abnormal sublingual collateral vessels in different portal hypertension models.
Objective
To observe the changes and study the formation mechanism of sublingual collateral vessels in three different portal hypertension models. And from the Angle of angiogenesis, explore the relationship between VEGF/VEGFR2, Apelin/APJ signaling pathways and abnormal sublingual collateral vessels mainly in order to provide data support for revealing the molecular mechanism of abnormal sublingual collateral vessels.
Methods
(1) Methods of making different portal hypertension models with dog. Beagle dogs were randomly divided into five groups. There are normal group, sham group, prehepatic portal hypertension group, intrahepatic portal hypertension group, and compound portal hypertension group. There were 6 dogs in each group. The portal veins of dogs were ligatured in prehepatic portal hypertension group. Canine model of intrahepatic portal hypertension was established by injecting dimethylnitrosamine (DMN) into portal vein once a week which would last for 7 weeks. The above two methods were used to make compound portal hypertension model together. During the process of each model production, portal pressure of dogs was determined with the bioelectricity recording system. After the dogs were sacrificed, The serum liver function was detected with reagent kits, and the liver pathological changes were observed with HE coloration.
(2) Methods of observing the features of tongue collaterals in different portal hypertension models. The changes of sublingual collaterals form and color were detected by macroscopic observation. The width of tongue root collaterals was measured by vernier caliper. CD31 protein expression of sublingual collateral vessels was detected by immunohistochemical coloration. The morphological changes the endothelial cells of sublingual collaterals were observed by transmission electron microscope.
(3) Methods of studying the formation mechanism of sublingual collateral vessels in different groups. Nitric oxide in serum was detected by nitrate reductase method. The vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α(HIF-1α) expression of serum was detected by ELISA. The proteins expression of VEGF, HIF-1α, VEGFR2, Apelin and APJ in sublingual tissues was detected by immunohistochemical coloration and western blotting. The mRNA expression level of VEGF, VEGFR2 and HIF-1αin sublingual tissues was detected by Real-Time PCR.
Results
(1) The portal vein pressure of dogs was beginning to rise after DMN induction 2 weeks in intrahepatic and compound groups compared with this in sham group(P<0.05). The portal vein pressures of dogs in model groups were a ladder shaped up in 4 weeks, six weeks, eight weeks with experimental progress(P<0.01),and were stable after 8 weeks. The pressure of dogs in compound group further increased after operation of portal vein ligation(P<0.01). After portal vein ligation, the pressure of dogs in prehapatic model group lifted to peal quickly, and gradually declined to a high level when the dogs were sacrificed(P<0.01).What’s more, the dog anomalous changes of the liver function and liver pathology were similar with human.
(2) The morphous and color of sublingual collateral vessels of the beagle dogs in intrahepatic and compound model groups changed obviously compared with those in normal control group and sham group. Sublingual collateral vessels appeared enlarged tortuous and increased diameters, the increased number of small collateral vessels and even bluish purple. While in prehepatic model group, sublingual collateral vessels appeared color changed only. Immunohistochemical coloration results showed that the expression of CD31 protein increased compared with this in normal and sham group (P<0.01), and the most obvious increase was in compound model group. Transmission electron microscopy showed that sublingual hemal walls of dogs in three model groups were thicken totally or partly, vascular cavity becomes small, new capillaries appeared, and membrane boundary of endothelial cell is not clear, cell nuclei lost or disappeared. For changes degree, it was heaviest in compound model group, second in intrahepatic model group, and lightest in prehepatic model group.
(3) NO in serum of the beagle dogs was significantly increased in model groups compared with normal and sham groups (P<0.01). VEGF, HIF-1αprotein expression in serum was significantly higher in two model groups (P<0.01), while no significant changes in prehehatic model group. Immunohistochemical coloration results showed that there is almost no expression of VEGF, VEGFR2 and HIF-1αof sublingual collateral vessels in the normal control group. But the expression of three model groups increased. The mRNA of VEGF, VEGFR2 and HIF-1αalso significantly increased. As for Apelin and APJ, their protein expression was same as others.
Conclusion
The dog sublingual collaterals vessels appeared macroscopic and microcosmic changes in three kinds of portal hypertension models. Portal hypertension caused by intrahepatic obstruction may be contributing to the formation of abnormal sublingual collaterals vessels. Extrahepatic jam often becomes the aggravation of factors inducing collaterals changes. Angiogenesis induced by VEGF and Apelin participated in the formation of abnormal sublingual collaterals. They reacted by HIF-1α/ VEGF/ VEGFR2/ NO and Apelin/APJ pathways respectively. It may be one of the important mechanisms of vascular remodeling in abnormal sublingual collaterals vessels.
引文
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