金属蛋白酶ADAMTS-7在肺动脉高压发病机制中的作用研究
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摘要
背景：肺动脉高压(Pulmonary arterial hypertension,PAH)在临床是一种多因素参与的病理生理过程,可由多种疾病引起共有的常见综合征,是严重的、具有潜在破坏力的慢性肺循环疾病,是以肺小动脉的血管痉挛、内膜及中膜增生和重构为主要特征的一种疾病。临床表现主要有劳力性呼吸困难、气促、乏力、胸痛、晕厥等,患者最终往往死于右心衰竭。目前尚缺乏有效治疗方法,确诊后患者平均生存时间与恶性肿瘤不相上下,自然存活时间2.8年,预后极差。
虽然近年来细胞分子生物学和遗传学的高速前进促进了肺动脉高压发病机制的研究,取得了一系列的研究成果,但目前认为,肺动脉高压的发病机制非常复杂,至今没有一个完整的阐述。到目前为止,其发生机制研究得出主要为：1、与Ⅱ型骨形成蛋白(Bone Morphogenetic Protein, BMP)受体(BMPR2)有关的基因异常突变；2、内皮系统受损及异常分泌；3、钾离子通道异常；4、炎症细胞的作用；5、基质金属蛋自酶(matrix metalloproteinases,MMPs)的上调。
基质金属蛋白酶及其特异性抑制物(tissue inhibitor of metallo-proteinases, TIMPs)在细胞处基质合成与降解的动态平衡中起着关键作用。在主动脉和冠状动脉壁病变中,基质金属蛋白酶的作用是研究热点。在肺动脉高压的研究中,早有专家研究显示MMP-1和MMP-2通过降解血管基底膜的胶原及变性胶原,后者又拮抗其它基质金属蛋白酶的降解,促进肺血管平滑肌的增殖与迁移,从而影响肺血管的结构重塑,参与各种肺动脉高压的形成。而TIMP-1是活性基质金属蛋白酶-1和-2的特异性抑制物,其可特异性的阻断后者与底物相结合,从而抑制相关胶原的降解过程,正常情况下,二者平衡维持稳态。布朗等证实一氧化氮的充分供给可以使基质金属蛋白酶抑制物质的分泌减少。动物研究显示,一氧化氮可通过TIMP-1/MMP-1平衡的调节来减少胶原的堆积,加强胶原的降解,从而影响肺血管重构。有人研究发现抑制基质金属蛋白酶可以明显降低肺动脉高压患者的肺动脉压力。肺动脉高压时平滑肌细胞增生,无肌化肺小动脉出现有肌化现象；严重肺动脉高压的肺动脉内皮细胞与内弹力板之间出现“新生内膜”,后者由肌纤维细胞及细胞外基质构成；内皮细胞、平滑肌细胞及成纤维细胞增殖与凋亡出现失平衡状态,导致肺动脉结构重构,最终形成肺动脉高压。在肺动脉高压血管重构过程中,首先是外膜成纤维细胞被激活增殖并合成基质成分；之后MMP2、MMP9等基质金属蛋白酶上调并介导成纤维细胞由外膜迁移进入血管中膜和内膜,再之出现肺血管中膜及内膜增生增殖、血管重构形成。
含Ⅰ型血小板反应蛋白的解聚素和金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs,ADAMTS)家族是一类新近被克隆的Zn依赖的分泌型金属蛋白酶家族,这个家族与基质金属蛋白酶家族及解聚蛋白样金属蛋白酶同属于金属蛋白酶家族,但由于其发现时间较晚(1997年),我们对它的了解远远不如后两个家族那么深入。该家族成员与基质金属蛋白酶家族一样,同样具有降解细胞外基质的能力。其于其它两个家族结构的主要不同点是羧基端含有至少一个TSP重复序列(又称之为TSP基序)。ADAMTS家族属于分泌型蛋白酶,可由体内多种细胞合成与分泌,如平滑肌细胞、血管内皮细胞、成纤维细胞、巨噬细胞等,其通常与基质相结合。其一共有19个家族成员组成,目前了解的较多是ADAMTS-1、ADAMTS-2、ADAMTS-4、ADAMTS-5、ADAMTS-9及ADAMTS-13这几个成员,它们通过对细胞外基质蛋白的降解或调节而参与了一系列的正常生理学功能,如发育、血管新生和凝血等。而它们的异常表达与细胞外基质蛋白相互作用后通常会引发各种疾病,如癌症、关节炎、动脉粥样硬化、皮肤脆性相关的疾病、凝血功能障碍性紫癜等疾病。而其更多的家族其它成员的功能仍不清楚,有待研究。
其中ADAMTS家族的第七个成员——ADAMTS-7对血管方面的精细调节随着人们的深入研究而逐渐让人所知,通过其底物软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)精细调控血管稳态。ADAMTS-7是1999年由Hurskainen TL团队首先报道,其结构包括一个锌离子催化结构域和其他几个非催化结构域,包括：1个disintegrin结构域、1个thrombospondin结构域、1个cysteine-rich结构域(cysteine-rich domain, CRD)、1个spacer-1结构域、3个thrombospondin motifs、1个spacer-2结构域和C-末端4个thrombospondin motifs。ADAMTS-7其与家族成员ADAMTS-12同属ADAMTS家族一个亚型,可直接结合并降解软骨基质蛋白。近十年来国际上研究其较多的有刘传聚教授(现在美国纽约大学医学院系)和北京大学医学部的王利教授研究团队,其中刘教授对其研究主要是从关节炎及软骨发育这一领域,如其研究显示通过体外试验,发现在风湿性软骨滑膜炎病人过表达与降解COMP相关的ADAMTS-7明显升高。在关节炎病人中发现的COMP降解碎片与ADAMTS-7降解COMP的碎片相同。另外,通过应用ADAMTS-7的抗体则可明显的抑制软骨培养中的肿瘤坏死因子和白介素-1β诱导的COMP降解。用siRNA沉默技术抑制ADAMTS-7也可明显的阻止COMP的降解。王利研究团队研究ADAMTS-7从血管出发,这其中也涉及到从COMP入手,发现ADAMT-7可以通过调节COMP的降解而有可能促进血管平滑肌细胞钙化的作用,其发现miRNA-29可干预ADAMTS-7的下游反应,从而调节血管钙化；另外其还通过球囊损伤大鼠颈动脉模型发现ADAMT-7在血管平滑肌细胞的增殖与迁移中起重要作用；其研究认为ADAMTS-7可能参与了动脉粥样硬化中血管基质的降解和重塑,从而加速了动脉粥样硬化的进程。也有研究报道ADAMTS-7其还参与了血管损伤后内膜的增生。国内有专家对非ST段抬高型心肌梗死的血液进行了相关检测,显示ADAMTS-7的水平与NSTEMI患者远期预后密切相关。ADAMTS-7的信使RNA之前报道称其是由人THP-1的单核/巨噬细胞中的TNF-α上调的,但其调节的精细机制仍不清楚；在王利等的研究认为其可能的机制是通过转录因子NF-κB和AP-1来让TNF-α上调而刺激ADAMTS-7诱导大鼠平滑肌增殖。
据研究报道,在成人样本中,通过检测分析ADAMTS-7,发现在心脏、胰腺、肾、骨骼肌和肝脏具有最高水平的表达,在大鼠肝脏、睾丸、肺、卵巢、肾、胚胎、心脏和胸腺组织中是均有不同程度地表达。在骨骼肌、半月板和脂肪也可较低水平的被检测到。但是在肺动脉高压疾病相关的肺组织中其表达情况暂时未见报道。
基于以上资料,我们已明确肺动脉高压的发病机制非常复杂,具体机制仍不是很清楚,但可以肯定的是基质金属蛋白酶的异常表达是肺动脉高压重要发病机制之一,而ADAMTS这一家族金属蛋白酶与基质金属蛋白酶一样,同样具有降解细胞外基质的能力,同时又广泛分布于哺乳动物的大部分脏器内；而既往研究资料显示其中ADAMTS-7与血管发育关系密切,特别是与体循环的动脉粥样硬化关系密切,我们推测ADAMTS-7可能与肺循环也有密切联系,如能加以证实有望成为肺动脉高压治疗的新靶点。本课题拟先在成人肺动脉高压肺组织和正常肺动脉压力肺组织行ADAMTS-7检测,以明确ADAMTS-7有无在肺组织中表达,有无在肺动脉高压患者肺组织中表达。再进行肺动脉平滑肌细胞培养,进行TNF-α干预后检测ADAMTS-7在其中的表达,以及相关增殖、凋亡、炎症指标的变化。最后,我们在肺动脉高压大鼠中再进行相关检测验证。本研究三部分分述如下：第一部分轻中度肺动脉高压患者肺组织中的ADAMTS-7的表达情况
目的：为明确ADAMTS-7在肺组织中的表达及肺动脉高压患者肺组织中的表达情况。方法：采用病例对照研究,随机选取在广东医学院附属医院住院并拟开胸手术患者,其中20例患者经右心导管测压显示有轻中度肺动脉高压,另20例患者肺动脉压力经右心导管检测正常。两组患者在手术中取1cm3肺组织,进行免疫组织化学法及免疫荧光RT-PCR法检测两组患者肺组织中ADAMTS-7的表达。结果：免疫组织化学法显示在正常压力对照组肺组织中可见少许ADAMTS-7的阳性表达,在肺动脉高压组肺组织中可见大量ADAMTS-7阳性表达,经图像分析软件计算并统计得出病例组IOD值明显高于对照组(P<0.05)。免疫荧光RT-PCR法测两组肺组织中的ADAMTS-7显示两组与空白组(规定其Folds值为1)比较均有统计学差异(P<0.05)；对照组与肺动脉高压组比较,两者具有统计学差异(P<0.05)。结论：ADAMTS-7可能参与肺动脉高压的发病过程。
第二部分ADAMTS-7在肺动脉平滑肌细胞中的表达及可能作用机制研究目的：为明确ADAMTS-7在肺动脉平滑肌细胞中的表达及可能的作用机制。方法：培养人原代肺动脉平滑肌细胞,先用免疫荧光细胞化学进行了ADAMTS-7在肺动脉平滑肌细胞中的表达的检测；再用CCK-8法检测不同时间、浓度的TNF-α处理肺动脉平滑肌细胞,以明确其增殖的最佳时间及浓度。后进行分组,分为空白组、TNF-α25ug/L干预组、ADAMTS-7siRNA沉默组,分别培养12小时、24小时、48小时、72小时,再用RT-PCR法检测各组肺动脉平滑肌细胞中ADAMTS-7、PCNA、NF-κB、PI3K、VEGF、MARKK4、ERK1、IL-1、IL-6等因子的表达相对含量。取其中干预组24小时点用免疫蛋白印迹法(Western Blot)检测ADAMTS-7、Bcl-2、COMP等因子的mRNA含量。结果：(1)通过细胞免疫荧光显示ADAMTS-7在肺动脉平滑肌细胞中丰富表达于平滑肌细胞胞浆内。(2)CCK-8法筛选出TNF-α在浓度为25μg/L,时间为24小时时对人肺动脉平滑肌细胞增殖的浓度时间为最优。(3)RT-PCR检测显示TNF-α25ug/L干预组ADAMTS-7、 PI3K、PCNA、VEGF、IL-1、IL-6等因子的mRNA表达量在各个时间点均明显高于siRNA沉默组(P<0.05);ADAMTS-7在TNF-α干预后24小时其表达量最高,PI3K、PCNA、IL-1等因子的mRNA在TNF-α干预后48小时其表达量最高,VEGF、IL-6等因子的mRNA在TNF-α干预后72小时其表达量最高；另显示TNF-α25ug/L干预组NF-κB、MARKK4等因子的mRNA表达量除第12小时段无统计学意义外(P>0.05),其它各个时间段(24小时、48小时、72小时)表达明显高于siRNA组,两者差异具有统计学意义(P<0.05),其中NF-κB等因子的mRNA在TNF-α干预后24小时其表达量最高,MARKK4等因子的mRNA在TNF-α干预后48小时其表达量最高;ERK1因子的mRNA在两组各个时间点均无统计学差异(P<0.05)。(4)免疫印迹检测结果显示TNF-α干预组其ADAMTS-7表达量明显高于siRNA沉默组及对照组,差异具有统计学意义(P<0.05),对照组表达量高于siRNA沉默组,差异具有统计学意义(P<0.05)；而COMP表达含量在siRNA沉默组及对照组明显高于TNF-α干预组,其差异具有统计学意义(P<0.05),siRNA沉默组及对照组相比较,二者无统计学差异(P>0.05)；在Bcl-2表达在三组相当,结果无统计学差异(P>0.05)。结论：(1)ADAMTS-7可能通过P13K、NF-κB、TNF-α、JNK等途径调节肺动脉平滑肌细胞的增殖或凋亡；(2)ADAMTS-7可能没有通过ERK、Bcl-2参与肺动脉平滑肌细胞的凋亡；(3)ADAMTS-7可能促进肺动脉平滑肌细胞COMP的降解及VEGF的表达。
第三部分ADAMTS-7在野百合碱诱导肺动脉高压大鼠中的表达情况
目的：通过干预野百碱诱导肺动脉高压大鼠,检测ADAMTS-7的表达情况及其对肺动脉平滑肌的增殖及炎症反应情况。方法：SD大鼠30只,随机分为正常对照组(对照组)、MCT诱导的肺动脉高压组(MCT组)、强力霉素组。21天后进行了平均肺动脉压力(mPAP)测定,右心肥大指数(RVHI)测定,并对大鼠肺组织进行ADAMTS-7免疫组织化学检测及TNF-α、NF-κB、IL-1、IL-6、PI3K、MARKK4等因子RT-PCR法检测,免疫印迹法检测各组ADAMTS-7、PCNA、Bcl-2的mRNA表达情况。结果：(1)MCT组及强力霉素组大鼠的mPAP和RVHI均高于对照组(P<0.05),强力霉素组mPAP比MCT组大鼠的mPAP低,但无统计学意义(P>0.05),其RVHI比MCT组RVHI低,并具有统计学意义(P<0.05)。(2)大鼠肺组织ADAMTS-7免疫组织化学显示MCT组明显高于对照组及强力霉素组(P<0.05),强力霉素组与对照组相比,前者高于后者,差值具有统计学差异(P<0.05)。(3)RT-PCR法测各因子mRNA示(规定对照组Folds值为1) TNF-α、NF-κB>、IL-1、 IL-6、PI3K、MARKK4的mRNA表达在MCT组与强力霉素组均明显高于对照组,两者差异具有统计学意义(P<0.05)；各因子在MCT组明显高于强力霉素组,其差异具有统计学意义(P<0.05)。(4)免疫印迹法结果显示：MCT组ADAMTS-7、PCNA、Bcl-2的mRNA表达高于对照组及强力霉素组,P<0.05；强力霉素组PCNA、Bcl-2的mRNA表达高于对照组,P<0.05；其ADAMTS-7的mRNA表达与对照组相当,无统计学意义,P>0.05。结论：强力霉素可能通过抑制ADAMTS-7来调节肺动脉平滑肌的增殖及炎症反应。单一强力霉素通过抑制ADAMTS-7等金属蛋白酶的作用机制来降低肺动脉压力效果不明显。
Research background:The Pulmonary arterial hypertension(PAH) is a common syndrome caused by various diseases, which is a pathophysiological process involved in a kind of multiple factors. PAH is a serious and potentially destructive chronic Pulmonary disease, which characteristics include small artery vasospasm and hyperplasia in endometrium and medial, then vascular remodelling. The PAH's main clinical symptoms include exertional dyspnea, shortness of breath, fatigue, chest pain, syncope et al. patients die eventually right heart failure. It is lack of effective treatment methods in currently. Just like malignant tumour, the prognosis so poor that the average survival time after diagnosis as PAH is2.8years.
In recent years, although the biotechnology are developed very fast and made a series of research results about the pathogenesis of pulmonary hypertension, this pathogenesis is so complex that there are not yet a complete explaining. Until now, the main mechanism of PAH include as follow:One is the abnormal gene mutation related to type Ⅱ Bone Morphogenetic proteins receptor (BMPR2); Two is the impaired endothelial system and abnormal secretion; Three is abnormal potassium channels; Four is the action of inflammatory cells; Five is up regulated of the matrix metalloproteinases(MMPs).
Tissue inhibitor of metalloproteinases(TIMPs), the MMP specific inhibitor, and Matrix metalloproteinases are dynamic equilibrium in cell matrix synthesis and degradation. In aortic and coronary artery, the effect of matrix metalloproteinases is hot research topic. In pulmonary arterial hypertension research, the earlier studies had shown that through the degradation of collagen and collagen denaturation of vascular basement membrane, which in turn antagonism other the degradation of matrix metalloproteinases, MMP-1and MMP-2promoted the proliferation and migration of pulmonary vascular smooth muscle and then affected the pulmonary vascular structural remodelling, so participated in the formation of pulmonary hypertension. The TIMP-1is specific inhibitor of the matrix metalloproteinases-1and-2which can specifically block the latter combined with the substrate, thus inhibiting the degradation of collagen, under normal circumstances, the balance maintain homeostasis. Brown who proved adequate supply of nitric oxide can decrease the secretion of inhibitor about matrix metalloproteinase. Animal studies have shown that nitric oxide can affect pulmonary vascular remodelling by reduced the accumulation of collagen through adjusting the TIMP-1/MMP-1balance and strengthened collagen degradation. It was found that inhibition of matrix metalloproteinase can significantly reduce pulmonary artery pressure in patients with pulmonary hypertension. The phenomenon will appear. For example the smooth muscle cell proliferation in pulmonary hypertension and the pulmonary no muscle arteries have muscle."Neointimal" appears between endothelial cells and internal elastic lamina while the severe pulmonary arterial hypertension, the neointimal are composed by the myofibroblasts and extracellular matrix. The proliferation and apoptosis appears loss of balance in the endothelial cells and smooth muscle cells and fibroblasts, then leading to pulmonary structural remodelling and ultimately causing the pulmonary arterial hypertension. In the process of pulmonary hypertension vascular remodelling, the first is to be activated adventitial fibroblasts to proliferate and synthesise matrix components. Then the matrix metalloproteinase, like MMP2and MMP9, are up regulated and mediated fibroblast migration from the outer membrane into the medial and intima of the blood vessels. Then the intimal and medial of pulmonary vascular will be hyperplasia and proliferation and formed vascular remodelling.
A disinterring and metalloproteinase with thrombospondin motifs(ADAMTS) family is recently cloned who is a class of secreted metalloprotease family with Zn2+-dependent. The family and the family of matrix metalloproteinases and Depolymerization polyprotein-like metalloprotease belong metalloproteinase family. Because it is found just in the last ten years (in1997), we understand it is far less than the two families. The family same as the matrix metalloproteinase family have the ability to degrade the extracellular matrix. Compare to the other two family, the main difference of ADAMT family in structures is a repeat sequences containing at least one TSP (also known as TSP motifs) in carboxy-terminal. ADAMTS family are secreted proteases, which can be synthesized and secreted in vivo by a variety of synthetic and secretory cells, such as smooth muscle cells, endothelial cells, fibroblasts, macrophages. And ADAMTS is usually combined with the matrix. There were19family members. We are known some of their function, like the ADAMTS-1, ADAMTS-2, ADAMTS-4, ADAMTS-5, ADAMTS-9and ADAMTS-13.They degrade or regulate the extracellular matrix proteins and participate in a series of normal physiological functions, such as growth, angiogenesis, and coagulation et al. While their express abnormally and interact with the extracellular matrix, they will often lead to various diseases, such as cancer, arthritis, atherosclerosis, skin fragility-related diseases, coagulation disorders purpura and other diseases. The function of other family members remains unclear and let us to be studied.
ADAMTS-7is the seventh of the members of the ADAMTS family, who is known gradually by people in-depth study due to the fine adjustment of blood vessel. Through its substrate cartilage oligomer matrix protein (COMP), ADAMTS-7maintains steady-state by fine regulation of blood vessels. ADAMTS-7is first reported by Hurskainen TL team in1999, whose structure is composed of a catalytic domain who have a zinc ions and several other non-catalytic domain structure, including:a disintegrin domain structure, a thrombospondin domain structure, a structure of cysteine-rich domain (CRD), a spacer-1structure domain, three thrombospondin motifs, a spacer-2domain structure, four thrombospondin motifs in the ends of the C. ADAMTS-7with the ADAMTS-12belong to one hypo type of ADAMTS family, who can be directly combined and degradation of the cartilage matrix proteins. The Professor Liu Chuan-Ju who are in New York University School of Medicine and Professor Wang Li's research team whose are in the medical department of Peking University have done many researches over the past decade in internationally. Professor Liu does it in this field from arthritis and cartilage development. His research shows that the ADAMS-7who was found in patients with rheumatoid synovitis cartilage increased significantly through in vitro tests and who is overexpression associated with COMP degradation. In arthritis patients, they found COMP degradation fragments same as the COMP fragments through degrading by ADAMTS-7. In addition, through implicating the antibody of ADAMTS-7, it can significantly inhibit the COMP degradation by induced through tumour necrosis factor and interleukin-1β who are in cartilage cultured. Inhibition ADAMTS-7by siRNA silencing technology can also significantly prevent the degradation of COMP. The team of Wang Li study ADAMTS-7from blood vessels and also involve the COMP. They found that ADAMT-7could promote vascular smooth muscle cells calcification by regulating the degradation of COMP. they found that the microRNAs-29could regulate vascular calcification through intervened downstream reaction of ADAMTS-7. They also found ADAMT-7plays an important role in vascular smooth muscle cell proliferation and migration in balloon injury of rat carotid artery model. This research shows the ADAMTS-7may participate in the atherosclerosis in vascular matrix degradation and remodelling, which accelerated the process of atherosclerosis. Some research also reported that ADAMTS-7is involved in the intima hyperplasia after vascular injury. Through some related detected on the blood of non ST segment elevation myocardial infarction, some experts thought that the ADAMTS-7levels are closely associated with long-term prognosis of NSTEMI patients. The messenger RNA of ADAMTS-7is up regulate by TNF alpha in THP1in the monocyte or macrophage of people. The fine mechanism of adjustment is still unclear. Some research thought that its possible mechanism is through the transcription factor of the nuclear factor kappa B and AP-1to let the TNF alpha up regulate and stimulate ADAMTS-7induced rat smooth muscle proliferation.
According to studies about detecting ADAMTS-7, it found that there are high expression levels in heart, pancreas, kidney, skeletal muscle and liver in the adult samples. It was found expressed in liver, testis, lung, ovary, kidney, fetal heart and thymus tissue in varying degrees. In skeletal muscle, meniscus and fat can also be detected lower levels. In the lung tissue of pulmonary hypertension patient, the expression of ADAMTS-7is not reported now.
Based on the above information, we have known that the pathogenesis of pulmonary hypertension is very complex. The exact mechanism of PAH is still not very clear. There is one thing could make sure that the abnormal expression of matrix metalloproteinase is important in the pathogenesis of pulmonary hypertension. And the ADAMTS same as the family of matrix metalloproteinase, it also has the ability to degrade extracellular matrix. At the same time, the ADAMTS is widely distributed in mammalian organs. Previous research data had showed that the ADAMTS-7is closely associated with vascular development, in particular with circulation of atherosclerosis closely related to atherosclerosis. We hypothesized that ADAMTS-7and pulmonary circulation may also have close ties, as can be confirmed is expected to become a new target for the treatment of pulmonary hypertension. This paper intends to test the ADAMTS-7expression of lung tissue between adult pulmonary hypertension and normal pulmonary artery pressure in first, then could to clarify whether the ADAMTS-7expression in lung tissue, with or without expression in the lung tissue of patients with pulmonary hypertension. Second, cultured pulmonary artery smooth muscle cells, we test the expression of ADAMTS-7and some index of associated with proliferation and apoptosis and inflammation after intervened of the TNF-a. Finally, we verify the correlation detection of pulmonary hypertension in rats. In this study, there are three parts as follows.
Part one. The expression of the ADAMTS-7in Patients lung tissue with mild-to-moderate pulmonary hypertension
Objective:To verify the expression of ADAMTS-7in lung tissue in patients with pulmonary hypertension. Methods:There was a case-control study. Selected40cases of patients randomly who want to do the open chest surgery in The Affiliated Hospital of Guangdong medical college,20cases showed mild-to-moderate pulmonary hypertension by tested pressure through right cardiac catheterization(as case group), the pulmonary artery pressure in other20cases was normal(as control group). Take a few lung tissue in the two groups of patients in the surgery, used the immune immunofluorescence histochemical method and RT-PCR method to detect the expression of ADAMTS-7in lung tissue of the two groups patients. Results: Through the immune histochemical methods, it showed that there is a little positive expression of ADAMTS-7in the lung tissue of the control group and numerous positive expressions of ADAMTS-7in the lung tissue of the case group. By the image analysis and statistical calculation software, the IOD value in case group was significantly higher than control group (P<0.05). Through immunofluorescence RT-PCR method to test the expression of ADAMTS-7in the lung tissue in the two groups, it showed that the two groups (control group and case group) compared with the blank group (rules its Folds value is1) were statistically significant (P<0.05). In the control group compared with the case group, which had a statistically significant difference (P<0.05). Conclusion:ADAMTS-7may be involved in the pathogenesis of pulmonary hypertension.
Part two. The expression and the possible mechanism of ADAMTS-7in pulmonary artery smooth muscle cells
Objective:to verify the expression and the possible mechanism of ADAMTS-7in pulmonary artery smooth muscle cells. Methods:Cultured the people primitive pulmonary artery smooth muscle cells, first we tested the expression of ADAMTS-7in pulmonary artery smooth muscle cells by immunofluorescence cytochemistry Then used the CCK8method to detect the TNF alpha intervene on cell proliferation, in order to make clear of the optimum time and concentration. Then divided into three groups:blank group, the25ug/L TNF alpha intervention group, the ADAMTS-7siRNA silence group. Cultured respectively for12hours,24hours,48hours,72hours and detected the relative content of ADAMTS-7, PCNA, NF-kappa B, PI3K, VEGF, MARKK4, ERK1, IL-1, IL-6by RT-PCR method in every groups. Chose the point of24hours in intervention group (the25ug/L TNF alpha intervention group, the ADAMTS-7siRNA silence group) with immune protein imprinting method (Western Blot) to detect the mRNA levels of ADAMTS-7, the Bcl-2and COMP. Results:(1) Through the cellular immune fluorescent method, there was numerous positive expression of ADAMTS-7in pulmonary smooth muscle cell cytoplasm.(2) Through CCK8method, the concentration of25ug/L TNF alpha and the cultured time of24hours were screen out as the optimal concentration and time for proliferation of pulmonary artery smooth muscle cell.(3) The RT-PCR detection showed that the expression levels of ADAMTS-7, PI3K, PCNA, VEGF, IL-1, IL-6in all time point in TNF alpha25ug/L intervention group were significantly higher than that of siRNA silent group (P<0.05). The time of the highest express quantity of ADAMTS-7was the24hours after intervening by25ug/L TNF alpha. The time of the highest express quantity of PI3K, PCNA and IL-1was the48hours after intervening by25ug/L TNF alpha. The time of the highest express quantity of VEGF, IL-6was the72hours after intervening by25ug/L TNF alpha. The RT-PCR detection showed that the expression levels of NF-kappa B, MARKK4in24hour and48hour and72hour in TNF alpha25ug/L intervention group were significantly higher than that of siRNA silent group (P<0.05). The two groups were no significant statistical significance (P>0.05) in the12hour time point. The time of the highest express quantity of NF-kappa B was the24hours after intervening by25ug/L TNF alpha. The time of the highest express quantity of MARKK4was the48hours after intervening by25ug/L TNF alpha. ERK1factor mRNA between two groups had no statistically significant difference in the various time points (P<0.05).(4) the western blot test results showed that the amount of ADAMTS-7express in TNF alpha intervention group was significant higher than that of ADAMTS-7siRNA silent group and control group(P<0.05), The amount of ADAMTS-7express in control group was significant higher than that of the ADAMTS-7siRNA silent group(P<0.05), While the COMP expression levels in TNF alpha intervention group was significance lower than that of ADAMTS-7siRNA silent group and control group(P<0.05), The COMP expression levels in the siRNA ADAMTS-7group was no statistical difference (P>0.05), the Bcl-2expression in three groups was no statistical difference (P>0.05). Conclusion:(1) The ADAMTS-7May regulate the pulmonary artery smooth muscle cell proliferation or apoptosis by the TNF alpha pathway, NF-kappa B pathway, JNK pathway, PI3K pathway.(2) The ADAMTS-7may not participate the apoptosis of pulmonary smooth muscle cells through ERK or Bcl-2pathway.(3) The ADAMTS-7may promote the migration of smooth muscle cells by promoting the degradation of COMP and the expression of the VEGF.
Part three. The expression of ADAMTS-7in monocrotaline (MCT) induced pulmonary hypertension rats
Objective:By intervening the monocrotaline induced pulmonary hypertension rats, detecting the expression of ADAMTS-7and the proliferation and inflammation in pulmonary artery smooth muscle. Methods:30SD rats were randomly divided into three group:the normal control group (control group), the group of pulmonary hypertension induced by MCT (MCT), the doxycycline group. Measured the average pulmonary artery pressure (mPAP) and the right heart hypertrophy index (RVHI) after21days. And detect the ADAMTS-7by immunohistochemical and the TNF alpha, NF-kappa B, IL-1, IL-6, PI3K, MARKK4by RT-PCR in the lung tissue of rats. And detected the mRNA expression of ADAMTS-7, PCNA and Bcl-2by western blot method at each group. Results:(1) The mPAP and RVHI of rats in MCT group and doxycycline group were significantly higher than that of control group (P<0.05). The mPAP of rats in doxycycline group was lower than MCT group, but no statistical significance (P>0.05). The RVHI of rats in doxycycline group was less than the MCT group and has statistical significance (P<0.05).(2) Through immunohistochemical method for the lung tissue of rat, the positive of ADAMTS-7in MCT group was significantly higher than the control group and doxycycline group (P<0.05). While the doxycycline group compared with the control group, the former was higher than the latter and the difference was statistically significant (P<0.05).(3) By measuring used the RT-PCR method, the mRNA expression of the TNF alpha, the NF-kappa B, IL-1, IL-6, PI3K, MARKK4in MCT group and doxycycline group were significantly higher than that of control group (regulation control Folds value of1)(P<0.05). Each factor in MCT group obviously higher than that of doxycycline group, the difference was statistically significant (P<0.05).(4) Western blot analysis showed that the mRNA expression of ADAMTS-7, PCNA, Bcl-2in MCT group was significantly higher than that of the doxycycline group and the control group (P <0.05). The mRNA expression of Bcl-2and PCNA in doxycycline group were significantly higher that of the control group (P<0.05). The mRNA expression of ADAMTS-7in doxycycline group was equivalent that of the control group. It was no statistical significance (P>0.05). Conclusion:Doxycycline may adjust the pulmonary artery smooth muscle proliferation and inflammation by inhibiting ADAMTS-7. Doxycycline alone through the mechanism inhibiting ADAMTS-7and other metal loproteinase reduces pulmonary artery pressure is not obvious.
虽然近年来细胞分子生物学和遗传学的高速前进促进了肺动脉高压发病机制的研究,取得了一系列的研究成果,但目前认为,肺动脉高压的发病机制非常复杂,至今没有一个完整的阐述。到目前为止,其发生机制研究得出主要为：1、与Ⅱ型骨形成蛋白(Bone Morphogenetic Protein, BMP)受体(BMPR2)有关的基因异常突变；2、内皮系统受损及异常分泌；3、钾离子通道异常；4、炎症细胞的作用；5、基质金属蛋自酶(matrix metalloproteinases,MMPs)的上调。
基质金属蛋白酶及其特异性抑制物(tissue inhibitor of metallo-proteinases, TIMPs)在细胞处基质合成与降解的动态平衡中起着关键作用。在主动脉和冠状动脉壁病变中,基质金属蛋白酶的作用是研究热点。在肺动脉高压的研究中,早有专家研究显示MMP-1和MMP-2通过降解血管基底膜的胶原及变性胶原,后者又拮抗其它基质金属蛋白酶的降解,促进肺血管平滑肌的增殖与迁移,从而影响肺血管的结构重塑,参与各种肺动脉高压的形成。而TIMP-1是活性基质金属蛋白酶-1和-2的特异性抑制物,其可特异性的阻断后者与底物相结合,从而抑制相关胶原的降解过程,正常情况下,二者平衡维持稳态。布朗等证实一氧化氮的充分供给可以使基质金属蛋白酶抑制物质的分泌减少。动物研究显示,一氧化氮可通过TIMP-1/MMP-1平衡的调节来减少胶原的堆积,加强胶原的降解,从而影响肺血管重构。有人研究发现抑制基质金属蛋白酶可以明显降低肺动脉高压患者的肺动脉压力。肺动脉高压时平滑肌细胞增生,无肌化肺小动脉出现有肌化现象；严重肺动脉高压的肺动脉内皮细胞与内弹力板之间出现“新生内膜”,后者由肌纤维细胞及细胞外基质构成；内皮细胞、平滑肌细胞及成纤维细胞增殖与凋亡出现失平衡状态,导致肺动脉结构重构,最终形成肺动脉高压。在肺动脉高压血管重构过程中,首先是外膜成纤维细胞被激活增殖并合成基质成分；之后MMP2、MMP9等基质金属蛋白酶上调并介导成纤维细胞由外膜迁移进入血管中膜和内膜,再之出现肺血管中膜及内膜增生增殖、血管重构形成。
含Ⅰ型血小板反应蛋白的解聚素和金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs,ADAMTS)家族是一类新近被克隆的Zn依赖的分泌型金属蛋白酶家族,这个家族与基质金属蛋白酶家族及解聚蛋白样金属蛋白酶同属于金属蛋白酶家族,但由于其发现时间较晚(1997年),我们对它的了解远远不如后两个家族那么深入。该家族成员与基质金属蛋白酶家族一样,同样具有降解细胞外基质的能力。其于其它两个家族结构的主要不同点是羧基端含有至少一个TSP重复序列(又称之为TSP基序)。ADAMTS家族属于分泌型蛋白酶,可由体内多种细胞合成与分泌,如平滑肌细胞、血管内皮细胞、成纤维细胞、巨噬细胞等,其通常与基质相结合。其一共有19个家族成员组成,目前了解的较多是ADAMTS-1、ADAMTS-2、ADAMTS-4、ADAMTS-5、ADAMTS-9及ADAMTS-13这几个成员,它们通过对细胞外基质蛋白的降解或调节而参与了一系列的正常生理学功能,如发育、血管新生和凝血等。而它们的异常表达与细胞外基质蛋白相互作用后通常会引发各种疾病,如癌症、关节炎、动脉粥样硬化、皮肤脆性相关的疾病、凝血功能障碍性紫癜等疾病。而其更多的家族其它成员的功能仍不清楚,有待研究。
其中ADAMTS家族的第七个成员——ADAMTS-7对血管方面的精细调节随着人们的深入研究而逐渐让人所知,通过其底物软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)精细调控血管稳态。ADAMTS-7是1999年由Hurskainen TL团队首先报道,其结构包括一个锌离子催化结构域和其他几个非催化结构域,包括：1个disintegrin结构域、1个thrombospondin结构域、1个cysteine-rich结构域(cysteine-rich domain, CRD)、1个spacer-1结构域、3个thrombospondin motifs、1个spacer-2结构域和C-末端4个thrombospondin motifs。ADAMTS-7其与家族成员ADAMTS-12同属ADAMTS家族一个亚型,可直接结合并降解软骨基质蛋白。近十年来国际上研究其较多的有刘传聚教授(现在美国纽约大学医学院系)和北京大学医学部的王利教授研究团队,其中刘教授对其研究主要是从关节炎及软骨发育这一领域,如其研究显示通过体外试验,发现在风湿性软骨滑膜炎病人过表达与降解COMP相关的ADAMTS-7明显升高。在关节炎病人中发现的COMP降解碎片与ADAMTS-7降解COMP的碎片相同。另外,通过应用ADAMTS-7的抗体则可明显的抑制软骨培养中的肿瘤坏死因子和白介素-1β诱导的COMP降解。用siRNA沉默技术抑制ADAMTS-7也可明显的阻止COMP的降解。王利研究团队研究ADAMTS-7从血管出发,这其中也涉及到从COMP入手,发现ADAMT-7可以通过调节COMP的降解而有可能促进血管平滑肌细胞钙化的作用,其发现miRNA-29可干预ADAMTS-7的下游反应,从而调节血管钙化；另外其还通过球囊损伤大鼠颈动脉模型发现ADAMT-7在血管平滑肌细胞的增殖与迁移中起重要作用；其研究认为ADAMTS-7可能参与了动脉粥样硬化中血管基质的降解和重塑,从而加速了动脉粥样硬化的进程。也有研究报道ADAMTS-7其还参与了血管损伤后内膜的增生。国内有专家对非ST段抬高型心肌梗死的血液进行了相关检测,显示ADAMTS-7的水平与NSTEMI患者远期预后密切相关。ADAMTS-7的信使RNA之前报道称其是由人THP-1的单核/巨噬细胞中的TNF-α上调的,但其调节的精细机制仍不清楚；在王利等的研究认为其可能的机制是通过转录因子NF-κB和AP-1来让TNF-α上调而刺激ADAMTS-7诱导大鼠平滑肌增殖。
据研究报道,在成人样本中,通过检测分析ADAMTS-7,发现在心脏、胰腺、肾、骨骼肌和肝脏具有最高水平的表达,在大鼠肝脏、睾丸、肺、卵巢、肾、胚胎、心脏和胸腺组织中是均有不同程度地表达。在骨骼肌、半月板和脂肪也可较低水平的被检测到。但是在肺动脉高压疾病相关的肺组织中其表达情况暂时未见报道。
基于以上资料,我们已明确肺动脉高压的发病机制非常复杂,具体机制仍不是很清楚,但可以肯定的是基质金属蛋白酶的异常表达是肺动脉高压重要发病机制之一,而ADAMTS这一家族金属蛋白酶与基质金属蛋白酶一样,同样具有降解细胞外基质的能力,同时又广泛分布于哺乳动物的大部分脏器内；而既往研究资料显示其中ADAMTS-7与血管发育关系密切,特别是与体循环的动脉粥样硬化关系密切,我们推测ADAMTS-7可能与肺循环也有密切联系,如能加以证实有望成为肺动脉高压治疗的新靶点。本课题拟先在成人肺动脉高压肺组织和正常肺动脉压力肺组织行ADAMTS-7检测,以明确ADAMTS-7有无在肺组织中表达,有无在肺动脉高压患者肺组织中表达。再进行肺动脉平滑肌细胞培养,进行TNF-α干预后检测ADAMTS-7在其中的表达,以及相关增殖、凋亡、炎症指标的变化。最后,我们在肺动脉高压大鼠中再进行相关检测验证。本研究三部分分述如下：第一部分轻中度肺动脉高压患者肺组织中的ADAMTS-7的表达情况
目的：为明确ADAMTS-7在肺组织中的表达及肺动脉高压患者肺组织中的表达情况。方法：采用病例对照研究,随机选取在广东医学院附属医院住院并拟开胸手术患者,其中20例患者经右心导管测压显示有轻中度肺动脉高压,另20例患者肺动脉压力经右心导管检测正常。两组患者在手术中取1cm3肺组织,进行免疫组织化学法及免疫荧光RT-PCR法检测两组患者肺组织中ADAMTS-7的表达。结果：免疫组织化学法显示在正常压力对照组肺组织中可见少许ADAMTS-7的阳性表达,在肺动脉高压组肺组织中可见大量ADAMTS-7阳性表达,经图像分析软件计算并统计得出病例组IOD值明显高于对照组(P<0.05)。免疫荧光RT-PCR法测两组肺组织中的ADAMTS-7显示两组与空白组(规定其Folds值为1)比较均有统计学差异(P<0.05)；对照组与肺动脉高压组比较,两者具有统计学差异(P<0.05)。结论：ADAMTS-7可能参与肺动脉高压的发病过程。
第二部分ADAMTS-7在肺动脉平滑肌细胞中的表达及可能作用机制研究目的：为明确ADAMTS-7在肺动脉平滑肌细胞中的表达及可能的作用机制。方法：培养人原代肺动脉平滑肌细胞,先用免疫荧光细胞化学进行了ADAMTS-7在肺动脉平滑肌细胞中的表达的检测；再用CCK-8法检测不同时间、浓度的TNF-α处理肺动脉平滑肌细胞,以明确其增殖的最佳时间及浓度。后进行分组,分为空白组、TNF-α25ug/L干预组、ADAMTS-7siRNA沉默组,分别培养12小时、24小时、48小时、72小时,再用RT-PCR法检测各组肺动脉平滑肌细胞中ADAMTS-7、PCNA、NF-κB、PI3K、VEGF、MARKK4、ERK1、IL-1、IL-6等因子的表达相对含量。取其中干预组24小时点用免疫蛋白印迹法(Western Blot)检测ADAMTS-7、Bcl-2、COMP等因子的mRNA含量。结果：(1)通过细胞免疫荧光显示ADAMTS-7在肺动脉平滑肌细胞中丰富表达于平滑肌细胞胞浆内。(2)CCK-8法筛选出TNF-α在浓度为25μg/L,时间为24小时时对人肺动脉平滑肌细胞增殖的浓度时间为最优。(3)RT-PCR检测显示TNF-α25ug/L干预组ADAMTS-7、 PI3K、PCNA、VEGF、IL-1、IL-6等因子的mRNA表达量在各个时间点均明显高于siRNA沉默组(P<0.05);ADAMTS-7在TNF-α干预后24小时其表达量最高,PI3K、PCNA、IL-1等因子的mRNA在TNF-α干预后48小时其表达量最高,VEGF、IL-6等因子的mRNA在TNF-α干预后72小时其表达量最高；另显示TNF-α25ug/L干预组NF-κB、MARKK4等因子的mRNA表达量除第12小时段无统计学意义外(P>0.05),其它各个时间段(24小时、48小时、72小时)表达明显高于siRNA组,两者差异具有统计学意义(P<0.05),其中NF-κB等因子的mRNA在TNF-α干预后24小时其表达量最高,MARKK4等因子的mRNA在TNF-α干预后48小时其表达量最高;ERK1因子的mRNA在两组各个时间点均无统计学差异(P<0.05)。(4)免疫印迹检测结果显示TNF-α干预组其ADAMTS-7表达量明显高于siRNA沉默组及对照组,差异具有统计学意义(P<0.05),对照组表达量高于siRNA沉默组,差异具有统计学意义(P<0.05)；而COMP表达含量在siRNA沉默组及对照组明显高于TNF-α干预组,其差异具有统计学意义(P<0.05),siRNA沉默组及对照组相比较,二者无统计学差异(P>0.05)；在Bcl-2表达在三组相当,结果无统计学差异(P>0.05)。结论：(1)ADAMTS-7可能通过P13K、NF-κB、TNF-α、JNK等途径调节肺动脉平滑肌细胞的增殖或凋亡；(2)ADAMTS-7可能没有通过ERK、Bcl-2参与肺动脉平滑肌细胞的凋亡；(3)ADAMTS-7可能促进肺动脉平滑肌细胞COMP的降解及VEGF的表达。
第三部分ADAMTS-7在野百合碱诱导肺动脉高压大鼠中的表达情况
目的：通过干预野百碱诱导肺动脉高压大鼠,检测ADAMTS-7的表达情况及其对肺动脉平滑肌的增殖及炎症反应情况。方法：SD大鼠30只,随机分为正常对照组(对照组)、MCT诱导的肺动脉高压组(MCT组)、强力霉素组。21天后进行了平均肺动脉压力(mPAP)测定,右心肥大指数(RVHI)测定,并对大鼠肺组织进行ADAMTS-7免疫组织化学检测及TNF-α、NF-κB、IL-1、IL-6、PI3K、MARKK4等因子RT-PCR法检测,免疫印迹法检测各组ADAMTS-7、PCNA、Bcl-2的mRNA表达情况。结果：(1)MCT组及强力霉素组大鼠的mPAP和RVHI均高于对照组(P<0.05),强力霉素组mPAP比MCT组大鼠的mPAP低,但无统计学意义(P>0.05),其RVHI比MCT组RVHI低,并具有统计学意义(P<0.05)。(2)大鼠肺组织ADAMTS-7免疫组织化学显示MCT组明显高于对照组及强力霉素组(P<0.05),强力霉素组与对照组相比,前者高于后者,差值具有统计学差异(P<0.05)。(3)RT-PCR法测各因子mRNA示(规定对照组Folds值为1) TNF-α、NF-κB>、IL-1、 IL-6、PI3K、MARKK4的mRNA表达在MCT组与强力霉素组均明显高于对照组,两者差异具有统计学意义(P<0.05)；各因子在MCT组明显高于强力霉素组,其差异具有统计学意义(P<0.05)。(4)免疫印迹法结果显示：MCT组ADAMTS-7、PCNA、Bcl-2的mRNA表达高于对照组及强力霉素组,P<0.05；强力霉素组PCNA、Bcl-2的mRNA表达高于对照组,P<0.05；其ADAMTS-7的mRNA表达与对照组相当,无统计学意义,P>0.05。结论：强力霉素可能通过抑制ADAMTS-7来调节肺动脉平滑肌的增殖及炎症反应。单一强力霉素通过抑制ADAMTS-7等金属蛋白酶的作用机制来降低肺动脉压力效果不明显。
Research background:The Pulmonary arterial hypertension(PAH) is a common syndrome caused by various diseases, which is a pathophysiological process involved in a kind of multiple factors. PAH is a serious and potentially destructive chronic Pulmonary disease, which characteristics include small artery vasospasm and hyperplasia in endometrium and medial, then vascular remodelling. The PAH's main clinical symptoms include exertional dyspnea, shortness of breath, fatigue, chest pain, syncope et al. patients die eventually right heart failure. It is lack of effective treatment methods in currently. Just like malignant tumour, the prognosis so poor that the average survival time after diagnosis as PAH is2.8years.
In recent years, although the biotechnology are developed very fast and made a series of research results about the pathogenesis of pulmonary hypertension, this pathogenesis is so complex that there are not yet a complete explaining. Until now, the main mechanism of PAH include as follow:One is the abnormal gene mutation related to type Ⅱ Bone Morphogenetic proteins receptor (BMPR2); Two is the impaired endothelial system and abnormal secretion; Three is abnormal potassium channels; Four is the action of inflammatory cells; Five is up regulated of the matrix metalloproteinases(MMPs).
Tissue inhibitor of metalloproteinases(TIMPs), the MMP specific inhibitor, and Matrix metalloproteinases are dynamic equilibrium in cell matrix synthesis and degradation. In aortic and coronary artery, the effect of matrix metalloproteinases is hot research topic. In pulmonary arterial hypertension research, the earlier studies had shown that through the degradation of collagen and collagen denaturation of vascular basement membrane, which in turn antagonism other the degradation of matrix metalloproteinases, MMP-1and MMP-2promoted the proliferation and migration of pulmonary vascular smooth muscle and then affected the pulmonary vascular structural remodelling, so participated in the formation of pulmonary hypertension. The TIMP-1is specific inhibitor of the matrix metalloproteinases-1and-2which can specifically block the latter combined with the substrate, thus inhibiting the degradation of collagen, under normal circumstances, the balance maintain homeostasis. Brown who proved adequate supply of nitric oxide can decrease the secretion of inhibitor about matrix metalloproteinase. Animal studies have shown that nitric oxide can affect pulmonary vascular remodelling by reduced the accumulation of collagen through adjusting the TIMP-1/MMP-1balance and strengthened collagen degradation. It was found that inhibition of matrix metalloproteinase can significantly reduce pulmonary artery pressure in patients with pulmonary hypertension. The phenomenon will appear. For example the smooth muscle cell proliferation in pulmonary hypertension and the pulmonary no muscle arteries have muscle."Neointimal" appears between endothelial cells and internal elastic lamina while the severe pulmonary arterial hypertension, the neointimal are composed by the myofibroblasts and extracellular matrix. The proliferation and apoptosis appears loss of balance in the endothelial cells and smooth muscle cells and fibroblasts, then leading to pulmonary structural remodelling and ultimately causing the pulmonary arterial hypertension. In the process of pulmonary hypertension vascular remodelling, the first is to be activated adventitial fibroblasts to proliferate and synthesise matrix components. Then the matrix metalloproteinase, like MMP2and MMP9, are up regulated and mediated fibroblast migration from the outer membrane into the medial and intima of the blood vessels. Then the intimal and medial of pulmonary vascular will be hyperplasia and proliferation and formed vascular remodelling.
A disinterring and metalloproteinase with thrombospondin motifs(ADAMTS) family is recently cloned who is a class of secreted metalloprotease family with Zn2+-dependent. The family and the family of matrix metalloproteinases and Depolymerization polyprotein-like metalloprotease belong metalloproteinase family. Because it is found just in the last ten years (in1997), we understand it is far less than the two families. The family same as the matrix metalloproteinase family have the ability to degrade the extracellular matrix. Compare to the other two family, the main difference of ADAMT family in structures is a repeat sequences containing at least one TSP (also known as TSP motifs) in carboxy-terminal. ADAMTS family are secreted proteases, which can be synthesized and secreted in vivo by a variety of synthetic and secretory cells, such as smooth muscle cells, endothelial cells, fibroblasts, macrophages. And ADAMTS is usually combined with the matrix. There were19family members. We are known some of their function, like the ADAMTS-1, ADAMTS-2, ADAMTS-4, ADAMTS-5, ADAMTS-9and ADAMTS-13.They degrade or regulate the extracellular matrix proteins and participate in a series of normal physiological functions, such as growth, angiogenesis, and coagulation et al. While their express abnormally and interact with the extracellular matrix, they will often lead to various diseases, such as cancer, arthritis, atherosclerosis, skin fragility-related diseases, coagulation disorders purpura and other diseases. The function of other family members remains unclear and let us to be studied.
ADAMTS-7is the seventh of the members of the ADAMTS family, who is known gradually by people in-depth study due to the fine adjustment of blood vessel. Through its substrate cartilage oligomer matrix protein (COMP), ADAMTS-7maintains steady-state by fine regulation of blood vessels. ADAMTS-7is first reported by Hurskainen TL team in1999, whose structure is composed of a catalytic domain who have a zinc ions and several other non-catalytic domain structure, including:a disintegrin domain structure, a thrombospondin domain structure, a structure of cysteine-rich domain (CRD), a spacer-1structure domain, three thrombospondin motifs, a spacer-2domain structure, four thrombospondin motifs in the ends of the C. ADAMTS-7with the ADAMTS-12belong to one hypo type of ADAMTS family, who can be directly combined and degradation of the cartilage matrix proteins. The Professor Liu Chuan-Ju who are in New York University School of Medicine and Professor Wang Li's research team whose are in the medical department of Peking University have done many researches over the past decade in internationally. Professor Liu does it in this field from arthritis and cartilage development. His research shows that the ADAMS-7who was found in patients with rheumatoid synovitis cartilage increased significantly through in vitro tests and who is overexpression associated with COMP degradation. In arthritis patients, they found COMP degradation fragments same as the COMP fragments through degrading by ADAMTS-7. In addition, through implicating the antibody of ADAMTS-7, it can significantly inhibit the COMP degradation by induced through tumour necrosis factor and interleukin-1β who are in cartilage cultured. Inhibition ADAMTS-7by siRNA silencing technology can also significantly prevent the degradation of COMP. The team of Wang Li study ADAMTS-7from blood vessels and also involve the COMP. They found that ADAMT-7could promote vascular smooth muscle cells calcification by regulating the degradation of COMP. they found that the microRNAs-29could regulate vascular calcification through intervened downstream reaction of ADAMTS-7. They also found ADAMT-7plays an important role in vascular smooth muscle cell proliferation and migration in balloon injury of rat carotid artery model. This research shows the ADAMTS-7may participate in the atherosclerosis in vascular matrix degradation and remodelling, which accelerated the process of atherosclerosis. Some research also reported that ADAMTS-7is involved in the intima hyperplasia after vascular injury. Through some related detected on the blood of non ST segment elevation myocardial infarction, some experts thought that the ADAMTS-7levels are closely associated with long-term prognosis of NSTEMI patients. The messenger RNA of ADAMTS-7is up regulate by TNF alpha in THP1in the monocyte or macrophage of people. The fine mechanism of adjustment is still unclear. Some research thought that its possible mechanism is through the transcription factor of the nuclear factor kappa B and AP-1to let the TNF alpha up regulate and stimulate ADAMTS-7induced rat smooth muscle proliferation.
According to studies about detecting ADAMTS-7, it found that there are high expression levels in heart, pancreas, kidney, skeletal muscle and liver in the adult samples. It was found expressed in liver, testis, lung, ovary, kidney, fetal heart and thymus tissue in varying degrees. In skeletal muscle, meniscus and fat can also be detected lower levels. In the lung tissue of pulmonary hypertension patient, the expression of ADAMTS-7is not reported now.
Based on the above information, we have known that the pathogenesis of pulmonary hypertension is very complex. The exact mechanism of PAH is still not very clear. There is one thing could make sure that the abnormal expression of matrix metalloproteinase is important in the pathogenesis of pulmonary hypertension. And the ADAMTS same as the family of matrix metalloproteinase, it also has the ability to degrade extracellular matrix. At the same time, the ADAMTS is widely distributed in mammalian organs. Previous research data had showed that the ADAMTS-7is closely associated with vascular development, in particular with circulation of atherosclerosis closely related to atherosclerosis. We hypothesized that ADAMTS-7and pulmonary circulation may also have close ties, as can be confirmed is expected to become a new target for the treatment of pulmonary hypertension. This paper intends to test the ADAMTS-7expression of lung tissue between adult pulmonary hypertension and normal pulmonary artery pressure in first, then could to clarify whether the ADAMTS-7expression in lung tissue, with or without expression in the lung tissue of patients with pulmonary hypertension. Second, cultured pulmonary artery smooth muscle cells, we test the expression of ADAMTS-7and some index of associated with proliferation and apoptosis and inflammation after intervened of the TNF-a. Finally, we verify the correlation detection of pulmonary hypertension in rats. In this study, there are three parts as follows.
Part one. The expression of the ADAMTS-7in Patients lung tissue with mild-to-moderate pulmonary hypertension
Objective:To verify the expression of ADAMTS-7in lung tissue in patients with pulmonary hypertension. Methods:There was a case-control study. Selected40cases of patients randomly who want to do the open chest surgery in The Affiliated Hospital of Guangdong medical college,20cases showed mild-to-moderate pulmonary hypertension by tested pressure through right cardiac catheterization(as case group), the pulmonary artery pressure in other20cases was normal(as control group). Take a few lung tissue in the two groups of patients in the surgery, used the immune immunofluorescence histochemical method and RT-PCR method to detect the expression of ADAMTS-7in lung tissue of the two groups patients. Results: Through the immune histochemical methods, it showed that there is a little positive expression of ADAMTS-7in the lung tissue of the control group and numerous positive expressions of ADAMTS-7in the lung tissue of the case group. By the image analysis and statistical calculation software, the IOD value in case group was significantly higher than control group (P<0.05). Through immunofluorescence RT-PCR method to test the expression of ADAMTS-7in the lung tissue in the two groups, it showed that the two groups (control group and case group) compared with the blank group (rules its Folds value is1) were statistically significant (P<0.05). In the control group compared with the case group, which had a statistically significant difference (P<0.05). Conclusion:ADAMTS-7may be involved in the pathogenesis of pulmonary hypertension.
Part two. The expression and the possible mechanism of ADAMTS-7in pulmonary artery smooth muscle cells
Objective:to verify the expression and the possible mechanism of ADAMTS-7in pulmonary artery smooth muscle cells. Methods:Cultured the people primitive pulmonary artery smooth muscle cells, first we tested the expression of ADAMTS-7in pulmonary artery smooth muscle cells by immunofluorescence cytochemistry Then used the CCK8method to detect the TNF alpha intervene on cell proliferation, in order to make clear of the optimum time and concentration. Then divided into three groups:blank group, the25ug/L TNF alpha intervention group, the ADAMTS-7siRNA silence group. Cultured respectively for12hours,24hours,48hours,72hours and detected the relative content of ADAMTS-7, PCNA, NF-kappa B, PI3K, VEGF, MARKK4, ERK1, IL-1, IL-6by RT-PCR method in every groups. Chose the point of24hours in intervention group (the25ug/L TNF alpha intervention group, the ADAMTS-7siRNA silence group) with immune protein imprinting method (Western Blot) to detect the mRNA levels of ADAMTS-7, the Bcl-2and COMP. Results:(1) Through the cellular immune fluorescent method, there was numerous positive expression of ADAMTS-7in pulmonary smooth muscle cell cytoplasm.(2) Through CCK8method, the concentration of25ug/L TNF alpha and the cultured time of24hours were screen out as the optimal concentration and time for proliferation of pulmonary artery smooth muscle cell.(3) The RT-PCR detection showed that the expression levels of ADAMTS-7, PI3K, PCNA, VEGF, IL-1, IL-6in all time point in TNF alpha25ug/L intervention group were significantly higher than that of siRNA silent group (P<0.05). The time of the highest express quantity of ADAMTS-7was the24hours after intervening by25ug/L TNF alpha. The time of the highest express quantity of PI3K, PCNA and IL-1was the48hours after intervening by25ug/L TNF alpha. The time of the highest express quantity of VEGF, IL-6was the72hours after intervening by25ug/L TNF alpha. The RT-PCR detection showed that the expression levels of NF-kappa B, MARKK4in24hour and48hour and72hour in TNF alpha25ug/L intervention group were significantly higher than that of siRNA silent group (P<0.05). The two groups were no significant statistical significance (P>0.05) in the12hour time point. The time of the highest express quantity of NF-kappa B was the24hours after intervening by25ug/L TNF alpha. The time of the highest express quantity of MARKK4was the48hours after intervening by25ug/L TNF alpha. ERK1factor mRNA between two groups had no statistically significant difference in the various time points (P<0.05).(4) the western blot test results showed that the amount of ADAMTS-7express in TNF alpha intervention group was significant higher than that of ADAMTS-7siRNA silent group and control group(P<0.05), The amount of ADAMTS-7express in control group was significant higher than that of the ADAMTS-7siRNA silent group(P<0.05), While the COMP expression levels in TNF alpha intervention group was significance lower than that of ADAMTS-7siRNA silent group and control group(P<0.05), The COMP expression levels in the siRNA ADAMTS-7group was no statistical difference (P>0.05), the Bcl-2expression in three groups was no statistical difference (P>0.05). Conclusion:(1) The ADAMTS-7May regulate the pulmonary artery smooth muscle cell proliferation or apoptosis by the TNF alpha pathway, NF-kappa B pathway, JNK pathway, PI3K pathway.(2) The ADAMTS-7may not participate the apoptosis of pulmonary smooth muscle cells through ERK or Bcl-2pathway.(3) The ADAMTS-7may promote the migration of smooth muscle cells by promoting the degradation of COMP and the expression of the VEGF.
Part three. The expression of ADAMTS-7in monocrotaline (MCT) induced pulmonary hypertension rats
Objective:By intervening the monocrotaline induced pulmonary hypertension rats, detecting the expression of ADAMTS-7and the proliferation and inflammation in pulmonary artery smooth muscle. Methods:30SD rats were randomly divided into three group:the normal control group (control group), the group of pulmonary hypertension induced by MCT (MCT), the doxycycline group. Measured the average pulmonary artery pressure (mPAP) and the right heart hypertrophy index (RVHI) after21days. And detect the ADAMTS-7by immunohistochemical and the TNF alpha, NF-kappa B, IL-1, IL-6, PI3K, MARKK4by RT-PCR in the lung tissue of rats. And detected the mRNA expression of ADAMTS-7, PCNA and Bcl-2by western blot method at each group. Results:(1) The mPAP and RVHI of rats in MCT group and doxycycline group were significantly higher than that of control group (P<0.05). The mPAP of rats in doxycycline group was lower than MCT group, but no statistical significance (P>0.05). The RVHI of rats in doxycycline group was less than the MCT group and has statistical significance (P<0.05).(2) Through immunohistochemical method for the lung tissue of rat, the positive of ADAMTS-7in MCT group was significantly higher than the control group and doxycycline group (P<0.05). While the doxycycline group compared with the control group, the former was higher than the latter and the difference was statistically significant (P<0.05).(3) By measuring used the RT-PCR method, the mRNA expression of the TNF alpha, the NF-kappa B, IL-1, IL-6, PI3K, MARKK4in MCT group and doxycycline group were significantly higher than that of control group (regulation control Folds value of1)(P<0.05). Each factor in MCT group obviously higher than that of doxycycline group, the difference was statistically significant (P<0.05).(4) Western blot analysis showed that the mRNA expression of ADAMTS-7, PCNA, Bcl-2in MCT group was significantly higher than that of the doxycycline group and the control group (P <0.05). The mRNA expression of Bcl-2and PCNA in doxycycline group were significantly higher that of the control group (P<0.05). The mRNA expression of ADAMTS-7in doxycycline group was equivalent that of the control group. It was no statistical significance (P>0.05). Conclusion:Doxycycline may adjust the pulmonary artery smooth muscle proliferation and inflammation by inhibiting ADAMTS-7. Doxycycline alone through the mechanism inhibiting ADAMTS-7and other metal loproteinase reduces pulmonary artery pressure is not obvious.
引文
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