摘要
背景:
主动脉夹层(aortic dissection,AD)是发生于主动脉的最常见灾难性疾病之一,致残致死率极高。近年随着人们生活水平的提高和社会人口老龄化程度的增加,同时由于经食管超声、MRA、CTA、DSA等影像检查技术的广泛应用,主动脉夹层的临床检出率增加,使得其发病率呈现上升趋势。传统开放手术技术上的不断改进逐步降低了主动脉夹层手术相关并发症率和死亡率,而近年兴起的腔内血管外科理论和技术更是实现了其治疗从巨创到微创的重大转变,进一步降低了相关并发症率和死亡率,将主动脉夹层的治疗推进到了一个新的台阶。所有这些都给主动脉夹层病人带来了福音,然而,仍有许多病人在明确诊断或手术治疗之前已经因破裂或相关并发症的发生而死亡或残疾,这是由该病的发病特点所决定的。因此,从发病机制水平出发来探讨主动脉夹层的预防、诊断和治疗的研究具有非常重要的意义。
目前对于主动脉夹层的发病和进展机理等问题仍不清楚,可能是血流动力学和组织病理学等多种因素综合作用的结果。通常认为主动脉壁本身结构异常是发病基础,而血流动力学异常则被认为是促发因素。糖胺聚糖(GAG)是一类重要的生物大分子,其重要生物学作用正越来越引起人们的重视。各种GAG均为多聚阴离子,分子量大,且无紫外吸收,这些特点给他们的分析检测造成极大困难,因此以往相关研究较少。然而,近年随着生物医学检测技术的快速发展和进步,对GAG的相关研究逐渐热门起来。目前已经在多种疾病中检测到GAG的异常改变,如骨科疾病和肿瘤疾病中,在大动脉疾病方面主要体现在对腹主动脉瘤的相关研究中。
以往已经在人主动脉壁中鉴定出了4种GAG,包括透明质酸(HA)、硫酸乙酰肝素(HS)、硫酸软骨素(CS)和硫酸皮肤素(DS),它们对于维持正常主动脉壁结构和功能的完整起着重要作用。结构决定功能,要想了解GAG在主动脉夹层发病和进展过程中所起的作用,首先必须了解它们的含量和结构改变。然而,目前我们还不清楚各种GAG在主动脉夹层中的准确含量,结构方面更是一片空白。基于这些背景,本课题将对各种GAG在主动脉夹层中的含量和细微结构改变进行阐述,为进一步探讨它们在该病的发病和进展过程中的可能作用提供理论依据。
目的:
1、阐明主动脉夹层主动脉壁中GAG的总含量改变,并讨论年龄和高血压等因素对其所产生的影响。
2、阐明主动脉夹层主动脉壁中CS、DS、HA和HS 4种GAG的相对含量改变,并讨论年龄和高血压等因素对它们所产生的影响。
3、阐明主动脉夹层主动脉壁中CS、DS、HA和HS 4种GAG的细微结构改变。
4、讨论各种GAG含量和分子结构异常在主动脉夹层发病过程中的可能作用,为探讨该病可能的发病机理提供理论依据。
方法:
1、本研究设实验组和正常对照组两个组。实验组共10例,为主动脉夹层病人升主动脉标本;对照组共7例,为与实验组年龄匹配的正常人相同位置的升主动脉标本;
2、组织学染色:通过HE染色和VB染色观察主动脉夹层和正常人主动脉壁的基本结构,通过AB-PAS染色观察GAG分布。
3、联合采用木瓜酶酶解法和氢氧化钠碱解法水解释放主动脉壁中的GAG,然后采用乙醇沉淀法进行初提,冷冻干燥后进行相关分析。
4、采用DEAE Sephacel离子交换层析法,以0.1～1.2 M氯化钠缓冲液进行分级梯度洗脱,对初提的HA、HS、CS和DS进行分离纯化:然后对分离纯化的各种GAG进行乙酸纤维素电泳,通过和GAG标准品对比进行鉴定。
5、采用浓硫酸-间羟联苯法对初提的各标本GAG糖醛酸总量进行测定,比较实验组和正常对照组之间的差异,并分析年龄和高血压等因素对其所产生的影响。
6、采用乙酸纤维素电泳法对初提的各标本GAG进行分离,采用0.02%阿利新蓝染色。然后分别切下各GAG条带,测定它们在677 nm下的吸光度值,计算HA、HS、CS和DS的相对含量。比较正常组和对照组之间的差异,并分析年龄和高血压等因素对它们所产生的影响。
7、分别采用硫酸软骨素酶ABC和B以及肝素酶Ⅰ、Ⅱ、Ⅲ,将各种GAG裂解为相应的不饱和二糖。采用高效毛细管电泳法,以二糖标准品作为对照,分别对样品中的各种二糖进行定性和定量,比较主动脉夹层和正常对照组之间各种GAG二糖组成的差异。
结果:
1、组织学染色:同正常人主动脉相比,HE染色和VB染色显示主动脉夹层主动脉壁基本结构发生明显改变,中膜可见假腔形成,弹力纤维断裂、减少,胶原纤维增生:AB-PAS染色显示主动脉夹层组酸性黏多糖于管壁全层增多。
2、实验组和对照组主动脉壁中GAG总含量分别为6.703±0.495和6.415±0.539mg/g干组织,两组之间的GAG总量无明显差异(P>0.05)。对年龄(以40岁分界)、病程(以7 d分界)、主动脉直径(以5.5 cm分界)和是否合并高血压等影响因素进行分析,结果表明主动脉壁中GAG总量随年龄增加明显降低(P<0.05),而其它3个因素对实验组的GAG总量无明显影响(P均大于0.05)。
3、实验组中各种GAG的比例较正常发生明显改变(CS:62.5%vs 54.7%;DS:9.8%vs 15.8%;HA:10.6%vs 7.8%;HS:17.1%vs 21.8%),其中CS和HA明显升高(P<0.01),而DS和HS明显降低(P<0.01)。随年龄增加,主动脉壁中DS和HS含量明显下降(P<0.05),而CS和HA无明显改变(P>0.05);病程、主动脉直径和高血压等因素对各种GAG的含量无明显影响(P>0.05)。
4、经高效毛细管电泳分析,实验组HA样品中仅检测到△di-nonS_(HA)1种二糖,其含量较正常对照组明显升高(P<0.05)。实验组DS样品中检测到5种二糖包括△di-di(2,6)S_(CS/DS)、△di-di(2,4)S_(CS/DS)、△di-di(4,6)S_(CS/DS)、△di-mono4S_+(CS/DS)和△di-nonS_(CS/DS),较正常对照组缺少△di-mono6S_(CS/DS),但增加了△di-di(4,6)S_(CS/DS),且其余4种二糖含量均明显降低(P<0.05)。实验组CS样品中检测到6种二糖包括△di-di(2,6)S_(CS/DS)、△di-di(2,4)S_(CS/DS)、△di-di(4,6)S_(CS/DS)、△di-mono6S_(CS/DS)、△di-mono4S_(CS/DS)和△di-nonS_(CS/DS),其中除△di-di(2,4)S_(CS/DS)含量较正常对照组明显降低(P<0.05)外,其余5种二糖均明显升高(P<0.05)。实验组HS样品中检测到7种二糖包括△di-tri(2,6,N)S_(HS)、△di-di(2,N)S_(Hs)、△di-di(6,N)S_(HS)、△di-di(2,6)S_(HS)、△di-monoNS_(HS)、△di-mono2S_(HS)和a△di-nonS_(HS),其中除a△di-nonS_(HS)含量较正常对照组明显升高(P<0.05)外,其余6种均无明显改变(P>0.05)。
结论:
1、我们发现主动脉夹层中GAG总含量较正常对照无明显改变,但各种GAG的相对比例却发生明显异常,提示各GAG相对比例的失调可能在主动脉夹层的发病和进展过程中起到了重要作用。
2、主动脉夹层中各种GAG的结构组成均发生明显异常,这可能会造成主动脉壁结构的薄弱,进而破坏其生物学功能的完整性。同时,我们还发现二糖组成的异常是造成各GAG之间相对比例失调的主要原因。因此,我们推测GAG细微结构的异常改变可能在主动脉夹层的发病和进展过程中起到了关键性作用。
Background:
Aortic dissection(AD) is one of the most common catastrophe involving the aortae, carrying high morbidities and mortalities.For the improvements of human life,age growing of the society and wide use of imaging studies such as transesophageal ultrasound, MRA and CTA,aortic dissections have been detected more and more requently in recent years,with its incidence rising.Improvements on techniques of traditional open surgeries have reduced operation-related morbidities and mortalities,while developments of endovascular theories and techniques in recent years have utilized their minimal invasion instead of the huge trauma of open surgery,furtherly reduced the morbidities and mortalities and improved the treatments of aortic dissections.Although all of the above have benefited patients suffering aortic dissection,many of them died or disabled from ruptures or major morbidities before final diagnosis and surgery made,which was determined by the characteristics of the disease itself.Thus,it is very important for us to achieve the prevention,diagnosis and treatment of aortic dissection at the level of pathogenesis.
Although aortic dissection could be initialized by many factors(e.g.hemodynamics and histopathology) together,we still don't know the exact pathogenesis now.Abnormal structures of the aortae are usually regarded as the basis of its pathogenesis,while abnormal hemodynamics as a trigger factor.Glycosaminoglycans(GAG) are important biomacromolecules and their important biological activities are attracting more and more people's attention.It is difficult to determine GAG for their polyanion,large molecular weight and inabsorption by UV,so there are little reports on them.With rapid advancements on biomedical detecting techonology in recent years,researches on GAG have gradually become more popular.We have observed many abnormal changes with GAG in many diseases such as orthopedics and tumors,while the abdominal aortic aneurysms are mainly focused on when it involves the aortic diseases.
So far we have found four kind of GAG in human aortae,including hyaluronic acid (HA),heparin sulfate(HS),chondroitin sulfate(CS) and dermatan sulfate(DS),all of which are important for maintaining the integrity of normal structures and functions of the aortae.Since structures usually determine functions,we should first determine the changes on the contents and structures of GAG in aortic dissections before elucidating their roles in the pathogenesis of the disease.However,we haven't known the exact contents of GAG in aortic dissection and there's little information about their structures.Based on the above backgrounds,we'll elucidate the changes on the contents and fine structures of GAG in aortic dissection in this study,which may benefit us on exploring the pathogenesis of the disease.
Objectives:
1.To elucidate the total contents of GAG in the aortae of aortic dissections and discuss the influences exerted by age and hypertension and so on.
2.To elucidate the relative amounts of HA,HS,CS and DS in the aortae of aortic dissections and discuss the influences exerted by age and hypertension and so on.
3.To elucidate the changes in fine structures of HA,HS,CS and DS in the aortae of aortic dissections.
4.To discuss the likely roles of abnormal contents and molecular structures of GAG in the pathogenesis of aortic dissection.
Methods:
1.Two groups were set up in this study.Ten ascending aortae in the experimental group were collected from patients with aortic dissections and seven specimens in the control group were collected from age-matched normal human ascending aortae at the same level.
2.Histopathological stains:The basic structures of the aortae in the experimental group and the control group were observed by HE stain and VB stain and the distribution of GAG in the aortae were observed by AB-PAS stain.
3.GAG in the aortic specimens were released by hydrolysis with combined papain and NaOH,and then extracted by ethanol precipitation and freeze-dried for further analytical uses.
4.HA,HS,CS and DS were separated and purified respectively through DEAE Sephacel by ion exchange chromatography,with 0.1～1.2 M NaCl as stepwise gradient elutions.Then the separated GAGs were identified by cellulose acetate electrophoresis comparing with standard GAG.
5.After the total contents of GAG of each sample being determined by concentrated sulfuric acid-hydroxydiphenyl method,they were compared between the experimental group and the control group and then the influences exerted by age and hypertension and so on were discussed.
6.The extracted GAG were separated by acetate cellulose electrophoresis and stained with 0.02%alcian.Each of the stained bands were cut down and dissolved with dimethylsulfoxide separately,and then their OD at 677 nm were determined and the relative contents of HA,HS,CS and DS were calculated.Then the relative contents of them in the experimental group were compared with these in the control group and the influences exerted by age and hypertension and so on were discussed.
7.After all the purified GAG being hydrolyzed into corresponding unsaturated disaccharides with chondroitinase ABC,B and heparinaseⅠ,Ⅱ,Ⅲ,each disaccharide was determined by high performance capillary electrophoresis with standard disaccharides as control.Then the disaccharide compositions of each GAG in the experimental group were compared with these in the control group.
Results:
1.Histopathological stains:Compared with normal human aortae,the basic structures in aortic dissections changed dramatically.We observed exist of false lumens,fracture and reduction of elastic fibers and hyperplasia of collagen fibers in the media by HE stain and VB stain.Increased acidic GAGs were observed throughout the full aortic wall in aortic dissections by AB-PAS stain.
2.The contents of GAG in the aortae in the experimental group and the control group were 6.703±0.495 and 6.415±0.539 mg/g dry defatted tissue respectively and there was no significant difference between the two groups(P>0.05).After influencing factors such as age(grouped by 40 y),history(grouped by 7 d),diameter of the aorta(grouped by 5.5 cm) and co-existed hypertension being discussed,significant decrease in the contents of total GAG with age growing was observed(P<0.05),while the other three factors had no significant impact(P>0.05).
3.Significant changes in the percentages of each GAG were observed in the experimental group(CS:62.5%vs 54.7%;DS:9.8%vs 15.8%;HA:10.6%vs 7.8%;HS: 17.1%vs 21.8%),among which CS and HA increased significantly(P<0.01) while DS and HS decreased significantly(P<0.01).Significant decreases in the contents of DS and HS(P<0.05) with age growing were observed and while CS and HA changed insignificantly(P>0.05).The other three factors such as history,diameter of the aorta and co-existed hypertension had no significant effects on the contents of each GAG(P>0.05).
4.By HPCE analysis,only one disaccharide named Adi-nonSHA was detected in HA samples in the experimental group,with its content increasing significantly(P<0.05).Five kind of disaccharides such as△di-di(2,6)S_(CS/DS),△di-di(2,4)S_(CS/DS),△di-di(4,6)S_(CS/DS),△di-mono4S_(CS/DS) and△di-nonS_(CS/DS) were detected in DS samples in the experimental group,with the absence of△di-mono6S_(CS/DS) instead of△di-di(4,6)S_(CS/DS) and the other four kind decreasing significantly(P< 0.05).Six kind of disaccharides such as△di-di(2,6)S_(CS/DS),△di-di(2,4)S_(CS/DS),△di-di(4,6)S_(CS/DS),△di-mono6S_(CS/DS),△di-mono4S_(CS/DS) and△di-nonS_(CS/DS) were detected in CS samples in the experimental group,with△di-di(2,4)S_(CS/DS) decreasing significantly(P<0.05) and the other five kind increasing significantly(P<0.05).Seven disaccharides such as△di-tri(2,6,N)S_(HS),△di-di(2,N)S_(HS),△di-di(6,N)S_(HS),△di-di(2,6)S_(HS),△di-monoNS_(HS),△di-mono2S_(HS) and a△di-nonS_(HS) were detected in HS samples,with the last one increasing significantly(P<0.05) and the other six not changed(P>0.05) in the experimental group.
Conclusions:
1.Although there was no significant difference in the total contents of GAG in the aortae between aortic dissections and normal human,the proportion of each GAG changed dramatically.So we think the proportional imblance of each GAG may play an important role in the pathegenesis and progression of aortic dissection.
2.Significant changes in structural compositions of each GAG were observed in aortic dissection,which may lead to weakness of the aortic wall and damage the functional integrity of the aorta.We aslo found that the proportional imbalance of each GAG was mainly resulted from their abnormal disaccharide compositions.Therefore,we consider the abnormal fine structures of GAG may play a key role in the initialization and progression of aortic dissection.
引文
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