富含α2巨球蛋白血清的分子生物学研究及其在创伤性关节炎早期干预治疗中的作用

发布时间：2018-01-03 20:45

本文关键词：富含α2巨球蛋白血清的分子生物学研究及其在创伤性关节炎早期干预治疗中的作用　出处：《山西医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:前交叉韧带(Anterior cruciate ligament,ACL)损伤是青壮年尤其是竞技体育运动者中最常见的膝关节韧带损伤之一。临床上以ACL重建术为其治疗的主要方法。但ACL重建术并不能降低ACL损伤所继发的创伤性关节炎(Post-traumatic osteoarthritis,PTOA)发生的风险和进程。最新的研究表明,在关节创伤后一周内关节腔即出现大量的炎症因子及氧自由基等,促使大量的软骨细胞凋亡,成为启动PTOA关节软骨退变和损伤的始动因素。因此及早抑制这些炎性因子的活性对阻止或者减缓PTOA的发展至为关键。α-2巨球蛋白(Alpha-2-macroglobulin,A2M)作为一种广谱的蛋白酶抑制剂在关节软骨损伤时被合成和分泌到关节液中,可有效地抑制IL-1诱导软骨基质降解的基质金属蛋白酶(MMP-3、9、13)和多种炎症因子(IL-1β、6、8,TNF-a和GM-CSF)的活性,但其在关节软骨损伤后血清和关节液中分泌情况却一直未见相关研究。本课题组在前期研究中也已经证实采用前交叉韧带切断术(Anterior cruciate ligament transection,ACLT)制作的SD大鼠的创伤性关节炎(PTOA)模型中,适量的补充外源性的A2M可以明显的减缓关节软骨的退变进程,具有非常显著的正性关节软骨保护作用。但所使用的A2M为大分子量蛋白质,免疫原性强,异种A2M长期关节腔注射极有可能引起免疫反应,因此寻求一种更加简单的提纯或富含A2M血清的方法并观察其对创伤性关节炎(PTOA)的治疗效果具有非常重要临床价值。研究目的:1)分析A2M在ACL损伤时血清、关节液中的分布变化,明确与关节软骨退化存在的关联,为外源性补充A2M提供理论依据;2)采用超滤离心法制备富含A2M血清(A2M-Rich Serum,A2MRS),检测A2MRS中的A2M的蛋白浓度及蛋白活性,并分析其分子生物学特性;3)观察富含A2M血清(A2M-Rich Serum,A2MRS)对创伤性关节炎(PTOA)的关节软骨是否具有明显的治疗效果或减缓关节软骨退化的作用。研究方法:1)首先以2月龄96只SD大鼠为实验对象,分为两组,每组48只。实验组通过前交叉韧带切断术(Anterior cruciate ligament transection,ACLT)建立PTOA动物模型,对照组采取假手术切开,不行ACLT。术后3天及1、2、4、8及12周每个时间点各组处死8只大鼠,收集血清及膝关节盥洗液。采用酶联免疫吸附试验(ELISA)检测大鼠ACL损伤后不同时间点的关节盥洗液、血清中A2M的浓度变化规律,同时采用番红O固绿染色观察PTOA随着时间的延长其关节软骨退化的病理进程与体液中A2M的含量变化是否存在某种关联,探讨A2M是否可以作为关节软骨退变早期诊断及监测病理进程的生物标记物,为外源性补充A2M提供理论依据。2)采用超滤离心法制备富含A2M血清(A2M-Rich Serum,A2MRS),采用酶联免疫吸附试验(ELISA)、蛋白免疫印迹法(Western Blot)等方法检测其分子生物学性质。3)选取80只健康成年SD大鼠,分为4组,每组20只:Sham+Saline(空白对照组)、ACLT+HA2MRS(高剂量组)、ACLT+LA2MRS(低剂量组)、ACLT+Saline(阳性对照组)。手术后3天、2周、4周依据分组的不同关节腔定时注射30ul生理盐水或30ul不同浓度A2MRS(高剂量组A2M为20ug/ul,低剂量组A2M为10ug/ul)。每个时间点治疗后24小时关节腔注射MMP680免疫荧光探针,采用荧光分子断层成像系统(Fluorescence Molecular Tomography,FMT)动态检测基质金属蛋白酶(Matrix metalloproteinase,MMP)在关节液中浓度的变化,观察A2MRS对基质金属蛋白酶是否具有抑制作用。术后8周统一处死动物,小动物X线片观察膝关节退变的影像学表现,印度墨水染色评估关节软骨大体损伤情况,并行Mankin’s评分;胫骨平台关节软骨采用甲苯胺蓝、番红O固绿染色、苏木精-伊红染色(HE染色)和免疫组化法观察A2MRS对PTOA软骨病理退变是否具有减缓或者修复作用,采用OARIS评分系统评估关节软骨的病理变化并作统计学分析;股骨髁关节软骨采用实时荧光定量PCR法(RTPCR)对软骨组织中II型胶原(COL-II)、X型胶原(COL-X)、基质金属蛋白酶3、13(Matrix metalloproteinase,MMP-3、13)、软骨聚集蛋白聚糖(Aggrecan)、Runt蛋白相关转录因子-2(Runt-related transcription factor,Runx2)等指标进行检测,评估富含A2M血清对关节软骨损伤在基因层面上的调控作用。采用ELISA检测关节液中基质金属蛋白酶(Matrix metalloproteinase,MMP-13)浓度。结果:1)ACLT术后实验组与对照组的关节软骨早期大体观察与组织病理学观察并无明显的区别,但术后8周开始,实验组的关节软骨退化明显加重。实验组术后3天、1周、8周关节盥洗液中A2M的浓度较血清中的浓度明显增高。并且实验组术后3天关节液盥洗液中的A2M的浓度与对照组关节盥洗液中A2M浓度相比较明显增高,术后1周开始迅速降低与对照组无差别。2)采用超滤离心法制备的富含A2M血清(A2M-Rich Serum,A2MRS)经ELISA检测结果为:人的血清中A2M的浓度为2.43±0.66mg/ml,富含A2M血清(A2MRS)中A2M的浓度为19.52±4.37mg/ml,A2MRS中的A2M的浓度较正常血清中的浓度提高了8.04倍。蛋白免疫印迹(Western Blot)结果显示:富含A2M血清(A2MRS)相比正常血清在180k D处有非常明显的蛋白浓聚现象。A2M蛋白活性检测表明:富含A2M血清(A2MRS)的A2M蛋白活性较正常血清A2M的活性提高2.13倍。3)动物体内实验观察A2MRS对PTOA关节软骨退化的治疗作用,通过荧光分子断层成像系统(Fluorescence Molecular Tomography,FMT)检测结果表明:富含A2M血清(A2MRS)早期可以明显抑制创伤性关节炎关节液中基质金属蛋白酶3、13(Matrix metalloproteinase,MMP-3、13)。膝关节的X光片的影像学表现:ACLT+Saline组的髌骨下缘有明显的骨赘增生,其余三组在影像学表现上均没有观察到明显的退行性改变。印度墨水染色表明:Sham+Saline组中软骨表面未见明显损伤,ACLT+Saline组软骨表面损伤最为严重,而ACLT+A2MRS组关节面损伤明显减轻,其中ACLT+HA2MRS组中软骨损伤在ACL切断组中最小,改良Mankin's评分显示:ACLT+HA2MRS组和ACLT+LA2MRS与ACLT+Saline组相比较均有明显统计学意义,但ACLT+HA2MRS组与ACLT+LA2MRS组相比较无统计学意义。胫骨平台番红O固绿染色、HE染色及甲苯胺蓝染色结果均显示:ACLT+Saline组软骨厚度明显变薄,软骨表面有较多小的裂隙,软骨细胞分布紊乱,糖胺多糖染色较差。ACLT+LA2MRS组软骨细胞聚集成团,软骨厚度降低,ACLT+HA2MRS组中软骨表面较为平滑,细胞排列较整齐,Sham+Saline组软骨表面完整,结构良好,细胞分布均匀,糖胺多糖染色良好。OARIS评分显示:ACLT+Saline组为10.15±2.88,ACLT+HA2MRS组为5.54±2.61,ACLT+LA2MRS组为4.87±2.74,Sham+Saline为3.05±1.39,统计学分析表明:两个实验组(ACLT+HA2MRS和ACLT+LA2MRS)与阳性对照组(ACLT+Saline)均有显著性差异(p0.0001),两个实验组(ACLT+HA2MRS和ACLT+LA2MRS)与空白对照组(Sham+Saline)均有显著性差异。而ACLT+HA2MRS组与ACLT+LA2MRS组之间经统计学分析不具有显著性差异(p=0.22)。在II型胶原的免疫组化染色上,ACLT+HA2MRS组和ACLT+LA2MRS组的阳性细胞明显比ACLT+Saline组染色强,即ACLT+HA2MRS组较ACLT+LA2MRS组阳性细胞要强,但两组II型胶原的免疫组化染色均低于Sham+Saline组。在X型胶原和MMP-13的免疫组化染色上,ACLT+Saline组阳性细胞在四组中表达最强,Sham+Saline组的阳性细胞表达最弱,而两个治疗组ACLT+HA2MRS组及ACLT+LA2MRS组在X型胶原表达上介于ACLT+Saline组与Sham+Saline组之间,并且同样存在剂量依赖性,即ACLT+HA2MRS的X型胶原的表达比ACLT+LA2MRS组弱。RT-PCR结果表明A2MRS具有较为明显的抑制负性基因X型胶原(COL-X)、基质金属蛋白酶3、13(Matrix metalloproteinase 3、13,MMP-3、13)、Runt蛋白相关转录因子-2(Runt-related transcription factor,Runxn2)的m RNA表达。关节液中MMP-13的ELISA测定结果表明:ACLT+HA2MRS组、ACLT+LA2MRS组与Sham+Saline组中关节液中MMP-13浓度均显著低于ACLT+Saline,统计学分析有显著性差异,但前三组之间组相比均无统计学差异。结论:1.关节液中A2M可以作为生物标记物早期诊断创伤性关节炎,术后一周关节液中A2M的迅速降低提示外源性的补充至关重要。2.超滤离心法可高效便捷的制备具有较高蛋白浓度和蛋白活性的富含A2M血清。3.富含A2M血清(A2MRS)具有较为明显减缓关节软骨退化的正性治疗作用。
[Abstract]:Background: anterior cruciate ligament (Anterior cruciate, ligament, ACL) in young adults especially injury is one of the most common knee ligament injury in sports. In clinical ACL reconstruction is the main treatment. But ACL reconstruction can not reduce ACL damage secondary to traumatic arthritis (Post-traumatic osteoarthritis PTOA), and the risk of the occurrence process. The latest research shows that in a week after trauma in joint articular cavity is the emergence of a large number of inflammatory cytokines and oxygen free radicals, promote the apoptosis of chondrocytes in great quantities, become the initiating factors start PTOA articular cartilage degeneration and injury. Therefore to inhibit these inflammatory cytokines activity is the key to the development of prevent or slow PTOA. Alpha -2 macroglobulin (Alpha-2-macroglobulin, A2M) as a broad-spectrum protease inhibitor in cartilage was synthesized and secreted The joint fluid, matrix metalloproteinases can effectively inhibit IL-1 induced cartilage matrix degradation (MMP-3,9,13) and a variety of inflammatory cytokines (IL-1 beta, 6,8, TNF-a and GM-CSF) activity in serum, but the articular cartilage and synovial fluid secretion but has no related research. The research group in the early stage research has also confirmed that the anterior cruciate ligament transection (Anterior cruciate ligament transection, ACLT) of traumatic arthritis in rats of the SD (PTOA) model, the amount of exogenous A2M can significantly reduce the articular cartilage degeneration process, has a significant protective effects of articular cartilage but. The use of A2M as a high molecular weight protein, immunogenicity, long-term intra-articular injection of xenogeneic A2M is likely to cause immune reactions, thus seeking a more simple purification or rich A2M serum and observe The traumatic arthritis (PTOA) treatment has very important clinical value. The purpose of the study: 1) analysis of A2M in ACL injury serum distribution in synovial fluid, clear associated with articular cartilage degeneration, and provide a theoretical basis for the exogenous A2M; 2) were rich in A2M serum by centrifugal ultrafiltration legal (A2M-Rich Serum, A2MRS, A2MRS) detection of A2M protein concentration and protein activity, and to analyze its molecular biological characteristics; 3) were rich in A2M serum (A2M-Rich Serum, A2MRS) of traumatic arthritis (PTOA) whether the articular cartilage has obvious therapeutic effect or reduce articular cartilage degradation effect. Methods: 1) first in February at the age of 96 SD rats were divided into two groups, 48 rats in each group. The experimental group by anterior cruciate ligament transection (Anterior cruciate ligament transection, ACLT) to establish an animal model of PTOA, the control group took The sham operation incision, no ACLT. and 1,2,4,8 after 3 days and 12 weeks each time point were sacrificed 8 rats, collect serum and knee joint lavage. Using enzyme-linked immunosorbent assay (ELISA) detection of joint lavage at different time points after ACL injury in rats, the concentration changes of serum A2M, at the same time using safranin O fast green staining of PTOA with the extension of time and the content changes of pathological process of humoral articular cartilage degradation in A2M if there is any connection, to investigate whether A2M can be used as biomarkers of articular cartilage degeneration in the early diagnosis and monitoring of pathological process, and provide a theoretical basis for.2 of exogenous A2M in preparation) A2M serum by centrifugal ultrafiltration method (A2M-Rich, Serum, A2MRS) by enzyme linked immunosorbent assay (ELISA), Western blotting (Western Blot) were used to detect the molecular biological properties of.3) select 80 healthy SD rats were divided into 4 groups, 20 rats in each group: Sham+Saline (control group), ACLT+HA2MRS (high dose group), ACLT+LA2MRS (low dose group), ACLT+Saline (positive control group). 3 days after surgery, 2 weeks, intra-articular injection of 30ul in different time saline or 30ul of different concentration of A2MRS on the basis of 4 weeks group (high dose group A2M 20ug/ul, low dose group A2M 10ug/ul). For each time point of 24 hours after intra-articular injection of MMP680 immuno fluorescence probe by fluorescence molecular tomography systems (Fluorescence Molecular Tomography, FMT) dynamic detection of matrix metalloproteinases (Matrix metalloproteinase, MMP) concentration changes in joint in the solution, to observe whether A2MRS has inhibitory effect on matrix metalloproteinases. 8 weeks after operation were unified animal performance, small animal X-ray observation of knee joint degeneration imaging, India ink staining in the assessment of articular cartilage damage, and Mankin 's score; articular cartilage by toluidine blue and safranin O fast green staining, hematoxylin eosin staining (HE staining) and immunohistochemistry method to observe the effect of A2MRS on PTOA cartilage pathological degeneration is slow or repair, OARIS was applied to evaluate the pathological changes of articular cartilage of the femoral and statistical analysis; condylar articular cartilage by using real-time fluorescence quantitative PCR method (RTPCR) for type II cartilage collagen (COL-II), type X collagen (COL-X), matrix metalloproteinase 3,13 (Matrix metalloproteinase MMP-3,13), cartilage proteoglycan aggregate (Aggrecan), Runt protein transcription factor -2 (Runt-related transcription factor, Runx2). The evaluation indexes were detected, A2M rich serum injury role in regulation of gene level of articular cartilage. ELISA was used to detect the matrix in the joint fluid metal protease (Matrix metalloproteinase, MMP-13). . results: 1) ACLT early after operation in experimental group and control group of articular cartilage gross observation and histological observation had no obvious difference, but at 8 week after operation, the experimental group of articular cartilage degeneration was significantly increased. The experimental group after 3 days, 1 weeks, 8 weeks of wash liquid A2M the concentration in serum concentration was significantly higher than the experimental group. And after 3 days of joint lavage fluid concentrations of A2M in the control group and the concentration of A2M in the joint lavage compared significantly increased, 1 weeks after the operation began to rapidly reduce differences between the two groups.2) were prepared by ultrafiltration (A2M in serum A2M-Rich Serum, A2MRS) by ELISA test results: the concentration of serum A2M was 2.43 + 0.66mg/ml, A2M in serum (A2MRS) concentration in A2M was 19.52 + 4.37mg/ml, the concentration of A2MRS in A2M compared with normal serum concentrations increased 8.04 times. Protein immunoblotting (Western Blot). The results show that the A2M rich serum (A2MRS) compared with normal serum 180K in D has very obvious protein concentration phenomenon of.A2M protein activity detection showed that A2M (A2MRS) in serum A2M protein activity compared with normal serum A2M activity increased 2.13 times.3) therapeutic effect of A2MRS in vivo animal experimental observation on articular cartilage degradation PTOA and by fluorescence molecular tomography system (Fluorescence Molecular Tomography, FMT) test results showed that the serum containing A2M (A2MRS) can inhibit matrix metalloproteinase early traumatic arthritis synovial fluid 3,13 (Matrix metalloproteinase MMP-3,13). The expression of X of the knee X-ray imaging: group ACLT+Saline significantly lower edge of patella the osteophyma, the other three groups showed in the image were observed no degenerative changes obviously. India ink staining showed that Sham+Saline group had no obvious loss of cartilage surface Group ACLT+Saline cartilage injury, surface damage is most serious, while in ACLT+A2MRS group, the articular surface was reduced, the cartilage injury in the ACL group ACLT+HA2MRS group in the minimum cut, the modified Mankin's score showed that group ACLT+HA2MRS and ACLT+LA2MRS compared with the ACLT+Saline group had significant statistical significance, but the ACLT+HA2MRS group and ACLT+LA2MRS group comparison was statistically significant tibial plateau. Safranin O fast green staining, HE staining and toluidine blue staining showed that the cartilage thickness was significantly thinner in group ACLT+Saline, the cartilage surface has many small cracks, the distribution of cartilage cell disorder, glycosaminoglycan poor staining of chondrocytes of.ACLT+LA2MRS group were clustered into groups, the thickness of articular cartilage in ACLT+HA2MRS group decreased, the cartilage surface is smooth, cells arranged was group Sham+Saline cartilage surface integrity, good structure, uniform cell distribution and glycosarninoglycan staining good.OARIS score Display: ACLT+Saline group is 10.15 + 2.88, 5.54 + 2.61 ACLT+HA2MRS group, ACLT+LA2MRS group is 4.87 + 2.74, 3.05 + 1.39 Sham+Saline, statistical analysis showed: two experimental groups (ACLT+HA2MRS and ACLT+LA2MRS) and positive control group (ACLT+Saline) had significant difference (P0.0001), two experimental groups (ACLT+HA2MRS and ACLT+LA2MRS) and control group (Sham+Saline). There were significant differences between ACLT+HA2MRS group and ACLT+LA2MRS group were analyzed statistically no significant difference (p=0.22). The immunohistochemistry of collagen II staining, positive cells of ACLT+HA2MRS group and ACLT+LA2MRS group were significantly higher than in group ACLT+Saline or ACLT+HA2MRS staining, compared with group ACLT+LA2MRS that group of positive cells, but the immune group of two groups of type II collagen staining were lower than those of Sham+Saline group. In the immunohistochemistry of collagen X and MMP-13 staining, ACLT+ positive cells in the Saline group in the four groups The expression of Sham+Saline positive cells were the strongest, the expression of the weakest, while the two treatment group ACLT+HA2MRS group and ACLT+LA2MRS group in the expression of type X collagen between ACLT+Saline group and Sham+Saline group, and the same is dose dependent, i.e. type X collagen ACLT+HA2MRS expression in ACLT+LA2MRS group than in the weak.RT-PCR results show that A2MRS has obvious inhibition negative type X collagen gene (COL-X), matrix metalloproteinase 3,13 (Matrix metalloproteinase 3,13, MMP-3,13), Runt protein transcription factor -2 (Runt-related transcription factor, Runxn2) expression of M RNA. The testing results of MMP-13 in synovial fluid ELISA showed that: ACLT+HA2MRS group, ACLT+LA2MRS group and Sham+Saline group in MMP-13 synovial fluid concentration were significantly lower than that of ACLT+Saline, has statistically significant difference between the three groups before, but there was no significant difference between the groups. Conclusion: 1. in synovial fluid A2M Can be used as a biomarker for early diagnosis of traumatic arthritis, one week after operation in the synovial fluid of A2M rapidly decreased vital.2. suggesting that the supplement of exogenous centrifugal ultrafiltration can be efficient and convenient preparation with high protein concentration and protein activity of A2M rich serum.3. A2M rich serum (A2MRS) has obvious positive effect to slow the degradation of articular cartilage.

【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R687.4

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