关节液Marker对早期诊断骨关节炎的作用
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摘要
随着关节创伤的日渐增多,骨性关节炎(Osteoarthritis OA)已成为临床多发病和常见病,严重影响着人类健康。由于本病发展缓慢,病程长,早期不引起症状或症状轻微,易被人忽视,而现有的诊断方法主要依靠病人的临床症状和影像学表现,才能做出诊断。所以,在依靠放射学诊断前,我们不能早期作出诊断,进而阻断OA的发展,这反映了我们缺乏适当的方法监测关节软骨的健康程度。而且现在的诊断方法和关节病分期只适合于术前计划,并不适合于早期诊断和监测疾病发展。如何在创伤后早期诊断骨性关节炎,预测其发展趋势,早期治疗,从而延长关节使用寿命,减轻病人痛苦,是一个亟待解决的问题。毫无疑问,关节软骨在病理状态下,生化改变要早于形态学改变。寻求反映软骨生化改变的生物标记物将对早期诊断有极大的帮助。关节液中的生物标记物(Biomarker)能够直接反映患病关节的关节软骨生化改变,且不受身体其它部位关节软骨变化的影响,并且可以动态观察。以往的研究中,结合关节镜下软骨损伤评分,我们分析了多个关节液Marker,如：糖胺多糖、透明质酸、金属蛋白酶-3及其抑制剂-1、二型胶原羧基端肽、二型胶原螺旋肽等,虽然这些Marker被证明与软骨损伤相关,特别是软骨损伤早期,但是,由于个体差异较大,无法形成有效的标准应用于临床。如何减少个体差异的影响,以及发现新的、特异性高的Marker成为关节液研究的方向。
关节镜下软骨损伤评分是界定软骨损伤的金标准,同时测定关节液中BM,能准确反映关节软骨的代谢情况,我们设计了如下研究内容：
Ⅱ型胶原是关节软骨的特征性蛋白,围绕Ⅱ型胶原的代谢,一方面测定了关节液中Ⅱ型胶原螺旋肽的含量,研究其与关节软骨损伤程度间的关系；另一方面,同时测定关节液中其分解代谢产物Ⅱ型胶原羧基端端肽和合成代谢产物Ⅱ型前胶原羧基端前肽,通过观察在关节软骨损伤过程中二者之间的相互关系,研究Ⅱ型胶原代谢规律,早期诊断关节软骨损伤；对于新发现的关节液Marker(印度刺猬蛋白)进行含量测定,结合软骨损伤评分,研究其在早期诊断关节软骨损伤中的作用；通过表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术结合弱阳离子芯片,研究不同损伤程度下关节液蛋白的差异位点,建立早期诊断关节软骨损伤的差异位点模型,并为进一步蛋白组学研究提供方向；结合关节镜下软骨损伤评分和滑膜炎评分,分析滑膜炎和软骨损伤之间的关系。
本课题分为五部分：
第一部分关节液中Ⅱ型胶原螺旋肽(HELIX-Ⅱ)含量测定早期诊断关节软骨损伤
目的：研究膝关节液中Ⅱ型胶原螺旋肽(HELIX-Ⅱ)的含量与关节镜下膝关节软骨损伤程度之间的关系,早期诊断关节软骨损伤。
方法：收集2008年3月至2008年10月在山西医科大学第二医院骨科行关节镜诊治和膝关节置换术的膝关节疾病患者关节液83例,采用ELISA法检测其关节液HELIX-Ⅱ含量,依据Outerbridge软骨损伤评分标准评价软骨损伤程度,以损伤最重区域评分为最高评分,累加6个区域的评分即为该膝关节软骨损伤累计评分。
结果：膝关节液中HELIX-Ⅱ含量与Outerbridge软骨损伤累计评分呈正相关(r=0.807,P<0.05),与Outerbridge软骨损伤最高评分呈正相关(r=0.794,P<0.05)；软骨损伤较重者(Outerbridge软骨损伤最高评分为2分、3分和4分)HELIX-Ⅱ的含量明显高于软骨损伤较轻者(0分和1分组)(P<0.05),而损伤较重者之间无明显差异。
结论：膝关节液中HELIX-Ⅱ的含量可作为生物标记物来早期判断关节软骨损伤,特别是可以反映软骨是否有表面破裂这一病理变化,但不能用来监测病情发展。
第二部分Ⅱ型胶原两种不同代谢BM同时测定早期诊断关节软骨损伤
目的：通过同时测定不同程度关节软骨损伤患者膝关节液中Ⅱ型胶原羧基端端肽(C-telopeptide of CⅡ,CTX-Ⅱ)与Ⅱ型前胶原羧基端前肽(Procollagen Ⅱ C-propeptide,PⅡ CP)的含量,通过观察二者之间的相互关系,一方面描述关节软骨损伤过程中的变化规律；另一方面研究二者在诊断关节软骨损伤中的作用。
方法：收集2009-2011年在我院行关节镜诊治或关节置换术,并且抽取关节液的患者47例共48膝,男25例(25膝),女22例(23膝),对纳入病例行关节镜手术或全膝关节表面置换术时,取髌下外侧入路行膝关节腔穿刺抽取关节液,将关节液于取出后2小时内,4500rpm,20分钟条件下离心,留取上清液分装于Eppendorf管中,快速冷冻,在-80℃深低温保存待测。根据膝关节镜检查或全膝关节表面置换术时所见,采用Outerbridge软骨损伤评分法对髌骨面、股骨髌面、股骨内侧髁、股骨外侧髁、胫骨内侧髁和胫骨外侧髁共6个区域的关节软骨损伤情况进行评分。按Outerbridge软骨损伤评分最高评分将样本分为0分、1分、2分、3分、4分5个组,按Outerbridge软骨损伤累计评分将样本分为<10分组和≥10分组两组,按性别将样本分为男性和女性两组。采用酶联免疫吸附试验(ELISA法)对关节液CTX-Ⅱ(武汉中美科技,E90686Hu)与PⅡCP(武汉中美科技,E90964Hu)浓度进行测定。
结果：关节液中CTX-Ⅱ和PⅡCP含量无论是作为单独指标还是二者之间的比值,在不同关节软骨损伤程度间的差异都不具有统计学意义(P>0.05),而且与软骨损伤程度无相关性(P>0.05)；但是从数据分布来看,二者相对于软骨损伤评分有明显的下降趋势。而且关节液中CTX-Ⅱ浓度和PIICP浓度具有正相关(P<0.05)。
结论：关节液中CTX-Ⅱ和PIICP的浓度无论作为独立指标,还是二者间比值,与软骨损伤程度间无明显相关关系；另外,总体上,CTX-Ⅱ和PIICP含量变化具有相关性,但每一个体,CTX-Ⅱ和PIICP含量变化并不一定同步。总之,CTX-Ⅱ和PIICP在描述骨关节炎病理生理过程具有一定的作用,但由于个体差异的影响,它们并不能作为标记软骨损伤程度的Marker。
第四部分SELDI-TOF-MS技术在骨关节炎早期诊断中的应用
目的：利用SELDI-TOF-MS技术寻找关节液中不同关节软骨损伤条件下的差异位点,建立早期诊断关节软骨损伤的诊断模型。
方法：收集2009年在我院行关节镜治疗患者44例,其中男性24例,女性20例。关节镜下评定软骨损伤程度,应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术结合弱阳离子芯片,寻找关节液中不同软骨损伤情况下的差异位点。
结果：软骨无损伤与软骨轻度损伤差异位点10个；软骨无损伤与软骨中度损伤差异
第三部分关节液Ihh含量变化与关节软骨损伤的关系
目的：结合关节镜下软骨损伤评分,测定关节液中的印度刺猬蛋白(Ihh)含量,研究Ihh含量与软骨损伤之间的关系,早期诊断关节软骨损伤。
方法：收集2010年67例在我院行关节镜手术患者,其中男32例(32膝),女35例(35膝)。关节镜手术时,行膝关节腔穿刺抽取关节液,将关节液于抽出后2小时内,以4500rpm,20分钟条件下离心,留取上清液分装于Eppendorf管中,快速冷冻,在-80℃深低温保存待测。根据膝关节镜检查,采用Outerbridge软骨损伤评分法对膝关节内6个区域的关节软骨损伤情况进行评分。按Outerbridge软骨损伤评分最高评分将样本分为0分、1分、2分、3分、4分5个组。采用酶联免疫吸附试验(ELISA法)对关节液中ihh含量进行测定(武汉中美科技)。
结果：在软骨损伤最高评分2分以内,Ihh含量成增高趋势,2分以外呈下降趋势；两组间对比分析,0分与2分、0分与3分之间有显著差异,p值分别是1.237E-05和1.237E-05；1分与2分、2分与4分之间有显著差异,p值分别是0.0250153和0.0017442。其它评分之间对比无显著差异。
结论：虽然Ihh含量在软骨无损伤和软骨极轻度损伤(Outerbridge评分=1)之间无明显差异,但是Ihh含量在软骨无损伤和软骨轻度损伤(Outerbridge评分=2)之间有显著差异。这样对于临床无症状或轻微,影像学检查没有表现的软骨损伤较早期患者具有十分重要的意义。Ihh含量升高提示关节软骨损伤损伤。位点30个；软骨轻度损伤与软骨中度损伤差异位点17个。每个差异位点蛋白相对含量都以均数±标准差表示,所有的标准差相对均数均较大,未发现特异性高的蛋白或多肽。
结论：运用SELDI-TOF-MS技术结合弱阳离子芯片研究关节液蛋白质组学,是一种快捷有效的关节液差异蛋白筛选方法,操作简单,高通量,重现性好。本实验未发现在软骨损伤早、中期产生特异性高的蛋白质或多肽；但是提示我们关节软骨在损伤初期(Outerbridge评分<4),关节软骨代谢以合成代谢升高为主,而随着软骨损伤进一步发展,软骨降解代谢迅速加强,这也许说明软骨损伤可以在很早阶段(Outerbridge评分>6)发生不可逆改变。虽然发现了多个差异蛋白位点,但由于所有的差异点特异性都很不高,不能有效形成特异性节点,无法建立早期诊断模型。
第五部分滑膜炎与关节软骨损伤程度间的关系
目的：研究不同关节软骨损伤情况下关节滑膜的表现,并用其来分析二型胶原羧基端端肽(C-telopeptide of Collagen Ⅱ,CTX-Ⅱ)和二型前胶原羧基端前肽(Procollagen Ⅱ C-propeptide,PⅡ CP)含量的数值分布情况,探讨滑膜炎对测定结果的影响。
方法：收集2001年至20008年关节镜手术337例,共339膝,其中男性161例,女性176例；年龄范围11～82岁,平均42.9岁。采用Outerbridge评分标准分别对髌骨面、股骨髌面、股骨内侧髁、股骨外侧髁、胫骨内侧髁、和胫骨外侧髁共6个区域的关节软骨损伤病理变化情况进行评分,并将评分累加。对滑膜采用Ayral评分法,对膝关节滑膜9个区域进行评分,按公式计算出滑膜炎评分。统计分析二者之间的关系。
结果：软骨损伤评分和滑膜炎评分做Spearman相关分析,结果表明软骨损伤评分与滑膜炎评分呈正相关(r=0.690,p<0.001)。当软骨损伤评分为零分时,将年龄和滑膜评分作Spearman相关分析,结果表明年龄与滑膜炎评分呈正相关(r=0.353,p<0.001)；性别与滑膜炎两者之间spearman相关系数为0.020,p值为0.827,提示两者之间无相关性。
结论：随着软骨损伤程度逐步加重,滑膜炎程度亦同时加重；然而,随着滑膜炎程度逐步加重,软骨损伤程度并不一定加重(出现最严重滑膜炎时,软骨也不一定有损伤)；并且,年龄因素对滑膜炎程度起重要作用。由此可以得出,软骨损伤对滑膜炎起决定作用,但同时,滑膜炎受多种因素影响,滑膜炎并不具备提示软骨损伤,并且加重骨关节炎的作用。
With the increasing number of joint trauma, osteoarthritis (Osteoarthritis OA) has become a common disease, serious impact on human health. Due to the slow development of the disease, it is not to cause symptoms or mild symptoms in early, so be easily ignored by people. Now to diagnosis mainly depends on the patient's clinical symptoms and imaging, especially on joint space narrowing. So, depending on the radiological diagnosis, we can not make the early diagnosis, and not to block the development of OA, which reflects the fact that we cannot monitor the health of articular cartilage. The diagnosis method and joint disease staging are only suitable for preoperative planning, are not suitable for early diagnosis and monitoring disease development. How in the early diagnosis of post-traumatic osteoarthritis, forecast the trend of its development, early treatment, thereby prolonging the service life of the joint, relieve the pain of patients, is an urgent problem to be solved.
There is no doubt, articular cartilage in pathological conditions, biochemical changes earlier than morphological changes. Seeking biomarkers reflect cartilage biochemical changes will be great helpful to the early diagnosis. Biomarkers in synovial fluid (Marker) can directly reflect the biochemical changes in articular cartilage in the joints, and is not affected by the change of articular cartilage in other parts of the body, and can dynamically observe. In the previous study, combined with arthroscopic cartilage injury score, we analyzed a plurality of synovial fluid Marker, such as:glycosaminoglycans, hyaluronic acid, metal protease and inhibitors of-3-1, type two collagen carboxyterminal propeptide, type two collagen helical peptide, and so on, although the Marker were proved to be associated with cartilage injury, because of individual differences, we can not form a standard effectively in clinical application. How to reduce the impact of individual differences, and to discover a new, high specificity for Marker become joint fluid research direction.
Arthroscopic cartilage injury score is the gold standard for defining the degree of cartilage injury, based on this standard, we designed the following contents:
Collagen II is a protein characteristic of articular cartilage, around the type II collagen metabolism,one hand,the type II collagen helical peptide of synovial fluid were determined, and to study the relationship between the articular cartilage injury degree and concentrations of HELIX-Ⅱ; on the other hand, by determine the Markers both catabolism and anabolism at the same time, to study the relationship between the two Markers, hope to be able to reduce the individual differences, more clearly defined the extent of cartilage damage; for synovial fluid Marker newly discovered (India hedgehog protein) were determined, with cartilage injury score, to determine whether it is helpful to early diagnosis of articular cartilage injury and to define the extent of cartilage damage; by surface enhanced laser desorption ionization mass spectrometry (SELDI-TOF-MS) technique combined with weak cation chip, to study the different sites in joint fluid protein under different damage degree, establishment of different site model for early diagnosis of joint cartilage injury, and provide direction for further research on proteomics; combined with cartilage injury score and synovitis arthroscopy score, analysis of the relationship between synovitis and cartilage injury, and based on the analysis of experimental data, to demonstrate that synovitis is the main factor causing the individual differences..
This paper is divided into five parts:
Part one:The determination of HELIX-Ⅱ in the synovial fluid
Objective:To investigate the Relationship between HELIX-Ⅱ (type Ⅱ collagen helical peptide, HELIX-Ⅱ) levels in the synovial fluid of the knee and the degree of cartilage injury. Methods:83patients with knee diseases were operated by arthroscopy and TKA were studied. The HELIX-Ⅱ levels were measured by enzyme linked immunoassay. The degree of cartilage injury was assessed with Outerbridge score system(0～4point). In6areas of one knee, if the cartilage of one area was destroyed most seriously, the area was maximum Outerbridge score. The summation of6areas scores was the accumulative Outerbridge score.
Results:The results show that the synovial HELIX-Ⅱ levels were correlated positively with accumulative Outerbridge score (r=0.807, P<0.05) and with maximum Outerbridge score(r=0.794, P<0.05). In maximum Outerbridge score groups, the levels in cartilage injury group(2、3、4point) were higher than the group (0、1point). Conclusion:Elevated synovial HELIX-Ⅱ levels are associated with cartilage injury. There is pertinency between the levels and degree of cartilage injury. Once the articular cartilage had breach, the HELIX-Ⅱ levels were increased rapidly. So the levels can clue on whether articular cartilage had been destroyed. Measurement of HELIX-Ⅱ could be useful for the clinical investigation of patients with cartilage injury in early stage.
Part two:The determination of type II collagen metabolism
Objective:By measuring the different degree of articular cartilage injury in patients with knee joint fluid of type two collagen C-terminal telopeptide (C-telopeptide of, C Ⅱ, CTX-Ⅱ) propeptide and type two procollagen C-terminal (Procollagen II C-propeptide, P II CP) content, through observation of two mutual relation, a description change of articular cartilage injury; on the other hand, hope to be able to reduce the individual differences, more clearly defined the extent of cartilage damage.
Methods:From2009-2011years in our hospital,the patients who had treated by arthroscopy or joint replacement, and47cases of patients with48knees,25male (25knees), female22cases (23knees), into the cases of arthroscopic operation or total knee arthroplasty, and patellar lateral approach underwent knee joint cavity puncture of synovial fluid, synovial fluid to remove the2hour,20minutes4500rpm, under the conditions of centrifugation, the supernatant of Eppendorf tube packing to leave, quick freezing, at the temperature of-70℃cryopreservation test. According to arthroscopy of knee or knee joint surface replacement see, score of articular cartilage injuries by Outerbridge cartilage injury score on the patellar surface, the surface of the femoral, medial femoral condyle, lateral femoral condyle, tibial medial condyle and lateral condyle of tibia in6areas. According to Outerbridge cartilage injury score the highest score divided the samples into0points,1points,2points,3points,4points5groups, according to the Outerbridge cartilage damage cumulative score divided the samples into<10group and≥10group two group by gender, the samples were divided into two groups in men and women. Using enzyme-linked immunosorbent assay (ELISA method) on the joint of liquid CTX-(Wuhan China Science and technology, E90686Hu) and PIICP (Wuhan USCN Sciences, E90964Hu) concentrations were measured.
Results:S±x representation of CTX-Ⅱ and content of PIICP in synovial fluid of both the ratio between as a separate index two, differences in different degree of articular cartilage injury are not statistically significant (P>0.05), but had no correlation with the degree of cartilage injury (P>0.05); but from the data distribution two, with respect to the cartilage injury score had significant downward trend. And the CTX-II concentration and PIICP concentration in synovial fluid has positive correlation (P<0.05).
Conclusion:The concentrations of CTX-Ⅱ and PIICP in the synovial fluid in both as an independent index, or between the two ratio, and there was no obvious correlation with the extent of cartilage damage; in addition, ratio of CTX-Ⅱ and PIICP did not reduce the individual differences. In conclusion, CTX-Ⅱ and PIICP have a certain role in describing the osteoarthritis pathophysiologic process, but due to the impact of individual differences, they are not as markers of cartilage injury degree.
Part three:The determination of Ihh content in synovia
Objective:Combined with arthroscopic cartilage injury score, determination of synovial fluid in Indian hedgehog protein (Ihh) content, to study the relationship between Ihh content and cartilage injury.
Methods:in2010,67cases of in patients in our hospital underwent arthroscopic operation, there were32male (32knees), female35cases (35knees). Arthroscopic operation, knee joint puncture from synovial fluid, synovial fluid to extract the after2hours,20minutes to4500rpm, under the conditions of centrifugation, the supernatant of Eppendorf tube packing to leave, quick freezing, at the temperature of-70℃cryopreservation test. According to knee arthroscopy, were evaluated using Outerbridge cartilage injury score of articular cartilage injury of knee joint in6areas. According to Outerbridge cartilage injury score the highest score divided the samples into0points,1points,2points,3points,4points5groups. The enzyme-linked immunosorbent assay (ELISA method) was used to determine the content of IHH in the synovial fluid (Wuhan Science and technology of China).
Results:the highest score in the cartilage injury within2points, Ihh content increased,2points outside a downward trend; analysis and comparison between the two groups, with0points and2 points,0points and3points between the significant difference, P values were1.237E-05and1.237E-05;1points and2points,2points and4divided between the significant difference, P values were0.0250153and0.0017442. No significant difference compared with other scores between.
Conclusion:Although in the cartilage without damage and cartilage very mild injury (Outerbridge score=1) there is no significant difference in the content of Ihh, but in the group without injury and the group with mild damage in cartilage (Outerbridge score=2), there was a significant difference between the values, and with centralized distribution, individual difference is smaller, the range of clear difference. So for clinically asymptomatic or mild, without cartilage damage imaging examination showed, there has very important significance for early patients. Ihh can be used as an effective index for clinical diagnosis of cartilage injury of articular cartilage injury in early stage.
Part four:The research of joint fluid proteomics
Objective:To find out the joint fluid specific markers for articular cartilage injury, and the establishment of diagnosis model of early diagnosis of articular cartilage injury. Methods:In2009,44patients in our hospital underwent arthroscopic treatment, including24male cases,20female cases. The extent of cartilage damage assessment under arthroscopy, surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) combined with weak cation chip, find the difference in the synovial fluid of cartilage damage under the site.
Results:the cartilage without damage and mild cartilage injury sites10; cartilage without damage and cartilage moderate injury sites30; mild cartilage injury and cartilage moderate injury sites17. The relative content of each difference of ectopic protein have show with mean±standard, all of the standard deviation of relative mean are large, not the high specificity of the protein or polypeptide found.
Conclusion:Using SELDI-TOF-MS technology combined with weak cation of joint chip fluid proteomics, is a screening method, with simple operation, high throughput, good reproducibility.
This experiment was not found in the cartilage injury in early, mid new specific protein or polypeptide; but reminds us that articular cartilage in early stage of injury (Outerbridge score<4), articular cartilage metabolism mainly by synthesis metabolism increased, but with the further development of cartilage injury, cartilage degradation up quickly, which might suggest that the cartilage injury can be very early stage (Outerbridge>6) irreversible changes. Although found a number of differences in protein loci, but because each site relative to the mean standard deviation is too large, the difference of all the specificity is low, can not form specific node, unable to establish early diagnosis model. There was not a significant difference between the values, and centralized distribution, individual difference is smaller, the range of clear difference. So for clinically asymptomatic or mild, cartilage damage imaging examination showed no has very important significance for early patients. Ihh can be used as an effective index for clinical diagnosis of cartilage injury of articular cartilage injury in early stage.
Part five:The relationship between synovitis and articular cartilage injury
Objective:To explore the synovial membrrane performance under the different articular cartilage injury cases, and to study its influence on the determination of synovial fluid Marker. Methods:337cases were studied from2001to20008under the arthroscopic operation, a total of339knees,161were male,176female; age range11～82years, mean42.9years.With Outerbridge standard respectively on the patellar surface, femoral patellar surface, medial femoral condyle, lateral condyle of femur, tibia, medial condyle and lateral condyle of tibia in all6regions of the articular cartilage injury pathological changes in score, and the cumulative score.The synovial membrane using Ayral score, on the knee joint synovial9regions according to the score, the formula to calculate the synovitis score. The data of concentrations of CTX-Ⅱ and PIICP.
Results:the cartilage injury score and synovitis score Spearman analysis, the results show that cartilage injury score and synovitis score were positively correlated (r=0.690,<0.001).When the cartilage injury score zero points, the age and synovial score for Spearman correlation analysis, the results showed that age was positively correlated with synovitis score (r=0.353,<0.001); gender and synovitis in Spearman correlation coefficient is0.020, P value was0.827, and there was no correlation between the tips of both. As the degree of cartilage injury, the degree of synovitis difference reducing; CTX-Ⅱ and P Ⅱ CP content was decreased, the numerical dispersion is reduced, especially for the cases with seriously articular cartilage damage.
Conclusion:with the cartilage injury severity, the degree of synovitis gradually increase; however, with the degree of synovitis gradually increase, cartilage damage degree does not necessarily increase (most severe synovitis, cartilage may not be injury); and, age factors plays an important role in degree of synovitis.Therefore, cartilage injury play a decisive role, but at the same time, synovitis is affected by many factors, synovitis have not the ability to prognosis the progress of osteoarthritis. The difference of synovial membrane hyperplasia is the main factor causing BM individual differences.
关节镜下软骨损伤评分是界定软骨损伤的金标准,同时测定关节液中BM,能准确反映关节软骨的代谢情况,我们设计了如下研究内容：
Ⅱ型胶原是关节软骨的特征性蛋白,围绕Ⅱ型胶原的代谢,一方面测定了关节液中Ⅱ型胶原螺旋肽的含量,研究其与关节软骨损伤程度间的关系；另一方面,同时测定关节液中其分解代谢产物Ⅱ型胶原羧基端端肽和合成代谢产物Ⅱ型前胶原羧基端前肽,通过观察在关节软骨损伤过程中二者之间的相互关系,研究Ⅱ型胶原代谢规律,早期诊断关节软骨损伤；对于新发现的关节液Marker(印度刺猬蛋白)进行含量测定,结合软骨损伤评分,研究其在早期诊断关节软骨损伤中的作用；通过表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术结合弱阳离子芯片,研究不同损伤程度下关节液蛋白的差异位点,建立早期诊断关节软骨损伤的差异位点模型,并为进一步蛋白组学研究提供方向；结合关节镜下软骨损伤评分和滑膜炎评分,分析滑膜炎和软骨损伤之间的关系。
本课题分为五部分：
第一部分关节液中Ⅱ型胶原螺旋肽(HELIX-Ⅱ)含量测定早期诊断关节软骨损伤
目的：研究膝关节液中Ⅱ型胶原螺旋肽(HELIX-Ⅱ)的含量与关节镜下膝关节软骨损伤程度之间的关系,早期诊断关节软骨损伤。
方法：收集2008年3月至2008年10月在山西医科大学第二医院骨科行关节镜诊治和膝关节置换术的膝关节疾病患者关节液83例,采用ELISA法检测其关节液HELIX-Ⅱ含量,依据Outerbridge软骨损伤评分标准评价软骨损伤程度,以损伤最重区域评分为最高评分,累加6个区域的评分即为该膝关节软骨损伤累计评分。
结果：膝关节液中HELIX-Ⅱ含量与Outerbridge软骨损伤累计评分呈正相关(r=0.807,P<0.05),与Outerbridge软骨损伤最高评分呈正相关(r=0.794,P<0.05)；软骨损伤较重者(Outerbridge软骨损伤最高评分为2分、3分和4分)HELIX-Ⅱ的含量明显高于软骨损伤较轻者(0分和1分组)(P<0.05),而损伤较重者之间无明显差异。
结论：膝关节液中HELIX-Ⅱ的含量可作为生物标记物来早期判断关节软骨损伤,特别是可以反映软骨是否有表面破裂这一病理变化,但不能用来监测病情发展。
第二部分Ⅱ型胶原两种不同代谢BM同时测定早期诊断关节软骨损伤
目的：通过同时测定不同程度关节软骨损伤患者膝关节液中Ⅱ型胶原羧基端端肽(C-telopeptide of CⅡ,CTX-Ⅱ)与Ⅱ型前胶原羧基端前肽(Procollagen Ⅱ C-propeptide,PⅡ CP)的含量,通过观察二者之间的相互关系,一方面描述关节软骨损伤过程中的变化规律；另一方面研究二者在诊断关节软骨损伤中的作用。
方法：收集2009-2011年在我院行关节镜诊治或关节置换术,并且抽取关节液的患者47例共48膝,男25例(25膝),女22例(23膝),对纳入病例行关节镜手术或全膝关节表面置换术时,取髌下外侧入路行膝关节腔穿刺抽取关节液,将关节液于取出后2小时内,4500rpm,20分钟条件下离心,留取上清液分装于Eppendorf管中,快速冷冻,在-80℃深低温保存待测。根据膝关节镜检查或全膝关节表面置换术时所见,采用Outerbridge软骨损伤评分法对髌骨面、股骨髌面、股骨内侧髁、股骨外侧髁、胫骨内侧髁和胫骨外侧髁共6个区域的关节软骨损伤情况进行评分。按Outerbridge软骨损伤评分最高评分将样本分为0分、1分、2分、3分、4分5个组,按Outerbridge软骨损伤累计评分将样本分为<10分组和≥10分组两组,按性别将样本分为男性和女性两组。采用酶联免疫吸附试验(ELISA法)对关节液CTX-Ⅱ(武汉中美科技,E90686Hu)与PⅡCP(武汉中美科技,E90964Hu)浓度进行测定。
结果：关节液中CTX-Ⅱ和PⅡCP含量无论是作为单独指标还是二者之间的比值,在不同关节软骨损伤程度间的差异都不具有统计学意义(P>0.05),而且与软骨损伤程度无相关性(P>0.05)；但是从数据分布来看,二者相对于软骨损伤评分有明显的下降趋势。而且关节液中CTX-Ⅱ浓度和PIICP浓度具有正相关(P<0.05)。
结论：关节液中CTX-Ⅱ和PIICP的浓度无论作为独立指标,还是二者间比值,与软骨损伤程度间无明显相关关系；另外,总体上,CTX-Ⅱ和PIICP含量变化具有相关性,但每一个体,CTX-Ⅱ和PIICP含量变化并不一定同步。总之,CTX-Ⅱ和PIICP在描述骨关节炎病理生理过程具有一定的作用,但由于个体差异的影响,它们并不能作为标记软骨损伤程度的Marker。
第四部分SELDI-TOF-MS技术在骨关节炎早期诊断中的应用
目的：利用SELDI-TOF-MS技术寻找关节液中不同关节软骨损伤条件下的差异位点,建立早期诊断关节软骨损伤的诊断模型。
方法：收集2009年在我院行关节镜治疗患者44例,其中男性24例,女性20例。关节镜下评定软骨损伤程度,应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术结合弱阳离子芯片,寻找关节液中不同软骨损伤情况下的差异位点。
结果：软骨无损伤与软骨轻度损伤差异位点10个；软骨无损伤与软骨中度损伤差异
第三部分关节液Ihh含量变化与关节软骨损伤的关系
目的：结合关节镜下软骨损伤评分,测定关节液中的印度刺猬蛋白(Ihh)含量,研究Ihh含量与软骨损伤之间的关系,早期诊断关节软骨损伤。
方法：收集2010年67例在我院行关节镜手术患者,其中男32例(32膝),女35例(35膝)。关节镜手术时,行膝关节腔穿刺抽取关节液,将关节液于抽出后2小时内,以4500rpm,20分钟条件下离心,留取上清液分装于Eppendorf管中,快速冷冻,在-80℃深低温保存待测。根据膝关节镜检查,采用Outerbridge软骨损伤评分法对膝关节内6个区域的关节软骨损伤情况进行评分。按Outerbridge软骨损伤评分最高评分将样本分为0分、1分、2分、3分、4分5个组。采用酶联免疫吸附试验(ELISA法)对关节液中ihh含量进行测定(武汉中美科技)。
结果：在软骨损伤最高评分2分以内,Ihh含量成增高趋势,2分以外呈下降趋势；两组间对比分析,0分与2分、0分与3分之间有显著差异,p值分别是1.237E-05和1.237E-05；1分与2分、2分与4分之间有显著差异,p值分别是0.0250153和0.0017442。其它评分之间对比无显著差异。
结论：虽然Ihh含量在软骨无损伤和软骨极轻度损伤(Outerbridge评分=1)之间无明显差异,但是Ihh含量在软骨无损伤和软骨轻度损伤(Outerbridge评分=2)之间有显著差异。这样对于临床无症状或轻微,影像学检查没有表现的软骨损伤较早期患者具有十分重要的意义。Ihh含量升高提示关节软骨损伤损伤。位点30个；软骨轻度损伤与软骨中度损伤差异位点17个。每个差异位点蛋白相对含量都以均数±标准差表示,所有的标准差相对均数均较大,未发现特异性高的蛋白或多肽。
结论：运用SELDI-TOF-MS技术结合弱阳离子芯片研究关节液蛋白质组学,是一种快捷有效的关节液差异蛋白筛选方法,操作简单,高通量,重现性好。本实验未发现在软骨损伤早、中期产生特异性高的蛋白质或多肽；但是提示我们关节软骨在损伤初期(Outerbridge评分<4),关节软骨代谢以合成代谢升高为主,而随着软骨损伤进一步发展,软骨降解代谢迅速加强,这也许说明软骨损伤可以在很早阶段(Outerbridge评分>6)发生不可逆改变。虽然发现了多个差异蛋白位点,但由于所有的差异点特异性都很不高,不能有效形成特异性节点,无法建立早期诊断模型。
第五部分滑膜炎与关节软骨损伤程度间的关系
目的：研究不同关节软骨损伤情况下关节滑膜的表现,并用其来分析二型胶原羧基端端肽(C-telopeptide of Collagen Ⅱ,CTX-Ⅱ)和二型前胶原羧基端前肽(Procollagen Ⅱ C-propeptide,PⅡ CP)含量的数值分布情况,探讨滑膜炎对测定结果的影响。
方法：收集2001年至20008年关节镜手术337例,共339膝,其中男性161例,女性176例；年龄范围11～82岁,平均42.9岁。采用Outerbridge评分标准分别对髌骨面、股骨髌面、股骨内侧髁、股骨外侧髁、胫骨内侧髁、和胫骨外侧髁共6个区域的关节软骨损伤病理变化情况进行评分,并将评分累加。对滑膜采用Ayral评分法,对膝关节滑膜9个区域进行评分,按公式计算出滑膜炎评分。统计分析二者之间的关系。
结果：软骨损伤评分和滑膜炎评分做Spearman相关分析,结果表明软骨损伤评分与滑膜炎评分呈正相关(r=0.690,p<0.001)。当软骨损伤评分为零分时,将年龄和滑膜评分作Spearman相关分析,结果表明年龄与滑膜炎评分呈正相关(r=0.353,p<0.001)；性别与滑膜炎两者之间spearman相关系数为0.020,p值为0.827,提示两者之间无相关性。
结论：随着软骨损伤程度逐步加重,滑膜炎程度亦同时加重；然而,随着滑膜炎程度逐步加重,软骨损伤程度并不一定加重(出现最严重滑膜炎时,软骨也不一定有损伤)；并且,年龄因素对滑膜炎程度起重要作用。由此可以得出,软骨损伤对滑膜炎起决定作用,但同时,滑膜炎受多种因素影响,滑膜炎并不具备提示软骨损伤,并且加重骨关节炎的作用。
With the increasing number of joint trauma, osteoarthritis (Osteoarthritis OA) has become a common disease, serious impact on human health. Due to the slow development of the disease, it is not to cause symptoms or mild symptoms in early, so be easily ignored by people. Now to diagnosis mainly depends on the patient's clinical symptoms and imaging, especially on joint space narrowing. So, depending on the radiological diagnosis, we can not make the early diagnosis, and not to block the development of OA, which reflects the fact that we cannot monitor the health of articular cartilage. The diagnosis method and joint disease staging are only suitable for preoperative planning, are not suitable for early diagnosis and monitoring disease development. How in the early diagnosis of post-traumatic osteoarthritis, forecast the trend of its development, early treatment, thereby prolonging the service life of the joint, relieve the pain of patients, is an urgent problem to be solved.
There is no doubt, articular cartilage in pathological conditions, biochemical changes earlier than morphological changes. Seeking biomarkers reflect cartilage biochemical changes will be great helpful to the early diagnosis. Biomarkers in synovial fluid (Marker) can directly reflect the biochemical changes in articular cartilage in the joints, and is not affected by the change of articular cartilage in other parts of the body, and can dynamically observe. In the previous study, combined with arthroscopic cartilage injury score, we analyzed a plurality of synovial fluid Marker, such as:glycosaminoglycans, hyaluronic acid, metal protease and inhibitors of-3-1, type two collagen carboxyterminal propeptide, type two collagen helical peptide, and so on, although the Marker were proved to be associated with cartilage injury, because of individual differences, we can not form a standard effectively in clinical application. How to reduce the impact of individual differences, and to discover a new, high specificity for Marker become joint fluid research direction.
Arthroscopic cartilage injury score is the gold standard for defining the degree of cartilage injury, based on this standard, we designed the following contents:
Collagen II is a protein characteristic of articular cartilage, around the type II collagen metabolism,one hand,the type II collagen helical peptide of synovial fluid were determined, and to study the relationship between the articular cartilage injury degree and concentrations of HELIX-Ⅱ; on the other hand, by determine the Markers both catabolism and anabolism at the same time, to study the relationship between the two Markers, hope to be able to reduce the individual differences, more clearly defined the extent of cartilage damage; for synovial fluid Marker newly discovered (India hedgehog protein) were determined, with cartilage injury score, to determine whether it is helpful to early diagnosis of articular cartilage injury and to define the extent of cartilage damage; by surface enhanced laser desorption ionization mass spectrometry (SELDI-TOF-MS) technique combined with weak cation chip, to study the different sites in joint fluid protein under different damage degree, establishment of different site model for early diagnosis of joint cartilage injury, and provide direction for further research on proteomics; combined with cartilage injury score and synovitis arthroscopy score, analysis of the relationship between synovitis and cartilage injury, and based on the analysis of experimental data, to demonstrate that synovitis is the main factor causing the individual differences..
This paper is divided into five parts:
Part one:The determination of HELIX-Ⅱ in the synovial fluid
Objective:To investigate the Relationship between HELIX-Ⅱ (type Ⅱ collagen helical peptide, HELIX-Ⅱ) levels in the synovial fluid of the knee and the degree of cartilage injury. Methods:83patients with knee diseases were operated by arthroscopy and TKA were studied. The HELIX-Ⅱ levels were measured by enzyme linked immunoassay. The degree of cartilage injury was assessed with Outerbridge score system(0～4point). In6areas of one knee, if the cartilage of one area was destroyed most seriously, the area was maximum Outerbridge score. The summation of6areas scores was the accumulative Outerbridge score.
Results:The results show that the synovial HELIX-Ⅱ levels were correlated positively with accumulative Outerbridge score (r=0.807, P<0.05) and with maximum Outerbridge score(r=0.794, P<0.05). In maximum Outerbridge score groups, the levels in cartilage injury group(2、3、4point) were higher than the group (0、1point). Conclusion:Elevated synovial HELIX-Ⅱ levels are associated with cartilage injury. There is pertinency between the levels and degree of cartilage injury. Once the articular cartilage had breach, the HELIX-Ⅱ levels were increased rapidly. So the levels can clue on whether articular cartilage had been destroyed. Measurement of HELIX-Ⅱ could be useful for the clinical investigation of patients with cartilage injury in early stage.
Part two:The determination of type II collagen metabolism
Objective:By measuring the different degree of articular cartilage injury in patients with knee joint fluid of type two collagen C-terminal telopeptide (C-telopeptide of, C Ⅱ, CTX-Ⅱ) propeptide and type two procollagen C-terminal (Procollagen II C-propeptide, P II CP) content, through observation of two mutual relation, a description change of articular cartilage injury; on the other hand, hope to be able to reduce the individual differences, more clearly defined the extent of cartilage damage.
Methods:From2009-2011years in our hospital,the patients who had treated by arthroscopy or joint replacement, and47cases of patients with48knees,25male (25knees), female22cases (23knees), into the cases of arthroscopic operation or total knee arthroplasty, and patellar lateral approach underwent knee joint cavity puncture of synovial fluid, synovial fluid to remove the2hour,20minutes4500rpm, under the conditions of centrifugation, the supernatant of Eppendorf tube packing to leave, quick freezing, at the temperature of-70℃cryopreservation test. According to arthroscopy of knee or knee joint surface replacement see, score of articular cartilage injuries by Outerbridge cartilage injury score on the patellar surface, the surface of the femoral, medial femoral condyle, lateral femoral condyle, tibial medial condyle and lateral condyle of tibia in6areas. According to Outerbridge cartilage injury score the highest score divided the samples into0points,1points,2points,3points,4points5groups, according to the Outerbridge cartilage damage cumulative score divided the samples into<10group and≥10group two group by gender, the samples were divided into two groups in men and women. Using enzyme-linked immunosorbent assay (ELISA method) on the joint of liquid CTX-(Wuhan China Science and technology, E90686Hu) and PIICP (Wuhan USCN Sciences, E90964Hu) concentrations were measured.
Results:S±x representation of CTX-Ⅱ and content of PIICP in synovial fluid of both the ratio between as a separate index two, differences in different degree of articular cartilage injury are not statistically significant (P>0.05), but had no correlation with the degree of cartilage injury (P>0.05); but from the data distribution two, with respect to the cartilage injury score had significant downward trend. And the CTX-II concentration and PIICP concentration in synovial fluid has positive correlation (P<0.05).
Conclusion:The concentrations of CTX-Ⅱ and PIICP in the synovial fluid in both as an independent index, or between the two ratio, and there was no obvious correlation with the extent of cartilage damage; in addition, ratio of CTX-Ⅱ and PIICP did not reduce the individual differences. In conclusion, CTX-Ⅱ and PIICP have a certain role in describing the osteoarthritis pathophysiologic process, but due to the impact of individual differences, they are not as markers of cartilage injury degree.
Part three:The determination of Ihh content in synovia
Objective:Combined with arthroscopic cartilage injury score, determination of synovial fluid in Indian hedgehog protein (Ihh) content, to study the relationship between Ihh content and cartilage injury.
Methods:in2010,67cases of in patients in our hospital underwent arthroscopic operation, there were32male (32knees), female35cases (35knees). Arthroscopic operation, knee joint puncture from synovial fluid, synovial fluid to extract the after2hours,20minutes to4500rpm, under the conditions of centrifugation, the supernatant of Eppendorf tube packing to leave, quick freezing, at the temperature of-70℃cryopreservation test. According to knee arthroscopy, were evaluated using Outerbridge cartilage injury score of articular cartilage injury of knee joint in6areas. According to Outerbridge cartilage injury score the highest score divided the samples into0points,1points,2points,3points,4points5groups. The enzyme-linked immunosorbent assay (ELISA method) was used to determine the content of IHH in the synovial fluid (Wuhan Science and technology of China).
Results:the highest score in the cartilage injury within2points, Ihh content increased,2points outside a downward trend; analysis and comparison between the two groups, with0points and2 points,0points and3points between the significant difference, P values were1.237E-05and1.237E-05;1points and2points,2points and4divided between the significant difference, P values were0.0250153and0.0017442. No significant difference compared with other scores between.
Conclusion:Although in the cartilage without damage and cartilage very mild injury (Outerbridge score=1) there is no significant difference in the content of Ihh, but in the group without injury and the group with mild damage in cartilage (Outerbridge score=2), there was a significant difference between the values, and with centralized distribution, individual difference is smaller, the range of clear difference. So for clinically asymptomatic or mild, without cartilage damage imaging examination showed, there has very important significance for early patients. Ihh can be used as an effective index for clinical diagnosis of cartilage injury of articular cartilage injury in early stage.
Part four:The research of joint fluid proteomics
Objective:To find out the joint fluid specific markers for articular cartilage injury, and the establishment of diagnosis model of early diagnosis of articular cartilage injury. Methods:In2009,44patients in our hospital underwent arthroscopic treatment, including24male cases,20female cases. The extent of cartilage damage assessment under arthroscopy, surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) combined with weak cation chip, find the difference in the synovial fluid of cartilage damage under the site.
Results:the cartilage without damage and mild cartilage injury sites10; cartilage without damage and cartilage moderate injury sites30; mild cartilage injury and cartilage moderate injury sites17. The relative content of each difference of ectopic protein have show with mean±standard, all of the standard deviation of relative mean are large, not the high specificity of the protein or polypeptide found.
Conclusion:Using SELDI-TOF-MS technology combined with weak cation of joint chip fluid proteomics, is a screening method, with simple operation, high throughput, good reproducibility.
This experiment was not found in the cartilage injury in early, mid new specific protein or polypeptide; but reminds us that articular cartilage in early stage of injury (Outerbridge score<4), articular cartilage metabolism mainly by synthesis metabolism increased, but with the further development of cartilage injury, cartilage degradation up quickly, which might suggest that the cartilage injury can be very early stage (Outerbridge>6) irreversible changes. Although found a number of differences in protein loci, but because each site relative to the mean standard deviation is too large, the difference of all the specificity is low, can not form specific node, unable to establish early diagnosis model. There was not a significant difference between the values, and centralized distribution, individual difference is smaller, the range of clear difference. So for clinically asymptomatic or mild, cartilage damage imaging examination showed no has very important significance for early patients. Ihh can be used as an effective index for clinical diagnosis of cartilage injury of articular cartilage injury in early stage.
Part five:The relationship between synovitis and articular cartilage injury
Objective:To explore the synovial membrrane performance under the different articular cartilage injury cases, and to study its influence on the determination of synovial fluid Marker. Methods:337cases were studied from2001to20008under the arthroscopic operation, a total of339knees,161were male,176female; age range11～82years, mean42.9years.With Outerbridge standard respectively on the patellar surface, femoral patellar surface, medial femoral condyle, lateral condyle of femur, tibia, medial condyle and lateral condyle of tibia in all6regions of the articular cartilage injury pathological changes in score, and the cumulative score.The synovial membrane using Ayral score, on the knee joint synovial9regions according to the score, the formula to calculate the synovitis score. The data of concentrations of CTX-Ⅱ and PIICP.
Results:the cartilage injury score and synovitis score Spearman analysis, the results show that cartilage injury score and synovitis score were positively correlated (r=0.690,<0.001).When the cartilage injury score zero points, the age and synovial score for Spearman correlation analysis, the results showed that age was positively correlated with synovitis score (r=0.353,<0.001); gender and synovitis in Spearman correlation coefficient is0.020, P value was0.827, and there was no correlation between the tips of both. As the degree of cartilage injury, the degree of synovitis difference reducing; CTX-Ⅱ and P Ⅱ CP content was decreased, the numerical dispersion is reduced, especially for the cases with seriously articular cartilage damage.
Conclusion:with the cartilage injury severity, the degree of synovitis gradually increase; however, with the degree of synovitis gradually increase, cartilage damage degree does not necessarily increase (most severe synovitis, cartilage may not be injury); and, age factors plays an important role in degree of synovitis.Therefore, cartilage injury play a decisive role, but at the same time, synovitis is affected by many factors, synovitis have not the ability to prognosis the progress of osteoarthritis. The difference of synovial membrane hyperplasia is the main factor causing BM individual differences.
引文
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[5]尹崑,卫小春.关节液中糖胺多糖含量与关节软骨损伤的关系.实用骨科杂志,2006；12(3)：216-219.
[6]李正南,卫小春.膝关节液中生物学标记物Ⅱ型胶原羧基端端肽的检测及临床意义.中华关节外科杂志,2008；2(2)：45-49.
[7]Gamero P, Convozier T, Christgau S, et al. Urinary type Ⅱ collagen C-telopeptide levels are increased in patients with rapidly destructive hip osteoarthritis. Ann Rheum Dis,2003,62 (10): 939-943.
[8]Quintana DJ, Gamero P, Huebner JL, et al. PⅡANP and HELIX-Ⅱ diurnal variation. Osteoarthritis Cartilage,2008,16(10):1192-1195.
[9]Murphy G, Lee M H. What are the roles of metalloproteinases in cartilage and bone damage? Ann Rheum Dis,2005;64(1):44-47.
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[11]Chami N, Juillet F, Garnero P. Urinary type Ⅱ collagen helical peptide (HELIX-Ⅱ) as a new biochemical marker of cartilage degradation in patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum,2005;52(4):1081-1090.
[12]Charni-Ben Tabassi N, Desmarais S, Garnero P, et al. The type Ⅱ collagen fragments Helix-Ⅱ and CTX-Ⅱ reveal different enzymatic pathways of human cartilage collagen degradation. Osteoarthritis Cartilage,2008,16(10):1183-1191.
[1]Xavier Chevalier, Thierry Conrozier. Biological markers for osteoarthritis:an update. Joint Bone Spine,2005,72:106-109.
[2]M. Reijman, et al. A New Marker for Osteoarthritis,Cross-Sectional andLongitudinal Approach. Arthritis & Rheumatism,2004,50(8):2471-2478.
[3]S. Cahue, et al. The ratio of type Ⅱ collagen breakdown to synthesis and its relationship with the progression of knee osteoarthritis. Osteoarthritis and Cartilage,2007,15(7):819-823.
[4]David J Hunter, et,al. Cartilage markers and their association with cartilageloss on magnetic resonance imaging in knee osteoarthritis:the Boston Osteoarthritis Knee Study. Arthritis Research & Therapy,2007,9:R108(doi:10.1186/ar2314).
[5]Johanne Martel-Pelletier, et al.Cartilage in normal and osteoarthritis conditions. Best Practice & Research Clinical Rheumatology,2008,22(2):351-384.
[6]Helga Lorenz, Wiltrud Richter. Osteoarthritis Cellular and molecular changes in degenerating cartilage. Progress in Histochemistry and Cytochemistry,2006,40:135-163.
[7]A. K. Olsen, et al. Anabolic and catabolic function of chondrocyte ex vivo is reflected by the metabolic processing of type Ⅱ collagen. OsteoArthritis and Cartilage,2007,15:335-342.
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