人类原发性膝关节骨性关节炎病变软骨全基因组表达与分析
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摘要
原发性膝关节骨性关节炎(KOA)是一种随年龄增长而发生率明显增加的退行性关节疾病,是老年人关节疼痛和致残的主要原因。据世界卫生组织(WHO)统计,骨性关节炎在女性患病率中占第四位。随年龄增大,患病率迅速上升,大于65岁人群中50%以上有骨性关节炎的X线片表现。它的主要改变是关节软骨面的退行性变和继发性的骨质增生。主要表现是关节疼痛和活动不灵活,X线表现关节间隙变窄,软骨下骨质致密,骨小梁断裂,有硬化和囊性变。关节边缘有唇样增生。后期骨端变形,关节面凹凸不平。关节内软骨剥落,骨质碎裂进入关节,形成关节内游离体。
KOA按病因可分为原发性KOA和继发性KOA。原发性KOA是指用目前所有的检查方法查不出病因的骨关节炎,通常所指的骨关节炎属于这一类;继发性KOA是指在其它各种病因或疾病的基础上,诱发的病变,如创伤、类风湿关节炎、神经及内分泌疾病等。这一类骨关节炎的病变比较局限,不伴发赫伯登结节。反复使关节劳损的人群是患骨关节炎的高危人群,如铸造工人、矿工和公共汽车司机等。但是进行长跑锻炼的人却不是患本病的高危人群。肥胖是造成骨关节炎的主要因素,但目前证据尚不充分。按照部位可分为局灶性KOA和全身性KOA,按照是否有临床症状可分为无症状性KOA和症状性KOA。临床工作中遇到的KOA大多是症状性KOA。主要侵害关节软骨和滑膜组织,导致关节疼痛、畸形和功能障碍,从而影响患者的活动能力。正常情况下,关节之间摩擦力很小不会造成磨损,除非过度使用或损伤。造成骨关节炎的最可能原因是合成软骨成分的异常,如胶原(是一种坚韧的、结缔组织中的纤维蛋白)和粘蛋白(一种产生软骨弹性的物质)的异常。另外,软骨虽然生长旺盛,但是很薄,其表面很容易发生破裂。关节边缘的骨过度生长,形成可以看见和摸到的包块(称为骨赘)。骨赘引起关节面不平,干扰正常关节的功能,引起疼痛。
该病从关节软骨起病,影响整个关节结构,包括软骨下骨、韧带、滑膜、关节囊及关节外肌肉,最终因关节软骨全部脱失而导致关节畸形和功能丧失的一种疾病。
关节软骨是其发病机制重要因素之一,关节软骨是由1-2mm厚度胶原纤维、糖蛋白、透明质酸酯聚集而成,当水合作用时就起了垫子样作用,以吸收和分散所承受的负重和机械力量。在生理状况下,关节软骨依靠关节周围及热的收缩及软骨下的骨质来完全上述的任务。肌肉的收缩除带动关节活动外,同时起着橡皮带样作用,吸收了大量传来的冲力,保护了关节。当发生意外(如摔跤)时,因为肌肉对此突发的震动不能及时出现保护性反应而使关节负重加重,可致致害关节损伤,因此肌肉对此突突发的震动不能及时出现保护性反映而使关节负重加重,可致关节损伤。此外,肌肉老化、周围神经病变时,肌肉吸收能量的功能也大大的减弱。协助软骨承负重的另一因素是软骨下呈现网状分布的骨质量,其质地虽较软骨应但比骨皮质软,故具有高度弹性,有利于承受压力。可以看出骨性关节炎多出新在以下两种情况:一是关节软骨、软骨下皮质地、关节周围肌肉有异常时,如老年性退行性变、骨质疏松、炎症、代谢性疾病等:二是关节软骨、关节下骨质、关节周围肌肉虽正常但因承受了过度性压力,如肥胖、外伤等。
中医药治疗膝骨性关节炎有着丰富的临床经验与独特的优势。随着中医药治疗膝骨性关节炎实验研究的不断深入,对于中医药治疗膝关节炎作用机制的认识,已经从简单的组织形态学分析发展到免疫组织化学、分子生物学等多学科、多层次、多环节的认识。近年来,各种实验研究发现中医药对膝骨性关节炎的影响主要表现在膝骨性关节炎的组织形态学、细胞因子、骨内压、氧自由基等方面。
KOA的诊断通常是依靠临床表现和放射线检查。其简单的诊断依据是
1、症状:
①疼痛:运动时疼痛主要由机械性或肌腱、韧带接头病所致;休息时疼痛为炎症所致。夜间痛提示骨内压增高,为病情严重和预后不良指征。
②僵硬:见于关节不活动之后,但一般持续时间不长。
③其他症状如关节肿胀、畸形和关节弹响等。
2、体征:关节活动响声,骨肥大,不同程度的滑膜炎可致压痛,腱反射异常,肌无力等。严重病例可见功能障碍甚至残废。
3、观察病人行走、弯腰和进行日常活动的能力。
4、影象学检查:包括X线、CT和MRI检查。
5、其他检查:血液检查、关节液检查和关节镜检查等。
然而,在KOA的早期并不能通过放射线检查发现关节软骨的变化,因为在放射线检查能够发现软骨有改变之前,软骨一定在分子代谢和超微结构上发生了非常显著的变化。
在原发性KOA的临床研究和实验研究中虽然作出了很多努力,研究表明关节滑膜和关节软骨在原发性KOA发病中起主导作用,但是其发病机制仍然不十分明了,目前多采取手术和对症治疗为主。
研究目的
本项研究以关节软骨为研究对象,目的在于通过全基因组基因谱的表达,研究与本病相关的全部差异表达基因,从而进一步探讨软骨差异表达基因与本病的相关性,寻找治疗本病的新靶点和新方法。同时,对部分差异基因进行复孔的实时定量PCR检测,进一步验证所获得的差异基因在原发性KOA病理过程中的意义。
研究方法
材料
1、本研究共采集16例人膝关节软骨样本进行实验。根据纳入标准及排除标准,原发性KOA病人12例,实验用软骨全部在膝关节镜手术和人工膝关节置换术中获得。并根据Kellgren和Laurence放射学分级标准,疾病组将每4个病人的软骨样本分为一组,分为轻(R2)、中(R3)、重(R4)三组;将4例健康人的膝关节软骨样本为健康组(RB1)。
方法
1、对上述16例软骨样本分别进行全基因组芯片分析,在每张芯片上所拥有的40000个点上检测出16例样本所共同拥有的基因表达。
2、按照预先的分组,在共有基因范围内,对每个组内每个对应基因运算其均量,将每组样本转变为一个个体(R2、R3、R4、RB1四组),进行组问比较。
3、RNA样品制备
(1)两相分离
(2)RNA沉淀
(3)RNA清洗
(4)重新溶解RNA沉淀
(5)RNA质量检测
4、aRNA样品标记和合成
(1)cDNA合成
(2)cDNA第二链合成
(3)aRNA合成
(4)aRNA纯化
(5)aa-UTP aRNA间接标记
(6)质量控制
5、芯片预杂交
6、杂交
7、洗片
8、结果扫描,数据读取
9、合成的cDNA用于实时定量PCR
10、进行Realtime PCR反应
研究结果
1、通过本次实验,在16例样本共有基因范围内,鉴定出很多差异表达的基因,而具有统计学意义的差异基因共70个,其中:上调3倍以上的基因46个,其中已知基因28个,未知基因18个。已知基因中,上调4倍以上的基因25个,上调5倍以上的基因15个,上调6倍以上的基因11个,上调7倍以上的基因8个,上调8倍以上的基因3个:下调3倍以上的基因24个,其中已知基因16个,未知基因8个。已知基因中,下调4倍以上的基因11个,下调5倍以上的基因8个,下调6倍以上的基因6个,下调7倍以上的基因6个,下调8倍以上的基因2个。
2、上调3倍以上的差异基因包括:参与软骨分解代谢的基因及具有免疫功能的基因。
3、下调3倍以上的差异基因包括:参与软骨合成代谢的基因及具有信息传递功能的基因。
4、在70个差异表达基因中任意选定10个差异基因,又进行了复孔的PCR实时定量检测,通过三次的重复实验,三次的结果经统计学处理,P＜0.001,有显著差异。
5、R2与R3、R3与R4、R2与R4比较发现,上调或下调3倍以上差异基因在骨性关节炎病变过程中无明显差异,与病变轻重无关。
结论
1、原发性膝关节骨性关节炎的发生与关节软骨中的差异表达基因有相关性。
2、原发性膝关节骨性关节炎的病变程度与关节软骨中的差异表达基因无关。
Osteoarthritis of the knee(KOA) with age is a significant increase in the incidence of degenerative joint disease,joint pain and the elderly are the main reasons for disability. According to the World Health Organization statistics,osteoarthritis prevalence rate in women in the fourth.Increases with age,the prevalence rate rising rapidly,more than 65-year-old more than 50%of people have osteoarthritis of the performance of X-ray film.It's main change is the surface of articular cartilage degeneration and secondary hyperplasia of the bone.The main performance and activities of joint pain is not flexible, X-ray manifestations of joint space narrowing,subchondral bone density,bone fracture, there is sclerosis and cystic change.Joint edge lip hyperplasia.The latter end of the deformation of bone,articular surface uneven.Off articular cartilage,bone fragments into the joints,the formation of intra-articular loose bodies.
KOA etiology can be divided into primary and secondary KOA,primary KOA, refers to the current method of examination of all the bone unable to cause arthritis, osteoarthritis is usually referred to fall into this category;secondary KOA is the other cause or basis of disease,-induced lesions,such as trauma,rheumatoid arthritis, neurological and endocrine diseases.This type of lesions of osteoarthritis more limited, not associated with Heberden nodules.Repeated strain of the crowd so that the joints suffering from osteoarthritis is high-risk groups,such as foundry workers,miners and bus drivers.But for long-distance train are not suffering from this disease in high-risk groups.Obesity is a major factor in osteoarthritis,but the evidence is not sufficient.In accordance with the KOA site can be divided into focal and generalized KOA,in accordance with whether there are clinical symptoms KOA can be divided into asymptomatic and symptomatic KOA.Encountered in clinical work KOA are symptomatic KOA.Mainly against articular cartilage,bone and synovial tissues,leading to joint pain,deformity and dysfunction,thus affecting the activities of patients.Under normal circumstances,very little friction between the joints will not cause wear and tear, unless the excessive use of or injury.Caused by osteoarthritis is the most likely cause of the abnormal synthesis of cartilage components,such as collagen(a tough,fibrous connective tissue protein) and mucin(a substance produced flexible cartilage) anomalies In addition,the growth of cartilage while strong,but thin,its surface is prone to rupture. Joints over the edge of the bone growth,can see and touch the formation of the mass (known as osteophyte).Osteopbyte caused by uneven articular surface,interfere with the normal function of the joints,causing pain.
Articular cartilage from disease onset,affects the entire joint structure,including the subchondral bone,ligaments,synovial membrane,joint capsule and muscles,the ultimate result of all the loss of articular cartilage leading to joint deformity and loss of function of a disease.
Articular cartilage is an important factor in the pathogenesis of the articular cartilage is a 1-2mm thickness of collagen fibers,glycoproteins,hyaluronic acid ester gathered together,when the hydration on a mat-like role in the absorption and dispersion the burden of weight-bearing and mechanical strength.In physiological conditions,relying on the articular cartilage and thermal contraction of the surrounding subchondral bone and to complete the above tasks.In addition to stimulate muscle contraction joint activities,at the same time plays the role of rubber band-like, absorbing a great deal of momentum from the protection of the joints.When an accident occurs(eg,wrestling),because this sudden shock the muscles can not react in time of emergence of protective weight-bearing joints increased virulence may be caused by joint injury,so this sudden burst of muscle vibration protection can not be there in time to reflect heavier load of the joints can be caused by joint injury.In addition,muscle aging,peripheral neuropathy,the muscle function of energy absorption also decreased significantly.Weight-bearing cartilage for the assistance of another factor is the network showing the distribution of subchondral bone quality,its texture,although more cartilage than bone cortex should be soft,it has a high degree of flexibility will help to bear the pressure.Osteoarthritis can be seen more new in the following two situations: First,articular cartilage,subchondral cortex and abnormal muscle around the joints, such as age-related degeneration,osteoporosis,inflammation,metabolic diseases,such as:The second is the articular cartilage of the joints under the bone,the muscles around the joints,although subject to normal due to excessive pressure,such as obesity,trauma, etc.
Chinese medicine treatment of knee osteoarthritis has a wealth of clinical experience and unique strengths.With the traditional Chinese medicine treatment of knee osteoarthritis Experimental study on the deepening of Chinese medicine for the treatment of knee arthritis awareness of the role of the mechanism,the organization has changed from simple morphological analysis of the development of immunohistochemistry,molecular biology,such as multi-disciplinary,multi-level, multi-session awareness.In recent years,a variety of experimental study found that traditional Chinese medicine for knee osteoarthritis mainly reflected the impact of knee osteoarthritis in the organization of morphology,cell factor,bone pressure,oxygen free radicals and so on.
KOA diagnosis usually relies on clinical manifestations and radiological examination.Its diagnosis is based on simple.
1.Symptoms:
①pain:pain during exercise or by mechanical tendon,ligament caused by joint disease;rest for the pain caused by inflammation.Night pain prompted increased intraosseous pressure,in order to be in a serious condition and poor prognosis indications.
②Rigid:in the joint after inactivity,it is generally not a long duration.③Other symptoms such as joint swelling,deformity and joint ring,such as shells.
2.Signs:the sound of joint,bone hypertrophy,various degrees of synovitis can be caused by tenderness,abnormal tendon reflexes,such as myasthenia gravis.Serious cases of dysfunction or disability can be seen.
3.To observe the patient walking,bending and the ability to carry out daily activities.
4.Imaging examination:including X-ray,CT and MRI examination.
5.Other tests:blood test,examination and synovial fluid,such as arthroscopy.
However,in the early and could not KOA radiological examination revealed the changes in articular cartilage,as can be found in the radiographic changes of cartilage prior to a certain cartilage metabolism and ultrastructure in the elements have taken place in very significant changes.
KOA in primary clinical research and experimental studies although many efforts have been made,the study shows that articular cartilage and synovium in the pathogenesis of primary KOA play a leading role,but its pathogenesis is still not very clear,the current operations and take more symptomatic therapy.
Objective
Articular cartilage in the study subjects for the purpose of the adoption of whole-genome gene expression,research and all the disease-related differences in gene expression,thus further explore differentially expressed genes in the cartilage and the relevance of this disease,for treatment of this disease new target and new methods.At the same time,some differences in gene-hole complex real-time quantitative PCR,to further verify the differences in access to primary KOA gene in pathological significance in the process.
Methods
Material
According to addition and exclusion standards,12 patients with OA were performed,and OA cartilage of the knee joint of humans was obtained at the time of arthroscopic surgery and total knee replacement.According to Kellgren and Laurence radiology classification standards 4 patients became a group,then mild(R2) and moderate(R3) and severe late-stage OA groups(R4) appeared.In addition,4 normal cartilage of the knee joint samples became RBl group.
Methods
1.All the 16 samples were performed the genome-wide analysis of chips,each chip has 40,000 point samples detected 16 cases of co-owned by the gene expression.
2.According to the foregoing groups,the genes within each group were corresponding calculated the mean,each group was considered as a entirety (R2,R3,R4,RBl four groups),comparing among the four groups.
3.RNA sample preparation
(1) two-dimensional separation
(2) PNA praecipitatum
(3) RNA emundation
(4) again dissolve RNA praecipitatum
(5) RNA mass detection
4.aRNA sample symbol and synthesis
(1) cDNA synthesis
(2) second-strand of cDNA synthesis
(3) aRNA synthesis
(4) aRNA depuration
(5) aa-UTP aRNA indirect labelling
(6) quality control
5.Chip prehybridization
6.Hybridisation
7.Chip emundation
8.Result scanning,data reading
9.Synthetical cDNA were used to real time quantitative polymerase chain reaction
10.Performing real time quantitative polymerase chain reaction Results
1.In this experiment,among the 41000 genes,many differential expression genes were identified,and 70 genes showed statistical significance,of which: 46 genes were up-regulation 3-fold,thereinto 28 known genes and 18 unknown genes,in known genes,25 genes were up-regulation 4-fold,15 genes were up-regulation 5-fold,11 genes were up-regulation 6-fold, 8 genes were up-regulation 7-fold,3 genes were up-regulation 8-fold;24 genes were down-regulation 3-fold,thereinto 16 known genes and 8 unknown genes,in known genes,11 genes were down-regulation 4-fold,8 genes were down-regulation 5-fold,6 genes were down-regulation 6-fold, 6 genes were down-regulation 7-fold,2 genes were down-regulation 8-fold.
2.More than up-regulation 3-fold of difference genes include:participating gene of cartilage's catabolism and gene with immunologic function.
3.More than down-regulation 3-fold of difference genes include: participating gene of cartilage's anabolism and gene with information transfer function.
4.In 70 differentially expressed genes in any differences in the selected 10 genes Were optional selected to perform real time quantitative polymerase chain reaction,through three times repetitive experiments,we miraculously observed the same results of the experiments.Through the statistical treatment, P values less than 0.01,There are significant differences.
5、During R2 and R3,R3 and R4,R2 and R4 comparison,up-regulation or down-regulation than 3-fold difference in gene expression in the course of osteoarthritis lesions in no significant difference has nothing to do with disease severity.
Conclusion
1.KOA's occurance is relate to differentially expressed genes of articular cartilage
2.KOA's pathological changes is irrelevance with differentially expressed genes of articular cartilage
KOA按病因可分为原发性KOA和继发性KOA。原发性KOA是指用目前所有的检查方法查不出病因的骨关节炎,通常所指的骨关节炎属于这一类;继发性KOA是指在其它各种病因或疾病的基础上,诱发的病变,如创伤、类风湿关节炎、神经及内分泌疾病等。这一类骨关节炎的病变比较局限,不伴发赫伯登结节。反复使关节劳损的人群是患骨关节炎的高危人群,如铸造工人、矿工和公共汽车司机等。但是进行长跑锻炼的人却不是患本病的高危人群。肥胖是造成骨关节炎的主要因素,但目前证据尚不充分。按照部位可分为局灶性KOA和全身性KOA,按照是否有临床症状可分为无症状性KOA和症状性KOA。临床工作中遇到的KOA大多是症状性KOA。主要侵害关节软骨和滑膜组织,导致关节疼痛、畸形和功能障碍,从而影响患者的活动能力。正常情况下,关节之间摩擦力很小不会造成磨损,除非过度使用或损伤。造成骨关节炎的最可能原因是合成软骨成分的异常,如胶原(是一种坚韧的、结缔组织中的纤维蛋白)和粘蛋白(一种产生软骨弹性的物质)的异常。另外,软骨虽然生长旺盛,但是很薄,其表面很容易发生破裂。关节边缘的骨过度生长,形成可以看见和摸到的包块(称为骨赘)。骨赘引起关节面不平,干扰正常关节的功能,引起疼痛。
该病从关节软骨起病,影响整个关节结构,包括软骨下骨、韧带、滑膜、关节囊及关节外肌肉,最终因关节软骨全部脱失而导致关节畸形和功能丧失的一种疾病。
关节软骨是其发病机制重要因素之一,关节软骨是由1-2mm厚度胶原纤维、糖蛋白、透明质酸酯聚集而成,当水合作用时就起了垫子样作用,以吸收和分散所承受的负重和机械力量。在生理状况下,关节软骨依靠关节周围及热的收缩及软骨下的骨质来完全上述的任务。肌肉的收缩除带动关节活动外,同时起着橡皮带样作用,吸收了大量传来的冲力,保护了关节。当发生意外(如摔跤)时,因为肌肉对此突发的震动不能及时出现保护性反应而使关节负重加重,可致致害关节损伤,因此肌肉对此突突发的震动不能及时出现保护性反映而使关节负重加重,可致关节损伤。此外,肌肉老化、周围神经病变时,肌肉吸收能量的功能也大大的减弱。协助软骨承负重的另一因素是软骨下呈现网状分布的骨质量,其质地虽较软骨应但比骨皮质软,故具有高度弹性,有利于承受压力。可以看出骨性关节炎多出新在以下两种情况:一是关节软骨、软骨下皮质地、关节周围肌肉有异常时,如老年性退行性变、骨质疏松、炎症、代谢性疾病等:二是关节软骨、关节下骨质、关节周围肌肉虽正常但因承受了过度性压力,如肥胖、外伤等。
中医药治疗膝骨性关节炎有着丰富的临床经验与独特的优势。随着中医药治疗膝骨性关节炎实验研究的不断深入,对于中医药治疗膝关节炎作用机制的认识,已经从简单的组织形态学分析发展到免疫组织化学、分子生物学等多学科、多层次、多环节的认识。近年来,各种实验研究发现中医药对膝骨性关节炎的影响主要表现在膝骨性关节炎的组织形态学、细胞因子、骨内压、氧自由基等方面。
KOA的诊断通常是依靠临床表现和放射线检查。其简单的诊断依据是
1、症状:
①疼痛:运动时疼痛主要由机械性或肌腱、韧带接头病所致;休息时疼痛为炎症所致。夜间痛提示骨内压增高,为病情严重和预后不良指征。
②僵硬:见于关节不活动之后,但一般持续时间不长。
③其他症状如关节肿胀、畸形和关节弹响等。
2、体征:关节活动响声,骨肥大,不同程度的滑膜炎可致压痛,腱反射异常,肌无力等。严重病例可见功能障碍甚至残废。
3、观察病人行走、弯腰和进行日常活动的能力。
4、影象学检查:包括X线、CT和MRI检查。
5、其他检查:血液检查、关节液检查和关节镜检查等。
然而,在KOA的早期并不能通过放射线检查发现关节软骨的变化,因为在放射线检查能够发现软骨有改变之前,软骨一定在分子代谢和超微结构上发生了非常显著的变化。
在原发性KOA的临床研究和实验研究中虽然作出了很多努力,研究表明关节滑膜和关节软骨在原发性KOA发病中起主导作用,但是其发病机制仍然不十分明了,目前多采取手术和对症治疗为主。
研究目的
本项研究以关节软骨为研究对象,目的在于通过全基因组基因谱的表达,研究与本病相关的全部差异表达基因,从而进一步探讨软骨差异表达基因与本病的相关性,寻找治疗本病的新靶点和新方法。同时,对部分差异基因进行复孔的实时定量PCR检测,进一步验证所获得的差异基因在原发性KOA病理过程中的意义。
研究方法
材料
1、本研究共采集16例人膝关节软骨样本进行实验。根据纳入标准及排除标准,原发性KOA病人12例,实验用软骨全部在膝关节镜手术和人工膝关节置换术中获得。并根据Kellgren和Laurence放射学分级标准,疾病组将每4个病人的软骨样本分为一组,分为轻(R2)、中(R3)、重(R4)三组;将4例健康人的膝关节软骨样本为健康组(RB1)。
方法
1、对上述16例软骨样本分别进行全基因组芯片分析,在每张芯片上所拥有的40000个点上检测出16例样本所共同拥有的基因表达。
2、按照预先的分组,在共有基因范围内,对每个组内每个对应基因运算其均量,将每组样本转变为一个个体(R2、R3、R4、RB1四组),进行组问比较。
3、RNA样品制备
(1)两相分离
(2)RNA沉淀
(3)RNA清洗
(4)重新溶解RNA沉淀
(5)RNA质量检测
4、aRNA样品标记和合成
(1)cDNA合成
(2)cDNA第二链合成
(3)aRNA合成
(4)aRNA纯化
(5)aa-UTP aRNA间接标记
(6)质量控制
5、芯片预杂交
6、杂交
7、洗片
8、结果扫描,数据读取
9、合成的cDNA用于实时定量PCR
10、进行Realtime PCR反应
研究结果
1、通过本次实验,在16例样本共有基因范围内,鉴定出很多差异表达的基因,而具有统计学意义的差异基因共70个,其中:上调3倍以上的基因46个,其中已知基因28个,未知基因18个。已知基因中,上调4倍以上的基因25个,上调5倍以上的基因15个,上调6倍以上的基因11个,上调7倍以上的基因8个,上调8倍以上的基因3个:下调3倍以上的基因24个,其中已知基因16个,未知基因8个。已知基因中,下调4倍以上的基因11个,下调5倍以上的基因8个,下调6倍以上的基因6个,下调7倍以上的基因6个,下调8倍以上的基因2个。
2、上调3倍以上的差异基因包括:参与软骨分解代谢的基因及具有免疫功能的基因。
3、下调3倍以上的差异基因包括:参与软骨合成代谢的基因及具有信息传递功能的基因。
4、在70个差异表达基因中任意选定10个差异基因,又进行了复孔的PCR实时定量检测,通过三次的重复实验,三次的结果经统计学处理,P＜0.001,有显著差异。
5、R2与R3、R3与R4、R2与R4比较发现,上调或下调3倍以上差异基因在骨性关节炎病变过程中无明显差异,与病变轻重无关。
结论
1、原发性膝关节骨性关节炎的发生与关节软骨中的差异表达基因有相关性。
2、原发性膝关节骨性关节炎的病变程度与关节软骨中的差异表达基因无关。
Osteoarthritis of the knee(KOA) with age is a significant increase in the incidence of degenerative joint disease,joint pain and the elderly are the main reasons for disability. According to the World Health Organization statistics,osteoarthritis prevalence rate in women in the fourth.Increases with age,the prevalence rate rising rapidly,more than 65-year-old more than 50%of people have osteoarthritis of the performance of X-ray film.It's main change is the surface of articular cartilage degeneration and secondary hyperplasia of the bone.The main performance and activities of joint pain is not flexible, X-ray manifestations of joint space narrowing,subchondral bone density,bone fracture, there is sclerosis and cystic change.Joint edge lip hyperplasia.The latter end of the deformation of bone,articular surface uneven.Off articular cartilage,bone fragments into the joints,the formation of intra-articular loose bodies.
KOA etiology can be divided into primary and secondary KOA,primary KOA, refers to the current method of examination of all the bone unable to cause arthritis, osteoarthritis is usually referred to fall into this category;secondary KOA is the other cause or basis of disease,-induced lesions,such as trauma,rheumatoid arthritis, neurological and endocrine diseases.This type of lesions of osteoarthritis more limited, not associated with Heberden nodules.Repeated strain of the crowd so that the joints suffering from osteoarthritis is high-risk groups,such as foundry workers,miners and bus drivers.But for long-distance train are not suffering from this disease in high-risk groups.Obesity is a major factor in osteoarthritis,but the evidence is not sufficient.In accordance with the KOA site can be divided into focal and generalized KOA,in accordance with whether there are clinical symptoms KOA can be divided into asymptomatic and symptomatic KOA.Encountered in clinical work KOA are symptomatic KOA.Mainly against articular cartilage,bone and synovial tissues,leading to joint pain,deformity and dysfunction,thus affecting the activities of patients.Under normal circumstances,very little friction between the joints will not cause wear and tear, unless the excessive use of or injury.Caused by osteoarthritis is the most likely cause of the abnormal synthesis of cartilage components,such as collagen(a tough,fibrous connective tissue protein) and mucin(a substance produced flexible cartilage) anomalies In addition,the growth of cartilage while strong,but thin,its surface is prone to rupture. Joints over the edge of the bone growth,can see and touch the formation of the mass (known as osteophyte).Osteopbyte caused by uneven articular surface,interfere with the normal function of the joints,causing pain.
Articular cartilage from disease onset,affects the entire joint structure,including the subchondral bone,ligaments,synovial membrane,joint capsule and muscles,the ultimate result of all the loss of articular cartilage leading to joint deformity and loss of function of a disease.
Articular cartilage is an important factor in the pathogenesis of the articular cartilage is a 1-2mm thickness of collagen fibers,glycoproteins,hyaluronic acid ester gathered together,when the hydration on a mat-like role in the absorption and dispersion the burden of weight-bearing and mechanical strength.In physiological conditions,relying on the articular cartilage and thermal contraction of the surrounding subchondral bone and to complete the above tasks.In addition to stimulate muscle contraction joint activities,at the same time plays the role of rubber band-like, absorbing a great deal of momentum from the protection of the joints.When an accident occurs(eg,wrestling),because this sudden shock the muscles can not react in time of emergence of protective weight-bearing joints increased virulence may be caused by joint injury,so this sudden burst of muscle vibration protection can not be there in time to reflect heavier load of the joints can be caused by joint injury.In addition,muscle aging,peripheral neuropathy,the muscle function of energy absorption also decreased significantly.Weight-bearing cartilage for the assistance of another factor is the network showing the distribution of subchondral bone quality,its texture,although more cartilage than bone cortex should be soft,it has a high degree of flexibility will help to bear the pressure.Osteoarthritis can be seen more new in the following two situations: First,articular cartilage,subchondral cortex and abnormal muscle around the joints, such as age-related degeneration,osteoporosis,inflammation,metabolic diseases,such as:The second is the articular cartilage of the joints under the bone,the muscles around the joints,although subject to normal due to excessive pressure,such as obesity,trauma, etc.
Chinese medicine treatment of knee osteoarthritis has a wealth of clinical experience and unique strengths.With the traditional Chinese medicine treatment of knee osteoarthritis Experimental study on the deepening of Chinese medicine for the treatment of knee arthritis awareness of the role of the mechanism,the organization has changed from simple morphological analysis of the development of immunohistochemistry,molecular biology,such as multi-disciplinary,multi-level, multi-session awareness.In recent years,a variety of experimental study found that traditional Chinese medicine for knee osteoarthritis mainly reflected the impact of knee osteoarthritis in the organization of morphology,cell factor,bone pressure,oxygen free radicals and so on.
KOA diagnosis usually relies on clinical manifestations and radiological examination.Its diagnosis is based on simple.
1.Symptoms:
①pain:pain during exercise or by mechanical tendon,ligament caused by joint disease;rest for the pain caused by inflammation.Night pain prompted increased intraosseous pressure,in order to be in a serious condition and poor prognosis indications.
②Rigid:in the joint after inactivity,it is generally not a long duration.③Other symptoms such as joint swelling,deformity and joint ring,such as shells.
2.Signs:the sound of joint,bone hypertrophy,various degrees of synovitis can be caused by tenderness,abnormal tendon reflexes,such as myasthenia gravis.Serious cases of dysfunction or disability can be seen.
3.To observe the patient walking,bending and the ability to carry out daily activities.
4.Imaging examination:including X-ray,CT and MRI examination.
5.Other tests:blood test,examination and synovial fluid,such as arthroscopy.
However,in the early and could not KOA radiological examination revealed the changes in articular cartilage,as can be found in the radiographic changes of cartilage prior to a certain cartilage metabolism and ultrastructure in the elements have taken place in very significant changes.
KOA in primary clinical research and experimental studies although many efforts have been made,the study shows that articular cartilage and synovium in the pathogenesis of primary KOA play a leading role,but its pathogenesis is still not very clear,the current operations and take more symptomatic therapy.
Objective
Articular cartilage in the study subjects for the purpose of the adoption of whole-genome gene expression,research and all the disease-related differences in gene expression,thus further explore differentially expressed genes in the cartilage and the relevance of this disease,for treatment of this disease new target and new methods.At the same time,some differences in gene-hole complex real-time quantitative PCR,to further verify the differences in access to primary KOA gene in pathological significance in the process.
Methods
Material
According to addition and exclusion standards,12 patients with OA were performed,and OA cartilage of the knee joint of humans was obtained at the time of arthroscopic surgery and total knee replacement.According to Kellgren and Laurence radiology classification standards 4 patients became a group,then mild(R2) and moderate(R3) and severe late-stage OA groups(R4) appeared.In addition,4 normal cartilage of the knee joint samples became RBl group.
Methods
1.All the 16 samples were performed the genome-wide analysis of chips,each chip has 40,000 point samples detected 16 cases of co-owned by the gene expression.
2.According to the foregoing groups,the genes within each group were corresponding calculated the mean,each group was considered as a entirety (R2,R3,R4,RBl four groups),comparing among the four groups.
3.RNA sample preparation
(1) two-dimensional separation
(2) PNA praecipitatum
(3) RNA emundation
(4) again dissolve RNA praecipitatum
(5) RNA mass detection
4.aRNA sample symbol and synthesis
(1) cDNA synthesis
(2) second-strand of cDNA synthesis
(3) aRNA synthesis
(4) aRNA depuration
(5) aa-UTP aRNA indirect labelling
(6) quality control
5.Chip prehybridization
6.Hybridisation
7.Chip emundation
8.Result scanning,data reading
9.Synthetical cDNA were used to real time quantitative polymerase chain reaction
10.Performing real time quantitative polymerase chain reaction Results
1.In this experiment,among the 41000 genes,many differential expression genes were identified,and 70 genes showed statistical significance,of which: 46 genes were up-regulation 3-fold,thereinto 28 known genes and 18 unknown genes,in known genes,25 genes were up-regulation 4-fold,15 genes were up-regulation 5-fold,11 genes were up-regulation 6-fold, 8 genes were up-regulation 7-fold,3 genes were up-regulation 8-fold;24 genes were down-regulation 3-fold,thereinto 16 known genes and 8 unknown genes,in known genes,11 genes were down-regulation 4-fold,8 genes were down-regulation 5-fold,6 genes were down-regulation 6-fold, 6 genes were down-regulation 7-fold,2 genes were down-regulation 8-fold.
2.More than up-regulation 3-fold of difference genes include:participating gene of cartilage's catabolism and gene with immunologic function.
3.More than down-regulation 3-fold of difference genes include: participating gene of cartilage's anabolism and gene with information transfer function.
4.In 70 differentially expressed genes in any differences in the selected 10 genes Were optional selected to perform real time quantitative polymerase chain reaction,through three times repetitive experiments,we miraculously observed the same results of the experiments.Through the statistical treatment, P values less than 0.01,There are significant differences.
5、During R2 and R3,R3 and R4,R2 and R4 comparison,up-regulation or down-regulation than 3-fold difference in gene expression in the course of osteoarthritis lesions in no significant difference has nothing to do with disease severity.
Conclusion
1.KOA's occurance is relate to differentially expressed genes of articular cartilage
2.KOA's pathological changes is irrelevance with differentially expressed genes of articular cartilage
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