单味中药骨碎补对兔膝骨关节炎软骨细胞凋亡作用的实验研究
摘要
目的:
骨关节炎(osteoarthritis,OA),是由于多种因素引起的多发于老年人的慢性退行性关节疾病,60岁以上的人群中,50%人群在X线上有骨性关节炎表现,其中35%-50%有临床表现;75岁以上的人群中,80%有骨性关节炎症状。随着人口老龄化的进展,骨关节炎的发病率逐年上升,发病年龄也有下降趋势,有资料报道45岁以上人群骨关节炎的发病率超过40%。随着我国步入老龄化人口社会,骨关节炎正日渐成为医患共同关注的焦点,为此世界卫生组织(WHO)将10月12日定为“世界骨关节炎日”,21世纪头十年为“骨关节病十年”。骨性关节炎是膝关节炎症中最常见的疾病。膝骨性关节炎在老年人群中最为常见,尤其是在肥胖的老年人群。男女均可发病,女性多于男性。临床观察证实,膝骨性关节炎患者已普遍扩展到35岁以上人群,严重困扰着人们的日常生活与劳动。
骨性关节炎是一种以关节软骨变性和丢失及关节边缘和软骨下骨骨质再生为特征的慢性关节炎疾病。该病的始发部位在软骨。骨关节炎的病因和发病机制尚不完全清楚,一般认为既有年龄、性别、家族易感性等全身因素的参与,又有局部生物力学、软骨细胞凋亡、细胞因子和降解酶等的作用,是多因素、多环节导致的疾病。有文献报道,软骨细胞凋亡可能是OA发病机制中的重要环节。目前,软骨细胞凋亡信号的诱导途径(Fax、NO),调控软骨细胞凋亡的信号因子(Bcl-2家族,caspases蛋白酶),软骨细胞凋亡的刺激信号等指标是实验研究中的热点。大量研究也证实,OA病程中白介素1(IL-1)、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶系(MMPs)[尤其是MMP-1、MMP-3、MMP-13和金属蛋白酶组织抑制剂-1(TIMP-1)]等显著变化。通过这些指标的测定可以间接了解OA的演进过程,推测它们在OA发病机制中对软骨细胞凋亡所起的作用,还有是用于评价OA干预措施的作用靶点与机理。
骨关节炎在中医学中属于“痹病”,“骨痹”范畴,肝肾不足,筋骨失养是重要发病机制。骨碎补性味苦、温,活血续伤,补肾强骨,为骨伤科要药,具有一定的改善软骨细胞功能,推迟软骨细胞退行性变的作用。
本实验主要利用细胞、分子生物学技术,采用反转录聚合酶链反应(RT-PCR)、蛋白印迹(Western-blot)等检测方法,通过软骨细胞凋亡相关因子的不同表达,进一步探讨OA发病机制,研究骨碎补作用机理,观察其对实验性兔膝骨关节炎软骨细胞凋亡的影响,并进行了理论分析,为中医药防治膝骨性关节炎的实验与临床提供理论与实际应用的依据。
材料与方法:
1、实验动物
成年新西兰大耳白兔24只,普通级,雌雄不分,重量2-3kg。辽宁中医药大学动物实验中心提供。
2、实验药物
2.1骨碎补辽宁中医药大学附属医院药剂科提供。水煎浓缩成含生药浓度1g/ml的药液备用,共计630ml。
2.2维固力爱尔兰Rottapharm Ltd,批号:H20040637,规格:0.25g(以硫酸氨基葡萄糖计),辽宁中医药大学附属医院药剂科提供。
3、实验主要试剂
(1) Western Blot检测用Ⅰ抗抗体:鼠抗兔单克隆抗体IL-1、羊抗兔TNF-α、鼠抗兔MMP3、鼠抗兔Bcl-2、羊抗兔BAX、鼠抗兔Caspase-3、羊抗兔β-actin(美国santa cruzbiotechnology,~(?)inc.)。Ⅱ抗抗体:碱性磷酸酶标记兔抗鼠、兔抗羊抗体(北京中山生物技术有限公司)。
(2)内源性过氧化物酶阻断剂:3%过氧化氢溶液
(3)封闭用正常小牛血清白蛋白工作液
(4)碱性磷酸酶标记的Ⅱ抗工作液
(5)浓缩PBS缓冲溶液
(6) 3,3-氨基联苯胺四盐酸盐(3,3-Diaminobenxidine tetrahydroehloride,DAB)
(2)-(6)福州迈新生物技术开发公司
(7)总RNA提取试剂异硫氰酸胍(Triozol Reagent):美国Invitrogen Lifetechnologies公司
(8)反转录酶(AMV)
(9) RNA酶抑制剂
(10) dNTP混合液
(11) Oligo(dT)15
(12)γ-Taq DNA聚合酶
(13)目的基因引物
(8)-(13)日本TaKaRa公司
(14)琼脂糖:美国Promaga公司
(15)考马斯亮蓝蛋白定量试剂盒:南京建成生物工程公司
(16)硝酸纤维素膜:购自Millopore公司
(17)其他试剂:6×凝胶上样缓冲液、DNA电泳缓冲液(TBE)、十二烷基磺酸钠(SDS)、三羟甲基氨基甲烷(Tris)、丙烯酰胺(Acr)、3%戊二醛溶液、甲醇、显影液、定影液、丙酮、二甲苯、无水乙醇、甲醛、脱脂奶粉等购于华美公司。
4、实验主要仪器与设备
(1) OLYMPUS显微镜(日本,CH)
(2)电热恒温水箱(河北省黄骅航天仪器厂,HH.W21.600型)
(3)切片机(德国Leitz,Kryostat 1720)
(4)制冰机(德国ZIGERA,2BE-70-25)
(5)小型台式离心机(美国SIGMA,1-13)
(6) PCR扩增仪(德国Biometra)
(7)紫外分光光度计(英国UV-visible Spectrometer,UV300)
(8)电泳仪(美国BIO-RAD,PowerPac200)
(9)半干转印仪(美国BIO-RAD Semidry Transfer System)
(10)水平摇床(美国GFL)
(11)垂直板电泳装置(美国BIO-RAD,Mini-ProteinⅢ)
(12) Chemi Imager5500凝胶电泳成像分析系统(美国Alphainnotech chemi Imager)
(13) HITACHI-7500型透射电镜(日本HITACHI公司)
5、兔膝OA的建立
兔左后肢伸直位管型石膏固定6周得到兔膝OA的模型。
6、动物分组及给药
新西兰大耳白兔24只,随机分为4组。
空白组6只,不造模,不用药,正常饲料喂养。
模型组6只,造模后不用药,正常饲料喂养。
治疗组(骨碎补组)6只,造模后灌胃,1次/天,用药4周。
对照组(维固力组)6只,造模后灌胃,1次/天,用药4周。
7、动物处死及取材
用药4周后,以耳缘静脉空气栓塞法处死各组动物。
外科操作取下每只家兔左后肢股骨髁(约1cm)和胫骨近端(约1cm)部分,精细分离关节软骨。
每只兔取下的关节软骨分成三部分。胫骨平台部分取一小块,用3%戊二醛溶液固定,4℃冷藏,用于电镜检测。股骨髁的一部分用Trizol液浸泡,4℃冷藏,用于RT-PCR检测;另一部分空置于塑料试管内,封盖,-80℃冷冻,用于Western blot检测。
8、电镜观察
在4℃条件下,置于3%戊二醛固定液中浸泡24小时后,环氧丙烷透明液脱钙,用0.1M磷酸缓冲液冲洗3次,再加0.1%四氧化锇固定60min,去离子水冲洗3次,终止锇酸反应,不同浓度乙醇逐级脱水,以Epon812环氧树脂浸透、包埋,硬化后修块半薄切片,定位后超薄切片,切片厚60nm,醋酸双氧铀及柠檬酸铅双重染色透射电镜观察并摄片。
9、RT-PCR检测
(1) TRIzol法抽提总RNA
(2) RNA浓度和纯度测定
(3)逆转录合成cDNA
(4) PCR扩增:应用Biometra PCR扩增仪
(5) RT-PCR产物电泳及分析定量
10、Western-blot检测
(1)蛋白的提取
(2)蛋白浓度的测定
(3)蛋白变性
(4)配胶
(5)电泳
(6)转印
(7)封闭
(8)一抗孵育
(9)二抗孵育
(10)清洗
(11)显色
(12)Western Blot结果量化分析:采用FlourChem V 2.0凝胶成像分析软件(America)分析,记录每条蛋白电泳带的灰度值,进行定量分析。
11、数据处理及统计
采用SPSS1 3.0软件包对RT-PCR和Western Blot结果进行方差分析和Pearson相关分析,数据以均值±标准差表示。P＜0.05有统计学意义。
结果:
1、大体形态观察
空白组关节软骨表面光滑,略呈淡蓝色。模型组软骨表面呈淡白色,仔细观察可见有散在小的点状和条形凹陷。治疗组和对照组软骨表面也呈淡白色或淡黄色,表面尚光滑,但均可见有散在的点状凹陷。
2、电镜下超微结构的变化
电镜下观察软骨表面的超微结构空白组表面较为平整,可见有匀称的细小微孔,覆盖有点状分布的白色圆球形凝胶样物质;模型组表面粗糙,凹凸不平,局部出现龟裂样改变,严重者表层脱失,其下的胶原纤维裸露,或者软骨表面出现裂隙,胶原纤维断裂;治疗组病变相对较轻,表面尚平整,表面覆盖有较大的圆形凝胶样物质,可见散在鳞屑样改变,局部有裂隙和小孔形成;对照组软骨表面欠平整,可见较多的裂隙和小孔和大小不一的浅溃疡形成,局部表层撕裂剥脱,呈片状卷起,露出其下的胶原纤维。
软骨细胞镜下观察显示,空白组细胞结构清楚,细胞膜完整,轮廓清晰,核内染色质均匀分布。细胞外可见纵横交错的胶原纤维。模型组细胞膜溶解,核膜不清,胞质消失,可见细胞肿胀,坏死明显增多。细胞周围的胶原纤维减少,排列紊乱,有的彼此聚集成团状或捆状,并出现交错现象,有的局部稀疏。治疗组核固缩,线粒体溶解,内质网肿胀;对照组核浓缩,核膜不清,线粒体溶解扩张。治疗组与对照组细胞周围胶原纤维排列欠规则。
3、RT-PCR检测结果
模型组较空白组的TNF-αmRNA、MMP3mRNA、caspase-3mRNA、BAXmRNA表达水平明显上升,差异有显著性(P＜0.05);对照组较模型组的TNF-αmRNA、MMP3mRNA、caspase-3mRNA、BAXmRNA表达水平有所下降,差异有显著性(P＜0.05);治疗组较模型组的TNF-αmRNA、MMP3mRNA、caspase-3mRNA、BAXmRNA表达水平明显下降,差异有显著性(P＜0.05);治疗组较对照组的TNF-αmRNA、MMP3mRNA、caspase-3mRNA、BAXmRNA表达差异无显著性。
模型组较空白组的bcl-2mRNA表达水平明显下降,差异有显著性(P＜0.05);对照组较模型组的bcl-2mRNA表达水平有所上升,差异有显著性(P＜0.05);治疗组较模型组的bcl-2mRNA表达水平明显上升,差异有显著性(P＜0.05):治疗组较对照组的bcl-2mRNA表达差异无显著性。
4、Western blot检测结果
模型组较空白组的IL-1、TNF-α、MMP3、caspase-3、BAX蛋白表达水平明显上升,差异有显著性(P＜0.05);对照组较模型组的IL-1、TNF-α、MMP3、caspase-3、BAX蛋白表达水平有所下降,差异有显著性(P＜0.05);治疗组较模型组的IL-1、TNF-α、MMP3、caspase-3、BAX蛋白表达水平明显下降,差异有显著性(P＜0.05);治疗组较对照组的IL-1、TNF-α、MMP3、caspase-3、BAX蛋白表达差异无显著性。
模型组较空白组的bcl-2蛋白表达水平明显下降,差异有显著性(P＜0.05);对照组较模型组的bcl-2蛋白表达水平有所上升,差异有显著性(P＜0.05);治疗组较模型组的bcl-2蛋白表达水平明显上升,差异有显著性(P＜0.05);治疗组较对照组的bcl-2蛋白表达差异无显著性。
结论:
1 IL-1、MMP3、TNF-α、caspase-3、BAX、bcl-2等因子与骨关节炎病变过程密切相关。
2本研究证实了在OA关节软骨中存在IL-1、MMP3、TNF-α、caspase-3、BAXmRNA和蛋白表达增强,bcl-2mRNA和蛋白表达减弱,可能是OA发病机制的一部分。因此,应用各种方法,抑制IL-1、MMP3、TNF-α、caspase-3、BAX的表达,增强bcl-2表达及其生物效应,有可能成为治疗OA的有效途径,具有重要的意义。
3通过RT-PCR、Western-blot检测,单味中药骨碎补较好的表达出对IL-1、MMP3、TNF-α、caspase-3、BAX、bcl-2等因子的调控作用,提示其应用对防治OA有重要作用,有必要进一步开展相关临床与基础方面的研究。
4西药维固力(硫酸氨基葡萄糖)与单味中药骨碎补同样表现出对软骨细胞凋亡相关因子的抑制或增强作用,且本组数据统计学显示无差异性,但在mRNA与蛋白表达上看到了明显的强弱差别,可以认为单味中药骨碎补防治OA具有相对优越性。
Object:
Osteoarthritis(osteoarthritis,OA),is caused by various factors in the elderly with multiple chronic degenerative joint disease,in the more than 60-year-old population,50%of people in the X-line has the performance of osteoarthritis,in which 35%-50%of clinical performance;in the more than 75%of the population,80 percent have symptoms of osteoarthritis.With the progress of the aging of the population,the incidence of osteoarthritis increases year by year,the age of onset is also a downward trend,with the data reported for more than 45-year-old population,the incidence of osteoarthritis is more than 40%.With the aging of our population into the community,osteoarthritis is becoming a focus of common concern to doctors and patients,for which the World Health Organization(WHO) will be October 12th as "the world of osteoarthritis Day" and the first ten years of the 21st.cencury as the "Decade of Bone and Joint Disease." Knee osteoarthritis is the most common inflammatory diseases.Knee osteoarthritis in the elderly population in the most common,especially in the obese elderly population.Incidence of both men and women,more women than men.Clinical observation that patients with knee osteoarthritis has been extended to the general population over the age of 35, seriously disturbing people's daily life and work.
Osteoarthritis is a degeneration and loss of articular cartilage and joint marginal and subchondral bone regeneration,which is characterized by chronic arthritis of the disease.The site of origin of the disease is the cartilage. The etiology and pathogenesis of Osteoarthritis is not entirely clear.It is generally believed that it is the participation of systemic factors such as the existing age,sex,familial susceptibility,and local biomechanics,cartilage cell apoptosis,cytokines,and degradation of the role of enzymes.It is a multi-factor,multi-link caused by the disease.The literature has reported the chondrocyte apoptosis may be an important part of the pathogenesis of OA.At present,the cartilage cell apoptosis induced by means of signals(Fax,NO), signal factor of controlling chondrocyte apoptosis(Bcl-2 family,caspases protease) and cartilage signal to stimulate apoptosis are hot spots in the experimental studies.A large number of studies have confirmed,OA course of IL-1 (IL-1),tumor necrosis factor-α(TNF-α),matrix metalloproteinase-line(MMPs) [particular MMP-1,MMP-3,MMP-13 and tissue inhibitor-1 of metalloproteinase (TIMP-1)]have significant change.These indicators can be indirectly measured to understand the evolution of OA,not only suggesting the role of chondrocyte apoptosis in the pathogenesis mechanism of OA,but also is used to evaluate the role of OA intervention's target and mechanism.
Osteoarthritis in the Traditional Chinese Medicine is "Bi disease," "Gubi" areas.Less than liver and kidney,Loss of nutrientsin the bones and tendons are important dystrophy pathogenesis.Drynariae is bitter taste,temperature, Huo xue continued injury,tonic kidney strong bones,as the important drugs of bone-setting,it is a certain improvement of cartilage cell function and it has a role of delaying degeneration of cartilage cells.
The experiment uses mainly cell,molecular biology techniques,using reverse transcription polymerase chain reaction(RT-PCR),Western blotting (Western-blot) and other detection methods,through the cartilage cells of different apoptosis-related factor expression,further exploring the pathogenesis of OA,studing the mechanism Drynariae,observing on cartilage cell apoptosis's influence of experimental osteoarthritis in rabbit knee,and the theoretical analysis.It provides experimental and clinical application of theory and practice basis oftraditional Chinese medicine for prevention and treatment of knee osteoarthritis.
Materials and Methods:
1,experimental animals
24 adult New Zealand rabbits,the general level,regardless of male and female, weight 2-3kg.Liaoning University of Traditional Chinese Medicine Center for animal experiments,animal permit number:SCXK(LU) 20030006.
2,the experimental drug
2.1 Drynariae Liaoning University of Traditional Chinese Medicine Pharmacy to provide.Condensed into a decoction with the liquid concentration 1g/ml reserve, for a total of 630ml.
2.2 dimensional solid edge Ireland Rottapharm Ltd,batch number:H20040637, specifications:0.25g(glucosamine sulfate),Liaoning University of Traditional Chinese Medicine Pharmacy to provide
3,experimental main reagents
(1) Western Blot Detection of Anti-Ⅰantibody used:mouse monoclonal antibody anti-rabbit IL-1,goat anti-rabbit TNF-α,rabbit anti-mouse MMP3,rabbit anti-rat Bcl-2,goat anti-rabbit BAX,rabbit anti-rat Caspase-3,goat anti-rabbitβ-actin(the United States santa cruz biotechnology,~(?) inc.). Anti-Ⅱantibodies:rabbit anti-mouse alkaline phosphatase marker,rabbit anti-sheep antibody(Beijing Zhongshan Biotechnology corporation).
(2) endogenous peroxidase blocking agent:3%hydrogen peroxide solution
(3) closed by normal bovine serum albumin working solution
(4) alkaline phosphatase labeled anti-working solutionⅡ
(5) concentrated PBS buffer solution
(6) 3,3 - Diaminobenzidine hydrochlorideⅣ(3,3-Diaminobenxidine tetrahydroehloride,DAB)
(2)-(6) Fuzhoumaixin the new biotechnology development companies
(7) total RNA extraction reagent guanidine isothiocyanate(Triozol Reagent): United States company Invitrogen Life technologies
(8) reverse transcriptase(AMV)
(9) RNA enzyme inhibitors
(10) dNTP mixture
(11) Oligo(dT) 15
(12)γ-Taq DNA polymerase
(13) target gene primers
(8)-(13) Japan TaKaRa Company
(14) agarose:The United States company Promaga
(15) Coomassie brilliant blue protein quantitative kit:Nanjing established bio-engineering company
(16) nitrocellulose membrane:purchased from the company Millopore
(17) Other reagents:6×gel sample buffer,DNA electrophoresis buffer(TBE), sodium dodecyl sulfate(SDS),Tris(Tris),acrylamide(Acr),3%glutaraldehyde, methanol,developer,fixed liquid,acetone,xylene,ethanol,formaldehyde, skimmed milk powder bought in the company humei.
4,the main experimental apparatus and equipment
(1) OLYMPUS microscope(Japan,CH)
(2) electric constant temperature water tank(Space Instrument Factory,Huanghua, Hebei Province,HH.W21.600 type)
(3) slicer(Germany Leitz,Kryostat 1720)
(4) The ice-making machine(Germany ZIGERA,2BE-70-25)
(5) small centrifuge desktop(U.S.SIGMA,1-13)
(6) PCR amplification instrument(Germany Biometra)
(7) UV spectrophotometer(UK UV-visible Spectrometer,UV300)
(8) Bio-Rad(USA BIO-RAD,PowerPac200)
(9) Semi-transfer instrument(USA BIO-RAD Semidry Transfer System)
(10) the level shaking bed(U.S.GFL)
(11) vertical slab electrophoresis unit(U.S.BIO-RAD,Mini-ProteinⅢ)
(12) Chemi Imager5500 gel electrophoresis image analysis system(United States Alphainnotech chemi Imager)
(13) HITACHI-7500 type transmission electron microscope(HITACHI,Japan Corporation)
5,the establishment of the rabbit knee OA
Rabbit left hindlimb extension position fixed plaster cast for 6 weeks has been the model of rabbit knee OA.
6,animal grouping and drug delivery
New Zealand 24 rabbits are randomly divided into 4 groups.
blank group 6,model not,do not use the normal diet.
Model group 6,after the model,do not use the normal diet.
Treatment group(Drynariae group) 6,gastric washing after the model,1 times / day,medication for 4 weeks.
The control group(solid peacekeeping force group) 6,gastric washing after the model,1 times / day,medication for 4 weeks.
7,animals killed and materials
After medication four weeks,the way of air ear vein embolization to kill animals in each group.
Surgical operation to remove the left hindlimb of each rabbit femoral condyle(about 1cm) and proximal tibia(about 1cm),the fine separation of articular cartilage.
Remove each of the articular cartilage in rabbits divided into three parts. A small part of the tibial plateau,3%glutaraldehyde solution fixed,4 C refrigerator for electron microscopy.Part of femur using Trizol soaking liquid, 4 C refrigerator for RT-PCR detection;another part in the plastic tube,the seal,-80 C frozen for Western blot detection.
8,electron microscopy
At 4 C condition,after the immersion in 3%glutaraldehyde fixative in 24 hours,Transparent liquid propylene oxide for decalcifying,with 0.1M phosphate buffer washing 3 times with 0.1%osmium tetroxide fixing for 60min,deionized water washing 3 times,the termination osmium tetroxide reaction,different concentrations of ethanol dehydration step by step,soaking using Epon812 epoxy resin,embedded,repair semi-thin slices after hardening,the ultra-thin slices after the positioning,slice thickness 6Ohm,dual-acetate uranium oxide and lead citrate double staining TEM and X-ray film.
9,RT-PCR detection
(1) TRIzol to extract total RNA
(2) RNA concentration and purity determination
(3) reverse transcriptase synthesis of cDNA
(4) PCR amplification:Application Biometra PCR amplification instrument
(5) RT-PCR product electrophoresis and apalysis of quantitative
10,Western-blot detection
(1) protein extraction
(2) Determination of protein concentration
(3) protein denaturation
(4) with plastic
(5) Electrophoresis
(6) Transfer
(7) closed
(8) incubated with anti- first
(9) incubated with anti-second
(10) cleaning
(11) Color
(12) Western Blot results of quantitative analysis:The FlourChem V 2.0 gel image analysis software(America) analysis,the grayscale value of protein electrophoresis of each record and quantitative analysis.
11,data processing and statistics
SPSS13.0 package used for RT-PCR and Western Blot results of variance analysis and Pearson correlation analysis,data indicated that the mean±standard deviation.The P＜0.05 has statistical significance.
Results:
1,the general morphology
In the blank group,Articular cartilage surface is smooth and a little blue. In the model group,Cartilage surface is a little white.Careful observation shows that there are scattered small punctate and linear depressions.Cartilage surface is also a little white or a little yellow in the Treatment group and control group,the surface is still smooth,but there are can be seen scattered punctate depressions.
2,the ultrastructural changes under the electron microscope
Under the electron microscope,the ultrastructure of the cartilage surface is relatively smooth in the blank group,we can see there is small pore symmetry, covering a little bit distribution of the white spherical gel-like substance; In the model group,surface is rough and uneven,partial part has crack-like changes.In severe cases,the surface is loss,under which collagen fibers are exposed or cartilage surface appears fissures,collagen fibers have the rupture;In the treatment group,lesions is relatively light,the surface is still smooth and the surface is covered with large round gel-like material,it can be seen scattered clastic-like change,cracks and small holes are formed in the local place;In the control group,the cartilage surface is not smooth, we can see more of the cracks and small holes and different sizes of shallow ulcers,the local surface which is flake up is tear and endarterectomy.Under it there is collagen fibers
Chondrocytes are observed shows that the structure of cells is clear in the blank group,cell membrane is integral,outline is clear and the nuclear chromatin is evenly distributed.Extracellular collagen fibers can be seen criss-cross.In the model group,Membrane is dissolved,nuclear membrane is unclear,cytoplasm is disappeared.It is seen that cell is swelling and necrosis is increased significantly.Collagen fibers decrease with disorder around Cells, and some gathered into one another corporation or a bundle-like shape,and a staggering phenomenon,and some sparse.In the treatment group,there is karyopyknosis,mitochondria dissolved and endoplasmic reticulum swollen;In the control group,there is nuclear enrichment,nuclear membrane unclear and the expansion of mitochondrial dissolved.The collagen fibers around Cells are arranged due to the rules in the treatment group and control group
3,RT-PCR test results
TNF-αmRNA,MMP3mRNA,caspase-3mRNA,BAXmRNA expression levels increase more in the Model group than in the blank group there was a significant difference (P＜0.05);TNF-αmRNA,MMP3mRNA,caspase-3mRNA,BAXmRNA expression levels decreased more significantly in the control group than in the model group, there was a significant difference(P＜0.05);TNF-αmRNA,MMP3mRNA, caspase-3mRNA,BAXmRNA expression level decreased more significantly in the treatment group than in the model group,there was a significant difference (P＜0.05);TNF-αmRNA,MMP3mRNA,caspase-3mRNA,BAXmRNA expression of the difference was not significant in the treatment group than in the control group the expression level of bcl-2mRNA decreased more in the Model group than in the blank group,there was a significant difference(P＜0.05);The expression level of bcl-2mRNA has increased more in the control group than in the model group,there was a significant difference(P＜0.05);the expression levels of bcl-2mRNA has a more marked increase in the treatment group than in the model group,the difference was significant(P＜0.05);The expression of bcl-2mRNA is no significant difference in the treatment group than in the control group.
4,Western blot test results
IL-1,TNF-α,MMP3,caspase-3,BAX protein expression levels were more significantly increased in the Model group than in the blank group,there was a significant difference(P＜0.05);IL-1,TNF-α,MMP3,caspase-3,BAX protein expression level decreased more significantly in the control group than in the model group(P＜0.05);IL-1,TNF-α,MMP3,caspase-3,BAX protein expression levels decreased more significantly in the treatment group than in the model group(P＜0.05),there was a significant difference;IL-1,TNF-α, MMP3,caspase-3,BAX protein expression had no significant difference in the treatment group than in the control group.
Bcl-2 protein expression level decreased more significantly in the model group than in the blank group(P＜0.05);bcl-2 protein expression level increased more in the control group than in the model group,there was a significant difference (P＜0.05);Bcl-2 protein expression was more significantly increased in the treatment group than in the model group of,there was a significant difference (P＜0.05);the treatment group than in the control group of bcl-2 protein expression was no significant difference.
Conclusion:
1 IL-1,MMP3,TNF-α,caspase-3,BAX,bcl-2 and other factors are closely related to the course of osteoarthritis.
2 The Study confirmed the existence of the reduction of OA articular cartilage in the IL-1,MMP3,TNF-α,caspase-3,BAXmRNA and protein expression,bcl-2mRNA and protein expression,which may be part of the pathogenesis of OA.Therefore, the application of various methods of inhibiting IL-1,MMP3,TNF-α,caspase-3, BAX expression,and enhance the expression of bcl-2 and its biological effects may be an effective way of great significance to the treatment of OA.It is important.
3 Through the RT-PCR,Western-blot detection,single herbs Drynaria better expresseS the regulation factor on the IL-1,MMP3,TNF-α,caspase-3,BAX,bcl-2, suggesting that its application plays an important role in the prevention and treatment of OA,it is necessary to further develop relevant clinical and basic research.
4 Western medicine dimensional solid-edge(glucosamine sulfate) and single herbs Drynaria cartilage showed the same inhibition or enhance to the apoptosis-related factors and The data of this set showed no difference statistically,but There is a marked difference in the mRNA and protein expression.It can be considered that a single herbs Drynaria has a comparative advantage of the prevention and treatment of OA.
骨关节炎(osteoarthritis,OA),是由于多种因素引起的多发于老年人的慢性退行性关节疾病,60岁以上的人群中,50%人群在X线上有骨性关节炎表现,其中35%-50%有临床表现;75岁以上的人群中,80%有骨性关节炎症状。随着人口老龄化的进展,骨关节炎的发病率逐年上升,发病年龄也有下降趋势,有资料报道45岁以上人群骨关节炎的发病率超过40%。随着我国步入老龄化人口社会,骨关节炎正日渐成为医患共同关注的焦点,为此世界卫生组织(WHO)将10月12日定为“世界骨关节炎日”,21世纪头十年为“骨关节病十年”。骨性关节炎是膝关节炎症中最常见的疾病。膝骨性关节炎在老年人群中最为常见,尤其是在肥胖的老年人群。男女均可发病,女性多于男性。临床观察证实,膝骨性关节炎患者已普遍扩展到35岁以上人群,严重困扰着人们的日常生活与劳动。
骨性关节炎是一种以关节软骨变性和丢失及关节边缘和软骨下骨骨质再生为特征的慢性关节炎疾病。该病的始发部位在软骨。骨关节炎的病因和发病机制尚不完全清楚,一般认为既有年龄、性别、家族易感性等全身因素的参与,又有局部生物力学、软骨细胞凋亡、细胞因子和降解酶等的作用,是多因素、多环节导致的疾病。有文献报道,软骨细胞凋亡可能是OA发病机制中的重要环节。目前,软骨细胞凋亡信号的诱导途径(Fax、NO),调控软骨细胞凋亡的信号因子(Bcl-2家族,caspases蛋白酶),软骨细胞凋亡的刺激信号等指标是实验研究中的热点。大量研究也证实,OA病程中白介素1(IL-1)、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶系(MMPs)[尤其是MMP-1、MMP-3、MMP-13和金属蛋白酶组织抑制剂-1(TIMP-1)]等显著变化。通过这些指标的测定可以间接了解OA的演进过程,推测它们在OA发病机制中对软骨细胞凋亡所起的作用,还有是用于评价OA干预措施的作用靶点与机理。
骨关节炎在中医学中属于“痹病”,“骨痹”范畴,肝肾不足,筋骨失养是重要发病机制。骨碎补性味苦、温,活血续伤,补肾强骨,为骨伤科要药,具有一定的改善软骨细胞功能,推迟软骨细胞退行性变的作用。
本实验主要利用细胞、分子生物学技术,采用反转录聚合酶链反应(RT-PCR)、蛋白印迹(Western-blot)等检测方法,通过软骨细胞凋亡相关因子的不同表达,进一步探讨OA发病机制,研究骨碎补作用机理,观察其对实验性兔膝骨关节炎软骨细胞凋亡的影响,并进行了理论分析,为中医药防治膝骨性关节炎的实验与临床提供理论与实际应用的依据。
材料与方法:
1、实验动物
成年新西兰大耳白兔24只,普通级,雌雄不分,重量2-3kg。辽宁中医药大学动物实验中心提供。
2、实验药物
2.1骨碎补辽宁中医药大学附属医院药剂科提供。水煎浓缩成含生药浓度1g/ml的药液备用,共计630ml。
2.2维固力爱尔兰Rottapharm Ltd,批号:H20040637,规格:0.25g(以硫酸氨基葡萄糖计),辽宁中医药大学附属医院药剂科提供。
3、实验主要试剂
(1) Western Blot检测用Ⅰ抗抗体:鼠抗兔单克隆抗体IL-1、羊抗兔TNF-α、鼠抗兔MMP3、鼠抗兔Bcl-2、羊抗兔BAX、鼠抗兔Caspase-3、羊抗兔β-actin(美国santa cruzbiotechnology,~(?)inc.)。Ⅱ抗抗体:碱性磷酸酶标记兔抗鼠、兔抗羊抗体(北京中山生物技术有限公司)。
(2)内源性过氧化物酶阻断剂:3%过氧化氢溶液
(3)封闭用正常小牛血清白蛋白工作液
(4)碱性磷酸酶标记的Ⅱ抗工作液
(5)浓缩PBS缓冲溶液
(6) 3,3-氨基联苯胺四盐酸盐(3,3-Diaminobenxidine tetrahydroehloride,DAB)
(2)-(6)福州迈新生物技术开发公司
(7)总RNA提取试剂异硫氰酸胍(Triozol Reagent):美国Invitrogen Lifetechnologies公司
(8)反转录酶(AMV)
(9) RNA酶抑制剂
(10) dNTP混合液
(11) Oligo(dT)15
(12)γ-Taq DNA聚合酶
(13)目的基因引物
(8)-(13)日本TaKaRa公司
(14)琼脂糖:美国Promaga公司
(15)考马斯亮蓝蛋白定量试剂盒:南京建成生物工程公司
(16)硝酸纤维素膜:购自Millopore公司
(17)其他试剂:6×凝胶上样缓冲液、DNA电泳缓冲液(TBE)、十二烷基磺酸钠(SDS)、三羟甲基氨基甲烷(Tris)、丙烯酰胺(Acr)、3%戊二醛溶液、甲醇、显影液、定影液、丙酮、二甲苯、无水乙醇、甲醛、脱脂奶粉等购于华美公司。
4、实验主要仪器与设备
(1) OLYMPUS显微镜(日本,CH)
(2)电热恒温水箱(河北省黄骅航天仪器厂,HH.W21.600型)
(3)切片机(德国Leitz,Kryostat 1720)
(4)制冰机(德国ZIGERA,2BE-70-25)
(5)小型台式离心机(美国SIGMA,1-13)
(6) PCR扩增仪(德国Biometra)
(7)紫外分光光度计(英国UV-visible Spectrometer,UV300)
(8)电泳仪(美国BIO-RAD,PowerPac200)
(9)半干转印仪(美国BIO-RAD Semidry Transfer System)
(10)水平摇床(美国GFL)
(11)垂直板电泳装置(美国BIO-RAD,Mini-ProteinⅢ)
(12) Chemi Imager5500凝胶电泳成像分析系统(美国Alphainnotech chemi Imager)
(13) HITACHI-7500型透射电镜(日本HITACHI公司)
5、兔膝OA的建立
兔左后肢伸直位管型石膏固定6周得到兔膝OA的模型。
6、动物分组及给药
新西兰大耳白兔24只,随机分为4组。
空白组6只,不造模,不用药,正常饲料喂养。
模型组6只,造模后不用药,正常饲料喂养。
治疗组(骨碎补组)6只,造模后灌胃,1次/天,用药4周。
对照组(维固力组)6只,造模后灌胃,1次/天,用药4周。
7、动物处死及取材
用药4周后,以耳缘静脉空气栓塞法处死各组动物。
外科操作取下每只家兔左后肢股骨髁(约1cm)和胫骨近端(约1cm)部分,精细分离关节软骨。
每只兔取下的关节软骨分成三部分。胫骨平台部分取一小块,用3%戊二醛溶液固定,4℃冷藏,用于电镜检测。股骨髁的一部分用Trizol液浸泡,4℃冷藏,用于RT-PCR检测;另一部分空置于塑料试管内,封盖,-80℃冷冻,用于Western blot检测。
8、电镜观察
在4℃条件下,置于3%戊二醛固定液中浸泡24小时后,环氧丙烷透明液脱钙,用0.1M磷酸缓冲液冲洗3次,再加0.1%四氧化锇固定60min,去离子水冲洗3次,终止锇酸反应,不同浓度乙醇逐级脱水,以Epon812环氧树脂浸透、包埋,硬化后修块半薄切片,定位后超薄切片,切片厚60nm,醋酸双氧铀及柠檬酸铅双重染色透射电镜观察并摄片。
9、RT-PCR检测
(1) TRIzol法抽提总RNA
(2) RNA浓度和纯度测定
(3)逆转录合成cDNA
(4) PCR扩增:应用Biometra PCR扩增仪
(5) RT-PCR产物电泳及分析定量
10、Western-blot检测
(1)蛋白的提取
(2)蛋白浓度的测定
(3)蛋白变性
(4)配胶
(5)电泳
(6)转印
(7)封闭
(8)一抗孵育
(9)二抗孵育
(10)清洗
(11)显色
(12)Western Blot结果量化分析:采用FlourChem V 2.0凝胶成像分析软件(America)分析,记录每条蛋白电泳带的灰度值,进行定量分析。
11、数据处理及统计
采用SPSS1 3.0软件包对RT-PCR和Western Blot结果进行方差分析和Pearson相关分析,数据以均值±标准差表示。P＜0.05有统计学意义。
结果:
1、大体形态观察
空白组关节软骨表面光滑,略呈淡蓝色。模型组软骨表面呈淡白色,仔细观察可见有散在小的点状和条形凹陷。治疗组和对照组软骨表面也呈淡白色或淡黄色,表面尚光滑,但均可见有散在的点状凹陷。
2、电镜下超微结构的变化
电镜下观察软骨表面的超微结构空白组表面较为平整,可见有匀称的细小微孔,覆盖有点状分布的白色圆球形凝胶样物质;模型组表面粗糙,凹凸不平,局部出现龟裂样改变,严重者表层脱失,其下的胶原纤维裸露,或者软骨表面出现裂隙,胶原纤维断裂;治疗组病变相对较轻,表面尚平整,表面覆盖有较大的圆形凝胶样物质,可见散在鳞屑样改变,局部有裂隙和小孔形成;对照组软骨表面欠平整,可见较多的裂隙和小孔和大小不一的浅溃疡形成,局部表层撕裂剥脱,呈片状卷起,露出其下的胶原纤维。
软骨细胞镜下观察显示,空白组细胞结构清楚,细胞膜完整,轮廓清晰,核内染色质均匀分布。细胞外可见纵横交错的胶原纤维。模型组细胞膜溶解,核膜不清,胞质消失,可见细胞肿胀,坏死明显增多。细胞周围的胶原纤维减少,排列紊乱,有的彼此聚集成团状或捆状,并出现交错现象,有的局部稀疏。治疗组核固缩,线粒体溶解,内质网肿胀;对照组核浓缩,核膜不清,线粒体溶解扩张。治疗组与对照组细胞周围胶原纤维排列欠规则。
3、RT-PCR检测结果
模型组较空白组的TNF-αmRNA、MMP3mRNA、caspase-3mRNA、BAXmRNA表达水平明显上升,差异有显著性(P＜0.05);对照组较模型组的TNF-αmRNA、MMP3mRNA、caspase-3mRNA、BAXmRNA表达水平有所下降,差异有显著性(P＜0.05);治疗组较模型组的TNF-αmRNA、MMP3mRNA、caspase-3mRNA、BAXmRNA表达水平明显下降,差异有显著性(P＜0.05);治疗组较对照组的TNF-αmRNA、MMP3mRNA、caspase-3mRNA、BAXmRNA表达差异无显著性。
模型组较空白组的bcl-2mRNA表达水平明显下降,差异有显著性(P＜0.05);对照组较模型组的bcl-2mRNA表达水平有所上升,差异有显著性(P＜0.05);治疗组较模型组的bcl-2mRNA表达水平明显上升,差异有显著性(P＜0.05):治疗组较对照组的bcl-2mRNA表达差异无显著性。
4、Western blot检测结果
模型组较空白组的IL-1、TNF-α、MMP3、caspase-3、BAX蛋白表达水平明显上升,差异有显著性(P＜0.05);对照组较模型组的IL-1、TNF-α、MMP3、caspase-3、BAX蛋白表达水平有所下降,差异有显著性(P＜0.05);治疗组较模型组的IL-1、TNF-α、MMP3、caspase-3、BAX蛋白表达水平明显下降,差异有显著性(P＜0.05);治疗组较对照组的IL-1、TNF-α、MMP3、caspase-3、BAX蛋白表达差异无显著性。
模型组较空白组的bcl-2蛋白表达水平明显下降,差异有显著性(P＜0.05);对照组较模型组的bcl-2蛋白表达水平有所上升,差异有显著性(P＜0.05);治疗组较模型组的bcl-2蛋白表达水平明显上升,差异有显著性(P＜0.05);治疗组较对照组的bcl-2蛋白表达差异无显著性。
结论:
1 IL-1、MMP3、TNF-α、caspase-3、BAX、bcl-2等因子与骨关节炎病变过程密切相关。
2本研究证实了在OA关节软骨中存在IL-1、MMP3、TNF-α、caspase-3、BAXmRNA和蛋白表达增强,bcl-2mRNA和蛋白表达减弱,可能是OA发病机制的一部分。因此,应用各种方法,抑制IL-1、MMP3、TNF-α、caspase-3、BAX的表达,增强bcl-2表达及其生物效应,有可能成为治疗OA的有效途径,具有重要的意义。
3通过RT-PCR、Western-blot检测,单味中药骨碎补较好的表达出对IL-1、MMP3、TNF-α、caspase-3、BAX、bcl-2等因子的调控作用,提示其应用对防治OA有重要作用,有必要进一步开展相关临床与基础方面的研究。
4西药维固力(硫酸氨基葡萄糖)与单味中药骨碎补同样表现出对软骨细胞凋亡相关因子的抑制或增强作用,且本组数据统计学显示无差异性,但在mRNA与蛋白表达上看到了明显的强弱差别,可以认为单味中药骨碎补防治OA具有相对优越性。
Object:
Osteoarthritis(osteoarthritis,OA),is caused by various factors in the elderly with multiple chronic degenerative joint disease,in the more than 60-year-old population,50%of people in the X-line has the performance of osteoarthritis,in which 35%-50%of clinical performance;in the more than 75%of the population,80 percent have symptoms of osteoarthritis.With the progress of the aging of the population,the incidence of osteoarthritis increases year by year,the age of onset is also a downward trend,with the data reported for more than 45-year-old population,the incidence of osteoarthritis is more than 40%.With the aging of our population into the community,osteoarthritis is becoming a focus of common concern to doctors and patients,for which the World Health Organization(WHO) will be October 12th as "the world of osteoarthritis Day" and the first ten years of the 21st.cencury as the "Decade of Bone and Joint Disease." Knee osteoarthritis is the most common inflammatory diseases.Knee osteoarthritis in the elderly population in the most common,especially in the obese elderly population.Incidence of both men and women,more women than men.Clinical observation that patients with knee osteoarthritis has been extended to the general population over the age of 35, seriously disturbing people's daily life and work.
Osteoarthritis is a degeneration and loss of articular cartilage and joint marginal and subchondral bone regeneration,which is characterized by chronic arthritis of the disease.The site of origin of the disease is the cartilage. The etiology and pathogenesis of Osteoarthritis is not entirely clear.It is generally believed that it is the participation of systemic factors such as the existing age,sex,familial susceptibility,and local biomechanics,cartilage cell apoptosis,cytokines,and degradation of the role of enzymes.It is a multi-factor,multi-link caused by the disease.The literature has reported the chondrocyte apoptosis may be an important part of the pathogenesis of OA.At present,the cartilage cell apoptosis induced by means of signals(Fax,NO), signal factor of controlling chondrocyte apoptosis(Bcl-2 family,caspases protease) and cartilage signal to stimulate apoptosis are hot spots in the experimental studies.A large number of studies have confirmed,OA course of IL-1 (IL-1),tumor necrosis factor-α(TNF-α),matrix metalloproteinase-line(MMPs) [particular MMP-1,MMP-3,MMP-13 and tissue inhibitor-1 of metalloproteinase (TIMP-1)]have significant change.These indicators can be indirectly measured to understand the evolution of OA,not only suggesting the role of chondrocyte apoptosis in the pathogenesis mechanism of OA,but also is used to evaluate the role of OA intervention's target and mechanism.
Osteoarthritis in the Traditional Chinese Medicine is "Bi disease," "Gubi" areas.Less than liver and kidney,Loss of nutrientsin the bones and tendons are important dystrophy pathogenesis.Drynariae is bitter taste,temperature, Huo xue continued injury,tonic kidney strong bones,as the important drugs of bone-setting,it is a certain improvement of cartilage cell function and it has a role of delaying degeneration of cartilage cells.
The experiment uses mainly cell,molecular biology techniques,using reverse transcription polymerase chain reaction(RT-PCR),Western blotting (Western-blot) and other detection methods,through the cartilage cells of different apoptosis-related factor expression,further exploring the pathogenesis of OA,studing the mechanism Drynariae,observing on cartilage cell apoptosis's influence of experimental osteoarthritis in rabbit knee,and the theoretical analysis.It provides experimental and clinical application of theory and practice basis oftraditional Chinese medicine for prevention and treatment of knee osteoarthritis.
Materials and Methods:
1,experimental animals
24 adult New Zealand rabbits,the general level,regardless of male and female, weight 2-3kg.Liaoning University of Traditional Chinese Medicine Center for animal experiments,animal permit number:SCXK(LU) 20030006.
2,the experimental drug
2.1 Drynariae Liaoning University of Traditional Chinese Medicine Pharmacy to provide.Condensed into a decoction with the liquid concentration 1g/ml reserve, for a total of 630ml.
2.2 dimensional solid edge Ireland Rottapharm Ltd,batch number:H20040637, specifications:0.25g(glucosamine sulfate),Liaoning University of Traditional Chinese Medicine Pharmacy to provide
3,experimental main reagents
(1) Western Blot Detection of Anti-Ⅰantibody used:mouse monoclonal antibody anti-rabbit IL-1,goat anti-rabbit TNF-α,rabbit anti-mouse MMP3,rabbit anti-rat Bcl-2,goat anti-rabbit BAX,rabbit anti-rat Caspase-3,goat anti-rabbitβ-actin(the United States santa cruz biotechnology,~(?) inc.). Anti-Ⅱantibodies:rabbit anti-mouse alkaline phosphatase marker,rabbit anti-sheep antibody(Beijing Zhongshan Biotechnology corporation).
(2) endogenous peroxidase blocking agent:3%hydrogen peroxide solution
(3) closed by normal bovine serum albumin working solution
(4) alkaline phosphatase labeled anti-working solutionⅡ
(5) concentrated PBS buffer solution
(6) 3,3 - Diaminobenzidine hydrochlorideⅣ(3,3-Diaminobenxidine tetrahydroehloride,DAB)
(2)-(6) Fuzhoumaixin the new biotechnology development companies
(7) total RNA extraction reagent guanidine isothiocyanate(Triozol Reagent): United States company Invitrogen Life technologies
(8) reverse transcriptase(AMV)
(9) RNA enzyme inhibitors
(10) dNTP mixture
(11) Oligo(dT) 15
(12)γ-Taq DNA polymerase
(13) target gene primers
(8)-(13) Japan TaKaRa Company
(14) agarose:The United States company Promaga
(15) Coomassie brilliant blue protein quantitative kit:Nanjing established bio-engineering company
(16) nitrocellulose membrane:purchased from the company Millopore
(17) Other reagents:6×gel sample buffer,DNA electrophoresis buffer(TBE), sodium dodecyl sulfate(SDS),Tris(Tris),acrylamide(Acr),3%glutaraldehyde, methanol,developer,fixed liquid,acetone,xylene,ethanol,formaldehyde, skimmed milk powder bought in the company humei.
4,the main experimental apparatus and equipment
(1) OLYMPUS microscope(Japan,CH)
(2) electric constant temperature water tank(Space Instrument Factory,Huanghua, Hebei Province,HH.W21.600 type)
(3) slicer(Germany Leitz,Kryostat 1720)
(4) The ice-making machine(Germany ZIGERA,2BE-70-25)
(5) small centrifuge desktop(U.S.SIGMA,1-13)
(6) PCR amplification instrument(Germany Biometra)
(7) UV spectrophotometer(UK UV-visible Spectrometer,UV300)
(8) Bio-Rad(USA BIO-RAD,PowerPac200)
(9) Semi-transfer instrument(USA BIO-RAD Semidry Transfer System)
(10) the level shaking bed(U.S.GFL)
(11) vertical slab electrophoresis unit(U.S.BIO-RAD,Mini-ProteinⅢ)
(12) Chemi Imager5500 gel electrophoresis image analysis system(United States Alphainnotech chemi Imager)
(13) HITACHI-7500 type transmission electron microscope(HITACHI,Japan Corporation)
5,the establishment of the rabbit knee OA
Rabbit left hindlimb extension position fixed plaster cast for 6 weeks has been the model of rabbit knee OA.
6,animal grouping and drug delivery
New Zealand 24 rabbits are randomly divided into 4 groups.
blank group 6,model not,do not use the normal diet.
Model group 6,after the model,do not use the normal diet.
Treatment group(Drynariae group) 6,gastric washing after the model,1 times / day,medication for 4 weeks.
The control group(solid peacekeeping force group) 6,gastric washing after the model,1 times / day,medication for 4 weeks.
7,animals killed and materials
After medication four weeks,the way of air ear vein embolization to kill animals in each group.
Surgical operation to remove the left hindlimb of each rabbit femoral condyle(about 1cm) and proximal tibia(about 1cm),the fine separation of articular cartilage.
Remove each of the articular cartilage in rabbits divided into three parts. A small part of the tibial plateau,3%glutaraldehyde solution fixed,4 C refrigerator for electron microscopy.Part of femur using Trizol soaking liquid, 4 C refrigerator for RT-PCR detection;another part in the plastic tube,the seal,-80 C frozen for Western blot detection.
8,electron microscopy
At 4 C condition,after the immersion in 3%glutaraldehyde fixative in 24 hours,Transparent liquid propylene oxide for decalcifying,with 0.1M phosphate buffer washing 3 times with 0.1%osmium tetroxide fixing for 60min,deionized water washing 3 times,the termination osmium tetroxide reaction,different concentrations of ethanol dehydration step by step,soaking using Epon812 epoxy resin,embedded,repair semi-thin slices after hardening,the ultra-thin slices after the positioning,slice thickness 6Ohm,dual-acetate uranium oxide and lead citrate double staining TEM and X-ray film.
9,RT-PCR detection
(1) TRIzol to extract total RNA
(2) RNA concentration and purity determination
(3) reverse transcriptase synthesis of cDNA
(4) PCR amplification:Application Biometra PCR amplification instrument
(5) RT-PCR product electrophoresis and apalysis of quantitative
10,Western-blot detection
(1) protein extraction
(2) Determination of protein concentration
(3) protein denaturation
(4) with plastic
(5) Electrophoresis
(6) Transfer
(7) closed
(8) incubated with anti- first
(9) incubated with anti-second
(10) cleaning
(11) Color
(12) Western Blot results of quantitative analysis:The FlourChem V 2.0 gel image analysis software(America) analysis,the grayscale value of protein electrophoresis of each record and quantitative analysis.
11,data processing and statistics
SPSS13.0 package used for RT-PCR and Western Blot results of variance analysis and Pearson correlation analysis,data indicated that the mean±standard deviation.The P＜0.05 has statistical significance.
Results:
1,the general morphology
In the blank group,Articular cartilage surface is smooth and a little blue. In the model group,Cartilage surface is a little white.Careful observation shows that there are scattered small punctate and linear depressions.Cartilage surface is also a little white or a little yellow in the Treatment group and control group,the surface is still smooth,but there are can be seen scattered punctate depressions.
2,the ultrastructural changes under the electron microscope
Under the electron microscope,the ultrastructure of the cartilage surface is relatively smooth in the blank group,we can see there is small pore symmetry, covering a little bit distribution of the white spherical gel-like substance; In the model group,surface is rough and uneven,partial part has crack-like changes.In severe cases,the surface is loss,under which collagen fibers are exposed or cartilage surface appears fissures,collagen fibers have the rupture;In the treatment group,lesions is relatively light,the surface is still smooth and the surface is covered with large round gel-like material,it can be seen scattered clastic-like change,cracks and small holes are formed in the local place;In the control group,the cartilage surface is not smooth, we can see more of the cracks and small holes and different sizes of shallow ulcers,the local surface which is flake up is tear and endarterectomy.Under it there is collagen fibers
Chondrocytes are observed shows that the structure of cells is clear in the blank group,cell membrane is integral,outline is clear and the nuclear chromatin is evenly distributed.Extracellular collagen fibers can be seen criss-cross.In the model group,Membrane is dissolved,nuclear membrane is unclear,cytoplasm is disappeared.It is seen that cell is swelling and necrosis is increased significantly.Collagen fibers decrease with disorder around Cells, and some gathered into one another corporation or a bundle-like shape,and a staggering phenomenon,and some sparse.In the treatment group,there is karyopyknosis,mitochondria dissolved and endoplasmic reticulum swollen;In the control group,there is nuclear enrichment,nuclear membrane unclear and the expansion of mitochondrial dissolved.The collagen fibers around Cells are arranged due to the rules in the treatment group and control group
3,RT-PCR test results
TNF-αmRNA,MMP3mRNA,caspase-3mRNA,BAXmRNA expression levels increase more in the Model group than in the blank group there was a significant difference (P＜0.05);TNF-αmRNA,MMP3mRNA,caspase-3mRNA,BAXmRNA expression levels decreased more significantly in the control group than in the model group, there was a significant difference(P＜0.05);TNF-αmRNA,MMP3mRNA, caspase-3mRNA,BAXmRNA expression level decreased more significantly in the treatment group than in the model group,there was a significant difference (P＜0.05);TNF-αmRNA,MMP3mRNA,caspase-3mRNA,BAXmRNA expression of the difference was not significant in the treatment group than in the control group the expression level of bcl-2mRNA decreased more in the Model group than in the blank group,there was a significant difference(P＜0.05);The expression level of bcl-2mRNA has increased more in the control group than in the model group,there was a significant difference(P＜0.05);the expression levels of bcl-2mRNA has a more marked increase in the treatment group than in the model group,the difference was significant(P＜0.05);The expression of bcl-2mRNA is no significant difference in the treatment group than in the control group.
4,Western blot test results
IL-1,TNF-α,MMP3,caspase-3,BAX protein expression levels were more significantly increased in the Model group than in the blank group,there was a significant difference(P＜0.05);IL-1,TNF-α,MMP3,caspase-3,BAX protein expression level decreased more significantly in the control group than in the model group(P＜0.05);IL-1,TNF-α,MMP3,caspase-3,BAX protein expression levels decreased more significantly in the treatment group than in the model group(P＜0.05),there was a significant difference;IL-1,TNF-α, MMP3,caspase-3,BAX protein expression had no significant difference in the treatment group than in the control group.
Bcl-2 protein expression level decreased more significantly in the model group than in the blank group(P＜0.05);bcl-2 protein expression level increased more in the control group than in the model group,there was a significant difference (P＜0.05);Bcl-2 protein expression was more significantly increased in the treatment group than in the model group of,there was a significant difference (P＜0.05);the treatment group than in the control group of bcl-2 protein expression was no significant difference.
Conclusion:
1 IL-1,MMP3,TNF-α,caspase-3,BAX,bcl-2 and other factors are closely related to the course of osteoarthritis.
2 The Study confirmed the existence of the reduction of OA articular cartilage in the IL-1,MMP3,TNF-α,caspase-3,BAXmRNA and protein expression,bcl-2mRNA and protein expression,which may be part of the pathogenesis of OA.Therefore, the application of various methods of inhibiting IL-1,MMP3,TNF-α,caspase-3, BAX expression,and enhance the expression of bcl-2 and its biological effects may be an effective way of great significance to the treatment of OA.It is important.
3 Through the RT-PCR,Western-blot detection,single herbs Drynaria better expresseS the regulation factor on the IL-1,MMP3,TNF-α,caspase-3,BAX,bcl-2, suggesting that its application plays an important role in the prevention and treatment of OA,it is necessary to further develop relevant clinical and basic research.
4 Western medicine dimensional solid-edge(glucosamine sulfate) and single herbs Drynaria cartilage showed the same inhibition or enhance to the apoptosis-related factors and The data of this set showed no difference statistically,but There is a marked difference in the mRNA and protein expression.It can be considered that a single herbs Drynaria has a comparative advantage of the prevention and treatment of OA.
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