今天是:2020-08-03 星期一

关节炎发生发展的糖基化机制研究
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注册号:

Registration number:

ChiCTR2000034414 

最近更新日期:

Date of Last Refreshed on:

2020-07-05 

注册时间:

Date of Registration:

2020-07-04 

注册号状态:

预注册  

Registration Status:

Prospective registration  

注册题目:

关节炎发生发展的糖基化机制研究 

Public title:

Study on glycosylation mechanism in the development of arthritis 

注册题目简写:

 

English Acronym:

 

研究课题的正式科学名称:

关节炎发生发展的糖基化机制研究 

Scientific title:

Study on glycosylation mechanism in the development of arthritis 

研究课题代号(代码):

Study subject ID:

 

在二级注册机构或其它机构的注册号:

The registration number of the Partner Registry or other register:

 

申请注册联系人:

吴子光 

研究负责人:

唐剑邦 

Applicant:

Wu Ziguang 

Study leader:

Tang Jianbang 

申请注册联系人电话:

Applicant telephone:

+86 13925689729 

研究负责人电话:

Study leader's telephone:

+86 13450943816 

申请注册联系人传真 :

Applicant Fax:

 

研究负责人传真:

Study leader's fax:

 

申请注册联系人电子邮件:

Applicant E-mail:

781751387@qq.com 

研究负责人电子邮件:

Study leader's E-mail:

85945915@qq.com 

申请单位网址(自愿提供):

Applicant website(voluntary supply):

 

研究负责人网址(自愿提供):

Study leader's website(voluntary supply):

 

申请注册联系人通讯地址:

广东省中山市康欣路3号中山中医院学生宿舍 

研究负责人通讯地址:

广东省中山市康欣路3号中山中医院住院部骨三科办公室 

Applicant address:

Student Dormitory of Zhongshan Hospital of Traditional Chinese Medicine, 3 Kangxin Road, Zhongshan, Guangdong 

Study leader's address:

Orthopaedic Office, 9th Floor, In-Patient Department, Zhongshan Hospital, 3 Kangxin Road, Zhongshan, Guangdong  

申请注册联系人邮政编码:

Applicant postcode:

 

研究负责人邮政编码:

Study leader's postcode:

 

申请人所在单位:

中山市中医院 

Applicant's institution:

Zhongshan Chinese Medicine Hospital 

是否获伦理委员会批准:

是 

Approved by ethic committee:

Yes 

伦理委员会批件文号:

Approved No. of ethic committee:

2020ZSZY-LLK-202 

伦理委员会批件附件:

Approved file of Ethical Committee:

查看附件View

批准本研究的伦理委员会名称:

中山市中医院伦理委员会 

Name of the ethic committee:

Ethics Committee of Zhongshan Chinese Medicine Hospital 

伦理委员会批准日期:

Date of approved by ethic committee:

2020-06-22 

伦理委员会联系人:

周小军 

Contact Name of the ethic committee:

Zhou Xiaojun 

伦理委员会联系地址:

广东省中山市西区康欣路3号中山市中医院行政楼2楼医学伦理委员会 

Contact Address of the ethic committee:

Medical Ethics Committee, 2nd Floor, Administration Building, Zhongshan Hospital, 3, Kangxin Road, West District, Zhongshan, Guangdong 

伦理委员会联系人电话:

Contact phone of the ethic committee:

 

伦理委员会联系人邮箱:

Contact email of the ethic committee:

 

研究实施负责(组长)单位:

中山市中医院 

Primary sponsor:

Zhongshan Chinese Medicine Hospital 

研究实施负责(组长)单位地址:

广东省中山市康欣路3号中山中医院住院部骨三科办公室 

Primary sponsor's address:

Orthopaedic Office, 9th Floor, In-Patient Department, Zhongshan Hospital, 3 Kangxin Road, Zhongshan, Guangdong 

试验主办单位(项目批准或申办者):

Secondary sponsor:

国家:

中国

省(直辖市):

广东

市(区县):

中山

Country:

China

Province:

Guangdong

City:

Zhongshan

单位(医院):

中山市中医院

具体地址:

西区康欣路3号

Institution
hospital:

Zhongshan Chinese Medicine Hospital

Address:

3 Kangxin Road, West District

经费或物资来源:

中山市中医院 

Source(s) of funding:

Zhongshan Chinese Medicine Hospital 

研究疾病:

骨关节炎 

Target disease:

Osteoarthritis 

研究疾病代码:

 

Target disease code:

 

研究类型:

基础科学研究 

Study type:

Basic Science 

研究所处阶段:

探索性研究/预试验 

Study phase:

研究目的:

OA广泛存在炎症、氧化应激通路异常,且多有合并2型糖尿病。糖基化在体内广泛存在,其变化也是动态平衡的,时刻在合成、降解。细胞也利用糖基化这个过程,调节细胞功能,对蛋白质进行糖修饰。关节软骨主要成分硫酸软骨素,是一种糖化蛋白;软骨调解素(Chm-1)、胰岛素样(IGF-I),对于关节软骨生发的调节均与糖基化位点相关;血管生成、炎症通路也均与蛋白的糖修饰相关。我们推测糖尿病患者糖修饰改变,也可能影响关节软骨性状,是糖尿病患者OA发生发展的重要机制。我们计划通过取膝关节置换手术后常规截下的骨块上病变侧的废弃软骨做观察组,取股骨颈骨折排除OA的患者行全髋置换术后废弃的股骨头上的健康软骨做对照组,检测N糖基化蛋白组学,尝试寻找并验证差异标志物。 

Objectives of Study:

Oa is characterized by inflammation, abnormal oxidative stress pathway and type 2 diabetes Mellitus. GLYCOSYLATION is widespread in the body, and its changes are also Dynamic equilibrium, and are constantly synthesized and degraded. Cells also use glycosylation, a process that regulates cell function and modifies proteins with sugars. The major components of articular cartilage, Chondroitin Sulfate, are glycosylated proteins, and the regulation of cartilage development by Chm-1 and IGF-I is related to glycosylation sites Angiogenesis and inflammatory pathways are also associated with glycoprotein modification. We hypothesized that changes in glucose modification may also affect articular cartilage properties, which is an important mechanism for the development of OA in diabetic patients. We planned to use the discarded cartilage on the diseased side of the bone block routinely amputated after knee replacement as an observation group, and the healthy cartilage on the femoral head discarded after total hip replacement as a control group for patients with femoral neck fracture excluding OA, to detect the N-GLYCOSYLATION proteomics and try to find and verify the differential markers. 

药物成份或治疗方案详述:

1、病例选择 选取广东省中山市中医院初次行单侧膝关节置换原发性退行性膝骨关节炎患者(K-L分级三、四级)10例,其中合并2型糖尿病者5例为OA-DM组,无糖尿病患者5例为OA组。另选取排除OA的股骨颈骨折行髋关节置换患者为非OA组。 2、样本采集 OA-DM组与OA组于膝关节置换手术后,取常规截下的骨块上病变侧的废弃软骨;非OA组股骨颈骨折排除OA的患者行全髋置换术后,取废弃的股骨头上的健康软骨。软骨样本生理盐水冲洗晾干后,液氮罐保存。 3、诊断标准 (1)膝关节骨性关节炎一般采取Kellgren-Lawrence分为5个级别,主要是根据膝关节骨性关节炎X片程度分级。 零级,就是关节表现正常,关节间隙正常,没有骨赘,也没有明显的畸形。 一级,表现为关节间隙疑似变窄,可能有骨质增生。 二级,关节周围有明显的骨赘,关节间隙疑似变窄。 三级,表现为关节周围有中等量的骨质增生,关节间隙变窄比较明确,并且有软骨下硬化的改变。 四级,表现为关节周围大量骨质增生,关节间隙的狭窄明显,伴有严重的硬化以及明显的关节畸形。 (2)糖尿病的诊断标准:空腹血糖大于或等于7.0mmol/L,和/或餐后两小时血糖大于或等于11.1 mmol/L即可确诊糖尿病。 4、N糖修饰蛋白组学试验。 取适量软骨样本在液氮中用研钵磨碎成细粉状,加入 5 倍体积的 TCA/丙酮(1:9),涡旋混匀,置于-20℃沉淀 4h 以上。4℃ 6000g 离心 40min,弃上清。加入预冷丙酮,洗涤三次。通风橱中干燥沉淀。称取 20-30mg 干燥后的粉末,加入 30 倍体积(m/v)的 SDT 裂解液,Votex 重悬沉淀,沸水浴 5min。超声破碎(80W,工作 10s,间歇 15s,循环 10 次),沸水浴 15min。14000g 离心 40min,取上清采用 0.22μm 滤膜过滤,收集滤液。采用 BCA 法进行蛋白质定量。分装样品,-80℃保存。各样品取蛋白质 20μg 分别加入 5X 上样缓冲液,沸水浴 5min,进行 12.5% SDS-PAGE电泳(恒流 14mA,90min),考马斯亮蓝染色。取 400μg 样品,分别加入 DTT 至终浓度为 100mM,沸水浴 5min,冷却至室温。加入200 μL UA buffer 混匀,转入 10kD 超滤离心管中,离心 14000g 15min,弃滤液(重复该步骤一次)。加入 100μL IAA buffer(100mM IAA in UA),600rpm 振荡 1min,室温避光反应30min,离心 14000g 15min。加入 100μL UA buffer 离心 14000g 15min,重复该步骤两次。加入 100μL 25mM NH4HCO3溶液,离心 14000g 15min,重复该步骤两次。加入 40μL Trypsin buffer(4μg Trypsin in 40μL 25mM NH4HCO3),600rpm 振荡 1min,37℃放置 16-18h。换新收集管,离心 14000g 15min;再加入 40μL 25mM NH4HCO3,离心 14000g 15min,收集滤液。将肽段转移至新 10kd 超滤离心管,加入 50μl 凝集素混合液 CWR( Concanavalin A、wheat germ agglutinin、RCA120 agglutinin),600rpm 振荡 1min,室温孵育 1h,离心 14000g 10min。加入 200μl 1×BB,离心 14000g 15min,重复 2 次。加入 50μl 用 H218O 配制的 25mM NH4HCO3 溶液,离心 14000g 15min,重复 3 次。加入 3μg PNGase F 糖苷酶(溶解于 40μl用 H218O 配制的 25mM NH4HCO3溶液),37℃反应 3h。换新收集管,离心 14000g 10min,加入 40μl 用 H218O 配制的 25mM NH4HCO3 溶液,离心 14000g 10min,收集滤液,冷冻干燥。每份样品加入适量0.1% FA复溶,采用纳升流速的HPLC液相系统Easy nLC进行分离。缓冲液A液为0.1%甲酸水溶液,B液为0.1%甲酸乙腈水溶液(乙腈为84%)。色谱柱以95%的A液平衡,样品由自动进样器上样到上样柱(Thermo Scientific Acclaim PepMap100, 100μm*2cm, nanoViper C18),经过分析柱(Thermo scientific EASY column, 10cm, ID75μm, 3μm, C18-A2)分离,流速为300 nL/min。样品经色谱分离后用 Q-Exactive 质谱仪进行质谱分析。质谱分析原始数据为RAW文件,采用MaxQuant软件(版本号1.3.0.5) 进行查库鉴定及定量分析。根据GO、KEGG、Cluster、PPI等分析结果,设计动物实验验证相关结果。 

Description for medicine or protocol of treatment in detail:

1. Case Selection Ten patients with primary degenerative knee osteoarthritis (Grade 3 and 4 K-L) were selected for the first time undergoing unilateral knee arthroplasty in Zhongshan Hospital of traditional Chinese Medicine of Guangdong Province. Five patients with type 2 diabetes were in OA-DM group, 5 Patients Without Diabetes Mellitus were in OA group. The patients with femoral neck fracture excluded from OA were selected as non-OA Group. 2. Sample Collection In OA-DM group and OA group, the discarded cartilage on the diseased side was taken after the knee replacement, and in non-OA group, the healthy cartilage on the femoral head was taken after the total hip replacement. The cartilage samples were washed and dried with normal saline and stored in liquid nitrogen tank. 3. DIAGNOSTIC CRITERIA (1) Kellgren-Lawrence is commonly used as a osteoarthritis/osteodystrophy to classify the knee into five grades, based primarily on the degree of osteoarthritis/osteodystrophy. Level Zero is when the joints are normal, the joint space is normal, there are no osteophytes, and there are no obvious deformities. Grade 1, presents with suspected narrowing of the joint space, possibly with bone hyperplasia. Grade two, obvious osteophytes around the joint, suspected narrowing of the joint space. Grade 3, there is moderate osteosis around the joint, narrowing of the joint space is more definite, and there is subchondral sclerosis. Grade 4, there is a lot of bone hyperplasia around the joint, obvious joint space narrowing, accompanied by severe sclerosis and obvious joint deformity. (2) criteria for the diagnosis of diabetes: Fasting Blood Glucose above or equal to 7.0 MMOL / l, and / or two hours after meal above or equal to 11.1 MMOL / l. 4.N-SUGAR-MODIFIED proteomics. Cartilage samples were ground into fine powder in liquid nitrogen in a mortar, then mixed with 5-fold volume of TCA / Acetone (1:9) and placed at-20 °C for more than 4 hours. 4 °C 6000G centrifugation for 40 Min, discard the supernatant. Add precooled acetone and wash three times. Dry Deposit in fume hood. The dried powder of 20-30 mg was added to SDT solution of 30 times volume (m / V) and precipitated by heavy suspension of Votex in boiling water for 5 min. Ultrasonic crushing (80W, 10s operation, 15s interval, 10 cycles) , boiling water bath 15min. After 14000G centrifugation for 40 Min, the filtrate was collected by 0.22 m filtration membrane. BCA method was used for protein quantification. Package the sample and keep at-80 °C. The protein of each sample was added into 5X buffer solution at 20 G for 5 min in boiling water for 12.5% SDS-PAGE (constant current 14 ma, 90 min) and stained with coomassie brilliant blue. DTT was added to 400 g samples until the final concentration was 100 mm, and the samples were cooled to room temperature in boiling water for 5 min. Add 200 L UA buffer to mix, transfer to 10 KD ultrafiltration centrifuge tube, centrifuge for 14000 g 15 min, discard filtrate (repeat this step once) . 100 L IAA buffer (100 mm IAA in u a) was added, 600 rpm was oscillated for 1 Min, room temperature dark reaction for 30 min, and centrifugation for 14000 G for 15 min. Add 100 l UA buffer and centrifuge for 14000 G for 15 min. Repeat twice. Add 100 l 25 mm NH4HCO3 solution, centrifuge for 14000 G for 15 Min, repeat twice. 40l Trypsin buffer (4g Trypsin 40l 25mM NH4HCO3) was added, and 600RPM was used to oscillate for 1 min at 37 °C for 16-18 H. The new collecting tube was centrifuged for 14000 G for 15 min, then 40 l 25 mm NH4HCO3 was added and centrifuged for 14000 G for 15 min to collect the filtrate. The peptide was transferred to a new 10kd ultrafiltration centrifuge tube, and 50 l lectin mixture CWR (CONCANAVALIN A, wheat germ agglutinin, RCA120 AGGLUTININ) was added, which was oscillated at 600 RPM for 1 min, incubated at room temperature for 1 H, and centrifuged at 14000 G for 10 min. Add 200 l 1 BB, Centrifuge 14000 G 15 min, repeat twice. 50 L 25MM NH4HCO3 solution prepared with H218o was centrifuged for 14000 G for 15 min and repeated 3 times. 3G PNGASE F glycosidase (dissolved in 40L H218o 25mM NH4HCO3 solution) was added and the reaction was carried out at 37 °C for 3H. The new collecting tube was centrifuged for 10 min at 14000g, the 25mM NH4HCO3 solution prepared with H218o was added into the 40l collecting tube and centrifuged for 10 min at 14000g, then the filtrate was collected and lyophilized. Each sample was redissolved with 0.1% FA, and separated by HPLC LIQUID-PHASE SYSTEM EASY NLC with up-flow rate. Buffer a is 0.1% formic acid aqueous solution, and B is 0.1% formic acid acetonitrile aqueous solution (84% acetonitrile) . The chromatographic column was in 95% a liquid equilibrium. The sample was transferred from thermos Scientific injector to the upper column (Thermo Scientific Injector Pepmap 100,100 m * 2cm, Nanoviper C18) . The column was separated by Thermos Scientific EASY column (10 cm, ID75 M, 3 m, C18-a2) . The flow rate was 300 nL / Min. The samples were separated by chromatography and analyzed by Q-active Mass Spectrometer. The RAW data for mass spectrometry is a RAW file, and the software, MaxQuant (version 1.3.0.5) , is used for library search and Quantitative analysis. According to the analysis results of GO, Kegg, Cluster, PPI and so on, animal experiments were designed to verify the related results. 

研究设计:

析因分组(即根据危险因素或暴露因素分组) 

Study design:

Factorial 

纳入标准:

(1)单侧膝骨关节炎诊断明确,分级为三、四级且非手术治疗无效,有明确手术适应症, 患者及其家属有手术意愿者。 (2)已获得患者及家属同意,依从性较高,接受术前术后各项测试检查。 (3)患者为初次行单侧全膝关节置换术。 (4)术前未见血小板、凝血功能异常者。 

Inclusion criteria

(1) The diagnosis of unilateral osteoarthritis of the knee is clear, and it is classified into three or four grades. The non-operative treatment is ineffective. (2) The patients and their families have consented, the compliance is high, and the patients have accepted all kinds of tests before and after operation. (3) The patient underwent unilateral total knee arthroplasty for the first time. (4) There were no abnormal platelet and coagulation before operation. 

排除标准:

(1)合并严重的肝肾功能异常; (2)合并有血栓形成高危因素的患者,如充血性心力衰竭、急性呼吸窘迫综合征等; (3)合并精神疾病等不能配合治疗; (4)有恶性肿瘤病史患者; (5)有深静脉血栓和肺栓塞(包括肌间静脉血栓)、脑栓塞和脑梗死等病史; (6)血红蛋白< 100g/L的患者。 

Exclusion criteria:

(1) Complicated with severe liver and renal dysfunction; (2) Patients with high risk factors for thrombosis, such as heart failure, Acute respiratory distress syndrome, etc. (3) Inability to cooperate with treatment for mental illness; (4) Patients with a history of malignant tumor; (5) A history of thrombosis and pulmonary embolism (including intramuscular vein thrombosis) , cerebral embolism and cerebral infarction; (6) Patients with Hemoglobin < 100g / L. 

研究实施时间:

Study execute time:

From2020-07-01To 2024-06-30 

征募观察对象时间:

Recruiting time:

From2020-07-01To 2024-06-30 

干预措施:

Interventions:

组别:

OA组

样本量:

5

Group:

OA Group

Sample size:

干预措施:

干预措施代码:

Intervention:

Nil

Intervention code:

组别:

OA-DM组

样本量:

5

Group:

OA-DM Group

Sample size:

干预措施:

干预措施代码:

Intervention:

Nil

Intervention code:

组别:

非OA组

样本量:

5

Group:

Non-OA group

Sample size:

干预措施:

干预措施代码:

Intervention:

Nil

Intervention code:

研究实施地点:

Countries of recruitment and research settings:

国家:

中国 

省(直辖市):

广东 

市(区县):

中山 

Country:

China 

Province:

Guangdong 

City:

Zhongshan 

单位(医院):

中山市中医院 

单位级别:

三级甲等 

Institution
hospital:

Zhongshan Chinese Medicine Hospital  

Level of the institution:

Tertiary A 

测量指标:

Outcomes:

指标中文名:

健康人N糖基化蛋白组学

指标类型:

主要指标 

Outcome:

N-GLYCOSYLATION proteomics in healthy volunteers

Type:

Primary indicator 

测量时间点:

测量方法:

Measure time point of outcome:

Measure method:

指标中文名:

OA患者软骨N糖基化蛋白组学

指标类型:

主要指标 

Outcome:

N-glycosylated proteomics of cartilage in patients with Oa

Type:

Primary indicator 

测量时间点:

测量方法:

Measure time point of outcome:

Measure method:

指标中文名:

患2型糖尿病者的OA患者软骨N糖基化蛋白组学

指标类型:

主要指标 

Outcome:

N-glycosylated proteomics of cartilage in patients with type 2 diabetes Mellitus and OA

Type:

Primary indicator 

测量时间点:

测量方法:

Measure time point of outcome:

Measure method:

采集人体标本:

Collecting sample(s)
from participants:

标本中文名:

OA-DM组病变侧的废弃软骨

组织:

Sample Name:

Waste cartilage in lesion side of oa-dm group

Tissue:

人体标本去向

使用后销毁 

说明

Fate of sample:

Destruction after use 

Note:

标本中文名:

OA组变侧的废弃软骨

组织:

Sample Name:

Waste cartilage with lateral change in OA group

Tissue:

人体标本去向

使用后销毁 

说明

Fate of sample:

Destruction after use 

Note:

标本中文名:

非OA组废弃软骨

组织:

Sample Name:

Disused cartilage in non-OA Group

Tissue:

人体标本去向

使用后销毁 

说明

Fate of sample:

Destruction after use 

Note:

征募研究对象情况:

Recruiting status:

尚未开始

Not yet recruiting

年龄范围:

Participant age:

最小 Min age years
最大 Max age years

性别:

男女均可

Gender:

Both

随机方法(请说明由何人用什么方法产生随机序列):

不适用

Randomization Procedure (please state who generates the random number sequence and by what method):

N/A

盲法:

N/A

Blinding:

N/A

试验完成后的统计结果(上传文件):

Calculated Results after
the Study Completed(upload file):

原始数据公开时间:

The time of sharing IPD:

即时公开/Real time access

共享原始数据的方式(说明:请填入公开原始数据日期和方式,如采用网络平台,需填该网络平台名称和网址):

原始数据上传至临床试验公共管理平台ResMan (www.medresman.org.cn),试验结束后即使公布。

The way of sharing IPD”(include metadata and protocol, If use web-based public database, please provide the url):

The raw data was uploaded to ResMan (www.medresman.org.cn) , the public administration platform for clinical trials, and released immediately after the trials ended.

数据采集和管理(说明:数据采集和管理由两部分组成,一为病例记录表(Case Record Form, CRF),二为电子采集和管理系统(Electronic Data Capture, EDC),如ResMan即为一种基于互联网的EDC:

暂未确定。

Data collection and Management (A standard data collection and management system include a CRF and an electronic data capture:

Not yet.

数据管理委员会:

Data Managemen Committee:

有/Yes

注册人:

Name of Registration:

 2020-07-04
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