CN107090501B - C1galt1c1在骨关节炎诊治中的应用 - Google Patents
C1galt1c1在骨关节炎诊治中的应用 Download PDFInfo
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Abstract
本发明公开了C1GALT1C1基因可以作为骨关节炎早期诊断的分子标志物。本发明使用QPCR实验证明与正常滑膜组织相比，骨关节炎患者滑膜组织中的C1GALT1C1基因表达显著升高，且经过RNA干扰实验证明C1GALT1C1能够影响滑膜细胞的增殖。根据本发明的研究成果，可以研发诊断骨关节炎的试剂盒以及治疗骨关节炎的药物。
Description
技术领域
本发明涉及生物技术领域，具体地涉及人C1GALT1C1基因在骨关节炎的诊断、治疗中的用途。
背景技术
骨关节炎(osteoarthritis，OA)是一种以关节软骨退行性病变和继发性骨质增生为特性的慢性关节疾病。多见于中老年人，女性多于男性。好发于负重较大的膝关节、髋关节、腰骶部脊柱关节(Lumbosacral joint)及第一跖趾关节(First MIP joints)等部位，以及手部的远端指间关节(DIP joints)、近端指间关节(PIP joints)。该病亦称为骨关节病、退行性关节炎、增生行关节炎、退化性关节炎、骨性关节炎等。
病因：由于关节腔中缺少了粘性的滑液(关节液)，导致原本应该充当骨关节中作为软垫的软骨不正常磨擦，造成破坏与退化。当软骨退化后，伴随肌肉萎缩韧带松驰。
分类：原发性，指发病原因不明，患者没有创伤、感染、先天性畸形病史，无遗传性缺陷，无全身代谢及内分泌异常。多见于50岁以上的中老年人。继发性，指由于先天性畸形，如先天性髋关节脱位；创伤，如关节内骨折；关节面后天性不平整，如骨的缺血性坏死；关节不稳定，如关节囊或韧带松弛等；关节畸形引起的关节面对合不良，如膝内翻、膝外翻等原因，在关节局部原有病变的基础上发生的骨关节炎。
目前临床上对于骨关节炎的诊断主要基于影像学的检查。X线表现主要为关节间隙狭窄，软骨下骨质硬化及囊性变，关节边缘骨赘形成，关节面萎陷、变形和关节半脱位等。MRI可示早期软骨、半月板等关节结构的病变，有利于早期诊断。CT用于椎间盘病的诊断，优于X线。关节间隙变窄，软骨下骨硬化，边缘性骨刺脊椎关节连成骨桥。亦可见骨囊肿以及畸形。如发现这些变化可作为诊断的依据和估计关节损伤的程度。OA一般在早期没有或很少有症状，只在继发炎症、疼痛或影响关节的活动时，病人才找医生。此时，关节损伤发生已久。因为开发用于早期骨关节炎诊断的方法是亟待解决的问题。
在分子水平上尤其是在基因水平上进行骨关节炎的早期诊断已经成为了骨关节炎诊断领域的发展趋势，在诊断方面，申请号为：201510548635.X、2015105495645、2015105486241、201510627048.X、2015107250040、201510724747.6、2015107257552专利文献均披露了可以用于骨关节炎诊断的基因标志物。本申请是在现有技术的启示下寻找新的可以用于骨关节炎诊断的生物标志物。
发明内容
为了弥补现有技术的不足，本发明的目的在于提供一种可用于骨关节炎(Osterarthritis，OA)早期诊断的分子标志物。本发明通过实验证明，在正常人和骨关节炎患者的滑膜组织中，C1GALT1C1基因表达存在差异，据此认为C1GALT1C1可以作为诊断骨关节炎的marker。
为了实现上述目的，本发明采用如下技术方案：
本发明提供了一种人C1GALT1C1基因及其表达产物在制备诊断骨关节炎的产品中的应用。
进一步，上面所提到的诊断产品包括：通过RT-PCR、实时定量PCR、免疫检测、原位杂交或芯片检测C1GALT1C1基因及其表达产物的表达水平以诊断骨关节炎的产品。
进一步，所述用RT-PCR诊断骨关节炎的产品至少包括一对特异扩增C1GALT1C1基因的引物；所述用实时定量PCR诊断骨关节炎的产品至少包括一对特异扩增C1GALT1C1基因的引物；所述用免疫检测诊断骨关节炎的产品包括：与C1GALT1C1蛋白特异性结合的抗体；所述用原位杂交诊断骨关节炎的产品包括：与C1GALT1C1基因的核酸序列杂交的探针；所述用芯片诊断骨关节炎的产品包括：蛋白芯片和基因芯片；其中，蛋白芯片包括与C1GALT1C1蛋白特异性结合的抗体，基因芯片包括与C1GALT1C1基因的核酸序列杂交的探针。
优选地，所述产品包括芯片、试剂盒。
本发明还提供了人C1GALT1C1基因在高通量测序平台中的应用。随着高通量测序技术的发展，对一个人的基因表达谱的构建将成为十分便捷的工作。通过对比疾病患者和正常人群的基因表达谱，容易分析出哪个基因的异常与疾病相关。因此，在高通量测序中获知人C1GALT1C1基因的异常与骨关节炎相关也属于人C1GALT1C1基因的用途，同样在本发明的保护范围之内。
本发明还提供了人C1GALT1C1基因及其表达产物在制备治疗骨关节炎的药物中的应用。
本发明所述的“治疗骨关节炎的药物”的主要活性成分包括抑制C1GALT1C1基因表达的物质、抑制C1GALT1C1基因表达产物稳定性的物质、和/或抑制C1GALT1C1基因表达产物活性的物质。
进一步，本发明所述的治疗骨关节炎的药物包括：通过干扰RNA抑制C1GALT1C1基因表达的双链核糖核酸，或用于抑制C1GALT1C1蛋白活性的蛋白质。
本发明还提供了一种用于治疗骨关节炎的药物组合物，所述药物组合物包含C1GALT1C1基因和/或其表达产物抑制剂。所述抑制剂包括抑制C1GALT1C1基因表达的物质、抑制C1GALT1C1基因表达产物稳定性的物质、和/或抑制C1GALT1C1基因表达产物活性的物质。
进一步，本发明所述抑制剂包括：通过干扰RNA抑制C1GALT1C1基因表达的双链核糖核酸，或用于抑制C1GALT1C1蛋白活性的蛋白质。
本发明还提供了上述C1GALT1C1基因和/或其表达产物抑制剂在制备治疗骨关节炎药物中的应用。
在本发明中，所述RNA干扰(RNA interference，RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA，dsRNA)诱发的、同源mRNA高效特异性降解的现象。使用RNAi技术可以特异性剔除或关闭特定基因的表达，该技术已被广泛用于探索基因功能和传染性疾病及恶性肿瘤的基因治疗领域。以细胞为基础的RNAi筛选在功能基因学研究方面具有许多优势，主要表现在大多数细胞类型都能使用RNAi方法，并且相对较容易下调或沉默任何目的基因的表达。
为了确保C1GALT1C1基因能够被高效剔除或沉默，根据C1GALT1C1基因的mRNA序列设计了siRNA特异性片段。siRNA的设计根据已发表的通用设计原则(Elbashir et.al2001，Schwarz et.al 2003，Khvorova et.al 2003，Reynolds et.al 2004，Hsieh et.al2004，Ui-Tei et.al 2004)，通过在线工具完成设计，该在线工具为：siRNASelectionProgram of Whitehead Institute(BingbingYuan et.al 2004，http://jura.wi.mit.edu/bioc/siRNAext/)和BLOCK-iTTM RNAi Designer ofINVITROGEN(winnerof the 2004Frost&Sullivan Excellence in Research Award，https://rnaidesigner.invitrogen.com/sirna/)。为了进一步提高siRNA片断的有效性，综合两个在线设计工具的优点来设计用于筛选的siRNA片断。最后，通过同源性比对(NCBI BLAST)来过滤siRNA序列，以提高siRNA片断的特异性并减少RNAi干扰的脱靶效应。
本发明的药物还包括药学上可接受的载体，载体，这类载体包括(但并不限于)：稀释剂、赋形剂如水等、填充剂如淀粉、蔗糖等；粘合剂如纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮；湿润剂如甘油；崩解剂如琼脂、碳酸钙和碳酸氢钠；吸收促进剂季铵化合物；表面活性剂如十六烷醇；吸附载体如高岭土和皂粘土；润滑剂如滑石粉、硬脂酸钙和镁、聚乙二醇等。
本发明的药物还可与其他治疗骨关节炎的药物联用，多种药物联合使用可以大大提到治疗的成功率。
本发明还提供了一种诊断骨关节炎的产品，所述产品包括但不限于芯片、试剂盒。
其中，所述芯片包括基因芯片、蛋白质芯片；所述基因芯片包括固相载体以及固定在固相载体的寡核苷酸探针，所述寡核苷酸探针包括用于检测C1GALT1C1基因转录水平的针对C1GALT1C1基因的寡核苷酸探针；所述蛋白质芯片包括固相载体以及固定在固相载体的C1GALT1C1蛋白的特异性抗体；所述基因芯片可用于检测包括人C1GALT1C1基因在内的多个基因(例如，与骨关节炎相关的多个基因)的表达水平。所述蛋白质芯片可用于检测包括人C1GALT1C1蛋白在内的多个蛋白质(例如与骨关节炎相关的多个蛋白质)的表达水平。通过将多个与骨关节炎的标志物同时检测，可大大提高骨关节炎诊断的准确率。
其中，所述试剂盒包括基因检测试剂盒和蛋白免疫检测试剂盒；所述基因检测试剂盒包括用于检测C1GALT1C1基因转录水平的试剂；所述蛋白免疫检测试剂盒包括C1GALT1C1蛋白的特异性抗体。进一步，所述试剂包括使用RT-PCR、实时定量PCR、免疫检测、原位杂交或芯片方法检测C1GALT1C1基因表达水平过程中所需的试剂。优选度，所述试剂包括针对C1GALT1C1基因的引物和/或探针。根据C1GALT1C1基因的核苷酸序列信息容易设计出可以用于检测C1GALT1C1基因表达水平的引物和探针。
与C1GALT1C1基因的核酸序列杂交的探针可以是DNA、RNA、DNA-RNA嵌合体、PNA或其它衍生物。所述探针的长度没有限制，只要完成特异性杂交、与目的核苷酸序列特异性结合，任何长度都可以。所述探针的长度可短至25、20、15、13或10个碱基长度。同样，所述探针的长度可长至60、80、100、150、300个碱基对或更长，甚至整个基因。由于不同的探针长度对杂交效率、信号特异性有不同的影响，所述探针的长度通常至少是14个碱基对，最长一般不超过30个碱基对，与目的核苷酸序列互补的长度以15-25个碱基对最佳。所述探针自身互补序列最好少于4个碱基对，以免影响杂交效率。
进一步，所述C1GALT1C1蛋白的特异性抗体包括单克隆抗体、多克隆抗体。所述C1GALT1C1蛋白的特异性抗体包括完整的抗体分子、抗体的任何片段或修饰(例如，，嵌合抗体、scFv、Fab、F(ab’)2、Fv等。只要所述片段能够保留与C1GALT1C1蛋白的结合能力即可。用于蛋白质水平的抗体的制备时本领域技术人员公知的，并且本发明可以使用任何方法来制备所述抗体。
在本发明的上下文中，“C1GALT1C1基因”包括人C1GALT1C1基因以及人C1GALT1C1基因的任何功能等同物的多核苷酸。C1GALT1C1基因包括与目前国际公共核酸序列数据库GeneBank中C1GALT1C1基因(NC_000023.11)DNA序列具有70％以上同源性，且编码相同功能蛋白质的DNA序列；
优选地，C1GALT1C1基因的编码序列包括以下任一一种DNA分子：
(1)序列表中SEQ ID NO.1所示的DNA序列；
(2)在严格条件下与1)限定的DNA序列杂交且编码相同功能蛋白质的DNA序列；
(3)与(1)或(2)限定的DNA序列具有70％、优选地，90％以上同源性，且编码相同功能蛋白质的DNA分子。
在本发明的具体实施方案中，所述C1GALT1C1基因的编码序列是SEQ ID NO.1所示的DNA序列。
在本发明的上下文中，C1GALT1C1基因表达产物包括人C1GALT1C1蛋白以及人C1GALT1C1蛋白的部分肽。所述C1GALT1C1蛋白的部分肽含有与骨关节炎相关的功能域。
“C1GALT1C1蛋白”包括人C1GALT1C1蛋白以及人C1GALT1C1蛋白的任何功能等同物。所述功能等同物包括人C1GALT1C1蛋白保守性变异蛋白质、或其活性片段，或其活性衍生物，等位变异体、天然突变体、诱导突变体、在高或低的严紧条件下能与人C1GALT1C1的DNA杂交的DNA所编码的蛋白质。
优选地，C1GALT1C1蛋白是具有下列氨基酸序列的蛋白质：
(1)由序列表中SEQ ID NO.2所示的氨基酸序列组成的蛋白质；
(2)将SEQ ID NO.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID NO.2所示的氨基酸序列具有相同功能的由SEQ ID NO.2所示的氨基酸序列衍生的蛋白质。取代、缺失或者添加的氨基酸的个数通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个。
(3)与SEQ ID NO.2所示的氨基酸序列具有至少80％同源性(又称为序列同一性)，更优选地，与SEQ ID NO.2所示的氨基酸序列至少约90％至95％的同源性，常为96％、97％、98％、99％同源性的氨基酸序列构成的多肽。
在本发明的具体实施方案中，所述C1GALT1C1蛋白是具有SEQ ID NO.2所示的氨基酸序列的蛋白质。
通常，已知的是，一个蛋白质中一个或多个氨基酸的修饰不会影响蛋白质的功能。本领域技术人员会认可改变单个氨基酸或小百分比的氨基酸或对氨基酸序列的个别添加、缺失、插入、替换是保守修饰，其中蛋白质的改变产生具有相似功能的蛋白质。提供功能相似的氨基酸的保守替换表是本领域公知的。
通过添加一个氨基酸或多个氨基酸残基修饰的蛋白质的例子是C1GALT1C1蛋白的融合蛋白。对于与C1GALT1C1蛋白融合的肽或者蛋白质没有限制，只要所得的融合蛋白保留C1GALT1C1蛋白的生物学活性即可。
本发明的C1GALT1C1蛋白也包括对SEQ ID NO.2所示的氨基酸序列的非保守修饰，只要经过修饰的蛋白质仍然能够保留C1GALT1C1蛋白的生物学活性即可。在此类修饰蛋白质中突变的氨基酸数目通常是10个或者更少，例如6个或者更少，例如3个或者更少。
在本发明的上下文中，“诊断骨关节炎”既包括判断受试者是否已经患有骨关节炎、也包括判断受试者是否存在患有骨关节炎的风险，也包括判断骨关节患者的预后，也包括判断骨关节炎患者的复发情况。
在本发明的上下文中，“治疗骨关节炎”从疾病的状态变化来分，可以包括疾病的缓解、疾病的完全治愈。
本发明的优点和有益效果：
本发明首次发现了C1GALT1C1基因表达与骨关节炎相关，通过检测受试者滑膜组织中C1GALT1C1的表达，可以判断受试者是否患有骨关节炎、或者判断受试者是否存在患有骨关节炎的风险，从而指导临床医师给受试者提供预防方案或者治疗方案。
附图说明
图1显示利用QPCR检测C1GALT1C1基因在滑膜细胞中的表达情况；
图2显示利用QPCR检测siRNA对C1GALT1C1基因的干扰效率；
图3显示干扰C1GALT1C1基因表达对OA成纤维样滑膜细胞增殖的抑制作用。
具体的实施方式
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring HarborLaboratoryPress,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1 OA滑膜组织和正常滑膜组织中C1GALT1C1基因的表达差异
60例OA患者的滑膜组织来自于青海大学附属医院骨科行膝关节置换或滑膜切除术的OA病人。所用病例符合Altam提出的关于OA的诊断标准。40例正常滑膜组织来自于青海大学附属医院骨科，为外伤手术病人关节滑膜组织。OA患者滑膜液(SF)高速离心后取上清，-80℃储存待用。本研究中所用临床样本，均对患者进行知情告知并经本院伦理委员会通过。
1、C1GALT1C1基因转录水平的检测
1.1滑膜组织细胞体外培养
将无菌获取的滑膜组织用PBS清洗后，用无菌手术剪刀反复剪切成约1mm x 1mm x1mm的组织块，加入胶原酶II(0.5mg/ml)37℃、消化2h后，经200目纱网过滤，离心去上清后，将细胞重悬于DMEM培养液，置于37℃，5％CO2细胞培养箱内培养。当细胞长成梭形并成片后，进行传代培养。当细胞传至第3代后，分别加入FITC标记的小鼠抗人CD3、CD14、CD19和PE标记的小鼠抗人CD11b进行标记，流式细胞仪检测鉴定。上述4种标记均为阴性的细胞为成纤维样滑膜细胞(Fibroblast-like Synoviocytes，FLS)用于本研究。
1.2总RNA提取
利用QIAGEN公司的组织/细胞总RNA提取试剂盒提取OA患者滑膜组织细胞和正常滑膜组织细胞的总RNA。
1.3逆转录
利用TAKARA公司的逆转录试剂盒进行RNA的逆转录。
1.4QPCR
(1)引物设计
根据Genbank中C1GALT1C1基因和GAPDH基因的编码序列设计QPCR扩增引物，由上海生工生物工程技术服务有限公司合成。具体引物序列如下：
C1GALT1C1基因：
正向引物为5’-TATAGAGACCAATACAACTG-3’(SEQ ID NO.3)；
反向引物为5’-CAATTCCTCCTTCCATAC-3’(SEQ ID NO.4),
GAPDH基因：
正向引物为5’-TTTAACTCTGGTAAAGTGGATAT-3’(SEQ ID NO.5)；
反向引物为5’-GGTGGAATCATATTGGAACA-3’(SEQ ID NO.6)。
(2)按照表1配制PCR反应体系：
其中，SYBR Green聚合酶链式反应体系购自Invitrogen公司。
表1PCR反应体系
| 试剂 | 体积 |
| 正向引物 | 1μl |
| 反向引物 | 1μl |
| SYBR Green聚合酶链式反应体系 | 12.5μl |
| 模板 | 2μl |
| 去离子水 | 补足25μl |
(3)PCR反应条件：95℃12min，(95℃15s，60℃55s)*45个循环。以SYBR Green作为荧光标记物，在Light Cycler荧光定量PCR仪上进行PCR反应，通过融解曲线分析和电泳确定目的条带，ΔΔCT法进行相对定量。
1.5统计学方法
实验都是按照重复3次来完成的，结果数据都是以平均值±标准差的方式来表示，采用SPSS13.0统计软件来进行统计分析的，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。
1.6结果
结果如图1所示，与正常滑膜组织相比，骨关节炎滑膜组织细胞中C1GALT1C1基因表达量显著增加，差异具有统计学意义(P<0.05)。
实施例2干扰C1GALT1C1基因的表达
1、siRNA设计合成
根据C1GALT1C1基因序列由上海吉玛制药技术有限公司设计并合成siRNA序列：
siRNA-C1GALT1C1：
正义链为5’-AAGAUAUCUUCUUUGUUAGGA-3’(SEQ ID NO.7)；
反义链为5’-CUAACAAAGAAGAUAUCUUGA-3’(SEQ ID NO.8)，
同时上海吉玛制药技术有限公司提供与C1GALT1C1基因序列无同源性的通用阴性对照siRNA序列(siRNA-NC)。
将OA成纤维样滑膜细胞按1×104/孔接种到24孔细胞培养板中，在37℃、5％CO2培养箱中细胞培养24h，在无双抗、含10％FBS的DMEM培养基中，转染按照脂质体转染试剂2000(购自于Invitrogen公司)的说明书转染，实验分为、阴性对照组(转染siRNA-NC)和实验组(20nM)(转染siRNA-C1GALT1C1)，siRNA浓度为20nM/孔，同时分别转染。
2、利用QPCR实验检测siRNA的干扰效率方法同实施例1。
3、统计学方法
实验都是按照重复3次来完成的，结果数据都是以平均值±标准差的方式来表示，采用SPSS13.0统计软件来进行统计分析的，干扰C1GALT1C1基因表达组与对照组之间的差异采用t检验，认为当P<0.05时具有统计学意义。
4、结果
结果如图2显示，siRNA-C1GALT1C1能够有效的抑制C1GALT1C1基因的表达，差异具有统计学意义(P<0.05)。
实施例3 C1GALT1C1基因对骨关节炎滑膜组织细胞增殖的抑制作用
1、细胞转染：按照实施例2的方法对OA成纤维样滑膜细胞进行siRNA-C1GALT1C1和siRNA-NC的转染。
2、转染24小时后加入3H-TdR(1μCi/孔)，再培养24小时，收集细胞，加液体闪烁液，β计数仪检测cpm值。
3、统计学方法
实验都是按照重复3次来完成的，结果数据都是以平均值±标准差的方式来表示，采用SPSS13.0统计软件来进行统计分析的，干扰C1GALT1C1基因表达组与对照组之间的差异采用t检验，认为当P<0.05时具有统计学意义。
4、结果
结果如图3显示，与siRNA-NC组相比，转染siRNA-C1GALT1C1的细胞增殖变慢了，差异具有统计学意义(P<0.05)。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。
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Claims (1)
1.抑制C1GALT1C1基因表达的试剂在制备治疗骨关节炎的药物中的应用；其特征在于，所述试剂是针对C1GALT1C1基因的siRNA，所述siRNA序列如SEQ ID NO.7和SEQ ID NO.8所示。
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| Woetzel D, et al..Accession number：GDS5403 / 219283_at，Rheumatoid arthritis and osteoarthritis: synovial tissues (Jena dataset).《GEO》.2014,第1页. * |
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