CN115120725B - Sdc1作为骨质破坏疾病药物治疗靶点的应用 - Google Patents
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Abstract
本发明提供一种SDC1作为骨质破坏疾病药物治疗靶点的应用,涉及生物医药技术领域。本发明还提供一种SDC1抑制剂在制备治疗骨质破坏疾病药物中的应用,SDC1抑制剂为SDC1的siRNA或单克隆抗体,SDC1抑制剂siRNA或单克隆抗体可用于制备治疗骨质破坏疾病的药物。
Description
技术领域
本发明涉及生物医药技术领域,特别是涉及SDC1作为骨质破坏疾病药物治疗靶点的应用。
背景技术
在正常骨组织中,破骨细胞(Osteoclasts,OCs)介导的骨吸收和成骨细胞(Osteoblasts,OBs)介导的骨形成之间维持动态平衡和偶联。但在骨质疏松、肿瘤骨转移、类分湿性关节炎和某些炎症性疾病中,破骨细胞被过度激活,平衡被打破而发生骨质丢失。人外周血中的单核细胞,在RANKL(receptor activation of nuclear factor NF-κBligand)及MCSF的作用下,能分化形成具有强骨吸收功能的多核破骨细胞,吸收骨质。破骨细胞过度激活会造成骨质丢失、引发骨折疼痛,继发反应性骨增生甚至残疾。在许多疾病比如绝经后骨质疏松、肿瘤骨转移、炎症相关骨破坏(如类风湿性关节炎、脓毒性关节炎、银屑病关节炎、牙周炎等)中均存在破骨细胞过度激活。
破骨细胞过度活化相关骨破坏病人数量庞大,仅原发性骨质疏松全世界就有上亿病人。此外,骨转移在进展期肿瘤十分常见。乳腺癌、前列腺癌等在疾病后期转移入骨后与成骨细胞、骨基质细胞和破骨细胞等相互作用,释放一系列细胞因子激活RANKL及非RANKL依赖性信号通路过度激活破骨细胞,造成溶骨性病变、引发病人剧烈疼痛及病理性骨折,降低病人活动能力和生活质量。
靶向抑制破骨细胞是治疗破骨细胞相关骨破坏的有效途径,然而现今临床上可供选择的药物较少,主要是RANKL单克隆抗体地诺单抗及双磷酸盐类。地诺单抗是2010年6月FDA批准上市的RANKL单克隆抗体,对破骨细胞增值、分化和生存起重要作用。但最近报导,地诺单抗可使高达11.4%的病人在用后2-52个月内出现颌骨坏死,另在停药后能导致自发性椎体骨折风险增加。双磷酸盐药物能抑制破骨细胞的附着和骨吸收,改善骨破坏。但长期使用可能导致颌骨坏死(可致残)等副作用。此外,多个针对RANKL之外新靶点破骨细胞抑制剂如组织蛋白酶K(CTSK)抑制剂等均仍处于临床研究阶段。因此,在破骨细胞和相关疾病治疗领域,探索新型临床可用的破骨细胞抑制剂及靶点仍具有较大的现实意义。
发明内容
基于此,有必要针对上述问题,提供一种SDC1作为骨质破坏疾病药物治疗靶点的应用。
细胞膜上的SDC1又称CD138,是含HSPG的硫酸乙酰肝素蛋白多糖。申请人发现SDC1可作为骨质破坏疾病药物治疗靶点,通过沉默或下调SDC1基因的表达、抑制SDC1活性、降解SDC1等方式可治疗骨质破坏疾病。可以理解的,沉默或下调SDC1基因的表达,包括但不限于针对SDC1表达基因进行基因编辑、降低mRNA的转录,抑制SDC1蛋白的翻译等手段实现。
在其中一个实施例中,所述药物抑制SDC1的表达。
在其中一个实施例中,所述骨质破坏疾病选自:骨质疏松、肿瘤转移骨破坏、风湿性关节炎、脓毒性关节炎、银屑病关节炎、牙周炎中的至少一种。
本发明还提供一种SDC1抑制剂在制备治疗骨质破坏疾病药物中的应用。
申请人发现SDC1在破骨细胞表达高,靶向抑制SDC1的抑制剂,如抑制SDC1活性的物质、或降解SDC1的物质、或降低SDC1表达水平的基因工具,可用于治疗骨质破坏疾病,缓解其所引起的疼痛。
在其中一个实施例中,所述SDC1抑制剂选自:siRNA、dsRNA、miRNA、核酶或shRNA。这些SDC1抑制剂均为降低SDC1表达水平的基因工具。
在其中一个实施例中,所述SDC1抑制剂为SDC1的siRNA。
在其中一个实施例中,所述siRNA为SDC1-Mus-siRNA,所述SDC1-Mus-siRNA正反义链的核苷酸序列为:
GGAAGGACGUGUGGCUGUUTT(SEQ ID NO.5),
AACAGCCACACGUCCUUCCTT(SEQ ID NO.6)。
在其中一个实施例中,所述siRNA为SDC1-homo-siRNA,所述SDC1-homo-siRNA正反义链的核苷酸序列为:
GACUUCACCUUUGAAACCUTT(SEQ ID NO.13),
AGGUUUCAAAGGUGAAGUCTT(SEQ ID NO.14)。
在其中一个实施例中,所述SDC1抑制剂为SDC1的单克隆抗体。SDC1的单克隆抗体可与SDC1特异性结合,抑制SDC1活性。
本发明还提供一种SDC1抑制剂在制备抑制破骨前体细胞分化成破骨细胞药物中的应用,所述SDC1抑制剂为本发明所述的SDC1抑制剂。
本发明还提供一种SDC1抑制剂在制备抑制破骨细胞骨吸收活性药物中的应用,所述SDC1抑制剂为本发明所述的SDC1抑制剂。
本发明还提供一种SDC1抑制剂在制备抑制破骨细胞生成药物中的应用,所述SDC1抑制剂为本发明所述的SDC1抑制剂。
本发明还提供一种用于治疗骨质破坏疾病的药物组合物,包括本发明所述的SDC1抑制剂,以及药学上可接受的辅料。
与现有技术相比,本发明具有以下有益效果:
发明人的研究团队发现SDC1直接参与破骨细胞的生成和骨吸收功能,并可能成为破骨细胞分化抑制剂新靶点。本发明首次发现SDC1-siRNA能靶向抑制RANKL诱导的小鼠破骨细胞生成及其骨吸收活性。此外,使用人SDC1-siRNA可显著抑制骨质疏松及肿瘤骨转移病人破骨细胞的过度生成和骨吸收活性;使用SDC1的单克隆抗体可显著抑制骨折病人破骨细胞生成。本发明的SDC1抑制剂SDC1-siRNA和单克隆抗体对破骨细胞生成和骨吸收活性具有抑制作用,可用于治疗骨质破坏相关的疾病。
附图说明
图1为三条SDC1-siRNA在RAW264.7细胞及BMMs中的沉默效果(基因及蛋白水平)。
其中,A:RAW264.7细胞SDC1基因敲低q-PCR统计结果;B:敲低效率最高的SDC1-siRNA-2序列BMMs中SDC1基因敲低q-PCR统计结果;C:SDC1-siRNA-2在BMMs中SDC1蛋白敲低结果。(注:与NC对照组比较,*P<0.05,**P<0.01,***P<0.001,****P<0.0001)。
图2为SDC-siRNA抑制RANKL诱导的BMMs分化成破骨细胞的实验结果。
其中,A:SDC-siRNA干扰的破骨细胞成像图;B-C:TRAP+破骨细胞生成数目统计结果图。(注:与NC对照组比较,###P<0.001;与NC-siRNA+RANKL组比较,**P<0.01)。
图3为SDC-siRNA抑制RANKL诱导的BMMs分化为破骨细胞骨吸收活性的实验结果。
其中,A:骨吸收陷窝成像图;B:骨吸收陷窝面积统计图。(注:与NC对照组比较,####P<0.001;与NC-siRNA+RANKL组比较,***P<0.001)。
图4为SDC1-homo-siRNA抑制骨质疏松病人过度破骨细胞生成的实验结果。
其中,A:骨质疏松病人破骨细胞骨吸收陷窝成像图;B:骨吸收陷窝面积统计图。(注:与NC-siRNA+RANKL组比较,***P<0.001)。
图5为SDC1-homo-siRNA抑制骨质疏松病人过度破骨细胞骨吸收活性的实验结果。
其中,A:骨质疏松病人破骨细胞骨吸收陷窝成像图;B:骨吸收陷窝面积统计图。(注:与NC-siRNA+RANKL组比较,*P<0.05)。
图6为SDC1-homo-siRNA抑制乳腺癌骨转移病人过度破骨细胞生成的实验结果。
其中,A:乳腺癌骨转移病人破骨细胞成像图;B:TRAP+破骨细胞生成数目统计结果图。(注:与NC-siRNA+RANKL组比较,*P<0.05)。
图7为SDC1-homo-siRNA抑制乳腺癌骨转移病人破骨细胞过度骨吸收活性的实验结果。
其中,A:乳腺癌骨转移病人破骨细胞骨吸收陷窝成像图;B:骨吸收陷窝面积统计图。
图8为SDC1单克隆抗体抑制骨折病人破骨细胞生成的实验结果。
其中,A:破骨细胞成像图及骨吸收陷窝成像图;B:TRAP+破骨细胞生成数目统计结果图。(注:与RANKL组比较,**P<0.01)。
具体实施方式
为了便于理解本发明,以下将给出较佳实施例对本发明进行更全面的描述。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
以下实施例中,未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。除非特殊说明,实施例中所用到的试剂、材料和设备,均为市售产品。
实施例1
SDC1-siRNA明显敲低BMMs(小鼠骨髓巨噬细胞)中SDC1基因及蛋白表达。
我们研究发现,SDC1基因表达在RANKL、肿瘤上清激活的破骨前体细胞中均出现显著升高,并推测靶向抑制SDC1基因表达后可能具有抑制破骨细胞形成的作用。针对鼠及人SDC1基因设计四条(鼠(Mus)三条,人(homo)一条)特异SDC1-siRNA序列,具体序列如表1所示,交由吉玛基因公司合成。
表1 RNA oligo序列表
1细胞培养
细胞来源:RAW264.7小鼠单核巨噬细胞白血病细胞购自中国科学院细胞库;C57BL/6小鼠购自广东省实验动物中心。
培养方法:(1)选用生长状态良好的RAW264.7细胞按2×104/孔密度接种于24孔板,于37℃、5%CO2培养箱孵育。(2)取8周的C57BL/6小鼠股骨和胫骨,剥离肌肉后剪去两端,用注射器吸取冰预冷的α-MEM反复冲洗骨髓腔直至发白,于37℃、5%CO2培养箱放置30分钟后取上清悬液,加入MCSF,37℃、5%CO2培养箱放置静置48h后消化得到BMMs。
2细胞转染
2.1取生长状态良好的RAW264.7细胞5×105个/孔铺板于六孔板中,稳定培养4-6小时后,按RNAiMAX Transfection Reagent说明书将SDC1-siRNA(吉玛基因)或对照NC-siRNA(吉玛基因)加入细胞,转染48小时后弃去上清液,收集RNA样品检测SDC1基因表达。
(1)RNA的提取:按照Promega的RNA提取试剂盒说明书提取RNA。
(2)RNA逆转录合成cDNA
按照Takara的试剂盒的逆转录说明书步骤,将总的RNA逆转录成cDNA。反应条件如下:37℃,15min;85℃,5s;4℃,∞。
表2 qRT-PCR反应液配制
(3)实时荧光定量PCR(Real-time PCR,qRT-PCR)
在冰上用Promega公司提供的Real-time PCR试剂盒,按照说明书配制qRT-PCR反应液,具体配方见表3。配制好上机反应液后,设置qRT-PCR扩增的程序:95℃,30秒;95℃,5秒;60℃,34秒;45个循环,上机检测。
表3 qRT-PCR反应液配制
实验结果以GAPDH作为内参基因,阴性对照组样本作为校准样本,不同刺激组作为待测样本,采用2-ΔΔCt方法计算比较目的基因的表达变化。
表4 SDC1基因引物
2.2 5×105个/孔BMMs铺板于六孔板中,过夜贴壁后,按照RNAiMAXTransfection Reagent说明书将SDC1-siRNA-2(吉玛基因)或对照NC-siRNA(吉玛基因)加入细胞,转染48小时后收集RNA样品检测SDC1基因表达。
2.3 5×105个/孔BMMs铺板于六孔板中,过夜贴壁后,按照RNAiMAXTransfection Reagent说明书将SDC1-siRNA(吉玛基因)或对照NC-siRNA(吉玛基因)加入细胞,转染48小时后收集样品检测SDC1蛋白表达。
(1)总蛋白的提取
①用预冷的PBS洗两遍细胞后加入新配好的裂解液100ul/孔,静置裂解10min后用刮子刮孔板底部的贴壁细胞。
②用移液枪吸取刮下的细胞液移入1.5ml EP管,冰上裂解15min。
③离心机4℃,12000g离心15min。
(2)蛋白定量
按照BCA蛋白定量试剂盒说明书进行蛋白定量。100℃金属浴中煮5min,使蛋白变性。
(3)SDS-PAGE电泳
①配胶:按表5配制分离胶和浓缩胶,再加蛋白样品(30μg/泳道)。按浓缩胶80V,15min,分离胶120V,80min进行电泳。
表5分离胶和浓缩胶配方
②转膜:按照下面的顺序排序压膜:(下)黑压膜夹→黑海绵→白滤纸→胶→PVDF膜(标记记号)→白滤纸→黑海绵→透明压膜夹(上)。PVDF膜和胶夹好后,100V恒压转膜90min。
③封闭:封闭盒中倒入用TBST配置的5%脱脂牛奶,常温封闭1h。
④一抗孵育:封闭后,将切好的膜卷进相应的SDC1和β-actin一抗中,4℃孵育过夜。
⑤洗膜孵二抗:TBST溶液洗膜,每10min洗1次,共3次。洗完后二抗孵育1h。用TBST溶液洗膜,每5min 1次,共6次。
⑥曝光显影:将现配置好的ECL发光液覆盖在PVDF膜表面,轻微左右摇晃将ECL发光液晃匀,避光孵育2min,放入仪器内进行图像的成像。
⑦灰度分析:将目的条带与内参条带用Image J软件进行光密度分析。
结果如附图1A-C,从附图1A中可以看出三条SDC1-siRNA序列中,SDC1-siRNA-2序列具有最高敲低SDC1基因表达的效率,基于此,后续转染实验将采用SDC1-siRNA-2序列进行;从附图1B中可以看出,与NC-siRNA组比较,加入SDC1-siRNA-2可明显敲低BMMs细胞中SDC1基因的表达,干扰效率具有统计学差异;从附图1C中可以看出:与NC-siRNA组比较,SDC1-siRNA-2可敲低BMMs细胞中SDC1蛋白的表达;这说明SDC1-siRNA可以在细胞水平敲低SDC1的基因和蛋白的表达。
实施例2
SDC1-siRNA抑制RANKL诱导破骨前体细胞分化成破骨细胞。
BMMs细胞1×105个/ml接种于孔板中,静置过夜后分别加入SDC1-siRNA-2和对照NC-siRNA进行转染后,加入RANKL、M-CSF诱导破骨细胞生成后行TRAP染色,在显微镜下观察拍照,并按TRAP阳性并且大于3个核的为破骨细胞进行计数(紫红色、多个细胞融合在一起的为破骨细胞),结果如图2所示。从图2A-C中可以看出,与NC-siRNA对照组相比,SDC1-siRNA-2组的紫红色破骨细胞显著减少,说明SDC1-siRNA能显著抑制体外RANKL诱导BMMs细胞生成破骨细胞。
实施例3
SDC1-siRNA抑制RANKL诱导的破骨细胞骨吸收活性。
BMMs细胞接种于包被人工骨片的Osteo Assay孔板中静置过夜,分别加入SDC1-siRNA-2和对照NC-siRNA转染后加入RANKL和M-CSF进行诱导,第7天洗去细胞,在40×Nikon倒置光学显微镜光镜观察拍照,结果如图3A所示,通过Image-Pro Plus软件计算每孔中骨吸收面积百分比,结果如图3B所示。从结果中可以看出,与NC-siRNA对照组相比,SDC1-siRNA-2组的白色骨凹陷面积显著减少,说明SDC1-siRNA能显著降低破骨细胞在骨片上形成的骨凹陷面积,即SDC1-siRNA对于破骨细胞的骨吸收活性具有抑制作用。
实施例4
SDC1-homo-siRNA抑制骨质疏松病人过度破骨细胞生成。
采用分选试剂盒分选出骨质疏松病人CD14+单核巨噬细胞用做破骨前体细胞,将细胞接种于孔板中贴壁,分别加入SDC1-homo-siRNA和对照NC-homo-siRNA进行转染,加入低剂量RANKL,M-CSF诱导破骨细胞生成后进行TRAP染色。在显微镜下观察拍照,并按TRAP阳性并且大于3个核的为破骨细胞进行计数。骨质疏松病人破骨细胞生成结果如图4所示,从图4A-B中可以看到,与NC-homo-siRNA组相比,SDC1-homo-siRNA的破骨细胞显著减少;说明SDC1-homo-siRNA能显著抑制骨质疏松病人破骨细胞过度生成。
实施例5
SDC1-homo-siRNA抑制骨质疏松病人过度破骨细胞骨吸收活性。
骨质疏松病人破骨前体细胞接种于孔板中贴壁稳定,分别加入SDC1-homo-siRNA和对照NC-homo-siRNA进行转染,加入低剂量RANKL,M-CSF诱导破骨细胞生成后洗去细胞并在40×Nikon倒置光学显微镜光镜观察拍照,通过Image-Pro Plus软件计算每孔中骨吸收面积百分比,结果如图5所示。图5A-B中可以看到,与NC-homo-siRNA组相比,SDC1-homo-siRNA组的白色骨凹陷面积显著减少,说明SDC1-homo-siRNA能显著降低骨质疏松病人破骨细胞过度的骨吸收活性。
实施例6
SDC1-homo-siRNA抑制肿瘤骨转移病人过度破骨细胞生成。
采用分选试剂盒分选出肿瘤(乳腺癌)骨转移病人CD14+单核巨噬细胞用做破骨前体细胞。将细胞接种于孔板中贴壁稳定,分别加入SDC1-homo-siRNA和对照NC-homo-siRNA进行转染,加入低剂量RANKL,M-CSF诱导破骨细胞生成后进行TRAP染色。在显微镜下观察拍照,并按TRAP阳性并且大于3个核的为破骨细胞进行计数。乳腺癌骨转移病人破骨细胞生成结果如图6所示,从图6A-B中可以看到,与NC-homo-siRNA组相比,SDC1-homo-siRNA的破骨细胞显著减少;说明SDC1-homo-siRNA能显著抑制乳腺癌骨转移病人破骨细胞过度生成。
实施例7
SDC1-homo-siRNA抑制肿瘤骨转移病人破骨细胞过度骨吸收活性。
乳腺癌骨转移病人破骨前体细胞接种于孔板中贴壁稳定,分别加入SDC1-homo-siRNA和对照NC-homo-siRNA进行转染,加入低剂量RANKL,M-CSF诱导破骨细胞生成后洗去细胞并在40×Nikon倒置光学显微镜光镜观察拍照,通过Image-Pro Plus软件计算每孔中骨吸收面积百分比,结果如图7所示。从图7A-B中可以看到,与NC-homo-siRNA组相比,SDC1-homo-siRNA组的白色骨凹陷面积显著减少,说明SDC1-homo-siRNA能显著降低乳腺癌骨转移病人破骨细胞过度的骨吸收活性。
实施例8
SDC1单克隆抗体抑制骨折病人破骨细胞生成。
采用分选试剂盒分选出骨折病人外周血CD14+单核巨噬细胞用作破骨前体细胞。将细胞接种于孔板中贴壁稳定,抗体组加入1ug/ml的SDC1-antibody(苏州缔码生物科技有限公司,BME1000014),溶剂组加入等体积PBS;加入RANKL及M-CSF刺激破骨细胞生成后进行TRAP染色,在显微镜下观察拍照,并按TRAP阳性并且大于3个核的为破骨细胞进行计数,结果如图8所示。从8A-B结果中可以看出,与RANKL及PBS组相比,SDC1-antibody组的紫红色破骨细胞显著减少,说明SDC1单克隆抗体能显著抑制体外RANKL诱导骨折病人破骨细胞生成。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 南方医科大学
<120> SDC1作为骨质破坏疾病药物治疗靶点的应用
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<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 20
acacattggg ggtaggaaca 20
Claims (1)
1. SDC1抑制剂在制备治疗骨质破坏疾病药物中的应用;
所述SDC1抑制剂为SDC1的siRNA;
所述siRNA为SDC1-Mus-siRNA,所述SDC1-Mus-siRNA正反义链的核苷酸序列为:
GGAAGGACGUGUGGCUGUUTT(SEQ ID NO.5),
AACAGCCACACGUCCUUCCTT(SEQ ID NO.6);
或者,
所述siRNA为SDC1-homo-siRNA,所述SDC1-homo-siRNA正反义链的核苷酸序列为:
GACUUCACCUUUGAAACCUTT(SEQ ID NO.13),
AGGUUUCAAAGGUGAAGUCTT(SEQ ID NO.14)。
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CN111183157A (zh) * | 2017-10-02 | 2020-05-19 | 威特拉公司 | Cd138抗体分子及其用途 |
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CN111183157A (zh) * | 2017-10-02 | 2020-05-19 | 威特拉公司 | Cd138抗体分子及其用途 |
CN111630182A (zh) * | 2018-01-24 | 2020-09-04 | 基因泰克公司 | 用于治疗类风湿性关节炎(ra)的诊断和治疗方法 |
WO2020162638A1 (ja) * | 2019-02-08 | 2020-08-13 | 学校法人近畿大学 | 悪性腫瘍疾患の改善用組成物 |
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金华萍 ; 单一旦 ; 严奉国 ; 陈关福 ; .上颌骨肿块为首发症状的多发性骨髓瘤1例.口腔医学.2017,(12),全文. * |
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