CN103536907B - 利拉鲁肽在骨质疏松治疗药物中的应用 - Google Patents
利拉鲁肽在骨质疏松治疗药物中的应用 Download PDFInfo
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Abstract
本发明属于医药制备领域，特别涉及利拉鲁肽在骨质疏松治疗药物中的应用。本发明以OVX大鼠简历骨质疏松模型，用两种GLP-1受体激动剂药物利拉鲁肽和Exendin-4连续8周经行皮下注射，以观察GLP-1类药物长期作用对骨质疏松大鼠的影响。本发明的结果证明了，对于经典的骨质疏松模型OVX大鼠，GLP-1受体激动剂利拉鲁肽和Exendin-4可以改善其骨质疏松状态，利拉鲁肽可以用于骨质疏松治疗药物的制备。
Description
技术领域
本发明属于医药制备领域，特别涉及利拉鲁肽在骨质疏松治疗药物中的应用。
背景技术
糖尿病作为综合性的代谢性疾病，随着病程的日益进展会逐渐累及多个脏器，并发多种慢性疾病，如糖尿病肾病、糖尿病心血管疾病、糖尿病神经病变和糖尿病视网膜病变等。骨骼除了对人体起到支撑和保护作用外，更因为其处于一种骨形成和骨吸收的动态平衡，因而成为人体最大的代谢器官。骨质疏松是累及绝经后妇女和老年人的一种常见慢性疾病，而糖尿病也是中老年人群中的高发性疾病，糖尿病并发骨质疏松性骨折严重影响糖尿病患者的预后和生活质量。因此，如果一种糖尿病治疗药物在降糖的同时若也能够兼顾到改善骨质量，将会在临床应用中有更为显著的意义。
GLP-1受体激动剂是一类新型的降糖药物，目前在临床应用较多的主要有利拉鲁肽和Exendin-4，它们以一种葡萄糖依赖的方式促进胰岛素的分泌。此外，GLP-1受体激动剂还可以通过直接作用于肝脏、脂肪和肌肉组织产生类胰岛素样的作用。细胞研究也证实，除了胰岛β细胞和α细胞外，甲状腺C细胞、成骨细胞和骨髓间充质干细胞等影响骨代谢的细胞上也存在GLP-1受体。GLP-1受体激动剂的广泛作用及其受体的在上述细胞中的表达使研究者们开始越来越多的关注这种新型降糖药物对骨的影响。
Yamada研究小组发现GLP-1受体敲除的小鼠表现为皮质骨密度降低，骨脆性增加，破骨细胞数量增加以及骨吸收加强。另有Nuche-Berenguer小组分别通过STZ刺激诱导形成的2型糖尿病大鼠（STZ-T2D大鼠）、果糖诱导形成的胰岛素抵抗大鼠（IR大鼠）和高脂喂养的肥胖大鼠模型证实3天连续泵入GLP-1和Exendin-4可以改善大鼠的骨损伤。但是，较少研究报道GLP-1受体激动剂对骨质疏松模型OVX大鼠的作用。
本发明通过构建OVX大鼠模型，研究GLP-1受体激动剂对骨代谢的直接作用，同时在细胞层面上探讨该类药物对原代骨髓间充质干细胞和成骨细胞系分化的影响。
发明内容
本发明的目的是通过研究GLP-1受体激动剂对OVX大鼠骨质量的影响，提供一种GLP-1受体激动剂利拉鲁肽在骨质疏松治疗药物中的应用。
本发明以OVX大鼠简历骨质疏松模型，用两种GLP-1受体激动剂药物利拉鲁肽和Exendin-4连续8周经行皮下注射，以观察GLP-1类药物长期作用对骨质疏松大鼠的影响。利拉鲁肽是一种人GLP-1同源类似物，通过对第26位346位氨基酸的替代和化学修饰，使其皮下注射后半衰期长达13小时，能够在9-14小时内维持很高的药物浓度，因此一天内仅需注射一次。Exendin-4是从巨蜥唾液中提取出的GLP-1类似物，能够与GLP-1受体结合，产生类似于GLP-1的降糖作用。Exendin-4由于结构的不同而对GLP-1降解酶DPP-4不敏感，因此半衰期明显延长，已获美国FDA批准成为一种新型降糖药。我们的实验设计共分四组，假手术组、OVX组、OVX后给予利拉鲁肽处理组和OVX后给予Exendin-4处理组。
实验选用8周龄的雌性Wistar大鼠随机分配入组后进行双侧卵巢切除术和假手术，术后3个月骨质疏松状态基本形成，行活体micro-CT检测验证相较于假手术组，卵巢切除后的大鼠其股骨和椎体的骨皮质变薄，骨密度下降，松质骨微细结构出现明显的紊乱，各参数均有下降。在验证骨质疏松模型已形成后，我们分别给予实验组相应的药物处理，在连续8周进行皮下注射利拉鲁肽、Exendin-4或生理盐水后对各组大鼠腰椎行离体micro-CT检测。通二维图像和三维立体重建后的参数分析结果显示，OVX大鼠给予利拉鲁肽和Exendin-4处理后，相比于OVX对照组，BV/TV和骨小梁厚度（Tb.Th）明显提高，BS/TV和骨小梁间隙（Tb.Sp）明显下降。
本发明的结果证明了，对于经典的骨质疏松模型OVX大鼠，GLP-1受体激动剂利拉鲁肽和Exendin-4可以改善其骨质疏松状态，利拉鲁肽可以用于骨质疏松治疗药物的制备。
附图说明
图1为两月龄Wistar雌性大鼠卵巢切除3个月后假手术（SHAM）组和卵巢切除（OVX）组大鼠腰椎及股骨的活体Micro-CT检测结果。A-C图:分别为大鼠腰椎侧面、第五腰椎矢状面和股骨CT扫描图；D图:骨体积分数（Bone Volume/Total Volume）、骨小梁厚度（Trabecular Thickness）、骨小梁间隙（Trabecular Spacing）、皮质骨厚度（Cortical Thickness）和骨小梁模型指数（Trabecular Patern Factor）结果柱状图。*代表P<0.05。
图2为大鼠BMSCs流式检测结果。上排显示BMSC的表面标记物CD90阳性率为96.5%，CD44阳性率为92.7%;下排显示造血干细胞的表面标记物CD34阳性率仅为0.281%，淋巴细胞的表面标记物CD45阳性率仅为0.328%，内皮细胞的表面标记物CD31阳性率仅为0.227%。红色曲线区域代表阴性对照IgG1,其他颜色曲线区域代表相应标记物抗体。
图3为利拉鲁肽对大鼠BMSC成骨分化的影响。A图：上排显示7天碱性磷酸酶染色情况，下排显示21天茜素红矿化结节染色情况，左侧为对照组，右侧为利拉鲁肽组；B-D图：成骨标志基因Runx2、ALP和Col-1在0天、3天、7天、14天和21天的表达情况，GAPDH作为内参基因进行校正。E：NaOH溶解茜素红矿化结节后进行的定量分析结果。*代表P<0.05,**代表P<0.01。
图4为利拉鲁肽对大鼠BMSC成脂分化的影响。A图：大鼠BMSC成脂诱导第14天进行油红O染色结果；B图：油红O染色提取定量分析结果；C：real-timePCR检测两组细胞成脂诱导第14天PPARγ的表达情况，GAPDH作为内参基因进行校正。**代表P<0.01。
具体实施方式
下面结合实施例，对本发明作进一步说明：
模型构建
32只6周龄未交配雌性Wistar购于上海斯莱克动物实验中心，饲养于上海交通大学医学院附属瑞金医院动物实验中心屏障环境中，受上海交通大学医学院动物中心伦理委员会监管。实验大鼠每两只喂养于塑料笼盒中，给予标准饮食和正常饮水，标准饲料中钙含量0.9%，磷含量0.7%。屏障环境控制在温 度20～25℃，相对湿度50-60％，12小时光照循环(8am---8pm)。
Wistar大鼠6周龄进入屏障环境后使其适应2周，于大鼠8周龄时开始实验处理。32只大鼠随机分配入假手术（SHAM）组，双侧卵巢切除（OVX）组，双侧卵巢切除加利拉鲁肽干预（OVX+L）组和双侧卵巢切除加Exendin-4干预（OVX+E）组。卵巢切除手术完成后给予实验大鼠3个月时间形成骨质疏松状态，并在药物干预前行活体显微CT（in-vivo micro CT）扫描以验证模型动物的骨质疏松状态。药物干预时间为8周，OVX+L组每只大鼠每日皮下注射利拉鲁肽0.6U/kg,OVX+E每只大鼠每日皮下注射Exendin-420ug/kg,OVX组每只大鼠每日皮下注射等体积生理盐水。
双侧卵巢切除手术
各组实验大鼠用5%水合氯醛腹腔注射麻醉（8ml/1kg）后背侧向上固定于无菌手术台上，待大鼠呼吸心跳平稳后行卵巢手术切除。手术切口位于肋弓下1～2cm,脊柱两侧各1cm处，切口处脱毛面积2～3cm2，70%酒精消毒。OVX组大鼠开腹后沿输卵管找到卵巢，以无菌手术线结扎输卵管血管后切除末端结缔组织包裹的卵巢，之后行双层间断缝合关闭腹腔。SHAM组大鼠切除与卵巢约相同体积的脂肪组织后缝合。整个手术过程遵循无菌操作原作，手术器械均高压蒸汽消毒灭菌，大鼠术后肌肉注射青霉素20U/(kg)抗感染。
活体显微CT扫描（In-vivo micro CT）
SHAM组8只大鼠及OVX组、OVX+L组、OVX+E组随机抽取3只共9只卵巢切除大鼠行活体micro CT扫描。实验大鼠吸收入2.5%的异氟烷和氧混合气体麻醉后固定于操作板中，期间持续进行气体麻醉。本实验所用显微CT为the InveonTMCT scanner（Siemens,German），该仪器最小分辨率可达8.89μmx8.89μm x8.89μm。选择扫描参数如下：电压80kv，电流500μA，360度旋转，360steps，每转1度拍照1次，曝光时间：200ms，sum frame：3，每一个位置曝光3次，总共曝光时间为600ms，binng2，system magnification：low，effective pixel size：50μm。以dsf2，校准好的HU值重建图像。定量分析使用系统自带的Explore MicroView version2.2+Adance Bone Analysis(OE Health Care Co．)InveonTM Research Workplace with Trabecular Bone Analysis Tool软件。分析参数包括：骨密度(BMD)、骨体积分数(bone volume fraction，BV/TV)、骨小梁厚度(trabecular thichness，Tb.Th．)、骨小梁间隔(trabecular separation，Tb.Sp．)、骨小梁数量(trabecular number，Tb.N．)、皮质骨厚度（mean thickness）、皮质骨模式系数（trabecular pattern factor）。
细胞实验
流式细胞分析
1）加入1ml PBS重悬细胞，轻轻吹散细胞，用细胞计数板进行计数。
2）进行换算，取含8×106细胞数量的细胞悬液，离心800rpm，4分钟。加入800微升PBS重悬细胞，轻轻吹散细胞，分别将200微升细胞悬液加入流式管中。
3）分别加入相应的荧光抗体，并设置空白对照。
4）4摄氏度避光孵育30分钟。
5）4摄氏度离心，速度1500rpm，离心时间7分钟，吸掉上清，200微升固定液重悬细胞。
6）流式细胞仪检测。
大鼠原代骨髓间充质干细胞的分离和成骨、成脂诱导培养
1)配制含10%FBS，1%抗生素的α-MEM培养液，置于37℃水浴锅中预热20min；
2)4周雄性Wistar大鼠两只，断颈处死，70%酒精浸泡15分钟；
3)无菌超净台中分离双侧股骨和胫骨，剔除周围肌肉及结缔组织；
4)去除长骨两端，用注射器抽取已配制好的培养液，反复冲洗骨髓腔多次，直至髓腔变白，留取冲洗液；
5)将得到的骨髓冲洗液离心，1000g，4min，移弃上清，重悬下层细胞沉淀，将细胞悬液种植入培养皿中，记为P0代。8字摇匀后放入37℃、5%CO2培养箱中进行培养；
6)24小时后待细胞基本贴壁后首次换液：移弃培养液后用PBS清洗，之后加入新鲜培养液继续培养；
7)3天后再次换液，以后每2-3天换液一次，约一周后细胞集落已较大，集落之间无空白区域；去掉原培养液，0.25%Trypsin-EDTA消化后以1:2比例传代。
8)取P3生长状态良好的细胞，诱导前进行流式细胞分析实验以验证细胞纯度。成骨诱导的细胞按3x103/cm2接种于六孔板，约70-80%汇合时，加入成骨诱导液，实验组同时加入10nmol/L利拉鲁肽，每2-3天换液一次，分别于诱导第7d、14d、21d收取细胞RNA。成脂诱导的细胞按2x104/cm2铺板，细胞完全汇合会继续生长1-2天后在加入成脂诱导液，实验组同时加入10nmol/L利拉鲁肽。3天后换为成脂诱导维持液，1天后再次换为成脂诱导液，如此进行三个循环，使用维持液培养至14天，收取细胞。
实验结果
去卵巢大鼠活体Micro CT检测结果
2月龄雌性Wistar大鼠随机分配入假手术（SHAM）组和卵巢切除（OVX）组，两组大鼠手术完成后给予3个月的时间，待OVX组骨质疏松状态基本形成后对两组大鼠进行活体Micro CT检测以验证造模成功。结果显示，与SHAM组相比较，OVX大鼠相同部位的椎体，其周围骨皮质变薄，髓腔质地疏松，呈现中空状态（图1A、B），股骨干骺端骨小梁数量明显减少，阴影减弱，骨髓腔面积增大，股骨皮质厚度变薄（图1C）。通过对影像结果的数据分析可以得到OVX大鼠骨体积分数（Bone Volume/Total Volume）、骨小梁厚度（Trabecular Thickness）、和皮质骨厚度(Cortical Thichness)均显著下降，骨小梁间隙明显增加。骨小梁模型指数（Trabecular Pattern Factor）提示骨小梁结构的改变，该指标由小变大提示骨小梁的结构由板状结构向杆状结构转变，而板状结构比杆状结构能够承受更大的骨应力，因此当骨小梁模型指数变大时代表骨质量的下降。由图1D可以看到OVX大鼠骨小梁模型指数明显变大。
GLP-1受体长效激动剂利拉鲁肽对大鼠骨髓间充质干细胞（BMSCs）成骨、成脂分化的影响
取4周龄雄性Wistar大鼠的双侧股骨和胫骨，采用全骨髓贴壁的方法获得大鼠骨髓间充质干细胞，随着传代次数增加其纯度逐渐增加。由于骨髓中不仅含有间充质干细胞，同时还有大量造血干细胞、淋巴细胞和内皮细胞等，我们进 而通过流式细胞实验对骨髓间充质干细胞的纯度进行鉴定。由图3可以看到，BMSC的表面标记CD90和CD44为阳性，而造血干细胞的表面标记CD34、淋巴细胞的表面标记CD45以及内皮细胞的表面标记CD31均为阴性。
取P4代的大鼠骨髓基质干细胞，以3×103/cm2密度接种于6孔板，待细胞融合至80%加入成骨诱导液进行诱导，实验组同时加入10nmol/L利拉鲁肽进行刺激。分别在0天、3天、7天、14天和21天五个时间点收集细胞RNA,通过real-timePCR检测实验组和对照组成骨标志基因的表达情况，同时在7天进行碱性磷酸酶染色，21天进行茜素红矿化结节染色。由图3可以看到，利拉鲁肽在早期阶段有促进成骨分化的作用，成骨诱导第7天的碱性磷酸酶染色，利拉鲁肽组明显强于对照组（图3A），成骨标志基因Runx2、ALP和Col-1的表达也是利拉鲁肽组高于对照组（图3B-D），但随着诱导时间延长，利拉鲁肽促进成骨的作用逐渐减弱，第14天和21天各基因的表达水平在利拉鲁肽组和对照组之间差异性消失，21天的茜素红矿化结节染色也显示两组间无明显差异（图3A、E）。
另取P4代大鼠BMSCs进行成脂诱导，通过向培养液中添加各种成脂诱导剂以启动成脂分化过程中的关键基因PPARγ。实验组同时加入10nmol/L利拉鲁肽。实验以三天诱导一天维持的方式连续进行三轮成脂诱导刺激后于第14天对细胞进行油红染色。由图4可以看到，利拉鲁肽显著抑制大鼠BMSCs的脂肪分化，14天时对照组大部分基质干细胞已经分化成熟为脂肪细胞，形成大量脂滴，而利拉鲁肽组中只有少量细胞出现脂肪分化，且脂滴较小(图4A)。油红O染色提取定量分析结果也表明对照组脂肪分化程度明显高于利拉鲁肽组（图4C）。Real-time PCR亦检测到利拉鲁肽抑制了脂肪分化的关键基因PPARγ(图4B)。
Claims (1)
1.利拉鲁肽在制备治疗骨质疏松的药物中的应用。
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