CN110777201A - 骨桥蛋白在缺氧缺血性脑损伤中的应用 - Google Patents
骨桥蛋白在缺氧缺血性脑损伤中的应用 Download PDFInfo
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Abstract
本发明提供骨桥蛋白在缺氧缺血性脑损伤中的应用，属于生物医药和分子生物学技术领域。本发明利用蛋白质组学发现在HI新生小鼠损伤皮层中骨桥蛋白变化最为明显，进一步通过试验证明骨桥蛋白可能是抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环的潜在靶点，由此证明骨桥蛋白可作为缺氧缺血性脑损伤中诊断、预后的标志物和潜在治疗靶点，因此具有良好的实际应用之价值。
Description
技术领域
本发明属于生物医药和分子生物学技术领域，具体涉及骨桥蛋白在缺氧缺血性脑损伤中的应用。
背景技术
本发明背景技术中公开的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
围产期缺氧缺血性(hypoxic-ischemic,HI)脑损伤是由于围产期缺氧缺血所致的胎儿、新生儿脑损伤，它可导致脑代谢和功能的破坏、脑血流异常、血管渗漏、组织损伤和坏死以及相应的炎症改变，缺血后引起能量衰竭、离子梯度维持失常、乳酸堆积、氧自由基产生增加、脂代谢改变、触发花生四烯酸连锁反应、释放兴奋性神经介质、钙离子自身稳定功能失衡等，因此其仍然是新生儿死亡和长期智力、运动障碍的重要原因之一，然而，其潜在的机制仍不清楚。
骨桥蛋白(osteopontin，OPN)是一种分泌型磷酸化糖蛋白，是一种重要的细胞粘附及趋化因子，可由体内多种细胞(如成骨细胞、上皮细胞、活化的T淋巴细胞、单核/巨噬细胞系、神经细胞等)合成并分泌。作为一种细胞因子，OPN参与多种生命现象，如免疫调节及炎症反应、肿瘤的浸润转移等。它主要通过两种机制发挥细胞信号分子的作用：一是以分子内谷氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp，RGD)基元与整合蛋白家族分子结合；二是与细胞表面黏附性糖蛋白CD44以非RGD依赖方式结合。两种作用方式均通过激活细胞内特异性信号传导系统而介导细胞粘附、迁移和增殖。目前，OPN在缺氧缺血性脑损伤中的作用和潜在机制尚不清楚。
发明内容
针对现有技术中的不足，本发明的目的在于提供骨桥蛋白在缺氧缺血性脑损伤中的应用。本发明利用蛋白质组学发现在HI新生小鼠损伤皮层中骨桥蛋白(OPN)变化最为明显，进一步通过试验证明骨桥蛋白可能是抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环的潜在靶点，因此骨桥蛋白可作为缺氧缺血性脑损伤中诊断、预后的标志物和潜在治疗靶点。
本发明的第一个方面，提供检测骨桥蛋白表达水平的物质在制备检测、诊断或预测缺氧缺血性脑损伤进展试剂中的应用。
根据蛋白质组学和免疫组化等试验证明，OPN在HI病灶区表达明显增多；同时免疫荧光显示在HI病灶区OPN与阳性小胶质细胞共定位，而星形胶质细胞与神经元不与OPN重合；而小胶质细胞免疫荧光染色显示缺氧缺血性脑损伤可引起小胶质细胞的激活并释放炎症因子，因此，OPN可用于检测、诊断或预测缺氧缺血性脑损伤进展。
其中，所述缺氧缺血性脑损伤进展包括缺氧缺血性脑损伤的不良预后。
本发明的第二个方面，提供一种用于检测、诊断或预测缺氧缺血性脑损伤进展的组合物，其包含基于高通量测序方法和/或基于定量PCR方法和/或基于探针杂交方法检测OPN基因及其表达产物表达情况的物质；或者基于免疫检测方法检测OPN蛋白表达情况的物质。
本发明的第三个方面，提供抑制OPN基因及其表达产物(包括OPN mRNA和OPN蛋白)和/或活性降低的物质在如下a)-h)至少一种中的应用：
a)抑制HI诱导的脑损伤；
b)抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环；
c)降低缺氧缺血性脑损伤导致的脑梗死面积；
d)减轻缺氧缺血性脑损伤导致的脑水肿情况；
e)抑制缺氧缺血性脑损伤导致的炎症因子表达；
f)降低外周入侵巨噬细胞的数量；
g)抑制外周入侵巨噬细胞产生的OPN；
h)抑制缺氧缺血性脑损伤诱导的CD11b+/CD45high免疫细胞的募集。
其中，所述e)应用中，
所述炎症因子包括但不限于CD16,CD11b，CD86,IL-1β和TNFα。
本发明的第四个方面，提供一种药物组合物，其包含抑制OPN基因及其表达产物和/或活性降低的物质。
所述抑制OPN基因及其表达产物和/或活性降低的物质，包括OPN蛋白特异的抗体、针对OPN mRNA的RNA干扰分子或反义寡核苷酸、小分子抑制剂、siRNA，以及实施慢病毒感染或基因敲除的物质；进一步的，所述抗体为人抗体或鼠抗体。
所述药物组合物具有治疗缺氧缺血性脑损伤的作用，具体的，所述治疗缺氧缺血性脑损伤至少包括如下a)-h)中任意一种或多种：
a)抑制HI诱导的脑损伤；
b)抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环；
c)降低缺氧缺血性脑损伤导致的脑梗死面积；
d)减轻缺氧缺血性脑损伤导致的脑水肿情况；
e)抑制缺氧缺血性脑损伤导致的炎症因子表达；
f)降低外周入侵巨噬细胞的数量；
g)抑制外周入侵巨噬细胞产生的OPN；
h)抑制缺氧缺血性脑损伤诱导的CD11b+/CD45high免疫细胞的募集。
其中，所述e)应用中，
所述炎症因子包括但不限于CD16,CD11b，CD86,IL-1β和TNFα。
进一步的，所述药物组合物还可以包含通常使用的适量载体、赋形剂及稀释剂。
本发明的有益技术效果：
本发明首次发现并证明了骨桥蛋白在缺氧缺血性脑损伤中的应用。具体的，本发明通过试验表明，缺氧缺血性脑损伤可引起小胶质细胞的激活并释放炎症因子，通过免疫组化显示OPN在HI病灶区表达明显增多，与蛋白组学结果一致；免疫荧光显示在HI病灶区OPN与阳性小胶质细胞共定位，而星形胶质细胞与神经元不与OPN重合；流式分析显示OPN主要由外周入侵的巨噬细胞表达，原驻小胶质细胞表达量较少。
同时利用OPN抗体阻断OPN的表达后，可明显降低脑梗死面积，抑制炎症因子的表达，降低外周入侵巨噬细胞的数量，以及外周入侵巨噬细胞产生的OPN。由此可证明OPN可能是抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环的潜在靶点。因此，骨桥蛋白可作为缺氧缺血性脑损伤中诊断、预后的标志物和潜在治疗靶点，具有良好的实际应用之价值。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为本发明实施例中差异蛋白表达相关图。其中，(A)热图显示HI组与Sham组的差异表达蛋白。(B)火山图，图中的每个点表示一个蛋白质。
图2为本发明实施例中HI激活小胶质细胞并释放炎症因子相关图。其中，(A)为免疫荧光染色图。标尺＝50μm，N＝4/组。(B)HI后72小时取损伤侧皮层，用qRT-PCR检测炎症因子CD16、CD11b、IL-1β表达图。N＝4/组，统计值＝平均值±标准差；*p<0.05,**p<0.01,数据使用独立样本T检验分析。
图3为本发明实施例中OPN的细胞定位相关图。其中，(A)为免疫组化结果图,标尺＝1mm。(A1)为(A)显示框内区域的放大图，标尺＝50μm。(B)为免疫荧光Iba-1与OPN共染色图，(B1)为(B)显示框内区域Iba-1+/OPN+染色放大图。(C)为免疫荧光GFAP、NeuN分别与OPN共染色图，(C1)为(C)显示框内区域GFAP+/OPN+或NeuN+/OPN+染色放大图。标尺＝50μm，N＝4/组。
图4为本发明实施例中OPN主要由浸润CD11b+/CD45high免疫细胞产生相关图。其中，(A)取HI后72小时损伤侧皮层细胞，使用流式分析仪根据细胞大小(FSC)和粒度(SSC)逐步对样本进行分析。(B)表示CD11b+/OPN+细胞。(C)通过CD11b+/OPN+画门圈出浸润的单核/巨噬细胞(CD11b+/OPN+/CD45high细胞)和原驻小胶质细胞(CD11b+/OPN+/CD45low细胞)。(D)表示CD11b+/OPN+细胞数量的定量分析。(E)表示CD11b+/OPN+/CD45high细胞数量的定量分析。(F)表示CD11b+/OPN+/CD45low细胞数量的定量分析。N＝4/组。统计值＝平均值±标准差；***p<0.001，数据使用独立样本T检验分析。
图5为本发明实施例中阻断OPN表达抑制HI诱导的脑损伤相关图。(A)HI后72小时，RT-PCR检测各组OPN mRNA水平，N＝4/组。(B)HI后72小时，各组用Western blot检测OPN蛋白水平，N＝3/组。(C)流式细胞仪分析HI后损伤侧皮层CD11b+/OPN+细胞群，N＝4/组。(D)HI后72小时测定脑含水量，N＝4/组。(E)TTC染色白色区域表示脑梗死面积，N＝4/组。统计值＝平均值±标准差；*p<0.05,**p<0.01,***p<0.001。数据使用单因素方差分析并用Bonferroni校正。
图6为本发明实施例中阻断OPN表达可抑制HI诱导的CD11b+/CD45high免疫细胞相关图。其中，(A)通过CD11b+/CD45low和CD11b+/CD45high门控识别原驻小胶质细胞和单核/巨噬细胞，右上象限为激活的单核/巨噬细胞(CD11b+/CD45high)，左下象限为原驻小胶质细胞(CD11b+/CD45low)，N＝4/组。(B)HI后72小时，RT-PCR检测各组炎症因子mRNA表达图。N＝4/组。统计值＝平均值±标准差；**p<0.01,***p<0.001，数据使用单因素方差分析并用Bonferroni校正。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
现结合具体实例对本发明作进一步的说明，以下实例仅是为了解释本发明，并不对其内容进行限定。如果实施例中未注明的实验具体条件，通常按照常规条件，或按照试剂公司所推荐的条件；下述实施例中所用的试剂、耗材等，如无特殊说明，均可从商业途径得到。
如前所述，目前，骨桥蛋白在缺氧缺血性脑损伤中的作用和潜在机制尚不清楚。
有鉴于此，本发明的一个具体实施方式中，提供检测骨桥蛋白表达水平的物质在制备检测、诊断或预测缺氧缺血性脑损伤进展试剂中的应用。
根据蛋白质组学和免疫组化等试验证明，OPN在HI病灶区表达明显增多；同时免疫荧光显示在HI病灶区OPN与阳性小胶质细胞共定位，而星形胶质细胞与神经元不与OPN重合；而小胶质细胞免疫荧光染色显示缺氧缺血性脑损伤可引起小胶质细胞的激活并释放炎症因子，因此，OPN可用于检测、诊断或预测缺氧缺血性脑损伤进展。
其中，所述缺氧缺血性脑损伤进展包括缺氧缺血性脑损伤的不良预后。
本发明的又一具体实施方式中，提供一种用于检测、诊断或预测缺氧缺血性脑损伤进展的组合物，其包含基于高通量测序方法和/或基于定量PCR方法和/或基于探针杂交方法检测OPN基因及其表达产物表达情况的物质；或者基于免疫检测方法检测OPN蛋白表达情况的物质。
优选采用Northern杂交方法、miRNA表达谱芯片、核酶保护分析技术、RAKE法、原位杂交检测OPN基因表达(转录)情况；
免疫检测方法优选包括但不限于免疫印迹法(Western Blot)、免疫荧光法、免疫酶标法(ELISA)、亲和免疫组织化学法和蛋白质芯片等；
本发明的又一具体实施方式中，提供抑制OPN基因及其表达产物(包括OPN mRNA和OPN蛋白)和/或活性降低的物质在如下a)-h)至少一种中的应用：
a)抑制HI诱导的脑损伤；
b)抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环；
c)降低缺氧缺血性脑损伤导致的脑梗死面积；
d)减轻缺氧缺血性脑损伤导致的脑水肿情况；
e)抑制缺氧缺血性脑损伤导致的炎症因子表达；
f)降低外周入侵巨噬细胞的数量；
g)抑制外周入侵巨噬细胞产生的OPN；
h)抑制缺氧缺血性脑损伤诱导的CD11b+/CD45high免疫细胞的募集。
本发明的又一具体实施方式中，所述e)应用中，
所述炎症因子包括但不限于CD16,CD11b，CD86,IL-1β和TNFα。
本发明的又一具体实施方式中，提供一种药物组合物，其包含抑制OPN基因及其表达产物和/或活性降低的物质。
本发明的又一具体实施方式中，所述抑制OPN基因及其表达产物和/或活性降低的物质，包括OPN蛋白特异的抗体、针对OPN mRNA的RNA干扰分子或反义寡核苷酸、小分子抑制剂、siRNA，以及实施慢病毒感染或基因敲除的物质；进一步的，所述抗体为人抗体或鼠抗体。
本发明的又一具体实施方式中，所述药物组合物具有治疗缺氧缺血性脑损伤的作用，具体的，所述治疗缺氧缺血性脑损伤至少包括如下a)-h)中任一种：
a)抑制HI诱导的脑损伤；
b)抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环；
c)降低缺氧缺血性脑损伤导致的脑梗死面积；
d)减轻缺氧缺血性脑损伤导致的脑水肿情况；
e)抑制缺氧缺血性脑损伤导致的炎症因子表达；
f)降低外周入侵巨噬细胞的数量；
g)抑制外周入侵巨噬细胞产生的OPN；
h)抑制缺氧缺血性脑损伤诱导的CD11b+/CD45high免疫细胞的募集。
本发明的又一具体实施方式中，所述e)应用中，
所述炎症因子包括但不限于CD16,CD11b，CD86,IL-1β和TNFα。
本发明的又一具体实施方式中，所述药物组合物还可以包含通常使用的适量载体、赋形剂及稀释剂。而且，根据通常的方法，可以制作成粉剂、颗粒剂、片剂、胶囊剂、混悬剂、乳剂、糖浆剂、喷雾剂等的口服剂、外用剂、栓剂及无菌注射溶液形式的剂型使用。
本发明的又一具体实施方式中，所述可以包含的载体、赋形剂及稀释剂等非药物活性成分在领域内是熟知的，本领域普通技术人员能够确定其符合临床标准。
本发明的又一具体实施方式中，所述载体、赋形剂及稀释剂包括但不限于有乳糖、葡萄糖、蔗糖、山梨糖醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石粉、硬脂酸镁和矿物油等。
本发明的又一具体实施方式中，本发明的药物组合物可通过已知的方式施用至体内。例如通过静脉全身递送或者局部注射递送到感兴趣组织中。可选地经由静脉内、经皮、鼻内、粘膜或其他递送方法进行施用。这样的施用可以经由单剂量或多剂量来进行。本领域技术人员理解的是，本发明中有待施用的实际剂量可以在很大程度上取决于多种因素而变化，如靶细胞、生物类型或其组织、待治疗受试者的一般状况、给药途径、给药方式等等。
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中为注明具体条件的试验方法，通常按照常规条件进行。
实施例1
1.动物模型建立和分组：孕15天以上C57BL/6J小鼠购自山东大学实验动物中心，将其在标准化的环境条件下饲养，该动物研究获得了山东大学动物伦理与福利委员会的批准。在小鼠出生后第7天，建立HI脑损伤模型，简言之，沿颈部中间偏右剪开皮肤暴露并结扎小鼠右侧颈总动脉，术后将其放回鼠笼休息1小时,之后小鼠被放置于8％氧气和92％氮气的缺氧箱缺氧1.5小时。
将小鼠随机分为3组，分别为Sham组、HI组、HI+OPN抗体组，术后72小时进行分子与病理检测。
2.蛋白组学分析技术：提取损伤侧皮层，用胰蛋白酶消化，TMT试剂标记，由诺禾致源生物公司使用气液联合色谱进行组学分析。
3.免疫组化检测OPN在HI表达：HI后72小时取脑组织并用多聚甲醛固定，进行石蜡切片。选择一抗(OPN；1:100)于4℃过夜孵育，用DAB试剂盒染色，使用显微镜观察OPN表达量。
4.免疫荧光检测OPN细胞定位：石蜡切片用二甲苯脱蜡，无水乙醇脱二甲苯，10％驴血清室温封闭1小时，一抗(OPN,1:100；NeuN,1:1000；Iba-1,1:400；GFAP,1:100)4℃孵育12小时以上，然后在37℃恒温箱孵育荧光二抗30分钟,DAPI室温染色5分钟，用荧光显微镜拍照分析。
5.流式分析技术：HI后72小时,取小鼠大脑损伤侧皮层并将其轻柔研磨,用70μm筛网过滤并用含有0.2％牛血清白蛋白(BSA)-磷酸盐缓冲液(PBS)洗涤细胞2次，400×g离心10分钟,用40％与70％percoll溶液根据密度梯度进一步纯化细胞。加入流式抗体(CD11b,CD45)在4℃条件下孵育30分钟，离心、清洗，细胞固定液室温孵育20分钟以固定已结合的抗体，破膜液与胞内抗体(OPN)4℃共同孵育30分钟，最后用PBS重悬细胞，使用流式细胞仪分析。
6.注射OPN抗体：HI前24小时用10μl微量注射器以每分钟1μl的速度侧脑室注射OPN抗体(2μg/3.3μl),注射完抗体停留2分钟后再将注射器移出脑内，注射部位为λ缝到眼睛2/5的距离处。
7.TTC检测脑梗死面积：HI后72小时将整个脑取出，并将其切成四片，用2％的2,3,5-三苯基四唑氯铵染色(TTC)，37℃孵育20分钟。梗死面积(％)＝(对侧半球面积-同侧半球未损伤面积)/对侧半球面积×100％。
8.采用qRT-PCR检测炎性细胞因子表达水平：根据制造商提供的说明书使用RNA提取试剂盒从损伤侧皮层提取总RNA，用逆转录试剂盒转录为cDNA，之后采用SYBR Green Mix和目的引物进行qRT-PCR。利用CFX Connect实时系统对扩增产物进行分析。
9.Western blot检测OPN表达量：取小鼠损伤侧皮层组织，加入由苯基甲烷磺酰氟(PMSF)、磷酸酶抑制剂(PI)和RIPA缓冲液组成的蛋白裂解液进行裂解，使用BCA蛋白检测试剂盒对总蛋白浓度进行定量。经10％SDS-PAGE凝胶电泳进行浓缩与分离目的蛋白，一抗(OPN,1:1000；β-actin,1:1000)孵育12小时以上，二抗孵育1小时，使用化学发光成像系统进行显影分析。
实验结果
1.差异蛋白表达
如图1所示，(A)热图显示HI组与Sham组的差异表达蛋白，蛋白组学显示HI后，受损皮层中93种蛋白表达量升高，8种蛋白表达量降低，这些差异表达蛋白参与氧化应激、免疫反应、补体激活和细胞凋亡等过程。(B)火山图，图中的每个点表示一个蛋白质，结果显示HI后，受损皮层中OPN上调最为明显。
2.HI激活小胶质细胞并释放炎症因子
如图2所示，(A)免疫荧光染色结果显示Sham组中小胶质细胞胞体较小，伸出数支突起，并且形态不规则，为未激活状态。HI组中，小胶质细胞已被激活，胞体较大，细胞形态呈阿米巴状。(B)HI后72小时取损伤侧皮层，用qRT-PCR检测炎症因子CD16、CD11b、IL-1β，结果显示与Sham组相比，HI组中炎症因子表达量升高。
3.OPN的细胞定位
如图3所示，(A)免疫组化结果显示OPN在HI损伤侧皮层中的表达大量增多，而在对侧半球中表达较少。(B)免疫荧光Iba-1与OPN共染色，结果显示OPN在病灶区与活化的小胶质细胞/巨噬细胞(Iba1+)共定位。(C)免疫荧光GFAP、NeuN分别与OPN共染色，结果显示病灶区可见少量OPN颗粒堆积，但是星形胶质细胞(GFAP+)、神经元(NeuN+)不与OPN重合。
4.OPN主要由浸润CD11b+/CD45high免疫细胞产生
如图4所示，(A)取HI后72小时损伤侧皮层细胞，使用流式分析仪根据细胞大小(FSC)和粒度(SSC)逐步对样本进行分析。(B)表示CD11b+/OPN+细胞。(C)通过CD11b+/OPN+画门圈出浸润的单核/巨噬细胞(CD11b+/OPN+/CD45high细胞)和原驻小胶质细胞(CD11b+/OPN+/CD45low细胞)。(D)表示CD11b+/OPN+细胞数量的定量分析，结果显示HI组与Sham组相比损伤侧皮层CD11b+/OPN+细胞数增加。(E)表示CD11b+/OPN+/CD45high细胞数量的定量分析，结果显示与Sham组相比，HI诱导浸润CD11b+/OPN+/CD45high细胞数增加。(F)表示CD11b+/OPN+/CD45low细胞数量的定量分析,结果显示与Sham组相比，HI组CD11b+/OPN+/CD45low细胞数降低。
5.阻断OPN表达抑制HI诱导的脑损伤。
如图5所示，(A)HI后72小时，RT-PCR检测各组OPN mRNA水平,结果显示注射抗体组OPN表达量较HI组明显下调。(B)HI后72小时，各组用Western blot检测OPN蛋白水平，结果显示注射抗体组OPN表达量较HI组明显下调。(C)流式细胞仪分析HI后损伤侧皮层CD11b+/OPN+细胞群(紫红色矩形)，结果显示注射OPN抗体降低HI诱导的CD11b+/OPN+细胞数量。(D)HI后72小时测定脑含水量，结果显示注射OPN抗体可显著减轻HI诱导的水肿情况。(E)TTC染色白色区域表示脑梗死面积，HI后72小时测量损伤侧半球梗死面积为60.0±18.8％，而OPN抗体组的梗死面积为26.4±5.6％，注射OPN抗体组梗死面积明显减轻。
6.阻断OPN表达可抑制HI诱导的CD11b+/CD45high免疫细胞
如图6所示，(A)通过CD11b+/CD45low和CD11b+/CD45high门控识别原驻小胶质细胞和单核/巨噬细胞，右上象限(深绿色矩形)为激活的单核/巨噬细胞(CD11b+/CD45high)，左下象限(红色矩形)为原驻小胶质细胞(CD11b+/CD45low)，结果显示利用OPN抗体阻断OPN的表达后，可降低HI诱导的CD11b+/CD45high细胞的募集，而上调CD11b+/CD45low细胞的数量。(B)HI后72小时，结果显示利用OPN抗体阻断OPN的表达后，可降低HI诱导CD16,CD11b，CD86,IL-1β和TNFα炎症因子的表达。
本发明未尽事宜为公知技术。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
Claims (10)
1.检测骨桥蛋白表达水平的物质在制备检测、诊断或预测缺氧缺血性脑损伤进展试剂中的应用。
2.如权利要求1所述应用，其特征在于，所述缺氧缺血性脑损伤进展包括缺氧缺血性脑损伤的不良预后。
3.一种用于检测、诊断或预测缺氧缺血性脑损伤进展的组合物，其特征在于，包含基于高通量测序方法和/或基于定量PCR方法和/或基于探针杂交方法检测OPN基因及其表达产物表达情况的物质；或者基于免疫检测方法检测OPN蛋白表达情况的物质。
4.如权利要求3所述的组合物，其特征在于，采用Northern杂交方法、miRNA表达谱芯片、核酶保护分析技术、RAKE法、原位杂交中的任意一种检测OPN基因表达情况。
5.如权利要求3所述的组合物，其特征在于，免疫检测方法包括免疫印迹法、免疫荧光法、免疫酶标法、亲和免疫组织化学法和蛋白质芯片。
6.抑制OPN基因及其表达产物和/或活性降低的物质在如下a)-h)至少一种或多种中的应用：
a)抑制HI诱导的脑损伤；
b)抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环；
c)降低缺氧缺血性脑损伤导致的脑梗死面积；
d)减轻缺氧缺血性脑损伤导致的脑水肿情况；
e)抑制缺氧缺血性脑损伤导致的炎症因子表达；
f)降低外周入侵巨噬细胞的数量；
g)抑制外周入侵巨噬细胞产生的OPN；
h)抑制缺氧缺血性脑损伤诱导的CD11b+/CD45high免疫细胞的募集。
7.如权利要求6所述应用，其特征在于，所述炎症因子包括CD16,CD11b，CD86,IL-1β和TNFα。
8.一种用于治疗缺氧缺血性脑损伤的药物组合物，其特征在于，包含抑制OPN基因及其表达产物和/或活性降低的物质；
优选的，所述治疗缺氧缺血性脑损伤包括如下a)-h)至少一种或多种：
a)抑制HI诱导的脑损伤；
b)抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环；
c)降低缺氧缺血性脑损伤导致的脑梗死面积；
d)减轻缺氧缺血性脑损伤导致的脑水肿情况；
e)抑制缺氧缺血性脑损伤导致的炎症因子表达；
f)降低外周入侵巨噬细胞的数量；
g)抑制外周入侵巨噬细胞产生的OPN；
h)抑制缺氧缺血性脑损伤诱导的CD11b+/CD45high免疫细胞的募集；
优选的，所述炎症因子包括CD16,CD11b，CD86,IL-1β和TNFα。
9.如权利要求8所述的药物组合物，其特征在于，所述抑制OPN基因及其表达产物和/或活性降低的物质，包括OPN蛋白特异的抗体、针对OPN mRNA的RNA干扰分子或反义寡核苷酸、小分子抑制剂、siRNA，以及实施慢病毒感染或基因敲除的物质；进一步的，所述抗体为人抗体或鼠抗体。
10.如权利要求8所述的药物组合物，其特征在于，所述药物组合物还包含载体、赋形剂和稀释剂；或，
所述组合物为粉剂、颗粒剂、片剂、胶囊剂、混悬剂、乳剂、糖浆剂、喷雾剂等的口服剂、外用剂、栓剂及无菌注射溶液形式的剂型中的任意一种或多种。
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| CN112067820A (zh) * | 2020-06-28 | 2020-12-11 | 暨南大学 | Cd74蛋白在制备识别缺血损伤后脑中巨噬细胞亚群试剂盒中的应用 |
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| CN108938631A (zh) * | 2018-05-29 | 2018-12-07 | 四川大学华西第二医院 | Sag在制备治疗发育期缺氧缺血脑损伤疾病的药物中的应用 |
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| CN104189909A (zh) * | 2014-09-18 | 2014-12-10 | 中国人民解放军第三军医大学 | 肾上腺素受体拮抗剂在制备缓解脑损伤引起钠电流增加的药物中应用 |
| CN108938631A (zh) * | 2018-05-29 | 2018-12-07 | 四川大学华西第二医院 | Sag在制备治疗发育期缺氧缺血脑损伤疾病的药物中的应用 |
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