CN112915196B - Creg1蛋白用于预防或治疗索拉非尼诱导的心肌损伤的医药用途 - Google Patents
Creg1蛋白用于预防或治疗索拉非尼诱导的心肌损伤的医药用途 Download PDFInfo
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- CN112915196B CN112915196B CN202110274042.4A CN202110274042A CN112915196B CN 112915196 B CN112915196 B CN 112915196B CN 202110274042 A CN202110274042 A CN 202110274042A CN 112915196 B CN112915196 B CN 112915196B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
本发明涉及E1A激活基因阻遏子蛋白的医药用途，具体涉及CREG1蛋白或其活性片段用于预防和/或治疗索拉非尼诱导的心肌损伤及相关疾病的医药用途。实验证明，索拉非尼干预引起野生型C57BL/6J小鼠心功能下降，同时心肌组织中CREG1基因的mRNA和蛋白表达均显著下降，心肌损伤标志物明显增加。而外源性给予CREG1重组蛋白后，可显著降低索拉非尼诱导的心肌损伤和心功能异常。索拉非尼诱导的小鼠心肌细胞程序性坏死标志蛋白表达显著增加，死亡方式为程序性坏死；外源性给予CREG1重组蛋白可显著抑制索拉非尼诱导的心肌细胞程序性死亡发生，改善心功能。重组CREG1蛋白可以用于预防或治疗索拉非尼引起的心肌损伤及相关疾病。
Description
技术领域
本发明涉及E1A激活基因阻遏子(Cellular Repressor of E1A-stimulatedGenes，CREG1)蛋白的医药用途，具体涉及CREG1蛋白或其活性片段用于预防和/或治疗索拉非尼诱导的心肌损伤及相关疾病的医药用途。
背景技术
随着临床肿瘤治疗药物的快速发展，肿瘤患者的生存率与生存质量得到了较大改善。然而，抗肿瘤药物导致的心脏毒性也同时存在且不可忽视。2016年8月，欧洲心脏病学会(ESC)发布了首部用于指导临床实践的《2016ESC癌症治疗与心血管毒性科学声明》，对肿瘤治疗相关的心血管并发症，例如心肌功能不全与心力衰竭、冠状动脉疾病、心脏瓣膜病、心律失常、高血压、血栓栓塞性疾病、周围血管疾病与脑卒中、肺动脉高压及心包并发症等的发病机制、临床评估与管理、预防缓解策略及长期随访流程进行了阐述和推荐。由于肿瘤性心脏病的研究开展较晚，抗肿瘤药物心脏毒性的发生机制尚未阐明，进行抗肿瘤合并心血管并发症治疗的药物研究成为临床肿瘤研究亟待解决的问题。
临床上容易导致心脏毒性的抗肿瘤药物主要有蒽环类药物、氟嘧啶类药物、人表皮生长因子受体2(ErbB2/HER2)抑制剂曲妥珠单抗和酪氨酸激酶抑制剂等。其中，靶向治疗药物VEGF通路抑制剂—索拉非尼，因其具有抑制丝氨酸和酪氨酸激酶活性，已被临床上广泛用于肾细胞癌、肝细胞癌及黑色素瘤的一线治疗。然而，已有报道发现索拉非尼存在如下心血管风险：QT间期延长、高血压、左室射血分数下降、进展性心力衰竭及急性冠脉综合征和心肌梗死等。在随机双盲III期临床试验中发现，索拉非尼治疗组与安慰剂组比较出现明显的心脏缺血事件(2.7vs.0.04％，p＝0.01)。此外，索拉非尼还能够增加坏死性心肌细胞死亡，抑制骨髓来源干细胞生长和线粒体复合物II，III，V及电子传递链等作用，抑制心梗后血管再生。因此，研发对抗索拉非尼心脏毒性又不影响其抗肿瘤治疗效果的药物具有重要的临床应用前景。
CREG1是哈佛大学医学院Gill教授在Hela细胞中发现的一种抑癌基因。后续研究证实，CREG1是一个高度保守的小分子糖基化修饰蛋白，含有3个6磷酸甘露糖糖结合位点，其主要细胞定位为溶酶体、线粒体及核周内质网，也能够分泌到细胞外发挥功能。CREG1在成体各个组织和细胞中均广泛表达，未分化的组织和细胞中表达显著减少，是维持组织和细胞的分化稳态调控的重要分子。
近年来研究提示，CREG1在维持心肌细胞分化及稳态调控中发挥重要作用。在主动脉缩窄所致的心肌肥厚模型中，CREG1蛋白表达明显下降，过表达CREG1蛋白能够显著降低心肌纤维化情况。发明人前期研究实验也证实，CREG1重组蛋白具有明确的心肌保护作用。外源性给予CREG1蛋白能够改善缺血再灌注损伤引起的心肌细胞凋亡，显著抑制血管紧张素II诱导的小鼠心肌肥厚及心肌纤维化对抗心肌梗死小鼠心肌细胞凋亡和心肌重构发生，改善心功能。综上，CREG1是非常重要的心肌细胞稳态调控蛋白。然而，CREG1是否调控索拉非尼诱导的心肌损伤中目前未见相关报道。
发明内容
本发明的目的在于提供CREG1蛋白或其活性片段用于预防和/或治疗索拉非尼诱导的心肌损伤及相关疾病的医药用途。本发明通过大量实验证明，索拉非尼干预引起野生型C57BL/6J小鼠心功能下降，同时心肌组织中CREG1基因的mRNA和蛋白表达均显著下降，心肌损伤标志物明显增加。而外源性给予CREG1重组蛋白后，可显著降低索拉非尼诱导的心肌损伤和心功能异常。进一步研究发现，索拉非尼诱导的小鼠心肌细胞程序性坏死标志蛋白表达显著增加，死亡方式为程序性坏死；外源性给予CREG1重组蛋白可显著抑制索拉非尼诱导的心肌细胞程序性死亡发生，改善心功能。以上结果表明，重组CREG1蛋白可以用于预防或治疗索拉非尼引起的心肌损伤及相关疾病。
为实现上述目的，本发明采用以下技术方案。
CREG1蛋白或其活性片段在制备药物中的用途，所述药物用于预防和/或治疗索拉非尼及其相关酪氨酸激酸抑制剂诱导的心肌损伤及相关疾病。
涉及编码CREG1蛋白或其活性片段的核酸分子、表达CREG1蛋白或其活性片段的重组载体或重组细胞在制备药物中的用途，所述药物用于预防和/或治疗索拉非尼及其相关酪氨酸激酸抑制剂诱导的心肌损伤及相关疾病。
能够抑制CREG1蛋白或其活性片段表达下调或促进CREG1蛋白或其活性片段表达上调的试剂在制备药物中的用途，所述药物用于预防和/或治疗索拉非尼及其相关酪氨酸激酸抑制剂诱导的心肌损伤及相关疾病。
检测CREG1蛋白或其活性片段表达水平的试剂在制备试剂盒中的用途，所述试剂盒用于索拉非尼及其相关酪氨酸激酸抑制剂诱导的心肌损伤及相关疾病的预测和/或治疗效果、预后的评估。
CREG1蛋白或其活性片段用于筛选预防和/或治疗索拉非尼及其相关酪氨酸激酸抑制剂诱导的心肌损伤及相关疾病的药物的用途。
一种组合物，其含有CREG1蛋白或其活性片段、编码CREG1蛋白或其活性片段的核酸分子、表达CREG1蛋白或其活性片段的重组载体或重组细胞、或者能够抑制CREG1蛋白或其活性片段表达下调的试剂，以及任选的药学上可接受的载体或赋形剂，所述组合物在制备用于预防和/或治疗索拉非尼及其相关酪氨酸激酸抑制剂诱导的心肌损伤及相关疾病药物中的应用。
一种试剂盒，其含有检测CREG1蛋白或其活性片段表达水平的试剂，所述试剂盒用于索拉非尼及其相关酪氨酸激酸抑制剂诱导的心肌损伤及相关疾病的预测和/或治疗效果、预后的评估。
在本发明中，所述哺乳动物例如可以为大鼠、小鼠、犬、小型猪、猴、人等。
在本发明中，所述CREG1蛋白为重组CREG1蛋白，来源于哺乳动物，特别是来源于人。在本发明的实施方案中，所述CREG1基因的GenBank号为NM_003851.2。
在本发明中，所述CREG1蛋白的活性片段是指具有CREG1蛋白功能的片段，其可以为CREG1蛋白的一部分，也可以为CREG1蛋白的氨基酸序列经过缺失、添加或替换后得到的片段；制备或得到CREG1蛋白活性片段的方法为本领域所公知，例如该活性片段为包含CREG1蛋白与配体或受体结合的部分的片段，或者经过氨基酸的缺失、添加或替换后仍保留CREG1蛋白功能的片段。本领域技术人员公知，CREG1蛋白上有一些关键的氨基酸和活性密切相关，突变后会影响蛋白的活性，例如，CREG1蛋白第136及137位赖氨酸突变为丙氨酸，或者CREG1蛋白第141-144位氨基酸缺失突变后，都会影响蛋白的活性和功能(Sacher M,PNAS,2005；102(51):18326-18331)。本领域技术人员可以根据需要避开上述这些可能影响活性的位点，对其它位点进行缺失、添加或替换等改造，使得改造后的CREG1蛋白仍具有CREG1蛋白的活性或功能。
在本发明中，所述索拉非尼及其相关酪氨酸激酸抑制剂心肌损伤具有本领域公知的含义，是指使用索拉非尼及其相关酪氨酸激酸抑制剂后出现的左心室功能障碍性疾病。
在本发明中，所述预防和/或治疗索拉非尼及其相关酪氨酸激酸抑制剂心肌损伤是指抑制或减缓索拉非尼及其相关酪氨酸激酸抑制剂心肌损伤及相关疾病的发生。
在本发明中，所述通过检测CREG1蛋白或其活性片段表达水平用于预测和/或评估是指当血液、组织或细胞中的CREG1蛋白或其活性片段表达水平低于参考值时，即可以预测索拉非尼及其相关酪氨酸激酸抑制剂心肌损伤及相关疾病发生，或者评估其治疗效果或预后。
在本发明中，可以通过本领域公知的方法检测CREG1蛋白或其活性片段的表达水平，例如通过聚合酶链式反应扩增CREG1的mRNA并进行定量反应，或者用Western Blot检测CREG1蛋白表达水平。
在本发明中，所述蛋白的表达水平是指mRNA的水平或者蛋白的水平。
在本发明中，所述上调/下调组织/细胞中蛋白的表达是指提高或降低组织/细胞中蛋白水平或mRNA水平的至少20％、30％、40％、50％、60％、70％、80％、90％、100％，或者提高大于100％。其中所述的上调或下调是与未干预的组织进行比较。
在本发明中，所述能够抑制CREG1蛋白或其活性片段表达下调或促进CREG1蛋白或其活性片段表达上调的试剂为本领域所公知，例如为能够与CREG1蛋白或其活性片段结合的配体，或者能够提高CREG1蛋白表达水平的调控分子，如启动子、增强子等。
与现有技术相比，本发明的有益效果如下。
本发明通过大量实验证明，索拉非尼干预引起野生型C57BL/6J小鼠心功能下降，同时心肌组织中CREG1基因的mRNA和蛋白表达均显著下降，心肌损伤标志物明显增加。而外源性给予CREG1重组蛋白后，可显著降低索拉非尼诱导的心肌损伤和心功能异常。进一步研究发现，索拉非尼诱导的小鼠心肌细胞程序性坏死标志蛋白表达显著增加，死亡方式为程序性坏死；外源性给予CREG1重组蛋白可显著抑制索拉非尼诱导的心肌细胞程序性死亡发生，改善心功能。以上结果表明，重组CREG1蛋白可以用于预防或治疗索拉非尼引起的心肌损伤及相关疾病。
附图说明
图1是C57BL/6J小鼠索拉非尼心功能损伤模型的建立，其中A为小动物超声评价左心室收缩功能EF％(n＝5，*p<0.05，**p<0.01，与对照组相比)；B为小动物超声评价左心室收缩功能FS％(n＝5，*p<0.05，**p<0.01，与对照组相比与对照组相比)；C为WGA染色检测心肌细胞面积(n＝3)。
图2A-2E是索拉非尼诱导心肌组织发生程序性死亡，其中2A-2B是Western blot检测心肌组织中程序性死亡标志蛋白p-RIP3表达(n＝3，**p<0.01，与对照组相比)；其中2C-2D为Western blot检测索拉非尼干预前后原代心肌细胞中程序性死亡标志蛋白p-RIP3表达(n＝3，**p<0.01，与对照组相比)；其中2E为给予不同死亡方式拮抗剂干预检测索拉非尼诱导的心肌细胞死亡情况(n＝3，**p<0.01，与索拉非尼干预组相比)。
图3A-3E是索拉非尼诱导心肌组织和心肌细胞中CREG表达下降。其中3A-3B为荧光定量PCR检测心肌组织CREG1基因mRNA表达(n＝3)；其中3B-3C为Western blot检测心肌组织CREG1蛋白表达(n＝3，p<0.001，与对照组相比)；其中3D-3E为Western blot检测心肌组织CREG1蛋白表达(n＝3，p<0.001，与对照组相比)。
图4A-4E是外源性给予CREG重组蛋白干预显著减轻索拉非尼诱导的C57BL/6J小鼠心肌损伤。其中，4A为小动物超声评价左心室收缩功能EF％(n＝5，p<0.05与WT-索拉非尼组比较)；4B为小动物超声评价左心室收缩功能FS％(n＝5，p<0.01与WT-索拉非尼组比较)；4C-4E为Western blot检测索拉非尼干预前后心肌组织中程序性死亡标志蛋白p-RIP3和CREG1表达(n＝4，p<0.001，与对照组相比较)。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明的实验数据均为百分数。两样本率的比较应用卡方检验，统计学处理均应用SPSS 22.0软件包处理。P<0.05为有统计学差异。
实施例1C57BL/6J小鼠索拉非尼干预的心肌损伤模型的建立。
1、C57BL/6J小鼠索拉非尼心肌损伤模型的建立。
采用随机表法，给予8周龄的雄性C57/BL6小鼠10只，随机分为实验组及对照组。实验组为灌胃索拉非尼(40mg/kg/d)，对照组采用生理盐水灌胃。灌胃21天后为实验终点。
2、小动物超声评价小鼠心脏舒张和收缩功能。
采用异氟烷麻醉小鼠，用Vevo2100型小动物心脏超声仪进行心脏舒张功能和收缩功能检测。同步记录小鼠心电、呼吸等生理参数，心率维持在450次/min左右，心率稳定1min后前胸涂抹耦合剂行超声检查并采集图像，经小动物超声仪自带心功能分析软件进行测量和分析心脏收缩功能指标EF％和FS％。
结果显示：与对照组相比，索拉非尼组左心室收缩功能指标EF％和FS％也显著低于对照组，如图1A-1B所示，表明索拉非尼干预可导致小鼠收缩功能下降。
3、麦胚凝集素(WGA，Triticum vulgaris lectin)染色评价心脏大体形态。
1)取材心肌组织，经4％甲醛固定后，常规石蜡包埋，5μm切片。
2)切片常规用二甲苯脱蜡，经各级乙醇至水洗：二甲苯(I)5min→二甲苯(II)5min→100％乙醇2min→95％的乙醇1min→80％乙醇1min→75％乙醇1min→蒸馏水洗2min。
3)WGA可以特异性的结合心肌细胞膜上的一种糖蛋白。WGA可以把心肌细胞膜染出来，然后用专门的软件对染出的细胞进行直径、面积等的测量，能够分析心肌细胞是否肥大。
4)切片-抗原修复-滴加WGA工作，温箱孵育30分子-染核封片-显微镜镜检。
5)显微镜下观察形态并照相保存用于统计分析。
结果显示：与对照组相比，索拉非尼干预小鼠心肌细胞面积显著增大，如图1C所示。
实施例2索拉非尼干预诱导心肌组织和细胞发生程序性死亡。
1、Western blot检测心肌组织程序性死亡蛋白表达。
Western Blot方法检测程序性死亡标志蛋白RIP3和P-RIP3表达情况。采用RIPA裂解液提取对照组和索拉非尼干预组小鼠心肌组织蛋白，采用BCA比色法试剂盒测定蛋白质浓度。用Western Blot方法检测CREG蛋白表达。
具体如下：将40μg蛋白在95℃煮沸5min后，经10％分离胶行SDS-PAGE电泳，判断电泳终止时间。以90V的电压将样品转到纤维素膜上，时间为2h；在TBS-T稀释的5％脱脂奶粉中常温封闭2h后加入一抗4℃孵育过夜。分别以抗RIP3抗体(1:1000，美国Abcam公司)、抗p-RIP3抗体(1:1000，美国Abcam公司)和抗GAPDH抗体(1：1 000，美国Abcam公司)作为一抗，以辣根过氧化物酶标记羊抗兔抗体(美国Cell signaling公司)作为二抗，行Western Blot检测，用ECL试剂盒(美国GE公司)发光显影。
结果显示：与对照组相比，索拉非尼干预组小鼠心肌组织中程序性坏死标志蛋白p-RIP3/RIP3蛋白比例显著升高，如图2A-2B所示，表明索拉非尼干预小鼠心肌组织发生程序性死亡。
2、Western blot检测索拉非尼干预后原代心肌细胞中程序性死亡蛋白表达。
1)原代心肌细胞培养：采用常规体外胶原酶消化和梯度贴壁方法制备乳鼠原代心肌细胞。
2)Western Blot方法检测程序性死亡标志蛋白RIP3和p-RIP3表达情况。采用RIPA裂解液提取对照组和索拉非尼(20μM,24小时)干预组小鼠原代心肌细胞蛋白，采用BCA比色法试剂盒测定蛋白质浓度。用Western Blot方法检测p-RIP3/RIP3蛋白表达。具体方法见实施例2-1。结果显示：与对照组相比，给予索拉非尼干预的小鼠原代心肌细胞发生程序性死亡，如图2C-2D所示。
3、应用CCK8细胞活性实验及干预剂抑制研究检测心肌细胞死亡方式。
1)将细胞种在96孔板中，干预后进行检测。
2)将细胞上清更换为90μL新鲜的无血清培养基，每孔加入10μL CCK8试剂，培养2小时后，在450nm波长处检测OD值。
3)在给予索拉非尼干预的同进，给予不同类型的心肌死亡抑制剂进行死亡方式的检测(Nec-1：程序性死亡抑制剂；Fer-1:铁死亡抑制剂；VX-765:细胞坏死抑制剂；VDAC:细胞凋亡抑制剂)。结果显示：加入程序性死亡抑制剂能够挽救索拉非尼干预引起的心肌细胞死亡。证实索拉非尼诱导心肌细胞发生程序性死亡，如图2E所示。
实施例3索拉非尼干预心肌组织中CREG1表达下降。
1.荧光定量PCR方法检测索拉非尼干预前后心肌组织CREG1基因mRNA表达。
采用Promega试剂盒提取组织RNA，并采用TakaRa逆转录试剂盒进行逆转录反应获取cDNA，之后用SYBGreen方法进行定量PCR反应。定量PCR引物序列如下：
结果显示：与对照组相比，索拉非尼组CREG基因mRNA表达水平无明显变化，如图2A所示。
2、Western blot检测心肌组织CREG蛋白表达。
为检测CREG在索拉非尼诱导的心肌损伤小鼠心肌组织中的表达情况，WesternBlot方法检测CREG蛋白表达。具体方法见实施例1。分别以抗CREG抗体(1:1000，美国Abcam公司)和抗GAPDH抗体(1：1 000，美国Abcam公司)作为一抗，以辣根过氧化物酶标记羊抗兔抗体(美国Cell signalling公司)作为二抗，行Western Blot检测，用ECL试剂盒(美国GE公司)发光显影。
结果显示：与对照组相比，索拉非尼干预组心肌组织CREG蛋白表达显著降低，如图2B-2C所示。
实施例4外源性给予CREG重组蛋白可显著减轻索拉非尼干预的C57BL/6J小鼠心功能损伤。
1、在索拉非尼诱导的心功能损伤小鼠模型上给予外源性CREG蛋白干预。
将8周龄雄性C57BL/6J小鼠皮下埋置CREG微量渗透泵(rCREG，300μg/kg.d)同时给予索拉非尼灌胃的心肌损伤模型。对照组小鼠皮下埋置等量PBS的微量渗透泵(WT)，每组5只。具体方法按照实施例1中方法建立索拉非尼小鼠心功能损伤模型。
2、小动物超声评价小鼠心脏收缩功能。
具体见实施例1。
结果显示：与WT-对照组小鼠相比，CREG过表达组小鼠心脏收缩功能均无明显变化如图4A-4B所示。当给予索拉非尼刺激后，WT组小鼠和CREG过表达组小鼠收缩功能如图4A-4B所示，均显著下降，但CREG过表达组小鼠心功能较WT组小鼠明显升高，具有显著的统计学意义，如图4A-4B所示，表明CREG过表达可改善索拉非尼诱导的心功能损伤。
3、Western blot方法检测CREG过表达组小鼠索拉非尼干预后心肌组织程序性死亡标志蛋白表达。具体见实施例2。
结果显示：与WT-对照组小鼠相比，重组CREG过表达组小鼠心肌组织CREG表达显著增加，如图4C-4D所示。当给予索拉非尼刺激后，WT组小鼠心肌组织中p-RIP3/RIP3比值显著升高，而重组CREG过表达小鼠心肌组织中p-RIP3/RIP3比值显著降低，具有显著的统计学意义，如图4C和4E所示。上述结表明，CREG过表达可改善索拉非尼诱导的心肌程序性死亡。
Claims (3)
1.CREG1蛋白在制备治疗索拉非尼诱导的心肌损伤药物中的用途，所述 CREG1 蛋白为重组 CREG1 蛋白，CREG1 基因的 GenBank 号为 NM_003851.2。
2.CREG1蛋白在制备用于抑制索拉非尼诱导的心肌细胞程序性死亡发生的药物的用途，所述 CREG1 蛋白为重组 CREG1 蛋白，CREG1 基因的 GenBank 号为 NM_003851.2。
3.根据权利要求1~2任一项所述的药物用途，其特征在于，所述药物包括CREG1蛋白以及任选的药学上可接受的载体或赋形剂。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101631861A (zh) * | 2006-12-08 | 2010-01-20 | 奥斯瑞根公司 | 作为治疗干预的靶标的miR-16调节基因和途径 |
| CN105056208A (zh) * | 2015-07-30 | 2015-11-18 | 中国人民解放军沈阳军区总医院 | Creg蛋白用于预防或治疗心肌梗死的医药用途 |
| CN105194651A (zh) * | 2015-07-30 | 2015-12-30 | 中国人民解放军沈阳军区总医院 | Creg蛋白用于保护心肌缺血再灌注损伤的医药用途 |
| WO2018028433A1 (zh) * | 2016-08-08 | 2018-02-15 | 中国人民解放军沈阳军区总医院 | Creg蛋白用于预防或治疗体重超重、肥胖及其相关疾病的医药用途 |
| CN111494605A (zh) * | 2020-04-13 | 2020-08-07 | 中国人民解放军北部战区总医院 | Creg蛋白用于预防或治疗阿霉素心肌损伤的医药用途 |
| CN111840515A (zh) * | 2020-08-19 | 2020-10-30 | 中国人民解放军北部战区总医院 | Creg蛋白在巨核细胞成熟分化促血小板生成中的医药用途 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017016244A1 (zh) * | 2015-07-30 | 2017-02-02 | 中国人民解放军沈阳军区总医院 | Creg蛋白的医药用途 |
-
2021
- 2021-03-15 CN CN202110274042.4A patent/CN112915196B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101631861A (zh) * | 2006-12-08 | 2010-01-20 | 奥斯瑞根公司 | 作为治疗干预的靶标的miR-16调节基因和途径 |
| CN105056208A (zh) * | 2015-07-30 | 2015-11-18 | 中国人民解放军沈阳军区总医院 | Creg蛋白用于预防或治疗心肌梗死的医药用途 |
| CN105194651A (zh) * | 2015-07-30 | 2015-12-30 | 中国人民解放军沈阳军区总医院 | Creg蛋白用于保护心肌缺血再灌注损伤的医药用途 |
| WO2018028433A1 (zh) * | 2016-08-08 | 2018-02-15 | 中国人民解放军沈阳军区总医院 | Creg蛋白用于预防或治疗体重超重、肥胖及其相关疾病的医药用途 |
| CN111494605A (zh) * | 2020-04-13 | 2020-08-07 | 中国人民解放军北部战区总医院 | Creg蛋白用于预防或治疗阿霉素心肌损伤的医药用途 |
| CN111840515A (zh) * | 2020-08-19 | 2020-10-30 | 中国人民解放军北部战区总医院 | Creg蛋白在巨核细胞成熟分化促血小板生成中的医药用途 |
Non-Patent Citations (2)
| Title |
|---|
| CREG1蛋白对血管紧张素Ⅱ诱导的小鼠心功能损伤的影响;宋海旭;李洋;孙鸣宇;彭程飞;田孝祥;闫承慧;韩雅玲;;解放军医学杂志(01);第22-27页 * |
| Targeting the insulin-like growth factor pathway in hepatocellular carcinoma;Mónica Enguita-Germán等;World J Hepatol;第6卷(第10期);第716-737页 * |
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