CN105288599A - 血红素氧化酶-2在制备抑制器官移植免疫排斥制剂中的应用 - Google Patents
血红素氧化酶-2在制备抑制器官移植免疫排斥制剂中的应用 Download PDFInfo
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- CN105288599A CN105288599A CN201510697954.7A CN201510697954A CN105288599A CN 105288599 A CN105288599 A CN 105288599A CN 201510697954 A CN201510697954 A CN 201510697954A CN 105288599 A CN105288599 A CN 105288599A
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Abstract
本发明涉及血红素氧化酶-2在制备抑制器官移植免疫排斥制剂中的应用。本发明公开了血红素氧化酶-2及其基因在制备抑制器官移植免疫排斥反应制剂中的应用。本发明的制剂可用于抑制器官移植免疫排斥反应，提高器官移植的存活率，为治疗肝脏、肾脏和心脏等慢性终末期疾病提供了有效方法。
Description
技术领域
本发明涉及血红素氧化酶-2在制备抑制器官移植免疫排斥制剂中的应用。
背景技术
器官移植是治疗肝脏、肾脏和心脏等慢性终末期疾病的主要方法，抑制排斥反应是移植器官长期存活的重要因素之一。随着研究的进展，移植器官自身保护性反应对阻止移植物排斥反应的作用逐渐引起重视。保护性作用主要通过保护性基因来介导，通过研究发现，在移植存活器官的平滑肌细胞和血管内皮细胞上有保护性基因的表达，在有排斥反应的移植器官上很少或没有表达。血红素氧化酶-2(HemeOxygenase-2,HO-2)HO-2是一种重要的内源性抗凋亡因子，对氧化应激和细胞因子介导的细胞凋亡起关键保护性作用，HO-2在调控新生猪和小鼠的脑血流量中扮演重要角色.本研究通过制备了HO-2的转基因小鼠，通过对小鼠皮肤的同种异体移植发现，HO-2转基因小鼠皮肤的存活率显著高于野生型小鼠皮肤存活率。
发明内容
为了解决移植器官因免疫排斥反应导致长期存活率低的难题，我们制备了HO-2的转基因小鼠，研究HO-2在小鼠皮肤同种异体移植中降低免疫排斥反应的作用，进而提供了HO-2及其基因在制备抑制器官移植免疫排斥反应制剂中的应用。
本发明公开了血红素氧化酶-2在制备抑制器官移植免疫排斥反应制剂中的应用。
本发明同时提供了血红素氧化酶-2基因在制备抑制器官移植免疫排斥反应制剂中的应用。
更具体地，提供了HO-2的真核表达载体在制备抑制器官移植免疫排斥反应制剂中的应用。
我们构建了HO-2的真核表达载体pEF1α-IRES-AcGFP，通过胚胎显微注射技术制备HO-2转基因小鼠。HO-2转基因小鼠皮肤移植到BALB/c小鼠后背上，同时野生型FVB小鼠作为阴性对照。观察HO-2转基因小鼠与阴性对照皮肤的存活时间、免疫组化分析炎性细胞的数量对比变化。
本发明的制剂可用于抑制器官移植免疫排斥反应，提高器官移植的存活率，为治疗肝脏、肾脏和心脏等慢性终末期疾病提供了有效方法。
附图说明
图1是HO-2转基因小鼠与野生型小鼠皮肤移植存活率对比。
图2是HO-2转基因小鼠与野生型小鼠皮肤移植后炎症反应。
具体实施方式
实施例1：HO-2基因的扩增及真核表达载体pEF1α-HO-2-AcGFP的构建
提取C57BL/6小鼠海马组织总RNA，用TaKaRa反转录试剂盒合成cDNA。以cDNA为模板。以F:5’-GTGAATTCATGTCTTCAGAGGTGGAGACC(EcoRI)-3’(SEQIDNO.1)，R:5’-TAGGATCCCATGTAGTACCAGGCCAATAG(BamHI)-3’(SEQIDNO.2)为上下游引物扩增HO-2的848bp片段，扩增条件：94℃4min，94℃40s，60℃40s，72℃40s，72℃10min，30个循环。扩增产物1％琼脂糖凝胶电泳，以确定扩增产物大小，然后将H0-2的PCR产物经过回收纯化后与载体pEF1α-IRES-AcGFP(购买于Clontech公司)同时用EcoRI和BamHI双酶切，酶切后进行1％琼脂糖凝胶电泳，并经过凝胶回收纯化试剂盒回收两者的酶切产物，将两者酶切纯化的产物按照3：1的摩尔比混合，用T4DNA连接酶37℃连接24h，转化DH5d感受态细胞中，然后进行阳性克隆的筛选，用质粒提取试剂盒提取阳性菌质粒进行酶切鉴定，送生工生物工程(上海)股份有限公司测序。
实施例2：HO-2转基因动物模型的建立
通过显微注射技术制备HO-2的转基因小鼠(由广州赛业生物公司协助完成)。
实施例3：小鼠皮肤移植实验
应用成年的HO-2转基因FVB小鼠及野生型FVB小鼠皮肤分别移植到成年BALB/c小鼠背部，每个时间段各取10只阳性鼠与野生型鼠观察皮肤的存活率，如图1所示，2d时HO-2转基因(Tg)移植皮肤与野生型(W)比较(A1，A2)，A1皮肤存活率较A2皮肤存活率差别不显著；第4d时，B1皮肤较B2皮肤，B1皮肤存活率为70％，B2皮肤存活率为40％，两者存活率差异达到显著水平；第8d，C1皮肤较C22皮肤，C1皮肤存活率为50％，B2皮肤存活率为20％，两者存活率差异达到显著水平。并对2d，4d，8dd的阳性皮肤和阴性皮肤进行免疫组化实验，观察二者的炎症反应，如图2所示，第2d，转基因(Tg)小鼠移植皮肤较野生型(W)小鼠无显著性差异；第4d，转基因小鼠移植皮肤的炎症细胞数显著小于野生型移植小鼠皮肤炎症细胞数；第8d，炎症细胞数明显增加，但转基因小鼠移植皮肤的炎症细胞数显著小于野生型移植小鼠皮肤炎症细胞数。结果显示，HO-2转基因小鼠的皮肤存活率显著高于对照组，并且皮肤炎症细胞数显著低于对照组。
Claims (3)
1.血红素氧化酶-2在制备抑制器官移植免疫排斥反应制剂中的应用。
2.血红素氧化酶-2基因在制备抑制器官移植免疫排斥反应制剂中的应用。
3.血红素氧化酶-2的真核表达载体在制备抑制器官移植免疫排斥反应制剂中的应用。
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| CN105727379A (zh) * | 2016-02-25 | 2016-07-06 | 顾宇春 | 血红素加氧酶药物洗脱支架 |
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| WO2000012118A2 (en) * | 1998-08-28 | 2000-03-09 | President And Fellows Of Harvard College | Inhibiting cardiomyocyte death |
| CN1507348A (zh) * | 2001-03-30 | 2004-06-23 | ɣ����ҽҩ��˾ | 用于治疗血管性、炎性和免疫疾病的一氧化碳生成性组合物 |
| CN1545548A (zh) * | 2001-06-21 | 2004-11-10 | ��˼����˹�����Ͽ���˹ҽҩ���� | 一氧化碳改善组织和器官移植结局并抑制细胞凋亡 |
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Patent Citations (3)
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| WO2000012118A2 (en) * | 1998-08-28 | 2000-03-09 | President And Fellows Of Harvard College | Inhibiting cardiomyocyte death |
| CN1507348A (zh) * | 2001-03-30 | 2004-06-23 | ɣ����ҽҩ��˾ | 用于治疗血管性、炎性和免疫疾病的一氧化碳生成性组合物 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105727379A (zh) * | 2016-02-25 | 2016-07-06 | 顾宇春 | 血红素加氧酶药物洗脱支架 |
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