CN109701020A - 免疫相关GTP酶Irgm1作为新靶点在治疗晚期动脉粥样硬化中的应用 - Google Patents
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Abstract
本发明公开了免疫相关GTP酶Irgm1作为新靶点在治疗晚期动脉粥样硬化中的应用。本发明利用临床分辨率最高的腔内影像学技术光学断层成像技术(Optical coherence tomography，OCT)和免疫学等技术，首次将免疫相关GTP酶Irgm1作为一种动脉粥样硬化治疗靶点应用于心血管领域。利用病理学技术和OCT技术，在动脉粥样硬化早期和晚期阶段，静态或者动态观察发现Irgm1通过调控巨噬细胞自噬和凋亡来达到调控斑块稳定性的作用。本发明将Irgm1作为调控斑块稳定性的靶点，将极大地避免患者造影及心血管有创性检测，为ACS发生机制及患者不稳定斑块的早期诊断提供新的思路，也为ACS患者的有效治疗提供理论依据。本发明的提出将有望让更多的心血管疾病的患者受益，有着很好的预防和治疗前景。
Description
技术领域
本发明涉及治疗动脉粥样硬化的新靶点，特别涉及免疫相关GTP酶Irgm1作为一种新的治疗靶点在治疗晚期动脉粥样硬化疾病中的应用。本发明属于基因治疗技术领域。
背景技术
急性冠状动脉综合征(Acute coronary syndrome，ACS)是导致全球居民死亡的首要原因。绝大多数患者是死于不稳定冠状动脉粥样斑块的破裂引起的血栓形成。研究发现，斑块破裂引起的心血管事件的临床表现和预后主要取决于斑块类型。斑块类型主要分为稳定斑块和不稳定斑块(又称易损斑块Vulnerable plaque，VP)。尽管研究者对不稳定斑块的形成机制做了大量的工作。但是目前不稳定斑块的提前诊断、提前预防及有针对性治疗仍未得到根本解决。因此，临床及时诊断不稳定斑块寻找影响不稳定斑块的形成机制和调控靶点仍然是心血管研究领域的重点。
基于目前对不稳定斑块的免疫学机制认识，其形成机制主要集中在斑块内免疫炎症细胞的募积、细胞凋亡和坏死、纤维帽变薄等引起的斑块破裂导致血栓生成等。巨噬细胞作为斑块中的主要免疫细胞，在斑块形成的过程中对其发展和稳定性均起重要的调节作用。不稳定斑块不是一成不变的，而是当外界微环境发生变化时会发生动态变化。这一特点意义重大。所以寻找能影响斑块稳定性最初的调控靶点具有非常重要的临床价值和意义。
目前，国际上对免疫相关GTP酶-Irgm1的研究多集中在炎症性肠病，肿瘤等疾病，在心血管领域的研究较少。本发明将Irgm1应用在心血管领域进行深入研究，证明并在国际上首次提出动脉粥样硬化(Atherosclerosis，AS)斑块中GTP酶Irgm1显著高表达，在AS发病过程中Irgm1可以介导巨噬细胞M1亚型(炎症亚型)的转化。该结论在临床AS患者血管斑块及Irgm1基因敲除的AS模型鼠斑块中均得以证明。
目前在心血管领域关于动脉粥样硬化斑块稳定性的研究中，诊断不稳定斑块寻找影响不稳定斑块的形成机制和调控靶点仍然是重中之重。因此，本发明将巨噬细胞中GTP酶Irgm1作为调控动脉粥样斑块稳定性的靶点的提出和证实将填补国内外研究中针对Irgm1影响不稳定斑块发生及破裂这一领域的空白。
发明内容
本发明的目的在于利用分子生物学、动物学、病理学及细胞学等几大研究平台的技术手段进行证明，首次将免疫相关GTP酶Irgm1作为一种治疗靶点应用于心血管领域。基于在动脉粥样硬化晚期阶段，Irgm1通过调控巨噬细胞自噬和凋亡并利用病理学技术，动态观察其调控斑块稳定性的作用。
为了达到上述目的，本发明采用了以下技术手段：
本发明所使用的实验组小鼠模型为Apoe-/-Irgm1+/-，对照组小鼠模型为Apoe-/-Irgm1+/+。利用Apoe-/-Irgm1+/-小鼠作为繁殖笼，小鼠2月龄后进行基因型鉴定，高脂喂养16周建立动脉粥样硬化晚期模型。我们利用ox-LDL刺激巨噬细胞模拟体内环境，通过转染方式使Irgm1低表达，利用qPCR和Western Blot等实验技术验证了自噬相关基因和蛋白的表达，发现在AS晚期，Irgm1能够通过促进巨噬细胞凋亡对AS斑块产生危害作用。
因此，在上述研究的基础上，本发明提出了免疫相关GTP酶Irgm1的抑制剂在制备治疗晚期动脉粥样硬化药物中的应用。
其中，优选的，所述的免疫相关GTP酶Irgm1的抑制剂为免疫相关GTP酶Irgm1的RNA干扰载体、免疫相关GTP酶Irgm1的抗体及其他能够抑制免疫相关GTP酶Irgm1表达的抑制剂中的一种。
其中，优选的，所述的免疫相关GTP酶Irgm1的抑制剂为Irgm1 siRNA.
其中，免疫相关GTP酶Irgm1的抑制剂通过抑制巨噬细胞凋亡对晚期动脉粥样硬化斑块起保护作用。
相较于现有技术，本发明的有益效果是：
本发明利用临床分辨率最高的腔内影像学技术光学断层成像技术(Opticalcoherence tomography，OCT)和免疫学等技术，首次将免疫相关GTP酶Irgm1作为一种动脉粥样硬化治疗靶点应用于心血管领域。首次将免疫相关GTP酶Irgm1作为一种动脉粥样硬化治疗靶点应用于心血管领域。利用病理学技术和OCT技术，在动脉粥样硬化晚期阶段，静态或者动态观察发现Irgm1通过调控巨噬细胞自噬和凋亡来达到调控斑块稳定性的作用。本发明将Irgm1作为调控斑块稳定性的靶点，将极大地避免患者造影及心血管有创性检测，为ACS发生机制及患者不稳定斑块的早期诊断提供新的思路，也为ACS患者的有效治疗提供理论依据。本发明的提出将有望让更多的心血管疾病的患者受益，有着很好的预防和治疗前景。
本发明将巨噬细胞中GTP酶Irgm1作为调控动脉粥样斑块稳定性的靶点的提出和证实将填补国内外研究中针对Irgm1影响不稳定斑块发生及破裂这一领域的空白。
附图说明
图1为琼脂糖凝胶电泳鉴定基因型示意图；
图2为HE和油红O染色分别检测动脉粥样硬化晚期小鼠模型主动脉窦斑块中脂质核心面积；
其中：左列图为HE染色结果及柱状图，HE染色发现ApoE-/-Irgm1+/-组主动脉窦斑块中脂质核心较对照组低表达；右列图为油红O染色结果及柱状图，结果显示ApoE-/-Irgm1+/-组主动脉窦斑块中脂质核心较对照组低表达((mean±SE)(n＝3)*p＜0.05,**p＜0.01)；
图3为免疫荧光和免疫组化实验结果；
显示ApoE-/-Irgm1+/-组主动脉窦斑块中巨噬细胞凋亡较对照组血管低；
图4为利用qPCR和Western Blot检测RAW264.7巨噬细胞中自噬相关基因和蛋白的表达；
其中：左图为荧光实时定量PCR检测ATG5、ATG7、Becclin1、LC3的表达(mean±SE)(n＝3for Si-Irgm1,Control groups)*p＜0.05,**p＜0.01，右图为Western blot实验检测ATG5、ATG7、Becclin1、LC3的表达，结果显示Si-Irgm1组LC3 Ⅱ较对照组低表达，P62较对照组高表达。
图5为利用Western Blot检测凋亡相关标志蛋白cleaved-caspase3，cleaved-caspase9的表达；
其中：流式细胞术和酶标仪结果显示实验组比对照组凋亡蛋白低表达(n＝3forSi-Irgm1,Control groups)*p＜0.05,**p＜0.01；Western blot实验检测结果显示Si-Irgm1组cleaved-caspase3，cleaved-caspase9较对照组低表达；
图6为利用免疫荧光检测DHE和TUNEL的表达。
其中：A图为免疫荧光结果，显示与对照组相比Si-Irgm1组DHE低表达；B图为免疫荧光标记Tunel，加入NAC抑制ROS后，凋亡进一步减少。
具体实施方式
下面结合具体实施例和附图来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1免疫相关GTP酶Irgm1治疗动脉粥样硬化的体内及体外实验
方法：
1.动脉粥样硬化动物模型的建立
本发明所使用的实验组小鼠模型为Apoe-/-Irgm1+/-，对照组小鼠模型为Apoe-/-Irgm1+/+。利用Apoe-/-Irgm1+/-小鼠作为繁殖笼，小鼠2月龄后进行基因型鉴定，高脂喂养16周建立动脉粥样硬化晚期小鼠模型。
使用HE和油红O染色检测动脉粥样硬化晚期小鼠模型中主动脉窦斑块中脂质核心面积。
利用免疫荧光和免疫组化实验检测动脉粥样硬化晚期小鼠模型主动脉窦斑块中巨噬细胞凋亡情况。
2.Raw264.7细胞培养
将RAW264.7巨噬细胞系接种在10％DMEM培养液中培养，用1xPBS清洗，用胰酶消化10秒，重悬后分别接种在24孔板中。为了诱导自噬和凋亡，用84μg/ml氧化LDL(oxLDL)处理细胞3小时和12h(37℃，95％空气/5％CO 2))。使用荧光标记超氧阴离子测定ROS产生DHE。立即通过荧光显微镜观察DHE阳性细胞，然后使用ImageJ软件定量。
3.巨噬细胞特定基因Irgm1进行siRNA沉默
Irgm1siRNA干扰载体购买于试剂公司，用Invitrogen公司Lipofectamine 3000转染试剂盒按指导手册在转染前一天调整培养细胞的密度为50％-70％，先用不含血清的优化培养基(opti-MEMI)稀释转染试剂，室温孵育5min。再用不含血清的优化培养基稀释，轻轻混匀。将目标基因-转染混合液加入培养细胞中，轻摇使之混匀，最后在37℃的CO2的培养箱中培养24小时。对转染后的细胞(Si-Irgm1)中自噬相关基因和基因以及凋亡相关标志蛋白的表达进行Western blot，real-time PCR检测。
4.Western Blot方法检测蛋白的表达
常规提取巨噬细胞蛋白，用紫外分光光度计测蛋白浓度；组装胶板，制备电泳凝胶：分离胶7ml(ddH2O 2.24ml，30％丙烯酰胺2.8ml，1.5molTris-PH8.8 1.82ml，10％SDS70μl，10％AP 70μl，TEMD 2.8μl)，浓缩胶3ml(ddH2O 2.1ml，30％丙烯酰胺495μl，1.0molTris-pH 6.8 375μl，10％SDS 30μl，10％AP 30μl，TEMD 3.0μl)；灌分离胶，水封，灌浓缩胶，插加样梳；5μl蛋白+1μl 6×loading buffer，沸水浴5min；将胶放入电泳槽中，倒入电泳缓冲液，上样，接通电源，恒压：100V 15mA，15min转为120V，20mA，100min；转膜：3层滤纸+膜+胶+3层滤纸，恒流：I＝面积×1.5，t＝1h；转完膜加封闭液，放在摇床摇1h；加一抗，放在摇床摇5min，4℃过夜；洗涤液洗膜TBST 10min×3，TBS10min；加二抗，放在摇床摇2h；洗涤液洗膜TBST 10min×3，TBS10min；染色，避光，凝胶图像处理系统分析。
5.病理学检测
(1)HE染色：冷丙酮(-20℃)固定10min；用苏木素染色3min，自来水冲洗；盐酸乙醇分化30sec；淡氨水返蓝5sec；伊红染色10min，自来水冲洗；常规脱水、脱酒精，二甲苯透明，封片。
(2)Masson染色：切片在室温下平衡20min；蒸馏水洗5min；Masson复合染液8min；0.2％醋酸水溶液稍洗；1％磷钨酸染30sec；0.2％醋酸水溶液洗2次；亮绿染液2min；0.2％醋酸水溶液洗2次；梯度酒精脱水；二甲苯透明；中性树胶封片。
(3)油红O染色：冰冻切片室温放置10min；4％PFA固定5min；PBS洗3次，每次5min；50％异丙醇孵育5min；油红孵育10min；蒸馏水洗3次，每次5min；苏木染核10min左右；自来水冲洗；水性封片剂封片。
(4)免疫荧光染色：取冰冻切片，冷丙酮固定10min，0.01M PBS浸洗；0.3％TritonX-100+0.5％BSA 37℃封闭30min；加一抗，4℃过夜；0.01M PBS洗去一抗；加二抗，室温1h，0.01M PBS浸洗；DAPI 1min，0.01M PBS浸洗；封片，激光共聚焦系统成像。
结果：
1、小鼠基因型鉴定
琼脂糖凝胶电泳鉴定基因型结果如图1所示，其中扩增出两条条带的鉴定为Apoe-/-Irgm1+/-，扩增出一条条带的鉴定为Apoe-/-Irgm1+/+。
2、晚期模型小鼠中Irgm1对AS斑块的影响
使用HE和油红O染色检测动脉粥样硬化晚期小鼠模型中主动脉窦斑块中脂质核心面积。结果如图2所示，左图为HE染色结果，HE染色发现ApoE-/-Irgm1+/-组主动脉窦斑块中脂质核心较对照组低表达；右图为油红O染色结果，结果显示ApoE-/-Irgm1+/-组主动脉窦斑块中脂质核心较对照组低表达，说明AS晚期模型小鼠中Irgm1对AS斑块具有危害作用。
3、免疫荧光和免疫组化实验检测动脉粥样硬化晚期小鼠模型主动脉窦斑块中巨噬细胞凋亡情况
结果如图3所示，显示ApoE-/-Irgm1+/-组主动脉窦斑块中巨噬细胞凋亡较对照组血管低。
4、qPCR和Western Blot检测RAW264.7巨噬细胞中自噬相关基因和蛋白的表达
结果如图4所示，左图为荧光实时定量PCR检测ATG5、ATG7、Becclin1、LC3的表达，右图为Western blot实验检测ATG5、ATG7、Becclin1、LC3的表达，结果显示Si-Irgm1组LC3Ⅱ较对照组低表达，P62较对照组高表达。
5、免疫荧光和Western Blot检测RAW264.7巨噬细胞中凋亡相关标志蛋白的表达
结果如图5所示，显示Si-Irgm1组cleaved-caspase3，cleaved-caspase9较对照组低表达。
6、利用免疫荧光检测DHE和TUNEL的表达
结果如图6所示，显示与对照组相比Si-Irgm1组DHE低表达；B图为免疫荧光标记Tunel，加入NAC抑制ROS后，凋亡进一步减少。
总结：
上述研究结果表明，在AS晚期，Irgm1能够通过促进巨噬细胞凋亡对AS斑块产生危害作用，使用Irgm1抑制剂能够降低其危害作用。
Claims (4)
1.免疫相关GTP酶Irgm1的抑制剂在制备治疗晚期动脉粥样硬化药物中的应用。
2.如权利要求2所述的应用，其特征在于，所述的免疫相关GTP酶Irgm1的抑制剂为免疫相关GTP酶Irgm1的RNA干扰载体、免疫相关GTP酶Irgm1的抗体及其他能够抑制免疫相关GTP酶Irgm1表达的抑制剂中的一种。
3.如权利要求1所述的应用，其特征在于，所述的免疫相关GTP酶Irgm1的抑制剂为Irgm1siRNA。
4.如权利要求1所述的应用，其特征在于，免疫相关GTP酶Irgm1的抑制剂通过抑制巨噬细胞凋亡对晚期动脉粥样硬化斑块起保护作用。
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