CN112402439A - miR-325核酸类似物在制备改善肝胶原沉积相关产品中的应用 - Google Patents
miR-325核酸类似物在制备改善肝胶原沉积相关产品中的应用 Download PDFInfo
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Abstract
本发明提供了miR‑325核酸类似物在制备改善肝胶原沉积相关产品中的应用。本发明是基于肝血窦内皮细胞的去内皮化是缓解肝胶原沉积的必要条件的原理。本发明以miR‑325为模板合成的miR‑325核酸类似物可以纠正肝纤维化环境下肝血窦内皮细胞内皮化病变，有效恢复肝血窦内皮细胞抑制胶原沉积的功能，进而缓解肝纤维化。
Description
技术领域
本发明涉及生物技术领域，尤其涉及miR-325核酸类似物在制备改善肝胶原沉积相关产品中的应用。
背景技术
肝硬化的过程就是肝受损后愈伤的过程，然而在这个过程中，肝脏的正常代谢受损，肝脏向着大量分泌胶原纤维而非向肝再生的方向发展。造成肝细胞受损，并造成坏死和炎症的因素都会导致肝纤维化进而发展成其重症肝硬化以至肝衰竭造成病人死亡。其中最为常见的因素有，过多的脂肪堆积，长期大量的酒精摄取，服用可造成肝损伤的药物（超过1000种药物已明确可引起肝损伤，如化疗药物，部分中药）等。由于肝脏本身是再生能力很强，可以在受损初期通过适当分泌胶原，具有一定自我保护能力的器官，导致慢性肝病在初期无明显症状，患者多于肝硬化后期到肝衰竭时才有表型出现。目前临床上治疗肝纤维化或肝硬化患者没有特效药，采用的常规方案只能针对肝病成因进行抗病毒等使肝脏胶原沉积进程减缓，或对肝纤维化的并发症进行保肝护肝治疗，而肝脏胶原沉积无法被逆转，肝脏正常功能难以恢复，发展到肝脏功能失代偿期的患者只有进行肝移植来尽可能延长生存期。然而健康的肝源紧张且伴随免疫排斥的副作用。
发明内容
本发明的目的在于，针对现有技术存在的问题，提供了miR-325核酸类似物在制备改善肝胶原沉积相关产品中的应用。本发明是基于肝血窦内皮细胞（Liver sinusoidalendothelial cell, LSEC）的去内皮化是缓解肝胶原沉积的必要条件的原理。本发明以miR-325为模板合成的miR-325核酸类似物可以纠正肝纤维化环境下肝血窦内皮细胞内皮化病变，有效恢复肝血窦内皮细胞抑制胶原沉积的功能，进而缓解肝纤维化。
本发明的目的是通过以下技术方案来实现的：
一方面，本发明提供一种miR-325核酸类似物在制备改善肝胶原沉积相关产品中的应用。
优选地，所述肝胶原沉积由脂肪肝、酒精性肝炎、药物性肝炎、细菌性肝炎、寄生虫性肝炎、化学毒物性肝炎、胆管炎、威尔逊病、自身免疫性肝炎、或肝血窦内皮细胞病理性功能异常中的至少一种疾病引起。
优选地，本发明提供一种miR-325核酸类似物在制备改善肝血窦内皮细胞内皮化产品中的应用。
优选地，所述血窦内皮细胞分化指细胞表面出现筛板结构，所述血窦功能指血窦超滤吞噬能力，供给营养和物质交换能力，抑制巨噬细胞能力，抑制星状细胞激活能力，以及响应和发出促进细胞再生信号的能力，所述促进细胞再生信号通路，指响应CXCR7激动剂，和恢复VEGFR2，VEGFR3表达。
优选地，导致所述肝胶原沉积的因素不包括病毒性肝炎。
第二方面，提供用于改善肝胶原沉积的药品，所述药品包括治疗有效量的miR-325核酸类似物。
第三方面，提供所述的药品在制备降低患有肝胶原沉积相关疾病个体血清中谷草转氨酶和谷丙转氨酶相关产品中的应用。
第四方面，提供一种组合物，所述组合物包括miR-325核酸类似物和CXCR7激动剂。
优选地，所述miR-325核酸类似物的浓度为20nM，所述CXCR7激动剂的浓度为300nM，所述CXCR7激动剂的纯度标准要求≥95%。
第五方面，提供上述的组合物在制备改善肝胶原沉积相关产品方面的应用。
第六方面，提供一种改善肝胶原沉积的方法，包括以下步骤，取上述产品或上述组合物，溶于无菌处理后的生理盐水，对患有上述疾病的个体进行静脉注射，降低个体肝胶原沉积。
第七方面，提供调控基因mannose receptor，stabilin-2，α-SMA, F4/80，PTPRM或YWHAQ 表达水平的方法，包括以下步骤：取所述产品或所述组合物溶于无菌处理后的生理盐水，对患有上述疾病的个体进行静脉注射，诱导个体血窦分化恢复功能。
相对于现有技术，本发明的有益效果在于：
肝纤维化个体的肝组织中肝血窦内皮细胞出现病理性的去分化，该去分化状态被逆转后可以显著降低肝纤维化，本发明通过实验证明，使用miR-325核酸类似物可以逆转肝血窦内皮细胞的病理去分化状态，本发明采用miR-325核酸类似物静脉注射作用于肝血窦内皮细胞去分化状态的个体或者肝纤维化的个体，通过miR-325直接和间接影响的信号通路，包括恢复细胞stabilin-2, PTPRM, YWHAQ等基因的表达，使肝血窦内皮细胞重新分化，促进肝血窦内皮细胞调节炎症，抗肝星状细胞激活的能力，从而降低肝纤维化；而且本发明采用了一种miR-325的核酸类似物，具有操作简单、成本低廉、效果显著等特点，实现了肝纤维化个体功能缺陷的肝血窦内皮细胞的重新分化，同时为临床上肝纤维化和其他具有血窦去分化相关疾病的患者治疗提供潜在的思路和方案。
附图说明
图1A为实施例中肝病组肝叶天狼猩红染色结果，红色胶原沉积显著；
图1B为实施例中miR-325静脉注射的肝病组天狼猩红染色结果，红色胶原沉积得到显著降低。
图2为实施例中使用miR-325治疗前后肝血窦内皮iTRAQ蛋白表达谱显著变化的信号通路。
图3为实施例中正常组（WT），肝病组（PBS+CCl4），miR-325静脉注射的肝病组(miR-325+CCl4)的肝功水平。
图4A为实施例中肝病组（PBS+CCl4）肝石蜡切片H&E染色结果，可见大量肝细胞坏死空泡，炎症浸润。
图4B为实施例中miR-325静脉注射的肝病组(miR-325+CCl4) 肝切片H&E染色结果，可见少量肝细胞坏死空泡，炎症浸润情况显著减轻。
图4C为实施例中CXCR7激动剂注射的肝病组（CXCR7agonist+CCl4）肝切片H&E染色结果，炎症细胞浸润，但形态正常肝细胞增多，坏死细胞减少。
图4D为实施例中miR-325及CXCR7组合物注射的肝病组（miR-325+CXCR7agonist+CCl4）肝切片H&E染色结果，炎症基本消除，肝细胞坏死空泡消失，细胞排列规律，肝脏基本恢复正常。
图 5A为实施例中肝病组（PBS+CCl4）肝冰冻切片的血窦分化标志物stabilin-2染色, 可见严重血窦损失和病理性去分化。
图 5B 为实施例中脐带间充质干细胞来源的细胞外囊泡静脉注射治疗肝病组（Extracellular vesicles+ CCl4）冰冻切片的血窦分化标志物stabilin-2染色，可见血窦密度和分化都有一定程度的恢复。
图5C为实施例中脐带间充质干细胞体外转染了miR-325核酸类似物后，使用其细胞外囊泡治疗肝病后，肝脏冰冻切片的血窦分化标志物stabilin-2染色，可见血窦密度和分化得到大幅提高。
图5D为实施例中脐带间充质干细胞体外转染了miR-325核酸抑制剂后，使用其细胞外囊泡治疗肝病后，肝脏冰冻切片的血窦分化标志物stabilin-2染色，可见血窦密度降低，分化被抑制。
具体实施例
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行详细地描述。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中细胞培养皿购自美国Corning公司。
下述实施例中的PBS缓冲液购自北京康为世纪生物科技有限公司。
下述实施例中的细胞培养基(DMEM培养基和α-MEM培养基)购自美国gibco公司。
下述实施例中的Liberase和OptiPrep购自sigma公司。
下述实施例中的MACS分选CD11b+磁珠和分选柱子购自Miltenyibiotec公司。
下述实施例中的stabilin-2抗体购自MBL International公司（D317-3）。
下述实施例中miR-325核酸类似物购自锐博生物。
下述实施例中细胞培养条件可采用：CO2浓度为5%，O2浓度为20%，温度为37℃的加湿细胞培养箱。
实施例
一、肝血窦功能异常的检测：
1.活检组织进行天狼猩红染色，检测肝血窦周胶原沉积
该方法可以使用冰冻长期保存的活检组织，也可以用新鲜组织。组织固定后进行常规天狼星红染色，结果如图1显示，肝病组肝叶天狼猩红染色后，红色胶原沉积显著； miR-325静脉注射的肝病组天狼猩红染色中，红色胶原沉积得到显著降低。
肝血窦原代细胞分离培养后再生关键信号通路的检测
将新鲜个体活检组织剪碎，用LiberaseTM溶解于2%胎牛血清的DMEM培养基中消化1小时，轻轻吹打成为单细胞，280rpm低速离心5分钟，去除肝实质细胞，接着上清经1800rpm离心10分钟获得包含肝血窦内皮细胞在内的肝非实质细胞，使用OptiPrep进行梯度离心获得肝血窦内皮细胞层，最后使用MACS磁珠分选，负选CD11b+细胞去除杂细胞，得到贴壁伸展开的原代血窦内皮细胞。使用iTRAQ技术进行蛋白质谱检测，如图2所示，经miR-325处理后肝原代血窦细胞中Hippo通路中关键蛋白显著变化提示：发育期表达，调节器官大小的信号通路Hippo signaling被激活。
表1为蛋白质谱检测的miR-325直接调节的YWHAQ和PTPRM蛋白表达水平的变化情况，蛋白ID为所列蛋白在Uniprot数据库的唯一编号，蛋白名称为所列为蛋白常用名称，Mean_Ratio为所列蛋白的表达量之比（miR-325处理的肝病组的均值比肝病组的均值），小于0.8为在miR-325注射后所列蛋白显著下调。
表1
| 蛋白ID | 蛋白名称 | Mean_Ratio | Pvalue | SD |
| P28828 | PTPRM | 0.80 | 0.02323 | 0.073 |
| P68254 | YWHAQ | 0.74 | 0.03696 | 0.122 |
二、miR-325核酸类似物或组合物治疗肝胶原沉积相关异常的应用
1.静脉注射恢复肝脏功能
可以根据实施范例步骤一中检测结果判断功能异常严重程度，决定单独使用miR-325核酸类似物，或是miR-325核酸类似物及CXCR7激动剂组合物。具体实施即对肝脏胶原沉积异常相关病患个体，进行静脉注射。miR-325核酸类似物浓度为20nM。所述CXCR7激动剂的纯度标准要求≥95%，等级要求细胞培养用；所述CXCR7激动剂使用前进行无菌处理，所述CXCR7激动剂的浓度为300nM。实施时所述产品和斯达汀类药物，以及褪黑素，和其他干预血管功能的药物不可混用。组合物不可用于患癌个体。取静脉血，常温静置半小时后，将析出的上层血清转移至无菌Eppendorf管，4℃，2000rpm离心20分钟，小心不碰到管底的红细胞沉淀，将最终获得的血清转移至无菌Eppendorf管。取5微升血清，使用AST活性检测试剂盒进行常规AST活性检测，结果如图3所示，miR-325静脉注射的肝病组较对照肝病组血清中AST活性显著下调，提示肝功得到缓解。联合用药结果如图4A-图4D所示，在各组肝脏的H&E染色中，图4A为肝纤维化病变组，图4B为单独使用miR-325核酸类似物治疗组，图4C为单独使用CXCR7激动剂治疗组，图4D为miR-325核酸类似物和CXCR7激动剂联合治疗组。染色结果指示，单独治疗较纤维化病变组肝实质细胞坏死空泡显著减少，炎症浸润缓解，肝单元排列有所恢复，更进一步，联合治疗组肝实质细胞坏死空泡和炎症基本消失，肝单元排列清晰有序，提示肝纤维化病理微环境显著逆转。
该方法也可以作为间充质干细胞有效性指标，在静脉注射间充质干细胞前检测间充质干细胞治疗能力。使用qPCR检测到间充质干细胞或者其细胞外囊泡中miR-325表达（表达水平参考：U6 Ct=19时，miR-325 Ct<30）可以保证间充质干细胞通过恢复血窦分化恢复肝功。
该方法也可以在间充质干细胞治疗中联合使用，体外培养的脐带间充质干细胞转染miR-325核酸类似物后使用转染的间充质干细胞或间充质干细胞外囊泡进行静脉注射，转染后培养液中miR-325核酸类似物终浓度应达到20nM。所述间充质干细胞培养方法为，取新鲜脐带，剪开小段，小心剥离血管神经，得到华氏胶后，剪碎成小块使用无血清α-MEM培养基进行原代培养，传代，扩增。所述传代应小于5代，即少于5次冻存或胰酶处理制作单细胞悬液。间充质干细胞外囊泡使用小于5代的细胞培养上清进行超速离心获取。超速离心步骤包括，常温800rpm离心去除细胞碎片，130,000rpm超速离心获得细胞外囊泡。结果如图5A-图5D所示，使用免疫荧光染肝血窦内皮细胞分化标志物stabilin-2，图5A为肝纤维化病变组，图5B为单独使用细胞外囊泡治疗组，图5C为使用转染了miR-325核酸类似物的间充质干细胞上清提取的细胞外囊泡治疗组，图5D为使用转染了miR-325核酸抑制剂的间充质干细胞上清提取的细胞外囊泡处理组。染色结果指示，上调细胞外囊泡中的miR-325核酸类似物的表达会诱导肝血窦的分化进而促进肝脏纤维化的消退，而抑制细胞外囊泡中的miR-325会抑制肝血窦的分化进而阻断肝纤维化的治疗效果。
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何在本申请揭露的技术范围内的变化或替换，都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以所述权利要求的保护范围为准。
Claims (7)
1.miR-325核酸类似物在制备改善肝胶原沉积相关产品中的应用。
2.根据权利要求1所述的应用，所述肝胶原沉积由脂肪肝、酒精性肝炎、药物性肝炎、细菌性肝炎、寄生虫性肝炎、化学毒物性肝炎、胆管炎、威尔逊病、自身免疫性肝炎或肝血窦内皮细胞病理性功能异常中的至少一种疾病引起。
3.一种用于改善肝胶原沉积的药品，其特征在于，所述药品包括治疗有效量的miR-325核酸类似物。
4.权利要求3所述的药品在制备降低患有肝胶原沉积相关疾病个体血清中谷草转氨酶和谷丙转氨酶相关产品中的应用。
5.一种组合物，其特征在于，所述组合物包括miR-325核酸类似物和CXCR7激动剂。
6.根据权利要求5所述的组合物，其特征在于，所述miR-325核酸类似物的浓度为15nM-30nM，所述CXCR7激动剂的浓度为200nM-500nM。
7.权利要求6所述的组合物在制备改善肝胶原沉积相关产品方面的应用。
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| LI ZHANG 等: "MiR‑325 Promotes Oxaliplatin‑Induced Cytotoxicity Against Colorectal Cancer Through the HSPA12B/PI3K/AKT/Bcl‑2 Pathway" * |
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