CN113198002A - 一种用于治疗非酒精性脂肪肝的肝细胞因子bmp9 - Google Patents
一种用于治疗非酒精性脂肪肝的肝细胞因子bmp9 Download PDFInfo
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Abstract
本发明公开肝细胞因子BMP9在制备延缓和/或治疗非酒精性脂肪肝药物的应用。本发明采用肝脏高亲和性的载体AAV8作为药物载体,通过基因治疗,可实现BMP9在肝脏组织的特异性的高效过表达,可减缓脂肪肝的相关症状,以可对脂肪肝进行有效治疗,其为非酒精性脂肪肝的治疗提供了新的治疗思路;且通过研究还发现BMP9在肝脏组织的特异性的高效过表达,还可对BMP9细胞因子低表达或缺失导致的肥胖症状进行有效的缓解。
Description
技术领域
本发明涉及分子生物学、细胞生物学领域,尤其涉及一种用于治疗非酒精性脂肪肝的肝细胞因子BMP9。
背景技术
肥胖诱导的非酒精性脂肪性肝病(Non-alcoholic fatty liver disease,NAFLD)主要的特征为肝脏脂肪变性。长期以来,NAFLD一直是一个亟待解决的全球性公共健康问题。其主要表现为甘油三酯(TGs)以脂滴的形式在肝细胞中异常积累。通常而言,肝脏脂肪变性可进一步发展为非酒精性脂肪肝炎、肝硬化、甚至肝癌。
骨形态发生蛋白(Bone morphogenetic proteins,BMPs)家族多个分子被报道与代谢综合征有着密切的关系。其中BMP2/BMP4/BMP7/BMP8b等分子已被证实可通过调节棕色脂肪产热,改善胰岛素抵抗等方式影响糖尿病和肥胖等慢性代谢疾病的发生。Bonemorphogenetic protein 9(BMP9),是一种由肝星状细胞特异性分泌的骨形态发生蛋白。主要功能为负责骨骼发育,成骨细胞分化以及血管生成。然而,有关于BMP9在肥胖,糖尿病,NAFLD等慢性代谢疾病中的功能仍然知之甚少。
发明内容
因此,基于以上背景,本发明经过研究,提供一种用于治疗非酒精性脂肪肝的肝细胞因子BMP9,其为脂肪肝的治疗提供了一种新的思路。
本发明的技术方案为:
本发明的发明点之一在于:肝细胞因子BMP9在制备延缓和/或治疗非酒精性脂肪肝药物的应用。
进一步地,所述药物包括在药学上可接受的载体。
进一步地,所述载体为AAV8。
进一步地,所述药物为注射剂。
本发明的发明点之二在于:肝细胞因子BMP9在制备治疗肥胖症状药物的应用。
进一步地,所述药物包括载体AAV8。
进一步地,所述药物为注射剂。
采取上述技术方案,具有的有益效果如下:
本发明采用肝脏高亲和性的载体AAV8作为药物载体,通过基因治疗,可实现BMP9在肝脏组织的特异性的高效过表达,可减缓脂肪肝的相关症状,以可对脂肪肝进行有效治疗,其为非酒精性脂肪肝的治疗提供了新的治疗思路;且通过研究还发现BMP9在肝脏组织的特异性的高效过表达,还可对BMP9细胞因子低表达或缺失导致的肥胖症状及其其他的代谢疾病进行有效的缓解。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1的AAV-BMP9可缓解非酒精性脂肪肝及相关并发症相关表征图;
其中图1A为BMP9敲除小鼠进行为期2个月或者4个月AAV-BMP9注射处理的操作示意图;
图1B为AAV-BMP9注射2个月后的小鼠中的不同组织中BMP9 mRNA表达水平;
图1C为AAV-BMP9注射后,BMP9敲除小鼠的肝脏中BMP9和PPARαmRNA表达水平(n=3);
图1D为AAV-BMP9注射后,BMP9敲除小鼠的肝脏中BMP9和PPARα蛋白表达水平;
图1E为AAV-BMP9注射后,BMP9敲除小鼠的血清中BMP9蛋白浓度(n=6);
图1F为H&E和油红染色检测肝脏脂肪变性程度。放大倍数,×200;
图1G为实验小鼠肝脏组织中甘油三酯含量;
图1H为AAV-BMP9处理4个月后,BMP9敲除小鼠的初始血糖值;
图1I为GTT;
图1J为ITT;
图1K为AAV-BMP9处理4个月,BMP9敲除小鼠的体重监测(n=6);
图1L为AAV-BMP9处理4个月,BMP9敲除小鼠的体态对比图(n=6),(*p<0.05,**p<0.01,***p<0.001)。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,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清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下面结合附图对发明做进一步说明。
实施例1:为验证BMP9对非酒精性脂肪肝及其肥胖、糖尿病等代谢疾病的影响,本实施例作出如下实验。
本实施例实验所用材料为:
C57BL/6小鼠(斯莱克,上海实验动物中心);BMP9敲除小鼠(赛业生物);93T细胞(Cobioer,CBP60439)。
本实施例实验所用实验仪器,见表1。
表1:实验仪器
本实施例所需实验试剂见表2。
表2:实验试剂
具体实验:
1)基因敲除小鼠模型的构建与鉴定
BMP9敲除小鼠(BMP9 KO)以C57BL/6野生型小鼠为背景,通过CRISPR/Cas9基因编辑手段进行构建。实验过程中,BMP9敲除小鼠与同窝同笼的野生型小鼠进行比较。BMP9敲除小鼠委托CYAGEN Bioscience公司进行构建。小鼠BMP9基因组信息参照数据库(GenBankaccession number:NM_019506.4;Ensemble:ENSMUSG00000072625)。小鼠BMP9基因位于小鼠14号染色体。具体而言,其遗传信息包含2个外显子。本实验针对小鼠BMP9基因1号外显子进行基因编辑实现基因敲除。本实验设计了靶向1号外显子的CRISPR向导RNA,gRNA1(TGTACAAGTCGATCATGTACTGG)和gRNA2(GCTGAAGCTCCGCACGATGTTGG)。Cas9 mRNA和guideRNA(gRNA)共转至小鼠受精卵用于BMP9敲除小鼠的生产。
通过PCR和DNA测序的手段进行基因型验证后,将其中获得的阳性幼鼠进行繁殖用于下一代扩繁。得到杂合子小鼠后,进一步对小鼠进行扩繁,
具体基因型鉴定方法如下:对新生小鼠进行剪尾处理,用Mouse Tissue DirectPCR Kit(TIANGEN,KG205)提取小鼠基因组DNA,并进行纯化。随后利用抽提的基因组DNA进行基因型鉴定,通过PCR扩增得到PCR扩增产物后,进行1.5%琼脂糖凝胶电泳(100V恒压,40分钟)。小鼠基因型鉴定所用PCR引物序列为:
(正向引物:AATGCCCACAGCTCCCTGGG;反向引物:CCACGCAAATGATGAACAGGGAAT)。
所有实验小鼠均饲养在恒温动物房内(22℃),并维持12小时光照和暗室交替环境,并提供充足的饲料与水。高脂饮食(HFD)实验小鼠需特别提供高脂饲料(SLACOM)。其中,高脂饮食饲料包含60%kcal的脂肪,20%kcal碳水化合物,以及20%kcal蛋白质。
2)BMP9过表达AAV构建
首先,合成小鼠BMP9基因序列,并将其克隆到腺相关病毒(AAV)AAV8表达载体GPAAV-WPRE(Genomeditech,Shanghai)中。Bam HI-EcoR I为克隆插入位点。简而言之,将获得的AAV空载和AAV-BMP9过表达载体通过Lipofectamine 2000reagent(Invitrogen,11668019)转染至293T细胞(Cobioer,CBP60439)。随后收集培养液以获得病毒上清液,并进行后续病毒纯化步骤。所获得的AAV病毒滴度进一步通过实时荧光定量PCR进行定量。
3)BMP9过表达AAV小鼠体内回补实验
首先,采用腹腔注射水合氯醛(Servicebio,WG)的方式,将16周龄的BMP9敲除小鼠进行麻醉。随后,分别向小鼠体内注射一定浓度的AAV-Vector和AAV-BMP9腺相关病毒,将注射模型分为AAV对照组合AAV-BMP9实验组。腺相关病毒需用0.9%生理盐水(Baxter,A6E1323)进行适当的稀释。按照小鼠体重为标准,具体的腺相关病毒的注射剂量为2×1012vg/kg。对于短期处理实验组(2个月),腺相关病毒处理2个月后,进行GTT和ITT测试。随后,处死小鼠并收集血样,肝脏以及其他组织用于后续实验。对于长期观察组(4个月),在注射腺相关病毒后,对小鼠的体重进行持续监测,实验监测周期为4个月。
4)甘油三酯(TG)含量测定
小鼠肝脏组织收集于液氮或者-80℃保存,测定甘油三酯含量时,首先将小鼠肝脏组织置于氯仿/甲醇(2:1v:v)中,经组织匀浆器(PRO Scientific,PRO200)研磨后用于后续实验。提取肝脏中的脂质物时,首先通过经典的Folch方法进行可溶性组织脂质物制备,随后将肝脏组织脂质提取物溶解于异丙醇中,用于后续操作。并通过BCA蛋白定量试剂盒(Beyotime,P0010)进行蛋白浓度测定。进行蛋白浓度校正后,根据甘油三酯检测试剂盒Triglyceride Assay kit(Dongou,V1103)进行甘油三酯含量测定。
5)胰岛素耐受(ITT)与葡萄糖耐受(GTT)测试
进行胰岛素耐受实验(ITT)前,先对实验小鼠进行5个小时的禁食处理。随后对禁食后的实验小鼠进行体重测量与记录。正式实验时,对小鼠进行腹腔注射胰岛素(NovoNordisk,Novolin R)处理,胰岛素的注射剂量为0.75IU/kg小鼠体重。随后用70%酒精擦拭小鼠尾部,并用手术剪剪开小鼠尾部,分别在15,30,60,90,120min处理时间点,用血糖仪Glucometer(ONETOUCH UltraEasy,SN-CFD8413CR)与配套的血糖试纸Glucose TestStrips(ONETOUCH Ultra,4251884)记录小鼠的血糖浓度。相类似的,进行葡萄糖耐受实验(GTT)时,首先对实验小鼠进行过夜(16个小时)的禁食处理,禁食处理结束后,记录小鼠体重,随后按照1g/kg小鼠体重比例,对小鼠进行腹腔注射葡萄糖(Sigma-Aldrich,G8270)处理,随后分别在15,30,60,90,120min处理时间点进行小鼠尾部采血,并用血糖仪记录相对应的血糖浓度。
6)NAFLD临床样本收集
临床样本包括32例健康对照样本,以及32例可诊断为慢性代谢疾病的实验样本。根据经典的临床诊断指标,肝脏脂质含量累积超过5%的样本被分级为非酒精性脂肪肝样本。肝脏脂质含量通过核磁共振波谱学(magnetic resonance spectroscopy,MRS)进行检测。血清中的BMP9浓度通过BMP9 ELISA试剂盒(RayBiotech,ELH-BMP9)进行检测。NAFLD人群中血样中BMP9含量偏低(表3),且与肝脏TG含量负相关。
表3:NAFLD临床样本基本信息
本实施例实验应用AAV对小鼠进行了BMP9体内过表达,并成功逆转BMP9敲除小鼠出现的脂肪肝等代谢综合征相关表型。注射剂量为2×1012vg/kg。
对BMP9敲除小鼠进行AAV-BMP9注射后,分别进行为期2个月的短期监测以及为期4个月的长期监测(见图1A)。小鼠体内完成AAV-BMP9注射2个月后,收集小鼠组织进行BMP9过表达效果验证,包括肌肉,肝脏,肺,棕色脂肪组织,白色脂肪组织,下丘脑,脾脏,眼球,肾脏,心脏,睾丸等。结果发现通过AAV介导了BMP9在小鼠体内成功回补,并且主要在小鼠肝脏中高表达(图1B)。AAV8是一种在肝脏中具有高度亲和性的血清型AAV载体,因而被认为是一种非常适合应用于靶向肝脏的基因治疗载体。为了评估AAV-BMP9的治疗效果,对各AAV-BMP9实验组以及AAV对照组的小鼠脾脏进行收集。通过进一步实验检测发现,在AAV-BMP9注射2个月后,小鼠肝脏中BMP9和PPARα的mRNA表达水平(图1C)以及蛋白表达水平(图1D)得到了明显的提升。小鼠血清中的BMP9含量也有了明显的增加(图1E)。
进一步对小鼠肝脏进行了形态学分析,以观察小鼠脂肪变性现象是否得到缓解。H&E以及油红染色结果证明,AAV-BMP9实验组的小鼠肝脏中的脂肪空泡减少,并且肝实质细胞中的红色脂滴物也有所减少(图1F)。最后,通过测定肝脏组织的甘油三酯的含量,结论也与上述结果保持一致(图1G)。此外,为了明确AAV-BMP9处理后,是否会对小鼠的血糖调节能力产生影响。本实验对小鼠进行了血糖浓度检测,结果表明,小鼠的高血糖症有了一定程度的缓解(图1H)。GTT结果显示小鼠的葡萄糖清除能力得到了改善(图1I)。ITT结果显示小鼠的胰岛素敏感性也有所增强(图1J)。
通过对BMP9敲除小鼠进行AAV-BMP9注射后,进行为期4个月的长期监测,结果发现,相对于AAV对照组小鼠,AAV-BMP9实验组小鼠的体重差异有逐渐缩小的趋势(图1K)。最终随着时间的推移,AAV-BMP9处理后的BMP9敲除小鼠的肥胖症状也得到了缓解(图1L)。
本实验的实验原理概况如下,通过利用AAV-BMP9对存在各种代谢综合征症状的BMP9敲除小鼠进行BMP9回补实验,可以实现BMP9在小鼠肝脏内特异性高表达。BMP9在小鼠肝脏中得到回补后,可以促进下游的p-smad 1/5蛋白的激活,进而促进PPARα的表达。由于受损的BMP9-smad-PPARα调控轴恢复了一定的功能,进而促进了下游相关功能基于的表达,。最终实现了肝脏脂肪变性,胰岛素抵抗以及肥胖等症状的缓解。上述结果表明,肝脏中的BMP9可以以一种PPARα介导的肝脏脂代谢调控的方式,对肝脏脂肪变性的发生发挥关键作用,因此BMP9可对非酒精性脂肪肝及其BMP9缺少或低表达动导致的肥胖具有良好的治疗效果。
以上对本发明及其实施方式进行了描述,这种描述没有限制性,附图中所示的也只是本发明的实施方式之一,实际的结构并不局限于此。总而言之如果本领域的普通技术人员受其启示,在不脱离本发明创造宗旨的情况下,不经创造性的设计出与该技术方案相似的结构方式及实施例,均应属于本发明的保护范围。
Claims (7)
1.肝细胞因子BMP9在制备延缓和/或治疗非酒精性脂肪肝药物的应用。
2.根据权利要求1所述的应用,其特征在于,所述药物包括在药学上可接受的载体。
3.根据权利要求2所述的应用,其特征在于,所述载体为AAV8。
4.根据权利要求1至3任一项所述的应用,其特征在于,所述药物为注射剂。
5.肝细胞因子BMP9在制备治疗肥胖症状药物的应用。
6.根据权利要求5所述的应用,其特征在于,所述药物包括载体AAV8。
7.根据权利要求5或6所述的应用,其特征在于,所述药物为注射剂。
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