CN116426633B - 一种载脂蛋白h在预防和/或治疗脂肪肝及相关疾病药物中的应用 - Google Patents
一种载脂蛋白h在预防和/或治疗脂肪肝及相关疾病药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种载脂蛋白H在筛选预防和/或治疗脂肪肝及相关疾病药物中的应用。研究表明,以野生型C57BL/6小鼠为对照,检测10周龄ApoH基因敲除小鼠(C57BL/6ApoH‑/‑)外周血血清转氨酶水平,结果示谷丙转氨酶(ALT)和谷草转氨酶(AST)水平明显升高,进一步检测肝组织甘油三酯(TG)含量,结果示ApoH‑/‑小鼠肝组织TG含量明显升高。本申请基于低表达载脂蛋白H诱导脂肪性肝炎,促进慢性肝病疾病进程,以及与终末期肝病预后相关的发现,有助于开发成改善脂肪肝和终末期肝病预后的精准靶向的治疗药物。
Description
技术领域
本发明涉及生物医药技术领域,特别涉及一种载脂蛋白H在预防和/或治疗脂肪肝及相关疾病药物中的应用。
背景技术
目前,我国成人脂肪肝患病率接近30%,已成为发病率最高的慢性肝病,超过病毒性肝病的发病率。而且随着慢性肝病的进展,伴随肝细胞脂肪变发生,将加速疾病进程,导致肝硬化和原发性肝癌,但机制仍未明确。
载脂蛋白H(APOH)也称为β2糖蛋白I(β2-GPI),由肝细胞合成和分泌,具有亲脂特性,能参与脂质代谢,也有报道认为是病毒感染的急性时相反应蛋白。我们前期工作发现APOH在慢性肝病进程中主要发挥代谢调控作用。但目前关于APOH在脂肪性肝病中的调控作用未见报道。
发明内容
本发明所要解决的技术问题是提供一种载脂蛋白H在预防和/或治疗脂肪肝及相关疾病药物中的应用,公开了在慢性肝病进程中肝细胞脂肪变的作用机制,提供一种全新的治疗脂肪肝及相关疾病的治疗方法。
本发明所要解决的技术问题是通过以下技术方案来实现的:
本申请的一个目的在于提供一种载脂蛋白H在筛选预防和/或治疗脂肪肝及相关疾病药物中的应用。
优选地,所述药物能够提高肝脏内载脂蛋白H的表达量。
优选地,所述载脂蛋白H用于调控肝脏代谢途径,降低肝脏脂肪变性程度,减轻炎症损伤和纤维化程度,改善终末期肝病的预后,延长生存时间。
优选地,所述脂肪肝及相关疾病包括:非酒精性和酒精性脂肪性肝病、病毒性肝病、自身免疫性肝病、肝纤维化、肝硬化、原发性肝癌。
本申请的另一个目的在于提供一种用于预防和/或治疗脂肪肝及相关疾病的药物,所述药物中包含载脂蛋白H或载脂蛋白H基因,或者所述药物中包括促进体内载脂蛋白表达的试剂,所述药物用于提高肝脏内的载脂蛋白H的表达量。
优选地,所述药物还包括医学上可接受的辅料,所述药物为注射剂、片剂、颗粒剂或口服剂、基因工程药物。
本申请的另一个目的在于提供一种药物在制备预防和/或治疗脂肪肝及相关疾病中的应用。
优选地,所述脂肪肝及相关疾病包括:非酒精性和酒精性脂肪性肝病、病毒性肝病、自身免疫性肝病、肝纤维化、肝硬化、肝癌。
本申请的另一个目的在于提供一种载脂蛋白H在制备脂肪肝及相关疾病不同进程的生物学诊断试剂盒中的应用。
本申请的另一个目的在于提供一种血清中载脂蛋白H的含量在作为判断脂肪肝及相关疾病进程的血清诊断标志物中的应用。
本发明上述技术方案,具有如下有益效果:
本发明发现,ApoH基因敲除小鼠存在自发性脂肪性肝炎,在终末期肝病患者中载脂蛋白H基因表达降低,主要参调控肝脏代谢途径,且与患者预后呈负相关,有助于开发成改善脂肪肝和终末期肝病预后的精准靶向治疗药物。
附图说明
被结合在说明书中并构成说明书的一部分的附图示出了本发明的实施例,并且连同其说明一起用于解释本发明的原理。
图1为小鼠ApoH基因guide RNA;
图2为模型小鼠肝组织基因测序结果;
图3为扩繁后实验用鼠的鉴定结果(其中:A为小鼠肝组织提RNA行RT-qPCR检测结果,B为模型小鼠外周血转氨酶ALT水平,C为模型小鼠外周血转氨酶AST水平,D为模型小鼠肝组织甘油三酯含量)。
图4为慢性肝病和原发性肝癌患者肝组织中APOH基因表达水平及肝癌患者预后分析(其中:A为非酒精性脂肪性肝炎(NASH)患者不同纤维化程度的肝组织中APOH基因表达水平,B为乙型肝炎肝硬化患者不同炎症分级的肝组织中APOH基因表达水平,C为乙型肝炎肝硬化患者不同纤维化程度的肝组织中APOH基因表达水平,D为TCGA数据库中乙肝病毒感染肝细胞癌患者肝组织中APOH基因表达水平,E为GEO数据库中乙肝病毒感染肝细胞癌患者肝组织中APOH基因表达水平,F为ICGC数据库中乙肝病毒感染肝细胞癌患者肝组织中APOH基因表达水平,G为CHCC数据库中乙肝病毒感染肝细胞癌患者肝组织中APOH基因表达水平,H为TCGA数据库中乙肝病毒感染肝细胞癌患者肝组织中APOH基因低表达与高表达的预后分析,I为GEO数据库中乙肝病毒感染肝细胞癌患者肝组织中APOH基因低表达与高表达的预后分析,J为ICGC数据库中乙肝病毒感染肝细胞癌患者肝组织中APOH基因低表达与高表达的预后分析,K为CHCC数据库中乙肝病毒感染肝细胞癌患者肝组织中APOH基因低表达与高表达的预后分析)。
图5为小鼠脂肪肝造模后的表型检测(其中:A为小鼠肝组织提RNA行RT-qPCR检测结果,B为模型小鼠外周血转氨酶ALT水平,C为模型小鼠外周血转氨酶AST水平)。
具体实施方式
现在将参照附图来详细描述本发明的各种示例性实施例。应注意到:除非另外具体说明,否则在这些实施例中阐述的部件和步骤的相对布置、数字表达式和数值不限制本发明的范围。
一种载脂蛋白H在筛选预防和/或治疗脂肪肝及相关疾病药物中的应用。
载脂蛋白H(APOH)也称为β2糖蛋白I(β2-GPI),由肝细胞合成和分泌,具有亲脂特性,能参与脂质代谢,也有报道认为是病毒感染的急性时相反应蛋白。我们前期工作发现APOH在慢性肝病进程中主要发挥代谢调控作用。但目前关于APOH在脂肪性肝病中的调控作用未见报道。本研究的实验结果可以为治疗脂肪肝及相关疾病的药物提供一个新的靶点,靶向肝脏,增加APOH表达,调节肝脏脂质代谢,使其恢复稳态,协助治疗脂肪肝及相关疾病。
本申请的药物可以是治疗性药物,也可以是预防性药物。
具体的,药物能够提高肝脏内载脂蛋白H的表达量。本申请通过外源补充载脂蛋白H复合物或基因工程药物,靶向肝脏发挥作用;或者外源应用保护肝细胞药物或应用人工肝支持系统治疗,以恢复或改善肝细胞功能,促进载脂蛋白H合成增加;或者通过干预调控肠-肝轴,进一步减轻肝脏炎症损伤,恢复或改善肝细胞功能,促进载脂蛋白H合成增加。
具体的,脂肪肝及相关疾病包括但不限于:非酒精性和酒精性脂肪性肝病、病毒性肝病、自身免疫性肝病、肝纤维化、肝硬化、原发性肝癌等。
一种用于预防和/或治疗脂肪肝及相关疾病的药物,所述药物中包含载脂蛋白H,或者所述药物中包括促进体内载脂蛋白表达的试剂,所述药物用于提高肝脏内的载脂蛋白H的表达量。所述药物还包括医学上可接受的辅料,所述药物为注射剂、片剂、颗粒剂或口服剂、基因工程药物。
一种药物在制备预防和/或治疗脂肪肝及相关疾病中的应用。载脂蛋白H用于调控肝脏代谢途径,降低肝脏脂肪变性程度,减轻炎症损伤和纤维化程度,改善终末期肝病患者预后,延长生存时间。
具体来说,该药物能够调节肝脏内载脂蛋白H的表达量。以载脂蛋白H作为靶点,针对不同患者的病情,调节体内的蛋白的表达量,维持体内载脂蛋白H含量正常,有助于肝脏脂代谢功能恢复稳态。
以野生型C57BL/6小鼠为对照,检测10周龄ApoH基因敲除小鼠(C57BL/6ApoH-/-)外周血血清转氨酶水平,结果示谷丙转氨酶(ALT)和谷草转氨酶(AST)水平明显升高(急慢性肝病时AST/ALT比值<1,常提示肝脏损害较轻,AST/ALT比值>1,则提示肝脏损害较重)。进一步检测肝组织甘油三酯(TG)含量,结果示ApoH-/-小鼠肝组织TG含量明显升高。我们可以明确编码ApoH基因敲除小鼠存在脂肪性肝炎。因此,提示我们,当肝脏载脂蛋白H合成分泌量降低时,可通过外源给药提高其含量,促进肝脏脂代谢恢复稳态,进一步减轻肝细胞脂肪性损伤,有助于恢复肝功能。
一种载脂蛋白H在制备脂肪肝及相关疾病不同进程的生物学检测试剂中的应用。一种血清中载脂蛋白H的含量在作为判断脂肪肝及相关疾病进程的血清诊断标志物中的应用。也就是说载脂蛋白H可作为脂肪肝及相关疾病的生物标记物。具体地,检测试剂可以通过载脂蛋白H的表达量来判定脂肪肝及相关疾病的病情严重程度。
分析不同慢性肝病肝组织转录组测序结果发现,非酒精性脂肪性肝炎患者和慢性乙型肝病患者肝组织中APOH基因表达水平,发现随着肝纤维化程度逐渐增强,APOH基因表达明显降低,差异有显著性。
进一步分析原发性肝癌队列的肝组织转录组测序结果,发现:与癌旁正常肝组织相比,肝癌组织中APOH基因表达明显降低。进一步生存分析表明,APOH低表达与患者预后呈负相关。
研究表明,当载脂蛋白H合成分泌量降低时,可通过外源药物补充或者改善肝功能,以提高其表达量,从而治疗脂肪肝及相关疾病。
以下以实施例详细说明:
实施例1构建ApoH基因敲除小鼠(C57BL/6)及表型鉴定
构建ApoH基因敲除小鼠(C57BL/6)及表型鉴定,模型构建在厦门大学实验动物中心完成。
构建包括以下步骤:
S1、设计并测试guide RNA效率:
Guide RNA(gRNA)设计:小鼠ApoH基因有两个异构体,挑选其共有的功能性区域所在比较靠前的外显子。本次实验设计针对两个外显子区域的4条gRNA,如图1所示;
S2、设计gRNA效率的测试:
构建合适抗性的Cas9 gRNA质粒,转染细胞,抗性药杀转染的细胞和未转染的细胞,未转染的细胞死完后取转染Cas9 gRNA的细胞,提取基因组。
设计在gRNA切割位点前后各300bp的引物,PCR,回收,连接到载体上,涂板,挑菌16个,送测序,统计16个菌中有KO的比例,选取敲除效率高于50%的进行下步实验。
在本实验中,我们最后选取作用在异构体X1第五个外显子(exon5)上的一对gRNA,序列如下:
gRNA-2-1:5’-tccaaagtttgcactcctta-3’;
gRNA-2-2:5’-gattgccagaatgcctgggt-3’。
S3、gRNA的订购:
从Thermo公司订购2OD的引物,序列如下:
加粗下划线序列为gRNA 5’-3’的序列,加粗斜体序列为T7。
S4、Cas9的PCR引物订购,序列为:
Cas9-F:5’-caccgactgagctccttaag-3’;
Cas9-R:5’-tagtcaagcttccatggctcga-3’。
S5、胞浆注射:
gRNA和Cas9体外转录后,按Cas9 100ng/μl和gRNA50ng/μl比例混合后进行0.5天受精卵的显微注射。共注射200个受精卵,移植10只ICR假孕母鼠,19天后获得39只仔鼠。
S6、小鼠基因敲除鉴定:
根据gRNA的位置设计PCR引物,序列为:
ApoH-F5’-TGGCATTGAA C TCACACT-3’;
ApoH-R5’-AACTAAGGCTACACAGAGAA-3’。
所得PCR产物进行核酸电泳,选择19只与野生型序列明显不同者送测序,确定缺失片段正确鼠进行繁殖。目前使用片段缺失140bp小鼠进行扩繁造模。
扩繁后实验用鼠的鉴定结果如图所示:
图2所示小鼠肝组织基因测序结果,图3中A所示小鼠肝组织提RNA,行RT-PCR检测结果。上述结果可确定小鼠体内ApoH基因已敲除。
S7、基因敲除小鼠表型鉴定:
转氨酶水平:以野生型C57BL/6小鼠为对照,检测10周龄ApoH基因敲除小鼠(C57BL/6ApoH-/-)外周血血清转氨酶水平,结果示ALT和AST水平明显升高(图3中B和C图)。
TG含量检测:进一步检测肝组织TG含量,结果示ApoH-/-小鼠肝组织TG含量明显升高(图3中的D图)。
综上,我们可以明确编码ApoH基因敲除小鼠存在自发性脂肪性肝炎。
实施例2慢性肝病和原发性肝癌患者肝组织中APOH基因表达水平及肝癌患者预后分析
S1、分析慢性肝病患者肝组织转录组测序结果,发现:非酒精性脂肪性肝炎患者肝纤维化程度增加,APOH基因表达水平明显降低(表1);慢性乙型肝病患者,随着肝脏炎症和纤维化程度增加,肝组织APOH基因表达水平也明显降低(表2)。图4中A示NASH患者不同纤维化程度的肝组织中APOH基因表达水平,差异有显著性。图4中B和C示乙型肝炎肝硬化患者,随着肝脏炎症和纤维化程度增加,APOH基因表达明显降低,差异有显著性。
表1.NASH患者肝组织不同纤维化分期的APOH基因表达水平
表2.慢性乙型肝病患者G和S分期的肝组织中APOH基因表达水平
S2、进一步分析原发性肝癌队列的肝组织转录组测序结果,发现:发现乙型病毒感染的原发性肝癌患者肝癌组织中APOH基因表达明显降低,与癌旁正常肝组织中APOH含量相比,差异有显著性(图4)。进一步生存分析表明,肝癌组织中APOH高表达与低表达相比,患者生存时间明显延长,也就是说肝癌患者肝组织中APOH低表达,与患者预后呈负相关(图4)。
综上,APOH低表达介导的脂肪性肝炎可加速慢性肝病疾病进程,且与肿瘤患者预后密切相关。进一步证实我们的研究对脂肪性肝病精准诊疗的重要临床意义。
实施例3提高载脂蛋白H表达量对小鼠脂肪性肝炎的影响
以6周龄C57BL/6ApoH-/-和野生型小鼠为研究对象,给予45%高脂饲料喂养12周,进一步构建脂肪肝模型。造模结束,收集小鼠血清和肝组织,检测血清转氨酶水平和肝组织中ApoH基因表达水平。
结果显示,脂肪肝造模组小鼠,野生型小鼠肝组织中ApoH基因表达水平明显降低,略高于基因敲除小鼠肝组织中ApoH表达水平(图5中A图);且与野生型小鼠相比,ApoH-/-小鼠血清ALT和AST水平无明显差异(图5中B和C图)。
因此,进一步证实ApoH基因低表达对脂肪性肝炎的关键调控作用。
下面,通过下述方法提高小鼠体内APOH含量,以明确其对肝脏脂肪性炎症损伤的影响。
(一)通过小鼠尾静脉外源补充重组APOH蛋白:将不同浓度的重组鼠源APOH蛋白通过尾静脉注入ApoH基因敲除小鼠体内,以未注射组为对照,选择注射后不同时间点收集小鼠外周血和肝组织,检测外周血转氨酶、血脂水平,肝组织APOH和甘油三酯含量,肝组织脂肪变性和炎症、纤维化程度等。
(二)靶向小鼠肝组织使ApoH基因过表达:以ApoH基因敲除小鼠为背景,进一步构建腺相关病毒介导的肝细胞特异性ApoH基因过表达模型。选择不同时间点收集小鼠外周血和肝组织,检测外周血转氨酶、血脂水平,肝组织APOH和甘油三酯含量,肝组织脂肪变性和炎症、纤维化程度等。
(三)构建无菌肠道微环境小鼠模型:以ApoH基因敲除小鼠为研究对象,采用DagHenrik Reikvamd等报道的抗生素复合物应用方案,抑制小鼠肠道细菌生长,使其处于无菌环境。以原肠道微生态环境小鼠为对照。检测外周血转氨酶、血脂水平,肝组织APOH和甘油三酯含量,肝组织脂肪变性和炎症、纤维化程度等。
初步研究结果显示,通过外源直接补充APOH蛋白,或通过肠-肝轴调控减轻肝脏炎症损伤,以促进肝细胞合成APOH能力增加,可使小鼠肝脏炎症损伤减轻,具体表现为:转氨酶水平显著降低,肝组织脂肪变性、炎症、纤维化程度减轻,甘油三酯(TG)含量下降。进一步提示:在慢性肝病进程中,检测APOH水平及提高体内APOH含量,使其维持正常,对脂肪性肝病精准诊疗,以及改善终末期肝病预后的重要意义。
同时,通过对载脂蛋白H在脂肪肝及相关疾病中的表达变化和作用分析研究,确定载脂蛋白H在疾病发生过程中的表达变化规律,可以通过血清中载脂蛋白H的含量来判断疾病的程度,解决临床上原发性肝癌早期难诊断困难的问题;而且更为重要的是,蛋白作为潜在的血清诊断标志物,辅助临床采用无创检测手段判断疾病分期,使疾病的无创性诊断成为可能。从疾病治疗角度,可以通过外源补充含载脂蛋白H的药物或补充能促进体内载脂蛋白H表达的药物来进行治疗,从而减轻或者延缓疾病进展。
本发明的技术方案明确了载脂蛋白在疾病发生中的作用以及变化规律,建立了脂肪肝及相关疾病不同进程的生物学诊断试剂盒以及疾病治疗的方案。通过提高患者体内载脂蛋白H含量,可以作为治疗脂肪肝及相关疾病的有效方案。本申请的技术方案明确了载脂蛋白H在治疗疾病中的作用。
虽然本发明已以实施例公开如上,然其并非用于限定本发明,任何本领域技术人员,在不脱离本发明的精神和范围内,均可作各种不同的选择和修改,因此本发明的保护范围由权利要求书及其等同形式所限定。
Claims (3)
1.载脂蛋白H在制备预防和/或治疗脂肪肝药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述药物中包含载脂蛋白H或载脂蛋白H基因,所述药物用于提高肝脏内的载脂蛋白H的表达量。
3.根据权利要求1所述的应用,其特征在于,所述药物还包括医学上可接受的辅料,所述药物为注射剂、片剂、颗粒剂或口服剂、基因工程药物。
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Alcohol-dependent downregulation of apolipoprotein H exacerbates fatty liver and gut microbiota dysbiosis in mice;Yaming Liu等;Lipids in Health and Disease;第89卷(第21期);第1-10页 * |
Apolipoprotein H induces sex-specific steatohepatitis and gut dysbiosis during chronic hepatitis B infection;Yaming Liu等;iScience(第26期);第1-16页 * |
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