CN111621561B - Olfm4在非酒精性脂肪肝(nafld)中的应用 - Google Patents
Olfm4在非酒精性脂肪肝(nafld)中的应用 Download PDFInfo
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Abstract
本发明公开了一种OLFM4在非酒精性脂肪肝(NAFLD)中的应用,具体包括OLFM4在制备NAFLD检测试剂中的应用以及OLFM4在制备NAFLD治疗药物中的应用,OLFM4表达变化对NAFLD具有调节作用,若治疗药物包含人源OLFM4序列重组蛋白特异性上调肝脏OLFM4表达,则可缓解NAFLD患者肝脏脂变。本发明还公开了一种NAFLD动物模型,该动物模型中敲除OLFM4基因,拟解决弥补现有技术在对NAFLD发生发展检测及治疗研究中对OLFM4的作用及评价方法的不足。本发明提供了OLFM4在NAFLD中的研究方法和应用,便于深入剖析NAFLD中OLFM4的作用,同时本技术方案的应用便于OLFM4作用机制的阐明,并为对OLFM4研究提供全新的角度。
Description
技术领域
本发明属于生物技术领域,具体是OLFM4在非酒精性脂肪肝(NAFLD)中Olfactomedin-4 (OLFM4)中的应用,具体包括用于制备检测试剂、治疗药物以及构建NAFLD动物模型。
背景技术
非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)是指除外酒精和其他明确肝损因素所致的以肝细胞脂肪变性和脂质贮积为特征的临床病理综合征。近年来,随着人们生活习惯和饮食结构的改变,我国成人NAFLD患病率快速增长,已高达29.2%,西方一些发达国家更是高于30%,已成为临床最常见的慢性肝病。根据病理学改变及临床表现,可将 NAFLD的自然病程分为单纯性NAFLD和非酒精性脂肪性肝炎(nonalcoholic steatohepatitis, NASH)。单纯性脂肪肝是一种良性疾病,而NASH却可以逐渐向肝硬化、肝癌等终末期肝病进展。脂变肝脏对很多药物和毒物的敏感性增加,增加了临床用药风险;再者,NAFLD可通过加剧机体代谢紊乱与2型糖尿病、冠心病、慢性肾脏疾病、肝外恶性肿瘤等疾病的高发密切相关,可能导致严重的临床后果。NAFLD发生发展的分子机制至今尚未明确,其治疗也缺乏特别有效方法。因此,加强NAFLD的相关研究,尤其是NAFLD发病机制的研究对推动NAFLD的防治工作有着重要的意义。
嗅质蛋白4(olfactomedin4,OLFM4,又称GW112或hGC-1)是一种具有多种生物学功能的糖蛋白,属于嗅质蛋白家族,该家族在C端特异性存在一个含有约250个氨基酸的嗅质蛋白结构域。OLFM4最初是在人造血髓细胞中用粒细胞集落刺激因子处理克隆得到,位于13q14.3号染色体上,编码了一个510个氨基酸的糖蛋白,其分子量为72kDa。OLMF4可在细胞膜、细胞质、细胞核、线粒体和成熟的中性粒细胞颗粒中表达。OLFM4的表达具有组织器官特异性,在消化道中表达相对较高,在胃、骨髓中有中度表达,在其他组织中表达较弱或不表达。OLFM4在炎症性肠病肠黏膜上皮细胞及胃癌等消化系统肿瘤组织中表达进一步升高,参与自身免疫调节以及肿瘤细胞周期与凋亡调控。目前尚未有研究报道OLFM4与非酒精性脂肪肝关系,本技术方案首次提出OLFM4可能与NAFLD相关,并通过预实验发现 OLFM4的表达水平在NAFLD的动物和细胞模型中显著上调,提示了OLFM4在NAFLD中的潜在调节作用。采用基因敲除技术进行OLFM4基因敲除小鼠的构建并以此基因背景研究 NAFLD发生发展,对揭示OLFM4在NAFLD中的潜在发病机制有重要意义,有助于启发后续研究从全新的角度解决NAFLD的治疗难题,具有重要的临床、科研意义和巨大的社会经济效益。
CRISPR/Cas(Clustered Regularly Interspaced Shot Palindromic repeats/CRISPR-associated) 系统,是一种基于细菌获得性免疫的由RNA介导CRISPR相关蛋白(Cas蛋白)对目的基因进行靶向DNA修饰的技术,广泛被应用于多种模式生物的基因敲除。在细菌及古细菌中, CRISPR系统分成3类,其中Ⅰ类和Ⅲ类需要多种CRISPR相关蛋白(Cas蛋白)共同发挥作用。而Ⅱ类系统只需一种Cas蛋白即可,这为其广泛应用提供了便利条件。CRISPR-Cas9系统应用最为广泛,具有适用物种范围广、载体构建快速、易操作优点。Cas9蛋白含有2个核酸酶结构域,可分别切割DNA两条单链。Cas9先与crRNA及tracrRNA结合为复合物,随后通过与PAM序列结合并侵入DNA,形成RNA-DNA复合结构,对目的DNA双链实现切割,使DNA双链断裂。由于PAM序列结构简单(5’-NGG-3’),序列选择限制较小,因此得到广泛的应用。CRISPR-Cas9系统已经成功应用于哺乳动物细胞、细菌、酵母、植物及鱼类等,是目前最高效的基因组编辑系统。因此,本技术方案选择基于此技术构建OLFM4基因敲除小鼠并应用于NALFD的研究。
发明内容
本发明的目的是提供OLFM4在非酒精性脂肪肝(NAFLD)中的应用及NAFLD动物模型。
为实现上述目标,本发明技术方案如下:
第一方面,公开一种OLFM4在制备NAFLD检测试剂中的应用,所述NAFLD检测试剂以OLFM4作为检测靶标。
进一步的,所述NAFLD检测试剂包含如SEQ ID NO.11和如SEQ ID NO.12所示的序列。
第二方面,本发明涉及OLFM4在制备NAFLD治疗药物中的应用,所述NAFLD治疗药物上调OLFM4的表达量。
进一步的,所述NAFLD治疗药物包含如SEQ ID NO.13所示的序列。
第三方面,本发明涉及一种NAFLD动物模型,该动物模型中敲除OLFM4基因。
进一步的,所述OLFM4基因敲除的位置为第四个外显子的位置。
进一步的,所述动物模型评估方法包括体重、血清学指标、胰岛素抵抗、油红染色、组织学特征等进行综合评估。
进一步的,通过特异性sgRNA靶向OLFM4基因第四个外显子进行基因敲除。
进一步的,所述特异性sgRNA序列如SEQ ID NO.1所示。
进一步的,第四个外显子的靶点附近检测引物序列如SEQ ID NO.6所示。
进一步的,所述的动物模型在NAFLD研究中的应用。
更进一步的,所述的动物模型在OLFM4研究中的应用。
第四方面,公开一种特异性sgRNA,特异性靶向小鼠OLFM4基因。
进一步的,所述sgRNA靶向序列如SEQ ID NO.1所示。
更进一步的,所述sgRNA靶点附近PCR检测引物序列SEQ ID NO.6所示。
本发明具有以下有益效果:
本发明研究非酒精性脂肪肝,于OLFM4基因缺失背景下构建NAFLD小鼠模型并应用,解决弥补现有技术在对NAFLD发生发展检测及治疗研究中对OLFM4的作用及评价方法的不足。通过检测证实模型构建达到了理想的效果并提供了其在NAFLD研究中的应用。本发明提供了OLFM4在NAFLD中的研究方法,便于深入剖析NAFLD中OLFM4的作用,同时本技术方案的应用便于OLFM4作用机制的阐明,并为对OLFM4研究提供全新的角度。另一方面本发明还提供了OLFM4非酒精性脂肪性肝病的检测、疗效评估、药物研发及潜在发病机制的研究中的应用。本发明具有重要价值。
附图说明
图1为NAFLD模型评估结果图,其中:
图1A为NAFLD模型小鼠体重评估结果图;
图1B为NAFLD模型小鼠血清甘油三酯测定结果图;
图1C为NAFLD模型小鼠血清总胆固醇测定结果图;
图1D为NAFLD模型小鼠血清谷丙转氨酶测定结果图;
图1E为NAFLD模型小鼠血清谷草转氨酶测定结果图;
图1F为NAFLD模型小鼠葡萄糖耐受实验结果图;
图1G为NAFLD模型小鼠肝脏H&E染色结果图;
图1H为NAFLD模型小鼠肝脏油红染色结果图;
图2为OLFM4-Cas9-KO小鼠策略设计图;
图3为saCas9/gRNA的活性检测结果图;
图4为F0代测序峰形图;
图5为基因敲除小鼠肝脏OLFM4基因表达检测图;
图6为OLFM4基因敲除小鼠NAFLD模型评估结果图,其中:
图6A为OLFM4基因敲除小鼠NAFLD模型小鼠体重评估结果图;
图6B为OLFM4基因敲除小鼠NAFLD模型小鼠肝脏甘油三酯测定结果图;
图6C为OLFM4基因敲除小鼠NAFLD模型小鼠血清谷丙转氨酶测定结果图;
图6D为OLFM4基因敲除小鼠NAFLD模型小鼠血清谷草转氨酶测定结果图;
图6E为OLFM4基因敲除小鼠NAFLD模型小鼠葡萄糖耐受实验结果图;
图6F为OLFM4基因敲除小鼠NAFLD模型小鼠肝脏H&E及油红染色结果图;
图7为NAFLD动物模型和人细胞模型中OLFM4表达改变图;
图8为NCBI基因表达数据库中基因芯片数据挖掘分析结果图;
图9为上调OLFM4表达调节NAFLD作用示意图。
具体实施方式
本发明构建了一种在非酒精性脂肪肝(NAFLD)中Olfactomedin-4(OLFM4)基因敲除动物模型的构建方法和应用。OLFM4在制备NAFLD检测试剂及治疗药物中的应用也属于本发明的保护范围。所述基于OLFM4基因敲除背景的非酒精性脂肪肝动物模型及OLFM4基因敲除小鼠模型的应用也属于本发明的保护范围。
下面结合实施例对本发明中的技术方案进行清晰、完整的说明。以下实施例将有助于本领域的技术人员进一步理解本发明,但不以任何形式限制本发明。应当指出的是,对本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干调整和改进。这些都属于本发明的保护范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的,油红O购自广州奥凯公司。统计学方法:采用SPSS11.5统计分析软件分析,各样本均数的比较采用Student t test分析,P<0.05 为差异有统计学意,*P<0.05,**P<0.01,***P<0.001。
实施例1:基于OLFM4基因敲除背景NAFLD动物模型
非酒精性脂肪肝(NAFLD)中Olfactomedin-4(OLFM4)基因敲除动物模型的构建方法通过以下步骤实现:
步骤1:构建NAFLD动物模型;
步骤2:构建OLFM4基因敲除动物模型;
步骤3:基于OLFM4基因敲除背景NAFLD动物模型;
步骤4:验证NAFLD中OLFM4基因敲除动物模型的成功构建。
具体如下:
步骤1-1:将8周龄野生型C57BL/6J小鼠随机分组如下:
正常饮食组(SCD):野生型C57BL/6J小鼠,正常饮水,普通饮食;
高脂饮食组(HFD):野生型C57BL/6J小鼠,正常饮水,包括高脂饮食4周、8周、12周。
其中,使用的高脂饲料:购自Research Diets,货号D12492,脂肪含量60kcal%,配方如下:
成份 | gm% | kcal% |
酷蛋白(80Mesh) | 200 | 800 |
L-胱氨酸 | 3 | 12 |
玉米淀粉 | 0 | 0 |
麦芽糊精10 | 125 | 500 |
蔗糖 | 68.8 | 275.2 |
纤维素BW200 | 50 | 0 |
豆油 | 25 | 225 |
猪油* | 245 | 2205 |
复合矿物质S10026 | 10 | 0 |
磷酸氢钙 | 13 | 0 |
碳酸钙 | 5.5 | 0 |
柠檬酸钾(1H<sub>2</sub>O) | 16.5 | 0 |
复合维生素V10001 | 10 | 40 |
重酒石酸氢胆碱 | 2 | 0 |
FD&C蓝色染料#1 | 0.05 | 0 |
总计 | 773.85 | 4057 |
步骤1-2:NAFLD模型评估
各组按计划喂养,喂养期间进行(1)-(2)实验,喂养至计划造模时间后,安乐处死,取各组肝脏组织样本及血样本,进行(3)-(6)实验。
(1)隔周称量体重,记录体重变化趋势。
(2)葡萄糖耐受实验(GTT)
收取样本前一周,进行葡萄糖耐受实验,检测肝脏胰岛素抵抗水平。禁食16小时,正常饮水,称各组小鼠体重,按小鼠体重腹腔注葡萄糖溶液0.1mg/10g,用生理盐水配制,于0,15min,30min,60min,90min,120min分别测血糖。
(3)血清生化指标检测。
眼眶取血,分离血清,检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平,以及血清甘油三酯(TG)及胆固醇(TC)含量。
(4)油红O染色
取小块冰冻肝脏组织,用冰冻切片机切8pm厚度的肝组织切片,用已稀释的油红O染液(储备液:ddH20=5:2,过滤)染色15min,蒸馏水洗15~30秒。苏木素染色5min,水洗 30~60秒。封片后于镜下观察。
(5)组织甘油三酯测定
按照甘油三酯测定试剂盒说明书进行操作。具体如下:称取一小块小鼠肝组织于2ml 离心管中,加入500μl裂解液,用组织研磨仪(加入磁珠)或者研磨棒手动匀浆裂解直至混合均匀得到混合液。4℃,12000rpm离心15min后,取50μl上清液采用BCA法蛋白定量试剂盒进行蛋白含量测定,其余混合液70℃加热10min,室温2000×g离心5min,上层清液用于酶学测定。96孔酶标板中将标准品和待测样品与工作液按说明书中列表所示体积混匀,37℃反应10min后用酶标仪在570nm波长下检测,用Excel作图分析数据,结合蛋白浓度以每mg蛋白浓度校正甘油三酯含量。
(6)肝脏组织H&E染色
取新鲜结肠组织,用预冷生理盐水清洗,4%多聚甲醛固定样本24小时以上,自动脱水机脱水后使用石蜡包埋样本后切片。染色前将切片置于60℃烘片机烘烤2小时。二甲苯脱蜡,梯度酒精脱水,苏木素染液染色5-15分钟,流水冲洗,洗去载玻片上多余染液;1×PBS溶液返蓝,1%盐酸酒精分色1-3s,流水冲洗15-30分钟;蒸馏水洗2s;0.1-0.5%伊红染液染色1-5分钟,蒸馏水洗2s。梯度酒精脱水,二甲苯透明,滴加中性树脂,加盖玻片封固,自然晾干后显微镜下观察。
根据如图1所示评估结果,HFD喂养8周小鼠呈现显著的NAFLD表现:小鼠毛发光泽度增加,动作迟缓,体重明显增加(图1A);HFD小鼠血清TG及TC含量与SCD小鼠相比明显升高(图1B,1C);HFD小鼠血清ALT及AST含量较SCD小鼠显著升高,提示有肝损伤(图 1D,1E);GTT实验结果显示HFD组小鼠葡萄糖耐受能力减弱,有胰岛素抵抗(图1F);光镜下 H&E切片显示,HFD小鼠弥漫性肝细胞脂肪变性,呈以小泡性为主的混合性脂肪变性,无肝细胞炎性表现(图1G);冰冻切片油红O染色显示,HFD小鼠有大量脂质沉积(图1H)。相比于HFD喂养8周,喂养4周小鼠未产生显著的脂肪肝改变;喂养16周小鼠肝脏脂变程度过高,肝脏损伤严重,影响小鼠生存状态,不利于实验进行,亦不同于常见人类脂肪肝状态。综上,HFD喂养8周小鼠呈现显著的NAFLD表现且更接近人类单纯性脂肪肝表现,成功构建稳定NAFLD模型,后续研究采用此模型构建方法。
实施例2:构建OLFM4基因敲除动物模型
步骤2-1:设计小鼠OLFM4基因CRISPR靶点并选择高活性gRNA靶点。
根据目标基因OLFM4的蛋白保守区和基因组结构,设计CRISPR靶点于转录产物OLFM4-201的exon7上或者在exon4上,如图2箭头所指位置。根据靶点,设计相应saCas9 的CRISPR靶点序列如下:
序列名称 | 序列 | PAM |
OLFM4-e4g1 | GACTCCAGCTTCTCTACCAAGAGGGT(SEQ ID NO.1) | GAGGGT |
OLFM4-e4g2 | GACCCTCTTGGTAGAGAAGCTGGAGT(SEQ ID NO.2) | TGGAGT |
OLFM4-e7g1 | TGTGATTCAGCTCAACTGGCTGGGGT(SEQ ID NO.3) | TGGGGT |
OLFM4-e7g2 | GGTTCTCTCTGGATGCTGAGGAGAGT(SEQ ID NO.4) | GAGAGT |
OLFM4-e7g3 | CACCCAGTACAGGGTTCTCTCTGGAT(SEQ ID NO.5) | CTGGAT |
使用saCas9/gRNA靶点效率检测试剂盒检测OFM4的gRNA靶点活性,试剂盒购自北京唯尚立德生物科技有限公司,货号:VK012。结果如图3所示,根据剪切DNA的活性结果,选用活性最高的gRNA,即靶向OLFM4-e4g1,GACTCCAGCTTCTCTACCAAGAGGGT(SEQ ID NO.1)的gRNA。
步骤2-2:显微注射首建OLFM4基因敲除小鼠。
将选中的OLFM4-e4g1体外转录成RNA,与saCas9蛋白一起显微注射到小鼠受精卵中,一共注射30个受精卵,注射结束后,将胚胎转移至含有M16培养液的培养皿中,放入37摄氏度、5%二氧化碳培养箱恢复0.5-1.0小时。将受精卵移植到假孕受体内。
步骤2-3:F0代OLFM4基因敲除小鼠的出生及鉴定。
移植后大约19-21天出生F0代小鼠13只。F0代小鼠两周龄后,对出生的小鼠进行基因型分析和鉴定,对基因组DNA CRISPR靶点位置附近序列进行PCR测序,确定首建小鼠是否发生移码突变,获得发生期望突变的首建小鼠。
测序条件如下:
引物选用前引物:Olfm4-F:ATTGCCTGAGATGTGACA(SEQ ID NO.6)
程序:
94℃——2min;
(98℃——10s,60℃——30s,68℃——20s)×35;
68℃——10min;
16℃保持
PCR片段长度为445bp。
检测结果共有2只小鼠发生移码突变,其中有1只为纯合子一只为杂合子,具体如下:
基因名 | 编号 | 性别 | 碱基变化 | 结论 |
OLFM4 | OLFM4-6# | 雄 | AGCCTGCTC前缺失139bp | 移码,纯合子 |
OLFM4 | OLFM4-13# | 雌 | GTCTCTAGACC前缺失22bp | 移码,杂合子 |
(1)OLFM4-6#:
基因型如下:
野生型(SEQ ID NO.7):
ATAAGGAATATGACCCTCTTGGTAGAGAAGCTGGAGTCTCTAGACCAAAACAATGTCC TTAGCATTCGCCGCCAGATCTTGGCTCTGAAGACCAAGCTGAAAGAATGTGAGGCCTCCAAAAGTGACCTTGTGCCTGCCACACCC CCTCCTCCTGCTCCTGGTAAGCCTGCTC
突变型:
ATAAGGAATATGACCCTCTTGGTA-------------------139bp------------------------AGCCTGCTC 测序峰图分析如下:
分析图4A所示测序峰形图,测序峰形图为单峰,表明为纯合子。通过峰形分析,可以将野生型的DNA序列(上方箭头所指峰形)和突变体的DNA序列(下方箭头所指峰形)分析出来。通过序列对比可以看出,突变体AGCCTGCTC前缺失139bp,发生移码突变。表明 OLFM4-6#为AGCCTGCTC前缺失139bp的突变体纯合子OLFM4(-/-)小鼠。
(2)OLFM4-13#:
基因型如下:
野生型(SEQ ID NO.8):
ATAAGGAATATGACCCTCTTGGTAGAGAAGCTGGAGTCTCTAGACC
突变型:
ATAAGGAATATGA---------22bp---------GTCTCTAGACC
测序峰图分析如下:
分析图4B所示测序峰形图,测序峰形图在突变位点开始出现双峰,表明为杂合子。通过峰形分析,可以将野生型的DNA序列(上方箭头所指峰形)和突变体的DNA序列(下方箭头所指峰形)分析出来,结果为GTCTCTAGACC前缺失22bp,发生移码突变。表明OLFM4-13# 为GTCTCTAGACC前缺失22bp的突变体杂合子OLFM4(+/-)小鼠
步骤2-4:OLFM4基因敲除稳定体系的建立
F0代OLFM4基因敲除小鼠性在8周龄性成熟后,与野生型C57BL/6J小鼠交配,出生的F1代小鼠,两周龄后进行基因型鉴定。OLFM4-6#品系共出生14只F1代小鼠,其中10 只小鼠带有移码突变。OLFM4-13#品系共出生9只F1代小鼠,其中7只小鼠带有移码突变。各品系F1代小鼠性成熟后,相互交配扩繁,出生F2代小鼠,在2周龄进行剪爪鉴定,仅保留纯合F2代雄鼠和雌鼠,性成熟后交配,得到OLFM4基因敲除纯合小鼠品系。
步骤3-1:分组。
将8周龄野生型C57BL/6J小鼠和OLFM4(-/-)基因敲除纯合小鼠分别随机分组如下:
正常对照组(WT SCD):野生型C57BL/6J小鼠,正常饮水,普通饮食
高脂对照组(WT HFD):野生型C57BL/6J小鼠,正常饮水,高脂饮食
正常实验组(KO SCD):OLFM4(-/-)小鼠,正常饮水,普通饮食
高脂实验组(KO HFD):OLFM4(-/-)小鼠,正常饮水,高脂饮食
高脂饲料配方:购自Research Diets,货号D12492,脂肪含量60kcal%。
步骤3-2:根据步骤3-1所述分组,给予各组不同饲料喂养8周后评估。
步骤4-1:OLFM4基因敲除动物模型验证
取OLFM4基因敲除纯合小鼠和野生型C57BL/6J小鼠肝脏组织样本,提取组织RNA,PCR检测肝脏中OLFM4含量,结果如图5所示。OLFM4(-/-)基因敲除纯合小鼠肝脏中OLFM4 成功敲除,且该基因敲除不影响小鼠生存状态,品系稳定遗传,OLFM4基因敲除动物模型构建成功。
步骤4-2:肝脏脂变评估。评估方法如步骤1-2所述,结果如图6所示。结果表明,HFD喂养8周后,高脂组体重均较正常饮食明显增加,且KO HFD组较WT HFD组体重上调更显著(图6A);KO HFD小鼠肝脏TG含量与WT HFD小鼠相比升高更显著(图6B);KO HFD小鼠血清ALT及AST含量较WT HFD小鼠显著升高,提示OLFM4敲除后有更明显的肝脏损伤(图6C,6D);GTT实验结果显示相比于WT HFD组小鼠,KO HFD小鼠葡萄糖耐受能力更弱(图1F);H&E染色和油红染色结果显示,KO HFD小鼠有更明显脂质沉积(图6F)。
以上结果表明,基于OLFM4基因缺失背景的NAFLD模型造模成功。且在NAFLD中,OLFM4基因敲除加重高脂饮食诱导的肝脏脂变和损伤。具体机制可通过此模型深入研究。由此可见,基于OLFM4基因缺失背景构建非酒精性脂肪肝动物模型在NAFLD的治疗和机制研究中具有重要作用,且为OLFM4的作用研究提供了新的方向。
实施例3:OLFM4作为NAFLD检测靶标的应用。
步骤1:实施例1步骤1中高脂饮食喂养C57BL/6J小鼠8周后,提取肝脏组织RNA,qPCR检测小鼠肝脏OLFM4 mRNA表达,检测所需引物序列如下:
F(SEQ ID NO.9):GTTAGGGTGAAGGAGGAATGA
R(SEQ ID NO.10):CTCCTAGACTTCCCTGAAGC
结果如图7A所示,高脂饮食喂养小鼠肝脏OLFM4在转录水平表达显著上调。
步骤2:人肝细胞株HepG2细胞(购自中国科学院上海细胞库)用含10%小牛血清、1%青 -链霉素的DMEM培养基(购自美国Gibco公司)传代培养,直至细胞达到70%~80%融合度。
步骤3:用软脂酸(PA)或游离脂肪酸(FFA)刺激细胞构建NAFLD细胞模型。软脂酸配置方法为:PA与小牛血清白蛋白以3:1(v/v)比例超声溶解,无菌过滤,加入DMEM培养基中;游离脂肪酸溶液配置方法为:油酸与软脂酸摩尔比为2:1,采用DMEM培养基稀释为1mM FFA工作液。用油红O染色或检测TG含量评价模型构建情况。软脂酸及油酸购于美国 Sigma-Aldrich公司。
步骤4:24小时后收集细胞,提取细胞RNA,qPCR检测人肝脏细胞中OLFM4 mRNA表达水平,检测所需引物序列如下:
F(SEQ ID NO.11):GAGGGACCAAATCTCCAACT
R(SEQ ID NO.12):ATCTGCCACATACAAAGCAT
结果如图7B所示,软脂酸刺激人肝细胞株HepG2细胞24小时建立的NAFLD细胞模型中,OLFM4 mRNA表达较对照细胞显著升高。
步骤5:我们进一步检索了NCBI基因表达数据库(www.ncbi.nlm.nih.gov/geoprofiles),以期寻找OLFM4与NAFLD存在联系的线索。结果发现,数据库中存在一些尚未被发掘的基因芯片结果支持OLFM4与NAFLD有关的推测。例如,有研究报道磷酸鞘氨醇裂解酶1 (Sphingosine 1-phosphate lyase,Sgpl1)基因敲除小鼠肝脏脂质沉积重于野生型小鼠,通过对他们的基因芯片数据深入挖掘,发现含有尚未被发掘的信息——Sgpl1基因敲除小鼠肝脏中 OLFM4表达水平显著高于野生型小鼠(NCBI GEO芯片数据登录号GSE18745),分析结果如图8A所示。再如,有研究者比较了肥胖症患者和非肥胖者肝脏基因表达的差异,他们的芯片数据中也包含着未被挖掘的信息——肥胖症患者肝脏OLFM4表达水平较非肥胖者有升高趋势(NCBI GEO芯片数据登录号GSE15653),分析结果如图8B所示。以上基因芯片结果提示OLFM4表达变化可能与人类肥胖及代谢性疾病相关,进一步提示可能与NAFLD有关。
以上结果表明,OLFM4在NAFLD小鼠模型肝脏和人肝细胞NAFLD模型中表达上调,且可能与人类肥胖及代谢性疾病相关,提示OLFM4表达的变化与NAFLD有关,OLFM4序列作为检测靶标包含于NAFLD检测试剂中有助于对NAFLD的检出。
实施例4:OLFM4在制备NAFLD治疗药物中的应用
步骤1:构建OLFM4过表达质粒,包含人源OLFM4基因序列,如SEQ ID NO.13所示(NCBI参考序列:NM_006418.5),本实施例中采用的质粒载体为pcdna3.1。
步骤2:取DMEM培养基培养的HepG2细胞,铺6孔板,细胞培养过夜,待每孔生长至50%-60%左右;
步骤3:根据lipo3000转染试剂盒说明书配制转染液,分别转染OLFM4过表达质粒和空载质粒到实验组和对照组HepG2细胞中,转染6小时后换成正常培养基,24小时后换成lmM FFA工作液刺激24小时。
步骤4:24小时后收集细胞,进行细胞TG检测,采用TG测定试剂盒(购自北京普利莱有限公司)及分光光度仪,根据GPO Trinder酶学反应原理检测细胞内TG浓度。先将试剂盒中不同浓度TG标准品加入TG工作液,然后用分光光度法检测570nm处吸光值(OD570) 并绘制TG浓度标准曲线。在待测细胞中加入甘油三酯检测裂解液,每个样本100μL,冰浴20分钟,取一半裂解液用于蛋白浓度测定,其余裂解液70℃水浴10分钟,室温2000 转离心5分钟,取上清加入TG工作液后检测OD570值,根据标准曲线获得TG浓度。检测中采用样本蛋白含量校准TG含量。结果如图9A所示。
步骤5:FFA刺激24小时后收集细胞,进行油红O染色,用已稀释得油红O染液(储备液:ddH20=5:2,过滤)液染色15min,蒸馏水洗15~30秒。苏木素染色5min,水洗30~60秒。封片后于镜下观察,结果如图9B所示。
如图9所示结果显示,运用融合质粒上调OLFM4的表达,可显著减轻游离脂肪酸诱导的HepG2细胞脂肪变。以上结果提示OLFM4表达变化对NAFLD具有调节作用,若治疗药物包含人源OLFM4序列重组蛋白特异性上调肝脏OLFM4表达,则可缓解NAFLD患者肝脏脂变。
序列表
<110> 浙江大学
<120> OLFM4在非酒精性脂肪肝(NAFLD)中的应用
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gactccagct tctctaccaa gagggt 26
<210> 2
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaccctcttg gtagagaagc tggagt 26
<210> 3
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgtgattcag ctcaactggc tggggt 26
<210> 4
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggttctctct ggatgctgag gagagt 26
<210> 5
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cacccagtac agggttctct ctggat 26
<210> 6
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
attgcctgag atgtgaca 18
<210> 7
<211> 172
<212> DNA
<213> Mus musculus
<400> 7
ataaggaata tgaccctctt ggtagagaag ctggagtctc tagaccaaaa caatgtcctt 60
agcattcgcc gccagatctt ggctctgaag accaagctga aagaatgtga ggcctccaaa 120
agtgaccttg tgcctgccac accccctcct cctgctcctg gtaagcctgc tc 172
<210> 8
<211> 46
<212> DNA
<213> Mus musculus
<400> 8
ataaggaata tgaccctctt ggtagagaag ctggagtctc tagacc 46
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gttagggtga aggaggaatg a 21
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctcctagact tccctgaagc 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gagggaccaa atctccaact 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atctgccaca tacaaagcat 20
<210> 13
<211> 2843
<212> DNA
<213> Homo sapiens
<400> 13
agcggctcca gctaagagga caagatgagg cccggcctct catttctcct agcccttctg 60
ttcttccttg gccaagctgc aggggatttg ggggatgtgg gacctccaat tcccagcccc 120
ggcttcagct ctttcccagg tgttgactcc agctccagct tcagctccag ctccaggtcg 180
ggctccagct ccagccgcag cttaggcagc ggaggttctg tgtcccagtt gttttccaat 240
ttcaccggct ccgtggatga ccgtgggacc tgccagtgct ctgtttccct gccagacacc 300
acctttcccg tggacagagt ggaacgcttg gaattcacag ctcatgttct ttctcagaag 360
tttgagaaag aactttccaa agtgagggaa tatgtccaat taattagtgt gtatgaaaag 420
aaactgttaa acctaactgt ccgaattgac atcatggaga aggataccat ttcttacact 480
gaactggact tcgagctgat caaggtagaa gtgaaggaga tggaaaaact ggtcatacag 540
ctgaaggaga gttttggtgg aagctcagaa attgttgacc agctggaggt ggagataaga 600
aatatgactc tcttggtaga gaagcttgag acactagaca aaaacaatgt ccttgccatt 660
cgccgagaaa tcgtggctct gaagaccaag ctgaaagagt gtgaggcctc taaagatcaa 720
aacacccctg tcgtccaccc tcctcccact ccagggagct gtggtcatgg tggtgtggtg 780
aacatcagca aaccgtctgt ggttcagctc aactggagag ggttttctta tctatatggt 840
gcttggggta gggattactc tccccagcat ccaaacaaag gactgtattg ggtggcgcca 900
ttgaatacag atgggagact gttggagtat tatagactgt acaacacact ggatgatttg 960
ctattgtata taaatgctcg agagttgcgg atcacctatg gccaaggtag tggtacagca 1020
gtttacaaca acaacatgta cgtcaacatg tacaacaccg ggaatattgc cagagttaac 1080
ctgaccacca acacgattgc tgtgactcaa actctcccta atgctgccta taataaccgc 1140
ttttcatatg ctaatgttgc ttggcaagat attgactttg ctgtggatga gaatggattg 1200
tgggttattt attcaactga agccagcact ggtaacatgg tgattagtaa actcaatgac 1260
accacacttc aggtgctaaa cacttggtat accaagcagt ataaaccatc tgcttctaac 1320
gccttcatgg tatgtggggt tctgtatgcc acccgtacta tgaacaccag aacagaagag 1380
attttttact attatgacac aaacacaggg aaagagggca aactagacat tgtaatgcat 1440
aagatgcagg aaaaagtgca gagcattaac tataaccctt ttgaccagaa actttatgtc 1500
tataacgatg gttaccttct gaattatgat ctttctgtct tgcagaagcc ccagtaagct 1560
gtttaggagt tagggtgaaa gagaaaatgt ttgttgaaaa aatagtcttc tccacttact 1620
tagatatctg caggggtgtc taaaagtgtg ttcattttgc agcaatgttt aggtgcatag 1680
ttctaccaca ctagagatct aggacatttg tcttgatttg gtgagttctc ttgggaatca 1740
tctgcctctt caggcgcatt ttgcaataaa gtctgtctag ggtgggattg tcagaggtct 1800
aggggcactg tgggcctagt gaagcctact gtgaggaggc ttcactagaa gccttaaatt 1860
aggaattaag gaacttaaaa ctcagtatgg cgtctaggga ttctttgtac aggaaatatt 1920
gcccaatgac tagtcctcat ccatgtagca ccactaattc ttccatgcct ggaagaaacc 1980
tggggactta gttaggtaga ttaatatctg gagctcctcg agggaccaaa tctccaactt 2040
ttttttcccc tcactagcac ctggaatgat gctttgtatg tggcagataa gtaaatttgg 2100
catgcttata tattctacat ctgtaaagtg ctgagtttta tggagagagg cctttttatg 2160
cattaaattg tacatggcaa ataaatccca gaaggatctg tagatgaggc acctgctttt 2220
tcttttctct cattgtccac cttactaaaa gtcagtagaa tcttctacct cataacttcc 2280
ttccaaaggc agctcagaag attagaacca gacttactaa ccaattccac cccccaccaa 2340
cccccttcta ctgcctactt taaaaaaatt aatagttttc tatggaactg atctaagatt 2400
agaaaaatta attttcttta atttcattat gaacttttat ttacatgact ctaagactat 2460
aagaaaatct gatggcagtg acaaagtgct agcatttatt gttatctaat aaagaccttg 2520
gagcatatgt gcaacttatg agtgtatcag ttgttgcatg taatttttgc ctttgtttaa 2580
gcctggaact tgtaagaaaa tgaaaattta attttttttt ctaggacgag ctatagaaaa 2640
gctattgaga gtatctagtt aatcagtgca gtagttggaa accttgctgg tgtatgtgat 2700
gtgcttctgt gcttttgaat gactttatca tctagtcttt gtctattttt cctttgatgt 2760
tcaagtccta gtctatagga ttggcagttt aaatgcttta ctcccccttt taaaataaat 2820
gattaaaatg tgctttgaaa aaa 2843
Claims (7)
1.OLFM4在制备NAFLD治疗药物中的应用,其特征在于,所述NAFLD治疗药物为OLFM4过表达质粒,上调OLFM4的表达量。
2.根据权利要求1所述的应用,其特征在于,所述NAFLD治疗药物包含如SEQ ID NO.13所示的序列。
3.一种NAFLD动物模型构建方法,其特征在于,在该动物模型中敲除OLFM4基因。
4.根据权利要求3所述动物模型构建方法,其特征在于,所述OLFM4基因敲除的位置为第四个外显子的位置。
5.根据权利要求4所述动物模型构建方法,其特征在于,通过特异性sgRNA靶向OLFM4基因第四个外显子进行基因敲除。
6.根据权利要求5所述动物模型构建方法,其特征在于,所述特异性sgRNA靶向序列如SEQ ID NO.1所示。
7.根据权利要求4所述动物模型构建方法,其特征在于,第四个外显子的靶点附近检测引物序列如SEQ ID NO.6所示。
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