FAM76B在小鼠各组织中的表达分布和FAM76B基因敲除对小鼠肝脏脂代谢影响的初步研究

【作者】 郑晓晶；

【导师】 夏海滨；

【作者基本信息】 陕西师范大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 FAM76B是由339个氨基酸组成的细胞核蛋白,分子量约为39kDa。人源的FAM76B基因定位于11q21染色体上,全长21468bp,生物学功能尚不明确。通过氨基酸序列比对,发现人的FAM76B氨基酸序列与大鼠、小鼠以及斑马鱼的FAM76B氨基酸序列相似度极高,说明FAM76B氨基酸序列高度保守,也暗示了FAM76B蛋白可能存在重要的生物学功能。目前已知FAM76B氨基酸序列中存在组氨酸重复序列(poly-His domain)和Lys-225的泛素化(ubiquitination)位点。通过基因本体(Gene Ontology,GO)分析表明,含有组氨酸重复序列的蛋白大多数为核蛋白,其生物学功能与基因转录和神经发育密切相关。FAM76B蛋白定位在细胞核的核散斑体(Nuclear speckles)中,而核散斑体属于细胞核亚结构,可对RNA剪切复合体进行保存和组装。FAM76B是本实验室以人颗粒蛋白前体(Progranulin,PGRN)作为诱饵蛋白进行酵母双杂交筛选,所获得的与PGRN具有相互作用的新的蛋白质。我们前期已经通过GST-pull down和CO-IP的方法进一步验证了两者的相互作用。PGRN是具有多重生物学功能的蛋白,主要涉及细胞增殖、伤口修复、生长发育以及神经系统发育等多种生理功能,同时PGRN与一系列疾病的发生密切相关,如肿瘤发生、神经退行性疾病、Ⅱ型糖尿病、脂类代谢疾病及炎症等。尽管FAM76B与PGRN存在相互作用,但FAM76B是否参与上述疾病的发生,目前还不清楚。为了深入研究FAM76B蛋白的生物学功能,本课题通过单克隆抗体制备技术,获得针对人FAM76B蛋白的单克隆抗体;由于人与小鼠FAM76B蛋白的氨基酸序列高度相似,本课题进一步验证了所获得针对人FAM76B的单克隆抗体可以特异识别小鼠的FAM76B蛋白。随后研究FAM76B在正常小鼠主要组织中的表达和分布;建立FAM76B基因敲除小鼠模型,初步探索FAM76B基因敲除小鼠表型的改变和相关疾病的发生机制,为探讨FAM76B蛋白的功能奠定了基础。本课题具体研究内容如下:1.FAM76B单克隆抗体的制备及鉴定:用纯化的原核人FAM76B-6His融合蛋白免疫Balb/c小鼠,取小鼠脾细胞与小鼠骨髓瘤细胞SP2/0进行融合;以纯化的人FAM76B-6His作为包被抗原,采用间接ELISA方法筛选出阳性克隆的杂交瘤细胞。通过有限稀释法对阳性克隆进行亚克隆,将最终阳性克隆扩大培养,制备针对人FAM76B的单克隆抗体6株,分别命名为FAM76B McAb No.1-6。采用Western blot、免疫沉淀以及免疫细胞化学染色等手段,对所获得的单克隆抗体进行一系列的检测。2.FAM76B蛋白在正常小鼠组织中的表达和分布:将人FAM76B蛋白的氨基酸序列与小鼠FAM76B蛋白的氨基酸序列进行比对,发现两者氨基酸序列相似度可达到98%,说明FAM76B蛋白高度保守。用上述所获的针对人FAM76B的单克隆抗体对鼠源FAM76B蛋白进行检测,同时通过Real-time PCR、Western blot及免疫组化的方法检测FAM76B在正常小鼠心、肝、脾、肺、肾、肌肉以及脑组织的表达和分布。3.FAM76B基因敲除小鼠的构建及鉴定:本实验室通过与公司合作,定制FAM76B基因敲除小鼠模型。通过PCR、Real-time PCR以及Western blot方法对本实验室繁育的FAM76B基因敲除小鼠后代,在基因组水平、RNA水平以及蛋白水平进行鉴定。4.FAM76B基因敲除小鼠表型的初步分析:对本实验室繁育的FAM76B基因敲除小鼠的雌雄比例、不同基因型比例进行统计。对不同时期小鼠的体质量、体长、肝脏、肾脏、脾脏以及脂肪组织称重,分析FAM76B基因敲除小鼠生长发育特征。5.FAM76B基因敲除小鼠肝脏和脂肪的组织病理学特征分析:对不同时期小鼠肝脏组织进行H&E染色和油红染色,对脂肪组织进行H&E染色,分析小鼠肝脏和脂肪的组织病理特征。6.FAM76B基因敲除小鼠相关血清指标分析:通过全自动生化分析仪检测FAM76B基因敲除小鼠血脂指标(TG、CHOL、HDL和LDL)和肝功能指标(ALT和AST),分析FAM76B基因敲除小鼠是否存在血脂异常及肝脏损伤。7.FAM76B基因敲除小鼠肝脏脂代谢相关基因表达的分析:利用Real-time PCR方法,检测1,3,6,9,12,15月龄FAM76B+/-小鼠肝脏组织脂代谢相关基因的表达水平:de novo lipogenesis 环节中 FASN、SCD、ACC、Dgat 1 和 Dgat 2;脂肪酸氧化途径中Cptl、Acox和Mcad;TG分泌过程中Mtp和Vldlr。同时检测lipin1、SREBP-1、PPARα和PGC-1α的mRNA相对表达量。通过上述研究结果,分析FAM76B基因敲除小鼠肝脏脂代谢异常的主要因素和途径。8.FAM76B基因敲除小鼠肝脏中F4/80、TNFα和PGRN基因表达的分析:检测1,3,6,9,12,15月龄FAM76B+/-小鼠肝脏组织中F4/80、TNFα和PGRN的mRNA水平,分析FAM76B在炎症反应中的作用。通过以上实验内容,本课题获得了以下结果:1.FAM76B单克隆抗体的制备及鉴定最终获得针对人FAM76B的单克隆抗体6株,分别命名为FAM76B McAb No.1-6。经过Western blot、免疫沉淀以及免疫细胞化学染色等手段研究发现,发现FAM76BMcAbNo.1,No.2和No.5抗体对人源FAM76B蛋白具有很好的亲和性,适用于人源FAM76B蛋白的Western blot、IP以及免疫细胞化学等检测,为研究FAM76B的功能提供了有利的工具。围绕组氨酸重复序列结构域构建多个的FAM76B截短体原核表达载体并进行小量诱导表达,通过Western blot检测发现FAM76B McAb No.3和No.6抗体所识别的表位在FAM76B蛋白的第97-163氨基酸之间;FAM76B McAb No.2抗体所识别的表位在第187-262氨基酸之间；FAM76B McAb No.5抗体所识别的表位在第263-265氨基酸之间;FAM76B McAb No.1抗体所识别的表位在第266-339氨基酸之间。同时构建相应的FAM76B截短体真核表达载体,转染HEK 293细胞后进行免疫细胞化学染色,发现FAM76B蛋白的核定位信号肽位于组氨酸重复序列结构域的后端,大概位于第187-265氨基酸之间。2.FAM76B蛋白在正常小鼠组织中的表达和分布用上述制备的针对人FAM76B的单克隆抗体对鼠源FAM76B蛋白进行检测,发现FAM76B McAb No.1,No.2和No.5抗体对鼠源FAM76B蛋白也具有很好的亲和性,适用于鼠源FAM76B蛋白的Western blot检测和免疫细胞化学染色。通过Real-time PCR和免疫组化染色方法检测FAM76B在正常小鼠心、肝、脾、肺、肾、肌肉以及脑组织的表达和分布,发现FAM76B在小鼠各组织中广泛分布,其中脑组织表达最高,其次是肝脏组织和脾脏组织。3.FAM76B基因敲除小鼠的鉴定对本实验繁育的FAM76B基因敲除小鼠子代,在基因组水平进行了鉴定,证实获得了三种基因型的小鼠子代;通过Real-time PCR实验在FAM76B的RNA水平进行鉴定,结果表明纯合子小鼠MEF细胞中FAM76B的mRNA几乎检测不到,杂合子小鼠MEF细胞中FAM76B的mRNA表达水平略高于50%;在蛋白水平,采用了本实验室制备的针对FAM76B的抗体对不同基因型的小鼠MEF中的FAM76B蛋白进行Western blot检测,结果表明纯合子小鼠MEF细胞不表达FAM76B蛋白,杂合子小鼠MEF细胞中FAM76B的蛋白表达量约为野生型的一半;上述实验结果表明FAM76B基因敲除小鼠已构建成功。4.FAM76B基因敲除小鼠表型的初步分析在生殖繁育方面,雌雄比1:1,野生型:杂合型的比例约为1:2,基本符合孟德尔遗传学定律,但纯合子小鼠比例明显下降(39.5%+/+,51.5%+/-,9%-/-),表明纯合型FAM76B基因敲除小鼠的产率较低,推测可能FAM76B基因对胚胎发育方面存在某种影响,造成部分纯合子小鼠胚胎死亡以及出生鼠死亡,最终导致出生比例发生明显的波动。在生长发育方面,1-5月龄杂合型FAM76B基因敲除小鼠的体质量相比野生型小鼠,没有明显的差异;6-8月龄杂合型FAM76B基因敲除小鼠的体质量高于野生型小鼠,并且体质量差逐步拉大;9月龄和15月龄,FAM76B杂合型基因敲除的小鼠体质量明显大于野生型,存在显著性差异。同时杂合型FAM76B基因敲除小鼠的肝脏和脂肪重量的变化的趋势与体质量相似。通过长期对杂合型FAM76B基因敲除小鼠生长发育的观察和测量,发现FAM76B基因敲除与小鼠肥胖有关。5.FAM76B基因敲除小鼠肝脏和脂肪的病理学特征对1-9月龄、12月龄和15月龄杂合型FAM76B基因敲除小鼠的肝脏HE染色结果表明:杂合型FAM76B基因敲除小鼠呈现进程性脂肪变性和炎症反应。HE实验结果表明:相比WT小鼠,在1-3月龄期间FAM76B+/-小鼠肝脏细胞在高倍镜下观察无明显的囊泡脂滴。4-5月龄期间,FAM76B+/-小鼠肝脏细胞在高倍镜下开始能够观察到多囊泡型脂滴。6-8月期间,FAM76B+/-鼠组肝脏细胞在高倍镜下能够观察到少量的大泡型脂滴和大量囊泡型脂滴。9和15月龄期间,WT鼠肝脏组织内出现零星的大泡脂滴,肝脏细胞内有少量囊泡型脂滴;FAM76B+/-小鼠组肝脏组织中出现大量空泡。肝细胞能观察到较多的大泡型脂滴并且比例随着月份的增加,同时细胞内含有大量囊泡型脂滴,尤其15月龄FAM76B+/-小鼠组观察到轻度肝小叶炎症。依据NAFLD病理学分析的半定量评分系统结合Matteoni等提出的建议,对不同月份FAM76B+/-小鼠组进行病理学评分,8和9月龄的FAM76B+/-小鼠分类为1级,15月龄的FAM76B+/-小鼠组分类为2级,其他组未到到病理级别。以上结果说明,随着年龄的增长,FAM76B+/-小鼠组肝脏非酒精性脂肪肝的病变程度逐步增强。提示FAM76B的缺失影响肝脏非酒精性脂肪肝的形成和发展。肝脏油红染色显示:杂合型FAM76B+/-小鼠相比WT小鼠,1-3月龄期间均无明显的有脂滴,染色无明显差异。4-5月龄期间,杂合型FAM76B+/-小鼠组,存在明显脂滴,但脂滴直径较小密度较低。6-8月龄期间,杂合型FAM76B+/-小鼠组,大泡型脂滴明显逐步数量增多。9和15月龄,大泡型脂滴明显。油红染色结果与病理学观察结果相符合。腹腔脂肪细胞形态学观察显示:FAM76B+/-小鼠与WT小鼠相比,1-3月龄期间腹腔脂肪细胞的面积没有明显差异。4-7月龄期间腹腔脂肪细胞的面积差距逐步变大,但差异不明显。8-15月龄期间,FAM76B+/-小鼠组腹腔脂肪细胞面积大于对照组。6.FAM76B基因敲除小鼠血清指标分析仅1和3月龄的FAM76B+/-小鼠血清中TG水平明显高于野生型小鼠,差异显著。其他月龄FAM76B+/-小鼠血清中TG水平略高于野生型小鼠,但差异不明显;1月、3月、9月和12月份,FAM76B+/-小鼠血清中CHOL水平明显高于野生型小鼠,差异显著,6和15月龄的FAM76B+/-小鼠血清中CHOL水平高于野生型小鼠,但没有显著性差异;FAM76B+/-小鼠血清中HDL水平仅在12月存在显著性差异外,其他月龄没有明显的差异;各组间LDL的水平没有显著性差异。检测FAM76B基因敲除小鼠肝功能指标,发现15月龄的FAM76B+/-小鼠血清中ALT达到较高值,并与野生组相比存在显著性差异,其他组ALT无明显差异。AST各组之间均无明显差异。上述结果表明,FAM76B的缺失对小鼠的血脂没有明显的影响,15月龄的FAM76B+/-小鼠存在一定的肝脏损伤。7.FAM76B基因敲除小鼠肝脏脂代谢相关基因的表达检测了不同时期FAM76B+/-小鼠和野生型小鼠肝脏的脂代谢相关基因的表达,实验结果表明:de novo lipogenesis环节中FASN和SCD的mRNA相对表达量上升,存在显著性差异。TG分泌过程中Vldlr的mRNA相对表达量上升。说明FAM76B+/-小鼠可能由于脂肪酸合成提高和TG分泌降低,造成肝脏细胞的TG沉积,最终导致非酒精性脂肪肝的形成。分别检测了不同时期FAM76B+/-小鼠和野生型小鼠肝脏中lipinl、SREBP-1、PPARα和PGC-1α的mRNA相对表达量,其中SREBP-1的相对表达量显著上升,FASN和SCD是SREBP-1转录调控的下游分子,相对表达量也升高,说明FAM76B可能通过影响SREBP-1的表达,从而影响脂肪酸的合成;另一方面,lipinl和PGC-1α的相对表达量明显下降。说明FAM76B可能通过影响lipin1和PGC-1α的相对表达,干扰vldl分泌途径,导致TG分泌降低。检测发现,PPARα的相对表达量没有明显的变化,说明FAM76B可能不通过PGC-1α/PPARα/lipinl途径影响脂肪酸的氧化反应,这与上述脂肪酸氧化途径中Cpt1、Acox和Mcad的mRNA相对表达量没有明显变化的实验结果相一致。8.FAM76B基因基因敲除小鼠肝脏中F4/80、TNFα和PGRN基因的表达1-3月龄时,FAM76B+/-小鼠和野生型小鼠肝脏中,F4/80和TNFα的mRNA没有明显差异。6月龄后,FAM76B+/-小鼠中F4/80和TNFα的mRNA明显升高,与同期野生型小鼠相比,差异极显著。15月龄时FAM76B+/-小鼠中F4/80和TNFα的mRNA相对表达量最高,这与15月龄FAM76B+/-小鼠病理学观察发现轻度肝小叶炎症型相符。F4/80是巨噬细胞表面分子,TNFa是促炎因子,说明FAM76B+/-小鼠的炎症反应逐步加强,这与非酒精性脂肪肝的进程相一致。同时,提示FAM76B具有抑制炎症反应的作用。1-12月龄时,FAM76B+/-小鼠相比野生型小鼠肝脏中PGRN的mRNA表达较高,但不存在明显差异。15月龄后,FAM76B+/-小鼠中PGRN的mRNA明显升高,与同期野生型小鼠相比,差异极显著,这与同一时期,FAM76B+/-小鼠中TNFa的mRNA相对表达量最高以及病理学观察发现轻度肝小叶炎症型一致。推测FAM76B和PGRN可能在非酒精性脂肪肝炎症反应中可能存在相互作用。综上所述,FAM76B在小鼠各组织中广泛分布,其中脑组织表达量最高,其次是肝脏组织和脾脏组织。FAM76B基因敲除会导致小鼠肝脏脂代谢异常和炎症反应升高。其分子机制可能是FAM76B基因敲除上调了小鼠肝脏中SREBP-1、FASN和SCD的基因表达,促进脂肪酸合成;下调lipin1、PGC-1α和上调vldlr的表达抑制TG分泌,导致脂质沉积。另一方面,FAM76B基因敲除上调小鼠肝脏中TNFα和PGRN表达,促进肝脏组织的炎症反应。通过以上因素协同诱导NAFLD病变。上述研究结果为进一步阐明FAM76B的生物学功能、FAM76B基因敲除对小鼠肝脏脂代谢异常的作用机制以及为深入研究FAM76B与PGRN相互作用在NAFLD发生过程的分子机制奠定基础。更多还原

【Abstract】 FAM76B is a novel nuclear localized protein and consists of 339 amino acids which encode a 39kDa protein.Human FAM76B gene in genomic is located on 11q21,which contains 21,468 bases.The function of FAM76B is unknown.By the analysis of the FAM76B proteins of human,rat,mouse and zebrafish,a sequence alignment of these proteins was found to have high degree of conservation,which suggested the important role of FAM76B.FAM76B is the significant number of poly His-containing protein and has an ubiquitination site at Lys225.Among proteins containing His-repeats with GO(Gene Ontology terms)annotations,there was a strong over-representation of nuclear proteins related to transcriptional regulation and nervous system development.FAM76B accumulates in nuclear speckles.The nuclear speckles are subnuclear structures defined as compartments in which components of the RNA splicing machinery are stored and assembled.FAM76B is novel PGRN-interacting protein identified from two yeast twohybrid screening and confirmed by GST-pull down and coimmunoprecipitation(Co-IP)in our previous study.PGRN as a multifunctional growth factor widely expressed in a variety of tissues is involved in many important physiological events,such as early embryogenesis,wound repair and nervous system development.Furthermore,PGRN plays an important role in the tumorigenesis,neurodegenerative diseases,type 2 diabetes,nonalcoholic fatty liver disease and inmflammatory.Although FAM76B interacts with PGRN,it remains unclear so far whether FAM76B involves in the developmemt of the related disease mentioned above.In this study,murine monoclonal antibodies(MAbs)against human FAM76B were generated by using purified prokaryotic recombinant human FAM76B protein.Due to the highly conserved protein sequences between mouse and human FAM76B,MAbs against human FAM76B were shown to react with mouse FAM76B.However,little is known of the role of FAM76B gene products in vivo.To understand the physiological role of FAM76B,We examined gene expression and distribution of FAM76B in normal mouse tissues.We generated of FAM76B knockout mice and the contributed phenotyping of FAM76B knockout mice will be explored.In addition,the mechanisms involved in the diseases casused by the deficient in FAM76B gene will lay a foundation for investigating the function of FAM76B protein.The contents of the project will include the following parts.1.Production and characterization of monoclonal antibodies against a novel human nuclear protein FAM76B.Six 5-week-old female Balb/c mice were immunized with FAM76B-6His three times at 3-week intervals.The fusion of SP2/0 myeloma cells with spleen cells isolated from immunized Balb/c mouse was carried out using standard methodology.About 5 days later,the supernatants from the 96-well plates were screened by ELISA using FAM76B-His as coating antigen for specific antibody-secreting clones.The hybridoma cells of positive wells were cloned at least three times by limiting dilution in aminopterin-free selection medium and were used for producing antibody-rich fluid called ascites fluid by inoculating enlarged hybridoma cell lines into the peritoneal cavity of Balb/c mice.MAbs specific for human FAM76B were obtained and characterized by using Western blot,immunoprecipitation(IP)and immunocytochemistry(ICH)applications.2.Gene expression and distribution of FAM76B in normal mouse tissues.Analyses of the mouse FAM76B and human FAM76B pair of protein,a sequence alignment of these two proteins highlighted their high degree of conservation.MAbs specific for human FAM76B were characterized by using Western blot and immunocytochemistry tested against mouse FAM76B protein.Then we examined FAM76B gene expression and distribution of FAM76B in normal mouse tissues by Real-time PCR and immunohistochemistry.3.Generation of FAM76B knockout mice.The function of the FAM76B is unknown.Accordingly,we chose to make FAM76B knockout mice cooperated with the company.Genomic DNA of offspring was confirmed by PCR.FAM76B expression monitored by Real-time PCR compared with GAPDH as a control.Western blotting using MAbs against human FAM76B confirmed that mouse FAM76B protein in MEF cells from different genotype mice.4.Phenotype of FAM76B knockout mice.The genotype ratio and the differences by sex were determined in offspring.The weights of mice tissues and the length of mice were determined in offspring.The development of mice was monitored.5.Histopathological features of liver and fat in FAM76B-deficient mice.Liver of FAM76B-deficient mice were stained with H&E and Oil Red O at different ages.At the same time,WAT adipocytes were stained with H&E.6.Biochemical Analysis in FAM76B-deficient mice.Plasma TQ TC,HDL,LDL,aspartate aminotransferase(AST),and alanine aminotransferase(ALT)levels were measured using amodel AU480 apparatus.7.Real-time PCR Analysis gene expression of lipogenesis in FAM76B-deficient mice.Gene expression of lipogenesis monitored by Real-time PCR compared with GAPDH as a control at 1,3,6,9,12 and 15 month,such as FASN,SCD,ACC,Dgat land Dgat 2 in de novo lipogenesis;Cptl,Acox,Acadv and Mcad in fatty acid beta oxidation;FABP and Apoa4 in uptake of free fatty acids into the liver;and Mtp,Vldlr in TG secretion.Lipin 1,SREBP-1,PPARa and PGC-la expression monitored by Real-time PCR compared with GAPDH as a control at 1,3,6,9,12 and 15 month.8.The expression of F4/80,TNFa and PGEN in FAM76B+/-mice livers.The expression of F4/80,TNFa and PGRN were monitored by Real-time PCR compared with GAPDH as a control at 1,3,6,9,12 and 15 month.Some results were obtained from the the project as follows:1.Production and characterization of monoclonal antibodies against a novel human nuclear protein FAM76BThe characterization of the six monoclonal antibodies investigated,mAbs No.1,No.2 and No.5 seem to have the highest affinities for human FAM76B and were effective in all assays tested.The study should provide a useful tool for investigating the biological function of FAM76B.In order to identify the epitope mapping,the regions of the FAM76B were determined by each mAb.Plasmids encoding FAM76B truncation mutants representing different regions of the protein were expressed in Escherichia coli BL21(DE3).They were separated by SDS-PAGE and analyzed by Western blot to test the FAM76B MAbs.Antibodies No.3 and No.6 reacted with epitopes in aa97-163.No.2 epitope in aa187-262 and No.5 might recognize aa263-265.No.1 reacted with aa266-339.HEK 293 cells were transfected with the vectors expressed the FAM76B truncation mutants were tested by different FAM76B MAbs immunohistochemically,which showed that nuclear localization signal was in aa187-265,C-terminal polyhistidine repeat domain.2.Gene expression and distribution of FAM76B in normal mouse tissues.MAbs No.1,No.2 and No.5 seemed to detect mouse FAM76B usefully and were effective in Western blot and immunocytochemistry tested.We examined FAM76B gene expression in mice by Real-time PCR and immunohistochemistry.Brain,liver and spleen expressed FAM76B at high levels,especially in brain,3.Generation of FAM76B knockout miceGenomic DNA of heterozygous,homozygous mice and wild type mice were confirmed by PCR.FAM76B expression was monitored by Real-time PCR.FAM76B mRNA levels in the MEF were undetectable from homozygous mice.Real-time PCR likewise detected MEF from heterozygous mice.About half of messenger for FAM76B compared to WT.Western blotting using MAbs against human FAM76B confirmed that FAM76B protein was markedly diminished in the MEF from homozygous mice.Protein contents of MEF from heterozygous mice were shows～50%reduction.These experiments demonstrate that FAM76B knockout mice had been successful.4.Phenotype of FAM76B knockout miceHowever,no significant differences by physical exanination could be detected.Homozygotes generated from offspring survived at a significantly lower frequency than the expected 1:2:1 Mendelian ratio(39.5%+/+,51.5%+/-,9%-/-).This data indicates that FAM76B-deficient mice have unkown lethal problems between the embryonic stage and weaning,caused the prenatal or postnatal death.Body weight was not significantly different between FAM76B+/-mice and WT animals when the feeding trials started.When maintained on for 5 months,body weight changes did not differ significantly between FAM76B+/-and WT mice.Until 8 months,FAM76B+/-mice showed higher body weight,but showed no significantly difference compared with WT mice.After 9 months,FAM76B+/-mice showed a significantly higher body weight compared with WT mice.This effect reached statistical significance from month 9 to month 15.Liver weight and fat mass had a similar trend during the feeding trials.In this study,we demonstrated that ablation of FAM76B induced obesity in mice.5.Histopathological features of liver and fat in FAM76B knockout miceHistological examination of livers from FAM76B knockout mice and WT mice demonstrated the progressive development of substantial steatosis and inflammatory reaction.Until 3 months,livers from FAM76B+/-mice demonstrated no evidence of steatosis at any time.At 4 months and 5 months,FAM76B+/-mice had substantially less steatosis.Small fat droplets were presented in hepatocytes at 6 months and 8 months,but these did not fill the cytoplasm.The amount of fat and size of the droplets increased in hepatocytes over time such that by 9 and 15months.Also by 9 months,small-droplet fat accrued in perivenous hepatocytes,and this was more pronounced at 15 months.Inflammatory foci were scattered across the lobule was seen at 15 months.The NAFLD activity score(NAS)was used to assess in FAM76B+/-mice and WT mice.FAM76B+/-mice evaluated nonalcoholic fatty liver disease activity score 1 at 8 months.and 9 months.FAM76B+/-mice classified nonalcoholic fatty liver disease activity score 2 at 15 months.Histological examination of livers from FAM76B mice demonstrated the progressive development of substantial steatosis.FAM76B deficient induces NAFLD in miceOil Red O staining of liver sections confirms raise of steatosis in FAM76B+/-mice.Until 3 months,livers from FAM76B+/-mice demonstrated no evidence of lipid droplets(LDs)at any time.At 4 months and 5 months,there were some FAM76B+/-mice had substantially less LDs.The diameter and density of the LDs were smaller.The amount of large LDs increased in hepatocytes at 6 months and 8 months.Large LDs were more marked at 9 and 15months.WAT adipocytes were stained with H&E.The areas of white adipocytes were in accordance with the morphological observations.Compared with WT mice,the areas of the white adipocytes in FAM76B+/-mice showed no significantly difference until 3 months.The areas of the white adipocytes in FAM76B+/-mice were increased from 4 months to 7 months,but had no significantly difference.After 8 months,the areas of the white adipocytes in FAM76B+/-mice were significantly increased6.Biochemical Analysis in FAM76B-deficient miceThe serum TG levels were significantly increased in FAM76B-deficient mice at 1 and 3months compared with WT mice.FAM76B-deficient mice display elevated levels of plasma CHOL at 1,3,9 and 12 months.HDL levels were not significantly increased in FAM76B-deficient mice except for 12 months.However,the FAM76B-deficient mice did not display significantly increased LDL levels.AST and ALT levels in FAM76B+/-mice showed no significantly difference compared with WT mice,except for ALT levels were significantly increased at 15 months.The above results suggested FAM76B deletion showed no noteworthy impacts on the lipid plasma level and induced liver injury in mice at 15 months.7.Real-time PCR Analysis gene expression of lipogenesis in FAM76B-deficient mice.Indeed,we found that the expression of Fasn,Scdl and vldlr were significantly induced in the livers of FAM76B+/-mice.Accumulation of lipid in the liver of FAM76B+/-mice can be traced by the increased incidence of de novo lipogenesis and the decreased of TG secretion.Accumulation of lipid in the liver resulted in the progression of non-alcoholic fatty liver disease(NAFLD).The expression of Srebpl was significantly induced in the livers of FAM76B+/-mice,consistenting with its lipogenic targets,Fasn and Scd1.We considered the possibility that FAM76B+/-mice livers might have an elavation in SREBPlc induction that could account for their increased TG levels by induced the fatty acid biosynthesis.On the other hand,the expression of lipinl and PGC-la were significantly reduced in the livers of FAM76B+/-mice.The studies have demonstrated that enhanced expression of lipinl or PGC-1α stimulated VLDL secretion.We considered the possibility that FAM76B+/-mice livers might have a defect in lipinl and PGC-la induction that could account for their increased TG levels by reduced TG secretion.The expression of PPARa did not display significantly decreased in the FAM76B+/-mice.FAM76B+/-mice livers had no effect on the PGC-la/PPARα/lipinl Pathway leading to decreased Fatty Acid Oxidation,consistenting with the expression of Cpt1,Acox,Acadv and Mead in Fatty acid oxidative pathways.8.The expression of F4/80,TNFaand PGRN in FAM76B+/-mice liversLiver F4/80 and TNFa mRNA levels did not display significantly decreased in the FAM76B+/-mice until 3 months.After 6 months,the expression of F4/80 and TNFa were significantly induced in the livers of FAM76B+/-mice,especially fold increased over WT mice at 15 months,consistenting with slight lobular inflammation.F4/80 is the phenotypic characterization of mouse tissue macrophages.TNFa is proinflammatory cytokines.At this time point,NASH in FAM76B+/-mice was characterized and by marked upregulation in liver levels of TNFa,consistenting with progressive non-alcoholic fatty liver disease.However,it suggests that FAM76B play a suppressor in liver.The expression of PGRN was significantly induced in the livers of FAM76B+/-mice at 15 months,consistenting with levels of TNFa and the progressive non-alcoholic fatty liver disease.It suggested FAM76B interacted with PGRN in inflammatory of NAFLD.In summary,brain,liver and spleen expressed FAM76B at high levels,especially brain.We examined abnormal hepatic lipid metabolism and inflammatory in FAM76B+/-mice.We delineated the molecular mechanisms for lipid accumulation in the liver of FAM76B+/-mice as increased the fatty acid biosynthesis and reduced VLDL secretion.The FAM76B+/-mice livers might have elavations in SREBP1,FASN and SCD induction that could account for their increased TG levels by induced the fatty acid biosynthesis.FAM76B+/-mice livers might have a defect in lipinl and PGC-la induction that could account for their increased TG levels by reduced TG secretion.On the other hand,the expression of TNFa and PGRN were significantly induced in the livers of FAM76B+/-mice.Inflammatory was upregulation in liver levels of TNFa.NAFLD was stemmed from induced the fatty acid biosynthesis,reduced TG secretion and inflammatory.All the findings above will lay a basis for the elucidation of FAM76B biological functions,its molecular mechanisms in nonalcoholic fatty liver disease and FAM76B interacted with PGRN in NAFLD.更多还原