PNPLA3基因与非酒精性脂肪肝的相关性及其机制研究
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摘要
遗传因素在非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）的形成中起重要作用。国外新近发现PNPLA3基因rs738409[C/G]多态性（编码I148M）与NAFLD显著相关，但其是否能独立导致NAFLD，以及影响肝脏脂肪代谢的作用机理不明。本研究通过“个体-细胞-分子”三个层面，研究了PNPLA3I148M多态性位点的作用机制，希望在临床水平验证PNPLA3基因I148M多态性与中国汉族人群NAFLD的相关性，揭示此多态性对细胞代谢水平的影响机制，阐明其对酶活性影响的分子机制。
首先，本文研究了青岛地区汉族人群中PNPLA3基因I148M多态性与NAFLD及慢性乙型肝炎（Chronic hepatitis B, CHB）的遗传易感性的相关性。采用多聚酶链反应（PCR）对315例NAFLD患者及336例健康对照和185例CHB患者及164例健康对照的血液样本进行基因型检测及分析。结果显示：rs738409G等位基因频率分布在NAFLD（65.40%）与正常对照组（33.18%）、NASH组（71.87%）与SS组（56.47%）、CHB组（31.9%）与对照组（21.9%）中分别比较差异均有统计学意义（P﹤0.05）。非条件Logistic回归模型分析，G等位基因与C等位基因相比较，前者发生NAFLD的比值比（OR）为3.81（95%CI：3.03~4.79，P﹤0.05），发生NASH的OR为1.97（95%CI=1.41~2.75， P＜0.05)，发生CHB的OR为1.67（95%CI=1.18—2.34, P=0.003）。调整性别、年龄后，发生CHB的OR为1.63（95%CI=1.17—2.68, P=0.04）。PNPLA3基因rs738409[C/G]多态性与血清ALT、γ-GT水平有关（P﹤0.05），对NASH组分层分析，GG基因型BMI、ALT、FINS均高于CC基因型（P﹤0.05），血清HDL水平低于CC基因型和GC基因型（P﹤0.05）。研究结果提示我们，在中国汉族人群中， PNPLA3基因I148M多态性与NAFLD及CHB的遗传易感性相关，是决定NAFLD及CHB个体遗传易感性的重要因素。
其次，本文探讨了PNPLA3基因I148M多态性在体外培养肝细胞中的作用机制。通过对经典的两步灌流法进行简化与改进，我们建立了一种能获得高纯度小鼠原代肝细胞的分离与培养方法，改进后的分离方法得到了足够数量的肝细胞；台盘蓝染色示细胞活率大于90%；细胞形态与活力可稳定保持1周。我们成功构建携带PNPLA3基因野生型和I148M突变型完整编码序列的慢病毒载体，两种慢病毒各自感染肝癌细胞系Huh-7和小鼠原代肝细胞，Western blot证实PNPLA3蛋白野生型和突变型成功过表达。油红O染色及生化分析表明：与空病毒组相比，过表达PNPLA3基因的细胞内甘油三酯、总胆固醇、脂蛋白含量明显升高，转氨酶水平也明显升高，过表达PNPLA3基因I148M突变型的细胞内以上指标升高更明显。经过不同时间、不同剂量的辛伐他汀处理后，表达不同基因型的Huh-7细胞内的甘油三酯水平均有不同程度的降低，且其降低呈现出时间和剂量依赖性，与空病毒和PNPLA3野生型组相比，过表达PNPLA3I148M突变型的细胞内甘油三酯水平降低程度更明显。研究结果提示我们，PNPLA3基因I148M可独立引起体外培养肝细胞脂肪变性，此基因型对辛伐他汀的降脂作用更敏感，在未来的工作中，我们可以根据患者的基因型选择不同的治疗方案，为非酒精性脂肪性肝病的个体化治疗提供了理论指导。
再次，利用转基因技术构建的特定基因在组织中特异表达的小鼠模型，已经成为当今后基因组时代研究基因功能的利器，而制备转基因小鼠最常用的一种方法是DNA原核显微注射，这需要我们首先构建PNPLA3I148M肝脏特异表达质粒。我们利用PCR技术成功得到了PNPLA3基因I148M多态性和肝脏特异性启动子ALB的完整编码序列，并将ALB与PNPLA3I148M成功连接，PCR产物转化GeneHogs化学感受态细菌进行质粒扩增，酶切及测序验证显示成功得到ALB-PNPLA3I148M质粒，将目的质粒转染肝癌细胞系SMMC-7721，westernblot结果显示，ALB-PNPLA3I148M在肝癌细胞SMMC-7721中成功过表达。PNPLA3I148M肝脏特异表达质粒构建的成功，为将来转基因小鼠的制备奠定了基础。
最后，为了深入了解其突变对酶活性的影响，我们利用分子动力学模拟和柔性对接进行了深入研究。数据表明突变组允许底物进入催化中心的通道明显减小，从而限制了软脂酸进入催化二联体。另外，酶与底物的结合能也表明了突变组对底物的低亲和力。另外，分子动力学模拟的的动态过程显示软脂酸在野生组和突变组的结合方式有显著不同。由于I148M突变发生位置在闭袢处导致底物识别特异性消失。该研究为I148M影响酶活性的机制提供了有力的证据，并且对酶的催化腔进行干扰可能成为治疗NAFLD的一个潜在途径。
Hereditary factors play an important role in the development of non-alcoholic fattyliver disease (NAFLD). A single nucleotide polymorphism (SNP) in the PNPLA3gene(rs738409, representing a substitution from cytosine to guanine, resulting in a switchfrom isoleucine to methionine at148residue, I148M) was reported to be significantlyassociated with NAFLD. It is unknown if the variant can induce NAFLDindependently, it is also unclear by which mechanism the variant affecting the lipidmetabolism of liver. In this study, we researched the physiological functions ofPNPLA3I148M variant at the individual, cellular and molecular levels. We hope toverify the correlation between this variant and NAFLD in Chinese Han population, toreveal the mechanism by which this variant affecting hepatocytes' metabolism, and toelucidate the molecular mechanism by which this variant affecting the activity of theenzyme.
Firstly, we explored the relationship between PNPLA3I148M variant andhereditariness to NAFLD and chronic hepatitis B (CHB) in Chinese Han population ofQingdao. The study included315NAFLD patients with336healthy controls and185CHB patients with164healthy controls. Genotypes of blood samples were examinedby polymerase chain reaction (PCR) and genotyping method. Allele frequencydistribution of rs738409G were65.40%,71.87%and56.47%in patients with NAFLD,NASH and SS and33.18%in control,31.9%in CHB patients and21.9%in controlgroup（P﹤0.05）, respectively, which had statistical signification(P﹤0.05). Non-conditional Logistic regression showed that, comparing with CC gene carriers, oddsratio of occurrence of NAFLD was3.81(95%CI:3.03to4.79, P <0.05), and the oddsratio of occurrence of NASH OR was1.97(95%CI=1.41~2.75, P <0.05) in GGgene carriers. A case-control analysis revealed a1.67-fold (95%CI=1.18—2.34, P=0.003) excess risk of developing CHB for rs738409G allele compared with C allele.Unconditional logistic regression model adjusted for age, sex, OR is1.63-fold (95%CI=1.17—2.68P=0.04). PNPLA3I148M variant was associated with the level ofserum ALT、γ-GT. Through stratified analysis of NASH group, the BMI, ALT, andFINS of GG genotype were all higher than that of the CC genotype (P <0.05), whilethe serum HDL level of GG genotype was lower than that of both CC genotype andGC genotype (P <0.05). The results suggested that, PNPLA3I148M variant wasassociated with NAFLD and CHB hereditary susceptibilities; the variant was asignificant factor which could determine NAFLD and CHB hereditary susceptibilitiesin Chinese Han population.
Secondly, we explored the mechanism of PNPLA3I148M variant in the culturedhepatocytes. We have improved the classic two-step perfusion technique andconstructed a simple method to isolate and culture primary mouse hepatocytes withhigh purity and high viability. Adequate hepatocytes were isolated by this method; themotility rate of the hepatocytes was higher than90%, the viability of the primaryhepatocyts lasted stably for7days. We constructed the recombinant lentivirusescontaining PNPLA3gene or PNPLA3gene I148M variant successfully. Huh-7cellswere infected by the recombinant lentiviruses containing PNPLA3or PNPLA3I148M.Western blot showed that PNPLA3protein was overexpressed in Huh-7cells. PNPLA3gene improved the level of triglyceride, total cholesterol, lipoproteins, as well as theaminotransferance in the cells; the I148M variant possessed a more enormousenhancing effect. Simvastatin depressed the triglyceride level in the cells, and theeffect showed a time and dose dependent tendency, the cells expressing I148M variantwere more sensitive to the simvastatin. The results suggested that, PNPLA3geneI148M variant can induce the liver steatosis independently, and the I148M variant wasmore sensitive to the simvastatin. In the future work, we can make individualtherapeutics to NAFLD according to the patients' genotypes.
Thirdly, transgenic mouse expressing definite gene in definite tissue has become apowerful tool to study gene function. DNA pronucleus microinjection is the mostlyused technique to construct transgenic mouse, and this need us to construct liverspecific expression plasmid of PNPLA3I148M variant. We got the complete codingsequences of PNPLA3I148M variant and the liver specific promoter ALB by PCR. The PNPLA3I148M and the ALB were connected by PCR. GeneHogs weretransformed by the PCR products and the plasmids were harvested and then verified byelectrophoresis and sequencing. SMMC-7721cells were infected by the ALB-PNPLA3I148M plasmids, western blot showed that ALB-PNPLA3I148M wasoverexpressed in SMMC-7721cells. The liver specific expression plasmid of PNPLA3I148M will lay a foundation for the construction of transgenic mouse.
Lastly, to glean insights into the variant’s effect on enzymatic activity, we performedmolecular dynamics simulation and flexible docking studies. Our data showed that thesize of the substrate-access entry site was significantly reduced in mutants, whichlimited the access of palmitic acid to the catalytic dyad. Besides, the binding freeenergy calculations suggested low affinity for substrate to mutant enzyme. Thesubstrate-bound system simulations revealed that the spatial arrangement of palmiticacid was distinct in wild-type from that in mutant. The substrate recognition specificitywas lost due to the loop where the I148M mutation was located. Our results providedstrong evidence for the mechanism by which I148M affected the enzyme activity andsuggested that mediating the dynamics might offer a potential avenue for NAFLD.
首先，本文研究了青岛地区汉族人群中PNPLA3基因I148M多态性与NAFLD及慢性乙型肝炎（Chronic hepatitis B, CHB）的遗传易感性的相关性。采用多聚酶链反应（PCR）对315例NAFLD患者及336例健康对照和185例CHB患者及164例健康对照的血液样本进行基因型检测及分析。结果显示：rs738409G等位基因频率分布在NAFLD（65.40%）与正常对照组（33.18%）、NASH组（71.87%）与SS组（56.47%）、CHB组（31.9%）与对照组（21.9%）中分别比较差异均有统计学意义（P﹤0.05）。非条件Logistic回归模型分析，G等位基因与C等位基因相比较，前者发生NAFLD的比值比（OR）为3.81（95%CI：3.03~4.79，P﹤0.05），发生NASH的OR为1.97（95%CI=1.41~2.75， P＜0.05)，发生CHB的OR为1.67（95%CI=1.18—2.34, P=0.003）。调整性别、年龄后，发生CHB的OR为1.63（95%CI=1.17—2.68, P=0.04）。PNPLA3基因rs738409[C/G]多态性与血清ALT、γ-GT水平有关（P﹤0.05），对NASH组分层分析，GG基因型BMI、ALT、FINS均高于CC基因型（P﹤0.05），血清HDL水平低于CC基因型和GC基因型（P﹤0.05）。研究结果提示我们，在中国汉族人群中， PNPLA3基因I148M多态性与NAFLD及CHB的遗传易感性相关，是决定NAFLD及CHB个体遗传易感性的重要因素。
其次，本文探讨了PNPLA3基因I148M多态性在体外培养肝细胞中的作用机制。通过对经典的两步灌流法进行简化与改进，我们建立了一种能获得高纯度小鼠原代肝细胞的分离与培养方法，改进后的分离方法得到了足够数量的肝细胞；台盘蓝染色示细胞活率大于90%；细胞形态与活力可稳定保持1周。我们成功构建携带PNPLA3基因野生型和I148M突变型完整编码序列的慢病毒载体，两种慢病毒各自感染肝癌细胞系Huh-7和小鼠原代肝细胞，Western blot证实PNPLA3蛋白野生型和突变型成功过表达。油红O染色及生化分析表明：与空病毒组相比，过表达PNPLA3基因的细胞内甘油三酯、总胆固醇、脂蛋白含量明显升高，转氨酶水平也明显升高，过表达PNPLA3基因I148M突变型的细胞内以上指标升高更明显。经过不同时间、不同剂量的辛伐他汀处理后，表达不同基因型的Huh-7细胞内的甘油三酯水平均有不同程度的降低，且其降低呈现出时间和剂量依赖性，与空病毒和PNPLA3野生型组相比，过表达PNPLA3I148M突变型的细胞内甘油三酯水平降低程度更明显。研究结果提示我们，PNPLA3基因I148M可独立引起体外培养肝细胞脂肪变性，此基因型对辛伐他汀的降脂作用更敏感，在未来的工作中，我们可以根据患者的基因型选择不同的治疗方案，为非酒精性脂肪性肝病的个体化治疗提供了理论指导。
再次，利用转基因技术构建的特定基因在组织中特异表达的小鼠模型，已经成为当今后基因组时代研究基因功能的利器，而制备转基因小鼠最常用的一种方法是DNA原核显微注射，这需要我们首先构建PNPLA3I148M肝脏特异表达质粒。我们利用PCR技术成功得到了PNPLA3基因I148M多态性和肝脏特异性启动子ALB的完整编码序列，并将ALB与PNPLA3I148M成功连接，PCR产物转化GeneHogs化学感受态细菌进行质粒扩增，酶切及测序验证显示成功得到ALB-PNPLA3I148M质粒，将目的质粒转染肝癌细胞系SMMC-7721，westernblot结果显示，ALB-PNPLA3I148M在肝癌细胞SMMC-7721中成功过表达。PNPLA3I148M肝脏特异表达质粒构建的成功，为将来转基因小鼠的制备奠定了基础。
最后，为了深入了解其突变对酶活性的影响，我们利用分子动力学模拟和柔性对接进行了深入研究。数据表明突变组允许底物进入催化中心的通道明显减小，从而限制了软脂酸进入催化二联体。另外，酶与底物的结合能也表明了突变组对底物的低亲和力。另外，分子动力学模拟的的动态过程显示软脂酸在野生组和突变组的结合方式有显著不同。由于I148M突变发生位置在闭袢处导致底物识别特异性消失。该研究为I148M影响酶活性的机制提供了有力的证据，并且对酶的催化腔进行干扰可能成为治疗NAFLD的一个潜在途径。
Hereditary factors play an important role in the development of non-alcoholic fattyliver disease (NAFLD). A single nucleotide polymorphism (SNP) in the PNPLA3gene(rs738409, representing a substitution from cytosine to guanine, resulting in a switchfrom isoleucine to methionine at148residue, I148M) was reported to be significantlyassociated with NAFLD. It is unknown if the variant can induce NAFLDindependently, it is also unclear by which mechanism the variant affecting the lipidmetabolism of liver. In this study, we researched the physiological functions ofPNPLA3I148M variant at the individual, cellular and molecular levels. We hope toverify the correlation between this variant and NAFLD in Chinese Han population, toreveal the mechanism by which this variant affecting hepatocytes' metabolism, and toelucidate the molecular mechanism by which this variant affecting the activity of theenzyme.
Firstly, we explored the relationship between PNPLA3I148M variant andhereditariness to NAFLD and chronic hepatitis B (CHB) in Chinese Han population ofQingdao. The study included315NAFLD patients with336healthy controls and185CHB patients with164healthy controls. Genotypes of blood samples were examinedby polymerase chain reaction (PCR) and genotyping method. Allele frequencydistribution of rs738409G were65.40%,71.87%and56.47%in patients with NAFLD,NASH and SS and33.18%in control,31.9%in CHB patients and21.9%in controlgroup（P﹤0.05）, respectively, which had statistical signification(P﹤0.05). Non-conditional Logistic regression showed that, comparing with CC gene carriers, oddsratio of occurrence of NAFLD was3.81(95%CI:3.03to4.79, P <0.05), and the oddsratio of occurrence of NASH OR was1.97(95%CI=1.41~2.75, P <0.05) in GGgene carriers. A case-control analysis revealed a1.67-fold (95%CI=1.18—2.34, P=0.003) excess risk of developing CHB for rs738409G allele compared with C allele.Unconditional logistic regression model adjusted for age, sex, OR is1.63-fold (95%CI=1.17—2.68P=0.04). PNPLA3I148M variant was associated with the level ofserum ALT、γ-GT. Through stratified analysis of NASH group, the BMI, ALT, andFINS of GG genotype were all higher than that of the CC genotype (P <0.05), whilethe serum HDL level of GG genotype was lower than that of both CC genotype andGC genotype (P <0.05). The results suggested that, PNPLA3I148M variant wasassociated with NAFLD and CHB hereditary susceptibilities; the variant was asignificant factor which could determine NAFLD and CHB hereditary susceptibilitiesin Chinese Han population.
Secondly, we explored the mechanism of PNPLA3I148M variant in the culturedhepatocytes. We have improved the classic two-step perfusion technique andconstructed a simple method to isolate and culture primary mouse hepatocytes withhigh purity and high viability. Adequate hepatocytes were isolated by this method; themotility rate of the hepatocytes was higher than90%, the viability of the primaryhepatocyts lasted stably for7days. We constructed the recombinant lentivirusescontaining PNPLA3gene or PNPLA3gene I148M variant successfully. Huh-7cellswere infected by the recombinant lentiviruses containing PNPLA3or PNPLA3I148M.Western blot showed that PNPLA3protein was overexpressed in Huh-7cells. PNPLA3gene improved the level of triglyceride, total cholesterol, lipoproteins, as well as theaminotransferance in the cells; the I148M variant possessed a more enormousenhancing effect. Simvastatin depressed the triglyceride level in the cells, and theeffect showed a time and dose dependent tendency, the cells expressing I148M variantwere more sensitive to the simvastatin. The results suggested that, PNPLA3geneI148M variant can induce the liver steatosis independently, and the I148M variant wasmore sensitive to the simvastatin. In the future work, we can make individualtherapeutics to NAFLD according to the patients' genotypes.
Thirdly, transgenic mouse expressing definite gene in definite tissue has become apowerful tool to study gene function. DNA pronucleus microinjection is the mostlyused technique to construct transgenic mouse, and this need us to construct liverspecific expression plasmid of PNPLA3I148M variant. We got the complete codingsequences of PNPLA3I148M variant and the liver specific promoter ALB by PCR. The PNPLA3I148M and the ALB were connected by PCR. GeneHogs weretransformed by the PCR products and the plasmids were harvested and then verified byelectrophoresis and sequencing. SMMC-7721cells were infected by the ALB-PNPLA3I148M plasmids, western blot showed that ALB-PNPLA3I148M wasoverexpressed in SMMC-7721cells. The liver specific expression plasmid of PNPLA3I148M will lay a foundation for the construction of transgenic mouse.
Lastly, to glean insights into the variant’s effect on enzymatic activity, we performedmolecular dynamics simulation and flexible docking studies. Our data showed that thesize of the substrate-access entry site was significantly reduced in mutants, whichlimited the access of palmitic acid to the catalytic dyad. Besides, the binding freeenergy calculations suggested low affinity for substrate to mutant enzyme. Thesubstrate-bound system simulations revealed that the spatial arrangement of palmiticacid was distinct in wild-type from that in mutant. The substrate recognition specificitywas lost due to the loop where the I148M mutation was located. Our results providedstrong evidence for the mechanism by which I148M affected the enzyme activity andsuggested that mediating the dynamics might offer a potential avenue for NAFLD.
引文
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