| Background:In recent years, people gradually realize that nonalcoholic fatty liver disease(NAFLD) is the result of a multifunctional accumulation of fat in liver cells had chronic progressive multiple lesions. With the change of social development,the way of life, environmental pollution and drug abuse is widespread,the incidence of NAFLD in the world is constantly rising, pose a serious hazard to human health and social development.The pregnane X receptor(PXR) as a nuclear receptor super family member,not only play an important role in metabolism, growth and development and pathophysiological processes,but also play an indispensable role in gluconeogenesis,lipid metabolism and the balance of bile. Previous study found that PXR defenses foreign invasion accelerated the metabolism and discharge at the same time, can also lead to hepatic steatosis and other injuries.Objective:Through the study, to investigate the relationship between polymorphisms of PXR gene and NAFLD, clear the key role of PXR in NAFLD.Methods:1、Collecting 100 cases of clinical peripheral blood, divided into NAFLD group and healthy control group.51 people in the NAFLD group and healthy control group of 49 people. Extraction of DNA from blood and then PCR,using 1% agarose gel electrophoresis to determine the 542 bp fragments, the PCR products were sequenced.Statistical analysis, calculate the difference of allele frequency and genotype frequency and the corresponding genotype distribution between the two groups,through the Hardy-Weinberg analysis of genetic equilibrium test whether the sample is representative of the population.2、Collecting 100 cases of peripheral blood, divided into groups of nonalcoholic fatty liver disease and healthy controls, grouping ditto. Using kit to extract DNA,using the ultramicro protein, nucleic acid analyzer to detect the extracted DNA and then PCR.Using 3% agarose gel electrophoresis to determine the 722 bp fragments,the PCR products were sequenced. Statistical analysis, calculate the difference of allele frequency and genotype frequency and the corresponding genotype distribution between the two groups, through the Hardy-Weinberg analysis of genetic equilibrium test whether the sample is representative of the population.3、Sample sources and groups are as above. The process is similar to above.Using kit to extract DNA and then PCR.Using 3% agarose gel electrophoresis to determine the 250 bp fragments, the PCR products were sequenced. Statistical analysis, calculate the difference of allele frequency and genotype frequency and the corresponding genotype distribution between the two groups, through the Hardy-Weinberg analysis of genetic equilibrium test whether the sample is representative of the population.4、Sample sources and groups are as above. DNA was extracted for PCR experiment, The PCR products were 3% agarose gel electrophoresis experiments to determine whether the 210 bp fragment. The PCR products were sequenced.Finally,through the Hardy-Weinberg analysis of genetic equilibrium test whether the sample is representative of the population, statistical analysis of the corresponding gene frequency and genotype frequency.5、Collecting 300 cases of clinical peripheral blood, divided into NAFLD group and healthy control group.153 people in the NAFLD group and healthy control group of 147 people. DNA extraction from different varieties, the selection of qualified DNA and then PCR.Using 3% agarose gel electrophoresis to determine the 383 bp fragments, the PCR products were sequenced. Statistical analysis, calculate the difference of allele frequency and genotype frequency and the corresponding genotype distribution between the two groups, through the Hardy-Weinberg analysisof genetic equilibrium test whether the sample is representative of the population.6、The source of sample grouping, DNA extraction and PCR method are as above. Using 3% agarose gel electrophoresis to determine the 256 bp fragments, the PCR products were sequenced. Statistical analysis of the above methods.7、Based on the target area to capture sequencing method PXR gene SNP loci and study the correlation of NAFLD.Clinical peripheral blood samples collected 20 cases, of which the NAFLD group of 10 people, 10 people healthy controls.Extract DNA from various varieties, carries on the target area for sequencing, capture and analysis of PXR gene polymorphism loci associated.Results:1、There are two SNPs were found in PXR gene exon 2, called mutation 1 and mutation 2. The results showed that the mutated 1: Two groups were compared the genotype distribution difference of overall no statistical significance(χ2=1.77, P >0.05). The Hardy-Weinberg equilibrium test(χ2=0.29, 0.75<P<0.9), indicating that the sample is representative of the population(P>0.05). Mutation 2:Two groups were compared with the general distribution differences did not reach statistical significance(χ2=0.10, P > 0.05). The Hardy-Weinberg equilibrium test(χ2=2.00,0.90<P<0.95), indicating that the sample is representative of the population.2、In the PXR gene exon 4, one SNP was found. Results showed that two groups compared with the general distribution differences did not reach statistical significance(χ2=0.58, P>0.05). The Hardy-Weinberg equilibrium test(χ2=0.12,0.90<P<0.95), the description of the sample is representative of the population(P>0.05).3、There is no SNP site was found in exons 6 and 8 of PXR gene.4、PXR gene rs2461823 by sequencing results analysis, between the two groups genotype distribution difference was statistically significant(χ2=71.43, P < 0.05). The distribution of each gene was compared between the 2 groups. The results showed that three genotypes in both groups showed significant difference CC(χ2=33.16, P<0.05), TT(χ2=25.86, P<0.05), CT(χ2=50.31(P<0.05). Allele frequency distribution have significant difference(P<0.0001, OR=0.555, 95%CI : 0.399-0.773). The Hardy-Weinberg equilibrium test(χ2=1.99, 0.25<P<0.5), description of the sample is representative of the population(P>0.05).5、PXR gene rs7643645 by sequencing of the results between the two groups genotype distribution difference was statistically significant(χ2=103.18, P<0.05). The distribution of each gene was compared between the two groups. The results showed that three genotypes in the two groups have significant difference, CC(χ2=42.27, P <0.05), TT(χ2=30.72, P<0.05), CT(χ2=52.91(P< 0.05). The Hardy-Weinberg equilibrium test(χ2=1.51,0.25<P<0.50), description of the sample is representative of the population(P>0.05).6、By sequencing high-throughput screening to the PXR 120 SNPS loci, the PXR gene polymorphism loci correlation analysis(P>0.05).Conclusion:1、In this article we found that PXR gene exon 2 and exon 4 of the mutation may be no associated with NAFLD.2、The PXR gene rs2461823 and rs7643645 may be associated with NAFLD,and rs2461823 loci alleles C genotypes(CC and CT) for NAFLD may be a protective factor, laid a solid foundation for the further study of PXR as NAFLD therapeutic targets and the related drug development. |
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