| 1 Research objective and backgroundBecause of the similarity of food habit, anatomy and physiology characteristics and drug metabolism characteristics of pigs and those of human, there are more and more researches about the pharmacology, toxicology, pharmacokinetics and pharmacodynamics of pigs. Accompanying the animal protection movement, 3R principle is becoming increasingly popularized, usage of higher animals such as non-human primates and dogs is restricted, and usage of edible animals such as pigs is gradually increasing. Therefore, pigs have a good prospect as experimental animals of drug evaluation. Because miniature pigs have the advantage of small body size, the experimental operation of them is convenient, the standardized quality control of them is easy, and the cost of feed is low. Therefore the application of them has a special advantage and they are routine experimental pigs in biomedicine at present.As drug evaluation models, the animals are required to have similarity compared with human in drug reaction mechanism and the interaction between the drug and the body. Thus the metabolic process of the drug in human body can be more exactly simulated through utilizing model animals. The metabolic process of drugs roughly includes four phases which are absorption, distribution, metabolism and excretion. The pharmacokinetics characters, bioavailability, toxic and side effects and the interaction of drugs are determined by the four phases. Drug metabolism is mainly in the liver. The biological transformation in the liver depends on multiple enzyme systems in microsomes, of which cytochrome P450 mixed-function oxidise system is the most important. Because the enzyme system is universally distributed in tissues and organs such as liver, kidney, brain, skin, lung, gastrointestinal tract and placenta, oxidation-reduction reaction catalyzed by P450 enzyme system can take place in many locations in vivo, of which liver is still the most important.P450 enzyme system is universally distributed in various vertebrate animals, invertebrate animals, plants, fungi and bacteria. The enzyme system which is called P450 gene superfamily has a very complex composition and is controlled by gene diversity. To this day, there are 101 P450 families and 2345 genes in animals according to the newest published data. Because P450 gene families are closely related with human personalized medicine and toxic and side effects, P450 gene families of human are relatively deeply studied, at present there are 57 functional genes having been discovered. While in mouse which is an mature animal model of organism and human diseases, there are 102 P450 functional genes having been discovered. Although pig is an drug evaluation and biomedical animal model having a good prospect, researches of P450 gene of pigs are relatively deficient, there are only 29 cytochrome P450 functional genes which have been named by Cytochrome P450(CYP) Allele Nomenclture Committee.The great magnanimity of increased sequences in the public database provide us sufficient resources for discovering new genes. As a part of cDNA sequence, EST is used to discover and clone new genes, which is an important route of acquiring new genes and carrying out function study. Large fragment or total length cDNA sequence can be directly acquired through classification, integration and composition of the great magnanimity of EST in the database. New members of gene families can be acquired fast and economically with this method. There are already more than 600,000 sequences of EST in the pig EST database. These data provide us an opportunity to find more information about P450 gene family.Genetic polymorphism of cytochrome P450 is very important to drug metabolism and it is one of the most important factors related with human personalized medicine at present. Genetic polymorphism of cytochrome P450 of different individuals may cause different drug reactions to the same drug. Sometimes because of the genetic difference of individuals, the difference of effects and toxic and side effects of the same drug in different individuals can achieve 300 fold. According to the information of Cytochrome P450(CYP) Allele Nomenclture Committee, there are 40 polymorphic alleles (or mutants) of CYP3A4 of human having been confirmed. The main mutant of human in China Mainland is CYP3A4*4, which decreases the enzymatic activity of CYP3A4. Researches have demonstrated that for CYP3A subfamily, which is with the highest content in liver and can metabolism maximum drugs, the most suitable animal model is (mini) pig. Microsomes in pig's liver have the same metabolic activity as human CYP3A4 probe substrate and the same characteristics of inhibitors as that of human, it is considered that CYP3A29 of pigs may be a homologous enzyme of CYP3A4 of human. Is CYP3A29 of pigs has a same polymorphism as that of human? If this polymorphism does exist, will it influence the metabolic activity of the metabolic pathway it participates? If this polymorphism does exist and dose influence the metabolic activity of the metabolic pathway, the influence effects of using pigs as metabolic models of human CYP3A4 substrate must be considered.There are plentiful and superior miniature pig strains. As two of the represent strains, Bama miniature pigs and Guizhou miniature pigs, whose source are both Xiang pigs, are mainly produced in habitation of minority ethnic groups such as Congjiang, Guizhou, Sandou, Guizhou, Huanjiang, Guangxi, and Bama, Guangxi. Most of these areas are remote mountain areas which have very poor traffic. Other pig strains can't enter, what's more, the crowd there are used to making high inbred self-breeding and self-cultivating of pigs through parent-offspring mating, sibling mating and half-sib mating of pigs. Because of long-term natural selection, although there is a high extent of inbreeding, there are few genetic diseases and deformities. Thus there is a unique group of miniature pigs. Bama miniature pigs and Guizhou miniature pigs are both oriently selected with a breeding object of miniaturization. Bama miniature pigs have a higher extent of inbreeding and have white hair, can bear inbreeding, have stable heredity and a consistent phenotype. They have been proved to be superior animal models. Also, there are much background information about anatomy, physiology, biochemistry and basic biological characteristics. Related researches of Cytochrome P450 of Bama miniature pigs and Guizhou miniature pigs will have a significant meaning on the application of animal models of drug evaluation.Therefore, this study makes use of the sequences in the public databse to predict new genes of Cytochrome P450 with a bioinformatics method. On the basis of this, related gene cloning of Bama miniature pig is carried out, which provides a foundation for taking Chinese miniature pig strain as an drug evaluation animal model. At the same time, the inter-strain and intra-strain polymorphism of the key subtype of Cytochrome P450 of Bama miniature pig and Guizhou miniature pig, which is CYP3A29, is studied, and the influence of the difference of polymorphism on the drug metabolism animal model. Detection and analysis of the microsatellite genetic background of the two miniature pigs is carried out to observe the mutual relationship between the difference of the inter-strain and intra-strain microsatellite genetic background and the gene polymorphism of CYP3A29.2 Content and results of the research2.1 Bioinformatics prediction and analysis of Cytochrome P450 gene of pigsOn the basis of homologous P450 gene sequences of other mammals and mainly taking advantage of the current EST database, silico cloning of Cytochrome P450 gene families of pigs was done with bioinformatics methods to acquire new genes of Cytochrome P450 gene families of pigs. Analysis of silico gene expression profile and alternative splicings of Cytochrome P450 of pigs were also done. Results: 37 possible new gene sequences of Cytochrome P450 of pigs were acquired with bioinformatics methods by prediction, gene expression data of gene expression in different tissues of pigs were acquired with EST silicon expression analysis, and 46 alternative splicings of Cytochrome P450 of pigs were discovered, which provided basic data and document of P450 gene for drug metabolism research of miniature pigs. 37 possible new gene sequences of Cytochrome P450 of pigs were acquired with bioinformatics methods. These sequences distributed in 11 families, 19 subfamilies and had 23 genes. Of which 9 sequences had full-length coding regions. Sequence analysis indicated that the sequences acquired by prediction all had a P450 structural domain or were highly similar to the corresponding P450 sequences of other species. Cluster analysis indicated that these sequences were homologous to the already-known P450 genes of pigs or corresponding P450 genes of human. 52 P450 gene expression data were acquired with EST silicon expression analysis on the already-known or predicted P450 genes. P450 genes which had the highest expression quantity in the liver of pigs were CYP2D25(50.26%), CYP2E1(31.09%), CYP2C33(3.69%), CYP2A19(3.11%) and CYP2D21(2.07%)in order, and these P450 gene expression data were compared with EST silicon expression of human. 46 alternative splicings of cytochrome P450 of pigs were discovered, of which there were 31 alternative splicings in coding regions, 3 alternative splicings in 5'UTR, 9 alternative splicings in 3'UTR, another two alternative splicings in coding regions and 3'UTR at the same time, and the other one alternative splicing in coding regions and 5'UTR at the same time.2.2 Clone and verification of predictive cytochrome P450 gene of Bama Miniature PigOne the basis of the prediction results of Cytochrome P450 gene sequences of pigs, corresponding gene sequences were amplificated with RT-PCR using designed primers, and then TA cloning and sequencing were done, results of which were compared to the predicted Cytochrome P450 gene sequences. Results: 18 Cytochrome P450 new gene sequences of Bama miniature pig were acquired by cloning. All the new gene sequences were registered in GeneBank, registration number was EF625344-EF625358. And the new gene sequences were presented to Cytochrome P450(CYP) Allele Nomenclture Committee to be assigned names. The research not only provided information about P450 gene sequences for drug metabolism study of miniature pigs, but also indicated that bioinformatics method combining experimental testing was a fast and effective route to acquire P450 new gene sequences. RT-PCR and TA cloning verification of the predicted 19 sequences of Cytochrome P450 gene sequences of pigs all acquired amplification products consistent with the expected fragment sizes. By sequencing and comparing, 17 cloned sequences of P450 gene were consistent with the predicted sequences by bioinformatics method, and 1 cloned sequence might be a neomorph of cyp4f6 gene of pig. 1 cloned sequence, cyp4f2v4 had a great difference compared with the predicted sequence. NCBI retrieval by Blastx didn't discover similar P450 sequences, so that this sequence was not regarded as a P450 sequence of pigs. Amplification sequence of cyp4f3v1 had a greater difference with the original sequence, but it was very similar to the cyp4f6v1 sequence, one contig could be attained when it was spliced with cyp4f6v1*1, cyp4f6v1*2 and cyp4f6v1*3. Although NCBI retrieval of this contig by Blastx didn't find an identical sequence, it highly resembled CYP4F6 subfamily, therefore it might be a new variant of the cyp4f6 gene of pigs. The other sequences were consistent with the predicted results on the whole. Amplification of Cyp39a1 was performed in 2 segment, cyp39a1*1 and cyp39a1*2 could be merged to form a contig, whose encoded protein was completely identical to the predicted results, and the protein should contain full-length of the coding region. All the sequences which were consistent with the predicted sequences were all registered in GeneBank, and the registration number is EF625344-EF625358. All the sequences have been presented to Cytochrome P450(CYP) Allele Nomenclture Committee to be assigned names. 2.3 Polymorphism of CYP3A29 of miniature pigsFull length coding region of CYP3A29 gene of Bama miniature pig and Guizhou miniature pig were amplificated with RT-PCR using designed primers, and then TA cloning and sequencing were done, polymorphism sites were acquired by sequence comparing and then their distribution and influence were analyzed. Results: 6 SNP sites and 1 alternative splicing of CYP3A29 gene of Bama miniature pig and Guizhou miniature pig were discovered. Distribution of SNP had strain and individual polymorphism. SNP could be of different types in different strains. SNP of Bama miniature pig had intra-strain consistent type, and SNP of Guizhou miniature pig had intra-strain different types. This demonstrated that P450 gene sequences of pigs had similar genetic polymorphism as those of human, therefore while using pigs as metabolic models, inter-strain and individual difference should be noted. CYP3A29 gene sequences of 13 Bama miniature pigs and 5 Guizhou miniature pigs were acquired. Contrasting results showed that there were 6 possible SNP sites, 1 alternative splicing, 1 possible new gene of CYP3A29 subfamily. Of the 6 possible SNP sites, 5 of them were samesense mutation, amino acids types weren't changed, and encoded protein sequences weren't influenced. Frameshift mutation and premature termination of translation caused by base deletion happened to one of them. Three SNP sites, CYP3A29*1A, CYP3A29*1B and CYP3A29*1C all displayed inter-strain difference, that was to say, there were different SNP subtypes in Bama miniature pig and Guizhou miniature pig. CYP3A29*1E and CYP3A29*2 sites displayed difference of miniature pig strain and other strain. For CYP3A29*1D site, there was 1 Guizhou miniature pig had a SNP type different from the others. There was one alternative splicing of G3a2902 in Guizhou miniature pig, 499-587 fragment deletion was found, at which site there was frame shift resulting interrupted protein. A non-specific sequence was found in one Bama miniature pig, it might be a new member of CYP3A subfamily of pigs.2.4 Microsatellite genetics detection in experimental miniature pig populationsWith the selected 21 microsatellite marks recommended by USA Pig Genome Cooperation Project for genetic diversity research of pigs worldwide and a fluorescence-based semi-automated genotyping method, genetics detection and evaluation was done to 2 miniature pig populations, which were Bama miniature pigs and Guizhou miniature pigs, to identify its genetic background and genetic relationship and analyze individual similarities and differences. Results: There was good genetic stability and genetic variations in both Bama miniature pig and Guizhou miniature pig. Individual homogenicity of Bama miniature pig was better than that of Guizhou miniature pig, there was a great genetic differences between the two groups. The genetic stability demonstrated that two strains of pigs were closed colonies suitable for biomecical research, having similar heredity character as human. At the same time genetic diversity inter-population and intra-population was consisitent with SNP polymorphism of P450 gene, which further demonstrated that inter-population and individual difference should be noted in application of pigs as drug metabolism models. There was good genetic diversity in the detected 21 microsatellite sites in the two populations of miniature pigs and Rong Chang pigs and large white pigs. Together 281 alleles were detected, and there were 7-22 alleles each locus. Those of Bama miniature pigs, Guizhou miniature pigs, Rong Chang pigs and large white pigs were 132, 125, 174 and 119, respectively. Alleles detected in Bama miniature pigs were more than detected in Guizhou miniature pigs, however, Shannon information index and polymorphism information content of Bama Miniature pigs were both lower than Guizhou miniature pigs, genetic heterozygosis of the former was lower than the latter and homozygosis of the former was higher than the latter, which indicated resemblance extent between individuals of Bama miniature pigs was higher than that between Guizhou miniature pigs. Genetic differentiation index between the two strains of miniature pigs, Fst was 0.2270. Nei standard genetic distance was 0.9134, which suggested there was great genetic differentiation between the two strains. Hardy-Weinberg genetic equilibrium detection suggested among the 21 sites of the two strains of minature pigs, there were 7 diverging from equilibrium. While in Rong Chang pigs and large white pigs, there were 10 and 11 sites diverging from equilibrium, respectively, which suggested they could maintain good genetic stability during breeding.3 conclusion3.1 37 possible new gene sequences of Cytochrome P450 of pigs were acquired with bioinformatics methods by prediction, gene expression data of gene expression in different tissues of pigs were acquired with EST silicon expression analysis, and 46 alternative splicings of Cytochrome P450 of pigs were discovered, which provided basic data and document of P450 gene for drug metabolism research of miniature pigs.3.2 18 Cytochrome P450 new gene sequences of Bama miniature pig were acquired by cloning. All the new gene sequences were registered in GeneBank, registration number was EF625344-EF625358. And the new gene sequences were presented to Cytochrome P450(CYP) Allele Nomenclture Committee to be assigned names. The research not only provided information about P450 gene sequences for drug metabolism study of miniature pigs, but also indicated that bioinformatics method combining experimental testing was a fast and effective route to acquire P450 new gene sequences.3.3 6 SNP sites and 1 alternative splicing of CYP3A29 gene of Bama miniature pig and Guizhou miniature pig were discovered. Distribution of SNP had strain and individual polymorphism. SNP could be of different types in different strains. SNP of Bama miniature pig had intra-strain consistent type, and SNP of Guizhou miniature pig had intra-strain different types. This demonstrated that P450 gene sequences of pigs had similar genetic polymorphism as those of human, therefore while using pigs as metabolic models, inter-strain and individual difference should be noted.3.4 There was good genetic stability and genetic variations in both Bama miniature pig and Guizhou miniature pig. Individual homogenicity of Bama miniature pig was better than that of Guizhou miniature pig, there was a great genetic differences between the two groups. The genetic stability demonstrated that two strains of pigs were closed colonies suitable for biomecical research, having similar heredity character as human. At the same time genetic diversity inter-population and intra-population was consisitent with SNP polymorphism of P450 gene, which further demonstrated that inter-population and individual difference should be noted in application of pigs as drug metabolism models.
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