Application of Kctd10 gene in treatment of liver diseases
阅读说明：本技术 Kctd10基因在治疗肝脏疾病中的应用 (Application of Kctd10 gene in treatment of liver diseases ) 是由 任凯群 樊安方 马涛 肖轶卉 张健 向双林 于 2019-12-16 设计创作，主要内容包括：本发明涉及基因的应用领域及试验用的医药配制品,具体涉及肝干细胞调控基因Kctd10在小鼠肝脏中特异性敲除模型的建立、鉴定方法、并应用于治疗肝脏疾病药物筛选以及治疗肝脏疾病。利用FLP/Frt,Cre/Loxp重组酶系统首先构建了Kctd10<Sup>flox/flox</Sup>的小鼠模型,再将该小鼠模型与Alb-cre+转基因小鼠品系杂交,获得了Kctd10肝脏特异性基因敲除品系,并予以证实。本发明为人类肝干细胞疾病研究提供在体动物模型,为深入了解肝病发生提供有效的研究途径和方法,为特异性治疗肝脏疾病提供了药物筛选方法,也为治疗肝脏疾病奠定基础。(The invention relates to the field of gene application and a pharmaceutical preparation for experiments, in particular to a method for establishing and identifying a specific knockout model of a hepatic stem cell regulatory gene Kctd10 in mouse liver, and application of the method in screening drugs for treating liver diseases and treating liver diseases. Kctd10 was first constructed using the FLP/Frt, Cre/Loxp recombinase system flox/flox The mouse model is then crossed with an Alb-cre + transgenic mouse strain to obtain a Kctd10 liver-specific gene knockout strain, and the results are confirmed. The invention provides an in-vivo animal model for the research of human liver stem cell diseases, provides an effective research approach and method for deeply knowing the occurrence of liver diseases, provides a drug screening method for specific treatment of liver diseases, and lays a foundation for the treatment of liver diseases.)
Application of Kctd10 gene in preparing medicine for treating liver diseases.
2. The application of the liver stem cell regulatory gene Kctd10 liver specificity knock-out non-human mammal model in screening liver disease treating medicine is not directly related to the disease treating and diagnosing application process.
3. The use according to claim 2, wherein the non-human mammal is a mouse, rat, guinea pig, rabbit, monkey, sheep, pig.
4. The use according to claim 2 or 3, wherein the method for preparing the liver stem cell regulatory gene Kctd10 liver-specific gene knockdown non-human mammal model comprises the following steps:
(I) at Kctd10 flox/floxIn the mouse model of (3), two Loxp sequences flank the second exon of the Kctd10 gene;
(II) mixing Kctd10 flox/floxThe resulting mice were mated with FLP ER mice to obtain Neo gene-deleted Kctd10 flox/floxA mouse;
(III) Kctd10 obtained in step (II) flox/+A mouse; mating with an Alb-Cre mouse, and screening Alb-Cre +; kctd10 flox /+A mouse;
(IV) Kctd10 obtained in step (II) flox/+And (III) the obtained Alb-cre +; kctd10 flox/+Mating the mice, and screening out Alb-cre +; kctd10 floxfloxA mouse, namely a Kctd10 gene liver-specific knockout homozygote mouse;
in step (I), the Loxp sequence is inserted into the intron on both sides of exon 2, so that the normal expression of the gene is not affected in the absence of Cre recombinase.
5. The use of claim 2 or 3, wherein the hepatic stem cell regulatory gene Kctd10 is used in a method for liver-specific knock-out of non-human mammal model, comprising the steps of:
(I) identifying the genotype of the Kctd10 liver-specific knockout non-human mammal model by PCR-agarose gel electrophoresis;
(II) determining the KCTD10 protein expression condition of a Kctd10 liver specificity knocking non-human mammal model by a protein imprinting experiment, thereby verifying the Kctd10 gene knocking-out effect;
(III) the experiment of the real-time fluorescent quantitative PCR technology verifies the KCTD10 mRNA expression condition of the Kctd10 liver-specific gene knockout non-human mammal model, thereby verifying the Kctd10 gene knockout effect.
Technical Field
The invention relates to the field of gene application, and relates to a medicinal preparation used in vivo tests. In particular to a method for establishing and identifying a specific knockout model of a hepatic stem cell regulatory gene Kctd10 in mouse liver, which is applied to screening drugs for treating liver diseases and treating liver diseases.
Background
The function and characteristics of hepatic stem cells are closely related to the development and progression of liver disease: in the case of liver damage, hepatic stem cells proliferate and differentiate to maintain the normal function of the liver, and hepatic stem cells are actually one functional state of hepatic cells in a specific environment. The hepatic stem cell has self-replication and bidirectional differentiation potential, is expected to become a serious liver disease cell replacement therapy and a donor cell constituting a bioartificial liver, and thus becomes one of the current research hotspots. The research on the molecular development mechanism of the hepatic stem cells and the establishment of related animal models have very important significance for clinical liver disease treatment, drug research and the like. The mouse model is an ideal model for researching the formation and differentiation of the hepatic stem cells. The international peers knock out key genes in mice to cause liver stem cells to excessively proliferate and stem cell marker gene expression changes to induce the generation of liver cancer of the mice.
KCTD10(potassium channel tetramerization domain-conjugation 10) is a potassium tetramer channel protein belonging to PDIP1(polymerase delta-interacting protein 1) gene family. This gene family is highly similar in protein structure, with common BTB/POZ and K-tetra domains at the N-terminus, and a well-conserved PCNA binding site at the C-terminus. The Kctd10 gene is highly conserved evolutionarily, and encodes a highly conserved protein in humans, cattle, mice, chickens, African toads, zebrafish, and the like. At present, the Cre/loxp recombinase system is a gene-targeted recombination system used in conditional gene knockdown. The inventor surprisingly finds that by conditional gene knockout mice, two loxP sites are firstly placed on two sides of one or more important exons of a target gene to construct chimeric mice, and then the chimeric mice are hybridized with mice expressing Cre enzyme in a specific tissue, sequences between two LoxP with the same direction can be knocked out, and tissues or cells not expressing Cre enzyme cannot generate the knocking-out phenomenon, so that the purpose of knocking out the gene in the specific tissue or cells is realized, and meanwhile, the expression of the gene in other tissues or cells is not influenced. Therefore, the influence of the embryo lethal gene on the growth and development of the mouse can be avoided, so that the relevance of the gene and the physiological and pathological functions of the gene in specific tissues or cells can be better researched, and the invention is created.
The inventor firstly establishes Kctd10 Flox (
The invention provides an in-vivo animal model for the research of human liver stem cell diseases, provides an effective research approach and method for deeply understanding the occurrence of liver diseases, provides a drug screening method for specific treatment of liver diseases, and lays a foundation for gene therapy of liver diseases.
Disclosure of Invention
The invention aims to provide a method for constructing a liver stem cell regulatory gene liver specificity knockout mouse model.
The invention also aims to provide application of the liver stem cell regulatory gene liver-specific gene knockout mouse model.
The invention aims to provide a drug screening method for specific treatment of liver diseases.
The application of the Kctd10 gene in treating liver diseases is a non-therapeutic and non-diagnostic application.
The application of the liver stem cell regulatory gene Kctd10 liver specificity knock-out non-human mammal model in screening liver disease treating medicine is not directly related to the disease treating and diagnosing application process.
Wherein the non-human mammal is mouse, rat, guinea pig, rabbit, monkey, sheep, pig.
A method for liver stem cell regulatory gene Kctd10 liver specific gene knockdown in a non-human mammalian model, comprising the steps of:
(I) at Kctd10 flox/floxIn the mouse model of (3), two Loxp sequences flank the second exon of the Kctd10 gene;
(II) mixing Kctd10 flox/floxThe resulting mice were mated with FLP ER mice to obtain Neo gene-deleted Kctd10 flox /floxA mouse;
(III) Kctd10 obtained in step (II) flox/+A mouse; mating with an Alb-Cre mouse, and screening Alb-Cre +; kctd10 flox/+A mouse;
(IV) Kctd10 obtained in step (II) flox/+And (III) the obtained Alb-cre +; kctd10 flox/+Mating the mice, and screening out Alb-cre +; kctd10 floxfloxThe mouse is a liver-specific knockout homozygote mouse with the Kctd10 gene.
In step (I), the Loxp sequence is inserted into the intron on both sides of
Liver stem cell regulatory gene Kctd10 application in liver-specific knockout of non-human mammal model.
The identification method of the liver stem cell regulatory gene Kctd10 liver specificity knocking non-human mammal model comprises the following steps:
(I) identifying the genotype of the Kctd10 liver-specific knockout non-human mammal model by PCR-agarose gel electrophoresis;
(II) determining the KCTD10 protein expression condition of a Kctd10 liver specificity knocking non-human mammal model by a protein imprinting experiment, thereby verifying the Kctd10 gene knocking-out effect;
(III) the experiment of the real-time fluorescent quantitative PCR technology verifies the KCTD10 mRNA expression condition of the Kctd10 liver-specific gene knockout non-human mammal model, thereby verifying the Kctd10 gene knockout effect.
The liver stem cell regulatory gene Kctd10 is used for specifically knocking non-human mammal model and finding the abnormal expression of liver stem cell marker gene.
The application of the hepatic stem cell regulatory gene Kctd10 in gene therapy of liver diseases.
The use of any of the foregoing, such that KCTD10 mRNA levels are reduced to around 30% of normal levels; KCTD10 positive cells were reduced to about 30% of normal levels.
The use of any one of the preceding, wherein the hepatic stem cell marker gene Bmi1, Nanog, CD44, ALDH1 mRNA level (A, B), protein level (C) is significantly reduced.
The use of any one of the preceding claims, wherein the number of positive cells of the hepatic stem cell marker gene Bmi1 is reduced to about 60% of the normal level.
Use according to any one of the preceding claims to modulate alterations in Notch signaling pathway protein levels and mRNA levels in stem cells.
The use of any one of the foregoing to modulate the level of stem cell Notch signaling pathway ligands DLL3, Jagged1, Jagged2 protein reduced to about 50% of normal levels.
The use of any one of the above, wherein the signal intensity of TACE protein, a gene downstream of the Notch signaling pathway in stem cells is reduced to about 40% of the normal level.
All of the above applications are for non-therapeutic, non-diagnostic purposes.
Accordingly, the invention provides a liver stem cell regulatory gene liver specificity knockout animal model which is used for determining biological functions of liver stem cells and livers, so that identification methods of a Kctd10 liver specificity knockout animal model and a Kctd10 liver specificity knockout animal model are provided, the application of the liver stem cell regulatory gene liver specificity knockout animal model and the identification methods of the Kctd10 liver specificity knockout animal model and the Kctd10 liver specificity knockout animal model are used for screening drugs for treating liver diseases, and a foundation is laid for gene therapy of the liver diseases.
Drawings
FIG. 1: schematic representation of mating protocol for Kctd10 liver-specific knockout mice.
FIG. 2: performing PCR agarose gel electrophoresis identification on Kctd10 liver-specific gene knockout mice genotype; alb cre +; kctd10 flox/floxRepresents KCTD10 liver-specific knockout mouse homozygote, Alb cre-; kctd10 flox/floxRepresents KCTD10 liver-specific knockout mouse wild type.
FIG. 3: and (3) specifically knocking out mouse liver Kctd10 gene, and then, expressing KCTD10 protein.
FIG. 4: after the Kctd10 gene of mouse liver is knocked out specifically, KCTD10 mRNA level is reduced to about 30% of normal level.
FIG. 5: after the mouse liver Kctd10 gene is specifically knocked out, KCTD10 positive cells are reduced to about 30% of normal level.
FIG. 6: the liver stem cell marker genes Bmi1, Nanog, CD44 and ALDH1 mRNA level (A, B) and protein level (C) in the mouse model obtained by the method are obviously reduced.
FIG. 7: the liver stem cell marker gene Bmi1 positive cells in the mouse model obtained by the method are reduced to about 60 percent of the normal level.
FIG. 8: the level of Notch signaling pathway protein and mRNA regulating stem cells in the mouse model obtained by the method are changed.
FIG. 9: the protein levels of Notch signal pathway ligands DLL3, Jagged1 and Jagged2 for regulating stem cells in the mouse model obtained by the method are reduced to about 50% of the normal level.
FIG. 10: the signal intensity of the TACE protein of the downstream gene of the Notch signal path of the regulatory stem cells in the mouse model obtained by the method is reduced to about 40 percent of the normal level.
Detailed Description
The inventor establishes a Kctd10 liver-specific gene knockout mouse model through extensive and intensive research, and has good guiding significance for identifying a method for knocking out a mouse genotype. The invention is further illustrated by the following examples.
Table 1: PCR primer set
Table 2: qPCR primer list
