CN113444730A - Screening and constructing method of primary hepatocyte klotho gene transduction stem cells - Google Patents
Screening and constructing method of primary hepatocyte klotho gene transduction stem cells Download PDFInfo
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- 238000010361 transduction Methods 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000012216 screening Methods 0.000 title claims abstract description 22
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Abstract
The invention discloses a primary hepatocyte klotho gene transduction stem cell screening and constructing method in the technical field of gene transduction stem cell screening and constructing methods, which is characterized in that a polymorphic DNA sequence of a klotho gene exon 4 region is obtained through PCR amplification as a basis, a T allelic site of rs648202 is used as a drug design target spot to screen out an active molecule capable of adjusting the klotho gene expression level, a polymorphic DNA fragment is adopted to transduce human hepatocytes, and dual vectors of hTERT and pLXSN are adopted to transduce vectors, so that pLXSN-hTERT expression vector transduction of hTERT is efficiently expressed, the effect of constructing stem cells efficiently expressing klotho genes is achieved, and the survival capability of the inter-marrow stem cells transducing exogenous klotho genes is improved in a low-solubility hygromycin dual screening culture system.
Description
Technical Field
The invention relates to the technical field of screening and constructing methods of gene transduction stem cells, in particular to a screening and constructing method of primary hepatocyte klotho gene transduction stem cells.
Background
Primary hepatocellular carcinoma (liver cancer, HCC for short) is one of the most common malignant tumors worldwide, and the hepatitis B virus is a hepadnavirus, can cause various diseases such as acute and chronic hepatitis, cirrhosis and the like, and has a very close relationship with the occurrence of hepatocellular carcinoma; in addition, Klotho is a gene discovered to be related to aging in 1997, is located on human chromosome 13q12, is about 50kb in length, consists of 5 exons and 4 introns, and the Klotho gene is lack of expression in mice to cause various phenotypes similar to human aging.
The existing stem cell in vitro amplification technical methods can be roughly classified into two types: the first method is to add different combinations of recombinant cell growth factors in a culture system, aiming at utilizing the cell growth factors to properly regulate and control the proliferation and differentiation of stem cells, and the technical method can improve the expansion multiple of the stem cells to a certain extent, but if the types of the cell factors in the combination are too few, the ideal expansion effect is difficult to achieve, and if the types of the cell factors are too many, the combination is difficult to apply in clinical transplantation because of high cost; the second type is a method developed in recent years, i.e. bone marrow stromal stem cells are used to simulate the in vivo cell growth and differentiation environment to promote the rapid expansion and functional activation, but in this method, because some cytokines (such as TPO and FL) in bone marrow stromal stem cells are expressed very slightly, exogenous cytokines are still added in an expansion culture system to assist, and in combination with the application of klotho gene in treating tumors, klotho gene is integrated and expressed in human target cell nucleus, which can lead to the change of cell proliferation activity and show various tumor-related transformation phenotypes, so that stem cells obtain the capability of unlimited proliferation and are immortalized, and the most commonly used technology for immortalizing cells is to support the in vitro expansion capability of stem cells by constructing an expression vector containing telomerase catalytic subunit (hTERT) and transfecting cells by various methods, therefore, a screening and constructing method of primary hepatocyte klotho gene transduction stem cells is provided.
Disclosure of Invention
The invention aims to provide a screening and constructing method of primary hepatocyte klotho gene transduction stem cells, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a primary hepatocyte klotho gene transduction stem cell screening and constructing method, the specific steps of the transduction stem cell method are:
s1: taking human whole genome DNA to be detected containing klotho gene as a template, then obtaining a polymorphic DNA sequence of a klotho gene exon 4 region through PCR amplification, wherein the DNA sequence contains rs648202 mononucleotide, then carrying out enzyme digestion on a product obtained after PCR amplification through restriction endonuclease HaeIII, and simultaneously carrying out rs648202 mononucleotide polymorphic typing through agarose gel electrophoresis to form DNA fragments with different lengths;
s2: separating mRNA from human liver cells, synthesizing corresponding cDNA by reverse transcription-polymerase chain reaction technology, connecting polymorphic DNA fragments with an Internal Ribosome Entry Site (IRES) sequence to form IRES-klotho fragments, cloning the IRES-klotho fragments into a pBluescript vector to construct IRES-klotho recombinant fragments, and transferring the IRES-klotho recombinant fragments from the pBluescript vector to a retrovirus vector pLXIN through an XhoI enzyme cleavage site to construct a pfl30 expression vector;
s3: according to the instruction manual of a LipofectAMINE liposome transduction kit, after a pLXSN-hTERT recombinant expression vector suspension is prepared by a facultative packaging cell line PA317, the pLXSN-hTERT expression vector is transduced to human stem cells for eight hours under the assistance of dimethyl dinitrogen undecyl methylene polymethine bromide based on the pLXSN vector, so that the human stem cells for efficiently expressing exogenous hTERT genes can be constructed;
s4: the technology of double screening the human stem cells transduced with the exogenous hTERT gene by adopting the hygromycin with low concentration improves the survival capability of the stem cells transduced with the exogenous hTERT gene.
The nucleotide polypeptide typing in the step S1 is specifically as follows: the length of the DNA fragment after the CC genotype is enzyme-cut is still 163bp, the DNA fragments with the three lengths of 163bp, 100bp and 63bp are generated after the CT genotype is enzyme-cut, and the DNA fragments with the two lengths of 100bp and 63bp are generated after the TT genotype is enzyme-cut.
The pBluescript vector used in the step S2, the human retrovirus vector, is pLXIN, and can obtain the pfl30 expression vector for efficiently expressing the klotho gene.
Compared with the prior art, the invention has the beneficial effects that: the invention adopts a polymorphic DNA sequence of a klotho gene exon 4 region obtained by PCR amplification as a basis, utilizes a T allelic site of rs648202 as a drug design target spot to screen drugs, screens out active molecules capable of regulating the klotho gene expression level, judges whether an individual has susceptibility to primary hepatocellular carcinoma by detecting the existence of the T allelic site, is beneficial to screening of susceptible population of the primary hepatocellular carcinoma and prevention of the primary hepatocellular carcinoma, and achieves the effect of obtaining stem cells by one-time gene transduction and simultaneously efficiently expressing exogenous klotho genes and the human hepatocytes by adopting polymorphic DNA fragment transduction based on the human hepatocytes, thereby reducing the cost of stem cell in-vitro amplification culture, effectively improving the in-vitro self-renewal, amplification and differentiation capacity of the stem cells, and adopting hTERT and pLXSN dual vectors to carry out vector transduction successively, the pLXSN-hTERT expression vector transduction for efficiently expressing hTERT is achieved, the effect of constructing the stem cell for efficiently expressing the klotho gene is achieved, the survival capability of the bone marrow mesenchymal stem cell for transducing the exogenous klotho gene is improved in a low-solubility hygromycin double-screening culture system, and the success rate of constructing the stem cell for exogenously expressing the klotho gene is improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A primary hepatocyte klotho gene transduction stem cell screening and constructing method, a primary hepatocyte klotho gene transduction stem cell screening and constructing method, the stem cell transduction method comprises the following steps:
s1: taking human whole genome DNA to be detected containing klotho gene as a template, then obtaining a polymorphic DNA sequence of a klotho gene exon 4 region through PCR amplification, wherein the DNA sequence contains rs648202 mononucleotide, then carrying out enzyme digestion on a product obtained after PCR amplification through restriction endonuclease HaeIII, and simultaneously carrying out rs648202 mononucleotide polymorphic typing through agarose gel electrophoresis to form DNA fragments with different lengths;
s2: separating mRNA from human liver cells, synthesizing corresponding cDNA by reverse transcription-polymerase chain reaction technology, connecting polymorphic DNA fragments with an Internal Ribosome Entry Site (IRES) sequence to form IRES-klotho fragments, cloning the IRES-klotho fragments into a pBluescript vector to construct IRES-klotho recombinant fragments, and transferring the IRES-klotho recombinant fragments from the pBluescript vector to a retrovirus vector pLXIN through an XhoI enzyme cleavage site to construct a pfl30 expression vector;
s3: according to the instruction manual of a LipofectAMINE liposome transduction kit, after a pLXSN-hTERT recombinant expression vector suspension is prepared by a facultative packaging cell line PA317, the pLXSN-hTERT expression vector is transduced to human stem cells for eight hours under the assistance of dimethyl dinitrogen undecyl methylene polymethine bromide based on the pLXSN vector, so that the human stem cells for efficiently expressing exogenous hTERT genes can be constructed;
s4: the technology of double screening the human stem cells transduced with the exogenous hTERT gene by adopting the hygromycin with low concentration improves the survival capability of the stem cells transduced with the exogenous hTERT gene;
the nucleotide polypeptide typing in the step S1 is specifically as follows: the length of the DNA fragment after the CC genotype enzyme digestion is still 163bp, DNA fragments with three lengths of 163bp, 100bp and 63bp are generated after the CT genotype enzyme digestion, and DNA fragments with two lengths of 100bp and 63bp are generated after the TT genotype enzyme digestion;
the pBluescript vector used in the step S2, the human retrovirus vector, adopts pLXIN, and can obtain pfl30 expression vector for high-efficiency expression of klotho gene;
the application comprises the following steps: the nucleic acid sequence of the polymorphism sites related to the primary hepatocyte can construct a kit for genetic screening of primary hepatocyte susceptible population, and the kit is used as a target point of drug design to find out active molecules capable of regulating klotho gene expression, thereby promoting the discovery of new antitumor drugs.
The invention adopts a polymorphic DNA sequence of a klotho gene exon 4 region obtained by PCR amplification as a basis, utilizes a T allelic site of rs648202 as a drug design target spot to screen drugs, screens out active molecules capable of regulating the klotho gene expression level, judges whether an individual has susceptibility to primary hepatocellular carcinoma by detecting the existence of the T allelic site, is beneficial to screening of susceptible population of the primary hepatocellular carcinoma and prevention of the primary hepatocellular carcinoma, and achieves the effect of obtaining stem cells by one-time gene transduction and simultaneously efficiently expressing exogenous klotho genes and the human hepatocytes by adopting polymorphic DNA fragment transduction based on the human hepatocytes, thereby reducing the cost of stem cell in-vitro amplification culture, effectively improving the in-vitro self-renewal, amplification and differentiation capacity of the stem cells, and adopting hTERT and pLXSN dual vectors to carry out vector transduction successively, the pLXSN-hTERT expression vector transduction for efficiently expressing hTERT is achieved, the effect of constructing the stem cell for efficiently expressing the klotho gene is achieved, the survival capability of the bone marrow mesenchymal stem cell for transducing the exogenous klotho gene is improved in a low-solubility hygromycin double-screening culture system, and the success rate of constructing the stem cell for exogenously expressing the klotho gene is improved.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A primary hepatocyte klotho gene transduction stem cell screening and constructing method is characterized in that: the method for transducing the stem cells comprises the following specific steps:
s1: taking human whole genome DNA to be detected containing klotho gene as a template, then obtaining a polymorphic DNA sequence of a klotho gene exon 4 region through PCR amplification, wherein the DNA sequence contains rs648202 mononucleotide, then carrying out enzyme digestion on a product obtained after PCR amplification through restriction endonuclease HaeIII, and simultaneously carrying out rs648202 mononucleotide polymorphic typing through agarose gel electrophoresis to form DNA fragments with different lengths;
s2: separating mRNA from human liver cells, synthesizing corresponding cDNA by reverse transcription-polymerase chain reaction technology, connecting polymorphic DNA fragments with an Internal Ribosome Entry Site (IRES) sequence to form IRES-klotho fragments, cloning the IRES-klotho fragments into a pBluescript vector to construct IRES-klotho recombinant fragments, and transferring the IRES-klotho recombinant fragments from the pBluescript vector to a retrovirus vector pLXIN through an XhoI enzyme cleavage site to construct a pfl30 expression vector;
s3: according to the instruction manual of a LipofectAMINE liposome transduction kit, after a pLXSN-hTERT recombinant expression vector suspension is prepared by a facultative packaging cell line PA317, the pLXSN-hTERT expression vector is transduced to human stem cells for eight hours under the assistance of dimethyl dinitrogen undecyl methylene polymethine bromide based on the pLXSN vector, so that the human stem cells for efficiently expressing exogenous hTERT genes can be constructed;
s4: the technology of double screening the human stem cells transduced with the exogenous hTERT gene by adopting the hygromycin with low concentration improves the survival capability of the stem cells transduced with the exogenous hTERT gene.
2. The method for screening and constructing primary hepatocyte klotho gene transduced stem cells according to claim 1, characterized in that: the nucleotide polypeptide typing in the step S1 is specifically as follows: the length of the DNA fragment after the CC genotype is enzyme-cut is still 163bp, the DNA fragments with the three lengths of 163bp, 100bp and 63bp are generated after the CT genotype is enzyme-cut, and the DNA fragments with the two lengths of 100bp and 63bp are generated after the TT genotype is enzyme-cut.
3. The method for screening and constructing primary hepatocyte klotho gene transduced stem cells according to claim 1, characterized in that: the pBluescript vector used in the step S2, the human retrovirus vector, is pLXIN, and can obtain the pfl30 expression vector for efficiently expressing the klotho gene.
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| CN112921009A (en) * | 2021-03-17 | 2021-06-08 | 昆明市延安医院 | Cell construction method of recombinant adenovirus carrying Klotho promoter gene and application thereof |
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