CN113201062B - Method for stably expressing secreted human insulin in mesenchymal stem cells - Google Patents
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Abstract
The invention discloses a method for stably expressing secretive human insulin in mesenchymal stem cells, which comprises the following steps: (1) constructing an mCherry-2A-insulin-His fusion gene lentiviral expression vector, and (2) expressing the constructed mCherry-2A-insulin-His fusion gene lentiviral expression vector in human mesenchymal stem cells to obtain the human insulin secreted to the outside of cells. After transfecting cells, a stable expression cell line is obtained by screening, the cells express fusion polypeptides, human insulin is separated from the mCherry red fluorescent gene under the action of self-cutting polypeptides 2A, and the human insulin can be secreted to the outside of the cells. The method solves the dilemma of expressing human insulin in mesenchymal stem cells at present, and has great practical significance and potential medical application value.
Description
Technical Field
The invention belongs to the field of tissue regeneration engineering, and particularly relates to a method for stably expressing secretive human insulin in mesenchymal stem cells.
Background
Tissue regeneration engineering and stem cell and cell therapy are a new discipline developed in the modern biomedical field, and hope is brought to the treatment of a plurality of serious diseases including cancer, heart disease, diabetes, neurodegeneration and the like. The differentiation of embryonic stem cells or mesenchymal stem cells into islet cells or islet-like cells with Insulin secretion (Insulin) functions is an important research direction for tissue regeneration engineering and cell therapy, and brings hope for cell therapy of diabetes. Although work on this aspect has been carried out for decades, progress has been slow because islet cells are obtained via the stem cell differentiation pathway, results are very unstable, and experimental reproducibility is poor. On the other hand, the use of stem cells to obtain islet cells is difficult and expensive to obtain in high yields.
The mesenchymal stem cells comprise bone marrow mesenchyme, umbilical cord mesenchyme and mesenchymal stem cells of other organs, are distributed at each part of a body, and have the advantage of low immunological rejection when the patient mesenchymal stem cells are used for cell therapy. Therefore, the mesenchymal stem cells are ideal biological materials for cell therapy. The direct expression of Insulin (Insulin) and the secretion of Insulin (Insulin) by mesenchymal stem cells are one of the ways in which cells treat diabetes. Insulin (insulin) is generally only expressed in islet Beta-cells, but is not expressed in other cells in a natural state, so that the forced expression difficulty is high, particularly the expression difficulty in mesenchymal stem cells is extremely high, and even the expression is unstable. Although literature reports indicate that insulin can be successfully expressed in mesenchymal stem cells (1, 2), the early-stage laboratory reported expression of human insulin was unsuccessful. Therefore, it is necessary to develop a new method for stably expressing human insulin in mesenchymal stem cells, and the expressed insulin can be secreted to the extracellular space, which is necessary and very critical for cell therapy of diabetes.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the problems that human insulin (insulin) is difficult to express, does not secrete, has poor repeatability and is unstable in mesenchymal stem cells, the invention provides a repeatable method for stably expressing and secreting insulin in mesenchymal stem cells.
The technical scheme of the invention is as follows: a method for stably expressing secretionable human insulin in mesenchymal stem cells, comprising the following steps:
(1) constructing mCherry-2A-insulin-His fusion gene lentiviral expression vector,
(2) the constructed mCherry-2A-insulin-His fusion gene lentiviral expression vector is expressed in human mesenchymal stem cells, and then the human insulin secreted to the outside of the cells can be obtained.
Further, the method for constructing the mCherry-2A-insulin-His fusion gene lentiviral expression vector comprises the following steps: performing PCR amplification by using a lentiviral expression vector pLV [ Exp ] -Neo-TRE3G > hINS [ NM-001185097.1 ]/EK/10XHis as a template and 2A-insulinF and His-insulinR as primers, performing double enzyme digestion on an amplification product and a lentiviral expression vector pLV-EF1 alpha-mCherry-Puro, and then connecting the amplification product and the lentiviral expression vector pLV-EF1 alpha-mChery-His to obtain the lentiviral vector pLV-EF1 alpha-mChery-2A-insulin-His-Puro for expressing the mChery-2A-insulinF fusion gene, wherein the nucleotide sequence of the 2A-insulinF is shown as SEQ ID No.1, and the nucleotide sequence of the His-insulinR is shown as SEQ ID No. 2.
Further, the reaction system of the PCR amplification is as follows:
1 μ LpLV [ Exp ] -Neo-TRE3G > hINS [ NM-001185097.1 ]/EK/10XHis vector at a concentration of 1 ng/. mu.L
1 μ L of 2A-insulinF primer at a concentration of 25 μ M
1 μ L His-InsulinR primer, concentration 25 μ M
25μL 2xPCR mastermix
22μL ddH2O
The total volume of the reaction system is 50 mu L;
the amplification procedure of the PCR amplification is as follows:
the first step is as follows: 95 ℃ for 3min
The second step is that: 1min at 95 ℃; 20sec at 55 ℃; 72 ℃ for 1min for 35 cycles
The third step is that the temperature is 72 ℃ for 10 min.
Further, the method for expressing the constructed mCherry-2A-insulin-His fusion gene lentiviral expression vector in the human mesenchymal stem cells comprises the following steps:
(a) co-transfecting 293T cells with a lentiviral vector pLV-EF1 alpha-mCherry-2A-insulin-His-puro and viral packaging helper vectors pH1 and pH 2;
(b) culturing the transfected 293T cells, collecting a culture medium containing viruses, and centrifugally collecting a virus-containing supernatant;
(c) culturing human mesenchymal stem cells, adding the supernatant obtained in the step (b) into the human mesenchymal stem cells for infection, and continuously culturing to obtain the human insulin secreted to the outside of the cells.
Further, while the supernatant was added to human mesenchymal stem cells for infection, Polybrene was added and the final concentration of Polybrene was controlled to 10. mu.g/mL.
Compared with the prior art, the invention has the following beneficial effects:
the method of the invention uses self-cutting polypeptide 2A gene to connect mCherry red fluorescence gene and human insulin gene into fusion gene which is cloned on slow virus expression vector, after transfecting cell, stable expression cell line is obtained by screening, human insulin and mCherry red fluorescence protein are separated under the action of self-cutting polypeptide 2A, human insulin can be secreted out of cell. The method solves the current dilemma of expressing human insulin in mesenchymal stem cells, and has great practical significance and potential medical application value. In addition, because the secretion signal peptide is positioned at the N-end of the insulin, the HIS label is added at the C-end of the insulin, so that the secretion of the insulin is not influenced, and the insulin is conveniently purified by using an agarose nickel column; for example, the agarose nickel column can be used for enriching the secreted insulin in vitro of the cell, and the effect of killing two birds with one stone can be achieved.
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FIG. 1 is a schematic diagram of the construction of pLV-mCherry-2A-insulin-His plasmid. Synthesizing a 2A-insulin-his fusion gene by a gene amplification method (PCR), preparing a chronic disease expression vector pLV-mCherry-puro, and cloning the 2A-insulin-his fusion gene at the downstream of the mCherry to form the mChery-2A-insulin-his fusion gene;
FIG. 2 is a schematic diagram of a method for expressing insulin (insulin) using human mesenchymal stem cells;
the method comprises the following steps of (1) transfecting a human mesenchymal stem cell with a pLV-mCherry-2A-Insulin-His expression vector, (2) expressing mCherry-2A-Insulin-His fusion protein by the human mesenchymal stem cell, (3) screening a stable expression cell line by using puromycin, (2) self-cutting the mCherry-2A-Insulin-His fusion protein into mChery, 2A and Insulin protein (Insulin-His) with a His label, and (5) secreting the Insulin protein (Insulin-His) with the His label to the outside of a cell.
FIG. 3 shows a stable human mesenchymal stem cell line expressing mCherry-2A-insulin fusion protein;
FIG. 4 detection of human insulin expression in mesenchymal stem cells, immunoblot detection indicating successful expression of human insulin in mesenchymal stem cells;
FIG. 5 shows the detection that human insulin can be secreted to the outside of cells after the expression of mesenchymal stem cells, and Elisa detects the content of human insulin in cell lysate and culture medium.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.
The method mainly comprises three main stages of work:
1. an mCherry-2A-insulin-His fusion gene lentiviral expression vector containing 2A is constructed based on a self-cutting polypeptide 2A technology.
Transfecting mCherry-2A-insulin-his fusion gene lentiviral expression vector and screening a mesenchymal stable cell line for expressing the fusion gene.
3. And (3) identifying the stable cell line of the mesenchymal stem cell expressing the fusion gene.
The steps involved in the operation of each stage are set forth below
1. An mCherry-2A-insulin-his fusion gene lentiviral expression vector containing 2A is constructed based on a self-cutting polypeptide 2A technology.
1) The search literature obtained sequences for self-cleaving peptide 2A are as follows:
GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT
2) the sequence of human Insulin (Insulin) gene obtained by searching using NCBI gene library is as follows:
ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC
3) a pair of primers for cloning mCherry-2A-insulin-His is designed according to the multiple cloning site of pLV-EF1 alpha-mCherry-Puro lentivirus expression vector (figure 1) and the DNA sequence of mCherry (a red fluorescent protein). The primer sequences are as follows:
2A-insulinF：
CGCGGAATTCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGCCCTGTGGATGCGCCTC
His-insulinR：
GCGCGGATCCCTAGTGGTGGTGATGAGTGTG
4) polymerase Chain Reaction (PCR) was performed using the lentiviral expression vector pLV [ Exp ] -Neo-TRE3G > hINS [ NM-001185097.1 ]/EK/10XHis (purchased from VectorBuilder Inc) with the cloned insulin gene as a template and primers in 3) as follows:
mu.L pLV [ Exp ] -Neo-TRE3G > hINS [ NM-001185097.1 ]/EK/10XHis vector (concentration 1 ng/. mu.L)
mu.L of 2A-insulinF primer (25. mu.M)
mu.L His-InsulinR primer (25. mu.M)
25μL 2xPCR mastermix
22μL ddH2O (deionized distilled water)
The total volume of the reaction system is 50 mu L;
the PCR reaction was performed in a PCR instrument according to the following thermal cycle
The first step is as follows: 95 ℃ for 3min
The second step is that: 1min at 95 ℃; 20sec at 55 ℃; 72 ℃ for 1min for 35 cycles
The third step is that the temperature is 72 ℃ for 10 min.
5) Because the reverse primer His-intulin R contains a His tag, the mCherry-2A-intulin-His fusion gene is obtained by PCR in the step 4), and a PCR product is purified by a DNA purification kit.
6) The lentiviral expression vector pLV-EF1 alpha-mCherry-Puro (purchased from Inovogen) and the PCR product were cleaved with EcoRI and BamHI and purified.
7) The PCR product was cloned downstream of the lentiviral expression vector (pLV-EF1 alpha-mCherry-Puro) mCherry according to standard methods for molecular cloning. Obtaining a lentivirus vector pLV-EF1 alpha-mCherry-2A-insulin-His-puro for expressing the mCherry-2A-insulin-His fusion gene (figure 1).
Transfecting mCherry-2A-insulin-his fusion gene lentiviral expression vector and screening mesenchymal stable cell line for expressing the fusion gene.
1) A large amount of lentiviral vector pLV-EF1 alpha-mCherry-2A-insulin-His-puro for expressing mCherry-2A-insulin-His fusion gene is extracted.
2) The lentiviral vector pLV-EF1 α -mCherry-2A-insulin-His-puro and viral packaging helper vectors pH1 and pH2 (purchased from Inovogen) were co-transfected into 293T cells.
3) The transfected 293T cells were cultured, and after 48 hours, the virus-containing medium was collected, transferred to a centrifuge and centrifuged at 8000rpm for 5min, and the supernatant was filtered through a 0.45 μm filter.
4) While culturing human mesenchymal stem cells (purchased from seiko corporation) in a 12-well plate, the medium was changed after 12 hours, and 1mL of the filtered virus-containing culture solution was added to the 12-well plate, while adding Polybrene to a final concentration of 10. mu.g/mL.
5) Culturing the mesenchymal stem cells for 48 hours to allow the virus to infect the mesenchymal stem cells.
6) Cells with resistance are screened by purine mildews, and the mesenchymal stem cells are observed to be red by using a common fluorescence microscope, if most of the cells are red fluorescence, the mCherry-2A-insulin-His fusion protein is successfully expressed (figure 2).
3. Functional identification of mesenchymal stem cell stable cell line expressing fusion protein
1) Identifying the mesenchymal stem cell stable cell line expressing the fusion protein by using a fluorescence microscope: the cells are passed through more than 10 passages, and the mesenchymal stem cells are placed under a fluorescence microscope for imaging. The mesenchymal stem cells still showed red fluorescence, indicating that the mesenchymal stem cells can continuously express the mCherry-2A-insulin fusion protein (FIG. 3).
2) Western blot to identify whether the fusion protein expressed by the mesenchymal stem cells has undergone self-cleavage: the red fluorescent mesenchymal stem cells are collected, lysed by using cell lysate, boiled for 10min, and then the sample is used for Western blot analysis, and the detection antibodies are His label antibodies (note: in the process of cloning genes, His labels are added at the tail end of the Insulin genes, see figures 1 and 2), Insulin antibodies and GAPDH antibodies. As shown in FIG. 4, Insulin (Insulin) expression was detected both by His antibody and by Insulin antibody, and was of the same molecular weight as the isolated Insulin molecule, about 14 kD. Indicating that the fusion protein self-cleaved under the action of 2A (FIG. 2). However, control mesenchymal stem cells not transfected with lentiviral vectors did not detect any Insulin signal.
3) The Elisa method identifies whether the Insulin expressed by the mesenchymal stem cells has been secreted extracellularly: insulin (Insulin) must be secreted extracellularly for clinical medical value. The aim of our invention is to prepare mesenchymal stem cells that can secrete Insulin extracellularly. Since the Insulin is secreted out of the cell, the concentration is diluted, and Western blot is not easy to detect, so I adopt an Elisa method (also called enzyme-linked immunosorbent assay) to detect the Insulin. Samples of mesenchymal stem cells and extracellular culture medium were collected and subjected to the enzymatic chain reaction using the Insulin Elisa kit (available from Biyuntian). And (4) measuring the content of the Insulin in the cytosol and the culture solution by using a microplate reader. The results are shown in FIG. 5, the signals of Insulin can be detected both intracellularly and extracellularly, which indicates that the Insulin expressed by the mesenchymal stem cells can be secreted extracellularly. Whereas no significant signal was detected for the control cells. The result shows that the method can be used for preparing human mesenchymal stem cells capable of secreting Insulin, has potential application value, provides technical support for biological or clinical research of treating diabetes by using the stem cells, and promotes the field to develop rapidly.
Sequence listing
<110> Wuhan university of science and technology
<120> method for stably expressing secretive human insulin in mesenchymal stem cells
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ggagaaccct ggacctatgg ccctgtggat gcgcctc 97
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gcgcggatcc ctagtggtgg tgatgagtgt g 31
Claims (3)
1. A method for stably expressing secretionable human insulin in mesenchymal stem cells, which is characterized by comprising the following steps:
(1) constructing mCherry-2A-insulin-His fusion gene lentiviral expression vector,
(2) expressing the constructed mCherry-2A-insulin-His fusion gene lentiviral expression vector in human mesenchymal stem cells to obtain human insulin secreted to the outside of cells;
the method for constructing the mCherry-2A-insulin-His fusion gene lentiviral expression vector in the step (1) comprises the following steps: carrying out PCR amplification by taking pLV [ Exp ] -Neo-TRE3G > hINS [ NM-001185097.1 ]/EK/10xHis as a template and 2A-insulinF and His-insulinR as primers, carrying out double enzyme digestion on an amplification product and a lentiviral expression vector pLV-EF1 alpha-mCherry-Puro, and then connecting the amplification product and the lentiviral expression vector pLV-EF1 alpha-mChery-2A-insulinl-His-Puro to obtain the lentiviral vector pLV-EF1 alpha-mCheliny-2A-insulin-His-Puro for expressing the mChery-2A-insulinl-His fusion gene, wherein the nucleotide sequence of the 2A-insulinF is shown as SEQ ID No.1, and the nucleotide sequence of the His-insulinR is shown as SEQ ID No. 2;
in the step (2), the method for expressing the constructed mCherry-2A-insulin-His fusion gene lentiviral expression vector in the human mesenchymal stem cells comprises the following steps:
(a) co-transfecting 293T cells with a lentiviral vector pLV-EF1 alpha-mCherry-2A-insulin-His-puro and viral packaging helper vectors pH1 and pH 2;
(b) culturing the transfected 293T cells, collecting a culture medium containing viruses, and centrifugally collecting a virus-containing supernatant;
(c) culturing human mesenchymal stem cells, adding the supernatant obtained in the step (b) into the human mesenchymal stem cells for infection, and continuously culturing to obtain the human insulin secreted to the outside of the cells.
2. The method for stably expressing secretionable human insulin in mesenchymal stem cells according to claim 1, wherein the reaction system of PCR amplification is:
1 μ L pLV [ Exp ] -Neo-TRE3G > hINS [ NM-001185097.1 ]/EK/10XHis vector at a concentration of 1 ng/. mu.L
1 μ L of 2A-insulinF primer at a concentration of 25 μ M
1 μ L His-InsulinR primer, concentration 25 μ M
25μL 2xPCR mastermix
22μL ddH2O
The total volume of the reaction system is 50 mu L;
the amplification procedure of the PCR amplification is as follows:
the first step is as follows: 95 ℃ for 3min
The second step is that: 1min at 95 ℃; 20sec at 55 ℃; 72 ℃ for 1min for 35 cycles
The third step is that the temperature is 72 ℃ for 10 min.
3. The method for stably expressing secretive human insulin in mesenchymal stem cells according to claim 1, wherein the supernatant is added into human mesenchymal stem cells for infection, and polybrene is added and the final concentration of polybrene is controlled to be 10 μ g/mL.
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