An immortalized stem cell line and a method for constructing the same

Abstract

The present invention provides a method for constructing an immortalized stem cell line, comprising the following steps: constructing a recombinant lentiviral overexpression vector Kozak-FLAG tag-SV40LT, wherein the base sequence of FLAG tag-SV40LT is shown in SEQ ID No: 1; packaging the recombinant lentiviral overexpression vector to obtain a recombinant lentivirus; and using the recombinant lentivirus to infect human umbilical cord mesenchymal stem cells to obtain the immortalized stem cell line. The immortalized stem cell line of the present invention has trilineage differentiation ability, no abnormalities in chromosome structure and number, and does not have the ability to form tumors.

Description

Technical Field

[0001] The present invention belongs to the field of gene engineering technology, and in particular relates to a carrier, a system and a use method thereof for preparing immortalized stem cells. Background Art

[0002] Umbilical cord mesenchymal stem cells (UCMSCs) are extracted from animal umbilical cord tissue. They are characterized by high yield, strong cell proliferation ability, low immunogenicity, and multidirectional differentiation potential. They can differentiate into various cell types: osteoblasts, chondrocytes, adipocytes, hepatocytes, neurons, islet-like cells, etc. They have broad application prospects in the fields of tissue engineering and regenerative medicine. UCMSCs have become an ideal seed cell source for tissue repair and organ reconstruction. However, the biggest problem with UCMSCs as an intervention therapy is that the number of passages in vitro culture is limited, and continuous culture in vitro may change the biological characteristics of stem cells, making them prone to aging, degeneration of multidirectional differentiation potential, and reduction of secretory ability.

[0003] Cell immortalization refers to the process in which cells avoid aging and death due to changes in their own genes or under the stimulation of external factors, and gain the ability to proliferate indefinitely. Immortalized cell lines can be used to study the pathogenesis of many diseases, including cancer, genetic diseases and immune system disorders.

[0004] CN112941034A discloses a method for constructing an immortalized human umbilical cord mesenchymal stem cell line, which is based on human umbilical cord mesenchymal stem cells, transfected with a homologous recombination vector carrying the SV40 large T antigen, and the SV40 large T antigen gene is integrated into the AAVS1 gene site of the human umbilical cord mesenchymal stem cell genome. After continuous passage and screening, a cell line expressing the SV40 large T antigen gene and having multidirectional differentiation potential is obtained. The cell line still has good vitality after more than 50 passages in vitro, maintains a strong proliferation ability, and maintains the characteristics of mesenchymal stem cells. It is an immortalized cell line. This patent conducts osteogenic differentiation and proliferation tests on the constructed immortalized cell line.

[0005] Umbilical cord mesenchymal stem cells (UCMSCs) change their morphology with the increase of passage times and the extension of culture time. The cell volume increases, the cells become flat, and giant nuclei appear. The structure is disordered, apoptosis and necrosis increase, and the secretion capacity is weakened, which limits the application of UCMSCs in the field of tissue engineering and regenerative medicine. The existing technology does not perform karyotype and tumorigenicity detection on the constructed immortalized cell lines. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for constructing an immortalized stem cell line, comprising the following steps:

[0007] A recombinant lentiviral overexpression vector Kozak-FLAG tag-SV40LT was constructed, and the base sequence of FLAG tag-SV40LT is shown in SEQ ID No: 1;

[0008] Packaging the recombinant lentiviral overexpression vector into a recombinant lentivirus;

[0009] The immortalized stem cell line is obtained by infecting human umbilical cord mesenchymal stem cells with the recombinant lentivirus.

[0010] In a further method for constructing an immortalized stem cell line, the recombinant lentiviral overexpression vector Kozak-FLAGtag-SV40LT expresses the SV40LT gene under the control of the EFS promoter.

[0011] A further method for constructing an immortalized stem cell line is to package the recombinant lentiviral overexpression vector into a recombinant lentivirus, comprising one or more of the following steps:

[0012] The 293T cells are inoculated into a culture dish and then placed in an incubator for culture;

[0013] The plasmid-liposome complex is placed in a culture dish and then placed in an incubator for culture. The raw materials for preparing the plasmid-liposome complex include Packaging Mix, lentiviral expression plasmid, Opti-MEM culture medium, and Lipofectamine 2000.

[0014] A further method for constructing an immortalized stem cell line is to use the recombinant lentivirus to infect human umbilical cord mesenchymal stem cells to obtain the immortalized stem cell line, comprising one or more of the following steps:

[0015] The human umbilical cord mesenchymal stem cells were 1×10 4 Pieces / cm 2 Cell density seeding;

[0016] The cell confluence was 30%-50%;

[0017] Infect cells at an MOI of 10 to 200, preferably at an MOI of 100; or

[0018] Use puromycin.

[0019] A further method for constructing an immortalized stem cell line is to package the recombinant lentiviral overexpression vector into a recombinant lentivirus, specifically comprising:

[0020] HEK 293T human embryonic kidney cells in good cell condition and in logarithmic growth phase were selected and counted. 5 / cm 2 Inoculate in a culture dish with DMEM + 10% FBS as the culture medium and culture in a 37°C, 5% CO2 incubator for 24 hours; remove the culture medium before infection and replace with 5 mL Opti-MEM TM Culture medium;

[0021] Take 9 μg Packaging Mix and 3 μg lentiviral expression plasmid and add 1.5 mL Opti-MEM TM Mix gently in the culture medium; take 36μL Lipofectamine TM 2000 Add 1.5mL Opti-MEM TM Gently mix the plasmid solution and Lipofectamine in the culture medium and place at room temperature for 5 minutes. TM 2000 dilution and placed at room temperature for 20 min to obtain 3 mL of plasmid-liposome complex;

[0022] Then carefully add 3 mL of plasmid-liposome complex to the replaced 5 mL Opti-MEM TM The culture dish was placed in a 37°C, 5% CO2 incubator for 6 hours, and then replaced with complete medium DMEM + 10% FBS; after 48 hours, the cell supernatant was collected, centrifuged at 3000 rpm for 10 minutes to remove cells and debris, and filtered with a 0.45 μm filter;

[0023] Finally, the virus stock solution was ultracentrifuged at a centrifugal force of 50,000 g for 2 h, the supernatant was removed, and the solution was resuspended in 1 mL of DMEM culture medium and dispensed into small tubes. A small amount was taken to determine the titer, and the rest was stored at -80°C for later use, thereby obtaining the recombinant lentivirus solution.

[0024] A further method for constructing an immortalized stem cell line is to use the recombinant lentivirus to infect human umbilical cord mesenchymal stem cells to obtain the immortalized stem cell line, which specifically includes:

[0025] Human umbilical cord mesenchymal stem cells were collected at 1x10 4 Pieces / cm 2 The cells were inoculated in a 6-well plate at a density of 30%-50%. When the cell confluence reached 30%-50%, the recombinant lentivirus solution was used to infect the cells at an MOI of 10-200. 12 hours after infection, the culture medium containing the virus was removed and replaced with a fresh complete culture medium for continued culture. 48 hours after infection, a fresh complete culture medium with an appropriate concentration of Puromycin was replaced, and the cell lines stably transduced were selected through continuous passage, which was the immortalized stem cell line.

[0026] A further method for constructing an immortalized stem cell line, wherein the immortalized stem cell line comprises immortalized human umbilical cord mesenchymal stem cells i-hUCMSCs, and the accession number of the i-hUCMSCs is CCTCC NO: C2023364.

[0027] The present invention also provides an immortalized stem cell line, which is constructed according to the aforementioned method for constructing an immortalized stem cell line.

[0028] A further immortalized stem cell line comprises immortalized human umbilical cord mesenchymal stem cells i-hUCMSCs, and the accession number of the i-hUCMSCs is CCTCCNO: C2023364.

[0029] The present invention also provides immortalized human umbilical cord mesenchymal stem cells. The name of the immortalized human umbilical cord mesenchymal stem cells is i-hUCMSCs, and the preservation number of the i-hUCMSCs is CCTCC NO: C2023364.

[0030] The beneficial effects of the present invention are: the immortalized stem cell line of the present invention has the ability of three-lineage differentiation, has no abnormality in chromosome structure and number, and has no ability to form tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Vector map;

[0032] Figure 2 Target gene expression in i-hUCMSCs (RT-qPCR);

[0033] Figure 3 Expression of tagged proteins in i-hUCMSCs (Western blot);

[0034] Figure 4 Analysis of proliferation ability of i-hUCMSCs;

[0035] Figure 5 :SA-β-gal (ONPG) method was used to detect cell senescence characteristics;

[0036] Figure 6 : Distribution of surface markers of i-hUCMSCs;

[0037] Figure 7 : i-hUCMSCs tri-lineage differentiation;

[0038] Figure 8 :Karyotype analysis of i-hUCMSCs;

[0039] Fig. 9:The clonal growth ability of i-hUCMSCs. DETAILED DESCRIPTION

[0040] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0041] Explanation of terms

[0042] MOI indicates multiplicity of infection.

[0043] Western blot indicates protein blotting.

[0044] Kozak refers to a nucleic acid sequence located after the cap structure at the 5' end of eukaryotic mRNA.

[0045] SV40 (Simian vacuolating virus 40) stands for simian vacuolating virus 40.

[0046] FLAG-tag represents a sequence consisting of 8 amino acid residues, N-DYKDDDDK-C (1012Da), which functions as a label.

[0047] Sources of cells, reagents, etc.

[0048] Lipofectamine TM 2000 (transfection reagent) (Invitrogen, 0.3 mL), SMMC-7721 (human liver cancer cells), HEK 293T (human embryonic kidney cells) (donated by Professor Zhu Lei of Xi'an Red Cross Hospital), Puromycin (Solebol, 25 mg), Packaging Mix (plasmid mixture for lentiviral packaging, Saiye Biotechnology), Opti-MEM TM (Reduced serum medium) (Gibco, 500mL), basal medium DMEM (Gibco, 500mL), fetal bovine serum (FBS) (Gibco, 500mL), human umbilical cord mesenchymal stem cell serum-free medium (without phenol red) (Tang Yi Huike, 475mL+25mL).

[0049] The above cells, reagents, etc. were all purchased commercially.

[0050] The embodiments of the present invention are described below.

[0051] In some embodiments of the present invention, a method for constructing an immortalized stem cell line is provided, comprising the following steps:

[0052] A recombinant lentiviral overexpression vector Kozak-FLAG tag-SV40LT was constructed, and the base sequence of FLAG tag-SV40LT is shown in SEQ ID No: 1;

[0053] Packaging the recombinant lentiviral overexpression vector into a recombinant lentivirus;

[0054] The immortalized stem cell line is obtained by infecting human umbilical cord mesenchymal stem cells with the recombinant lentivirus.

[0055] In these embodiments, the present invention does not specifically limit the method for constructing the recombinant lentivirus overexpression vector of the present invention, and the known methods for constructing the recombinant lentivirus are within the scope of the present invention. The method of virus packaging is not specifically limited, and the virus packaging method that can package the target gene into a virus vector to infect cells and achieve the expression of the exogenous gene is within the scope of the present invention.

[0056] In some further embodiments of the present invention, it relates to a method for constructing an immortalized stem cell line, wherein the recombinant lentiviral overexpression vector Kozak-FLAG tag-SV40LT expresses the SV40LT gene under the control of the EFS promoter.

[0057] In some further embodiments of the present invention, a method for constructing an immortalized stem cell line is provided, characterized in that the recombinant lentiviral overexpression vector is subjected to viral packaging to obtain a recombinant lentivirus, comprising one or more of the following steps:

[0058] The 293T cells are inoculated into a culture dish and then placed in an incubator for culture;

[0059] The plasmid-liposome complex is placed in a culture dish and then placed in an incubator for culture. The raw materials for preparing the plasmid-liposome complex include Packaging Mix, lentiviral expression plasmid, Opti-MEM culture medium, and Lipofectamine 2000.

[0060] In some further embodiments of the present invention, a method for constructing an immortalized stem cell line is provided, wherein the immortalized stem cell line is obtained by infecting human umbilical cord mesenchymal stem cells with the recombinant lentivirus, and the method comprises one or more of the following steps:

[0061] The human umbilical cord mesenchymal stem cells were 1×10 4 Pieces / cm 2 Cell density seeding;

[0062] The cell confluence was 30%-50%;

[0063] Infect cells at an MOI of 10 to 200, preferably at an MOI of 100; or

[0064] Use puromycin.

[0065] In some further embodiments of the present invention, a method for constructing an immortalized stem cell line is provided, wherein the recombinant lentiviral overexpression vector is subjected to viral packaging to obtain a recombinant lentivirus, specifically comprising:

[0066] HEK 293T human embryonic kidney cells in good cell condition and in logarithmic growth phase were selected and counted. 5 / cm 2 Inoculate in a culture dish with DMEM + 10% FBS as the culture medium and culture in a 37°C, 5% CO2 incubator for 24 hours; remove the culture medium before infection and replace with 5 mL Opti-MEM TM Culture medium;

[0067] Take 9 μg Packaging Mix and 3 μg lentiviral expression plasmid and add 1.5 mL Opti-MEM TM Gently mix in the culture medium; take 36 μL Lipofectamine TM 2000 Add 1.5mL Opti-MEM TM Gently mix the plasmid solution and Lipofectamine in the culture medium and place at room temperature for 5 minutes. TM 2000 dilution and placed at room temperature for 20 min to obtain 3 mL of plasmid-liposome complex;

[0068] Then carefully add 3 mL of plasmid-liposome complex to the replaced 5 mL Opti-MEM TM The culture dish was placed in a 37°C, 5% CO2 incubator for 6 hours, and then replaced with complete medium DMEM + 10% FBS; after 48 hours, the cell supernatant was collected, centrifuged at 3000 rpm for 10 minutes to remove cells and debris, and filtered with a 0.45 μm filter;

[0069] Finally, the virus stock solution was ultracentrifuged at a centrifugal force of 50,000 g for 2 h, the supernatant was removed, and the solution was resuspended in 1 mL of DMEM culture medium and dispensed into small tubes. A small amount was taken to determine the titer, and the rest was stored at -80°C for later use, thereby obtaining the recombinant lentivirus solution.

[0070] In some further embodiments of the present invention, a method for constructing an immortalized stem cell line is provided, wherein the immortalized stem cell line is obtained by infecting human umbilical cord mesenchymal stem cells with the recombinant lentivirus, and specifically comprises:

[0071] Human umbilical cord mesenchymal stem cells were collected at 1x104 Pieces / cm 2 The cells were inoculated in a 6-well plate at a density of 30%-50%. When the cell confluence reached 30%-50%, the recombinant lentivirus solution was used to infect the cells at an MOI of 10-200. 12 hours after infection, the culture medium containing the virus was removed and replaced with a fresh complete culture medium for continued culture. 48 hours after infection, a fresh complete culture medium with an appropriate concentration of Puromycin was replaced, and the cell lines stably transduced were selected through continuous passage, which was the immortalized stem cell line.

[0072] In some further embodiments of the present invention, it relates to a method for constructing an immortalized stem cell line, wherein the immortalized stem cell line includes immortalized human umbilical cord mesenchymal stem cells i-hUCMSCs, and the preservation number of the i-hUCMSCs is CCTCC NO: C2023364.

[0073] Example 1

[0074] 1. Construction of SV40LT lentiviral vector:

[0075] A recombinant lentiviral overexpression vector Kozak-FLAGtag-SV40LT (1833 bp) expressing the SV40LT gene under the control of the EFS promoter was established. The vector map is as follows ( Figure 1 ):

[0076] 2. Virus Packaging

[0077] Take 293T cells (HEK 293T human embryonic kidney cells) in good cell condition and in logarithmic growth phase, count the cells, and use 6×10 5 / cm 2 Inoculate in a culture dish (the culture medium in this step is DMEM + 10% FBS) and culture in a 37°C, 5% CO2 incubator for 24 hours. Remove the culture medium before infection and replace it with 5 mL Opti-MEM TM Culture medium.

[0078] Take 9 μg Packaging Mix and 3 μg lentiviral expression plasmid (Kozak-FLAG tag-SV40LT) and add 1.5 mL Opti-MEM TM Gently mix in the culture medium (preheated at 37°C). Take 36 μL of Lipofectamine TM 2000 Add 1.5mL Opti-MEM TM Gently mix the plasmid solution and Lipofectamine in the culture medium and place at room temperature for 5 minutes. TM 2000 dilution and placed at room temperature for 20 min to obtain 3 mL of plasmid-liposome complex.

[0079] Then carefully add 3 mL of plasmid-liposome complex to the replaced 5 mL Opti-MEM TM The culture dish was placed in a 37°C, 5% CO2 incubator for 6 hours, and then replaced with complete medium DMEM + 10% FBS. After 48 hours, the cell supernatant was collected, centrifuged at 3000 rpm for 10 minutes to remove cells and debris, and filtered with a 0.45 μm filter.

[0080] Finally, the virus stock solution was ultracentrifuged at a centrifugal force of 50,000 g for 2 h, the supernatant was removed, and the solution was resuspended in 1 mL of DMEM culture medium and dispensed into small tubes. A small amount was taken for titer determination, and the rest was stored at -80°C for later use, thereby obtaining a recombinant lentivirus solution (recombinant lentivirus).

[0081] 3. Recombinant Lentivirus Infection

[0082] Human umbilical cord mesenchymal stem cells were collected at 1x10 4 Pieces / cm 2 The cells were inoculated in a 6-well plate at a density of 1:1 and cultured in a serum-free medium for human umbilical cord mesenchymal stem cells. When the cell confluence reached 30%-50%, the recombinant lentivirus solution was used to infect the cells at an MOI of 100. After 12 hours of infection, the culture medium containing the virus was removed and replaced with a fresh complete culture medium for continued culture. After 48 hours of infection, the culture medium was replaced with a fresh complete culture medium containing an appropriate concentration of Puromycin, and the cell lines that were stably transduced were selected through continuous passage, and named immortalized human umbilical cord mesenchymal stem cells (i-hUCMSCs).

[0083] IV. Identification of Immortalized Cell Lines

[0084] 1. Detection of target gene SV40LT expression in i-hUCMSCs by RT-qPCR

[0085] i-hUCMSCs (experimental group) and hUCMSCs (control group, prepared as follows: resuscitated P3 human umbilical cord mesenchymal stem cells were cultured in human umbilical cord mesenchymal stem cell serum-free medium, and passaged when the confluence reached 80%-90%. Each passage was digested with 0.25% trypsin. When most cells became round under a microscope, an equal volume of complete medium (containing 10% fetal bovine serum by volume) was added to terminate the digestion. The cells were passaged at a ratio of 1:3 and the medium was changed every 2 days.) and total cellular RNA was extracted. After quantification, 1 μg RNA was used for reverse transcription to obtain cDNA. Primer sequences: SV40LT F: 5'-AAAAGAACAGCCCAGCCACT-3' (20 bp), SV40LT R: 5'-CAGCAGAGCCTGTAGAACCA-3' (20 bp); internal reference β-actin F: 5'-AGAGCCTCGCCTTTGCC-3' (17 bp), β-actin R: 5'-GGGGTACTTCAGGGTGAGGA-3' (20 bp). RT-qPCR results showed that the expression of SV40LT in P15 i-hUCMSCs was significantly increased compared with that in P15 hUCMSCs (P<0.001).

[0086] The test results are as follows ( Figure 2 ):

[0087] 2. Western blot detection of the expression of tagged proteins in i-hUCMSCs

[0088] i-hUCMSCs (experimental group) and hUCMSCs (control group, preparation method: resuscitated P3 human umbilical cord mesenchymal stem cells were cultured in human umbilical cord mesenchymal stem cell serum-free medium, and subcultured when the confluence reached 80% to 90%, and digested with 0.25% trypsin each time. When most cells became round under the microscope, an equal volume of complete medium (containing 10% fetal bovine serum) was added to terminate the digestion, and the cells were subcultured at a ratio of 1:3, and the medium was changed every 2 days.), the total cell protein was extracted, and the label protein was detected. Western blot results showed that P15hUCMSCs had no expression of Flag label protein, while P20 i-hUCMSCs had obvious protein expression.

[0089] The test results are as follows ( Figure 3 ):

[0090] 3. Detection of i-hUCMSCs proliferation using holographic label-free cell imaging analysis system

[0091] The cell morphology was observed under phase contrast microscopy, and i-hUCMSCs (P15 and P20) and hUCMSCs (P5, P15, P20) were cultured at 10000 / cm 2 The cells were inoculated in 6-well plates and incubated for 72 hours. The holographic label-free cell imaging analysis system was used to dynamically monitor the cell proliferation status in real time. P5 hUCMSCs proliferated fastest, the proliferation rate decreased significantly at P15, and basically stopped proliferating at P20. Compared with P15 hUCMSCs, P15 i-hUCMSCs still had a strong proliferation ability (P<0.01), and until P20, the proliferation was still significantly higher than that of P20 hUCMSCs (P<0.05). The above results indicate that i-hUCMSCs have good proliferation ability and can be propagated for more than 40 generations.

[0092] The test results are as follows ( Figure 4 ):

[0093] 4. Detection of i-hUCMSCs senescence by β-galactosidase method

[0094] The results of β-galactosidase (β-gal) showed that the positive rates of senescent cells in P9 hUCMSCs were 9.45% and 45.75%, and most cells in P21 showed morphological characteristics of senescence, such as flat and slender cells with giant nuclei, and the positive rate of senescent cells was as high as 45.75%. However, the positive rate of senescent cells in P21 i-hUCMSCs was only 18.25%, and the cell viability was significantly better than that of P21 hUCMSCs (P<0.01), indicating that i-hUCMSCs have good anti-aging ability. The test results are as follows ( Figure 5 ):

[0095] 5. Distribution of surface markers of i-hUCMSCs

[0096] Flow cytometry was used to detect relevant cell surface marker molecules, and the results showed that CD29 (98.82%), CD73 (96.52%), CD90 (95.14%), and CD105 (97.92%) were positive in P22i-hUCMSCs, and CD14 (1.2%), CD34 (0.13%), CD45 (0.28%), CD79a (2.12%), and HLA-DR (0.3%) were negative, indicating that i-hUCMSCs still have good stemness. The test results are as follows ( Figure 6 ):

[0097] 6. Identification of Tri-lineage Differentiation of i-hUCMSCs

[0098] 1x10 4 Pieces / cm2 The cells were inoculated at a high density in a 6-well culture plate, and the cells were induced to differentiate into three lines using the corresponding culture medium when the cells reached about 70% fusion. After 14 days of adipogenic induction, the accumulated oil droplets were detected by Oil Red O staining. After 14 days of osteogenic induction, the calcium nodules were detected by Alizarin Red staining. After 14 days of chondrogenic induction, the cells were stained with Alicin Blue staining, and the staining results were observed under a microscope and photographed. The results are as follows ( Figure 7 ):

[0099] 7. Karyotype analysis of i-hUCMSCs

[0100] i-hUCMSCs did not detect cell clones with abnormal chromosome structure or number. The results are as follows ( Figure 8 ):

[0101] 8. Soft agar colony formation assay

[0102] The negative control hUCMSCs, positive tumor control group (SMMC-7721 and HEK-293T) and i-hUCMSCs were subjected to soft agar cloning experiments. SMMC-7721 and HEK-293T both formed large clones, while i-hUCMSCs and hUCMSCs did not form clones. i-hUCMSCs do not have the ability to form tumors. The results are as follows ( Fig. 9 ):

[0103] Although the present invention has been described in detail above by general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for constructing an immortalized stem cell line, characterized in that: The steps include: A recombinant lentiviral overexpression vector Kozak-FLAG tag-SV40LT was constructed, and the base sequence of FLAG tag-SV40LT is shown in SEQ ID No: 1; Packaging the recombinant lentiviral overexpression vector into a recombinant lentivirus; The immortalized stem cell line is obtained by infecting human umbilical cord mesenchymal stem cells with the recombinant lentivirus.

2. The method for constructing an immortalized stem cell line according to claim 1, characterized in that: The recombinant lentiviral overexpression vector Kozak-FLAG tag-SV40LT expresses the SV40LT gene under the control of the EFS promoter.

3. The method for constructing an immortalized stem cell line according to claim 2, characterized in that: The recombinant lentiviral overexpression vector is packaged into a recombinant lentivirus, comprising one or more of the following steps: The 293T cells are inoculated into a culture dish and then placed in an incubator for culture; The plasmid-liposome complex is placed in a culture dish and then placed in an incubator for culture. The raw materials for preparing the plasmid-liposome complex include Packaging Mix, lentiviral expression plasmid, Opti-MEM culture medium, and Lipofectamine2000.

4. The method for constructing an immortalized stem cell line according to claim 3, characterized in that: Using the recombinant lentivirus to infect human umbilical cord mesenchymal stem cells to obtain the immortalized stem cell line comprises one or more of the following steps: The human umbilical cord mesenchymal stem cells were 1×10 4 Pieces / cm 2 Cell density seeding; The cell confluence was 30%-50%; Infect cells at an MOI of 10 to 200, preferably at an MOI of 100; or Use puromycin.

5. The method for constructing an immortalized stem cell line according to any one of claims 1 to 4, characterized in that: The recombinant lentiviral overexpression vector is packaged into a recombinant lentivirus, specifically comprising: HEK 293T human embryonic kidney cells in good cell condition and in logarithmic growth phase were selected and counted. 5 / cm 2 Inoculate in a culture dish with DMEM + 10% FBS as the culture medium and culture in a 37°C, 5% CO2 incubator for 24 hours; remove the culture medium before infection and replace with 5 mL Opti-MEM TM Culture medium; Take 9 μg Packaging Mix and 3 μg lentiviral expression plasmid and add 1.5 mL Opti-MEM TM Gently mix in the culture medium; take 36 μL Lipofectamine TM 2000 Add 1.5mL Opti-MEM TM Gently mix the culture medium and place at room temperature for 5 minutes; Gently mix the plasmid solution and Lipofectamine TM 2000 dilution and placed at room temperature for 20 min to obtain 3 mL of plasmid-liposome complex; Then carefully add 3 mL of plasmid-liposome complex to the replaced 5 mL Opti-MEM TM The culture dish was placed in a 37°C, 5% CO2 incubator for 6 h, and then replaced with complete medium DMEM + 10% FBS; after 48 h, the cell supernatant was collected, centrifuged at 3000 rpm for 10 min to remove cells and debris, and filtered with a 0.45 μm filter; Finally, the virus stock solution was ultracentrifuged at a centrifugal force of 50,000 g for 2 h, the supernatant was removed, and the solution was resuspended in 1 mL of DMEM culture medium and dispensed into small tubes. A small amount was taken to determine the titer, and the rest was stored at -80°C for later use, thereby obtaining the recombinant lentivirus solution.

6. The method for constructing an immortalized stem cell line according to any one of claims 1 to 4, characterized in that: Using the recombinant lentivirus to infect human umbilical cord mesenchymal stem cells to obtain the immortalized stem cell line specifically comprises: Human umbilical cord mesenchymal stem cells were collected at 1x10 4 Pieces / cm 2 The cells were inoculated in a 6-well plate at a density of 30%-50%. When the cell confluence reached 30%-50%, the recombinant lentivirus solution was used to infect the cells at an MOI of 10-200. 12 hours after infection, the culture medium containing the virus was removed and replaced with a fresh complete culture medium for continued culture. 48 hours after infection, a fresh complete culture medium with an appropriate concentration of Puromycin was replaced, and the cell lines stably transduced were selected through continuous passage, which was the immortalized stem cell line.

7. The method for constructing an immortalized stem cell line according to any one of claims 1 to 4, characterized in that: The immortalized stem cell line includes immortalized human umbilical cord mesenchymal stem cells i-hUCMSCs, and the preservation number of the i-hUCMSCs is CCTCC NO: C2023364.

8. An immortalized stem cell line, characterized in that: The immortalized stem cell line is constructed according to the method for constructing an immortalized stem cell line according to any one of claims 1 to 7.

9. The immortalized stem cell line according to claim 8, characterized in that: The immortalized stem cell line includes immortalized human umbilical cord mesenchymal stem cells i-hUCMSCs, and the preservation number of the i-hUCMSCs is CCTCC NO: C2023364.

10. An immortalized human umbilical cord mesenchymal stem cell, characterized in that: The immortalized human umbilical cord mesenchymal stem cells are named i-hUCMSCs, and the accession number of the i-hUCMSCs is CCTCC NO: C2023364.

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