Construction method of notch1 knockdown stable cell line of immortalized human umbilical cord mesenchymal stem cells and application thereof in hepatocyte differentiation

Abstract

The application discloses a kind of immortalization human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable cell line construction method and its application in liver differentiation, it is related to immortalized cell technical field.The application is first to connect the gene of interest to vector, and the lentiviral plasmid containing the gene of interest is obtained, then the lentiviral plasmid containing the gene of interest is introduced into 293T cell, and high titer lentivirus containing the gene of interest is generated;The immortalized human umbilical cord mesenchymal stem cell is infected with lentivirus again, and mixed clone cell strain is screened, and the mixed clone cell strain is screened, and the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable cell line is obtained.The application proves that the differentiation efficiency of immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown to hepatocyte-like cell can be effectively improved by glycogen staining and western blotting experiment, and the research result is expected to provide new technical thought for the next step development to improve the yield of MSCs liver differentiation.

Description

Technical Field

[0001] This invention relates to the field of immortalized cell technology, specifically to a method for constructing an immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable cell line and its application in hepatic differentiation. Background Technology

[0002] Stem cells are cells with multi-lineage differentiation potential and have potential applications in gene therapy and cell therapy research. Studies have shown that the differentiation of mesenchymal stem cells (MSCs) into hepatocyte-like cells (HLCs) is considered one of the most promising strategies to replace hepatocyte transplantation for the treatment of end-stage liver disease. Current methods for obtaining hepatocyte-like cells include cytokine induction, cell co-culture induction, transgenic induction, and induction based on the liver microenvironment. However, existing methods generally suffer from low differentiation efficiency, making it difficult to generate a sufficient number of immature HLCs, severely limiting their basic research and clinical applications. Furthermore, after limited cell passage, human umbilical cord mesenchymal stem cells (hUCMSCs) exhibit cell proliferation arrest, senescence, and death, resulting in low differentiation levels of primary hUCMSCs and functionally immature hepatocyte-like cells obtained during in vitro differentiation into hepatocyte-like cells via cytokine induction. Therefore, obtaining a sufficient number of functionally mature hepatocyte-like cells is a pressing technical problem to be solved in this field.

[0003] The applicant discovered that the expression level of NOTCH1 protein was significantly reduced during the in vitro hepatic differentiation of hUCMSCs, and that the Notch signaling pathway inhibitor DAPT could promote differentiation, indicating that NOTCH1 may be a key target regulating the hepatic differentiation of hUCMSCs. However, simply modifying the genes of primary hUCMSCs cannot overcome their rapid in vitro aging and loss of stemness, making it difficult to achieve long-term stable research and large-scale expansion. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a method for constructing an immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable cell line. Based on the existing technology for in vitro directed differentiation of mesenchymal stem cells into hepatocyte-like cells, there is a problem that it cannot be expanded on a large scale. Based on the immortalized human umbilical cord mesenchymal stem cells constructed by the research group in the previous stage, this invention provides a method for constructing an immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable cell line.

[0005] To address the aforementioned technical challenges, this invention employs a lentivirus-mediated method to transfect P3 generation hUCMSCs with recombinant plasmids carrying the hTERT and EGFP genes, obtaining immortalized human umbilical cord mesenchymal stem cells capable of stable proliferation and maintaining a pluripotent differentiation state. Based on this, shRNA targeting NOTCH1 is designed, a lentivirus knockdown vector is constructed, and this vector is transfected into P21 generation immortalized hUCMSCs. Stable NOTCH1 knockdown cell lines are screened, and their differentiation efficiency into hepatocyte-like cells is verified. This invention aims to overcome the shortcomings of low differentiation efficiency and easy senescence in primary MSCs, providing a new technical means for efficiently obtaining functionally mature hepatocyte-like cells.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for constructing an immortalized human umbilical cord mesenchymal stem cell line with NOTCH1 knockdown and stable transformation, characterized by comprising the following steps: S1. The target gene is ligated into a vector to construct a lentiviral plasmid containing the target gene. The target gene is a DNA fragment from the human NOTCH1 gene. The DNA fragment includes at least one of fragment 1, fragment 2, and fragment 3. The sequence of fragment 1 is 5'-GCTCCCTCAACTTCAATGACC-3', the sequence of fragment 2 is 5'-CCGGGACATCACGGATCATAT-3', and the sequence of fragment 3 is 5'-GGACAAGATCGATGGCTACGA-3'. S2. The lentiviral plasmid was introduced into 293T cells, and high-titer lentiviruses containing the target gene were screened. S3. Infect immortalized human umbilical cord mesenchymal stem cells with the lentivirus to obtain a hybrid clone cell line; S4. The mixed clone cell line is screened to obtain an immortalized human umbilical cord mesenchymal stem cell line with NOTCH1 knockdown and stable transformation.

[0007] Preferably, in S1, the target gene is directionally inserted into the vector using the BamHI + EcoRI double digestion method, wherein the vector is pLVX-shRNA2-Puro.

[0008] Preferably, in S1, The amplification primers for fragment 1 are: hNotch1-1-F: 5-GATCCGCTCCCTCAACTTCAATGACCCTCGAGGGTCATTGAAGTTGAGGGAGCTTTTTG-3', hNotch1-1-R: 5'-AATTCAAAAAGCTCCCTCAACTTCAATGACCCTCGAGGGTCATTGAAGTTGAGGGAGCG-3'; The amplification primers for fragment 2 are: hNotch1-2-F: 5'-GATCCGCCGGGACATCACGGATCATATCTCGAGATATGATCCGTGATGTCCCGGTTTTTTG-3', hNotch1-2-R: 5'-AATTCAAAAAACCGGGACATCACGGATCATATCTCGAGATATGATCCGTGATGTCCCGGCG-3'; The amplification primers for fragment 3 are: hNotch1-3-F: 5'-GATCCGGACAAGATCGATGGCTACGACTCGAGTCGTAGCCATCGATCTTGTCCTTTTTG-3', hNotch1-3-R: 5'-AATTCAAAAAGGACAAGATCGATGGCTACGACTCGAGTCGTAGCCATCGATCTTGTCCG-3'.

[0009] Preferably, S1 includes the following specific steps: Dissolve the amplification primers in 100 μl of annealing buffer, take 2 μl of each amplification primer and add them to 16 μL of annealing buffer, mix thoroughly, anneal at 100°C and cool naturally to room temperature, and dilute the annealed products 100 times with DEPC water. The vector plasmid pLVX-shRNA2-Puro was double-digested with BamHI and EcoRI, and the digestion reaction was carried out in a water bath at 37°C for 3 hours. The large fragment of plasmid pLVX-shRNA2-Puro BamHI+EcoRI digested by agarose gel electrophoresis was recovered. The large fragment of plasmid pLVX-shRNA2-Puro was recovered and ligated with the annealing product. The ligation reaction was carried out at 22°C for 3 hours. Take 10 μL of ligation product and mix with 100 μL of JM109 competent bacteria, incubate on ice for 30 min, heat shock at 42°C for 45 s, immediately place on ice for 2 min, add 400 μL of LB medium preheated to room temperature, incubate at 37°C in a shaker for 1 h, centrifuge at 4000 rpm for 1 min, discard 400 μL of culture supernatant, mix the remaining 100 μL with a pipette and spread evenly on LB plates containing 100 μg / ml Ampicillin resistance, and incubate in an inverted incubator at 37°C overnight. Single colonies were picked and inoculated into LB medium containing 5 ml of 100 μg / ml Ampicillin resistance. The culture was incubated overnight at 37°C and 250 rpm. The bacterial culture was then sent for sequencing verification to construct a lentiviral plasmid containing the target gene.

[0010] Preferably, S2 includes the following specific steps: The day before transfection, 293T cells were digested with trypsin, and the cell density was adjusted with serum-containing DMEM medium. The cells were then incubated at 37°C in a 5% CO2 incubator. After 16-24 hours, when the cell density reached 80%-90%, the cells were ready for transfection. 2-4 hours before transfection, the medium was changed with 5 ml of complete medium without P / S. Perform transfection as follows: Add 500 μL of CPT Buffer A to a clean 1.5 mL EP tube. In another clean 1.5 mL EP tube, add Vector reagent, Lentiviral packaging mixture, CPT Buffer B, and ddH2O to make a final volume of 500 μL. Mix the solutions in the two EP tubes and let the mixture stand at room temperature for 30 min. Then, add the mixture dropwise to the cell culture dish, mix gently, and incubate at 37°C in a 5% CO2 incubator. After culturing for 12-16 hours, discard the culture medium and replace it with 10 ml of fresh complete culture medium containing FBS and P / S. 24 hours after changing the medium, collect the supernatant and store it in a 4°C refrigerator. Then add 10 ml of fresh complete culture medium containing FBS and P / S to the cell culture dish and continue culturing in a 37°C, 5% CO2 incubator. Collect the supernatant again, mix it with the supernatant collected the first time, centrifuge at 1000 rpm for 5 min, discard cell debris, filter the supernatant through a 0.45 μm PVDF filter, centrifuge, and collect the virus pellet. The viral precipitate was resuspended, and high-titer lentiviruses containing the target gene were obtained by screening.

[0011] Preferably, S3 includes the following specific steps: Revive immortalized human umbilical cord mesenchymal stem cells, passage the cells when they grow to 80-90% cell density, and passage them 2-3 times consecutively. Culture immortalized human umbilical cord mesenchymal stem cells. When the cells reach 80-90% confluence density, digest the cells and count them, then divide them into groups of 5 × 10⁻⁶. 5 Cells are seeded into the well plate at a cell density per well. The lentiviruses were added at MOI=20 and incubated overnight at 37°C in a 5% CO2 incubator. After adding the lentivirus, the medium was replaced with human mesenchymal stem cell complete culture medium 24 hours later. After adding the lentivirus for 48 hours, Puromycin was added at a final concentration of 5ug / ml for resistance screening, and the medium was changed every 2-3 days. Digest the cells when they reach 80-90% confluence and seed them at T-25cm. 2 Continue culturing in cell culture flasks; After 2-3 consecutive passages, a stable hybrid clone cell line was obtained.

[0012] Secondly, the present invention provides an immortalized human umbilical cord mesenchymal stem cell line with NOTCH1 knockdown and stable transformation, which is obtained by the construction method described above.

[0013] Thirdly, the present invention provides the application of the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable cell line in liver differentiation.

[0014] This invention induces the hepatic differentiation of the aforementioned immortalized human umbilical cord mesenchymal stem cells (UCMSCs) with NOTCH1 knockdown in vitro using a cytokine induction method. During the differentiation process, changes in cell morphology, glycogen production capacity, and liver function-related indicators of the NOTCH1 knockdown stable cell line and the empty vector group were observed. Glycogen staining and Western blotting experiments confirmed that NOTCH1 knockdown of immortalized UCMSCs effectively improves their differentiation efficiency into hepatocyte-like cells. Therefore, NOTCH1 knockdown is beneficial for the differentiation of human umbilical cord mesenchymal stem cells into hepatocytes.

[0015] Preferably, the application method includes: pretreating the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell line, and inducing and maturing the pretreated immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell line to obtain liver-like cells.

[0016] Preferably, the pretreatment method includes: culturing immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell lines in IMDM medium containing EGF and bFGF for 1-3 days; The induction culture method includes: culturing immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell lines in IMDM medium containing HGF, bFGF and nicotinamide for 6-8 days; The method for mature culture includes culturing immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell lines in IMDM medium containing dexamethasone, ITS+premix and OSM.

[0017] This invention has at least one of the following beneficial effects: This invention provides a method for constructing an immortalized human umbilical cord mesenchymal stem cell (UC-MSC) NOTCH1 knockdown stable transgenic line. Further research by the applicant revealed that RNA-seq analysis of UC-MSC cells before and after differentiation showed a significant decrease in the expression levels of the receptor NOTCH1, its ligand JAG1, and its downstream factor HES1. Western blotting further confirmed that NOTCH1 expression decreased during differentiation. Therefore, this immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable transgenic line can be applied to hepatic differentiation. Compared with existing technologies, this invention's immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable transgenic line can effectively improve the efficiency of hepatic differentiation and the maturity of hepatocyte-like cells. Attached Figure Description

[0018] Figure 1 It is a Vector Map; Figure 2 This is an RNA-seq analysis of human umbilical cord mesenchymal stem cells before and after induced differentiation. The results show that the expression level of the NOTCH1 gene was significantly reduced; Figure 3 This is a graph showing the expression of NOTCH1 protein in human umbilical cord mesenchymal stem cells during differentiation. The results indicate that NOTCH1 protein expression decreases at days 7, 14, and 21 of differentiation. Figure 4 This is a sequencing result of the bacterial culture pLVX-shRNA2-Puro-hNotch1-1-16#. Figure 5 This is a sequencing result of the bacterial culture pLVX-shRNA2-Puro-hNotch1-2-10#. Figure 6 This is a sequencing result of the bacterial culture pLVX-shRNA2-Puro-hNotch1-3-19#. Figure 7 This is a diagram of 293T cells transfected with plasmid pLVX-shRNA2-Puro-hNotch1-1; Figure 8 This is a diagram of 293T cells transfected with plasmid pLVX-shRNA2-Puro-hNotch1-2; Figure 9 This is a diagram of 293T cells transfected with plasmid pLVX-shRNA2-Puro-hNotch1-3; Figure 10 This is a fluorescence image of HEK293 cells infected with lentivirus rLV-shRNA2-Puro-hNotch1-1 72 hours ago. Figure 11 This is a fluorescence image of HEK293 cells infected with lentivirus rLV-shRNA2-Puro-hNotch1-2 for 72 hours. Figure 12 This is a fluorescence image of HEK293 cells infected with lentivirus rLV-shRNA2-Puro-hNotch1-3 for 72 hours. Figure 13 This is a diagram of MSCs-shRNA2-Puro-hNOTCH1-3 knockdown cells; Figure 14 This is a diagram of MSCs-shRNA2-Puro knockdown control cells; Figure 15 This is a validation graph showing the knockdown efficiency of MSCs-shRNA2-Puro-hNOTCH1-1, MSCs-shRNA2-Puro-hNOTCH1-2, MSCs-shRNA2-Puro-hNOTCH1-3 and the control group. Figure 16 These are PAS staining images of immortalized human umbilical cord mesenchymal stem cells in the NOTCH1 knockdown group and the control group; Figure 17 This is a graph showing the protein expression of liver function indicators AFP and ALB in the NOTCH1 knockdown group and the control group before and after induction at three stages of induced differentiation of immortalized human umbilical cord mesenchymal stem cells at 7, 14 and 21 days. Figure 18 This is a graph showing the expression of NOTCH1 protein and liver-related indicators before and after induction of normal immortalized human umbilical cord mesenchymal stem cells, and after 28 days of induction differentiation in the NOTCH1 knockdown group and the control group. Detailed Implementation

[0019] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0020] Example 1: Identification of NOTCH1 target This embodiment mainly includes two parts: transcriptome sequencing verification and Western blotting detection.

[0021] Part 1: Verification via transcriptome sequencing before and after induction: Mesenchymal stem cells (MSCs) were obtained from the umbilical cords of healthy mothers. MSCs were acquired from the collected umbilical cords using the tissue adherence method. Highly viable P4 generation human umbilical cord MSCs were hepatically induced to differentiate after 28 days. Total RNA was extracted from the cells before and after induction using the RNeasy Plus Mini Kit (Qiagen). Sequencing data were provided by Guangzhou Kidnova RNA, and the quantity and integrity of RNA were verified by agarose gel electrophoresis, NanoDrop microspectrophotometer, and Agilent 2100 bioanalyzer. RNA-seq libraries were constructed using Illumina's NEBNext Ultra RNA library preparation kit according to the manufacturer's protocol. The libraries were then evaluated using an Agilent 2100 bioanalyzer. Finally, 150 bp paired-end sequencing was performed on an Illumina NovaSeq 6000 HiSeq sequencer. The raw data from the Illumina HiSeq sequencing underwent quality control processing, and the resulting clean reads were used for downstream bioinformatics analysis. Clean reads were aligned to a reference genome using HISAT2 software, and gene expression levels were determined by calculating the number of fragments per kilobase transcript per million mapped reads (FPKM). Differentially expressed genes (DEGs) were identified using the DESeq R package, with criteria of |log2 fold change| > 1 and p < 0.05. Differential expression analysis was performed using the Omicamart website (https: / / www.omicsmart.com), and Fisher's exact test was applied to identify gene ontology (GO) terms and significantly enriched pathways significantly associated with differentially expressed genes (DEGs), with p < 0.05 considered statistically significant.

[0022] Part Two: Notch1 protein detection during induction: Step S1: Total protein was extracted from each cell population of human umbilical cord mesenchymal stem cells induced to differentiate to 7, 14, and 28 days using RIPA buffer and the addition of protease inhibitors.

[0023] In step S2, the protein is separated by SDS-PAGE at a volume concentration of 8% to 15% (depending on the protein molecular weight) and then transferred to a PVDF membrane.

[0024] Step S3: The PVDF membrane is blocked for 1 hour at room temperature with TBST buffer containing 5% bovine serum albumin by volume.

[0025] Step S4: The PVDF membrane is incubated overnight at 4°C with anti-NOTCH1 antibody and anti-GAPDH antibody.

[0026] Step S5: The PVDF membrane is inoculated with goat anti-rabbit IgG secondary antibody (Proteintech, USA) at room temperature at a dilution of 1:5000 and incubated for 1 hour.

[0027] In step S6, the membrane was visualized using an enhanced chemiluminescence system, and the grayscale values ​​of the image were analyzed using Image Lab software.

[0028] like Figure 2-3 As shown, RNA sequencing results confirmed that NOTCH1 levels significantly decreased after hepatic differentiation of human umbilical cord mesenchymal stem cells. Furthermore, Western blotting analysis of NOTCH1 protein expression during induction revealed a gradual decrease in NOTCH1 protein levels. Therefore, NOTCH1 is a key target regulating hepatic differentiation of human umbilical cord mesenchymal stem cells.

[0029] Example 2: Construction of pLVX-shRNA2-Puro-hNotch1 plasmid The target gene was ligated into a vector to construct lentiviral plasmids pLVX-shRNA2-Puro-hNotch1-1, pLVX-shRNA2-Puro-hNotch1-2, and pLVX-shRNA2-Puro-hNotch1-3 containing the target gene. The specific construction method is as follows: (1) Construction information: (2) Gene Name: human Notch1 (NM_017617.5); Cloning Vector: pLVX-shRNA2-Puro; Cloning Strategy: BamHI+EcoRI; Vector Map: Figure 1 ; DNA primer design; hNotch1-1 sequence: 5'-GCTCCCTCAACTTCAATGACC-3'; hNotch1-2 sequence: 5'-CCGGGACATCACGGATCATAT-3'; hNotch1-3 sequence: 5'-GGACAAGATCGATGGCTACGA-3'; hNotch1-1-F: 5'-GATCCGCTCCCTCAACTTCAATGACCCTCGAGGGTCATTGAAGTTGAGGGAGCTTTTTG-3'; hNotch1-1-R: 5'-AATTCAAAAAGCTCCCTCAACTTCAATGACCCTCGAGGGTCATTGAAGTTGAGGGAGCG-3'; hNotch1-2-F: 5'-GATCCGCCGGGACATCACGGATCATATCTCGAGATATGATCCGTGATGTCCCGGTTTTTTG-3' hNotch1-2-R: 5'-AATTCAAAAAACCGGGACATCACGGATCATATCTCGAGATATGATCCGTGATGTCCCGGCG-3'; hNotch1-3-F: 5'-GATCCGGACAAGATCGATGGCTACGACTCGAGTCGTAGCCATCGATCTTGTCCTTTTTG-3'; hNotch1-3-R: 5'-AATTCAAAAAGGACAAGATCGATGGCTACGACTCGAGTCGTAGCCATCGATCTTGTCCG-3'; The synthesized DNA primer fragments were thoroughly dissolved in 100 μl of annealing buffer (10 mM Tris-HCl, 50 mM NaCl, pH 8.0). 2 μl of each DNA primer fragment was then added to 16 μL of annealing buffer, mixed thoroughly, annealed at 100 °C, and allowed to cool naturally to room temperature. The annealed products were then diluted 100 times with DEPC water.

[0030] (1) Double digestion of the vector plasmid pLVX-shRNA2-Puro with BamHI and EcoRI: The enzyme digestion reaction was carried out in a water bath at 37°C for 3 hours. The vector enzyme digestion system is as follows: The large fragment of plasmid pLVX-shRNA2-Puro BamHI+EcoRI was recovered by 1% agarose gel electrophoresis; (2) Ligation of the large fragment recovered from plasmid pLVX-shRNA2-Puro with the annealing product. The ligation reaction was carried out at 22℃ for 3 hours. The ligation reaction system is as follows: (3) Transformation of ligation products: Take 10 μL of ligation product and 100 μL of JM109 competent bacteria, mix well and incubate on ice for 30 min, heat shock at 42℃ for 45 s, immediately place on ice for 2 min, add 400 μL of LB medium preheated to room temperature, incubate at 37℃ in a shaker for 1 h, centrifuge at 4000 rpm for 1 min, discard 400 μL of culture supernatant, mix the remaining 100 μL with a pipette and spread evenly on LB plates containing 100 μg / ml Ampicillin resistance, and incubate in an inverted incubator at 37℃ overnight.

[0031] Three single colonies from each culture were inoculated into LB medium containing 5 ml of 100 μg / ml Ampicillin-resistant culture medium and cultured overnight at 37°C with a constant temperature shaker at 250 rpm. The bacterial cultures pLVX-shRNA2-Puro-hNotch1-1-16#, pLVX-shRNA2-Puro-hNotch1-2-10#, and pLVX-shRNA2-Puro-hNotch1-3-19# were then sequenced for verification. Sequencing results are shown below. Figure 4-6 Sequencing primers: U6-F (5'-TACGATACAAGGCTGTTAGAGAG-3').

[0032] like Figure 4-6 As shown, the sequencing results of the bacterial cultures pLVX-shRNA2-Puro-hNotch1-1-16#, pLVX-shRNA2-Puro-hNotch1-2-10#, and pLVX-shRNA2-Puro-hNotch1-3-19# are consistent with the designed sequences. Therefore, the pLVX-shRNA2-Puro-hNotch1 plasmid was successfully constructed.

[0033] Example 3: Lentiviral Packaging The lentiviral plasmids pLVX-shRNA2-Puro-hNotch1-1, pLVX-shRNA2-Puro-hNotch1-2, and pLVX-shRNA2-Puro-hNotch1-3 containing the target gene from Example 2 were introduced into 293T cells to generate high-titer lentiviruses rLV-shRNA2-Puro-hNotch1-1, rLV-shRNA2-Puro-hNotch1-2, and rLV-shRNA2-Puro-hNotch1-3 containing the target gene. The specific method is as follows: The day before transfection, cells were prepared for passage: 293T cells were digested with 0.25% trypsin, and the cell density was adjusted with DMEM medium containing 10% serum. Cells were then seeded at a rate of 6–8 × 10⁶ cells per 10 cm² cell culture dish. 6 Transfer the cells to a 10cm cell culture dish and incubate at 37℃ in a 5% CO2 incubator. After 16-24 hours, when the cell density reaches 80%-90%, the cells are ready for transfection. On the second day, 2–4 hours before transfection, replace the medium with 5 ml of complete culture medium without P / S (DMEM + 10% FBS).

[0034] Transfection was performed following these experimental steps: A. Add 500ul CPT Buffer A to a clean 1.5ml EP tube (which can be labeled as tube A); B. In another clean 1.5ml EP tube (which can be labeled as tube B), add the following reagents, with a final volume of 500ul; C. Slowly add the DNA warming solution from tube B dropwise into tube A, and gently mix with a pipette for 10 minutes. D. Let the mixed mixture stand at room temperature for 30 minutes, then add the mixture dropwise evenly to a 10cm cell culture dish and mix gently. Incubate at 37℃ in a 5% CO2 incubator.

[0035] On the third day, after 12 to 16 hours of incubation, the culture medium was discarded and replaced with 10 ml of fresh complete culture medium (DMEM + 10% FBS + P / S). On the fourth day, 24 hours after changing the medium, the supernatant was collected and stored in a 4°C refrigerator. Then, 10 ml of fresh complete culture medium (DMEM + 10% FBS + P / S) was added to a 10 cm cell culture dish and placed in a 37°C, 5% CO2 incubator for further culture. Day 5: Collect the supernatant again, mix it with the supernatant collected the first time, centrifuge at 1000 rpm for 5 min, discard cell debris, and filter the supernatant through a 0.45 μm PVDF filter into a 50 ml round-bottom centrifuge tube. Centrifuge at 4℃, 50000g for 2.5h. After centrifugation, the virus precipitate can be marked with a marker. Carefully discard the supernatant, air dry, add DMEM (without serum or antibiotics) or PBS to resuspend the virus pellet at a volume of 100ul / 10cm culture dish, let stand at room temperature for 2 hours, then gently pipette to mix (avoiding air bubbles), continue to stand at room temperature for 30 minutes, aliquot the amount of virus used each time into clean 1.5ml EP tubes, and store at -80℃.

[0036] Plasmids pLVX-shRNA2-Puro-hNotch1-1, pLVX-shRNA2-Puro-hNotch1-2, and pLVX-shRNA2-Puro-hNotch1-3 were transfected into 293T cells. Figure 7-9 ), resulting in lentiviruses rLV-shRNA2-Puro-hNotch1-1, rLV-shRNA2-Puro-hNotch1-2, and rLV-shRNA2-Puro-hNotch1-3.

[0037] like Figure 7-9 As shown, after plasmid transfection into 293T cells, the expression of green fluorescent protein in the cells can be observed, indicating that the lentivirus packaging was successful.

[0038] Example 4: Determination of lentivirus biological titer The biological titers of the lentiviruses rLV-shRNA2-Puro-hNotch1-1, rLV-shRNA2-Puro-hNotch1-2, and rLV-shRNA2-Puro-hNotch1-3 in Example 3 were determined using the following method: One day before the titer determination, take a 48-well plate and prepare 5 × 10⁵ samples per well. 4 HEK 293 cells were seeded at the appropriate density. Add polybrene to fresh complete culture medium (DMEM + 10% FBS + P / S) to a final concentration of 8 μg / ml.

[0039] The virus was serially diluted 10-fold using complete culture medium containing polybrene, as follows: Take a 48-well plate, with 90 μL of medium in each well. Add 10 μL of the stock virus solution to the first well, mix well, and then transfer 10 μL of the virus mixture to the second well (avoiding air bubbles during mixing). Continue this dilution until the sixth well (i.e., a 10-fold dilution). 5 times).

[0040] Pipette 10 μL of virus dilution into each corresponding 48-well plate and incubate overnight at 37°C in a 5% CO2 incubator. After 16-24 hours, replace the medium with fresh complete culture medium (DMEM + 10% FBS + P / S).

[0041] After incubating at 37℃ in a 5% CO2 incubator for 48 hours, count the number of fluorescent cells using a fluorescence microscope (if the observation is unclear, change the medium with PBS (pH 7.4) before observing). Generally, if N (N<10) fluorescent cells appear in the wells of the highest dilution gradient of 10m, the viral titer is Nx10mTU / ul (10m is the dilution factor), that is, the viral titer is Nx10m+3TU / ml. If N>10, further dilution is required.

[0042] Example 5: Construction of stable cell lines Immortalized human umbilical cord mesenchymal stem cells were infected with high-titer lentiviruses containing the target gene, rLV-shRNA2-Puro-hNotch1-1, rLV-shRNA2-Puro-hNotch1-2, and rLV-shRNA2-Puro-hNotch1-3, as screened in Example 4, to obtain hybrid clonal cell lines. These were then screened to obtain stable NOTCH1 knockdown transgenic lines of immortalized human umbilical cord mesenchymal stem cells. The specific method is as follows: Revive immortalized human umbilical cord mesenchymal stem cells, passage the cells when they grow to 80-90% cell density, and passage them 2-3 times consecutively. Culture immortalized human umbilical cord mesenchymal stem cells. When the cells reach 80-90% confluence density, digest the cells and count them, then divide them into groups of 5 × 10⁻⁶. 5 Cells were seeded into 6-well plates at a cell density per well. On the second day, lentiviruses rLV-shRNA2-Puro-hNotch1-1, rLV-shRNA2-Puro-hNotch1-2, and rLV-shRNA2-Puro-hNotch1-3 were added at MOI=20 respectively; and incubated overnight at 37°C in a 5% CO2 incubator. 24 hours after adding the virus, the medium was replaced with complete culture medium for human mesenchymal stem cells. 48 hours after adding the virus, Puromycin was added at a final concentration of 5 ug / ml for resistance screening. The medium was changed every 2-3 days (complete medium containing 5 ug / ml Puromycin). Digest the cells when they reach 80-90% confluence and seed them at T-25cm. 2 Continue culturing in cell culture flasks; After 2-3 consecutive passages, stable hybrid clonal cell lines were obtained. The core of this process was "single-cell isolation and screening." The hybrid cell pool was then monocloned using limiting dilution to obtain MSCs-shRNA2-Puro-hNOTCH1-1 knockdown cell lines, MSCs-shRNA2-Puro-hNOTCH1-2 knockdown cell lines, and MSCs-shRNA2-Puro-hNOTCH1-3 knockdown cell lines.

[0043] Fluorescence images of HEK293 cells infected with lentiviral rLV-shRNA2-Puro-hNotch1-1, rLV-shRNA2-Puro-hNotch1-2, and rLV-shRNA2-Puro-hNotch1-3 for 72 hours are shown in the image. Figure 10-12 ; Images of MSCs-shRNA2-Puro-hNOTCH1-3 knockdown cells and MSCs-shRNA2-Puro knockdown control cells are shown below. Figure 13-14 ; Verification of the knockdown efficiency of MSCs-shRNA2-Puro-hNOTCH1-1 knockdown cells, MSCs-shRNA2-Puro-hNOTCH1-2 knockdown cell lines, and MSCs-shRNA2-Puro-hNOTCH1-3 knockdown cells can be found in [link to documentation]. Figure 15 .

[0044] like Figure 10-15 As shown, after 72 hours of infection with HEK293 cells using lentiviruses rLV-shRNA2-Puro-hNotch1-1, rLV-shRNA2-Puro-hNotch1-2, and rLV-shRNA2-Puro-hNotch1-3, green fluorescent protein (GFP) expression was observed under a microscope, indicating successful lentiviral transfection. Furthermore, GFP expression was observed in MSCs-shRNA2-Puro-hNOTCH1-3 knockdown cells and MSCs-shRNA2-Puro knockdown control cells, and the knockdown efficiency was verified using Western blotting. The results confirm the successful construction of the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown cell line.

[0045] Example 6: Liver-induced differentiation experiment In vitro, the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable transgene (MSCs-shRNA2-Puro-hNOTCH1-3) obtained in Example 5 was induced to differentiate into a liver variant using a cytokine induction method, as follows: Step S1: Resuscitation of immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable transgenes a. Take one cell from the liquid nitrogen and place it in a pre-prepared 37°C constant temperature water bath. Stir rapidly to thaw the frozen cell as quickly as possible.

[0046] b. Wipe the surface of the cryovial with an alcohol swab and transfer it to a clean bench. Take 9 ml of PBS into a 15 ml centrifuge tube, add 1 ml of the cryovial suspension to the centrifuge tube, and centrifuge at 1100 rpm for 4 min.

[0047] c. Carefully aspirate the supernatant after centrifugation, add 1 ml of low-glucose complete culture medium, resuspend, transfer to a 10 cm culture dish, add 9 ml of low-glucose complete culture medium, mix well with a cross-shaped incubator and mark with a MAKER pen, and incubate in a 37℃, 5% CO2 incubator.

[0048] d. Observe cell morphology under a microscope daily. Generally, when the cells have grown to more than 90%, passage them.

[0049] Step S2: Induction of immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable transgenes into hepatoid cells I) Cell pretreatment stage (days 1-2) a. When the pre-coated MSCs have grown to 80%-90%, begin preparing the pretreatment medium. Dissolve the pre-prepared and aliquoted EGF and FGF4 at room temperature, add them to IMDM medium (final cytokine concentration of 20 ng / ml for EGF and 10 ng / ml for FGF4). Gently invert the reagent tubes to ensure thorough mixing. Prepare just enough for one medium change each time.

[0050] b. Carefully aspirate the complete culture medium from the six-well plate and add 2 mL of the prepared pretreatment culture medium to each well.

[0051] c. Incubate the cells in a 37°C, 5% CO2 incubator for 2 days.

[0052] (ii) Hepatocyte induction phase (days 2-9) a. Dissolve the pre-prepared and aliquoted 20 ng / ml HGF, 10 ng / ml FGF4, and 0.061 g / mL nicotinamide at room temperature, add them to IMDM medium, and gently invert the reagent tubes to ensure that the reagents are mixed evenly. Prepare just enough for one medium change each time.

[0053] b. Carefully aspirate the pretreatment medium from the six-well plate, carefully rinse the cells once with PBS, add 2 mL of the prepared hepatocyte induction medium to each well, and change the medium every 3 days.

[0054] c. Incubate the cells in a 37°C, 5% CO2 incubator for 7 days.

[0055] (III) Hepatoid cell maturation stage (days 9-28).

[0056] a. Dissolve the pre-amplified 0.1 μmol / ml dexamethasone, ITS+premix (100X), and 10 μg / ml OSM at room temperature, add them to IMDM medium, and gently invert the reagent tube to ensure that the reagents are mixed evenly. Prepare just enough for one medium change each time.

[0057] b. Carefully aspirate the hepatocyte induction medium from the six-well plate, carefully rinse the cells once with PBS, and then add 2 ml of the prepared hepatocyte maturation medium to each well.

[0058] Efficiency assay of liver-directed differentiation of immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable transgenes: The morphological changes of immortalized human umbilical cord mesenchymal stem cells in the NOTCH1 knockdown group and control group were observed by electron microscopy on days 7, 14, and 21 after induction, and glycogen production during hepatic differentiation was detected by PAS staining kit.

[0059] like Figure 16 As shown, there was no significant difference in cell morphology between the NOTCH1 knockdown group and the control group on day 7 of induction differentiation. However, on day 14, the cell morphology of the NOTCH1 knockdown group was closer to the cobblestone morphology of hepatocytes. This trend was more pronounced on day 21. Therefore, morphologically, the NOTCH1 knockdown group differentiated into hepatocytes earlier than the control group.

[0060] Example 7: Glycogen Staining Test The glycogen synthesis capacity of the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown cell line (MSCs-shRNA2-Puro-hNOTCH1-3) in Example 6 was assessed at 7, 14, and 21 days after differentiation using cyclic acid-Schiff (PAS) staining (Beyotime, China). The specific method is as follows: Step S1: Sample processing, HLCs fixed with anhydrous ethanol Step S2: PAS staining a. Oxidation with periodic acid solution: Remove the periodic acid solution and equilibrate to room temperature. Add 100 μl of periodic acid solution to each sample and react in a humidified chamber in the dark for 10 minutes. After that, remove the periodic acid solution, soak in distilled water, and wash on a shaker for 5 minutes.

[0061] b. Schiff reagent staining: Add 100 μl of Schiff reagent to each sample, place in a humidified chamber, and stain in a 37°C oven in the dark for 30 minutes to 1 hour. Remove the staining solution, soak in distilled water, and wash on a shaker for 5 minutes.

[0062] c. Hematoxylin staining: Add 100 μl of hematoxylin staining solution to each sample and stain for 30 seconds. Remove the staining solution and rinse with distilled water at least twice, 3 seconds each time, until the surface stain is removed. Then, take pictures under a microscope.

[0063] The results are as follows Figure 16 As shown, on day 7 of induced differentiation, neither the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown group nor the empty vector group showed the ability to generate glycogen. However, on day 14, glycogen was stained magenta in both groups, and the cell nuclei were stained blue by hematoxylin. However, a comparison of the glycogen staining area between the two groups revealed that the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown group had a superior glycogen generation capacity compared to the empty vector group, and this advantage became more pronounced on day 21.

[0064] Example 8: Western blot detection of liver function related indicators In Example 6, total protein was extracted from the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown cell line and the control group on days 7, 14, and 21 of induced differentiation. The expression of ALB and AFP was detected. The Western blot procedure has been described above, and the results are as follows: Figure 17 As shown in the figure. The results revealed that NOTCH1 knockdown was beneficial for the differentiation of human umbilical cord mesenchymal stem cells into hepatocytes.

[0065] On day 28 of induced differentiation, the expression of NOTCH1 protein and liver function-related indicators (ALB, AFP, and HNF-4α) in normal immortalized cells in the uninduced group, induced group, empty vector induced group, and NOTCH1 knockdown induced group were detected. The Western blot operation method has been described above.

[0066] Test results as follows Figure 18 As shown, after 28 days of induced differentiation, normal immortalized human umbilical cord mesenchymal stem cells (HLCs) exhibited decreased NOTCH1 protein expression and increased ALB, AFP, and HNF-4α expression. NOTCH1 knockdown further reduced NOTCH1 expression, leading to a more significant increase in ALB and HNF-4α expression. Notably, at day 28, AFP protein expression levels in the NOTCH1 knockdown group were lower than those in the empty vector group. This decrease is attributed to the gradual decline in AFP expression as HLCs mature.

[0067] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing an immortalized human umbilical cord mesenchymal stem cell line with NOTCH1 knockdown and stable transformation, characterized in that, Includes the following steps: S1. The target gene is ligated into a vector to construct a lentiviral plasmid containing the target gene; wherein the target gene is a DNA fragment from the human NOTCH1 gene, and the DNA fragment includes at least one of fragment 1, fragment 2, and fragment 3, wherein the sequence of fragment 1 is 5'-GCTCCCTCAACTTCAATGACC-3', the sequence of fragment 2 is 5'-CCGGGACATCACGGATCATAT-3', and the sequence of fragment 3 is 5'-GGACAAGATCGATGGCTACGA-3'; S2. The lentiviral plasmid was introduced into 293T cells, and high-titer lentiviruses containing the target gene were screened. S3. Infect immortalized human umbilical cord mesenchymal stem cells with the lentivirus to obtain a hybrid clone cell line; S4. The mixed clone cell line is screened to obtain an immortalized human umbilical cord mesenchymal stem cell line with NOTCH1 knockdown and stable transformation.

2. The construction method according to claim 1, characterized in that, In S1, the target gene was directionally inserted into the vector pLVX-shRNA2-Puro using the BamHI + EcoRI double digestion method.

3. The construction method according to claim 2, characterized in that, In S1, The amplification primers for fragment 1 are: hNotch1-1-F: 5-GATCCGCTCCCTCAACTTCAATGACCCTCGAGGGTCATTGAAGTTGAGGGAGCTTTTTG-3', hNotch1-1-R: 5'-AATTCAAAAAGCTCCCTCAACTTCAATGACCCTCGAGGGTCATTGAAGTTGAGGGAGCG-3'; The amplification primers for fragment 2 are: hNotch1-2-F: 5'-GATCCGCCGGGACATCACGGATCATATCTCGAGATATGATCCGTGATGTCCCGGTTTTTTG-3', hNotch1-2-R: 5'-AATTCAAAAAACCGGGACATCACGGATCATATCTCGAGATATGATCCGTGATGTCCCGGCG-3'; The amplification primers for fragment 3 are: hNotch1-3-F: 5'-GATCCGGACAAGATCGATGGCTACGACTCGAGTCGTAGCCATCGATCTTGTCCTTTTTG-3', hNotch1-3-R: 5'-AATTCAAAAAGGACAAGATCGATGGCTACGACTCGAGTCGTAGCCATCGATCTTGTCCG-3'.

4. The construction method according to claim 3, characterized in that, S1 includes the following specific steps: Dissolve the amplification primer fragments in 100 μl of annealing buffer, take 2 μl of each amplification primer and add them to 16 μL of annealing buffer, mix thoroughly, anneal at 100°C and cool naturally to room temperature, and dilute the annealed products 100 times with DEPC water. The vector plasmid pLVX-shRNA2-Puro was double-digested with BamHI and EcoRI, and the digestion reaction was carried out in a water bath at 37°C for 3 hours. The large fragment of plasmid pLVX-shRNA2-Puro BamHI+EcoRI digested by agarose gel electrophoresis was recovered. The large fragment of plasmid pLVX-shRNA2-Puro was recovered and ligated with the annealing product. The ligation reaction was carried out at 22°C for 3 hours. Take 10 μL of ligation product and mix with 100 μL of JM109 competent bacteria, incubate on ice for 30 min, heat shock at 42°C for 45 s, immediately place on ice for 2 min, add 400 μL of LB medium preheated to room temperature, incubate at 37°C in a shaker for 1 h, centrifuge at 4000 rpm for 1 min, discard 400 μL of culture supernatant, mix the remaining 100 μL with a pipette and spread evenly on LB plates containing 100 μg / ml Ampicillin resistance, and incubate in an inverted incubator at 37°C overnight. Single colonies were picked and inoculated into LB medium containing 5 ml of 100 μg / ml Ampicillin resistance. The culture was incubated overnight at 37°C and 250 rpm. The bacterial culture was then sent for sequencing verification to construct a lentiviral plasmid containing the target gene.

5. The construction method according to claim 1, characterized in that, S2 includes the following specific steps: The day before transfection, 293T cells were digested with trypsin, and the cell density was adjusted with serum-containing DMEM medium. The cells were then incubated at 37°C in a 5% CO2 incubator. After 16-24 hours, when the cell density reached 80%-90%, the cells were ready for transfection. 2-4 hours before transfection, the medium was changed with 5 ml of complete medium without P / S. Perform transfection as follows: Add 500 μL of CPT Buffer A to a clean 1.5 mL EP tube. In another clean 1.5 mL EP tube, add Vector reagent, Lentiviral packaging mixture, CPT Buffer B, and ddH2O to make a final volume of 500 μL. Mix the solutions in the two EP tubes and let the mixture stand at room temperature for 30 min. Then, add the mixture dropwise to the cell culture dish, mix gently, and incubate at 37°C in a 5% CO2 incubator. After culturing for 12-16 hours, discard the culture medium and replace it with 10 ml of fresh complete culture medium containing FBS and P / S. 24 hours after changing the medium, collect the supernatant and store it in a 4°C refrigerator. Then add 10 ml of fresh complete culture medium containing FBS and P / S to the cell culture dish and continue culturing in a 37°C, 5% CO2 incubator. Collect the supernatant again, mix it with the supernatant collected the first time, centrifuge at 1000 rpm for 5 min, discard cell debris, filter the supernatant through a 0.45 μm PVDF filter, centrifuge, and collect the virus pellet. The viral precipitate was resuspended, and high-titer lentiviruses containing the target gene were obtained by screening.

6. The construction method according to claim 1, characterized in that, S3 includes the following specific steps: Revive immortalized human umbilical cord mesenchymal stem cells, passage the cells when they grow to 80-90% cell density, and passage them 2-3 times consecutively. Culture immortalized human umbilical cord mesenchymal stem cells. When the cells reach 80-90% confluence density, digest the cells and count them, then divide them into groups of 5 × 10⁻⁶. 5 Cells are seeded into the well plate at a cell density per well. The lentiviruses were added at MOI=20 and incubated overnight at 37°C in a 5% CO2 incubator. After adding the lentivirus, the medium was replaced with human mesenchymal stem cell complete culture medium 24 hours later. After adding the lentivirus for 48 hours, Puromycin was added at a final concentration of 5ug / ml for resistance screening, and the medium was changed every 2-3 days. Digest the cells when they reach 80-90% confluence and seed them at T-25cm. 2 Continue culturing in cell culture flasks; After 2-3 consecutive passages, a stable hybrid clone cell line was obtained.

7. An immortalized human umbilical cord mesenchymal stem cell line with NOTCH1 knockdown and stable transformation, characterized in that... It is obtained by using the construction method described in any one of claims 1 to 6.

8. The application of the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown stable cell line according to claim 7 in liver differentiation.

9. The application according to claim 8, characterized in that, The application method includes: pretreating the immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell line, and then inducing and maturing the pretreated immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell line to obtain liver-like cells.

10. The application according to claim 9, characterized in that, The pretreatment method includes: culturing immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell lines in IMDM medium containing EGF and bFGF for 1-3 days; The induction culture method includes: culturing immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell lines in IMDM medium containing HGF, bFGF and nicotinamide for 6-8 days; The method for mature culture includes culturing immortalized human umbilical cord mesenchymal stem cell NOTCH1 knockdown transgenic cell lines in IMDM medium containing dexamethasone, ITS+premix and OSM.

Images