Immortalized Uterine Blood Stem Cell Line

Abstract

The present invention discloses an immortalized human uterine blood stem cell line and a preparation method and use thereof. The immortalized human uterine blood stem cell line provided by the present invention has the main functions of primary human uterine blood stem cells, and is obtained by transfecting SV40Tag and hTERT genes into isolated normal human uterine blood stem cells through lentiviral vectors, and screening using a cloning method. The immortalized human uterine blood stem cell line can be amplified in vitro on a large scale. The immortalized human uterine blood stem cell line described in the present invention provides a sufficient source of MenSCs for basic research and gene therapy, and has broad clinical application prospects in cell replacement, gene therapy and regenerative medicine.

Description

[0001] This application is a divisional application of the invention patent application "Immortalized Uterine Blood Stem Cell System" with application date of November 7, 2018 and application number 201811322705.X. Technical Field

[0002] The present invention relates to an immortalized human uterine blood stem cell, and in particular to a uterine blood stem cell line capable of stably expressing human telomerase catalytic subunit hTERT and simian virus 40 large T antigen SV40Tag. The present invention also relates to a method for establishing and using the stem cell line. The present invention also relates to the construction of a recombinant lentiviral vector containing human telomerase catalytic subunit gene hTERT and simian virus 40 large T antigen SV40Tag. Background Art

[0003] The application of stem cells in disease treatment is a new medical technology developed in recent years. Compared with traditional treatment methods, it has the following advantages: ① Stem cells can repair and replace damaged cells and tissues; ② Stem cells secrete a variety of nutritional factors that can effectively promote tissue growth.

[0004] Embryonic stem cells are an important type of stem cells. Many literatures have reported the use of embryonic stem cells for cell therapy. However, embryonic stem cells still have the potential for tumorigenesis and immune rejection, and it is currently difficult to overcome this problem.

[0005] Mesenchymal stem cell therapy has no rejection reaction, no medical ethical controversy, is safe and reliable, and has significant efficacy. Human uterine blood stem cells (MenSCs) can be used in regenerative medicine and as target cells for gene therapy, and have broad clinical application prospects in cell replacement and gene therapy. However, there are many problems to be solved in MenSCs, especially the limited proliferation ability of autologous MenSCs in transplantation applications. The morphology of primary mesenchymal stem cells gradually changes with the increase in the number of passages, the proliferation and differentiation ability decreases, and eventually they age and die. It is impossible to obtain the amount of cells required for clinical research and treatment.

[0006] The main ways to solve this problem should include two aspects: one is to enhance the proliferation activity of MenSCs, and the other is to use allogeneic or xenogeneic MenSCs. However, allogeneic transplantation has immunological problems, so the establishment of immortalized human uterine blood stem cells is a very meaningful work.

[0007] Telomeres are repeated TTAGGG sequences at the ends of chromosomes, which gradually shorten during normal cell division. When telomeres shorten to a certain limit in one or more chromosomes, cell aging and growth arrest occur. Telomere length is maintained by telomerase. Telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase. Experiments have shown that the introduction of exogenous hTERT gene into cells can increase telomerase activity, delay cell aging, maintain multidirectional differentiation potential and have no tumorigenic tendency. This shows that modifying MenSCs with hTERT gene transfection can achieve the purpose of extending the life cycle of MenSCs and maintaining multidirectional differentiation potential. Simonsen JL et al. transferred hTERT into human bone marrow mesenchymal stem cells hMSCs through retroviral vectors. The selected bone marrow hMSCs cell clones were cultured in vitro for multiple generations. The cells showed no signs of aging and maintained multidirectional differentiation potential.

[0008] SV40 Tag is a small DNA tumor virus in the genus Polyomavirus of the family Papovaviridae. SV40 can immortalize certain human cells, allowing cells to survive and be subcultured for 20-30 generations after the M1 phase, and then enter a crisis phase (M2 phase), with increased cell death rate and chromosomal abnormalities, a gradual decrease in the number of dividing cells, and the vast majority of cells dying. The early protein T antigen (SV40 Tag) of this virus has Rb and p53 binding domains, which causes Rb and p53 to lose their tumor suppressor protein functions and lose the regulation of the cell cycle, resulting in accelerated cell division and unlimited growth. Summary of the invention

[0009] The present invention has successfully screened hTERT and SV40Tag-positive uterine blood stem cell lines, which have reached 100 generations in vitro culture, far exceeding the in vitro proliferation capacity of uterine blood stem cells, proving that an immortalized human uterine blood stem cell line has been successfully obtained. The cell morphology and cell surface antigens of the screened cell line are consistent with the characteristics of human uterine blood stem cells. Karyotype analysis shows that the cells are still diploid cells, maintain differentiation potential, and have no tumorigenic tendency. The immortalized human uterine blood stem cell line established by the present invention solves the problems of difficult cell collection and limited cell division, provides a sufficient source of MenSCs for basic research and gene therapy, and has broad clinical application prospects in cell replacement and gene therapy.

[0010] Therefore, the present invention provides an immortalized human uterine blood stem cell line, characterized in that it can express hTERT and SV40Tag.

[0011] Preferably, the immortalized human uterine blood stem cell line of the present invention is an immortalized human uterine blood stem cell line obtained by transfecting SV40Tag and hTERT into isolated normal human uterine blood stem cells via lentiviral vectors and screening.

[0012] More specifically, the present invention provides an immortalized human uterine blood stem cell line, which was deposited at the General Microbiology Center of the China Microorganism Culture Collection Committee on April 8, 2018, with the deposit number CGMCC No.15584, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] The immortalized human uterine blood stem cell line of the present invention preferably has the following biological characteristics: (1) capable of inducing osteoblasts; (2) capable of expressing PD-L1 protein; (3) non-tumorigenic; and / or (4) the cell karyotype is diploid after multiple subcultures.

[0014] Another aspect of the present invention provides a method for preparing an immortalized human uterine blood stem cell line, wherein SV40T antigen and hTERT gene are transfected into isolated normal human uterine blood stem cells via a lentiviral vector, and the immortalized human uterine blood stem cell line is obtained by screening.

[0015] In the method for preparing the immortalized human uterine blood stem cell line of the present invention, the lentiviral vector is preferably a recombinant vector obtained by transfecting the lentivirus with pLVX-Puro carrying the hTERT-SV40Tag gene.

[0016] In the method for preparing the immortalized human uterine blood stem cell line of the present invention, the human normal uterine blood stem cells are preferably human adult uterine blood stem cells.

[0017] In the method for preparing the immortalized human uterine blood stem cell line of the present invention, it optionally further comprises the step of expanding and culturing the immortalized human uterine blood stem cell line in vitro after obtaining the immortalized human uterine blood stem cell line through screening.

[0018] In another aspect of the present invention, it provides use of the immortalized human uterine blood stem cell line of the present invention or the immortalized human uterine blood stem cell line prepared according to the method of the present invention in preparing a cell therapeutic agent.

[0019] In another aspect of the present invention, it provides use of the immortalized human uterine blood stem cell line of the present invention or the immortalized human uterine blood stem cell line prepared according to the method of the present invention in expressing exogenous proteins.

[0020] The present invention constructs a recombinant lentiviral vector pLVX-hTERT-SV40Tag, introduces hTERT and SV40Tag genes into uterine blood stem cells, and screens out a uterine blood stem cell line (hereinafter referred to as MenSC-hTERT-SV40Tag) that can stably express hTERT and SV40Tag. After cell culture and various levels of identification, it is proved that an immortalized uterine blood stem cell line has been obtained. The cell line maintains differentiation ability and has no tumorigenic tendency, and can be used as a gene vector for expressing various antigens and antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : pEGFP-N1 vector map.

[0022] Figure 2 : pLVX-Puro vector map.

[0023] Figure 3 : RT-QPCR gel electrophoresis results of hTERT and SV40Tag of MenSC-hTERT-SV40Tag cell line and MenSCs, wherein: lane 1 is SV40Tag; lane 2 is hTERT; and lane 3 is β-actin.

[0024] Figure 4 : Cell morphology of MenSCs-P2, MenSC-hTERT-SV40Tag-P50 and MenSC-hTERT-SV40Tag-P100 (from left to right).

[0025] Figure 5 :Surface antigen analysis of MenSC-hTERT-SV40Tag cell line by flow cytometry. Results of analysis of surface antigens CD11b, CD19, CD34, CD45, CD73, CD90, CD105, and HLA-DR on P50 and P100 MenSC-hTERT-SV40Tag cells.

[0026] Figure 6 : Osteogenic induction results of MenSCs-P2, MenSC-hTERT-SV40Tag-P50 and MenSC-hTERT-SV40Tag-P100 (from left to right).

[0027] Figure 7 : The results of nude mouse tumorigenicity experiments using MenSCs-P2, MenSC-hTERT-SV40Tag cells at the 50th and 100th passages.

[0028] Figure 8: Immunohistochemistry was used to detect the expression of PD-L1 in MenSC-hTERT-SV40Tag cell line. Among them: (A) negative control; (B) MenSC-hTERT-SV40Tag after infection with adenovirus carrying PD-L1 gene. DETAILED DESCRIPTION

[0029] In the present invention, based on the fact that mesenchymal stem cells cannot be immortalized in vitro, exogenous telomerase reverse transcriptase gene and SV40Tag gene are introduced to maintain telomerase activity and telomere length without inducing tumorigenesis, thereby obtaining in vitro immortalized stem cells, and verifying their proliferation and passage ability and tumorigenicity.

[0030] Lentiviral vectors have high infection efficiency, can infect both dividing and non-dividing cells, and can be expressed in cells for a long time with high safety. The present invention constructs a recombinant lentiviral vector pLVX-hTERT-SV40Tag, introduces hTERT and SV40Tag genes into uterine blood stem cells, integrates them into the uterine blood stem cell genome, and enables uterine blood stem cells to continuously express hTERT and SV40Tag.

[0031] The inventors of the present invention have specifically conducted the following research:

[0032] 1. A recombinant lentiviral vector containing hTERT and SV40Tag was constructed and co-transfected with the viral backbone plasmid into 293T cells. The recombinant lentivirus containing hTERT and SV40Tag was successfully packaged, and the titer of the recombinant lentivirus was determined.

[0033] 2. Screening out positive hTERT and SV40Tag cell lines. The second generation of human uterine blood stem cells were infected with recombinant lentivirus containing hTERT and SV40Tag. Under the selection of puromycin, the MenSC-hTERT-SV40Tag cell line was successfully screened out and continued to be cultured. The MenSC-hTERT-SV40Tag cell line has been successfully cultured to the 100th generation.

[0034] 3. The biological activity and biological function of the MenSC-hTERT-SV40Tag cell line obtained above were demonstrated by various methods:

[0035] 1) Real-time quantitative PCR and conventional PCR techniques detected a significant increase in the expression levels of hTERT and SV40Tag genes at the DNA and RNA levels;

[0036] 2) Using the Trapeze telomerase detection kit, the MenSC-hTERT-SV40Tag cell line was identified to have telomerase activity;

[0037] 3) Morphological comparison of MenSCs-P2, MenSC-hTERT-SV40Tag-P50 and MenSC-hTERT-SV40Tag-P100;

[0038] 4) Flow cytometry analysis showed that the surface markers of this cell line were consistent with the characteristics of MenSCs;

[0039] 5) The MenSC-hTERT-SV40Tag cell line was induced with osteogenic induction reagents, and the calcium crystal level increased, proving that osteoblasts can be induced, indicating that the MenSC-hTERT-SV40Tag cell line still has multidirectional differentiation ability;

[0040] 6) Karyotype analysis showed that the MenSC-hTERT-SV40Tag cell line had normal diploid characteristics;

[0041] 7) The safety of the cell line was confirmed. The mouse tumorigenicity experiment showed that the established MenSC-hTERT-SV40Tag cell line did not cause tumors in mice, confirming that the cell line was not tumorigenic and was relatively safe.

[0042] 8) PD-L1 was successfully expressed in the MenSC-hTERT-SV40Tag immortalized cell line.

[0043] Experiments have shown that the immortalized uterine blood stem cells of the present invention (1) can induce osteoblasts; (2) can express PD-L1 protein; (3) are non-tumorigenic; and / or (4) have a diploid karyotype after multiple subcultures. Therefore, the immortalized human uterine blood stem cells screened by the present invention can solve the problem of limited stem cell division and subculture, and provide a sufficient cell source for basic research and gene therapy.

[0044] The terms and expressions that have been adopted herein are used as descriptive rather than limiting terms, and in the use of such terms and expressions, it is not intended to exclude any equivalents of the features shown and described or parts thereof, but it should be recognized that various modifications are possible within the scope of the invention claimed. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0045] In order to more clearly illustrate the present invention, it is now described in detail in conjunction with the following examples, but these examples are merely exemplary descriptions of the present invention and are not to be construed as limiting the present application.

[0046] The present invention is further described in conjunction with the accompanying drawings and embodiments.

[0047] Example 1 Establishment and identification of immortalized human uterine blood stem cell line

[0048] 1. Cell Isolation and Culture

[0049] Uterine blood stem cells were isolated from menstrual blood by density gradient centrifugation and routinely cultured in DMEM / F-12+10% FBS. When the cells grew to 80%-90%, they were passaged at a ratio of 1:3.

[0050] 2. Construction and identification of recombinant lentiviral vector hTERT-SV40Tag

[0051] Using pcl-neo-hTERT plasmid and SV40Tag plasmid as templates, PCR was used to amplify the complete gene fragments of hTERT (containing Xho I and EcoR I restriction sites) and SV40Tag (containing Sal I and BamH I restriction sites). After purification, double digestion with Xho I and EcoR I and double digestion with Sal I and BamH I were performed to purify and recover the hTERT and SV40Tag target fragments. Figure 1 ) is used as the backbone vector. After double digestion with Xho I and BamH I, it is connected with the purified and recovered hTERT and SV40Tag target fragments by enzyme digestion and ligation. After ligation with T4 ligase, the competent Escherichia coli is transformed, and the high-purity pEGFP-hTERT-SV40Tag plasmid is extracted. Double digestion with Xho I and BamH I is performed for identification. The digestion product is about 6300bp of hTERT-SV40Tag fragment. The purified hTERT-SV40Tag fragment is recovered from the digestion product of the pEGFP-hTERT-SV40Tag plasmid using a gel recovery kit. pLVX-Puro vector ( Figure 2 ) Xho I and BamH I were double-digested at the same time, and the linearized pLVX-Puro vector was purified and recovered. The purified hTERT-SV40Tag fragment was connected to the linearized pLVX-Puro vector with 16°C T4 ligase, and the ligation product was transformed into DH5α Escherichia coli and inoculated into ampicillin-resistant LB medium and cultured at 37°C overnight. The bacteria were cultured overnight, the plasmid was extracted, and sequencing was performed for identification. After correct identification, the pLVX-hTERT-SV40Tag recombinant lentiviral vector was obtained, and the recombinant vector was transiently transfected into HEK293 cells. The plasmid was identified by immunofluorescence to determine whether it could express the telomerase catalytic subunit hTERT and SV40Tag.

[0052] 3. Purification of recombinant lentivirus

[0053] The high-purity pLVX-hTERT-SV40Tag recombinant plasmid was extracted using a plasmid extraction kit and the concentration was determined. One day before the experiment, 293T cells were cultured at 4 × 10 6 The amount was inoculated into a culture dish with a diameter of 10 cm, and the cells were cultured in high-glucose DMEM culture medium containing 10% FBS. On the day of the experiment, when the cell density grew to 85%-90%, 7μg of the extracted high-quality plvx-hTERT-SV40Tag plasmid was mixed with 36μl of lentiviral packaging plasmid mixture and 75μl of FuGene HD transfection reagent, incubated for 30 minutes and co-transfected into 293T cells. The cell supernatant was harvested 72 hours after transfection, centrifuged at 300g, and filtered with a 0.45μm filter. The recombinant virus titer obtained was detected using the Clontech P24 quantitative detection kit. Conclusion: The recombinant lentivirus containing hTERT and SV40Tag genes was successfully packaged, and the virus titer can reach 1×10 7 .

[0054] 4. Infection of MenSCs with Recombinant Lentivirus

[0055] DMEM / F-12 culture medium containing 10% FBS was used for culture. One day before the experiment, the P1 generation MenSC in the logarithmic growth phase was inoculated into a six-well plate. When the cell confluence was 70-90%, the obtained recombinant lentivirus was used to infect the cells at an MOI of 20:1. 8μg / mL polybrene was added during infection. The cells were replaced with fresh antibiotic-free serum-free medium containing puromycin 24-48h after infection for screening. Fresh antibiotic-free serum-free medium containing puromycin was used every other day to replace the medium containing a large number of dead cells. Until the resistant colony can be identified, that is, when all the control cells die, the MenSC-hTERT-SV40Tag cell line is obtained, and the cells screened are continued to be subcultured. The cell was deposited in the General Microbiology Center of China Microorganism Culture Collection on April 8, 2018, with the deposit number CGMCC No.15584. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0056] Example 2 Bioactivity and biological function verification of MenSC-hTERT-SV40Tag cell line

[0057] The following tests were performed on the P2 MenSCs that were not infected with the lentivirus, and the 50th and 100th MenSC-hTERT-SV40Tag cells after infection obtained according to Example 1:

[0058] 1. RT-QPCR method was used to identify the MenSC-hTERT-SV40Tag cell line.

[0059] Total cellular RNA was extracted, and RT-QPCR (primer sequences are shown in Table 1 ) was performed to identify the differences in hTERT and SV40Tag expression levels in the MenSC-hTERT-SV40Tag cell line and MenSCs.

[0060] The results of RT-QPCR gel electrophoresis showed that β-actin of about 396 bp could be amplified in both MenSCs and MenSC-hTERT-SV40Tag. In the MenSC-hTERT-SV40Tag cell line, a 422 bp SV40Tag gene fragment and a 244 bp hTERT gene fragment could be amplified (e.g. Figure 3 ), while the corresponding fragment could not be amplified in MenSCs without hTERT gene and SV40Tag gene, indicating that the exogenous hTERT gene and SV40Tag gene in MenSC-hTERT-SV40Tag have been successfully integrated into the cell genome.

[0061] RT-QPCR results showed that the detection Ct value of hTERT in MenSC-hTERT-SV40Tag was significantly lower than that of conventional MenSC cells, indicating that the expression level of hTERT gene at the mRNA level in MenSC-hTERT-SV40Tag cell line was much greater than that in MenSC without recombinant lentivirus infection.

[0062] Table 1 RT-QPCR primer sequences

[0063]

[0064]

[0065] 2. Telomerase activity detection in MenSC-hTERT-SV40Tag cells

[0066] According to the instructions of Roche's TeloTAGGG Telomerase PCR ELISA Telomerase Activity Assay Kit, the telomerase activity of P2 MenSCs, P50 and P100 MenSC-hTERT-SV40Tag cells was detected, and P2 MenSCs were used as the control. 2×10 cells of the above cells in good condition were collected. 5Cells were washed twice with cold PBS, lysed on ice for 30 min, centrifuged at 16000×g for 20 min, 3 μl of supernatant was added to TRAP reaction solution and supplemented with nuclease-free water to 50 μl for PCR amplification. The reaction conditions were: 25°C, 30 min; 94°C, 5 min; 94°C, 30 s; 50°C, 30 s; 72°C, 90 s, 30 cycles. Take 2.5μl of amplified product, add 10μl of denaturing solution, let it stand at room temperature for 10 minutes, add 100μl of hybridization solution, add the mixture to the streptavidin-coated microplate, incubate at 37℃ for 2h at 300r / min, discard the supernatant, wash the plate 3 times, add 100μl of digoxigenin peroxidase to each well, incubate at room temperature for 30min at 300r / min, discard the supernatant, wash the plate 5 times, add 100μl of TMB substrate solution, add stop solution after 15-30min, detect the absorbance at 450nm with an enzyme reader, and the reference wavelength is 690nm. Repeat 3 times for each sample.

[0067] Results: It was shown that telomerase could be expressed in MenSC-hTERT-SV40Tag cell line and the enzyme had telomerase activity.

[0068] Table 2 Telomerase activity test results of each cell strain

[0069]

[0070] 3. MenSC-hTERT-SV40Tag Cell Culture

[0071] The cell line was cultured in DMEM-F12 medium containing 10% FBS until the 100th generation, and the cell morphology did not change (e.g. Figure 4 ), which has far exceeded the number of cell passages of ordinary human uterine blood stem cells, indicating that we have obtained an immortalized human uterine blood stem cell line.

[0072] 4. Identification of Cell Surface Markers

[0073] The surface antigens of MenSC-hTERT-SV40Tag cell line were analyzed by flow cytometry. The surface antigens CD11 b, CD19, CD34, CD45, CD73, CD90, CD105, and HLA-DR of MenSC-hTERT-SV40Tag cells of P50 and P100 were analyzed respectively. The results are shown in Figure 5 and as shown in Table 3. The results showed that the established cell lines still had the cell phenotype of MenSCs.

[0074] Table 3 Detection results of cell surface markers of various strains (%)

[0075]

[0076] 5. Karyotype analysis According to conventional chromosome preparation techniques, chromosome analysis was performed on cells of the 50th and 100th passages of the MenSC-hTERT-SV40Tag cell line. The results showed that the cell karyotype was diploid.

[0077] 6. Osteogenic Induction Identification

[0078] After inducing MenSCs-P2, MenSC-hTERT-SV40Tag-P50, and MenSC-hTERT-SV40Tag-P100 with osteogenic induction medium for 4 weeks, the cells were stained with Alizarin red to determine whether the cells could be induced into osteoblasts.

[0079] The induction process was carried out according to the instructions of the Cyagen Adult Bone Marrow Mesenchymal Stem Cell Osteogenesis Induction Kit.

[0080] The results are as follows Figure 6 As shown. After induction, the cells are stained red or dark red, indicating that a large number of calcium nodules are produced, which is an important feature of osteoblasts. Figure 6 The results show that MenSC-hTERT-SV40Tag-P50 and MenSC-hTERT-SV40Tag-P100 still have strong osteogenic differentiation ability.

[0081] 7. Nude mouse tumorigenesis experiment

[0082] In order to test whether the established MenSC-hTERT-SV40Tag cell line has tumorigenic tendency and the safety of cell application, a nude mouse tumorigenic experiment was conducted. Whether a malignant tumor can be formed after being inoculated into nude mice is an important indicator for testing the malignancy of cancer cells.

[0083] Experimental method: MenSCs-P2, MenSC-hTERT-SV40Tag cells at passage 50 and 100 were digested to prepare single cell suspensions and the cell concentration was adjusted to 2×10 7 / ml. Male 5-week-old nude mice were selected. Each group of mice was subcutaneously injected with 0.25 ml of cell suspension in the forelimb. The positive control was Hela cells, with an inoculation volume of 1×10 6 The negative control was phosphate buffered saline (PBS). The growth of nude mice and tumors were observed regularly.

[0084] The results are as follows Figure 7As shown: 25 days after nude mice were inoculated with various cells, tumors began to appear on the back of the neck of mice in the Hela cell group, while no tumors appeared in the MenSCs group, MenSC-hTERT-SV40Tag cell group and PBS group. After two months of observation, the tumors in the tumor cell group continued to grow and the weight decreased, while the MenSC-hTERT-SV40Tag cell group and PBS group still showed no signs of tumors. The photos were taken after the mice were anesthetized with sodium phenobarbital 45 days after the cells were inoculated. Two months after the MenSC-hTERT-SV40Tag cell line was inoculated, the nude mice still had no tendency to develop tumors, indicating that MenSC-hTERT-SV40Tag has no tendency to cause tumors and is safe.

[0085] Example 3 Expression of PD-L1 virus in MenSC-hTERT-SV40Tag Adenovirus carrying the PD-L1 gene was purchased from Hanheng Biotechnology.

[0086] Experimental process: MenSC-hTERT-SV40Tag cell line was cultured in DMEM / F-12 medium containing 10% FBS. One day before the experiment, cells were inoculated into six-well plates (with glass slides in the wells). The next day, when the cells reached 70-90%, adenovirus carrying the PD-L1 gene was infected with cells at an MOI of 10:1. The medium was changed after 4 hours. On the third day after infection, the cell slides in the six-well plate were taken out, and the expression of PD-L1 protein in the MenSC-hTERT-SV40Tag cell line was detected by immunohistochemistry.

[0087] The experimental results are as follows Figure 8 As shown, after DAB staining, positive expression is brown or brown. The results show that PD-L1 protein can be effectively expressed in the MenSC-hTERT-SV40Tag cell line.

[0088] Although the present invention is described using the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and these modifications and variations are all within the scope of protection of the present invention.

Claims

1. An immortalized human uterine blood stem cell line, characterized in that It can express hTERT and SV40Tag.

2. The immortalized human uterine blood stem cell line according to claim 1 is obtained by transfecting SV40Tag and hTERT into isolated normal human uterine blood stem cells via lentiviral vectors and screening.

3. An immortalized human uterine blood stem cell line, which was deposited at the General Microbiology Center of China Microorganism Culture Collection Administration on April 8, 2018, with the deposit number CGMCC No.15584.

4. The immortalized human uterine blood stem cell line according to any one of claims 1-3, wherein the biological characteristics of the immortalized human uterine blood stem cell line are: (1) capable of inducing osteoblasts; (2) capable of expressing PD-L1 protein; (3) non-tumorigenic; and / or (4) the cell karyotype is diploid after multiple subcultures.

5. The method for preparing the immortalized human uterine blood stem cell line according to any one of claims 1 to 4, wherein the SV40T antigen and the hTERT gene are transfected into the isolated normal human uterine blood stem cells by a lentiviral vector, and the immortalized human uterine blood stem cell line is obtained by screening.

6. The method for preparing an immortalized human uterine blood stem cell line according to claim 5, wherein the lentiviral vector is a recombinant vector obtained by transfecting lentivirus with pLVX-Puro carrying the hTERT-SV40Tag gene.

7. The method for preparing an immortalized human uterine blood stem cell line according to claim 5 or 6, wherein the human normal uterine blood stem cells are human adult uterine blood stem cells.

8. The method for preparing the immortalized human uterine blood stem cell line according to claim 5 or 6, further comprising expanding and culturing the immortalized human uterine blood stem cell line in vitro after obtaining the immortalized human uterine blood stem cell line through screening.

9. Use of the immortalized human uterine blood stem cell line according to any one of claims 1 to 4 or the immortalized human uterine blood stem cell line prepared by any one of claims 5 to 8 in the preparation of a cell therapeutic agent.

10. Use of the immortalized human uterine blood stem cell line according to any one of claims 1 to 4 or the immortalized human uterine blood stem cell line prepared by any one of claims 5 to 8 in expressing foreign proteins.

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