Mouse immortalized jawbone mesenchymal stromal cell line WZL-2 and construction method thereof

Abstract

The application belongs to the technical field of biotechnology, and specifically discloses a mouse jawbone mesenchymal stromal cell immortalized cell line WZL-2 and a construction method thereof. The application first proposes and proves that Krt14 can be used as a specific gene marker of mouse jawbone-derived mesenchymal stromal cells (MSCs), for accurate identification, marking and separation of the cell population; and on this basis, a cascading process flow of "Krt14 sorting + genetic engineering immortalization transformation + monoclonal screening" is adopted to obtain an immortalized cell line. The cell line has unlimited proliferation capacity, maintains Krt14 positive expression, and retains the clonogenicity of mesenchymal stromal cells, in-vitro three-dimensional differentiation potential and in-vivo tissue formation capacity. Thus, the application solves the problems of lack of standard markers, strong cell heterogeneity, easy aging in vitro, unstable supply and the like of the existing mouse jawbone MSCs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and involves the interdisciplinary application of cell biology, genetic engineering and regenerative medicine research. Specifically, it relates to an immortalized mouse jawbone mesenchymal matrix cell line WZL-2 and its construction method. Background Technology

[0002] In cutting-edge scientific research on jawbone regeneration and bone metabolism, mouse jawbone-derived mesenchymal stromal cells (MSCs) are indispensable key experimental subjects due to their self-renewal capacity and multi-lineage differentiation potential. Currently, researchers generally obtain these cells by directly extracting and culturing a limited number of primary mesenchymal stromal cells from jawbone tissues of multiple mice from different batches and individuals using traditional techniques such as enzymatic digestion and tissue block adhesion methods for subsequent experimental research.

[0003] These primary cells are of significant research value for understanding the development, repair, and pathological mechanisms of maxillofacial bones and related diseases. However, this technical approach, which relies on the direct extraction of primary cells, has revealed a series of insurmountable bottlenecks in practical applications, severely restricting the efficiency, reliability, and standardization of related research. The following is a systematic analysis of these shortcomings and their underlying causes:

[0004] The process is cumbersome and complex: the extraction of primary cells involves multiple steps such as meticulous dissection, tissue processing, enzyme digestion and cell culture. In addition, the use of primary cells is limited due to aging, and repeated preparation is often required, which is time-consuming and labor-intensive, and requires high technical skills from the experimenters.

[0005] In vitro senescence limitations: When primary cells are passaged and expanded in vitro, the number of divisions is limited by the inherent biological characteristics of the cells. Cells inevitably enter a state of replicative senescence, a phenomenon known as the "Hayflick limit." This results in a very limited number of uses for cells extracted in a single batch, making it impossible to support long-term or large-scale experimental needs.

[0006] Lack of identification criteria and high population heterogeneity: Due to the lack of standardized biomarkers, mainstream methods such as enzymatic digestion and tissue block adhesion lack specificity when isolating cells, failing to accurately distinguish cells from different anatomical sites such as the periosteum and bone marrow. Furthermore, the lack of standardized biomarkers results in a mixed population of cells with diverse phenotypes and extremely low purity, severely impacting the accuracy and interpretability of experimental results.

[0007] Poor stability and reproducibility: Because cells originate from individuals with different genetic backgrounds and physiological states, their biological characteristics exhibit significant individual differences. Coupled with the heterogeneity of cell populations, it is difficult to compare and replicate experimental results from different batches and sources of cells, greatly hindering the standardization of research and the translation of findings.

[0008] Ambiguity in attribute confirmation: In the absence of marker guidance, researchers find it difficult to confirm whether the obtained population is a true mesenchymal stromal cell in the early stages of cell sorting, and often have to spend a lot of time culturing before retrospective identification can be carried out. Summary of the Invention

[0009] Addressing the core technical challenges of existing technologies for mouse jawbone MSCs, such as the lack of standardized biomarkers, high cellular heterogeneity, easy in vitro aging, and unstable supply, this invention aims to:

[0010] 1. Provide standardized biomarker solutions: For the first time, Krt14 is proposed and used as one of the specific biomarkers for mouse jawbone-derived mesenchymal stromal cells, thereby enabling precise identification, sorting and purification of this cell population.

[0011] 2. Fully verifying mesenchymal properties: This invention not only uses Krt14 for sorting, but also fully verifies that Krt14 positive expression cells have typical mesenchymal matrix cell properties from two dimensions: cellularity and in vivo biological function through systematic clonogenic experiments, in vitro three-way differentiation experiments (osteogenesis, adipogenesis, and chondrogenesis) and in vivo renal capsule experiments.

[0012] 3. Addressing Cellular Heterogeneity: This method provides a way to specifically label, sort, and purify mouse jawbone-derived mesenchymal matrix cells using a single, well-defined genetic marker (Krt14). This aims to eliminate confounding cell populations, significantly improve cell homogeneity and purity, and thus ensure the reliability of experimental results.

[0013] 4. Overcoming Cellular Senescence and Supply Limitations: This invention provides a method for preparing a stable, Krt14-positive mouse jawbone mesenchymal matrix cell line that can be passaged indefinitely through genetic engineering (immortification technology). This aims to completely eliminate the fundamental technical bottleneck of limited primary cell usage and the need for repeated sampling, providing scientific research with an inexhaustible, standardized, and high-quality cell source.

[0014] 5. Construct an immortalization tool platform: Based on the identification of the Krt14 marker, prepare an immortalized cell line that can be passaged indefinitely and has stable properties to solve the problem of unstable supply.

[0015] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0016] In a first aspect, the present invention provides the application of Krt14 as a specific genetic marker in the identification and isolation of mesenchymal stromal cell populations derived from mouse jawbone.

[0017] This invention is the first to propose and demonstrate that Krt14 can serve as a specific gene marker for mouse jawbone-derived mesenchymal stromal cells (MSCs) for the precise identification, labeling, and isolation of this cell population.

[0018] Secondly, the present invention provides a mouse jawbone mesenchymal matrix cell immortalized cell line WZL-2. This cell line is a mouse jawbone-derived Krt14-positive expressed mesenchymal matrix cell immortalized cell line, deposited at the China Center for Type Culture Collection, classified and named as mouse jawbone mesenchymal matrix cell immortalized cell line WZL-2 Mus musculus, with accession number CCTCC NO:C202647 and deposit date of April 14, 2026.

[0019] This invention provides a genetically engineered mouse jawbone-derived mesenchymal stem cell line that highly expresses Krt14 and possesses unlimited proliferative capacity. Even after immortalization, this cell line retains the core biological properties of MSCs, namely clonogenic ability, in vitro tri-lineage differentiation potential (osteogenic, adipogenic, and chondrogenic), and in vivo tissue-forming ability (such as osteogenic ability confirmed by kidney capsule experiments). This cell line maintains high homogeneity during long passages, overcoming the technical bottlenecks of strong heterogeneity and susceptibility to aging in primary cells. Furthermore, this cell line does not exhibit tumorigenicity in vivo and demonstrates good biocompatibility.

[0020] Thirdly, this invention provides a method for constructing the aforementioned mouse jawbone mesenchymal matrix cell immortalized cell line WZL-2, employing a cascade process of "specific gene marker (Krt14) sorting + genetic engineering immortalization transformation + monoclonal screening" to establish a standardized cell line with genetic consistency. The construction method includes the following steps:

[0021] S1. Specific isolation and culture of high-purity primary cells

[0022] Cell source: Maxillary and mandibular bone tissues from Krt14Cre R26LSL-tdTomato transgenic mice were used as the starting material for cells. The key to selecting this specific mouse model is that all cells expressing the Krt14 gene and their progeny cells stably express tdTomato red fluorescent protein, thus providing an ideal endogenous marker for subsequent specific sorting.

[0023] Separation method: A combined technique of enzymatic digestion and tissue block method was used to process the jawbone tissue to efficiently release primary mesenchymal cells from the tissue.

[0024] Purification method: Flow cytometry sorting (FACS) was used to specifically sort out cell populations expressing tdTomato fluorescent protein. This is a key step to ensure that the final cell line originates from Krt14 positive cells and guarantees its high homogeneity.

[0025] S2. Construction and screening of immortalized cell lines (immortality and cloning)

[0026] Immortalization gene delivery: The SV40 large T (SV40 LT) antigen gene is introduced into purified Krt14-positive primary cells via a highly efficient and safe lentiviral transduction system. The SV40 LT antigen can bypass the Hayflick limit of cells by inhibiting key regulatory proteins in the cell cycle (such as p53 and Rb), thereby endowing cells with the ability to proliferate indefinitely.

[0027] Positive clone screening: Using the resistance gene (such as puromycin resistance gene) carried on the lentiviral vector, puromycin is added to the culture medium for drug screening, thereby eliminating cells that have not successfully integrated the SV40 LT gene and obtaining a cell population that stably expresses immortalization genes.

[0028] Monoclonalization: The selected cells are cultured and expanded using a limiting dilution method. Cells are diluted to extremely low concentrations and seeded into multi-well plates, ensuring that only clones originating from a single cell grow in each well. Once the single cells have proliferated to form clearly visible clones, they are transferred to larger culture containers for expansion. This step is the ultimate guarantee that the final cell lines have a completely consistent genetic background.

[0029] S3. Identification of mesenchymal properties in immortalized cell lines

[0030] To verify that the constructed immortalized cell line retains its key biological characteristics as mesenchymal stromal cells while achieving unlimited proliferation, this invention includes a series of systematic functional identification experiments:

[0031] Clonalization assay: used to verify the self-renewal and proliferation capacity of a single cell, and is one of the basic characteristics of stem / progenitor cells.

[0032] In vitro tri-directional differentiation experiment: By culturing cells in a specific induction medium, the potential of cells to differentiate into osteoblasts, adipocytes, and chondrocytes is assessed. This is one of the standards for identifying mesenchymal stromal cells.

[0033] In vivo renal capsule assay: Transplanting cells under the renal capsule of immunodeficient mice to assess their tissue-forming ability in the in vivo microenvironment is a key experiment to verify their in vivo function.

[0034] Furthermore, in step S1, it is preferred to use Krt14Cre R26LSL-tdTomato transgenic mice for sorting by endogenous fluorescence, but the scope of protection of the present invention also covers: the method of using Krt14 specific antibodies to immunofluorescence label primary jawbone cells of ordinary mice or related animal models, and then obtaining Krt14 positive cell populations by flow cytometry (FACS) or magnetic bead sorting (MACS).

[0035] Furthermore, in step S2, the SV40 LT antigen gene used in the immortalization gene introduction process can be replaced by other genes with similar cell cycle regulation functions, including but not limited to hTERT (human telomerase reverse transcriptase), human papillomavirus E6 / E7 gene, Bmi-1 gene, or a combination of the above genes.

[0036] Furthermore, in step S2, the immortalization gene introduction process preferably uses a lentiviral transduction system, but alternatives include using adenovirus, retrovirus, electroporation, liposome transfection, and using CRISPR / Cas9 technology to integrate the immortalization gene into a specific site in the cell genome (such as the AAVS1 site).

[0037] Furthermore, in step S2, the puromycin resistance screening used in the positive clone screening process can be replaced by antibiotic screening schemes such as neomycin (G418), blasticidin, and hygromycin; or resistance screening can be completely omitted, and cells that stably express immortalized vector markers can be directly sorted again by flow cytometry (FACS).

[0038] Fourthly, this invention provides the application of the aforementioned mouse jawbone mesenchymal matrix cell immortalized cell line WZL-2 as a standardized biomaterial in jawbone regeneration, bone metabolism research, and related drug screening.

[0039] The core innovation of this invention lies in the first-ever use of Krt14 as a specific genetic marker for identifying and isolating mesenchymal stromal cell populations derived from mouse jawbones. Furthermore, based on this, the invention successfully immortalized this high-purity cell population for the first time, ultimately obtaining a novel cell line tool. More importantly, through systematic functional identification, this invention has confirmed that the immortalized cell line fully retains the core biological properties of mesenchymal stromal cells, demonstrating the success of this technical solution.

[0040] Compared with the prior art, the technical solution provided by the present invention brings the following significant beneficial effects:

[0041] 1. High Homogeneity and High Purity: Through specific labeling of the Krt14 gene and flow cytometry sorting technology, the cell line obtained by this invention fundamentally solves the problem of mixed cell populations in existing technologies, exhibiting unprecedented high homogeneity. This greatly improves the reliability, accuracy, and comparability of experimental results based on this cell line.

[0042] 2. Unlimited Proliferation Capacity and Stable Supply: The successful application of immortalization technology has enabled this cell line to break through the Hayflick limit and achieve unlimited passage expansion. This provides the scientific community with a stable and continuously available cell resource, completely solving the ethical and technical challenges of limited use of primary cells due to aging and the need for repeated animal injury for material harvesting.

[0043] 3. Simplified experimental procedures and reduced costs: Researchers can directly obtain the cell line from reliable sources for culture and experiments, eliminating the need to invest significant time and effort in the tedious, complex, and costly process of primary cell isolation and extraction. This significantly improves research efficiency and lowers the experimental threshold and overall cost.

[0044] 4. Promoting Standardization and Large-Scale Application: The establishment of this stable, homogeneous cell line lays a solid foundation of cellular materials for large-scale drug screening, high-throughput functional genomics research, and the development of regenerative medicine applications in related fields. It is a crucial step in moving related research fields from a workshop-style approach to standardization and industrialization. Attached Figure Description

[0045] Figure 1 : Flowchart of the technical route for constructing immortalized cell lines in this invention.

[0046] Figure 2 Schematic diagram of flow cytometry results for sorting Krt14-positive (tdTomato-positive) cells. The diagram shows the sorted tdTomato cells. + The cells are Krt14-positive mesenchymal matrix cells extracted and isolated from mouse jawbones and sorted by flow cytometry.

[0047] Figure 3 Image of SV40 LT lentivirus vector.

[0048] Figure 4Microscopic images of primary mesenchymal stromal cells transfected with SV40LT-GFP lentivirus and expressing Krt14. In the image, A shows the fluorescence microscopy of polyclonal Krt14-positive primary mesenchymal stromal cells transfected with SV40LT lentivirus, including images under the red fluorescence channel (left) and the green fluorescence channel (right). + ZsGreen represents Krt14 positive expression. + A represents SV40LT positive expression (i.e., successful transfection); B is a fluorescence micrograph of primary mesenchymal stromal cells monoclonal with Krt14 positive expression selected from polyclonal Krt14-transfected SV40LT lentivirus using the "limiting dilution method".

[0049] Figure 5 Simultaneously, microscopic images of aging phenotypes were obtained from primary cells of generation P10 and immortalized cell lines of generation P10.

[0050] Figure 6 Microscopic images of immortalized cell lines of different generations (P10 and P50). In the image, A is a low-power microscopic view of the P10 and P50 immortalized cell lines; B is a high-power microscopic view of the P50 immortalized cell line at 100× (left) and 200× (right).

[0051] Figure 7 Figure 1 shows the results of in vitro detection of the proliferation and colony-forming ability of the immortalized cell line (SV40 LT), with primary cells as the control. In the figure, A represents the cell line proliferation ability detected by the CCK8 assay; B represents the cell line colony-forming ability detected by the CFU assay.

[0052] Figure 8 Figure 1 shows the results of in vitro three-dimensional (osteogenic, adipogenic, and chondrogenic) differentiation induction and specific staining identification. In the figure, A shows the results of alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining; B shows the results of Oil Red O staining; and C shows the results of Toluidine Blue staining.

[0053] Figure 9 Micro-CT results of in vivo subrenal transplantation experiment of immortalized cell lines.

[0054] Figure 10 Results of tumorigenesis experiments on immortalized cell lines. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents used in the following embodiments are all commercially available conventional reagents, and the experimental procedures involved are all conventional procedures in the art unless otherwise specified.

[0056] Example 1

[0057] This embodiment provides a process for constructing and identifying an immortalized mesenchymal stromal cell line derived from mouse jawbone and expressing Krt14 positively. The procedure is as follows: Figure 1 As shown, the specific steps are as follows:

[0058] 1. Isolation and purification of Krt14-positive primary mesenchymal stromal cells

[0059] Krt14Cre R26LSL-tdTomato transgenic mice (obtained from B6N.Cg-Tg(KRT14-cre)1Amc / J (number: 018964, from The Jackson Laboratory) and B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze / J (number: 007909, obtained from The Jackson Laboratory) was obtained by aseptic separation of the maxilla and mandible tissues. After removing soft tissue, the mandible was cut into tissue blocks. Enzymatic digestion (containing collagenase and trypsin) was performed at 37°C. Subsequently, the digested tissue blocks were seeded in complete culture medium (a-MEM medium + 1% penicillin-streptomycin + 10% fetal bovine serum) and cultured under standard cell culture conditions (37°C, 5% CO2) for adherence. When cells had migrated from around the tissue blocks and reached 80% confluence, they were digested with trypsin and collected. The collected cells were resuspended in flow cytometry loading buffer and sorted using a flow cytometer, using tdTomato fluorescent protein expression as the gating criterion, to identify tdTomato-positive cell populations ( Figure 2 medium td + The cells are high-purity Krt14-positive primary mesenchymal stromal cells.

[0060] 2. Lentiviral-mediated cell immortalization

[0061] 2.1 Preparation of Lentiviral Concentrate

[0062] 2.1.1 Construction of SV40 LT Lentiviral Vector

[0063] Based on the target gene (SV40 LT) sequence information, Hanheng Biotechnology was commissioned to construct the SV40 LT lentiviral vector using molecular cloning experiments.

[0064] Target gene acquisition: PCR amplification to obtain the target gene fragment, followed by electrophoresis gel recovery and purification.

[0065] Double digestion of vector and fragment: The target gene and the lentiviral empty vector shuttle plasmid are digested with the same restriction endonuclease, and the linear vector and target fragment are recovered and purified.

[0066] Construction of recombinant plasmids: The vector and the target fragment are mixed in proportion, and DNA ligase is used to ligate them to form recombinant lentiviral plasmids.

[0067] Transformation and amplification: The ligation product was transformed into competent E. coli cells, plated on antibiotic-resistant plates, and single colonies were picked for amplification.

[0068] Positive clone identification: The insertion of the target gene and the correct sequence were confirmed by colony PCR, enzyme digestion and sequencing.

[0069] High-purity plasmid extraction: Expand the culture of positive strains, extract endotoxin-free plasmids, complete the preparation of lentiviral recombinant vectors for subsequent virus packaging.

[0070] The constructed lentiviral recombinant vector map is shown below. Figure 3 .

[0071] The target gene (SV40 LT) sequence is as follows:

[0072]

[0073] 2.1.2 SV40 LT Lentiviral Packaging

[0074] Cell preparation: Culture 293T cells and plate them to a confluence of 70%~80% before transfection.

[0075] Plasmid transfection: The recombinant lentiviral shuttle plasmid (i.e., the lentiviral vector obtained in the previous step), packaging plasmid, and envelope plasmid are mixed in a 2:2:1 ratio and transfected using the Lipofiter transfection reagent. TM (Purchased from Hanbio Biotechnology, catalog number HB-TRCF-1000) Co-transfected cells.

[0076] Medium replacement and culture: Replace with fresh complete medium 6-8 hours after transfection and continue culturing.

[0077] Virus collection: Cell supernatant was collected at 48h and 72h, and impurities were removed by filtration.

[0078] Concentration and aliquoting: The viral supernatant is concentrated and purified, the viral titer is determined, and the virus is aliquoted and stored at low temperature.

[0079] The obtained lentivirus concentrate is HBLV-SV40T-3xflag-ZsGreen-PURO lentivirus concentrate.

[0080] 2.2 Lentiviral transfection of Krt14-positive primary cells

[0081] Purified Krt14-positive primary cells were seeded into culture dishes. After cell adhesion, HBLV-SV40T-3xflag-ZsGreen-PURO lentiviral concentrate carrying the SV40LT gene and puromycin resistance gene was added to infect the cells. After a period of infection, the culture medium was replaced with fresh medium for further culture. Subsequently, puromycin was added for resistance selection, and the drug-containing medium was changed regularly for a sufficient period of time until all uninfected control cells died, leaving only cells in the culture dish that had stably integrated the exogenous gene. Microscopic images of the morphology of polyclonal Krt14-positive primary mesenchymal stromal cells transfected with SV40LT lentivirus are shown below. Figure 4 As shown in Figure A.

[0082] 3. Establishment of monoclonal cell lines

[0083] Cells that survived the puromycin selection were digested and prepared into a single-cell suspension. The cell suspension was diluted using a limiting dilution method and seeded into 96-well plates, theoretically ensuring only one cell per well. Under a microscope, wells containing only a single cell were selected, labeled, and cultured continuously. Once the single cell proliferated to form a clearly visible clone (…),… Figure 4 (B) The cells were transferred stepwise to 24-well plates, 6-well plates, and culture flasks for amplification, ultimately establishing an immortalized cell line derived from a single cell, namely the mouse jawbone mesenchymal matrix cell immortalized cell line WZL-2. This cell line is deposited at the China Center for Type Culture Collection, classified and named as mouse jawbone mesenchymal matrix cell immortalized cell line WZL-2 Mus musculus, with accession number CCTCC NO:C202647 and deposit date of April 14, 2026.

[0084] 4. Verification of cell line immortalization characteristics

[0085] (1) Detection of aging phenotypes

[0086] Simultaneously, senescence phenotypes were detected in primary cells of generation P10 (i.e., cells normally cultured to generation P10 after sorting in step 1) and immortalized cell lines of generation P10. Senescence-associated β-galactosidase staining was used to detect cellular senescence phenotypes (senescent cells typically increase in size and express β-galactosidase, which exhibits high enzyme activity at pH 6.0; using X-Gal as a substrate, a deep blue product is generated under the catalysis of senescence-specific β-galactosidase, making the blue-colored β-galactosidase-expressing cells easily observable under a light microscope). Microscopic observation revealed that primary cells of generation P10 (Ctrl-P10) widely exhibited senescence phenotypes, while no cellular senescence phenotypes were observed in the immortalized cell line of generation P10 (SV40 LT-P10). Figure 5 ).

[0087] (2) Morphological observation

[0088] Immortalized cell lines were continuously passaged, and the morphology of cells at different passage numbers (passage 10 and passage 50) was observed under a microscope. Microscopic observation confirmed that with increasing passage number, cells grew stably, and the morphology of P10 and P50 cells did not change significantly. Figure 6 A). Under high magnification, P50 generation cells showed stable morphology. Figure 6 B).

[0089] 5. Identification of the mesenchymal properties of immortalized cell lines

[0090] (1) Identification of proliferation capacity and colony formation capacity

[0091] The proliferative capacity of immortalized cells was detected using the CCK8 assay. The results showed that ( Figure 7 A), the proliferation capacity of immortalized cell lines is significantly enhanced compared to primary cells (i.e., the high-purity Krt14-positive primary mesenchymal stromal cells obtained in step 1).

[0092] Colony formation assay (CFU): Immortalized cells were seeded at low density in 12-well plates and cultured for a period of time. The cells were then stained with dyes such as crystal violet, and the resulting cell colonies were observed and counted to assess their self-renewal capacity. Results showed ( Figure 7 B), immortalized cell lines have a significantly enhanced ability to form clones compared to primary cells.

[0093] (2) Identification of tri-directional differentiation ability

[0094] Cells were seeded in osteogenic induction medium, adipogenic induction medium, and chondrogenic induction medium and cultured for several weeks, respectively. After culture, specific staining methods such as alkaline phosphatase staining (to detect early osteogenic capacity), alizarin red staining (to detect calcium nodules), Oil Red O staining (to detect lipid droplets), and toluidine blue staining (to detect glycosaminoglycans) were used to determine whether the cells had successfully differentiated into the corresponding lineages.

[0095] The information on the different staining methods and the positive detection methods are shown in the table below:

[0096]

[0097] Staining results as follows Figure 8 As shown in the table above, all results are positive. ALP and ARS staining results indicate that the cells have the potential to differentiate into osteoblasts; Oil Red O staining results indicate that the cells have the potential to differentiate into adipocytes; and Toluidine Blue staining results indicate that the cells have the potential to differentiate into chondrocytes.

[0098] The above three-way differentiation results confirm that the immortalized cell line has completely preserved the multi-directional differentiation potential of mesenchymal matrix cells.

[0099] (3) Identification of ectopic osteogenic capacity in vivo

[0100] Immortalized cells were mixed with Matricene (40184ES08, Yisheng) and transplanted under the renal capsule of C57BL / 6J mice. After 4 weeks of in vivo culture, the mouse kidneys were removed for micro-CT scanning to observe whether new tissue formation occurred. Results are as follows: Figure 9 As shown, the high-density shadow on micro-CT (marked with a yellow arrow) indicates the formation of osteoid tissue under the renal capsule, proving that these immortalized cells have the ability to form osteoids in ectopic environments in vivo.

[0101] 6. In vivo tumorigenesis experiment of immortalized cell lines

[0102] Immortalized cells were injected subcutaneously into nude mice, and tumor formation was observed one month later. Figure 10 As shown, no tumors formed under the skin, indicating that the transplanted immortalized cells do not have the ability to autonomously form progressively growing masses in the body. This excludes the possibility that the unlimited proliferation capacity of the immortalized cell line comes from malignant transformation (tumoricity), and the biosafety is good.

[0103] In summary, this embodiment successfully isolated and purified Krt14-positive primary cells from the jawbone of Krt14Cre R26LSL-tdTomato mice, and successfully immortalized them by transfecting the SV40 LT gene with lentivirus. Finally, a stable cell line was established using monoclonal technology. Subsequent functional identification experiments confirmed that this cell line, while possessing unlimited proliferative capacity, fully retains the clonogenic ability, in vitro tri-lineage differentiation potential, and in vivo tissue formation ability unique to mesenchymal stromal cells. This embodiment fully verifies the completeness, feasibility, and effectiveness of the technical solution of this invention.

[0104] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. Application of Krt14 as a specific gene marker in the identification and isolation of mesenchymal stromal cell populations derived from mouse jawbone.

2. An immortalized mouse jawbone mesenchymal matrix cell line, WZL-2, is deposited at the China Center for Type Culture Collection (CCTCC), classified and named as Mouse Jawbone Mesenchymal Matrix Cell Immortalized Cell Line WZL-2 Mus musculus, with accession number CCTCC NO:C202647 and deposit date of April 14, 2026.

3. The mouse jawbone mesenchymal matrix cell immortalization cell line WZL-2 according to claim 2, characterized in that, This cell line possesses unlimited proliferative capacity, maintains Krt14 positive expression, and retains the clonogenic ability of mesenchymal stromal cells, in vitro tri-lineage differentiation potential, and in vivo tissue formation ability.

4. The method for constructing the immortalized mouse jawbone mesenchymal matrix cell line WZL-2 according to claim 2, characterized in that, Includes the following steps: S1. Specific isolation and culture of high-purity primary cells: Using maxillary and mandibular bone tissues of Krt14Cre R26LSL-tdTomato transgenic mice as starting material, primary cells were isolated by enzymatic digestion combined with tissue block adhesion method. Flow cytometry was used to specifically sort out the cell population expressing tdTomato fluorescent protein to obtain Krt14 positive primary mesenchymal matrix cells. Alternatively, Krt14-specific antibodies can be used to immunofluorescently label primary cells of the jawbone of ordinary mice, and then Krt14-positive cells can be obtained by flow cytometry or magnetic bead sorting. S2. Immortification and Cloning Treatment: The immortalization gene was introduced into the Krt14-positive primary cells obtained in step S1. After positive clone screening and single-clone culture, an immortalized cell line was obtained.

5. The construction method according to claim 4, characterized in that, In step S2, the immortalization gene is one or more of the following: SV40 LT gene, hTERT gene, human papillomavirus E6 / E7 gene, and Bmi-1 gene; And / or, the immortalized gene is introduced into purified Krt14-positive primary cells using a lentiviral transduction system, adenovirus, retrovirus, electroporation, liposome transfection, or CRISPR / Cas9 technology.

6. The construction method according to claim 5, characterized in that, In step S2, the immortalized gene is the SV40 LT gene, which is introduced into purified Krt14 positive primary cells using a lentiviral transduction system.

7. The construction method according to claim 4, characterized in that, In step S2, the positive clone screening method is as follows: using the antibiotic resistance gene carried on the lentiviral vector, antibiotics are added to the culture medium for drug screening, cells that have not successfully integrated the SV40 LT gene are eliminated, and a cell population that stably expresses the immortalization gene is obtained. And / or, the monoclonal culture method is as follows: the cells obtained from the positive clone screening are diluted and seeded into a multi-well plate to ensure that only clones derived from a single cell grow in each well. After the single cell proliferates to form a clearly visible clone, it is transferred to a larger culture container for amplification, and finally an immortalized cell line derived from a single cell is established.

8. The construction method according to claim 7, characterized in that, In step S2, the antibiotic is puromycin, neomycin, blastomycin, or hygromycin.

9. The construction method according to claim 4, characterized in that, It also includes: Step S3. Identification of mesenchymal properties of immortalized cell lines: Identification of the cell lines obtained in step S2 in terms of clonogenic ability, in vitro tri-lineage differentiation potential and in vivo tissue formation ability.

10. The application of the immortalized mouse jawbone mesenchymal matrix cell line WZL-2 as described in claim 2 in jawbone regeneration, bone metabolism research and related drug screening.

Images