Preparation method and application of conditionally immortalized human myocardial fibroblast model

Abstract

The invention relates to the field of cell engineering, and particularly discloses a preparation method and application of a conditionally immortalized human myocardial fibroblast model. According to the method, a Tet-on system driven by a cardiac fibroblast specific promoter is constructed to regulate and control a lentiviral vector of an SV40LT-tsA58 temperature-sensitive mutant gene, and after primary human cardiac fibroblasts are transduced, conditional immortalization of the cells is realized in the presence of an inducer doxycycline. The model can be stably amplified under an induction condition, the immortalized gene expression is closed after an inducer is removed, the primary physiological state of cells is recovered, and the high-fidelity response capability to fibrosis promoting and anti-fibrosis drugs is reserved. According to the method, the in-vitro proliferation limit of primary cells is broken through, the problems of species difference and ethics are avoided, and a humanized, high-fidelity and high-controllability standardized tool is provided for myocardial fibrosis pathological mechanism research and drug screening.

Description

A method for preparing a conditionally immortalized human cardiomyocyte fibroblast model and its application. Technical Field

[0001] This invention belongs to the field of cell engineering, specifically relating to a method for preparing a conditionally immortalized human cardiomyocyte fibroblast model and its application. Background Technology

[0002] Cardiac fibrosis is a core pathological feature in the development of various heart diseases (such as myocardial infarction, hypertensive heart disease, cardiomyopathy, etc.) and a key factor ultimately leading to heart failure. Its pathological essence lies in the overactivation of cardiac fibroblasts (CFBs) under pathological stimulation, leading to differentiation into myofibroblasts (CMFBs), resulting in excessive deposition of extracellular matrix (ECM), cardiac structural remodeling, and functional impairment. Therefore, inhibiting abnormal CFB activation is a key strategy for preventing and treating cardiac fibrosis, and the development of new anti-fibrotic drugs has significant clinical implications. Existing research and drug screening for myocardial fibrosis mainly rely on the following models: 1. Primary cardiac fibroblasts (pCFB) in animals (mice, rats): Isolation and culture are time-consuming, labor-intensive, and costly; different batches of cells exhibit significant heterogeneity; and animal models cannot accurately simulate human disease characteristics, resulting in low clinical translation efficiency of drug screening results. 2. Human pCFB: Human heart tissue sources are extremely limited, difficult to obtain, and involve ethical review issues. Human pCFB has limited in vitro expansion capacity and a small cell count, making it difficult to meet the needs of high-throughput drug screening. At the same time, individual differences between different donors lead to high cellular heterogeneity, making it difficult to guarantee the reproducibility of experimental results.

[0003] Current technologies address cell expansion through immortalization, but sustained expression of proto-oncogenes alters fundamental cell characteristics, leading to significant functional differences compared to primary cells and failing to accurately simulate CFB behavior under physiological / pathological conditions. Therefore, developing a standardized cell model that overcomes primary cell passage limitations, maintains a humanized background, accurately simulates key characteristics of pCFB under physiological and pathological conditions, and is simple to prepare and highly reproducible is crucial for research on the mechanisms of myocardial fibrosis and drug screening. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing a conditionally immortalized human cardiomyocyte fibroblast model and its application.

[0005] The present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing a conditionally immortalized cardiac fibroblast (iCFB) model, comprising: constructing a lentiviral vector carrying the SV40LT-tsA58 gene regulated by the Tet-on system;

[0007] Human cardiomyocytes were transduced using the lentiviral vector. After transduction, the SV40LT-tsA58 gene was induced to express under the action of an inducer. When the cell confluence reached 85% to 90%, the cells were passaged to obtain a conditionally immortalized human cardiomyocyte model.

[0008] Furthermore, the Tet-on system includes the cardiomyocyte-fibroblast-specific promoter COL1A2, which drives the expression of the tetR-KRAB fusion protein.

[0009] Among them, the specific promoter COL1A2 can control the expression site of tetR-KRAB, ensuring that it is expressed only in cardiomyocytes, thereby strictly limiting the activation of the entire immortalization system to cardiomyocytes and avoiding misconversion of non-target cells.

[0010] Furthermore, the Tet-on system also includes the SV40LT-tsA58 gene, with tetO regulatory elements located upstream and downstream of it.

[0011] The SV40LT-tsA58 gene is a core element for immortalization, and its expression is controlled by the tetO regulatory element: in the presence of doxycycline (dox), the tetO element allows transcription factors to bind and initiate SV40LT-tsA58 expression, enabling cells to proliferate; when dox is removed, the tetO element blocks transcription through steric hindrance, shutting down SV40LT-tsA58 expression. Therefore, under dox-containing culture conditions, SV40LT-tsA58 is continuously expressed, and cells acquire unlimited proliferative capacity; under dox-free conditions, SV40LT-tsA58 expression is inhibited, cell proliferation stops, and the morphology and function of primary cardiomyocytes are restored. Furthermore, SV40LT-tsA58 introduces a temperature-sensitive mutation in tsA58 (i.e., tsA438A-V) (see Loeber G, et al. Journal of virology, 1989, 63(10): 4426-4430.). At permissible temperatures (≤37°C), the antigen structure is stable and can normally promote cell proliferation; however, at non-permissible temperatures (≥39°C), the antigen conformation changes, and its ATP-binding capacity is lost, thereby inactivating its function of driving cell proliferation. This design provides dual protection for the safety and controllability of the conditional immortalization system.

[0012] Furthermore, the specific steps for transducing human cardiomyocytes using the lentiviral vector are as follows:

[0013] (1) Primary human cardiomyocytes were isolated from human heart tissue and cultured in vitro;

[0014] (2) The primary human cardiomyocytes obtained in step (1) were seeded in a culture plate and transduced using a lentiviral vector encoding the SV40LT-tsA58 gene regulated by the Tet-on system.

[0015] (3) After transduction, the culture medium containing dox was replaced to induce the expression of the SV40LT-tsA58 gene, and the cells were cultured and passaged under the condition containing dox, which is the conditionally immortalized human cardiomyocyte model.

[0016] Furthermore, the preparation method of the lentiviral vector carrying the Tet-on system-regulated SV40LT-tsA58 gene includes: firstly, using the oligonucleotide hybridization products shown in SEQ ID NO: 1 and SEQ ID NO: 2, a temperature-sensitive mutation of tsA58 is introduced into the SV40 large T antigen gene through homologous recombination to obtain the mutant gene sequence; subsequently, using this sequence as a template, PCR amplification is performed using primers shown in SEQ ID NO: 3 and SEQ ID NO: 4, the PCR product is purified, and inserted into the pJET1.2 vector using the TA cloning method to generate a recombinant plasmid. This plasmid is then inserted into the lentiviral shuttle plasmid pLV.iCOL1A2, which is derived from pLVET-tTR-KRAB (Addgene, Cambridge, MA, plasmid number: 116444), whose original promoter and fluorescent protein coding sequences have been replaced by a cardiomyocyte-fibroblast-specific COL1A2 promoter. In the expression system, the expression of SV40LT-tsA58 was driven by the COL1A2 promoter, resulting in the final recombinant transfer plasmid pLV.iCOL1A2.LT-tsA58. The obtained recombinant transfer plasmid and lentiviral packaging plasmid were co-transfected into 293T cells. After culturing, the cell supernatant was collected and concentrated to obtain infective recombinant lentiviral particles.

[0017] Furthermore, the extraction method of primary human cardiomyocytes in step (1) is as follows: under sterile conditions, ventricular tissue is separated from the heart, and the atria, blood vessels and fibrotic areas are removed to ensure that there are no blood clots in the tissue; the ventricular tissue is cut into small pieces, a digestive enzyme mixture is added, stirred, and the digestive fluid containing free cells is collected. Fresh digestive enzymes are added to the remaining tissue pieces, and the above digestion steps are repeated 1-2 times until the tissue is basically digested. All digestive fluids are combined to separate the cells.

[0018] Furthermore, the stirring temperature mentioned above is 37°C. o C, each digestion session takes 30-50 minutes.

[0019] Furthermore, the in vitro culture method described in step (1) is as follows: the separated cells are transferred to DMEM medium containing 10% fetal bovine serum, centrifuged to make a cell suspension, and then inoculated into a cell culture dish for culture.

[0020] Furthermore, the culture conditions are: 37°C, in a 5% CO2 saturated humidity incubator. Culture for 1-3 hours, then remove the suspension and replace with fresh culture medium to remove cardiomyocytes.

[0021] Furthermore, when the cell fusion rate reaches 85-90%, the cells are digested with trypsin and passaged.

[0022] Furthermore, during transduction in step (2), the multiplicity of infection (MOI) of the lentivirus is 5-30.

[0023] Furthermore, in step (3), the concentration of the dox is 50-200 ng / mL.

[0024] Furthermore, in step (3), when the cell fusion rate reaches 85-90%, the cells are digested with 0.25% trypsin and passaged at a ratio of 1:4; after 2 weeks, the cells are passaged at a ratio of 1:8 to obtain a conditionally immortalized human cardiomyocyte model.

[0025] Furthermore, the conditionally immortalized human cardiomyocyte model is characterized by exhibiting both pro-fibrotic and anti-fibrotic responses.

[0026] Secondly, this invention provides an application of a conditionally immortalized human myocardial fibroblast model in a drug screening model for myocardial fibrosis.

[0027] Further, this includes: constructing a lentiviral vector carrying the SV40LT-tsA58 gene, which is regulated by the Tet-on system;

[0028] Human cardiomyocytes were transduced using the lentiviral vector. After transduction, the SV40LT-tsA58 gene was induced to express under the action of an inducer. When the cell confluence reached 85% to 90%, the cells were passaged to obtain a conditionally immortalized human cardiomyocyte model.

[0029] Furthermore, the conditionally immortalized human cardiomyocyte model has the characteristics of high fidelity, high stability, high sensitivity, high homogeneity, low cost, and standardization.

[0030] Furthermore, the application of the conditionally immortalized human myocardial fibroblast model in myocardial fibrosis drug screening models includes its application in screening for drugs that promote or inhibit myocardial fibrosis.

[0031] Furthermore, the pro-myocardial fibrosis drug is any one of transforming growth factor β1 (TGFβ1), angiotensin II (AngII), and palmitic acid (PA).

[0032] Furthermore, the anti-myocardial fibrosis drug is either pirfenidone (PFD) or NS8593.

[0033] The present invention has the following beneficial effects:

[0034] This invention provides a method for preparing a conditionally immortalized human cardiomyocyte fibroblast model. By constructing a lentiviral vector carrying the SV40LT-tsA58 gene regulated by the Tet-on system and transducing it into human cardiomyocytes, conditional immortalization of the cells was successfully achieved. The obtained immortalized human cardiomyocyte fibroblast model achieves conditional immortalization under the induction of the inducer dox. Under dox-containing culture conditions, the obtained conditionally immortalized human cardiomyocytes continuously express SV40LT-tsA58, enabling the cells to acquire stable and robust unlimited proliferation capacity, significantly increasing the population multiplication rate. This completely breaks through the limits of in vitro passage proliferation of primary human cardiomyocytes, solving the fundamental bottleneck of their scarcity and inability to meet high-throughput requirements, and reducing usage costs. Simultaneously, this model uses a humanized background, avoiding the species difference risks associated with using animal-derived cells (such as mice and rabbits) and avoiding the ethical issues associated with repeatedly obtaining human tissues.

[0035] This invention utilizes the COL1A2 promoter, specific to cardiac fibroblasts, to drive the expression of the tetR-KRAB fusion protein, ensuring that the activation of the entire immortalization system is strictly limited to the target cells, thus preventing the misconversion of non-cardiac fibroblasts at the source. Secondly, the Tet-on system achieves precise and reversible regulation through chemical induction: in the presence of dox, SV40LT-tsA58 expression is activated, driving cell proliferation; upon dox removal, its expression is reversibly deactivated, cell proliferation ceases, and the physiological state and key biomarker expression of primary cells are restored, thereby completely avoiding the phenotypic distortion caused by the continuous expression of proto-oncogenes in traditional immortalization technologies. Furthermore, the introduced tsA58 temperature-sensitive mutation provides another independent safety guarantee for the system. SV40LT-tsA58 is constructed by introducing a point mutation into the tsA58 point in the ATP-binding domain of the Large T antigen, creating a temperature-sensitive protein. Its mutation site is located in the key ATP-binding domain, which causes the protein function to be regulated by temperature: at the permitted temperature (≤37°C), the antigen structure is stable and can promote cell proliferation normally; while at the unpermitted temperature (≥39°C), the antigen conformation changes and the ATP-binding ability is lost, thereby inactivating its function of driving cell DNA replication and proliferation. This constitutes a dual control switch of inducer and temperature, which greatly enhances the reliability and safety of the system.

[0036] This immortalized human cardiomyocyte fibroblast model, after restoring its physiological state, exhibited fibrosis response characteristics highly consistent with primary cells. It could respond to different cardiomyocyte fibrosis inducing agents (such as TGFβ1, Ang II, and PA), efficiently differentiating into myofibroblasts and accurately mimicking the pathological fibrosis process. Furthermore, treatment with antifibrotic drugs (such as pirfenidone and NS8593) effectively inhibited myofibroblast transformation, achieving dedifferentiation. This demonstrates that the model possesses both highly efficient expansion capacity and high-fidelity pathophysiological function.

[0037] The cell model prepared in this invention provides a human-derived, high-fidelity, high-stability, high-sensitivity, highly homogeneous, low-cost, standardized, and reproducible drug screening tool for myocardial fibrosis research. This tool can accurately simulate the transdifferentiation pathway and molecular regulatory mechanisms of primary cells, respond rapidly to drugs, and ensure that the screening results are consistent with the physiological and pathological processes, thus providing an efficient and reliable standardized solution for the study of the pathological mechanisms of myocardial fibrosis and drug discovery. Attached Figure Description

[0038] To more clearly illustrate the technical solutions of the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 is a schematic diagram of the lentivirus shuttle plasmid structure constructed in an embodiment of the present invention.

[0040] Figure 2 is a flowchart of the construction process of the conditionally immortalized human cardiomyocyte fibroblast model in an embodiment of the present invention.

[0041] Figure 3 shows the immunofluorescence image of SV40LT-tsA58 protein and the cell growth curve. Specifically, Figure 3A is a comparison of the immunofluorescence of SV40LT-tsA58 protein in primary cardiomyocytes (pCFB) and transduced cardiomyocytes (iCFB) in this embodiment of the invention; Figure 3B is a comparison of the growth curves of pCFB cells and iCFB cells in this embodiment of the invention; Figure 3C is the immunofluorescence image of SV40LT-tsA58 protein in iCFB cells after dox removal on days 0, 2, 4, 6, and 8 in this embodiment of the invention; Figure 3D is the dox-dependent growth curve of iCFB cells in this embodiment of the invention; and Figure 3E is the temperature-dependent growth curve of iCFB cells in this embodiment of the invention.

[0042] Figure 4 shows the immunofluorescence comparison of COL-1 and CX43 proteins in pCFB cells and iCFB cells in the embodiments of the present invention; human foreskin fibroblasts (FFB) were used as negative controls for CX43 expression.

[0043] Figure 5 shows the differentiation of iCFB cells in response to profibrotic and antifibrotic drugs in this embodiment of the invention; wherein, A in Figure 5 is the immunofluorescence image of iCFB cells and iCFB cells after treatment with TGFβ1 and TGFβ1+NS8593; B in Figure 5 is the relative gene expression level of ACTA2 in iCFB cells after treatment with profibrotic drugs (TGFβ1, Ang II and PA) and antifibrotic drugs (TGFβ1+NS8593, TGFβ1+PFD) by RT-qPCR.

[0044] All results involving numerical values ​​are expressed as mean ± standard error. # P>0.05 (no significant difference); **P<0.01. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0046] Example 1

[0047] 1. Construction of a conditionally immortalized human cardiomyocyte model

[0048] As shown in Figure 2, the specific steps are as follows:

[0049] (1) Generate the thermosensitive mutant tsA58 of the SV40 large T antigen

[0050] First, the plasmid pAT153.SV40ori(-) (Yunzhou Biotechnology (Guangzhou) Co., Ltd.) was digested with the restriction endonuclease PvuII. Then, the intermediate plasmid pAT153.SV40ori(-).dPvuII was obtained by cyclization reaction catalyzed by T4 DNA ligase.

[0051] Subsequently, a temperature-sensitive mutant, tsA58, was introduced using molecular cloning technology. The hybridization products of oligodeoxynucleotides SEQ ID NO: 1 and SEQ ID NO: 2 (wherein, the nucleotides GTG from positions 3 to 5 from the 5' end in SEQ ID NO: 1 and CAC from positions 28 to 30 from the 5' end in SEQ ID NO: 2 are the mutant LT codon and their complementary sequence) were homologously recombinated with the aforementioned circular plasmid (for specific preparation methods, see Loeber G, et al. Journal of virology, 1989, 63(10): 4426-4430.) to introduce the key mutation. Positive clones were screened after transformation, and the mutation site was verified by sequencing. The plasmid pAT153.SV40ori(-).dPvuII.LT-tsA58 containing the mutant was constructed.

[0052] (2) Amplification and cloning of the coding sequence of the SV40LT-tsA58 gene

[0053] The mutant SV40LT-tsA58 gene coding sequence was amplified using polymerase chain reaction (PCR). Specifically, the constructed plasmid pAT153.SV40ori(-).dPvuII.LT-tsA58 was used as a template, and amplification was performed using primers shown in SEQ ID NO: 3 and SEQ ID NO: 4. The forward primer SEQ ID NO: 3 contained the start codon ATG of the SV40LT-tsA58 gene, and the 5' end sequence of the reverse primer SEQ ID NO: 4 was designed to be complementary (TTA) to the region containing the stop codon on the template strand. The PCR reaction conditions were as follows: pre-denaturation at 98°C for 30 seconds, followed by 34 cycles, each cycle consisting of denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, and extension at 72°C for 5 minutes. After amplification, the PCR product was purified and inserted into the pJET1.2 vector using the TA cloning method to generate the recombinant plasmid pJet1.2.LT-tsA58. To verify the accuracy of cloning, the recombinant plasmid was transformed into DH5α competent cells, positive clones were screened by ampicillin, the size of the inserted fragment was verified by colony PCR, and the integrity and accuracy of the gene sequence were ensured by sequencing.

[0054] (3) Assembly of lentiviral shuttle plasmids

[0055] The above fragment was inserted into the lentiviral shuttle plasmid pLV.iCOL1A2 to obtain the final constructed vector pLV.iCOL1A2.LT-tsA58, the structure of which is shown in Figure 1. pLV.iCOL1A2 is derived from pLVET-tTR-KRAB (Addgene, Cambridge, MA, plasmid number: 116444), whose original promoter and fluorescent protein coding sequences have been replaced by a cardiomyocyte-fibroblast-specific COL1A2 promoter. The insertion was performed via restriction enzyme digestion (EcoRI and BamHI sites) and ligation. After construction, the vector integrity was verified by enzyme digestion and full-length sequencing to ensure correct layout.

[0056] The lentiviral vector used was designed with the Tet-on system plasmid as the basic backbone to create an expression system for the immortalized gene SV40LT-tsA58 controlled by the Tet-on system. The Tet-on system plasmid was derived from pLVET-tTR-KRAB (Addgene, Cambridge, MA, plasmid number: 116444). In the expression system, the expression of SV40LT-tsA58 was driven by the COL1A2 promoter, forming a recombinant transfer plasmid.

[0057] (4) Packaging and collection of lentiviruses

[0058] 80% confluenced 293T cell monolayers were co-transfected with the aforementioned lentiviral shuttle vector and packaging plasmid psPAX2 (Addgene; plasmid number: 12260) and pLP / VSVG (Thermo Fisher Scientific) at a molar ratio of 2:1:1. 293T cells were cultured in DMEM containing 10% fetal bovine serum. The transfection mixture (35 μg plasmid DNA and 105 μg polyethyleneimine per 175 cm² cell culture flask, dissolved in 2 mL of 150 mM NaCl solution) was added directly to the culture medium. The following morning, the medium was replaced with 15 mL of fresh high-glucose DMEM containing 5% fetal bovine serum and 25 mM HEPES-NaOH (pH 7.4).

[0059] Approximately 48 hours after transfection, the culture supernatant was collected and centrifuged at 3,750 × g for 10 minutes at room temperature to remove cell debris. The supernatant was then filtered through a Millex-HP syringe filter with a 0.45 μm pore size and a 33 mm diameter. To concentrate and purify the lentiviral particles, 30 mL of the vector suspension was added to a 38.5 mL polypropylene ultracentrifuge tube, with 5 mL of phosphate-buffered saline (PBS) containing 20% ​​(w / v) sucrose added to the bottom. The tube was centrifuged in an SW32 rotor at 15,000 rpm for 120 minutes at 4°C (slow acceleration without braking). The supernatant was then discarded, and the precipitate was resuspended overnight in PBS containing 1% bovine serum albumin with gentle shaking at 4°C. The concentrated vector suspension was aliquoted into 50 μL portions on ice and stored at -80°C.

[0060] (5) Cell isolation, culture and lentivirus transduction

[0061] S1. Under aseptic conditions, ventricular tissue of the aborted fetus was taken (ethical review approved by the Ethics Committee of Shenzhen Longgang District People's Hospital, ethics approval number: 2025101), and the atria, blood vessels and fibrotic areas were removed.

[0062] S2, cut the ventricular tissue into small pieces, add a mixture of type I collagenase (Worthington Biochemical, Lakewood, NJ) and DNase I (Sigma-Aldrich) digestive enzymes, stir and digest at 37°C for 35 minutes, repeat twice to fully separate the cells;

[0063] S3: Centrifuge the cell suspension at room temperature (1000 rpm, 10 min) to obtain cell pellet. Resuspend the cells in DMEM medium containing 10% fetal bovine serum, pipette to form a cell suspension, transfer to culture dishes, and incubate at 37°C, 5% CO2 saturated humidity for 2 hours to allow CFB preferential adhesion. Discard the suspension to remove non-adherent cells (mainly cardiomyocytes), add fresh medium, and continue culturing.

[0064] S4. When the cell confluence reaches 90%, digest with 0.25% trypsin for 3 minutes, and passage the cells at a ratio of 1:4.

[0065] S5, second- and third-generation pCFB cells were seeded into 6-well plates. After cell adhesion, lentiviral transduction was performed. The specific procedure was as follows: Calculate the required volume of lentiviral suspension based on MOI=10 and the viral concentration. Add the lentiviral suspension prepared in step 1.1 directly to the cell supernatant and gently shake to mix. After culturing the cells for another 24 hours, the medium was replaced with fresh, virus-free complete medium. On the third day, the medium was changed with culture medium containing 100 ng / mL dox to induce SV40LT-tsA58(LT) expression.

[0066] S6. Change the DMEM medium containing 100 ng / ml dox every other day and repeat the steps in S4. That is, when the cell confluence reaches 90%, perform passage.

[0067] After continuous expansion for 1-2 weeks, the cells were passaged at a ratio of 1:8 to obtain a stable conditionally immortalized cardiomyocyte fibroblast model (iCFB).

[0068] Table 1 Primer sequence listing

[0069] Numbering sequence SEQ ID NO: 15'-CTGTGCTTCTAGAATTATGTGGGGGGAA-3'SEQ ID NO: 25'-AGCTTTCCCCCCACATAATTCTAGAAGCACAG-3'SEQ ID NO: 35'-AGGTTTAAACTACGGGATCCGTGCACCATGGATAAAGTTTTAAACAGAGAGGA-3'SEQ ID NO: 45'-CCGAATTCTTTATGTTTCAGGTTCAGGG-3' surface

[0070] 2. Expression of the immortalization gene SV40LT-tsA58 in iCFB

[0071] 2.1 Experimental Methods

[0072] The expression of SV40LT-tsA58 in pCFB and iCFB cells was detected using an immunofluorescence microscopy system. SV40LT-tsA58 (LT) antigen expression was labeled with red fluorescence, and the cell nucleus (DNA) was labeled with blue fluorescence.

[0073] 2.2 Experimental Procedure

[0074] The specific steps are as follows:

[0075] (1) Cell fixation and permeabilization: Cell slides from each group were fixed with 4% paraformaldehyde at room temperature for 15 minutes, and then permeabilized with 0.1% Triton X-100 for 10 minutes;

[0076] (2) Blocking: Wash with PBS 3 times, 5 min each time, and then block with PBS solution containing 10% goat serum at room temperature for 1 hour to reduce non-specific staining;

[0077] (3) Primary antibody incubation: Add mouse anti-SV40LT primary antibody (dilution ratio 1:200) and incubate overnight at 4°C;

[0078] (4) Secondary antibody incubation: Wash 3 times with PBS, 5 min each time, then add Alexa Fluor 594-labeled goat anti-mouse IgG (red fluorescence, dilution ratio 1:200) and incubate at room temperature in the dark for 1 hour;

[0079] (5) Nuclear counterstaining: stain with Hoechst 33342 (10 μg / mL) for 5 minutes to label the cell nuclei (blue fluorescence);

[0080] (6) Image acquisition: Wash with PBS 3 times, 5 min each time, and then observe and acquire images using an immunofluorescence microscope or confocal microscope.

[0081] 3. Assays on the proliferation capacity of iCFB cells and their ability to regulate doxorubicin or temperature.

[0082] The proliferative capacity of pCFB and iCFB cells was tested using cell counting. The study assessed whether iCFB cells maintained long-term proliferative capacity and whether this capacity was regulated by dox and temperature.

[0083] 3.1 Experimental Group Design

[0084] Table 2 Experimental Group Design Table

[0085] Experimental grouping and treatment conditions and objectives pCFB-37 o C primary human cardiomyocytes, at 37 o Cells were cultured under standard C conditions. As a control, this verified the basal proliferative capacity of cells under normal conditions. iCFB-37 o C conditionally immortalized cells, at 37 o C. Cultured under conditions without dox to assess the basal proliferation capacity of iCFB in a non-induced state. iCFB-37 o C+ dox conditionally immortalized cells, at 37 o C. Cultured under conditions containing dox, the basal proliferative capacity of iCFB under induced conditions was evaluated. iCFB-39 o C+ dox conditionally immortalized cells, at 39 o C. Cultured under conditions containing dox (100 ng / mL) to evaluate the inhibitory effect of high temperature on iCFB cell proliferation. surface

[0086] 3.2 Experimental Procedure

[0087] (1) Cell plating and treatment: Cells from each experimental group were seeded into culture plates at the same density. After 24 hours of plating (until the cells were fully attached to the plate), the culture medium was replaced with fresh medium.

[0088] (2) Inducer addition: According to the grouping, add iCFB-37o C+dox group and iCFB-39 o In the C+dox group, a specific concentration of dox (final concentration 100 ng / mL) was added to the culture medium, while other groups were replaced with an equal amount of fresh culture medium without dox.

[0089] (3) Cell culture and result observation: pCFB-37 o C, iCFB-37 o C and iCFB-37 o Place the C+ dox group in 37 o Incubate iCFB-39 in a 5% CO2 incubator. o Place the C+ dox group in 39 o Incubate in a 5% CO2 incubator at C for culture and cell counting.

[0090] Time point setting: Perform cell counting or population doubling count on specific days of culture.

[0091] 4. iCFB cell characterization assay

[0092] The expression of CX43 and COL-1 proteins in iCFB cells and pCFB cells was analyzed by immunocytological staining to assess whether iCFB retains the characteristics of pCFB.

[0093] First, iCFB cells were cultured for 4 days in dox-free medium to metabolize SV40LT-tsA58. Then, antibodies were added to label COL-1 with green fluorescence (recommended dilution 1:200) and CX43 with red fluorescence (recommended dilution 1:200). Human foreskin fibroblasts (FFB) were used as a negative control, as FFB cells do not express the myocardial-specific marker CX43. Specific procedures are detailed in section 2.2.

[0094] 5. Response test to pro-fibrotic and anti-fibrotic drugs

[0095] 5.1 Response test to pro-fibrotic drugs

[0096] 5.1.1 Experimental Objective

[0097] To verify whether iCFB, after dox withdrawal, can respond to profibrotic factors TGFβ1, Ang II, and PA, and differentiate into activated myofibroblasts (CMFB), just like primary pCFB.

[0098] 5.1.2 Cell Grouping and Processing

[0099] Table 3 Treatment groupings in the fibrotic drug response test

[0100] Groups, Treatment Conditions, Control Group, Conventional Culture Medium, TGFβ1 Group, TGFβ1 Group, Ang II Group, Ang II Group, PA Group, PA Group, PA Group surface

[0101] 5.1.3 Detection Indicators and Methods

[0102] 5.1.3.1 Immunofluorescence

[0103] The control group and the TGFβ1 group were stained with α-SMA (α-smooth muscle actin, red, a key marker of myofibroblasts, gene name ACTA2) using the same method as in 3.2. The expression of α-SMA was observed to be upregulated after TGFβ1 treatment, leading to the formation of stress fibers.

[0104] 5.1.3.2 Real-time quantitative PCR (qPCR mRNA) detection

[0105] RNA extraction: Four groups of cells were collected, and total RNA was extracted using the TRIzol method.

[0106] Reverse transcription: Reverse transcription of RNA into cDNA.

[0107] Real-time quantitative PCR: The relative expression level of ACTA2 mRNA was detected using the SYBR Green assay with GAPDH as an internal reference gene. The 2^(-ΔΔCt) value was calculated and statistical analysis was performed.

[0108] 5.2 Antifibrotic drug response test

[0109] 5.2.1 Experimental Objective

[0110] To evaluate whether antifibrotic drugs can reverse the fibrotic model of iCFB and to verify the application value of this model in drug screening.

[0111] 5.2.2 Experimental Methods

[0112] To evaluate the response of iCFB cells to anti-fibrotic drugs, iCFB cells induced by the pro-fibrotic drug TGFβ1 in section 5.1 were randomly divided into three groups, with two groups receiving intervention with the anti-fibrotic drugs NS8593 and PFD, respectively.

[0113] Table 4 Treatment Groups in Antifibrotic Drug Response Tests

[0114] The treatment conditions for each group were as follows: The control group was the same as the control group in Table 3. The TGFβ1+NS8593 group was treated with medium containing 10 ng / mL TGFβ1 and 10 μM NS8593 (TRPM7 ion channel inhibitor) for 48 hours. The TGFβ1+PFD group was treated with medium containing 10 ng / mL TGFβ1 and 1 mg / mL PFD (pirfenidone) for 48 hours. surface

[0115] 5.2.3 Detection Indicators

[0116] After treatment, the following tests were performed on the three groups of cells:

[0117] (1) Immunofluorescence: The protein expression level of α-SMA in cells of the control group and the TGFβ1+NS8593 group was detected;

[0118] (2) qPCR: detect the mRNA expression level of ACTA2 in cells of each group.

[0119] 6. Test Results

[0120] 6.1 Results of iCFB proliferation capacity and dox regulation performance testing

[0121] As shown in Figure 3A, immunocytological staining revealed that SV40LT-tsA58 (LT) was expressed in iCFB cells but not in pCFB cells. As shown in Figure 3B, pCFB cells gradually ceased proliferation after approximately 10 population doublings (PD), while transduced iCFB cells expressing SV40LT-tsA58 continued to proliferate for at least 70 PDs without a significant decrease in proliferation rate; that is, one primary cell could produce at least approximately 2 69 The results showed that the expression of SV40LT-tsA58 enabled CFB cells to successfully immortalize, overcome senescence, and continue to proliferate.

[0122] Immunocytological staining analysis of SV40LT-tsA58 expression in iCFB cells before dox removal (day 0) and on days 2, 4, 6, and 8 after dox removal (Figure 3C) showed that SV40LT-tsA58 expression was efficiently induced in the presence of dox; however, its expression level gradually decreased with prolonged dox removal time and became undetectable after day 4, confirming that SV40LT-tsA58 expression is strictly regulated by dox. Consistent with this, cell proliferation experiments (Figure 3D) showed that iCFB cells proliferated rapidly in the presence of dox, with a doubling time of 28 hours; after dox removal, the cell number only increased slightly in the first two days, then entered a state of stagnation. This slight increase in the initial stage of proliferation is likely due to the slow degradation of residual SV40LT-tsA58 protein within the cells. Furthermore, as shown in Figure 3E, even under dox-containing conditions, increasing the culture temperature from 37°C to 39°C effectively inhibited iCFB cell proliferation. This demonstrates that the function of the temperature-sensitive mutant SV40LT-tsA58 it carries is also strictly regulated by temperature. In summary, these results collectively indicate that the proliferation of iCFB cells can be precisely and reliably controlled via a dual mechanism of dox and temperature, constituting a robust system for the regulation of gene expression and function.

[0123] 6.2 Results of iCFB cell model characteristic detection

[0124] As shown in Figure 4, COL-1 protein in both pCFB and iCFB cells exhibits characteristic fibrillary expression, and the FFB group also expresses a similar COL-1 protein signal. CX43 protein shows strong signal expression in both CFB and iCFB cells, while the FFB group shows no expression at all. The merged channel map shows that COL-1 and CX43 are co-localized in pCFB and iCFB cells, while there is no overlapping signal in the FFB group, indicating that iCFB cells can maintain the characteristics of pCFB cells after the immortalization gene SV40LT-tsA58 is removed.

[0125] 6.3 Results of Response Testing to Pro-fibrotic and Anti-fibrotic Drugs

[0126] As shown in Figure 5A, untreated iCFB cells (group Ctrl) exhibit a spindle-shaped morphology. Cells induced by TGFβ1 showed significant shrinkage and deformation, while cells treated with the antifibrotic drug NS8593 partially recovered their spindle shape. Furthermore, as shown in Figure 5B, RT-qPCR results showed that the ACTA2 mRNA expression level was significantly increased in cells induced by TGFβ1, AngII, or PA, while the ACTA2 mRNA expression level was significantly decreased in cells treated with the antifibrotic drugs PFD or NS8593. This indicates that iCFB cells can respond to antifibrotic drugs, and both drugs can significantly inhibit TGFβ1-induced fibrosis. Therefore, iCFB cells can be used as a screening model for pro-fibrotic and anti-myocardial fibrosis drugs.

[0127] In summary, this study successfully constructed a regulated, conditionally immortalized human cardiomyocyte fibroblast (iCFB) model. The iCFB cell model stably expressed the SV40LT-tsA58 antigen under dox induction, achieving long-term stable proliferation. Within 4 days of dox withdrawal, SV40LT-tsA58 was completely degraded, and cell proliferation was controlled and stopped, demonstrating its strict reversible regulation. Immunofluorescence assays showed that after removing the immortalization gene, iCFB cells completely retained the core characteristics of primary cardiomyocytes. Regarding drug response, the iCFB cells responded to the pro-fibrotic factor TGFβ1 completely in the same way as primary cells, with a significant increase in α-SMA expression after stimulation, successfully differentiating into myofibroblasts. Simultaneously, they exhibited high sensitivity to anti-fibrotic drugs, significantly reducing ACTA2 mRNA levels. All of these findings collectively validate that this model possesses stable expansion capabilities, precise preservation of primary cardiomyocyte function, and drug response sensitivity, providing an efficient and standardized tool for studying the pathological mechanisms of myocardial fibrosis and screening drugs for myocardial fibrosis.

[0128] The present invention has been further described above with reference to specific embodiments. However, it should be understood that the specific description herein should not be construed as limiting the nature and scope of the present invention. Various modifications made to the above embodiments by those skilled in the art after reading this specification are all within the scope of protection of the present invention.

Claims

1. A method for preparing a conditionally immortalized human cardiomyocyte fibroblast model, characterized in that, include: Construct a lentiviral vector carrying the SV40LT-tsA58 gene regulated by the Tet-on system; Human cardiomyocytes were transduced using the lentiviral vector. After transduction, the SV40LT-tsA58 gene was induced to express under the action of an inducer. When the cell confluence reached 85% to 90%, the cells were passaged to obtain a conditionally immortalized human cardiomyocyte model.

2. The preparation method according to claim 1, characterized in that, The Tet-on system includes the cardiomyocyte-fibroblast-specific promoter COL1A2, which drives the expression of the SV40LT-tsA58 and tetR-KRAB fusion protein, with SV40LT-tsA58 and tetR-KRAB linked by an IRES element.

3. The preparation method according to claim 1, characterized in that, The Tet-on system also includes the SV40LT-tsA58 gene, with tetO regulatory elements located upstream and downstream of it.

4. The preparation method according to claim 1, characterized in that, The inducer is doxycycline.

5. The preparation method according to claim 4, characterized in that, The concentration of doxycycline is 50-200 ng / mL.

6. A conditionally immortalized human cardiomyocyte model prepared according to any one of claims 1-5, characterized in that, The population doubling number of the conditionally immortalized human cardiomyocyte fibroblast model is no less than 70.

7. A conditionally immortalized human cardiomyocyte model as described in claim 6, characterized in that, It exhibits both pro-fibrotic and anti-fibrotic responses.

8. The application of the conditionally immortalized human myocardial fibroblast model as described in claim 7 in the preparation of a drug screening model for myocardial fibrosis.

9. The application of the conditionally immortalized human cardiomyocyte model as described in claim 8 in a drug screening model for myocardial fibrosis, characterized in that, The drug screening includes screening for drugs that promote or inhibit myocardial fibrosis.

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