Method for establishing a cell model for detecting the selectivity of fgf19 and fgf21 analogs for fgf receptors

Abstract

This invention discloses a method for establishing a cell model to detect the selectivity of FGF19 and FGF21 analogs for the FGF receptor. The method involves constructing cell lines stably expressing β-Klotho and FGFR through viral infection, screening and sorting positively expressing cells, and establishing a rapid and efficient method for detecting the binding specificity of FGF19 / FGF21 analog drugs to different subtypes of FGFR. This method solves the problems of low cell transfection efficiency, low viability, and poor experimental reproducibility in existing FGFR specificity identification methods. It effectively addresses the problem of poor drug response caused by low transfection efficiency. Furthermore, the established stable transfected cell lines exhibit high cell viability and stable protein expression, overcoming the problems of mass cell death and poor reproducibility associated with transient transfection methods.

Description

Technical Field

[0001] This invention relates to a method for establishing a cell model, and more particularly to a method for establishing a cell model for detecting the selectivity of FGF19 and FGF21 analogs for FGF receptor. Background Technology

[0002] Human fibroblast growth factor (FGF) comprises 22 members, among which FGF19, FGF21, and FGF23 belong to the FGF19 subfamily, also known as endocrine FGF. Unlike FGF23, which requires Klotho as an essential cofactor to activate the FGF signaling pathway, FGF19 and FGF21 require β-Klotho as a cofactor to enhance their binding ability to the FGF receptor (FGFR), thereby activating the FGF signaling pathway. The FGF19 / FGF21 complex with β-Klotho activates FGFR1c, FGFR2c, and FGFR3c in vitro; however, only FGF19 can activate FGFR4. Activated FGF receptors are coupled to intracellular signaling pathways, primarily including RAS-MAPK, PI3K-AKT, PLC, and STAT pathways. The FGF19 subfamily regulates various metabolic processes, with FGF19 primarily inhibiting bile acid synthesis and postprandial release in the liver. FGF21 is primarily produced in the liver, promoting lipolysis in adipocytes and ketone body production in the liver; it also promotes glucose uptake and lowers blood sugar. Therefore, FGF21 has become a very promising therapeutic agent for metabolic diseases such as obesity and type 2 diabetes. However, FGF19 and FGF21, as protein drugs, have many limitations, so analogs requiring smaller doses, longer durations of action, and higher specificity have become a research hotspot in recent years.

[0003] The evaluation of FGF21 analogues may require assessing their activation of different FGFR isoforms. Currently, the method for detecting the binding specificity of FGF19 / FGF21 analogues to different FGFR is mainly to simultaneously transfect cells with plasmids expressing different FGFR isoforms and β-Klotho plasmids. When stimulated with FGF19 / FGF21 or its analogues, the activation of the signaling pathway is evaluated by detecting the level of downstream phosphorylated ERK protein. However, this method requires re-transfection of the plasmid for each different drug, which may lead to poor reproducibility due to low transfection efficiency, varying efficiency, decreased cell viability, and cell death caused by transfection. Furthermore, the experimental procedure is complex and cannot meet the needs of high-throughput drug detection. Summary of the Invention

[0004] The purpose of this invention is to address the problems of low cell transfection efficiency, low viability, and poor reproducibility in existing FGFR specificity identification methods, and to establish a rapid and efficient method for detecting the binding specificity of FGF19 / FGF21 analog drugs to different FGFR subtypes. This invention constructs cell lines stably expressing β-Klotho and FGFR through viral infection, and screens and sorts positively expressing cells, effectively solving the problem of poor drug response caused by low transfection efficiency. Simultaneously, the established stable transfected cell lines exhibit high cell viability and stable protein expression, overcoming the problems of massive cell death and poor reproducibility caused by transient transfection methods.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for establishing a cell model to detect the selectivity of FGF19 and FGF21 analogs for FGF receptor selection includes the following steps:

[0007] (1) Construction of lentiviral transfer vectors containing the target gene β-Klotho and four types of FGFR-FGFR1c, FGFR2c, FGFR3c, and FGFR4:

[0008] A lentiviral plasmid system was used, including the psPAX2 lentiviral packaging plasmid, the pMD2.G envelope protein plasmid, and the pLVX-Puro and pLVX-IRES-ZsGreen1 viral expression vectors. Two viral expression vectors were constructed: the pLVX-Puro-β-Klotho expression vector expressing β-Klotho; and the pLVX-IRES-ZsGreen1-FGFR1c, pLVX-IRES-ZsGreen1-FGFR2c, pLVX-IRES-ZsGreen1-FGFR3c, and pLVX-IRES-ZsGreen1-FGFR4 expression vectors expressing four FGFRs. All plasmids underwent high-purity endotoxin-free extraction.

[0009] (2) Preparation of viruses expressing β-Klotho:

[0010] HEK293T cells were cultured in 15cm culture dishes until confluence reached approximately 70%. Following the instructions for the FuGENE transfection reagent, 60µl of FuGENE transfection reagent, 8µg of psPAX2, 4µg of pMD2.G, and 8µg of pLVX-Puro-βKlotho plasmid were transfected. After 24 hours, the medium was replaced with complete medium, and the cells were cultured for another 24 hours and 48 hours. Cell culture medium containing virus particles was collected at both times. The two collections of virus-containing cell culture medium were then combined.

[0011] (3) Establishment of stable cell lines overexpressing β-Klotho:

[0012] L6 rat myoblasts were cultured in 6-well plates, with 2 × 10⁶ cells per well. 5 Cells were cultured in a medium containing 8 μg / ml polybrene and an appropriate amount of virus expressing β-Klotho virus particles. After 24 hours of infection, the medium was replaced with complete medium for further culture. 48 hours after infection, the medium was replaced with 10 μg / ml puromycin for selection over a week until all uninfected control cells were killed by puromycin. Cells resistant to puromycin were expanded, and the expression efficiency of β-Klotho was detected by flow cytometry to obtain the L6-β-Klotho cell line.

[0013] (4) Preparation of viruses expressing FGFR1c, FGFR2c, FGFR3c, and FGFR4:

[0014] 8 μg psPAX2, 4 μg pMD2.G and 8 μg pLVX-IRES-ZsGreen1-FGFR1c or pLVX-IRES-ZsGreen1-FGFR2c or pLVX-IRES-ZsGreen1-FGFR3c or pLVX-IRES-ZsGreen1-FGFR4 plasmids were transfected into HEK293T cells, respectively. Four types of viral fluids were collected, and the specific method was the same as that used to prepare the virus expressing β-Klotho.

[0015] (5) Establishment of stable cells overexpressing β-Klotho and FGFR:

[0016] L6 cells overexpressing β-Klotho (L6-β-Klotho cells) were cultured in 6-well plates at 2 × 10⁶ cells per well. 5 Cells were cultured and, after adhesion, replaced with fresh medium containing 8 μg / ml polybrene and an appropriate amount of viral solution expressing FGFR1c, FGFR2c, FGFR3c and FGFR4 viral particles. After 24 hours of infection, the medium was replaced with complete medium for further culture.

[0017] Cells were labeled with the Humanβ-Klotho fluorescent antibody, and cells expressing high green fluorescence were sorted by flow cytometry to obtain L6-β-Klotho-FGFR1c, L6-β-Klotho-FGFR2c, L6-β-Klotho-FGFR3c and L6-β-Klotho-FGFR4 cell lines, respectively.

[0018] In step (1) above, the pLVX-Puro-β-Klotho expression vector expressing β-Klotho carries the puromycin resistance gene, and the pLVX-IRES-ZsGreen1-FGFR1c, pLVX-IRES-ZsGreen1-FGFR2c, pLVX-IRES-ZsGreen1-FGFR3c, and pLVX-IRES-ZsGreen1-FGFR4 expression vectors expressing four FGFRs carry the ZsGreen1 green fluorescent protein gene.

[0019] In step (3) above, the cells used are L6 rat myoblasts. Positive cells were screened using puromycin.

[0020] In step (4) above, the four transfected plasmids express the receptors of four subtypes: FGFR1c, FGFR2c, FGFR3c, and FGFR4, respectively.

[0021] In step (5) above, cells that highly express green fluorescence are sorted by flow cytometry.

[0022] The advantages of this invention are: the stable cell lines of different isoforms of β-Klotho and FGFR established do not require transfection each time, have stable protein expression, high cell viability, and good experimental reproducibility; at the same time, the experimental operation is relatively simple and convenient for high-throughput drug screening and detection. Attached Figure Description

[0023] Figure 1 This is the dose-response curve of FGF19 and FGF21 stimulation of L6-β-Klotho-FGFR1c / 2c / 3c / 4 cell lines. Detailed Implementation

[0024] Establishment of stable cell lines expressing β-Klotho and FGFRs:

[0025] A lentiviral plasmid system was used, including the psPAX2 lentiviral packaging plasmid, the pMD2.G envelope protein plasmid, and the pLVX-Puro and pLVX-IRES-ZsGreen1 viral expression vectors. Two viral expression vectors were constructed: the pLVX-Puro-β-Klotho expression vector expressing β-Klotho; and the pLVX-IRES-ZsGreen1-FGFR1c, pLVX-IRES-ZsGreen1-FGFR2c, pLVX-IRES-ZsGreen1-FGFR3c, and pLVX-IRES-ZsGreen1-FGFR4 expression vectors expressing four FGFRs. All plasmids underwent high-purity endotoxin-free extraction.

[0026] HEK293T cells were cultured in 15cm culture dishes until confluence reached approximately 70%. Following the instructions for the FuGENE transfection reagent, 60µl of FuGENE transfection reagent, 8µg of psPAX2, 4µg of pMD2.G, and 8µg of pLVX-Puro-β-Klotho plasmid were transfected. After 24 hours, the medium was replaced with complete medium, and the cells were cultured for another 24 hours and 48 hours. Cell culture medium containing virus particles was collected at both times. The two collections of virus-containing cell culture medium were then combined.

[0027] L6 rat myoblasts were cultured in 6-well plates, with 2 × 10⁶ cells per well. 5 Cells were cultured and, after adhesion, replaced with fresh medium containing 8 μg / ml polybrene and an appropriate amount of virus expressing β-Klotho virus particles. After 24 hours of infection, the medium was replaced with complete medium for further culture. 48 hours after infection, the medium was replaced with 10 μg / ml puromycin for selection over a week, until all uninfected control cells were killed by puromycin. Cells resistant to puromycin were expanded, and the expression efficiency of β-Klotho was detected by flow cytometry to obtain the L6-β-Klotho cell line.

[0028] 8 μg psPAX2, 4 μg pMD2.G and 8 μg pLVX-IRES-ZsGreen1-FGFR1c or pLVX-IRES-ZsGreen1-FGFR2c or pLVX-IRES-ZsGreen1-FGFR3c or pLVX-IRES-ZsGreen1-FGFR4 plasmids were transfected into HEK293T cells, respectively. Four types of viral fluids were collected, and the specific method was the same as that used to prepare the virus expressing β-Klotho.

[0029] L6 cells overexpressing β-Klotho (L6-β-Klotho cells) were cultured in 6-well plates at 2 × 10⁶ cells per well. 5 Cells were cultured and, after adhesion, replaced with fresh medium containing 8 μg / ml polybrene and an appropriate amount of viral solution expressing FGFR1c, FGFR2c, FGFR3c and FGFR4 viral particles. After 24 hours of infection, the medium was replaced with complete medium for further culture.

[0030] Cells were labeled with the Humanβ-Klotho fluorescent antibody, and cells expressing high green fluorescence were sorted by flow cytometry to obtain L6-β-Klotho-FGFR1c, L6-β-Klotho-FGFR2c, L6-β-Klotho-FGFR3c and L6-β-Klotho-FGFR4 cell lines, respectively.

[0031] Validation of cell models with FGF19 and FGF21 analogs exhibiting FGF receptor selectivity: Four cell lines—L6-β-Klotho-FGFR1c, L6-β-Klotho-FGFR2c, L6-β-Klotho-FGFR3c, and L6-β-Klotho-FGFR4—were seeded in 96-well plates at a density of 10,000 cells per well. The next day, the medium was replaced with serum-free medium (containing 0.2% BSA) and the cells were starved overnight. Each cell line was then stimulated with seven concentrations of FGF19 and FGF21 (300 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, and 0.032 nM) for 10-15 minutes. The supernatant was discarded, and the expression level of phosphorylated ERK protein in the cells was detected using HTRF. Figure 1 As shown, the detection results indicate that FGF19 and FGF21 can selectively bind to FGFR1c, FGFR2c, and FGFR3c and activate downstream signaling pathways; however, their binding and activation effects on FGFR4 are relatively weak.

Claims

1. A method for establishing a cell model to detect the selectivity of FGF19 and FGF21 analogs for FGF receptor, characterized in that, Includes the following steps: (1) Construct a transfer vector containing the target gene β-Klotho and four lentiviruses, wherein the four lentiviruses are FGFR-FGFR1c, FGFR2c, FGFR3c and FGFR4; Viral expression vectors were constructed using a lentiviral plasmid system. The lentiviral plasmid system included the psPAX2 lentiviral packaging plasmid, the pMD2.G envelope protein plasmid, the pLVX-Puro expression vector, and the pLVX-IRES-ZsGreen1 expression vector. The viral expression vectors included: the pLVX-Puro-β-Klotho expression vector expressing β-Klotho, the pLVX-IRES-ZsGreen1-FGFR1c expression vector expressing FGFR1c, the pLVX-IRES-ZsGreen1-FGFR2c expression vector expressing FGFR2c, and the pLVX-IRES-ZsGreen1-FGFR3c expression vector. The R3c expression vector and the pLVX-IRES-ZsGreen1-FGFR4 expression vector expressing FGFR4 were used; all plasmids were extracted with high purity and free of endotoxin; the pLVX-Puro-β-Klotho expression vector expressing β-Klotho carried the puromycin resistance gene; the pLVX-IRES-ZsGreen1-FGFR1c, pLVX-IRES-ZsGreen1-FGFR2c, pLVX-IRES-ZsGreen1-FGFR3c and pLVX-IRES-ZsGreen1-FGFR4 expression vectors expressing four FGFRs carried the ZsGreen1 green fluorescent protein gene. (2) Preparation of viruses expressing β-Klotho: HEK293T cells were cultured in 15cm culture dishes until the confluence reached approximately 70%. Following the instructions for the FuGENE transfection reagent, 60µl of FuGENE transfection reagent, 8µg of psPAX2, 4µg of pMD2.G, and 8µg of pLVX-Puro-β-Klotho plasmid were transfected. After 24 hours, the medium was replaced with complete medium, and the cells were cultured for another 24 hours and 48 hours. Cell culture medium containing virus particles was collected at each time, and the two collections of virus-containing cell culture medium were combined. (3) Establishment of stable cell lines overexpressing β-Klotho: L6 rat myoblasts were cultured in 6-well plates, with 2 × 10⁶ cells per well. 5 Cells were cultured in a medium containing 8 μg / ml polybrene and an appropriate amount of virus expressing β-Klotho virus particles. After 24 hours of infection, the medium was replaced with complete medium for further culture. 48 hours after infection, the medium was replaced with one week of selection using puromycin-containing medium. The uninfected control cells were killed by puromycin. Cells resistant to puromycin were then expanded, and the expression efficiency of β-Klotho was detected by flow cytometry to obtain the L6-β-Klotho cell line. Positive cells were selected using puromycin. (4) Preparation of viruses expressing FGFR1c, FGFR2c, FGFR3c and FGFR4: HEK293T cells were transfected with 8 μg psPAX2, 4 μg pMD2.G, and 8 μg FGFR viral expression vectors, respectively. Viral fluid was collected using the same method as for preparing the virus expressing β-Klotho. The FGFR viral expression vectors were pLVX-IRES-ZsGreen1-FGFR1c, pLVX-IRES-ZsGreen1-FGFR2c, pLVX-IRES-ZsGreen1-FGFR3c, or pLVX-IRES-ZsGreen1-FGFR4 plasmids. The four transfected plasmids expressed FGFR1c, FGFR2c, FGFR3c, and FGFR4, respectively. (5) Establishment of stable cells overexpressing β-Klotho and FGFR: L6 cells overexpressing β-Klotho (L6-β-Klotho cells) were cultured in 6-well plates at 2 × 10⁶ cells per well. 5 Cells were cultured and attached to the culture medium. After attachment, the medium was replaced with fresh medium containing 8 μg / ml polybrene and an appropriate amount of virus solution expressing FGFR1c, FGFR2c, FGFR3c and FGFR4 virus particles was added. After 24 hours of infection, the medium was replaced with complete medium and cultured for another 24 hours. (6) Human β-Klotho fluorescent antibody was used to label cells, and cells expressing high green fluorescence were sorted out to obtain L6-β-Klotho-FGFR1c cell line, L6-β-Klotho-FGFR2c cell line, L6-β-Klotho-FGFR3c cell line and L6-β-Klotho-FGFR4 cell line, respectively; cells expressing high green fluorescence were sorted out by flow cytometry.

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