Adipose cell, white fat browning cell model, construction method and application

Abstract

The invention provides an adipocyte, a white adipose browning cell model and a construction method and application, and relates to the technical field of biological medicine, the adipocyte contains an exogenous nucleic acid construct for specifically inhibiting Kdm7a gene expression; expression of the endogenous Kdm7a gene in fat cells is inhibited and an uncoupling protein 1 is expressed. By specifically inhibiting the expression of Kdm7a, the adipocyte can obviously express the uncoupling protein 1 and presents a stable brown adipotype, so that the defects of limited cell source, complex induction and high heterogeneity in the prior art are effectively overcome; a standardized model which is simple and convenient to operate, wide in source and definite in function is provided for metabolic disease mechanism research and drug screening.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a kind of fat cell, white fat brown cell model and construction method and application. BACKGROUND

[0002] Obesity and its triggered type 2 diabetes, fatty liver and cardiovascular diseases and other metabolic syndromes have evolved into a global major public health challenge. The adipose tissue in mammals is mainly divided into white adipose tissue (WAT) and brown adipose tissue (BAT). The former is responsible for storing excess energy, and the latter consumes energy to produce heat through high expression of uncoupling protein 1 (UCP1). Activating or increasing brown fat in the body is considered one of the ideal strategies for treating obesity. However, the number of metabolically active brown fat in adult humans is extremely limited, and how to effectively obtain functional brown adipocytes in vitro or in vivo is the focus of current metabolic research.

[0003] The existing technology to obtain brown adipocytes mainly includes: isolating primary brown precursor adipocytes from the shoulder region of animals; using pluripotent stem cells for directional differentiation; and inducing white adipocytes to "brown" by drug stimulation. In addition, at the molecular mechanism research level, epigenetic regulatory factors such as lysine demethylase 7A (KDM7A) are known to remove methylation modifications at specific histone sites, and existing literature reports that it is involved in neural differentiation and the occurrence of some tumors.

[0004] However, the above-mentioned existing means has limitations in actual application. The primary cell separation method is limited by tissue source, and the cell acquisition amount is small, the expansion ability is poor, and the individual difference is large, which is not suitable for large-scale screening; although the pluripotent stem cell induction differentiation has high theoretical yield, the process is complicated, the cycle is long, the cost is high, and the efficiency is unstable; the existing drug-induced "browning" scheme often has low conversion efficiency, and the obtained cell function is not perfect. For KDM7A, although its biochemical activity in the field of nerves or tumors has been recorded, the existing technology has not disclosed its specific function in adipocyte differentiation, especially directional differentiation into brown adipocytes, and the related application is still blank.

[0005] In summary, there is a lack of a widely sourced, highly efficient, simple to operate and stable function brown adipocyte acquisition method in the prior art. There is an urgent need in the field to develop new experimental models to overcome the limitations of cell source, high cost and large heterogeneity in the prior art, thereby providing a reliable tool for high-throughput screening of anti-obesity drugs and in-depth research on the pathogenesis of metabolic diseases.

[0006] Therefore, the present application is proposed. SUMMARY

[0007] The present application aims to provide a cell model for white adipocyte browning and a construction method and application, wherein the adipocyte is induced to present a high expression of uncoupling protein 1 by specific inhibition of Kdm7a expression, thereby solving the problems of difficulty in obtaining brown adipocytes and instability of functions, and providing a standardized biological tool for metabolic disease research and drug screening.

[0008] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted: In a first aspect, the present application provides a genetically modified adipocyte, wherein the adipocyte comprises an exogenous nucleic acid construct for specifically inhibiting expression of a Kdm7a gene. The expression of the endogenous Kdm7a gene in the adipocyte is inhibited, and the adipocyte expresses uncoupling protein 1.

[0009] In an optional embodiment, the nucleic acid construct is an expression vector for expressing small interfering RNA or short hairpin RNA, or a CRISPR-Cas system capable of knocking out or inhibiting the Kdm7a gene through gene editing technology.

[0010] In an optional embodiment, the nucleic acid construct is a lentivirus vector for expressing shRNA.

[0011] In an optional embodiment, the shRNA comprises a nucleotide sequence targeting the Kdm7a gene. Further, the coding sequence of the shRNA comprises a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] In an optional embodiment, the adipocyte is derived from a white precursor adipocyte of a mammal. Preferably, the adipocyte is derived from 3T3-L1 cells of a mouse.

[0013] In an optional embodiment, the adipocyte further expresses at least one brown fat specific marker selected from type II iodothyronine deiodinase and cell death-inducing DFFA-like effector a.

[0014] In a second aspect, the present application provides a cell model for white adipocyte browning, wherein the cell model is composed of the genetically modified adipocyte according to the foregoing embodiments.

[0015] In a third aspect, the present application provides a construction method of the genetically modified adipocyte according to any one of the foregoing embodiments, comprising: providing a nucleic acid construct capable of specifically inhibiting expression of the Kdm7a gene; The nucleic acid construct was introduced into mammalian preadipocytes; The precursor adipocytes were cultured and induced to differentiate. Adipocytes with suppressed Kdm7a gene expression and gene modification expressing uncoupling protein 1 were obtained through screening, thus obtaining the cell model.

[0016] Fourthly, the present invention provides a recombinant expression vector for inducing brown adipocyte differentiation, wherein the recombinant expression vector contains an shRNA coding sequence targeting the Kdm7a gene; Preferably, the coding sequence of the shRNA comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] Fifthly, the present invention provides the use of the white adipose tissue browning cell model as described in any of the foregoing embodiments in screening drugs for the treatment of obesity, type 2 diabetes or metabolic syndrome.

[0018] In a sixth aspect, the present invention provides the use of the cell model of white adipose tissue browning as described in any of the foregoing embodiments in the preparation of a kit for studying lipid metabolism mechanisms or screening drugs that promote brown adipose tissue browning.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: By introducing exogenous nucleic acid constructs into adipocytes to specifically inhibit the expression of lysine demethylase 7A (Kdm7a), the level of endogenous Kdm7a in cells can be effectively reduced, thereby relieving transcriptional repression of a specific gene at the epigenetic level and inducing significant expression of uncoupling protein 1 (UCP1). This gene modification strategy directly promotes the transformation of adipocytes into brown adipocytes with thermogenic function, successfully establishing a functionally defined brown adipocyte model.

[0020] Compared to traditional primary tissue isolation or complex stem cell-directed differentiation methods, this gene-modified cell method avoids the cumbersome and unstable induction process, solving the problems of scarce brown adipocyte sources, high heterogeneity, and difficulty in large-scale expansion in existing methods. The obtained cells possess stable molecular characteristics and functional phenotypes, serving as a high-fidelity, standardized in vitro research tool. This provides a new experimental vehicle for elucidating the pathological mechanisms of obesity and metabolic syndrome, and also offers widely available and homogeneous biological materials for high-throughput screening of related therapeutic drugs. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 A schematic diagram of the shRNA lentiviral vector (PlKO.1-shKdm7a) ​​targeting the Kdm7a gene constructed in an embodiment of the present invention; Figure 2 This is a Sanger sequencing alignment result of the shRNA insert fragment in the recombinant plasmid in an embodiment of the present invention; wherein, the above sequence is the sequencing result, and the following sequence is the designed shRNA antisense strand template sequence, and the results show that the sequences are completely identical; Figure 3 This is a bar chart showing the relative expression level of Kdm7a mRNA in 3T3-L1 cells after lentiviral infection, detected by real-time quantitative PCR (qRT-PCR) in an embodiment of the present invention; the results show that the shKdm7a group was significantly lower than the shControl group; Figure 4 This is a bar chart showing the relative expression levels of brown adipose-specific markers (Ucp1, Dio2, and Cidea) mRNA in 3T3-L1 cells after Kdm7a knockdown, as detected by real-time quantitative PCR (qRT-PCR) in an embodiment of the present invention. Figure 5 This is a microscopic fluorescence image of UCP1 protein expression and localization in 3T3-L1 cells after Kdm7a knockdown, as shown in an embodiment of the present invention; it shows that UCP1 protein (specific fluorescence signal) is highly expressed in the knockdown group cells. Detailed Implementation

[0023] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] This application provides a genetically modified adipocyte containing an exogenous nucleic acid construct that specifically inhibits the expression of the Kdm7a gene; the expression of the endogenous Kdm7a gene in the adipocyte is inhibited, and the adipocyte expresses uncoupling protein 1.

[0025] The aforementioned "genetically modified adipocytes" refer to adipocytes that have undergone artificial genetic manipulation, distinguishing them from naturally occurring wild-type cells.

[0026] The aforementioned "exogenous nucleic acid constructs that specifically inhibit Kdm7a gene expression" refers to genetic material fragments or vector systems artificially introduced into cells. "Exogenous" indicates that the substance is not native to the cell but introduced through transfection, infection, or other means; "specifically inhibits Kdm7a" means that the construct can specifically reduce or silence the gene function of lysine demethylase 7A (Kdm7a) ​​without significantly affecting other genes.

[0027] The aforementioned "inhibition of endogenous Kdm7a gene expression" is a result of the effects of the aforementioned constructs, meaning that the level of Kdm7a mRNA or protein that the cell should normally express is significantly reduced or absent.

[0028] The aforementioned expression of uncoupling protein 1 (UCP1) represents the final functional phenotype exhibited by this cell. UCP1 is a key marker protein in brown adipocytes, responsible for thermogenic function.

[0029] By introducing an exogenous inhibitor into the cell to block Kdm7a expression, the regulation (such as inhibition) of specific gene programs within the cell by Kdm7a is relieved, thereby activating the brown adipose-specific molecular program and enabling the cell to express UCP1. This setup transforms cells that originally lacked or expressed low levels of UCP1 into cells with brown adipose-like characteristics, solving the problem of difficulty in obtaining functional brown adipocytes.

[0030] In some embodiments, the nucleic acid construct is an expression vector expressing small interfering RNA or short hairpin RNA, or a CRISPR-Cas system capable of knocking out or inhibiting the Kdm7a gene using gene editing technology.

[0031] The aforementioned "expression vectors for small interfering RNA (siRNA) or short hairpin RNA (shRNA)" refer to tools that utilize RNA interference (RNAi) mechanisms. These vectors can transcribe specific RNA fragments within cells, which then degrade Kdm7a mRNA through complementary pairing, thereby inhibiting gene expression at the posttranscriptional level.

[0032] The aforementioned "CRISPR-Cas system" refers to a tool that utilizes gene editing technology (such as CRISPR-Cas9). This system can cut or modify the Kdm7a gene at the genomic DNA level, thereby disrupting its gene structure or inhibiting its transcription.

[0033] This embodiment provides a specific and mature technical implementation path for achieving Kdm7a inhibition. Whether through RNA-level degradation (siRNA / shRNA) or DNA-level editing (CRISPR), the aforementioned inhibition effect can be effectively achieved, providing flexibility and diversity in the implementation of the technical solution.

[0034] In some embodiments, the nucleic acid construct is a lentiviral vector expressing shRNA.

[0035] The aforementioned "lentiviral vector" is a viral vector tool capable of integrating exogenous genes into the host cell genome. Compared to vectors used for transient transfection, lentiviral vectors can stably integrate sequences encoding shRNA into the genome of adipocytes, achieving long-term, sustained expression of shRNA. This ensures that the inhibition of the Kdm7a gene is persistent and stable, thereby enabling the obtained genetically modified adipocytes to maintain the brown adipose phenotype for a long period, facilitating the construction of stable cell line models.

[0036] In some embodiments, the shRNA contains a nucleotide sequence that targets the Kdm7a gene.

[0037] Furthermore, the coding sequence of the shRNA comprises a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0038] The aforementioned "nucleotide sequence containing the Kdm7a gene" refers to a core sequence in the shRNA that is complementary to the Kdm7a gene sequence, which is the key to achieving "specificity".

[0039] For specific examples of preferred nucleotide sequences mentioned above, please refer to Table 1: Table 1. Nucleotide Sequences

[0040] In Table 1, NO. represents the sequence number "SEQ ID NO:".

[0041] This embodiment identifies specific target sequences that can efficiently identify and bind to Kdm7a mRNA. Using these validated sequences maximizes the knockdown efficiency of Kdm7a while minimizing off-target effects, ensuring uniform and reliable cell model traits.

[0042] In some embodiments, the adipocytes are derived from mammalian white precursor adipocytes; Furthermore, the adipocytes are derived from mouse 3T3-L1 cells.

[0043] The aforementioned "white precursor adipocytes of mammals" indicates that the initial state of the cell is the precursor cell of the white adipocyte lineage, which is responsible for energy storage.

[0044] The aforementioned "3T3-L1 cells from mice" is a specific preferred cell line, which is a standard proadipocyte line that is easy to culture and induce.

[0045] This embodiment demonstrates that the technical solution can achieve cell fate conversion, that is, using widely available and easily cultured white precursor adipocytes (such as 3T3-L1) as a "chassis" and modifying them to transform them into rare brown adipocytes. This takes advantage of the growth of common cell lines and overcomes the shortcomings of directly obtaining a small number of brown adipocytes and the difficulty in expansion.

[0046] In some embodiments, the adipocytes also express at least one brown fat-specific marker selected from type II iodothyronine deiodinase and cell death-inducing DFFA-like effector protein a.

[0047] The aforementioned "type II iodothyronine deiodinase (Dio2)" and "cell death-induced DFFA-like effector protein a (Cidea)" are both gene products that are unique to or highly expressed in brown adipocytes, and together with UCP1, they constitute the molecular characteristic profile of brown adipose tissue.

[0048] In this embodiment, the cells were limited to expressing not only UCP1 but also Dio2 or Cidea, meaning that the cells possessed more comprehensive characteristics of brown adipocytes at the molecular level. This further confirms that the cells induced by Kdm7a knockdown do not simply express UCP1, but rather initiate a more complete brown adipose tissue differentiation program, thus ensuring the realism and accuracy of this cell model in simulating brown adipose tissue function in vivo.

[0049] This application also provides a cell model of white adipose tissue turning brown, the cell model being composed of genetically modified adipocytes as described in the foregoing embodiments.

[0050] This application embodiment also provides a method for constructing a cell model of white adipose tissue turning brown as described in the foregoing embodiments, including: Step S1: Provide a nucleic acid construct that can specifically inhibit the expression of the Kdm7a gene.

[0051] This step refers to preparing the core "tools" used for gene manipulation. The "nucleic acid construct" is an artificially designed vector of genetic material specifically designed to target the Kdm7a gene.

[0052] Through molecular cloning or gene synthesis techniques, plasmids or viral vectors containing interfering sequences (such as shRNA coding sequences) or editing components (such as CRISPR guide RNA) targeting the Kdm7a gene are obtained. This provides the material basis for subsequent transfection or infection.

[0053] Specifically, a specific shRNA sequence can be designed based on the known Kdm7a gene sequence and inserted into a lentiviral backbone vector (such as PlKO.1) using restriction endonucleases and ligases; or an expression plasmid containing Cas9 protein and specific sgRNA can be constructed.

[0054] Step S2: The nucleic acid construct is introduced into the precursor fat cells of mammals.

[0055] This step refers to the "delivery" process, which involves introducing the prepared genetic tool into the target cell. The target cell is limited to "mammalian preadipocytes," that is, undifferentiated adipocytes with differentiation potential. Biological, physical, or chemical methods are used to break down the cell membrane barrier, allowing the nucleic acid construct to enter the nucleus or cytoplasm. The result is that the target preadipocyte contains exogenous genetic material capable of inhibiting Kdm7a.

[0056] If the construct is a lentiviral vector, viral particles can be packaged into packaging cells (such as HEK293T) and then used to infect precursor adipocytes (such as 3T3-L1). Polybrene is usually used in conjunction to improve infection efficiency. If it is a regular plasmid, transfection can be performed using liposome transfection reagents (such as Lipo3000) or electroporation technology.

[0057] Step S3: Culture and induce differentiation of the precursor adipocytes.

[0058] This step refers to the "transformation" process of cells. After introducing the construct, a specific growth environment and signals are needed to induce the precursor cells to initiate a differentiation process and develop into mature adipocytes. This is achieved by adding culture medium containing differentiation-inducing factors (such as insulin, dexamethasone, isobutylmethylxanthine, and other common inducers) to cell culture dishes. During this process, cell morphology changes, and due to the effect of the construct introduced in step S2, the expression of Kdm7a within the cells is continuously suppressed.

[0059] Specifically, a strategy of "induction after contact inhibition" can be adopted, that is, after the cells grow to the point of contact inhibition, the culture medium is switched to induction medium for several days, and then the culture medium is switched to maintenance medium until lipid droplets appear in the cells.

[0060] Step S4: Select adipocytes with suppressed Kdm7a gene expression and gene modification expressing uncoupling protein 1 to obtain the cell model of white adipose tissue turning brown.

[0061] This step is the "verification and acquisition" process. Specific detection indicators are used to confirm whether the construct is effective and whether the cell fate has undergone the expected change, thus obtaining the processed cell population. On one hand, the mRNA or protein level of Kdm7a is detected to confirm whether it is significantly lower than the control group (i.e., "expression is inhibited"); on the other hand, the expression of UCP1 is detected (i.e., whether brown adipose tissue is acquired). Only cells that simultaneously meet both conditions are the target product required by this method.

[0062] This step uses dual-indicator screening to exclude false-positive cells that have failed transfection or have not been fully induced, ensuring that the final cell model has consistency and reliability in genotype (Kdm7a KD) and phenotype (UCP1 High).

[0063] Specifically, gene expression levels can be detected using real-time quantitative PCR (qRT-PCR), or protein expression levels can be detected using immunofluorescence staining and Western blotting.

[0064] This application embodiment also provides a recombinant expression vector for inducing brown adipocyte differentiation, wherein the recombinant expression vector contains an shRNA coding sequence targeting the Kdm7a gene; Furthermore, the coding sequence of the shRNA comprises a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0065] The aforementioned "recombinant expression vector" refers to DNA molecules (such as plasmids and viral vectors) that have been modified by artificial gene recombination technology and possess functional elements (such as promoters and terminators) that can replicate and transcribe within host cells.

[0066] The aforementioned "shRNA coding sequence containing the Kdm7a gene" is the core functional component of this vector. It refers to a specific DNA sequence that, after transcription, can form a short hairpin structure (shRNA). This structure, after intracellular enzymatic cleavage, can produce small interfering RNAs that are complementary to Kdm7a mRNA.

[0067] This application also provides the application of the white adipose tissue browning cell model as described in any of the foregoing embodiments in screening drugs for the treatment of obesity, type 2 diabetes or metabolic syndrome.

[0068] The genetically modified adipocytes provided in this embodiment can serve as a highly efficient and stable in vitro cell model for screening drugs for obesity and metabolic diseases. Specifically, because these cells have established a stable brown adipose tissue-like phenotype (highly expressing UCP1) by inhibiting Kdm7a expression, they can mimic the thermogenesis and metabolic functions of brown adipose tissue in vivo. In drug screening applications, candidate compounds are contacted with these cells, and by detecting changes in UCP1 expression levels, mitochondrial functional indicators (such as oxygen consumption), lipid metabolism, or the activation status of browning-related signaling pathways, active molecules that can further enhance browning, improve mitochondrial function, or mimic the inhibitory effect of Kdm7a can be rapidly identified. This application overcomes the problem of low drug screening efficiency caused by the lack of high-quality human or mouse brown adipocyte models, providing a reliable biological evaluation platform for developing novel drugs for treating obesity, type 2 diabetes, or metabolic syndrome.

[0069] This application also provides the application of the cell model of white adipose tissue browning as described in any of the foregoing embodiments in the preparation of kits for studying lipid metabolism mechanisms or screening drugs that promote brown adipose tissue browning.

[0070] The genetically modified adipocytes provided in this embodiment can also be used to prepare research kits specifically for studying lipid metabolism mechanisms or screening drugs that promote brown adipose tissue formation. In this application, the genetically modified cells serve as the core component of the kit (e.g., provided in cryopreserved and thawed form or in situ culture form) and can be combined with specific induction media, detection primers, antibodies, or staining reagents. Based on the unique molecular characteristics of these cells—namely, the suppression of endogenous Kdm7a expression and the presence of brown adipose tissue-specific markers—kits prepared using these cells can provide researchers with a standardized experimental system for in-depth analysis of the specific molecular mechanisms of Kdm7a-mediated epigenetic regulation in adipocyte fate determination, or for standardized evaluation of the browning potential of different compounds. This not only lowers the barrier to building experimental models but also significantly improves the reproducibility and comparability of experimental data from different batches by providing a homogeneous cell source.

[0071] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0072] Example 1: Construction of a lentiviral vector for shRNA targeting the Kdm7a gene This embodiment describes the process of constructing a recombinant lentiviral vector that specifically inhibits the expression of the mouse Kdm7a gene.

[0073] 1. shRNA sequence design and synthesis: Specific shRNA target sequences were designed for the mouse Kdm7a gene, and the corresponding sense (F) and antisense (R) oligonucleotides were synthesized. The sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0074] mKdm7a-shRNA-F1 (as shown in SEQ ID NO: 1); mKdm7a-shRNA-R1 (as shown in SEQ ID NO: 2).

[0075] 2. Oligonucleotide annealing: Dissolve the synthesized dry powdered sense and antisense oligonucleotides to a concentration of 50 μM. Take 5 μL of each into a 0.2 mL EP tube for denaturation and annealing.

[0076] The annealing system is shown in Table 2: Table 2. Annealing System

[0077] PCR reaction conditions: 95℃ for 1 min, 50℃ for 2 min, then naturally cooled to room temperature. After incubation at 5℃ for 10 min, the concentration was detected using NanoDrop, and the sample was stored at 4℃ for later use.

[0078] 3. Vector digestion and recovery: The PlKO.1 plasmid was selected as the backbone vector and double-digested with EcoRI and AgeI restriction endonucleases.

[0079] The enzyme digestion system is shown in Table 3: Table 3. Enzyme digestion system

[0080] After overnight enzyme digestion in a 37°C water bath, gel extraction was performed to obtain the linearized vector fragment.

[0081] 4. Ligation and transformation: The annealed shRNA fragments were ligated into the linearized PlKO.1 vector using T4 DNA ligase.

[0082] The connection system is shown in Table 4: Table 4. Connection System

[0083] Ligation procedure: Incubate at 25°C for 4 hours, incubate at 65°C for 10 minutes to inactivate, and store at 4°C. Transform the ligation product into competent E. coli, and select single colonies for plasmid mini-prep (Tiangen, DP103).

[0084] 5. Identification: The extracted plasmids were verified by Sanger sequencing.

[0085] The results are as follows Figure 1 The image shown is a map of the successfully constructed Kdm7a-shRNA viral vector. Figure 2 The sequencing alignment results shown indicate that the shRNA sequence has been correctly inserted into the vector and the sequence is error-free, and the recombinant plasmid shKdm7a-1 has been successfully obtained.

[0086] Example 2: Packaging and establishment of an adipocyte model of Kdm7a knockdown lentivirus In this embodiment, the recombinant plasmid obtained in Example 1 was used to package lentivirus and infect 3T3-L1 precursor adipocytes.

[0087] 1. Cell preparation: Day 1: HEK293T cells were prepared at a rate of 2 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 70%-90% per well in 6-well plates and cultured until the second day when the confluence reached 70%-90%. Simultaneously, 3T3-L1 cells were prepared, with their density reaching approximately 70% the day before infection.

[0088] 2. Virus packaging (transfection): Day 2: Transfection was performed using a three-plasmid system (shKdm7a-1 : psPAX2 : pMD2.G) in conjunction with Lipo3000 reagent.

[0089] Transfection system (6-well plate, per well): (1) EP tube 1: 125 μL Opti-MEM + 3.7 μL Lipo3000 (2) EP tube 2: 125 μL Opti-MEM + 1250 ng shKdm7a-1 (or control shControl) + 832.5 ng psPAX2 + 417.5 ng pMD2.G + 5 μL P3000 Reagent Add the mixture from EP tube 1 dropwise into EP tube 2, vortex for 5 seconds, centrifuge for 30 seconds, and let stand at room temperature for 15 minutes.

[0090] Medium change: Aspirate HEK293T medium and add 2 ml of antibiotic-free DMEM. Add the transfection mixture dropwise to the cells, mix well, and then place in an incubator.

[0091] Day 3: Replace with fresh antibiotic-free DMEM 6 hours after transfection.

[0092] 3. Virus collection and infection: Day 4: Collect the supernatant of transfected HEK293T cells, centrifuge at 1,250 rpm for 5 minutes to remove cell debris, and filter through a 0.45 μm filter.

[0093] Add polybrene to the filtrate to a final concentration of 8 μg / ml and mix thoroughly.

[0094] Remove the old culture medium from the 3T3-L1 cells, wash them once with PBS, and then add the above-mentioned culture medium containing virus and Polybrene for infection.

[0095] 4. Model cell acquisition: Day 5: 24 hours after infection, replace with fresh complete culture medium. Continue culturing and add puromycin as needed for selection to obtain stably transduced Kdm7a knockdown adipocyte lines.

[0096] Example 3: Detection of Kdm7a knockdown efficiency and browning marker expression in a cell model using qPCR This embodiment verifies the model construction effect through real-time quantitative PCR (qRT-PCR).

[0097] 1. Experimental Method: 3T3-L1 cells from the shKdm7a-1 infection group constructed in Example 2 and control cells were collected. Total RNA was extracted from the cells using the Trizol method and cDNA was synthesized by reverse transcription.

[0098] qRT-PCR was performed using the SYBR Green dye method. The reaction procedure was performed according to the kit instructions.

[0099] The primer sequences used are shown in Table 5 below: Table 5. Primer sequences

[0100] In Table 5, NO. represents the sequence number "SEQ ID NO:".

[0101] 2. Experimental Results: Kdm7a expression detection: such as Figure 3 As shown, compared with the control group, the mRNA expression level of Kdm7a in the shKdm7a-1 group was significantly reduced, proving that the gene knockdown model was successfully constructed.

[0102] Detection of browning markers: such as Figure 4 As shown, after Kdm7a was knocked down, the mRNA expression levels of brown adipose tissue-specific marker genes Ucp1, Dio2, and Cidea all increased significantly. This indicates that inhibiting Kdm7a successfully induced the differentiation of white precursor adipocytes into brown adipocytes.

[0103] Example 4: Detection of UCP1 protein expression and localization in a cell model using immunofluorescence. This embodiment further verifies the browning characteristics of cells at the protein level using immunofluorescence technology.

[0104] 1. Experimental Methods: 3T3-L1 cells successfully infected with shKdm7a-1 were cultured at 6×10⁻⁶ cells / year. 4 Cells / wells were seeded in 24-well plates with pre-placed climbing plates.

[0105] Fixation and permeabilization: Wash with PBS, fix with 4% PFA at room temperature for 15 min; permeabilize with 0.3% Triton X-100 for 15 min.

[0106] Closed: Incubate with 5% BSA at room temperature for 1 hour.

[0107] Primary antibody incubation: Add diluted primary antibody (Ucp1, ABclonal, A21979, 1:100) dropwise and incubate overnight at 4°C in a humidified chamber.

[0108] Secondary antibody incubation: After washing with PBS, add fluorescent secondary antibody (Proteintech, SA00013-4, 1:500) and incubate at room temperature in the dark for 1 hour.

[0109] Nuclear staining and mounting: Incubate with Hochest staining solution at room temperature for 10 min, wash with PBS, and mount with anti-fluorescence quenching mounting medium.

[0110] 2. Experimental Results: The prepared slides were observed under a fluorescence microscope. For example... Figure 5 As shown, blue fluorescence indicates nuclear localization (Hochest), while red / green fluorescence (depending on the secondary antibody type) indicates UCP1 protein. The results showed that UCP1 protein was highly expressed in Kdm7a knockdown cells and located in the mitochondrial / cytoplasmic region, further confirming that this cell model possesses typical brown adipocyte characteristics.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A genetically modified adipocyte, characterized in that, The adipocytes contain an exogenous nucleic acid construct that specifically inhibits the expression of the Kdm7a gene; The expression of the endogenous Kdm7a gene in the adipocytes was suppressed, and the adipocytes expressed uncoupling protein 1.

2. The genetically modified adipocytes as described in claim 1, characterized in that, The nucleic acid construct is an expression vector that expresses small interfering RNA or short hairpin RNA, or a CRISPR-Cas system that can knock out or inhibit the Kdm7a gene through gene editing technology.

3. The genetically modified adipocytes as described in claim 2, characterized in that, The nucleic acid construct is a lentiviral vector expressing shRNA.

4. The genetically modified adipocytes as described in claim 3, characterized in that, The shRNA contains a nucleotide sequence that targets the Kdm7a gene; Preferably, the coding sequence of the shRNA comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

5. The genetically modified adipocytes as described in claim 1, characterized in that, The adipocytes are derived from mammalian white precursor adipocytes; preferably, the adipocytes are derived from mouse 3T3-L1 cells; and / or, The adipocytes also express at least one brown fat-specific marker selected from type II iodothyronine deiodinase and cell death-inducing DFFA-like effector protein a.

6. A cell model of white adipose tissue turning brown, characterized in that, The cell model is composed of genetically modified adipocytes as described in any one of claims 1-5.

7. A method for constructing a cell model of white adipose tissue browning as described in claim 6, characterized in that, include: Provide nucleic acid constructs that can specifically inhibit Kdm7a gene expression; The nucleic acid construct was introduced into mammalian preadipocytes; The precursor adipocytes were cultured and induced to differentiate. Adipocytes with suppressed Kdm7a gene expression and gene modification expressing uncoupling protein 1 were obtained through screening, thus obtaining the cell model.

8. A recombinant expression vector for inducing brown adipocyte differentiation, characterized in that, The recombinant expression vector contains an shRNA coding sequence targeting the Kdm7a gene; Preferably, the coding sequence of the shRNA comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

9. The use of the white adipose tissue browning cell model as described in claim 7 in screening drugs for the treatment of obesity, type 2 diabetes or metabolic syndrome.

10. The use of the cell model of white adipose tissue browning as described in claim 7 in the preparation of a kit for studying lipid metabolism mechanisms or screening drugs that promote brown adipose tissue browning.

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