Application of Sdccag3 gene in preparation of medicine for regulating adipose tissue generation and metabolism

Abstract

The invention relates to application of an Sdccag3 gene in preparation of a medicine for regulating adipose tissue generation and metabolism. According to the application, the Sdccag3 gene serves as an action target to be applied to preparation or screening of drugs for regulating and controlling adipose tissue generation and metabolism, and the nucleotide sequence of the Sdccag3 gene is shown as SEQ ID No.1. The invention firstly verifies the influence of Sdccag3 overexpression on precursor fat cell differentiation, and finds that the overexpressed Sdccag3 gene can significantly promote adipogenic differentiation of precursor fat cells. The influence of the Sdccag3 adipose tissue specific overexpression adeno-associated virus vector on adipose tissue generation and metabolism in a mouse body is verified, the overexpression Sdccag3 gene is found to be capable of remarkably promoting adipose generation and metabolism of an overnutrition mouse, and the adipose tissue specific overexpression Sdccag3 can also remarkably promote cell proliferation of the adipose tissue; therefore, a theoretical basis is provided for preparing or screening medicines or preparations for promoting the expression of the Sdccag3 gene as alternative medicines or preparations for regulating and controlling the generation and metabolism of adipose tissues.

Description

Technical Field

[0001] The present invention relates to the application of the Sdccag3 gene in the preparation of drugs for regulating adipose tissue generation and metabolism, and belongs to the fields of molecular biology and biotechnology. Background Art

[0002] Human adipose tissue is divided into brown adipose tissue and white adipose tissue. Among them, brown adipose tissue is an organ mainly for generating heat; white adipose tissue, as the largest energy storage depot in the body, is the main adipose tissue regulating the whole-body energy balance homeostasis. White adipose tissue stores excess energy when the body's energy supply is greater than demand and supplies it when the body needs it.

[0003] White adipose tissue is further divided into subcutaneous fat and visceral fat. In contrast, visceral fat is a better predictor of metabolic diseases (such as diabetes, hypertension, and dyslipidemia). The possible reason is that visceral fat can directly enter the portal vein system, while subcutaneous white fat cannot. The expansion caused by the increase in adipose tissue can be driven by the increase in the size of adipocytes (hypertrophy) or the differentiation of new adipocytes from precursor cells during adipogenesis (hyperplasia). The balance between the two has an important impact on metabolic health. Multiple studies have pointed out that hypertrophic adipocytes are related to systemic insulin resistance, and the increased mechanical pressure and hypoxia in themselves cause inflammation in adipose tissue; while small adipocytes show an important functional role in counteracting the metabolic disorders related to overnutrition. The hypertrophy of adipocytes is considered to increase the risk of diseases. Currently, more and more studies have proposed that the metabolic decline related to overnutrition is not simply caused by itself, but due to the lack of the ability of adipose tissue to further expand, which indicates that it is particularly important to selectively promote the hyperplasia rather than the hypertrophy of adipocytes.

[0004] Peroxisome proliferator-activated receptors (PPARs) belong to the subfamily of transcription factors in the nuclear hormone receptor superfamily, and their family members play their respective functional roles in the regulation of energy homeostasis, including three subtypes: PPARα, PPARγ, and PPARδ. Among them, PPARα is mainly expressed in the liver, heart, skeletal muscle, brown adipose tissue (BAT), intestine, and kidney, and its function is to affect fatty acid transport, esterification, and oxidation; PPARβ / δ is expressed throughout the body and affects the regulation of fatty acid oxidation and blood glucose levels; while in white adipose tissue, PPARγ has the highest expression level. By participating in adipogenesis and lipid synthesis, it promotes energy storage, affects lipid metabolism and energy homeostasis. PPARγ can regulate a variety of impaired biological processes, including inflammation, lipid and glucose metabolism, and overall energy homeostasis. Mice lacking PPARγ cannot develop all types of fat. Therefore, PPARγ is crucial for the development and function of adipose tissue and has become a target for the treatment of various lipid metabolism diseases in humans. Clinically, PPARγ agonists have been used to treat lipid metabolism disorders and control insulin sensitivity in diabetic patients. The most typical drugs are thiazolidinediones (TZD), including pioglitazone and rosiglitazone. However, due to controversial reports that they increase the risk of myocardial infarction (rosiglitazone) and cause bladder cancer (pioglitazone), as well as weight gain, fluid retention, atypical fractures, etc., these unacceptable side effects limit their use. Therefore, developing a class of drugs with effective PPARγ activation and avoiding the side effects caused by activation remains a great challenge.

[0005] Serologically defined colon cancer antigen 3 (sdccag3) has currently been found to have biological functions such as protein transport, mitosis, ciliogenesis, and promoting osteogenesis. The applicant's previous work has confirmed that sdccag3 can regulate the expression of PPARγ in different cells. In terms of metabolic diseases, only in a literature report in 2021, the author used a dual-system genetics method to determine that SDCCAG3 is one of the new possible common candidate genes for type 1 diabetes and type 2 diabetes. However, there is no research on how SDCCAG3 functions in adipose metabolism-related diseases, and there is no research on the effect of Sdccag3 on adipose tissue generation and metabolism. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides the application of the Sdccag3 gene in the preparation of drugs for regulating adipose tissue generation and metabolism. The present invention verified that it can regulate the generation and metabolism of adipose tissue in mice by constructing an adipose tissue-specific overexpression adenovirus-associated virus vector of Sdccag3, and the specific overexpression adenovirus-associated virus vector can be used as a drug for regulating adipose tissue generation and metabolism and promoted and applied in related fields.

[0007] The technical solution of the present invention is as follows:

[0008] Use of the Sdccag3 gene in the preparation of drugs for regulating adipose tissue generation and metabolism, wherein the nucleotide sequence of the Sdccag3 gene is as shown in SEQ ID No.1.

[0009] Preferably according to the present invention, the use of the Sdccag3 gene in the preparation of drugs for regulating adipose tissue generation and metabolism includes the following two aspects:

[0010] (1) Using the Sdccag3 gene as a target for use in the preparation of drugs for regulating adipose tissue generation and metabolism;

[0011] (2) Using the Sdccag3 gene as a target for use in the screening of drugs for regulating adipose tissue generation and metabolism.

[0012] Preferably according to the present invention, the use of the Sdccag3 gene as a target for use in the preparation of drugs for regulating adipose tissue generation and metabolism means: using the Sdccag3 gene as a target of a drug or preparation to increase the expression level of the Sdccag3 gene in adipose tissue, so as to develop drugs or preparations for regulating adipose tissue generation and metabolism.

[0013] Preferably according to the present invention, the use of the Sdccag3 gene as a target for use in the screening of drugs for regulating adipose tissue generation and metabolism means: using the Sdccag3 gene as a target of a drug or preparation to screen the drug or preparation, so as to find a drug or preparation that can promote the expression of the Sdccag3 gene in adipose tissue as an alternative drug or preparation for regulating adipose tissue generation and metabolism.

[0014] Preferably according to the present invention, the drugs for regulating adipose tissue generation and metabolism include but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or viruses.

[0015] More preferably, the drug for regulating adipose tissue generation and metabolism is an Sdccag3 adipose tissue-specific overexpression adeno-associated virus vector, the active ingredient of which contains the nucleotide sequence as shown in SEQ ID No.1, specifically prepared by co-transfecting a host cell with a recombinant expression plasmid and a packaging system, regulating the adipogenic differentiation and lipid droplet formation of preadipocytes, and the generation and metabolism of adipose tissue in mice under conditions of nutritional excess, thereby promoting the proliferation of newly formed adipocytes in adipose tissue of nutritionally excessive mice and improving metabolism, and inhibiting the pathological expansion of adipose tissue.

[0016] Preferably according to the present invention, the drugs for regulating adipose tissue generation and metabolism further contain a pharmaceutically acceptable excipient.

[0017] Further preferably, the excipient is one or more of glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, and water.

[0018] Preferably according to the present invention, the drug for regulating adipose tissue generation and metabolism is a tablet, pill, powder or injection.

[0019] Technical features and beneficial effects of the present invention:

[0020] 1. The present invention first verified the effect of Sdccag3 overexpression on the differentiation of preadipocytes, and found that overexpression of the Sdccag3 gene can significantly promote the adipogenic differentiation of preadipocytes. Then, the effect of adipose tissue-specific overexpression of the Sdccag3 adeno-associated virus vector on adipose tissue generation and metabolism in mice was verified, and it was found that overexpression of the Sdccag3 gene can significantly promote adipose tissue generation and metabolism in nutritionally excessive mice, and adipose tissue-specific overexpression of Sdccag3 can also significantly promote adipose tissue cell proliferation and inhibit adipocyte hypertrophy, thus providing a theoretical basis for preparing or screening drugs or preparations that promote the expression of the Sdccag3 gene as alternative drugs or preparations for regulating adipose tissue generation and metabolism.

[0021] 2. The present invention prepared an adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3. By injecting the adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3 and a control adeno-associated virus vector, it was found that specific overexpression of Sdccag3 in adipose tissue in mice can significantly promote adipose tissue cell proliferation, inhibit adipocyte hypertrophy, and effectively regulate adipose tissue generation and metabolism.

[0022] 3. The present invention uses the Sdccag3 gene as a target to prepare or screen drugs for regulating adipose tissue generation and metabolism. Drug screening is mainly for unknown drugs. The drug is applied to the target gene, and drugs for regulating adipose tissue generation and metabolism are screened according to whether the drug can promote the expression of the target gene; drug preparation is mainly based on the target gene, and drugs for regulating adipose tissue generation and metabolism are prepared or constructed specifically to promote the expression of the target gene; the drugs screened or prepared are of great significance in regulating adipose tissue generation and metabolism. Description of the Drawings

[0023] Figure 1 Schematic diagram of the results of real-time quantitative PCR detection after 6 days of adipogenic induction and differentiation of mouse preadipocytes after overexpressing Sdccag3 and inhibiting the expression of Sdccag3;

[0024] Figure 2Schematic diagram of the Western Blot experimental results after adipogenic induction and differentiation for 6 days in mouse preadipocytes with overexpression and inhibition of Sdccag3 expression.

[0025] Figure 3 Schematic diagram of the results for evaluating the differentiation ability during the differentiation process of preadipocytes by morphological Oil Red O staining.

[0026] Figure 4 Schematic diagram of the results for evaluating the differentiation ability during the differentiation process of preadipocytes by morphological BODIPY staining.

[0027] Figure 5 Agarose gel electrophoresis map of the digested plasmid vector GV585;

[0028] In the figure, lane 1 is 10kb Marker, and the bands from top to bottom are 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp; lane 2 is the product after digestion of the plasmid vector GV585; lane 3 is the undigested plasmid vector GV585.

[0029] Figure 6 Agarose gel electrophoresis map of the PCR amplification product;

[0030] In the figure, lane 1 is Marker, and the bands from top to bottom are 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp, 100bp; lane 2 is the PCR amplification product.

[0031] Figure 7 Agarose gel electrophoresis map of the colony PCR identification;

[0032] In the figure, lane 1 is the negative control (ddH2O); lane 2 is the negative control (empty vector self-ligation control group); lane 3 is the positive control (GAPDH); lane 4 is Marker, and the bands from top to bottom are 5kb, 3kb, 2kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, 100bp; lanes 5 - 12 are transformants 1 - 8.

[0033] Figure 8 Schematic diagram of the RT-PCR detection results of the inguinal adipose tissue in mice after specific overexpression of Sdccag3 in adipose tissue using the Sdccag3 adipose tissue-specific overexpression adeno-associated virus vector.

[0034] Figure 9Schematic diagram of the RT-PCR detection results of epididymal adipose tissue after specific overexpression of Sdccag3 in mouse adipose tissue using an adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3.

[0035] Figure 10 Schematic diagram of the Western Blot detection results of liver, spleen and kidney tissues after specific overexpression of Sdccag3 in mouse adipose tissue using an adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3.

[0036] Figure 11 Schematic diagram of the Western Blot detection results of inguinal adipose tissue after specific overexpression of Sdccag3 in mouse adipose tissue using an adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3.

[0037] Figure 12 Schematic diagram of the Western Blot detection results of epididymal adipose tissue after specific overexpression of Sdccag3 in mouse adipose tissue using an adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3.

[0038] Figure 13 Schematic diagram of the Oil Red O staining results of inguinal adipose tissue sections after specific overexpression of Sdccag3 in mouse adipose tissue using an adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3.

[0039] Figure 14 Schematic diagram of the Oil Red O staining results of epididymal adipose tissue sections after specific overexpression of Sdccag3 in mouse adipose tissue using an adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3. Detailed implementation methods

[0040] The present invention will be further described below in conjunction with embodiments. The embodiments are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0041] For the drugs and reagents involved in the embodiments, unless otherwise specified, they are all ordinary commercially available products; for the experimental operations involved in the embodiments, unless otherwise specified, they are all carried out according to the conventional operations in the art.

[0042] Example 1: Culture and induction of preadipocytes

[0043] 1. Culture of preadipocytes

[0044] The preadipocytes were cultured in a complete medium containing 10% calf serum and 1% double antibiotics. When the cells grew to contact inhibition, the old medium was discarded, and the cells were rinsed twice by shaking with sterile PBS solution. After discarding the PBS solution, 2 ml of trypsin was added and incubated at 37°C for 1 minute. Then, the pre-prepared complete medium was added to terminate the digestion. The cells were pipetted and mixed evenly, collected into a 15-ml centrifuge tube, centrifuged at 1000 r / min for 5 min, the old medium was discarded, and after adding fresh complete medium, the cells were pipetted and mixed evenly. The obtained cell suspension was placed in a culture dish, and the suspension was slowly pipetted multiple times to make it uniform. After making a mark, the cells were cultured in an incubator at 37°C with 5% carbon dioxide to obtain passaged preadipocytes.

[0045] 2. Preparation and induction culture of adipogenic induction medium

[0046] ① Solution A was prepared from a complete medium containing 10% fetal bovine serum, 10 μg / ml insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX).

[0047] ② Solution B was prepared from a complete medium containing 10% fetal bovine serum and 10 μg / ml insulin.

[0048] ③ Solution C was a complete culture medium containing 10% fetal bovine serum.

[0049] ④ When the passaged preadipocytes continued to grow to confluence, it was recorded as day 0, and they were further cultured for 2 days with a complete medium containing 10% calf serum and 1% double antibiotics.

[0050] ⑤ After 2 days, the old medium was discarded, and the cells were rinsed twice by shaking with sterile PBS solution. After discarding the PBS solution, an appropriate amount of freshly prepared Solution A was added, and the cells were cultured in an incubator at 37°C with 5% carbon dioxide for 48 h.

[0051] ⑥ After 48 h, the old medium was discarded, and the cells were rinsed twice by shaking with sterile PBS solution. After discarding the PBS solution, an appropriate amount of freshly prepared Solution B was added, and the cells were cultured in an incubator at 37°C with 5% carbon dioxide for 48 h.

[0052] ⑦ After 48 h, the old medium was discarded, and the cells were rinsed twice by shaking with sterile PBS solution. After discarding the PBS solution, an appropriate amount of freshly prepared Solution C was added, and the cells were maintained in culture in an incubator at 37°C with 5% carbon dioxide, and the medium was changed every other day to obtain induced mature adipocytes.

[0053] Example 2. Roles of overexpressing Sdccag3 and inhibiting Sdccag3 expression during preadipocyte differentiation

[0054] 1. Transfection of preadipocytes

[0055] The Sdccag3 overexpression plasmid and its control blank plasmid were respectively transfected into preadipocytes, induced as described in Example 1, grouped after transfection, and were respectively recorded as the Sdccag3 overexpression negative control group (Vector group) and the Sdccag3 overexpression group (HA-SDCCAG3 group); the Sdccag3 small interfering RNA and the irrelevant sequence control vector were transfected into preadipocytes, grouped after transfection, and were respectively recorded as the Sdccag3 inhibitory expression negative control group (siR-Control group) and the Sdccag3 inhibitory expression group (siR-SDCCAG3 group). Transfection was carried out on the 0th day of preadipocyte induction, and adipogenic induction and differentiation were carried out 2 days after transfection.

[0056] The Sdccag3 overexpression plasmid was artificially synthesized and constructed by Jinan Anbot Biotechnology Co., Ltd. according to the Sdccag3 sequence information (SEQ ID NO.1).

[0057] 2. Role of overexpressing Sdccag3 and inhibiting Sdccag3 expression during the differentiation of preadipocytes

[0058] After the adipogenic induction was completed, RT-PCR was used to detect the expression levels of Sdccag3, peroxisome proliferator-activated receptor (PPARγ), enhancer-binding protein α (CEBPα), enhancer-binding protein 4 (FABP4), lipoprotein lipase (LPL), and adiponectin (ADIPOQ) in preadipocytes of different groups. The detection results are as Figure 1 shown.

[0059] As Figure 1 can be seen, the expression level of the Sdccag3 gene in the siR-SDCCAG3 group was down-regulated, and the expression levels of the corresponding PPARγ, CEBPα, FABP4, LPL, and ADIPOQ genes were also down-regulated. That is, inhibiting Sdccag3 expression can effectively inhibit the adipogenic differentiation of preadipocytes. The expression level of the Sdccag3 gene in the HA-SDCCAG3 group was up-regulated, and the expression levels of the corresponding PPARγ, CEBPα, FABP4, LPL, and ADIPOQ genes were also up-regulated. That is, overexpressing Sdccag3 can effectively promote the adipogenic differentiation of preadipocytes. That is, when the expression of adipogenesis-related factors PPARγ, CEBPα, FABP4, LPL, and ADIPOQ in the overexpression group increased, the expression level of Sdccag3 increased synchronously; when the expression of adipogenesis-related factors PPARγ, CEBPα, FABP4, LPL, and ADIPOQ in the inhibition group decreased, the expression level of Sdccag3 decreased synchronously.

[0060] After the adipogenic induction was completed, Western blot was used to detect the expression levels of Sdccag3, PPARγ, and CEBPα proteins in preadipocytes of different groups, and the detection results are as Figure 2 shown.

[0061] As Figure 2 can be seen, the expression levels of PPARγ and CEBPα proteins in the siR-SDCCAG3 group were down-regulated, that is, inhibiting the expression of Sdccag3 could effectively inhibit the adipogenic differentiation of preadipocytes. The expression levels of PPARγ and CEBPα proteins in the HA-SDCCAG3 group were up-regulated, that is, overexpressing Sdccag3 could effectively promote the adipogenic differentiation of preadipocytes.

[0062] 3. Identification of adipogenic differentiation of preadipocytes

[0063] After the adipogenic induction was completed, Oil Red O staining was used to detect the adipogenic differentiation ability of preadipocytes in different groups, and the detection results are as Figure 3 shown.

[0064] As Figure 3 can be seen, the number of red lipid droplets in the siR-SDCCAG3 group was less than that in the siR-Control group; the number of red lipid droplets in the HA-SDCCAG3 group was more than that in the Vector group, indicating that overexpressing Sdccag3 could effectively promote the adipogenic differentiation of preadipocytes. That is, overexpressing Sdccag3 significantly promoted the differentiation ability of preadipocytes and affected the differentiation process of preadipocytes into mature adipocytes; inhibiting the expression of Sdccag3 significantly weakened the differentiation ability of preadipocytes and affected the differentiation process of preadipocytes into mature adipocytes.

[0065] After the adipogenic induction was completed, BPDIPY staining was used to detect the adipogenic differentiation ability of preadipocytes in different groups, and the detection results are as Figure 4 shown.

[0066] As Figure 4 can be seen, the number of green fluorescent lipid droplets in the siR-SDCCAG3 group was significantly less than that in the siR-Control group, and the number of green fluorescent lipid droplets in the HA-SDCCAG3 group was significantly more than that in the Vector group, indicating that overexpressing Sdccag3 could effectively promote the adipogenic differentiation of preadipocytes. That is, overexpressing Sdccag3 promoted the formation of mature adipocytes and lipid droplet formation, and inhibiting the expression of Sdccag3 significantly weakened the formation of mature adipocytes and lipid droplet formation.

[0067] Example 3. Preparation of adipose tissue-specific overexpression adenovirus-associated virus vector of Sdccag3

[0068] Target gene: Sdccag3 gene, as shown in SEQ ID No.1;

[0069] Cloning site: AgeI / BamHI;

[0070] Plasmid vector: GV585, purchased from Shanghai GeneChem Co., Ltd.

[0071] 1. Digestion of the vector

[0072] Prepare a 50 μl digestion system: 42 μl of ddH2O, 5 μl of 10× CutSmart Buffer, 2 μl of the DNA of purified plasmid vector GV585 (1 μg / μL), 1 μl of AgeI enzyme (10 U / μl), a total of 50 μl.

[0073] Gently pipette and mix the digestion system, briefly centrifuge, place it at 37 °C for 3 h or overnight, perform agarose gel electrophoresis on the product after digesting the plasmid vector GV585, and recover the target band. The results are as Figure 5 shown.

[0074] As Figure 5 can be seen, the digestion of the plasmid vector GV585 was successful.

[0075] 2. Obtaining the target gene fragment

[0076] The upstream primer and downstream primer were artificially synthesized by Shanghai GeneChem Co., Ltd., and then PCR amplification was performed using the plasmid as a template. Agarose gel electrophoresis was performed on the amplified product, and the target band was recovered. The results are as Figure 6 shown.

[0077] Upstream primer:

[0078] 5′-GGAGGTAGTGGAATACCGGTCGCCACCATGTCGGGCTACGCGCGGCG-3′ (SEQ ID No.2),

[0079] Downstream primer: 5′-ACCATGGTGGCGGGATCCACAGAGTCCACCTCATCTTTAACTTC-3′

[0080] (SEQ ID No.3).

[0081] PCR amplification system: 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of template, 0.5 μL of PrimeSTAR HS DNA polymerase, 10 μL of 5× PS Buffer, 4 μL of dNTP Mix (2.5 mM each), 32.5 μL of ddH2O, a total of 50 μL.

[0082] PCR amplification procedure: pre-denaturation at 98°C for 5 min; denaturation at 99°C for 10 sec; annealing at 55°C for 10 sec; extension at 72°C for 90 sec, for 30 cycles; final extension at 72°C for 8 min; finally hold at 4°C.

[0083] It can be seen from Figure 5 that the target gene fragment Sdccag3 was successfully amplified.

[0084] 3. Transformation of the target gene

[0085] Prepare the reaction system according to Table 1 respectively, then gently pipette and mix well, centrifuge briefly to avoid generating bubbles, react at 37°C for 30 min, and then immediately cool in an ice-water bath for 5 min and then transform.

[0086] The transformation steps are as follows: Add 10 μL of the above reaction system to 100 μL of Escherichia coli competent cells respectively, flick the tube wall several times to mix well, place on ice for 30 min, heat shock at 42°C for 90 s, and incubate in an ice-water bath for 2 min. Add 500 μL of LB liquid medium, place on a shaker at 37°C and shake for 1 h. Take the bacterial solution and spread it evenly on a plate containing the corresponding antibiotic, and incubate it upside down in a constant temperature incubator for 12 - 16 h, and pick out the transformed single colonies. Label them as 1 - 8 and set aside.

[0087] Table 1

[0088] Reaction system Positive control (μL) Self-ligation control (μL) Experimental group (μL) <![CDATA[ddH2O]]> 2.5 4.5 3.5 5×CE II Buffer 2 2 2 Digested vector DNA 2.5 2.5 2.5 Purified PCR product 2 0 1 ExnaseTMII 1 1 1 Total 10 10 10

[0089] 4. Colony PCR identification

[0090] The upstream and downstream primers were artificially synthesized by Shanghai Genechem Co., Ltd. Prepare a 20 μL identification system, mix well by shaking and centrifuge. In a laminar flow hood, use a sterile pipette tip to pick the transformed single colonies obtained in step 3 into the identification system, pipette and mix well, perform colony PCR identification, carry out agarose gel electrophoresis on the PCR product, and recover the target band. The results are as Figure 7 shown.

[0091] Upstream primer: 5′-TAGAGACACACAGGACTCAC-3′ (SEQ ID No.4),

[0092] Downstream primer: 5′-CGTCGCCGTCCAGCTCGACCAG-3′ (SEQ ID No.5).

[0093] PCR amplification system: 0.4 μL of upstream primer, 0.4 μL of downstream primer, 10 μL of 2×Taq Plus Master Mix, 9.2 μL of ddH2O, a total of 20 μL.

[0094] PCR amplification procedure: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec; annealing at 55°C for 30 sec; extension at 72°C for 30 sec for 22 cycles; final extension at 72°C for 5 min; and finally incubation at 4°C.

[0095] It can be seen from Figure 7 that the target gene fragment Sdccag3 was successfully transformed.

[0096] 5. Sequencing identification

[0097] Inoculate the identified positive clone transformants into LB liquid medium containing the corresponding antibiotic, culture at 37°C for 12 - 16 h, and take the bacterial liquid for sequencing. Analyze the alignment of the target gene sequence of the sequencing results. Then transfer the bacterial liquid with correct sequencing to 10 ml of LB liquid medium containing the corresponding antibiotic, culture overnight at 37°C, and extract the plasmid using the Tiangen Endotoxin-Free Plasmid Mini Kit (Midiprep) to obtain the recombinant expression plasmid of the Sdccag3 gene. Please refer to the instruction manual of the Tiangen Endotoxin-Free Plasmid Mini Kit (Midiprep) for the specific operation method.

[0098] Finally, after co-transfecting the recombinant expression plasmid and the packaging system into the host cells, the adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3 was obtained. The packaging system and the reagents used in the transfection process are all conventional reagents, and the packaging system and the transfection method are both publicly known conventional methods and can be carried out according to the existing technology.

[0099] Example 4. Establishment of animal model and collection of adipose tissue

[0100] 1. Randomly divide 32 male C57BL / 6J mice at 6 weeks of age into groups. Those fed with normal diet are NCD mice, and those fed with high-fat diet are HFD mice. All mice in the experiment are raised in sterilized standard mouse cages with a 12-hour light-dark cycle, provided with free drinking water and the corresponding standard diet. After 16 weeks of feeding, two groups of mouse models are obtained, namely normal mice and high-fat mice.

[0101] 2. After successful mouse modeling, sacrifice the mice by cervical dislocation. Use ophthalmic scissors to bluntly separate the skin and muscle of the hind limbs of the mice. Hold the skin with the tip of the scissors to avoid cutting the subcutaneous fat. Then longitudinally cut the skin to expose the inguinal fat, strip the inguinal lymph nodes therein, and cut off the adipose tissue; find the testis of the mouse in the lower abdomen, lift it with forceps, and the white fat attached to it is epididymal fat. Strip it along the vas deferens to the end of the testis and then cut it off. After each sample is taken out, rinse it with sterile PBS solution and store it in liquid nitrogen and 4% paraformaldehyde respectively for later detection.

[0102] Example 5. Effect of Adipose Tissue-Specific Overexpression Adeno-Associated Virus Vector of Sdccag3 on Adipogenesis and Metabolism in Mice

[0103] Thirty-two 6-week-old male C57BL / 6J mice were randomly divided into 4 groups and intraperitoneally injected with the adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3 constructed in Example 3 and the irrelevant sequence control virus vector. A mouse model was constructed according to the method of Example 4, denoted as the negative control group of adipose tissue-specific overexpression of Sdccag3 in mice fed a normal diet (NCD-AAV-NC group), the adipose tissue-specific overexpression group of Sdccag3 in mice fed a normal diet (NCD-AAV-Sdccag3 group), the negative control group of adipose tissue-specific overexpression of Sdccag3 in mice fed a high-fat diet (HFD-AAV-NC group), and the adipose tissue-specific overexpression group of Sdccag3 in mice fed a high-fat diet (HFD-AAV-Sdccag3 group).

[0104] At the same time, thirty-two HFD mice constructed according to the method of Example 4 were randomly divided into 2 groups and intraperitoneally injected with the adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3 constructed in Example 3 and the irrelevant sequence control virus vector, denoted as the AAV-NC group and the AAV-Sdccag3 group.

[0105] All the above mice were sacrificed by cervical dislocation to obtain samples of liver, spleen, kidney tissues, inguinal adipose tissue, and epididymal adipose tissue. The irrelevant sequence control virus vector was purchased from Shanghai Genechem Co., Ltd.

[0106] Samples of inguinal adipose tissue and epididymal adipose tissue of the mice in the NCD-AAV-NC group, NCD-AAV-Sdccag3 group, HFD-AAV-NC group, and HFD-AAV-Sdccag3 group were taken, and RT-PCR was used to detect the differential gene expression of Sdccag3 and lipid metabolism-related factors PPARγ, CEBPα, FABP4, LPL, and ADIPOQ. The results are as Figures 8 - 9 shown.

[0107] By Figures 8 - 9It can be seen that there is no significant difference in the expression levels of adipogenesis-related factors PPARγ, CEBPα, FABP4, LPL, and ADIPOQ between the NCD-AAV-NC group and the NCD-AAV-Sdccag3 group. At the same time, the expression level of Sdccag3 increases, indicating that Sdccag3 does not change adipogenesis and metabolism under normal nutritional conditions. The expression of adipogenesis-related factors PPARγ, CEBPα, FABP4, LPL, and ADIPOQ increases in the HFD-AAV-Sdccag3 group compared with the HFD-AAV-NC group. At the same time, the expression level of Sdccag3 increases, promoting adipogenesis and metabolism in mice with nutritional excess. That is, under normal diet feeding, there is no significant difference in the expression of adipogenesis-related factors PPARγ, CEBPα, FABP4, LPL, and ADIPOQ, and the expression level of Sdccag3 increases. Under high-fat diet feeding, the expression of adipogenesis-related factors PPARγ, CEBPα, FABP4, LPL, and ADIPOQ increases in the adipose tissue-specific overexpression group of Sdccag3 compared with the control group, and the expression level of Sdccag3 increases.

[0108] Take the liver, spleen, and kidney tissues of the mice in the above AAV-NC group and AAV-Sdccag3 group, and the inguinal adipose tissue and epididymal adipose tissue samples of the mice in the above NCD-AAV-NC group, NCD-AAV-Sdccag3 group, HFD-AAV-NC group, and HFD-AAV-Sdccag3 group. Western blot was used to detect the differential expression of Sdccag3, PPARγ, and CEBPα proteins. The results are as Figures 10 - 12 shown.

[0109] It can be Figure 10 seen that there is no significant difference in the expression of Sdccag3 in the liver, spleen, and kidney tissues between the AAV-NC group and the AAV-Sdccag3 group, confirming the specificity of Sdccag3 overexpression. It shows that there is no significant difference in the expression of Sdccag3 in the liver, spleen, and kidney tissues between the adipose tissue-specific overexpression group of Sdccag3 and the control group, confirming the specificity of Sdccag3 overexpression.

[0110] It can be Figures 11 - 12 seen that there is no significant difference in the protein expression of PPARγ and CEBPα in the NCD-AAV-Sdccag3 group, and the protein expression of Sdccag3 increases significantly, indicating that Sdccag3 does not change adipogenesis and metabolism under normal nutritional conditions. The protein expressions of Sdccag3, PPARγ, and CEBPα all increase significantly in the HFD-AAV-Sdccag3 group, that is, adipose tissue-specific overexpression of Sdccag3 promotes adipogenesis and metabolism.

[0111] Example 6, Paraffin Tissue Sections and Staining

[0112] After fixing the samples of inguinal adipose tissue and epididymal adipose tissue of the mice in the NCD-AAV-NC group, NCD-AAV-Sdccag3 group, HFD-AAV-NC group, and HFD-AAV-Sdccag3 group described in Example 5 with 4% paraformaldehyde, rinse them with running water for 4 hours, place them in an embedding cassette, and mark them with a pencil. Use ethanol with a concentration of 50% to 100% for gradient dehydration, xylene for clearing, and paraffin immersion and embedding. Cut into 10-μm-thick tissue sections on a paraffin tissue slicer, and use them for later staining after baking. The specific steps are as follows:

[0113] ① Dewaxing and rehydration of paraffin sections: Dewax in xylene (Ⅰ) for 10 min; dewax in xylene (Ⅱ) for 10 min; absolute ethanol (Ⅰ) for 2 min; absolute ethanol (Ⅱ) for 2 min; 95% ethanol for 2 min; 80% ethanol for 2 min; 70% ethanol for 2 min; distilled water for 2 min;

[0114] ② Stain with hematoxylin solution for 1 min, and rinse with tap water for 5 - 10 s;

[0115] ③ Differentiate with differentiating solution for 1 - 5 s, and rinse with tap water for 20 - 30 s;

[0116] ④ Blue return with blue returning solution for 10 s - 1 min, and rinse with tap water for 20 - 30 s;

[0117] ⑤ Stain with eosin for 2 min, and rinse with tap water for 1 - 5 s;

[0118] ⑥ Dehydration, clearing, and mounting: 80% ethanol (Ⅰ) for 2 - 3 s; 90% ethanol (Ⅱ) for 2 - 3 s; 95% ethanol (Ⅰ) for 2 - 3 s; 95% ethanol (Ⅱ) for 2 - 3 s; absolute ethanol (Ⅰ) for 2 - 3 s; absolute ethanol (Ⅱ) for 1 min; xylene (Ⅰ) for 1 min; xylene (Ⅱ) for 1 min; mount with neutral gum, and place under a microscope for photographing and recording.

[0119] Place the mounted sections under an inverted microscope, observe the staining of adipocytes, and show the results under a 200-fold microscope as Figures 13 - 14 shown.

[0120] From Figures 13 - 14It can be seen that in the NCD group, there were no significant differences in the inguinal adipose tissue and epididymal adipose tissue between the NCD-AAV-NC group and the NCD-AAV-Sdccag3 group; in the HFD group, compared with the HFD-AAV-NC group, in the HFD-AAV-Sdccag3 group, the adipocyte size in the inguinal adipose tissue and epididymal adipose tissue was significantly reduced, and the number was significantly increased. This further indicates that adipose tissue-specific overexpression of Sdccag3 promotes cell proliferation in adipose tissue and inhibits adipocyte hypertrophy. That is, under normal diet feeding, there was no significant difference in the adipocyte size between the two groups; under high-fat diet feeding, compared with the control group, in the Sdccag3 adipose tissue-specific overexpression group, the adipocyte size in adipose tissue was significantly reduced and the number increased.

[0121] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. Use of the Sdccag3 gene in the preparation of drugs for regulating adipose tissue generation and metabolism, characterized in that, The nucleotide sequence of the Sdccag3 gene is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The application of the Sdccag3 gene in the preparation of drugs for regulating adipose tissue generation and metabolism includes the following two aspects: (1) Using the Sdccag3 gene as a target for the preparation of drugs for regulating adipose tissue generation and metabolism; (2) Using the Sdccag3 gene as a target for screening drugs for regulating adipose tissue generation and metabolism.

3. The application according to claim 2, characterized in that, Using the Sdccag3 gene as a target for the preparation of drugs for regulating adipose tissue generation and metabolism means: using the Sdccag3 gene as the target of a drug or preparation to increase the expression level of the Sdccag3 gene in adipose tissue, so as to develop drugs or preparations for regulating adipose tissue generation and metabolism.

4. The application according to claim 2, wherein Using the Sdccag3 gene as a target for screening drugs for regulating adipose tissue generation and metabolism means: using the Sdccag3 gene as the target of a drug or preparation to screen the drug or preparation, so as to find drugs or preparations that can promote the expression of the Sdccag3 gene in adipose tissue as alternative drugs or preparations for regulating adipose tissue generation and metabolism.

5. The application according to claim 1, characterized in that The drugs for regulating adipose tissue generation and metabolism are nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or viruses.

6. The application according to claim 5, wherein The drug for regulating adipose tissue generation and metabolism is an adipose tissue-specific overexpression adeno-associated virus vector of Sdccag3, and its active ingredient contains the nucleotide sequence shown in SEQ ID No.

1. Specifically, it is prepared by co-transfecting a host cell with a recombinant expression plasmid and a packaging system. It regulates the adipogenic differentiation and lipid droplet formation of preadipocytes, as well as the generation and metabolism of adipose tissue in mice under conditions of nutritional excess, thereby promoting the proliferation of new adipocytes in the adipose tissue of nutritionally-excess mice and improving metabolism, and inhibiting the pathological expansion of adipose tissue.

7. The application according to claim 1, characterized in that, The drugs for regulating adipose tissue generation and metabolism also contain pharmaceutically acceptable excipients.

8. The application according to claim 1, characterized in that The excipients are one or more of glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, and water.

9. The application according to claim 8, characterized in that, The drugs for regulating adipose tissue generation and metabolism are tablets, pills, powders or injections.

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