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. The Sdccag3 gene is used 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 as shown in 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 invention relates to application of Sdccag3 gene in preparing 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. Brown adipose tissue is the organ whose main function is to generate heat. White adipose tissue is the largest energy storage depot in the body and is the main adipose tissue that regulates the body's energy balance. White adipose tissue stores excess energy when the body's energy supply exceeds demand, and supplies it when the body needs it.

[0003] White adipose tissue is divided into subcutaneous fat and visceral fat. In contrast, visceral fat is a better indicator for predicting metabolic diseases (such as diabetes, hypertension, and dyslipidemia), possibly because visceral fat can directly enter the portal venous system, while subcutaneous white fat cannot. The expansion caused by increased adipose tissue can be driven by an increase in adipocyte size (hypertrophy) or by the differentiation of new adipocytes from precursor cells during adipogenesis (hyperplasia). The balance between the two has an important impact on metabolic health. Several studies have pointed out that enlarged adipocytes are associated with systemic insulin resistance, and their own increased mechanical pressure and hypoxia cause inflammation in their adipose tissue; while small adipocytes show an important functional role in combating metabolic disorders associated with overnutrition. The hypertrophy of adipocytes is considered to increase the risk of disease. At present, more and more studies have suggested that the metabolic decline associated with overnutrition is not simply caused by itself, but is caused by the lack of further expansion of adipose tissue, which indicates that it is particularly important to selectively promote the proliferation of adipocytes rather than hypertrophy.

[0004] Proteosome proliferator-activated receptors (PPARs) belong to the transcription factor subfamily of the nuclear hormone receptor superfamily. Its family members play their respective functional roles in the regulation of energy homeostasis. There are three subtypes: PPARα, PPARγ and PPARδ. PPARα is mainly expressed in the liver, heart, skeletal muscle, brown adipose tissue (BAT), intestines and kidneys, and its function is to affect fatty acid transport, esterification and oxidation; PPARβ / δ is expressed throughout the body, affecting the regulation of fatty acid oxidation and blood glucose levels; and in white adipose tissue, PPARγ is expressed at the highest level, promoting energy storage by participating in lipogenesis and lipid synthesis, affecting 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 with PPARγ knockout 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 a variety of human lipid metabolism diseases. In clinical practice, PPARγ agonists have been used to treat lipid metabolism disorders and control insulin sensitivity in diabetic patients. The most typical drugs are thiazolidinediones (TZDs), including pioglitazone and rosiglitazone. However, 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., limit their use due to unacceptable side effects. Therefore, it is still a great challenge to develop a class of drugs that have the ability to activate PPARγ effectively and avoid the side effects caused by activation.

[0005] Serologically defined colon cancer antigen 3 (Sdccag3) is a 45kd protein with a coiled-coil domain. Studies have shown that it has biological functions such as protein transport, mitosis, ciliogenesis, and osteogenesis. Currently, there are no reports on the functional performance of Sdccag3 in fat metabolism-related diseases, especially the effect of Sdccag3 on adipose tissue generation and metabolism. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides the use of the Sdccag3 gene in the preparation of a drug for regulating the generation and metabolism of adipose tissue. The present invention constructs an adeno-associated virus vector specifically overexpressing Sdccag3 adipose tissue, verifies that it can regulate the generation and metabolism of adipose tissue in mice, and can promote and apply the specific overexpression adeno-associated virus vector as a drug for regulating the generation and metabolism of adipose tissue in related fields.

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

[0008] The use of the Sdccag3 gene in preparing a drug for regulating adipose tissue generation and metabolism, wherein the nucleotide sequence of the Sdccag3 gene is shown in SEQ ID No.1.

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

[0010] (1) Use the Sdccag3 gene as a target for the preparation of drugs that regulate adipose tissue production and metabolism;

[0011] (2) The Sdccag3 gene was used as a target for the screening of drugs that regulate adipose tissue production and metabolism.

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

[0013] Preferably according to the present invention, the application of the Sdccag3 gene as a target in the screening of drugs for regulating adipose tissue production and metabolism means: using the Sdccag3 gene as a target for drugs or preparations, screening the drugs or preparations 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 production and metabolism.

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

[0015] Further preferably, the drug for regulating adipose tissue production and metabolism is a Sdccag3 adipose tissue-specific overexpression adeno-associated virus vector, whose effective ingredient contains the nucleotide sequence shown in SEQ ID No.1, and is specifically prepared by co-transfecting host cells with a recombinant expression plasmid and a packaging system. It regulates the adipogenic differentiation and lipid droplet formation of precursor adipocytes, as well as the production and metabolism of mouse adipose tissue under conditions of overnutrition by overexpressing the Sdccag3 gene, thereby promoting the proliferation of newly formed adipocytes in the adipose tissue of overnutrition mice and improving metabolism, thereby inhibiting the pathological expansion of adipose tissue.

[0016] Preferably according to the present invention, the drug for regulating adipose tissue production and metabolism further contains a pharmaceutically acceptable excipient.

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

[0018] Preferably according to the present invention, the drug for regulating adipose tissue production and metabolism is in the form of tablets, pills, powder or injection.

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

[0020] 1. The present invention first verifies the effect of Sdccag3 overexpression on the differentiation of preadipocytes, and finds that overexpression of the Sdccag3 gene can significantly promote the adipogenic differentiation of preadipocytes. Then, the effect of adeno-associated virus vectors with specific overexpression of Sdccag3 adipose tissue on adipose tissue generation and metabolism in mice is verified, and it is found that overexpression of the Sdccag3 gene can significantly promote the adipogenesis and metabolism of overnourished mice, and that adipose tissue-specific overexpression of Sdccag3 can also significantly promote cell proliferation in adipose tissue and inhibit fat cell hypertrophy, thereby providing a theoretical basis for the preparation or screening of 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 adeno-associated virus vector specifically overexpressing Sdccag3 in adipose tissue. By injecting an adeno-associated virus vector specifically overexpressing Sdccag3 in adipose tissue of mice and a control adeno-associated virus vector, it was found that specific overexpression of Sdccag3 in adipose tissue of mice can significantly promote cell proliferation of adipose tissue, inhibit fat cell 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 that regulate the production and metabolism of adipose tissue. Drug screening is mainly aimed at unknown drugs, and the drugs are acted on the target gene. The drugs that regulate the production and metabolism of adipose tissue are screened according to whether the drugs can promote the expression of the target gene; drug preparation is mainly based on the target gene, and drugs that regulate the production and metabolism of adipose tissue are prepared or constructed in a targeted manner to promote the expression of the target gene; the screened or prepared drugs are of great significance in regulating the production and metabolism of adipose tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the real-time quantitative PCR detection results of mouse preadipocytes after overexpression of Sdccag3 and inhibition of Sdccag3 expression, after 6 days of adipogenic differentiation;

[0024] Figure 2Schematic diagram of Western Blot experimental results of mouse preadipocytes after overexpression of Sdccag3 and inhibition of Sdccag3 expression, followed by adipogenic differentiation for 6 days.

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

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

[0027] Figure 5 This is the agarose gel electrophoresis diagram after the plasmid vector GV585 was digested;

[0028] In the figure, lane 1 is a 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, and 250bp; lane 2 is the product of enzyme digestion of plasmid vector GV585; lane 3 is the undigested plasmid vector GV585.

[0029] Figure 6 Agarose gel electrophoresis of PCR amplification products;

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

[0031] Figure 7 Agarose gel electrophoresis diagram for 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 the Marker, from top to bottom are 5kb, 3kb, 2kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, 100bp; lanes 5 to 12 are transformants No. 1 to 8.

[0033] Figure 8 This is a schematic diagram of the RT-PCR detection results of inguinal adipose tissue after Sdccag3 was overexpressed specifically in mouse adipose tissue using adeno-associated virus vector specifically overexpressing Sdccag3.

[0034] Fig. 9This is a schematic diagram of the RT-PCR detection results of epididymal adipose tissue after Sdccag3 was overexpressed specifically in mouse adipose tissue using adeno-associated virus vector specifically overexpressing Sdccag3.

[0035] Fig.10 Schematic diagram of Western Blot detection results of liver, spleen and kidney tissues after Sdccag3 adipose tissue-specific overexpression adeno-associated virus vector was used to overexpress Sdccag3 in mouse adipose tissue.

[0036] Fig.11 This is a schematic diagram of the Western Blot detection results of inguinal adipose tissue after overexpression of Sdccag3 in mouse adipose tissue using adeno-associated virus vector specifically overexpressing Sdccag3.

[0037] Fig.12 This is a schematic diagram of the Western Blot detection results of epididymal adipose tissue after overexpression of Sdccag3 in mouse adipose tissue using adeno-associated virus vector specifically overexpressing Sdccag3.

[0038] Fig.13 This is a schematic diagram of the Oil Red O staining results of inguinal adipose tissue sections after Sdccag3 was overexpressed specifically in mouse adipose tissue using adeno-associated virus vector specifically overexpressing Sdccag3.

[0039] Fig.14 This is a schematic diagram of the Oil Red O staining results of epididymal adipose tissue sections after Sdccag3 adipose tissue-specific overexpression adeno-associated virus vector was used to overexpress Sdccag3 in mouse adipose tissue. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with examples, which are only used to illustrate the present invention but not to limit the scope of protection of the present invention.

[0041] Unless otherwise specified, the drugs and reagents involved in the examples are all common commercially available products; the experimental operations involved in the examples are all carried out according to routine operations in the art unless otherwise specified.

[0042] Example 1: Cultivation 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 antibody. When the cells grew to contact inhibition, the old medium was discarded, and the cells were washed twice with a sterile PBS solution by shaking. After discarding, 2 ml of trypsin was added, and the cells were incubated at 37°C for 1 minute. The complete medium prepared in advance was added to terminate the digestion. The cells were mixed by blowing with a pipette, collected in a 15 ml centrifuge tube, and centrifuged at 1000 r / min for 5 minutes. The old medium was discarded, and fresh complete medium was added and mixed by blowing. The obtained cell suspension was placed in a culture dish, and the suspension was slowly blown several times to make it uniform. After marking, it was cultured in an incubator at 37°C and containing 5% carbon dioxide to obtain the preadipocytes for subculture.

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

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

[0047] ② Complete culture medium containing 10% fetal bovine serum and 10 μg / ml insulin was prepared into solution B;

[0048] ③ Complete culture medium containing 10% fetal bovine serum is medium C;

[0049] ④ The subcultured preadipocytes were cultured until confluence, which was recorded as day 0, and then cultured for 2 more days using complete medium containing 10% calf serum and 1% double antibody;

[0050] ⑤ After 2 days, discard the old culture medium, shake and rinse twice with sterile PBS solution, discard it, add an appropriate amount of freshly prepared A solution, and culture in an incubator at 37°C and 5% carbon dioxide for 48 hours;

[0051] ⑥ After 48 hours, discard the old culture medium, shake and rinse twice with sterile PBS solution, discard it, add an appropriate amount of freshly prepared B solution, and culture in an incubator at 37°C and 5% carbon dioxide for 48 hours;

[0052] ⑦ After 48 hours, discard the old culture medium, shake and rinse twice with sterile PBS solution, discard it and add an appropriate amount of freshly prepared C solution, maintain culture in an incubator at 37°C and containing 5% carbon dioxide, and replace it every other day to obtain induced mature adipocytes.

[0053] Example 2: Effects of overexpression of Sdccag3 and inhibition of Sdccag3 expression on preadipocyte differentiation

[0054] 1. Transfection of preadipocytes

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

[0058] After adipogenesis induction, 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 test results were as follows: Figure 1 shown.

[0059] Depend on Figure 1 It can be seen that the expression level of Sdccag3 gene in siR-Sdccag3 group was downregulated, and the corresponding expression levels of PPARγ, CEBPα, FABP4, LPL and ADIPOQ genes were downregulated. That is, inhibiting Sdccag3 expression can effectively inhibit the adipogenic differentiation of preadipocytes. The expression level of Sdccag3 gene in HA-Sdccag3 group was upregulated, and the corresponding expression levels of PPARγ, CEBPα, FABP4, LPL and ADIPOQ genes were upregulated. That is, overexpression of Sdccag3 can effectively promote the adipogenic differentiation of preadipocytes. That is, when the expression of overexpressed lipid-related factors PPARγ, CEBPα, FABP4, LPL and ADIPOQ increased, the expression of Sdccag3 increased synchronously; when the expression of lipid-related factors PPARγ, CEBPα, FABP4, LPL and ADIPOQ decreased, the expression of Sdccag3 decreased synchronously.

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

[0061] Depend on Figure 2 It can be seen that the expression level of Sdccag3 protein in the siR-Sdccag3 group was downregulated, and the corresponding expression levels of PPARγ and CEBPα proteins were downregulated, that is, inhibiting the expression of Sdccag3 can effectively inhibit the adipogenic differentiation of preadipocytes. The expression level of Sdccag3 protein in the HA-Sdccag3 group was upregulated, and the corresponding expression levels of PPARγ and CEBPα proteins were upregulated, that is, overexpression of Sdccag3 can effectively promote the adipogenic differentiation of preadipocytes.

[0062] 3. Identification of adipogenic differentiation of preadipocytes

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

[0064] Depend on Figure 3 It can be seen that the red lipid droplets in the siR-Sdccag3 group were less than those in the siR-Control group; the red lipid droplets in the HA-Sdccag3 group were more than those in the Vector group, indicating that overexpression of Sdccag3 can effectively promote the adipogenic differentiation of preadipocytes. That is, overexpression of Sdccag3 significantly promoted the differentiation ability of preadipocytes and affected the differentiation process of preadipocytes into mature adipocytes; inhibition of Sdccag3 expression significantly weakened the differentiation ability of preadipocytes and affected the differentiation process of preadipocytes into mature adipocytes.

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

[0066] Depend on Figure 4 It can be seen that the green fluorescent lipid droplets in the siR-Sdccag3 group were significantly less than those in the siR-Control group, and the green fluorescent lipid droplets in the HA-Sdccag3 group were significantly more than those in the Vector group, indicating that overexpression of Sdccag3 can effectively promote the adipogenic differentiation of preadipocytes. That is, overexpression of Sdccag3 promoted the formation of mature adipocytes and lipid droplet formation, while inhibition of Sdccag3 expression significantly weakened the formation of mature adipocytes and lipid droplet formation.

[0067] Example 3: Preparation of adeno-associated virus vector for adipose tissue-specific overexpression 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 Jikai Technology Co., Ltd.

[0071] 1. Vector digestion

[0072] Prepare 50 μl of enzyme digestion system: 2 μl of ddH2O4, 5 μl of 10×CutSmart Buffer, 2 μl of purified plasmid vector GV585 DNA (1 μg / μL), 1 μl of AgeI enzyme (10 U / μl), a total of 50 μl.

[0073] The enzyme digestion system was gently pipetted and mixed, centrifuged briefly, and placed at 37°C for 3 h or overnight. The products of the plasmid vector GV585 digestion were subjected to agarose gel electrophoresis to recover the target bands. The results were as follows: Figure 5 shown.

[0074] Depend on Figure 5 It can be seen that the plasmid vector GV585 was successfully digested.

[0075] 2. Acquisition of target gene fragments

[0076] Shanghai Jikai Technology Co., Ltd. artificially synthesized upstream primers, downstream primers and Sdccag3 gene sequence, and then used Sdccag3 gene sequence as template for PCR amplification. The amplified product was subjected to agarose gel electrophoresis to recover the target band. The results are as follows: 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: upstream primer 1 μL, downstream primer 1 μL, template 1 μL, PrimeSTAR HS DNA polymerase 0.5 μL, 5× PS Buffer 10 μL, dNTP Mix (2.5 mM each) 4 μL, ddH2O3 2.5 μL, a total of 50 μL.

[0082] PCR amplification program: pre-denaturation, 98℃ for 5 min; denaturation, 99℃ for 10 sec; annealing, 55℃ for 10 sec; extension, 72℃ for 90 sec, 30 cycles; termination of extension, 72℃ for 8 min; and finally incubation at 4℃.

[0083] Depend on Figure 6 It can be seen that the target gene fragment Sdccag3 was amplified successfully.

[0084] 3. Transformation of target gene

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

[0086] The transformation steps are as follows: add 10 μL of the above reaction system to 100 μL of E. coli competent cells, flick the tube wall several times to mix, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and incubate in an ice-water bath for 2 minutes. Add 500 μL of LB liquid culture medium and place in a 37°C shaker for 1 hour. Take the bacterial solution and spread it evenly on a plate containing the corresponding antibiotics, invert and culture in a constant temperature incubator for 12 to 16 hours, and pick a single colony of the transformant. Record it as 1 to 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 Vector DNA after enzyme digestion 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] Shanghai Jikai Technology Co., Ltd. artificially synthesized upstream primers and downstream primers, prepared 20 μL identification system, oscillated and centrifuged, picked up the transformant colony obtained in step 3 into the identification system with a sterile pipette tip in the clean bench, pipetted and mixed, performed colony PCR identification, performed agarose gel electrophoresis on the PCR product, recovered the target band, and the results were as follows: 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: upstream primer 0.4 μL, downstream primer 0.4 μL, 2×Taq Plus Master Mix 10 μL, ddH2O 9.2 μL, a total of 20 μL.

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

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

[0096] 5. Sequencing and identification

[0097] The identified positive clone transformants were inoculated into LB liquid medium containing the corresponding antibiotics, cultured at 37°C for 12-16h, and the bacterial solution was taken for sequencing. The target gene sequence of the sequencing results was compared and analyzed. Then the bacterial solution with correct sequencing was transferred to 10ml LB liquid medium containing the corresponding antibiotics, cultured at 37°C overnight, and the plasmid was extracted using the Tiangen Endotoxin-Free Plasmid Mini-Extraction Kit to obtain the recombinant expression plasmid of the Sdccag3 gene. For specific operation methods, please refer to the instructions of the Tiangen Endotoxin-Free Plasmid Mini-Extraction Kit.

[0098] Finally, the recombinant expression plasmid and the packaging system are co-transfected into the host cell to obtain the Sdccag3 adipose tissue-specific overexpression adeno-associated virus vector. The packaging system and the reagents used in the transfection process are conventional reagents, and the packaging system and the transfection method are both publicly known conventional methods and can be performed according to the prior art.

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

[0100] 1. 32 C57BL / 6J male mice aged 6 weeks were randomly divided into two groups: NCD mice fed with normal feed and HFD mice fed with high-fat feed. All mice were kept in sterilized standard cages with a 12-hour light-dark cycle and provided with free drinking water and corresponding standard diet. After 16 weeks of feeding, two groups of mouse models were obtained, namely normal mice and high-fat mice.

[0101] 2. After the mouse model was successfully established, the mice were killed by cervical dislocation. The skin and muscles of the mouse hind limbs were bluntly separated with ophthalmic scissors. The tip of the scissors was pressed against the skin to avoid cutting the subcutaneous fat. The skin was then cut longitudinally to expose the inguinal fat, the inguinal lymph nodes were peeled off, and the adipose tissue was cut off. The mouse testicles were found in the lower abdomen and lifted with tweezers. The white fat attached to them was epididymal fat, which was peeled off along the vas deferens to the end of the testicle and then cut off. After each sample was taken out, it was rinsed with sterile PBS solution and stored in liquid nitrogen and 4% paraformaldehyde for later detection.

[0102] Example 5 Effects of adeno-associated virus vectors specifically overexpressing Sdccag3 on adipose tissue generation and metabolism in mice

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

[0104] At the same time, 32 HFD mice constructed according to the method of Example 4 were randomly divided into two groups and intraperitoneally injected with the Sdccag3 adipose tissue-specific overexpression adeno-associated virus vector constructed in Example 3 and an irrelevant sequence control virus vector, respectively, and were recorded as AAV-NC group and AAV-Sdccag3 group.

[0105] All the mice were killed by cervical dislocation, and liver, spleen, kidney tissue, inguinal fat tissue and epididymal fat tissue samples were obtained. The irrelevant sequence control virus vector was purchased from Shanghai Genetech Technology Co., Ltd.

[0106] Inguinal adipose tissue and epididymal adipose tissue samples were collected from the NCD-AAV-NC group, NCD-AAV-Sdccag3 group, HFD-AAV-NC group, and HFD-AAV-Sdccag3 group of mice, and the differential gene expressions of Sdccag3 and lipid metabolism-related factors PPARγ, CEBPα, FABP4, LPL, and ADIPOQ were detected by RT-PCR. The results are shown in Figures 8-9 shown.

[0107] Depend on Figures 8-9 It can be seen that there was no obvious 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, and the expression of Sdccag3 increased, indicating that Sdccag3 does not change the adipogenesis and metabolism under normal nutritional conditions; the expression of adipogenesis-related factors PPARγ, CEBPα, FABP4, LPL and ADIPOQ in the HFD-AAV-Sdccag3 group was increased compared with the HFD-AAV-NC group, and the expression of Sdccag3 increased, which promoted the adipogenesis and metabolism of overnourished mice. That is, under normal diet feeding, there was no obvious difference in the expression of adipogenesis-related factors PPARγ, CEBPα, FABP4, LPL and ADIPOQ, while the expression of Sdccag3 increased; under high-fat diet feeding, the expression of adipogenesis-related factors PPARγ, CEBPα, FABP4, LPL and ADIPOQ in the Sdccag3 adipose tissue-specific overexpression group was increased compared with the control group, while the expression of Sdccag3 increased.

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

[0109] Depend on Fig.10 It can be seen that there was no significant difference in the expression of Sdccag3 in 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 was no significant difference in the expression of Sdccag3 in liver, spleen and kidney tissues between the Sdccag3 adipose tissue-specific overexpression group and the control group, confirming the specificity of Sdccag3 overexpression.

[0110] Depend on Figures 11-12 It can be seen that there was no significant difference in the expression of PPARγ and CEBPα proteins in the NCD-AAV-Sdccag3 group, and the expression of Sdccag3 protein was significantly increased, indicating that Sdccag3 did not change the adipogenesis and metabolism under normal nutritional conditions; in the HFD-AAV-Sdccag3 group, the expression of Sdccag3, PPARγ and CEBPα proteins were all significantly increased, that is, the adipose tissue-specific overexpression of Sdccag3 promoted adipogenesis and metabolism.

[0111] Example 6: Paraffin tissue sections and staining

[0112] The inguinal adipose tissue and epididymal adipose tissue samples of the NCD-AAV-NC group, NCD-AAV-Sdccag3 group, HFD-AAV-NC group, and HFD-AAV-Sdccag3 group of mice described in Example 5 were fixed in 4% paraformaldehyde, rinsed with running water for 4 hours, placed in an embedding box, and marked with a pencil. Use 50% to 100% ethanol for gradient dehydration, xylene for transparency, and paraffin immersion and embedding. Cut into 10 μm thick tissue sections on a paraffin tissue slicer, and use them for later staining after drying. The specific steps are as follows:

[0113] ① Dewaxing and hydration of paraffin sections: dewaxing with xylene (Ⅰ) for 10 min; dewaxing with xylene (Ⅱ) for 10 min; anhydrous ethanol (Ⅰ) for 2 min; anhydrous 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 minute, then rinse with tap water for 5-10 seconds;

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

[0116] ④ The bluing solution will turn blue for 10s-1min, and then rinse with tap water for 20-30s;

[0117] ⑤Stain with eosin for 2 minutes and rinse with tap water for 1-5 seconds;

[0118] ⑥ Dehydration, transparency, and sealing: 80% ethanol (Ⅰ) 2-3s; 90% ethanol (Ⅱ) 2-3s; 95% ethanol (Ⅰ) 2-3s; 95% ethanol (Ⅱ) 2-3s; anhydrous ethanol (Ⅰ) 2-3s; anhydrous ethanol (Ⅱ) 1min; xylene (Ⅰ) 1min; xylene (Ⅱ) 1min; seal with neutral gum and take pictures under a microscope.

[0119] The above sealed sections were placed under an inverted microscope to observe the staining of fat cells. The results were shown under a 200x microscope. Figures 13-14 shown.

[0120] Depend on Figures 13-14It can be seen that in the NCD group, there was no significant difference in the inguinal adipose tissue and epididymal adipose tissue of the NCD-AAV-NC group and the NCD-AAV-Sdccag3 group; in the HFD group, the size of the adipocytes in the inguinal adipose tissue and epididymal adipose tissue of the HFD-AAV-Sdccag3 group was significantly reduced, and the number was significantly increased compared with the HFD-AAV-NC group. This further shows 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 size of adipose tissue cells between the two groups; under high-fat diet feeding, the size of adipose tissue cells in the Sdccag3 adipose tissue-specific overexpression group was significantly reduced, and the number was increased compared with the control group.

[0121] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. Application 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 use 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) Use the Sdccag3 gene as a target for the preparation of drugs that regulate adipose tissue production and metabolism; (2) The Sdccag3 gene was used as a target for the screening of drugs that regulate adipose tissue production and metabolism.

3. The use according to claim 2, characterized in that The use of the Sdccag3 gene as a target for the preparation of drugs for regulating adipose tissue production and metabolism means: using the Sdccag3 gene as a target for drugs or preparations, increasing the expression level of the Sdccag3 gene in adipose tissue, and developing drugs or preparations for regulating adipose tissue production and metabolism.

4. The use according to claim 2, characterized in that The use of the Sdccag3 gene as a target for screening drugs that regulate adipose tissue production and metabolism means: using the Sdccag3 gene as a target for drugs or preparations, screening the drugs or preparations 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 production and metabolism.

5. The use according to claim 1, characterized in that The drug for regulating adipose tissue production and metabolism is a nucleic acid molecule, a carbohydrate, a lipid, a small molecule chemical drug, an antibody drug, a polypeptide, a protein or a virus.

6. The use according to claim 5, characterized in that The drug for regulating adipose tissue production and metabolism is a Sdccag3 adipose tissue-specific overexpression adeno-associated virus vector, the active ingredient of which contains the nucleotide sequence shown in SEQ ID No.1, and is specifically prepared by co-transfecting a host cell with a recombinant expression plasmid and a packaging system. The drug regulates the adipogenic differentiation and lipid droplet formation of precursor adipocytes, as well as the production and metabolism of mouse adipose tissue under conditions of overnutrition by overexpressing the Sdccag3 gene, thereby promoting the proliferation of newly formed adipocytes in the adipose tissue of overnutrition mice and improving metabolism, thereby inhibiting the pathological expansion of adipose tissue.

7. The use according to claim 1, characterized in that The drug for regulating adipose tissue production and metabolism also contains a pharmaceutically acceptable excipient.

8. The use according to claim 1, characterized in that The excipient is one or more of glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose and water.

9. The use according to claim 8, characterized in that The drug for regulating adipose tissue production and metabolism is in the form of tablets, pills, powders or injections.

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