CN110973062A - Construction method of mature adipocyte specific β -catenin knockout mouse - Google Patents

Construction method of mature adipocyte specific β -catenin knockout mouse Download PDF

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CN110973062A
CN110973062A CN201911362176.0A CN201911362176A CN110973062A CN 110973062 A CN110973062 A CN 110973062A CN 201911362176 A CN201911362176 A CN 201911362176A CN 110973062 A CN110973062 A CN 110973062A
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宁光
王计秋
芦鹏
刘瑞欣
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SHANGHAI INSTITUTE OF ENDOCRINE AND METABOLIC DISEASES
Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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Abstract

The invention relates to a construction method of a mature adipocyte specificity β -catenin knockout mouse, which proves that β -catenin can inhibit transcription expression of Saa3, and discloses how a classical Wnt signal path influences the function of a mature adipocyte to influence obesity occurrence or provides a new idea for obesity treatment.

Description

Construction method of mature adipocyte specific β -catenin knockout mouse
Technical Field
The invention belongs to the field of obesity treatment, and particularly relates to a construction method of a mature adipocyte specific β -catenin knockout mouse.
Background
MacDougald published a study in 2000 to find that overexpression of WNT1 or activated β -catenin in 3T3-L1 cells could inhibit adipogenesis and that inhibition of GSK3 β with drugs could also block adipocyte differentiation.
However, it is still unclear how the in vivo level of the Wnt signaling pathway core molecule β -catenin regulates fat function.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a construction method of a mature adipocyte specificity β -catenin knockout mouse, and the method proves that β -catenin can inhibit transcription expression of Saa3, and discloses how a classical Wnt signal path influences the function of a mature adipocyte to influence obesity occurrence, or provides a new idea for obesity treatment.
The invention provides a construction method of a mature adipocyte specificity β -catenin knockout mouse, which comprises the following steps:
β -catenin with C57BL/6 backgroundflox/floxMating the mouse with an adipoectin-Cre mouse to obtain
Adiponectin-Cre；β-cateninflox/wtAfter the mice, the mice were reacted with β -cateninflox/floxMice were mated and control mice β -cateninf were obtained in the offspringlox/floxAnd knock-out mouse adipoectin-Cre, β -cateninflox/floxThus, the mature adipocyte-specific β -catenin knockout mouse is obtained and is recorded as an ABKO knockout mouse.
The mice were all housed in an SPF grade barrier environment.
The ambient temperature is 20-22 ℃.
Advantageous effects
The invention proves that β -catenin can inhibit transcription expression of Saa3, and discloses how a classical Wnt signal pathway influences the function of mature fat cells to influence obesity, or provides a new idea for obesity treatment.
Drawings
FIG. 1A shows aP2-cre, β -cateninflox/floxFat cell condition knockout mice construct a pattern map.
Fig. 1B is body weight of control and APBKO mice at 3 and 8 weeks of age.
Fig. 1C is a representative gross photograph of 8 week old control mice and APBKO mice.
FIG. 1D shows adipinectin-cre, β -cateninflox/floxFat cell condition knockout mice construct a pattern map.
FIGS. 1E-G show the expression level of β -catenin in mature adipocytes and SVF cells in comparison of subcutaneous adipose tissue, visceral adipose tissue and brown adipose tissue.
FIGS. 1H-I are body weight curves of control and ABKO mice fed normal diet and high fat diet.
Fig. 1J is a representative photomicrograph of 24-week-old, high-fat-fed control and ABKO mice.
FIG. 1K shows fat and lean mass of control and ABKO mice fed a high fat diet.
Fig. 2A is a glucose tolerance experiment performed after 16 hours of starvation in control and ABKO mice fed a high fat diet.
Fig. 2B is the area under the curve statistical result of the glucose tolerance experiment.
Figure 2C is an insulin tolerance experiment performed after 6 hours of starvation in control and ABKO mice fed a high fat diet.
Fig. 2D is a statistical result of the area under the curve of the insulin tolerance test.
Figure 2E is control and ABKO mice fed a high fat diet with fasting plasma insulin levels.
FIG. 2F, left panel, shows the results of Western blot analysis of phosphorylated Akt and total Akt in subcutaneous adipose tissue of control and ABKO mice fed a high fat diet; the right panel is the statistical results of the phosphorylated Akt and total Akt gray levels.
FIGS. 2G-H are plasma triglyceride and total cholesterol levels of control and ABKO mice fed a high fat diet.
FIG. 2I is a representative picture of HE staining of liver of control and ABKO mice fed a high fat diet, at a scale of 100 μm. Fig. 2J is a graph of control and ABKO mouse liver triglyceride quantification results with high fat diet feeding.
FIG. 2K is mRNA expression levels of macrophage markers F4/80, CD68, and CD11c in subcutaneous adipose tissue of control and ABKO mice fed a high fat diet.
FIG. 2L, left panel, is a representative picture of flow analysis of macrophages in subcutaneous fat of control and ABKO mice fed a high fat diet; the right panel shows the flow analysis statistics.
FIG. 2M is the mRNA expression level of macrophage marker type M1 in subcutaneous adipose tissue of control and ABKO mice fed a high fat diet.
FIG. 2N-P are control and ABKO mice plasma Leptin, Resistin and PAI-1 levels fed on a high fat diet.
Fig. 3A is a representative photograph of brown adipose tissue, subcutaneous adipose tissue, and visceral adipose tissue of control and ABKO mice.
FIGS. 3B-C are the absolute weight of adipose tissue and the tissue/weight percentage of control mice and ABKO mice fed a high fat diet. Fig. 3D is a representative picture of HE staining of subcutaneous adipose tissue in control and ABKO mice fed a high fat diet.
FIG. 3E, left panel, shows the distribution frequency of subcutaneous adipose tissue adipocyte diameters of control mice fed a high fat diet and ABKO mice; the right panel is a boxed graph of the diameter of the subcutaneous adipose tissue adipocytes of two groups of mice.
Fig. 3F is the number of adipocytes in a single field of view of HE stained subcutaneous adipose tissue in control and ABKO mice fed with a high fat diet.
FIG. 3G is a left graph representing the results of flow analysis of PDGFR α + cell ratio in subcutaneous adipose tissues of control mice and ABKO mice fed with high-fat diet, and a right graph representing the statistics of the ratio of PDGFR α + cells in SVF cells in subcutaneous adipose tissues of control mice and ABKO mice.
FIG. 3H is a graph showing the PDGFR α and BrdU staining representative of subcutaneous adipose tissues of control mice and ABKO mice on the left, and the PDGFR α + BrdU + cells in PDGFR α cells in the subcutaneous adipose tissues of control mice and ABKO mice on the right.
Fig. 4A is a profile of subcutaneous adipose tissue gene expression in control and ABKO mice fed a high fat diet, with heat maps showing two groups of differentially expressed genes with fold change greater than 1.5, corresponding to a P value less than 0.05.
FIG. 4B shows QPCR verification of Saa3 expression in subcutaneous adipose tissue in two groups of mice.
FIG. 4C shows the expression of Saa3 in 3T3-L1 cells induced to differentiate after the knock-down of β -catenin by QPCR assay.
FIG. 4D shows that 3T3-L1 cells induced to differentiate were treated with Wnt3a for 16 hours and Saa3 expression was detected.
FIG. 4E shows that the expression of Saa3 was detected by treating SVF cells induced to differentiate with Wnt signaling pathway inhibitor PKF115 for 48 hours. FIG. 4F, left panel, is a schematic diagram of a potential TCF4 binding site of the promoter region of SAA 3; the right panel shows the results of reporter gene detection 48 hours after transfection of the reporter plasmid in the 3T3-L1 cell line. pRL-TK (expressing Renilla luciferase) was used as an internal control. FIG. 4G shows chromatin co-immunoprecipitation using TCF-4 antibody in 3T3-L1 cells and detection of SAA3 promoter DNA fragments by PCR.
FIG. 4H shows chromatin co-immunoprecipitation using β -catenin antibody in 3T3-L1 cells, and detection of Saa3 promoter DNA fragment by PCR.
FIG. 4I is a schematic diagram of the experimental design of 3T3-L1 cells treated with supernatant after the recombinant protein Saa3 stimulates Raw264.7 cells.
FIGS. 4J-L are the concentrations of PDGF-AA, Mcp-1 and Cxcl2 in the supernatant after different concentrations of Saa3 recombinant protein stimulated Raw264.7 cells.
FIG. 4M shows the results of viability assays of 3T3-L1 cells further processed with supernatants obtained by stimulating Raw264.7 cells with different concentrations of Saa3 recombinant protein.
FIG. 4N is the results of viability assay of 3T3-L1 cells treated with different concentrations of PDGF-AA recombinant protein.
FIGS. 4O-P show the F4/80 expression level and PDGFR α + cell ratio for each group.
Figure 4Q is a significant increase in Saa3 expression level during long-term high fat diet.
FIG. 4R shows that the expression level of Saa3 is significantly related to β -catenin.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
1.Adiponectin-Cre；β-cateninflox/floxBreeding the knockout mice:
β -catenin with C57BL/6 backgroundflox/floxMating the mouse with Adiponectin-Cre mouse to obtain Adiponectin-Cre, β -cateninflox/wtAfter the mice, the mice were reacted with β -cateninflox/floxMice were mated and control mice β -cateninf were obtained in the offspringlox/floxAnd knock-out mouse adipoectin-Cre, β -cateninflox/floxTo obtain mature adipocyte specific β -catenin knockout mouse, which is recorded as ABKO knockout mouse, and the adipoectin-Cre and β -catenin are used in the subsequent reproduction processflox/floxMating with β -catenflox/flox mice.
Mice were housed in the SPF grade barrier environment of the department of zoology of the medical college of shanghai transportation university and of shanghai's square model biotechnology limited, all animals were housed in standard mouse cages under the following conditions: the ambient temperature is 20-22 ℃; free food intake and water, standard commercial mouse Diet (4.5% fat, 4% cellulose, 21% protein, 1.404kcal/g), high fat Diet purchased from Research Diet (60% fat, 20% carbohydrate, 20% protein); stable 12 hour day and night cycle. Brdu was dissolved in PBS (20mg/ml) and subsequently diluted into mouse drinking water to a final concentration of 0.4 mg/ml. The ABKO high-fat mice are treated by Brdu for one week, drinking water containing Brdu is replaced every other day, and after treatment, subcutaneous adipose tissues of the mice are taken to detect the Brdu labeling condition through immunofluorescence. Control and ABKO knockout mice were high fat fed for 24 weeks with clodronate liposomes (110mg/kg) injected at multiple points at the inguinal subcutaneous fat site on one side of each mouse, and the same amount of control liposomes injected at multiple points at the inguinal subcutaneous fat site on the other side of the mouse. The injection is carried out 3 days after the first injection, and subcutaneous adipose tissues of the mice are fixed in 4% paraformaldehyde 6 days after the first injection.
2. Specific knockout of β -catenin by mature fat cells can resist obesity induced by high-fat diet
To reveal how β -catenin regulates the lipoectasis process in mature adipocytes, a knockout mouse of β -catenin, which is relatively specific in adipose tissue, was first constructed using aP2-cre (fig. 1A). although this mouse is significantly lighter in weight than a control mouse (fig. 1B), it is difficult to exclude the effect of developmental disorders on body weight due to the presence of nonspecific expression of aP2-cre in tissues such as hypothalamus, bone, and muscle other than adipose tissue (fig. 1C).
In order to better elucidate the effects of β -catenin on fat and obesity, adipoectin-Cre and β -catenin were usedflox/floxThe mating of mice constructs an adipose tissue specific knockout mouse (FIG. 1D), β -catenin, which is specifically and effectively knocked out in mature adipocytes, and does not affect the expression of β -catenin in SVF cells (FIG. 1E-G).
The ABKO mice did not change significantly in body weight under normal diet conditions (fig. 1H), but in the case of long-term high fat feeding, the ABKO mice exhibited a weight, fat content lower than the obesity resistant phenotype of the control mice (fig. 1I-K).
The knockout of β -catenin in mature adipocytes can significantly reduce mouse body weight and obesity, which is an important risk factor for abnormal glycolipid metabolism, and thus it is presumed that metabolic disorders caused by high-fat diet in ABKO mice can also be greatly improved, glucose tolerance test (fig. 2A-B) indicates that glucose tolerance of ABKO mice is significantly enhanced, and insulin tolerance test results (fig. 2C-D), plasma insulin level (fig. 2E), and phosphorylation level of Akt in subcutaneous adipose tissue (fig. 2F) all indicate that insulin sensitivity of ABKO mice is significantly improved, besides, plasma lipid level (fig. 2G-H) of ABKO mice is lower than that of control mice, fatty liver (fig. 2I-J) also alleviates the adipogenesis process, and macrophages are important factors involved in the fat tissue macrophage infiltration, and macrophage infiltration, thus macrophage markers F4/80, CD 82, and CD11 (fig. 2I-J) in subcutaneous adipose tissue are examined, and macrophage infiltration map of ABKO 2K 1-B is a significant factor for improvement, macrophage infiltration, macrophage-2K 34 (fig. 2K) is an important factor for improving macrophage metabolism, and macrophage infiltration map 2K is a significant map of ABKO 2P 2, thus it is examined that macrophage infiltration of subcutaneous adipose tissue, macrophage metabolism is improved, and macrophage infiltration of ABKO-B2.
3. The deficiency of β -catenin in mature adipocytes during high-fat diet feeding process can result in the reduction of the number of adipose precursor cells and mature adipocytes
It is known that adipose tissue expansion is one of the most typical features in the development of obesity, and includes two processes of cellular hypertrophy (cell volume becomes larger) and cell proliferation (cell number becomes larger). ABKO was found to exhibit a significantly lower body weight, fat content than the obesity resistant phenotype of control mice on a high fat diet, but the tissue weighing results further showed that the subcutaneous adipose tissue weight of ABKO mice was significantly less than the control mice, while visceral fat weight and brown fat weight did not exhibit significant differences (fig. 3A-C). By HE staining sections of subcutaneous adipose tissue and observing it, it was found that although the subcutaneous adipose weight of the knockout mice was significantly smaller than that of the control mice, the two adipose cells did not differ significantly in size (fig. 3D-F).
Flow analysis of adipose precursor cells (SVF) cells in subcutaneous adipose tissue of control and knockout mice isolated on a high fat diet revealed that the number of PDGFR α positive adipose precursor cells was significantly reduced in knockout mice (FIG. 3G), and that Brdu-labeled cells in the PDGFR α cell population was significantly less than in control mice (FIG. 3H), suggesting that the reduction in the number of adipose precursor cells and the reduction in proliferative capacity in subcutaneous adipose tissue of knockout mice are key causes in limiting expansion of subcutaneous adipose tissue.
The Wnt/β -catenin signaling pathway realizes the dialogue between 'mature fat cell-macrophage-fat precursor cell' by regulating the expression of Saa3
The inhibition or activation of the canonical signaling pathway in adipocytes can correspondingly inhibit or activate Saa3 expression (fig. 4C-E) before and without research report on the expression of Saa3, so that the promoter region of Saa3 is firstly analyzed, the promoter region of Saa3 is further found to have a potential effect of binding to a luciferase promoter region in a mature gene promoter region, and the promoter region of Saa-468 is further found to have a potential effect of binding to a luciferase promoter region in a mature gene promoter region, and a promoter region of Saa-468 is further combined with a luciferase promoter region of a rat promoter region, so that the promoter region of Saa-468 is further combined with a luciferase promoter region, and a promoter region of a gene promoter region, namely a promoter region of Saa promoter region, a promoter region of a promoter region, a promoter region of Saa promoter region, a promoter.
In an in vitro experiment, firstly, a macrophage cell line Raw264.7 is stimulated by recombinant Saa3 protein, the level of various growth factors and inflammatory factors in macrophages is found to be remarkably enhanced (FIG. 4I-L), macrophage culture supernatant after Saa3 treatment is collected, 3T3L1 cells are treated by the supernatant, the growth rate of 3T3L1 cells can be remarkably increased (FIG. 4M), PDGF-AA direct treatment can also remarkably increase the growth rate of 3T3L1 cells (FIG. 4N), similar effects to the supernatant treatment are achieved, in order to further confirm whether macrophages in subcutaneous fat tissues affect the maintenance of PDGFR α positive adipose precursor cells or not, subcutaneous fat on one side of a high-fat diet fed control mouse and a knockout mouse are injected with clodronate liposome to clear macrophages, on the other side of subcutaneous fat tissues are injected with the same amount of macrophage, a contrast liposome is injected in the control mouse under the expression level of a high-fat diet fed control mouse 4/80, the decrease of the Saa knockout mouse, and the macrophage proliferation of a macrophage cell proliferation marker F6380/9680 is further reduced in the contrast tissue of the mouse, and the macrophage proliferation of a macrophage proliferation inhibition of macrophage proliferation in the macrophage proliferation of macrophage proliferation in the mouse, the macrophage proliferation of the macrophage proliferation of proliferation in tissue (FIG. 7 mice is found to decrease of proliferation.
5. Conclusion
In addition, by means of animal models, a dialogue (crostalk) exists between 'mature white fat cells' and 'non-fat cells' in the process of obesity induced by high fat diet, and a new round of adipogenesis is started through the Wnt/β -catenin/Saa3 signal pathway.
The invention proves that β -catenin can inhibit transcription expression of Saa3, and discloses how a classical Wnt signal pathway influences the function of mature fat cells to influence obesity, or provides a new idea for obesity treatment.

Claims (3)

1. A construction method of a mature adipocyte-specific β -catenin knockout mouse comprises the following steps:
β -catenin with C57BL/6 backgroundflox/floxMating the mouse with Adiponectin-Cre mouse to obtain Adiponectin-Cre, β -cateninflox/wtAfter the mice, the mice were reacted with β -cateninflox/floxMice were mated and control mice β -cateninf were obtained in the offspringlox/floxAnd knock-out mouse adipoectin-Cre, β -cateninflox/floxThus, the mature adipocyte-specific β -catenin knockout mouse is obtained and is recorded as an ABKO knockout mouse.
2. The construction method according to claim 1, characterized in that: the mice were all housed in an SPF grade barrier environment.
3. The construction method according to claim 2, wherein: the ambient temperature is 20-22 ℃.
CN201911362176.0A 2019-12-26 2019-12-26 Construction method of mature adipocyte specific β -catenin knockout mouse Pending CN110973062A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040109866A1 (en) * 2001-03-21 2004-06-10 Chumas Nicola Jane Method and composition
CN103173413A (en) * 2012-12-07 2013-06-26 天津三箭生物技术有限公司 Mouse anti-human beta-catenin monoclonal antibody and hybridoma cell strain for secreting monoclonal antibody
CN104957094A (en) * 2015-07-08 2015-10-07 上海交通大学医学院附属瑞金医院 Method for building multi-tissue beta-catenin knockout mouse model
CN105145470A (en) * 2015-07-08 2015-12-16 上海交通大学医学院附属瑞金医院 Construction method for mature adipose tissue beta-catenin knocked-out mouse model

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040109866A1 (en) * 2001-03-21 2004-06-10 Chumas Nicola Jane Method and composition
CN103173413A (en) * 2012-12-07 2013-06-26 天津三箭生物技术有限公司 Mouse anti-human beta-catenin monoclonal antibody and hybridoma cell strain for secreting monoclonal antibody
CN104957094A (en) * 2015-07-08 2015-10-07 上海交通大学医学院附属瑞金医院 Method for building multi-tissue beta-catenin knockout mouse model
CN105145470A (en) * 2015-07-08 2015-12-16 上海交通大学医学院附属瑞金医院 Construction method for mature adipose tissue beta-catenin knocked-out mouse model

Non-Patent Citations (2)

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Title
崔梦莹等: "脂肪干细胞来源外泌体通过Wnt/-catenin信号通路促进脂肪干细胞成骨分化", 《中国医科大学学报》 *
邹译娴等: "自体脂肪干细胞局部注射对糖尿病足创面约合及细胞因子、Wnt/-catenin通路的影响", 《临床和实验医学杂志》 *

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Application publication date: 20200410