CN101259266B - A kind of preadipocyte heterogeneous vaccine and its preparation method and application - Google Patents

Abstract

The invention relates to a preadipocyte heterogeneous vaccine and its preparation method and application, belonging to the field of biotechnology. The preadipocyte heterogeneous vaccine of the present invention uses the mouse preadipocyte cell line 3T3-L1 as the source of the vaccine cells, and the vaccine is prepared by culturing and amplifying the cells in vitro, fixing with paraformaldehyde, and then washing and discarding the residual paraformaldehyde , prepared as a cell suspension. The preadipocyte heterogeneous vaccine of the present invention can carry out active immunization, can prevent the occurrence of obesity and is used for treating obesity.

Description

A kind of preadipocyte heterogeneous vaccine and its preparation method and application

technical field

The invention relates to a preadipocyte heterogeneous vaccine and its preparation method and application, belonging to the field of biotechnology.

Background technique

In recent years, with the development of my country's social economy, the improvement of people's material living standards and the change of lifestyle, the incidence of obesity has been increasing year by year. Obesity is a global chronic disease that seriously threatens human health. It is now known that obesity is an important risk factor for many chronic diseases and psychosocial disorders such as hypertension, hyperlipidemia, diabetes, cardiovascular disease, and sleep apnea syndrome. Important global health problem with national financial burden.

According to the regulations of the World Health Organization, when the calorie intake exceeds the body's consumption and is stored in the body in the form of fat, resulting in a body mass index (BMI): weight (Kg)/ ^height2 (m ² ) greater than 25-29.9 is considered excess. Body weight ≥30.0 is called obesity. There are 32 million women and 26 million men in the United States who are overweight. The World Health Organization believes that obesity, AIDS, drug abuse, and alcoholism will become the four major global health problems in the 21st century, and the prevention and treatment of obesity is the biggest epidemic challenge that countries in the world will face in the first 50 years of this century.

The main cause of obesity is that the body's energy intake exceeds consumption, and the excess energy is stored in the form of fat. The energy balance of the body is affected by multiple factors such as diet, exercise, endocrine diseases and genetics. A combination of energy restriction and increased exercise is recommended as an effective weight loss strategy. In the U.S., 29% of men and 44% of women believe they are trying to lose weight, but only 20% admit to doing a combination of energy restriction and increased exercise, which, while leading to weight loss, is difficult to maintain over the long term Effect. It can be seen that for most obese patients, weight loss is difficult, and long-term weight loss will face more severe challenges. Therefore, appropriate selection of weight loss drugs is an inevitable trend in the future.

Currently, drugs for the treatment of obesity can be divided into three categories according to their mechanism of action: appetite suppressants acting on the central body, lipase inhibitors acting on the periphery to reduce nutrient absorption, and drugs increasing energy expenditure. As far as clinical application is concerned, only the central appetite suppressant sibutramine and the lipase inhibitor orlistat, which acts on the periphery to reduce nutrient absorption, have been widely used without serious side effects, but the application time is not limited. Long-term follow-up clinical evaluation is still needed. The main side effects of sibutramine are dry mouth, anorexia, constipation, insomnia, nausea, abdominal pain, dizziness and other gastrointestinal tract and central nervous system stimulation. A small number of patients have slightly increased heart rate and slightly increased blood pressure. The difference is generally tolerable. The main side effect of orlistat is gastrointestinal discomfort, including fatty stool, steatorrhea, abdominal pain, abdominal distension, increased anal gas and fecal incontinence, etc. A few people give up using it because of gastrointestinal adverse reactions. To sum up, people are now eager to develop a safer and more effective weight-loss drug.

Studies have now shown that obesity is not only an increase in the volume of fat cells, but also an increase in the number of fat cells. The change in fat cell volume is mainly through lipogenesis or lipolysis. The increase in the number of adipocytes is achieved through the proliferation and differentiation of preadipocytes and the proliferation of mature adipocytes; the decrease in the number is achieved through the apoptosis of adipocytes, preadipocytes, and other possible mechanisms such as dedifferentiation of adipocytes. After dedifferentiation of preadipocytes or adipocytes of obese people, they have the characteristics of excessive proliferation under the conditions of culture in vitro. Usually, a balance is maintained between the increase and decrease of the number of fat cells. When the number of fat cells increases significantly more than the decrease, it may lead to the occurrence of obesity. Studies have shown that the apoptosis rate of adipocytes in simple obese children is significantly lower than that in non-obese children, and this abnormality may be involved in the occurrence of obesity. Obesity is thought to be partly the result of uncontrolled proliferation and differentiation of preadipocytes and proliferation of mature adipocytes. Therefore, it can be deduced that trying to induce the apoptosis of preadipocytes and mature adipocytes will be an important strategy for the treatment of obesity.

Neuropeptide Y (neuropeptide Y, NPY) is the most effective substance found so far to stimulate appetite in the central nervous system. Some studies have found that intracerebroventricularly (i.c.v.) injection of neuropeptide Y-Y5 receptors through lateral ventricle intubation. Antisense oligodeoxyribonucleic acid can induce apoptosis and lipolysis in adipocytes. Leptin (also known as cellulite) is a peptide hormone produced by fat cells, which may act on the corresponding receptors in the hypothalamus to regulate the metabolism of fat tissue and the number of fat cells. Leptin has no direct effect on the catabolism of adipocytes, which is supported by the fact that no receptors for leptin have been found in adipose tissue so far. Gullicksen et al. found that intracerebroventricular (i.c.v.) injection of leptin not only induced apoptosis of adipocytes, but also reduced the size of adipocytes.

It can be seen that the neuropeptide Y and leptin that can induce adipocyte apoptosis found in the current research require surgical intraventricular (i.c.v.) injection to have the therapeutic effect of inducing adipocyte apoptosis. This kind of complicated brain surgery Not only is it expensive, but it is also risky, unacceptable to most patients, and basically not feasible for clinical implementation. Therefore, finding an effective and clinically feasible method for inducing apoptosis of preadipocytes and mature adipocytes is a key issue in current obesity treatment research and an international research hotspot.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a preadipocyte heterogeneous vaccine and its preparation method and application, providing a new method for inducing preadipocyte apoptosis.

Summary of the invention

We use pre-adipocyte xenogeneic immunity to induce the body to generate an immune response to its own pre-adipocytes, prevent the proliferation and differentiation of its own pre-adipocytes into mature adipocytes, and effectively inhibit the generation of adipose tissue. Under normal circumstances, the body does not produce an immune response to self-antigens, that is, self-tolerance; therefore, we combine heterogeneous homologous genes in biological evolution, heterogeneous immune rejection and autoimmune responses to overcome the immune response to self-antigens. tolerance. In this invention, we will design a method for inducing apoptosis of preadipocytes and explore its feasibility.

The present invention uses fixed heterogeneous preadipocytes as a vaccine for active immunization, and uses the same kind of preadipocytes and heterogeneous fibroblasts as controls to break the immune tolerance of the body to its own preadipocytes and induce the body to produce anti- The cross-immune reaction of self-preadipocytes leads to the apoptosis of preadipocytes, achieves the purpose of treating obesity, and finally achieves a breakthrough in new ways of treating obesity.

Detailed description of the invention

Technical scheme of the present invention is as follows:

The preadipocyte heterogeneous vaccine of the present invention is a preadipocyte heterogeneous vaccine produced by using mouse 3T3-L1 preadipocytes as the preadipocyte source. It can be used as a cross-antigen for heterogeneous immunity, stimulate the body's immune system, and generate an immune response to the body's preadipocytes.

Preadipocyte heterogeneous vaccine preparation method of the present invention, the steps are as follows:

Mouse 3T3-L1 preadipocytes were cultured on DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. When the mouse 3T3-L1 preadipocytes reached 70 %, digested with trypsin and passaged. The preadipocytes cultured in vitro were collected and washed 3 times with PBS. 3% paraformaldehyde was prepared in PBS, pH 7.4, as a fixative solution, and the cells were fixed at 4°C for 24 h. Wash 3 times with PBS and incubate at 37°C for 2h to remove residual paraformaldehyde. The preadipocyte xenogeneic vaccine is obtained after resuspended in PBS.

Carry out immunoprevention with the heterogeneous preadipocyte vaccine prepared by the present invention, through the research of anti-rat preadipocyte autoantibody and the detection of preadipocyte apoptosis, the result shows that the preadipocyte heterogeneous vaccine of the present invention can break the body to self preadipocyte. The immune tolerance of the cells induces the body to produce a cross-immune reaction against its own pre-adipocytes, thereby leading to the apoptosis of the pre-adipocytes, achieving the purpose of treating obesity, and can be used for the treatment of obesity.

The application of the preadipocyte heterogeneous vaccine of the present invention is used to prepare the medicine for preventing or treating obesity. The preadipocyte heterogeneous vaccine of the present invention can carry out active immunization and can prevent the occurrence of obesity and treat obesity.

Description of drawings

Figure 1 is the body weight-time curves of male rats in the treatment group and the control group.

Figure 2 is the body weight-time curves of female rats in the treatment group and the control group.

Figure 3 is an immunohistochemical photograph of autoantibody storage in rat preadipocytes.

Figure 4 is an immunofluorescence photograph of apoptosis of preadipocytes in vivo.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Example 1:

(1) Cultivation and vaccine preparation of vaccine cells and control cells:

1. Reagents:

(1) Phosphate buffered saline (PBS): Dissolve ZLI-9061PBS in 1000ml of distilled water and mix well. The measured pH should be between 7.2-7.4. If it deviates from this range, adjust it with 0.1N HCL or NaOH.

(2) Fixative solution: prepare 3% paraformaldehyde with PBS, pH 7.4.

2. Materials:

Preadipocytes were derived from human and mouse preadipocyte cell lines cultured in vitro, among which mouse preadipocyte cell line 3T3-L1, human fibroblast cell line MRC-5 and NIH/3T3 cell line were purchased from ATCC (American Type Culture Collection ), it is convenient to purchase, therefore, we used mouse preadipocyte cell line 3T3-L1 as the source of vaccine cells, and human fibroblast cell line MRC-5 and mouse fibroblast cell line NIH/3T3 as control cells.

3. Test method:

Culture of mouse 3T3-L1 preadipocytes: cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. When the mouse 3T3-L1 preadipocytes reached 70% confluence, they were digested with trypsin and passaged. Both the human fibroblast cell line MRC-5 and the NIH/3T3 cell line were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin according to the conventional cell line culture method. It is placed in a 37°C incubator containing 5% CO ₂ for massive amplification.

The cells after in vitro culture and expansion were collected in centrifuge tubes, including various preadipocytes and control cells, and washed 3 times with PBS. Cells were fixed with fixative at 4°C for 24 h. Wash 3 times with PBS and incubate at 37°C for 2h to remove residual paraformaldehyde. Add PBS to prepare a cell suspension, count the cells with a hemocytometer, and prepare a cell suspension with 0.9% physiological saline at a concentration of 10 ⁶ /ml. That is, the preadipocyte xenogeneic vaccine was prepared, placed in a centrifuge tube, sealed, and stored in a -80°C refrigerator for later use.

(2) Effect of preadipocyte xenogeneic vaccine immunization on preventing obesity in rats after eating high-energy diet:

6-8 weeks old, female SD rats were randomly divided into a treatment group and a control group (10 rats in each group). Each rat in the treatment group was treated with 1 ml of mouse preadipocytes at a concentration of 10 ⁶ /ml. Mice were also intraperitoneally injected with 1 ml of human fibroblast cell line MRC-5, NIH/3T3 cells and 0.9% normal saline, once a week for 4 consecutive weeks. After the 4th injection immunization was completed, SD rats were fed high-energy feed (higher fat and protein content), and the high-energy feed consisted of 25% crude protein, 11% fat, and 64% carbohydrates. The same method was used to study male rats. The weight changes of male and female rats in the vaccine immunization group and control group were observed, and the non-parametric rank sum test was used for statistical analysis to analyze whether immunization with xenogeneic preadipocytes could prevent the occurrence of obesity.

The body weight-time curves in the female rat treatment group and the control group were plotted, and the results are shown in Figure 1. The body weight-time curve of male rats is shown in Figure 2.

It can be seen that the body weight of rats immunized with heterogeneous mouse preadipocytes, regardless of male or female, was significantly suppressed, statistical analysis P<0.05. However, the body weight of rats immunized with human fibroblast cell line MRC-5, NIH/3T3 cells and 0.9% normal saline did not change significantly, statistical analysis P>0.05.

(3) Detection of autoantibody storage in preadipocytes of rats

The preadipocytes and mature adipocytes of immunized rats can be stained by immunohistochemical techniques using the serum of rats immunized with xenogeneic preadipocytes and the serum of rats not immunized with xenogeneic preadipocytes (as a control) as the primary antibody. Autoantibody stores were identified in preadipocytes of immunized rats (shown in Figure 3).

(4) Detection of apoptosis of preadipocytes:

The apoptosis of preadipocytes in vivo was detected by terminal deoxynucleotidyl transferase (TUNEL method) labeled immunofluorescent staining (shown in Figure 4).

Claims (2)

1. a preadipocyte heterogeneous vaccine, is characterized in that, is to use mouse 3T3-L1 preadipocyte as preadipocyte source, makes by the following method: Culture mouse 3T3-L1 preadipocytes on DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, when the confluence of mouse 3T3-L1 preadipocytes reaches 70% , digested with trypsin and passaged; collected preadipocytes cultured in vitro, washed 3 times with PBS; prepared 3% paraformaldehyde with PBS, pH 7.4, as a fixative, fixed cells at 4°C for 24h; washed 3 times with PBS , incubate at 37°C for 2 hours to remove residual paraformaldehyde; resuspend in PBS to obtain the preadipocyte xenogeneic vaccine. 2. the purposes of a kind of preadipocyte xenogeneic vaccine described in claim 1, for the preparation of the medicament of preventing or treating obesity.

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