CN-116676253-B - Working fluid with cell cycle regulating and cell immunity functions and using method thereof

Abstract

The invention relates to the technical field of novel application of compounds, and aims to provide a working solution with cell cycle regulating and cell immunity functions and a use method thereof. The active ingredients of the working solution are butyric acid and palmitic acid, and the balance of DMSO and ddH2O used as solvents and a cell culture medium used for providing nutrition for cells and promoting growth and proliferation of cells, wherein the mass ratio of butyric acid to the cell culture medium is 0.013..100, and the mass ratio of palmitic acid to the cell culture medium is 0.00064..100. The reagent provided by the invention has very obvious effects of enhancing cellular immunity and relieving cell cycle arrest when being used for cell culture. The reagent components are fatty acid, which is one of the components of the metabolic products of the organism cells, has no obvious toxic or side effect on the organism cells, and is safer, more convenient and less in side effect when being used as a reagent for regulating the cell cycle and enhancing the cellular immunity.

Inventors

• SHAN YING
• MAO JUNYONG
• Zhang Chuni
• XU JIDONG
• LI XIAOLIANG
• FANG WEIHUAN

Assignees

• 浙江大学

Dates

Publication Date

20260508

Application Date

20230529

Claims (6)

1. 1. A working solution with cell cycle regulating and cell immunity functions is characterized in that the working solution comprises the active ingredients of butyric acid and palmitic acid, the balance of DMSO and ddH 2 O used as solvents and a cell culture medium used for providing cell nutrition and promoting cell growth and proliferation, the palmitic acid plays a role in promoting the coordination of the innate immunity of the cells directly enhanced by the butyric acid, and the cell growth and replication are promoted by regulating CyclinA protein to relieve the cell cycle retardation caused by the butyric acid, wherein the mass ratio of the butyric acid to the cell culture medium is 0.013..100, and the mass ratio of the palmitic acid to the cell culture medium is 0.00064..100.
2. 2. The working fluid of claim 1, wherein the butyric acid has cas number 107-92-6, molecular formula C 4 H 8 O 2 and molecular weight 88.11, and the palmitic acid cas number 57-10-3, molecular formula C 16 H 32 O 2 and molecular weight 256.424.
3. 3. The working fluid according to claim 1, wherein the cell culture medium is any one of DMEM medium, 1640 medium, F12 medium, and MEM medium, and the active ingredients thereof include amino acids, sugars, minerals, vitamins, oxidants, and antibiotics.
4. 4. The method for preparing a working fluid with cell cycle and cellular immune function according to claim 1, comprising the steps of: (1) Dissolving palmitic acid in DMSO according to the proportion of 25.65 mg:10 mL to obtain 10 mM-concentration palmitic acid mother liquor, and dissolving butyric acid in ddH 2 O according to the proportion of 0.881: 0.881 g:10: 10 mL to obtain 1M-concentration butyric acid mother liquor; (2) The ratio of the cell culture medium is 2.5 mu L/mL and 1.5 mu L/mL, the mother solution of palmitic acid and the mother solution of butyric acid are respectively taken, and then the mother solution and the mother solution of butyric acid are sequentially added into the cell culture medium and are uniformly mixed to prepare working solution, wherein the percentage content of butyric acid and palmitic acid in the working solution is 0.013 percent and 0.00064 percent respectively.
5. 5. The method of using a working fluid having a cell cycle regulating and cellular immune function according to claim 1, wherein the working fluid is used for cell culture.
6. 6. The method according to claim 5, wherein the working fluid is used in any of the following ways: (1) Pretreatment: Adding 2 mL working solution to pretreat cells, then removing the working solution, washing twice with Hank's solution, and then carrying out subsequent operation on the cells according to an experimental scheme; (2) And (3) simultaneous treatment: For the cell culture test in progress, the original cell culture medium in the pore plate is discarded, and the cell culture is carried out by using 2mL working solution while the subsequent operation is carried out on the cells according to the experimental scheme; (3) Post-treatment: after the cells were manipulated according to the protocol, they were washed twice with Hank's solution and cultured by adding 2 mL working solution.

Description

Working fluid with cell cycle regulating and cell immunity functions and using method thereof Technical Field The invention belongs to the technical field of novel application of compounds, and relates to a working solution with cell cycle regulating and cell immunity functions and a use method thereof. Background Butyric acid, which is a short chain fatty acid, can provide energy to intestinal cells, promote differentiation, proliferation and maturation of intestinal cells, enhance intestinal immunity, and have a wide range of pharmacological actions including anti-inflammatory, antioxidant, antitumor activity, metabolic regulation, etc. Studies report that butyric acid has an inhibitory effect on the inflammatory response induced in the intestinal tract of mice, and that butyric acid activates immune cells such as macrophages in the colon, promotes the differentiation of T lymphocytes, and finally inhibits colonic inflammation. Butyrate may also protect digestive gland cells from apoptosis by inhibiting the PERK-CHOP pathway of endoplasmic reticulum stress. Palmitic acid, which is used as a long-chain saturated fatty acid and is often used as a feed additive, can directly stimulate a host to generate immune response, especially IgA antibody, through various ways of promoting the generation of pro-inflammatory cytokines IL-6 and TNF-alpha, and the like, so as to form intestinal mucosa immunity, thereby guaranteeing the intestinal immune barrier function. In the prior art, the application research of butyric acid and palmitic acid is limited to the stimulation or promotion of two fatty acids on host body related cells respectively so as to improve the immunocompetence of host body cells or organisms. The method for combining and using two fatty acids, namely butyric acid and palmitic acid, and the corresponding effect research are not reported at present. Disclosure of Invention The invention aims to solve the technical problems, overcome the defects in the prior art, provide a new application of the combination of two fatty acids, namely butyric acid and palmitic acid, and particularly provide a working solution with the functions of regulating cell cycle and cell immunity and a use method thereof. In order to solve the technical problems, the technical scheme of the invention is as follows: A working solution having cell cycle and cellular immunity regulating functions is provided, wherein the working solution comprises butyric acid and palmitic acid as effective components, and the balance of DMSO and ddH 2 O used as solvents, and a cell culture medium for providing cell nutrition and promoting cell growth and proliferation, wherein the mass ratio of butyric acid to cell culture medium is 0.013..100, and the mass ratio of palmitic acid to cell culture medium is 0.00064..100. In the invention, the cas number of the butyric acid is 107-92-6, the molecular formula is C 4H8O2, the molecular weight is 88.11, the cas number of the palmitic acid is 57-10-3, the molecular formula is C 16H32O2, and the molecular weight is 256.424. As a preferred embodiment of the present invention, the cell culture medium is any one of DMEM medium, 1640 medium, F12 medium and MEM medium, which are commercially available and mature products. The effective components of the cell culture medium include amino acids, sugar, minerals, vitamins, oxidants and antibiotics. Amino acids include, by way of example, glycine, tyrosine, alanine, histidine, phenylalanine, threonine, cysteine, valine, glutamic acid, and the like. The saccharide includes glucose, galactose, cytose, polylactic acid, polydextrose, etc. The main components of the mineral substances include calcium, phosphorus, magnesium, sodium, potassium, etc. Vitamins include vitamin B12, nicotinamide, acetylcholine, inositol, folic acid, vitamin E, and vitamin C. The oxidant comprises protease remover, glutathione, hydrogen peroxide, and indoletriol. Antibiotics include streptomycin, amphotericin B, and the like. The invention also provides a preparation method of the working solution, which comprises the following steps: (1) Dissolving palmitic acid in DMSO at a ratio of 25.65mg..10mL to obtain 10mM palmitic acid mother liquor, and dissolving butyric acid in ddH 2 O at a ratio of 0.881g..10mL to obtain 1M butyric acid mother liquor; (2) The ratio of the cell culture medium is 2.5 mu L/mL and 1.5 mu L/mL, the mother solution of palmitic acid and the mother solution of butyric acid are respectively taken, and then the mother solution and the mother solution of butyric acid are sequentially added into the cell culture medium and are uniformly mixed to prepare working solution, wherein the percentage content of butyric acid and palmitic acid in the working solution is 0.013 percent and 0.00064 percent respectively. The invention further provides a using method of the working fluid, and the working fluid is used for cell culture. In use, the working concentrations of butyric and palmi