阅读说明：本技术 一种脂质体转染细胞保护剂 (Liposome transfected cell protective agent ) 是由 杨大旺 张帆 于 2019-12-03 设计创作，主要内容包括：本发明提供了一种脂质体转染过程中的细胞活性保护剂,其特征在于所述细胞保护剂含有以下组分：终浓度为1～10mmol/L的维生素A,终浓度为5～15g/L的甘油,终浓度为10～40g/L的葡萄糖,终浓度为3～20g/L的人血白蛋白。本发明配制的细胞保护剂易于保存且具有较长的货架期,配制好的细胞保护剂在4～8℃下可以保存1年。与在转染容器,本保护剂与转染DNA和脂质体转染试剂的混合液进行转染,不仅可以保持细胞的活性,而且极大提高转染效率,特别是对于比较难转染的免疫细胞,转染效率提高了40％以上。(The invention provides a cell activity protective agent in a liposome transfection process, which is characterized by comprising the following components: vitamin A with the final concentration of 1-10 mmol/L, glycerol with the final concentration of 5-15 g/L, glucose with the final concentration of 10-40 g/L and human serum albumin with the final concentration of 3-20 g/L. The cell protective agent prepared by the invention is easy to store and has a long shelf life, and the prepared cell protective agent can be stored for 1 year at 4-8 ℃. When the protective agent is transfected with the mixed solution of transfected DNA and liposome transfection reagent in a transfection container, the activity of cells can be maintained, the transfection efficiency is greatly improved, and particularly for immune cells which are difficult to transfect, the transfection efficiency is improved by more than 40%.)

1. A cytoprotective agent for use in lipofection, characterized in that said cytoprotective agent comprises the following components: vitamin A with the final concentration of 1-10 mmol/L, glycerol with the final concentration of 5-15 g/L, glucose with the final concentration of 10-40 g/L and human serum albumin with the final concentration of 3-20 g/L.

2. The cytoprotective agent of claim 1, characterized in that the cytoprotective agent comprises the following components: vitamin A with the final concentration of 4-8 mmol/L, glycerol with the final concentration of 10-15 g/L, glucose with the final concentration of 15-20 g/L and human serum albumin with the final concentration of 10-17 g/L.

3. Cytoprotective agent according to claim 1 or 2, characterized in that it comprises the following components: vitamin A with a final concentration of 5mmol/L, glycerol with a final concentration of 10g/L, glucose with a final concentration of 16g/L and human serum albumin with a final concentration of 14 g/L.

4. The cytoprotective agent of claim 1, characterized in that the pH of the cytoprotective agent is 7.1-7.5.

5. The cell protective agent of claim 1 transfected with a mixture of transfected DNA and lipofectin.

Technical Field

The invention relates to the technical field of cell biology, in particular to a cell activity protective agent in a liposome transfection process.

Background

Transfection is a special technology for transferring exogenous genetic materials into eukaryotic cells, and along with the deep research on the functions of genes and proteins, transfection becomes a basic method frequently involved in the modern molecular biology research, solves the problem of introducing exogenous genetic materials into cells, and lays a foundation for the gene function research on the cell level. With the recent development of genetic engineering techniques, a variety of transfection techniques have emerged. The general transfection methods mostly adopt liposome method and electroporation method. The cationic liposome has positive charges on the surface, wraps DNA molecules into a DNA-liposome compound through electrostatic interaction with phosphate radicals of nucleic acid, can be adsorbed on cell membranes with negative charges on the surface, and enters cell bodies through fusion of the membranes or endocytosis of the cells. DNA is also sometimes delivered into cells by direct osmosis through small pores in the cell membrane. Electroporation or electroporation is a method in which exogenous molecules, such as DNA, mRNA, proteins, saccharides, etc., are delivered into the cytoplasm of a host cell by instantaneously increasing the permeability of cell membranes by the action of a high-intensity electric field. Viral methods often require higher virus titers to infect primary cells and are generally less than optimal. The electroporation method has more advantages in operation simplicity and experimental period than the virus method for infecting primary cells. However, the popularity of this method is limited by factors such as electrotransfer efficiency, cell viability, and experimental instrumentation. In addition, primary cells are highly resistant to a variety of transfection methods, which makes the means for efficient gene editing of cells lacking.

It has been found that the state of cell viability during transfection has a large effect on transfection efficiency. Especially in the transfection process of immune cells, the cell activity has a great influence on the transfection efficiency. At present, the cell activity protective agent on the market is generally compound sodium citrate preservative solution (ACD), EDTA or heparin. In order to ensure the activity of immune cells, anticoagulation blood added with compound sodium citrate preservative solution (ACD), EDTA or heparin must be used within 6 hours. If the preservation time of the anticoagulation blood exceeds 10 hours, the lymphocyte therein can be partially or totally dead, and the subsequent transfection experiment can not be carried out. Therefore, there is a need to develop a new cytoprotective agent for protecting the viability of immune cells, particularly lymphocytes and T cells, in blood.

Disclosure of Invention

The invention aims to provide a cell activity protective agent in a liposome transfection process, which comprises the following components: vitamin A with the final concentration of 1-10 mmol/L, glycerol with the final concentration of 5-15 g/L, glucose with the final concentration of 10-40 g/L and human serum albumin with the final concentration of 3-20 g/L;

the cytoprotective agent preferably comprises the following components: vitamin A with the final concentration of 4-8 mmol/L, glycerol with the final concentration of 10-15 g/L, glucose with the final concentration of 15-20 g/L and human serum albumin with the final concentration of 10-17 g/L;

the cytoprotective agent, more preferably, the cytoprotective agent comprises the following components: vitamin A with a final concentration of 5mmol/L, glycerol with a final concentration of 10g/L, glucose with a final concentration of 16g/L and human serum albumin with a final concentration of 14 g/L;

the cytoprotective agent, more preferably, the pH value of the cytoprotective agent is 7.1-7.5;

the cell protective agent is transfected with a mixed solution of transfection DNA and a liposome transfection reagent.

The cell protective agent prepared by the invention is easy to store and has a long shelf life, and the prepared cell protective agent can be stored for 1 year at 4-8 ℃. When the protective agent is transfected with the mixed solution of transfected DNA and liposome transfection reagent in a transfection container, the activity of cells can be maintained, the transfection efficiency is greatly improved, and particularly for immune cells which are difficult to transfect, the transfection efficiency is improved by more than 40%.

Drawings

FIG. 1 is a photomicrograph of T cells transfected with pEGFP-C1 using reagents under white light (left) and GFP excitation light (right);

FIG. 2 is a fluorescent micrograph of T cells transfected with GFP overexpressing retroviral vectors using the present reagent in combination with a TransLipidHL transfection reagent under GFP excitation light;

FIG. 3 is a fluorescence micrograph of T cells transfected with shRNA retroviral vectors using the present reagents under RFP excitation light.

Detailed Description