阅读说明：本技术 一种肿瘤细胞培养信息分析方法 (Tumor cell culture information analysis method ) 是由 刘译远 劉瑞宸 拉斯洛谢克利 于 2020-06-02 设计创作，主要内容包括：本发明公开了一种肿瘤细胞培养信息分析方法,包括以下步骤：肿瘤细胞培养、药理培养、细胞膜磷脂提取、细胞膜磷脂甲酯化、气相技术分析、信息对比。将血管内皮细胞接种于培养容器的渗透支持膜上形成血管内皮细胞层；起始细胞放置于渗透支持膜血管内皮细胞层上方并形成半固态凝胶状的混合体,在对一类、二类及三类培养过程中,对样品中添加抗肿瘤药物,培养后收集培养容器的细胞液,对其进行细胞计数,根据培养容器的细胞数量可检测出细胞迁移能力的强弱。(The invention discloses a tumor cell culture information analysis method, which comprises the following steps: tumor cell culture, pharmacological culture, cell membrane phospholipid extraction, cell membrane phospholipid methyl esterification, gas phase technical analysis and information comparison. Inoculating the vascular endothelial cells on the permeable support membrane of the culture container to form a vascular endothelial cell layer; the method comprises the steps of placing initial cells above an infiltration supporting membrane vascular endothelial cell layer to form a semi-solid gelatinous mixture, adding an anti-tumor drug into a sample in the processes of culturing a first type, a second type and a third type, collecting cell sap of a culture container after culturing, counting cells, and detecting the migration capacity of the cells according to the cell number of the culture container.)

1. A method for analyzing information of tumor cell culture, which is characterized in that: the method comprises the following steps:

s1, culturing tumor cells, namely providing the tumor cells as initial cells, culturing the tumor cells from the same tumor tissue of the same object in a culture medium, and classifying culture samples into one type, two types and three types as subsequent information comparison;

s2, performing pharmacological culture, namely, firstly, inoculating vascular endothelial cells on a permeable support membrane of a culture container to form a vascular endothelial cell layer; placing the initial cells above the vascular endothelial cell layer of the osmotic support membrane to form a semi-solid gelatinous mixture, adding an anti-tumor drug into a sample in the processes of culturing one type, two types and three types, collecting cell sap of a culture container after culturing, counting the cells, and detecting the migration capacity of the cells according to the cell number of the culture container;

s3, extracting cell membrane phospholipid, removing a culture medium after the tumor cell culture is finished, taking out tumor cell fluid, extracting the tumor cell membrane phospholipid, suspending the collected cells in PBS, breaking the membrane of the cell suspension in an ultrasonic cell crusher under the ice bath condition for 30 minutes, taking a certain volume of membrane breaking fluid in a mixed solution of methanol and chloroform, shaking uniformly, standing at room temperature, and centrifuging to take the lower phase;

s4, performing methyl esterification on cell membrane phospholipid, adding a potassium hydroxide-methanol solution into a cell membrane phospholipid extracting solution, performing methyl esterification to obtain methyl esterified fatty acid, adding hydrochloric acid, shaking, standing, adding a proper amount of anhydrous sodium sulfate, drying, and taking the lower layer clear liquid to be tested;

s5, performing gas phase technical analysis, namely performing gas phase analysis on the clear liquid extracted in the step 3, and making maps for the first class, the second class and the third class of samples;

and S6, comparing the information, and comparing the cell characteristic information and the cell characteristic difference to obtain an analysis result.

2. The method of claim 1, wherein the method comprises: the culture medium in the S1 consists of 10% of acellular ascites, 3% of fetal calf serum, 1% of double antibody and the balance of DMEM culture medium.

3. The method of claim 1, wherein the method comprises: when shaking and shaking are carried out in the S3, magnetic beads are enriched in a centrifugal tube, and the centrifugal tube is combined in a rotating mode for 20-40min at the temperature of 2-8 ℃; and then placing the centrifugal tube on a magnet for 3min, and removing the supernatant to obtain the magnetic bead suspension.

4. The method of claim 1, wherein the method comprises: the permeable support membrane in S2 is made of an organic material.

5. The method of claim 1, wherein the method comprises: one, two and three of the samples in S1 correspond to early, metaphase and late stage cells of tumor cells, respectively.

6. The method of claim 1, wherein the method comprises: the cellular characteristic differences in S6 include: differences in methylation levels of single or multiple genes, differences in methylation levels of single or multiple genomic regions.

7. The method of claim 1, wherein the method comprises: in the step S3, the culture medium is removed, the cell sediment is washed, then cell digestive juice is added, when the contraction of the interstitial cells becomes round but the epithelial cells do not contract, the culture bottle is tapped and tapped to separate the interstitial cells, and the interstitial cells are removed.

8. The method of claim 1, wherein the method comprises: in the S2, the culture temperature was set to 36.5 ℃ during the culture.

Technical Field

The invention belongs to the field of cell culture, and particularly relates to a tumor cell culture information analysis method.

Background

Malignant tumor has become one of the main diseases seriously threatening human life and health, and has the characteristics of high morbidity and high mortality, and the occurrence of tumor increasingly affects economic development and human health. Therefore, the development of malignant tumor is urgently inhibited by adopting an effective method for preventing and treating tumor.

Many diseases, including tumors, are accompanied by changes in membrane phospholipid metabolism, which affects the physicochemical properties of cell membranes and, in turn, the biological functions of various membrane proteins. The study of changes in cell membrane phospholipids not only helps to elucidate the pathogenesis of these diseases and tumors, but also helps in the diagnosis and treatment of diseases. Since the turnover rate of cell membrane phospholipids is extremely fast, it degrades rapidly with cell death and the structure and kind of fatty acids vary from tumor cell to tumor cell. Due to the limitation of detection technology, the analysis of membrane lipid is mostly limited to the analysis and identification of the total amount of the whole lipid, and the research on the phospholipid composition difference of different tumor cells is difficult due to the overlapping of chromatographic peaks of different fatty acids.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a tumor cell culture information analysis method.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for analyzing tumor cell culture information comprises the following steps:

s1, culturing tumor cells, namely providing the tumor cells as initial cells, culturing the tumor cells from the same tumor tissue of the same object in a culture medium, and classifying culture samples into one type, two types and three types as subsequent information comparison;

s2, performing pharmacological culture, namely, firstly, inoculating vascular endothelial cells on a permeable support membrane of a culture container to form a vascular endothelial cell layer; placing the initial cells above the vascular endothelial cell layer of the osmotic support membrane to form a semi-solid gelatinous mixture, adding an anti-tumor drug into a sample in the processes of culturing one type, two types and three types, collecting cell sap of a culture container after culturing, counting the cells, and detecting the migration capacity of the cells according to the cell number of the culture container;

s3, extracting cell membrane phospholipid, removing a culture medium after the tumor cell culture is finished, taking out tumor cell fluid, extracting the tumor cell membrane phospholipid, suspending the collected cells in PBS, breaking the membrane of the cell suspension in an ultrasonic cell crusher under the ice bath condition for 30 minutes, taking a certain volume of membrane breaking fluid in a mixed solution of methanol and chloroform, shaking uniformly, standing at room temperature, and centrifuging to take the lower phase;

s4, performing methyl esterification on cell membrane phospholipid, adding a potassium hydroxide-methanol solution into a cell membrane phospholipid extracting solution, performing methyl esterification to obtain methyl esterified fatty acid, adding hydrochloric acid, shaking, standing, adding a proper amount of anhydrous sodium sulfate, drying, and taking the lower layer clear liquid to be tested;

s5, performing gas phase technical analysis, namely performing gas phase analysis on the clear liquid extracted in the step 3, and making maps for the first class, the second class and the third class of samples;

and S6, comparing the information, and comparing the cell characteristic information and the cell characteristic difference to obtain an analysis result.

Preferably, the culture medium in S1 consists of 10% of acellular ascites, 3% of fetal bovine serum, 1% of double antibody, and the balance of DMEM culture medium.

Preferably, when shaking and shaking are carried out in S3, magnetic beads are enriched in a centrifuge tube, and the centrifuge tube is combined in a rotating mode for 20-40min at the temperature of 2-8 ℃; and then placing the centrifugal tube on a magnet for 3min, and removing the supernatant to obtain the magnetic bead suspension.

Preferably, the permeable support membrane in S2 is made of an organic material.

Preferably, one, two and three of the samples in S1 correspond to early stage cells, metaphase cells and late stage cells of tumor cells, respectively.

Preferably, the cellular characteristic difference in S6 includes: differences in methylation levels of single or multiple genes, differences in methylation levels of single or multiple genomic regions.

Preferably, in S3, after removing the culture medium and washing the cell pellet, a cell digest is added, and when the mesenchymal cells shrink and become round but the epithelial cells do not shrink, the culture flask is tapped and tapped to detach the mesenchymal cells, thereby removing the mesenchymal cells.

Preferably, in the S2, the culture temperature is set to 36.5 ℃ during the culture.

The invention has the technical effects and advantages that:

1. inoculating the vascular endothelial cells on the permeable support membrane of the culture container to form a vascular endothelial cell layer; placing the initial cells above the vascular endothelial cell layer of the osmotic support membrane to form a semi-solid gelatinous mixture, adding an anti-tumor drug into a sample in the processes of culturing one type, two types and three types, collecting cell sap of a culture container after culturing, counting the cells, and detecting the migration capacity of the cells according to the cell number of the culture container;

2. the culture samples are divided into a first class, a second class and a third class which are used for subsequent information comparison, and the first class, the second class and the third class respectively correspond to early cells, metaphase cells and late cells of tumor cells, so that the culture comparison of the tumor cells at different stages is facilitated.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.