阅读说明：本技术 一种通过免疫荧光检测药物对胰岛细胞摄脂影响的方法 (Method for detecting influence of drug on pancreatic islet cell lipid uptake through immunofluorescence ) 是由 余琦 张云婷 卫梦倩 于 2019-08-29 设计创作，主要内容包括：本发明公开了一种通过免疫荧光检测药物对胰岛细胞摄脂影响的方法,按照以下步骤具体实施：先将胰岛细胞培养长至3×10<Sup>6</Sup>～5×10<Sup>6</Sup>个,将胰岛细胞进行传代培养,传代培养后吸取一定量的细胞悬液,接种至共聚焦小皿,待细胞贴壁后进行饥饿处理,再用药物单剂量干预细胞；再采用Dil-LDL孵育干预后的细胞,使用PBS清洗三次、使用多聚甲醛固定,弃去液体,用去离子进行水洗；水洗后使用DAPI染色,染色后弃去液体,用去离子水洗,水洗后对细胞进行干燥处理,并在荧光显微镜下观察细胞摄入低密度脂蛋白胆固醇的情况。本发明的实验过程采样均匀,结果可信度高。(The invention discloses a method for detecting the influence of a medicament on the lipid uptake of islet cells through immunofluorescence, which is specifically implemented according to the following steps: firstly, islet cells are cultured to grow to 3 × 10 6 ～5×10 6 Secondly, subculturing the islet cells, sucking a certain amount of cell suspension after subculturing, inoculating the islet cells to a confocal dish, performing starvation treatment after the cells adhere to the wall, and interfering the cells with single dose of the medicine; then incubating the cells for stem prognosis by using Dil-LDL, washing the cells for three times by using PBS, fixing the cells by using paraformaldehyde, discarding liquid, and washing the cells by using deionized water; washing with water, staining with DAPI, removing liquid after staining, washing with deionized water, drying the cells after washing, and observing the condition of low density lipoprotein cholesterol uptake of the cells under a fluorescence microscope. The experimental process of the invention has uniform sampling and high result reliability.)

1. A method for detecting the influence of a medicament on the lipid uptake of islet cells through immunofluorescence is characterized by comprising the following steps of:

step 1, culturing islet cells to 3 × 10⁶～5×10⁶Secondly, subculturing the islet cells, sucking a certain amount of cell suspension after subculturing, inoculating the islet cells to a confocal dish, performing starvation treatment after the cells adhere to the wall, and interfering the cells with single dose of statins;

step 2, incubating the cells obtained in the step 1 by using Dil-LDL, washing the cells for three times by using PBS, fixing the cells by using paraformaldehyde, discarding liquid, and washing the cells by using deionized water;

step 3, dyeing the cells washed in the step 2 by using DAPI, discarding liquid after dyeing, washing for 3 times by using deionized water, and washing for 3min each time;

step 4, drying the cells obtained in the step 3;

and 5, observing the condition that the cells obtained in the step 4 take in the low-density lipoprotein cholesterol under a fluorescence microscope.

2. The method for detecting the effect of drugs on the lipid uptake of pancreatic islet cells through immunofluorescence according to claim 1, wherein the subculture in step 1 is a one-fourth culture, the volume of cells inoculated into a confocal dish is 500 μ L, the starvation treatment time is 12h, and the intervening cell time is 48 h.

3. The method for detecting the effect of a drug on the lipid uptake of islet cells via immunofluorescence according to claim 1, wherein the islet cells in step 1 are islet β cells MIN6 cells.

4. The method for detecting the effect of a drug on the lipid uptake of pancreatic islet cells through immunofluorescence according to claim 1, wherein the Dil-LDL incubation time of the cells in the step 2 is 4-6 h, the concentration of paraformaldehyde is 4%, the volume of the paraformaldehyde-immobilized cells is 1-2 mL, the time of the paraformaldehyde-immobilized cells is 20-30 min, and the number of times of washing with deionized water is 3-5, wherein each time of washing with water is 5 s.

5. The method for detecting the effect of a drug on the lipid uptake of pancreatic islet cells through immunofluorescence according to claim 1, wherein the volume of DAPI staining in step 3 is 1-2 mL, and the time of DAPI staining is 5 min.

6. The method for detecting the influence of the drug on the lipid uptake of the islet cells through immunofluorescence according to claim 1, wherein the drying temperature in the step 4 is 40-80 ℃, and the drying time is 2-5 min.

7. The method for detecting the effect of drugs on the lipid uptake of pancreatic islet cells through immunofluorescence according to claim 1, wherein in the step 5, during the fluorescence detection, the cover of the confocal dish is divided into the squared region for fluorescence photographing.

8. The method for detecting the effect of a drug on the lipid uptake of pancreatic islet cells by immunofluorescence according to any one of claims 1 to 7, wherein the fluorescence detection in step 5 is performed at a magnification of 20 times and a sensitivity of ISO 800.

Technical Field

The invention belongs to the technical field of basic medical research equipment, and particularly relates to a method for detecting the influence of a medicament on pancreatic islet cell lipid uptake through immunofluorescence.

Background

Abnormal blood lipid levels are one of the most important factors for the development of diabetes. Among them, the inflow of low density lipoprotein cholesterol (LDL-C) and accumulation of intracellular cholesterol are potential risk factors causing dysfunction of islet cells. Impaired islet cell function can lead to problems with insulin secretion, and absolute or relative insufficiency can cause blood glucose elevation and even diabetes. The invention detects the amount of low-density lipoprotein taken in by islet cells through immunofluorescence, thereby detecting the influence of the drug on the lipid taken in by the islet cells.

Disclosure of Invention

The invention aims to provide a method for detecting the influence of a medicament on the lipid uptake of islet cells through immunofluorescence, and the method can be used for detecting the condition of low-density lipoprotein cholesterol uptake of the islet cells.

The technical scheme adopted by the invention is that a method for detecting the influence of a medicament on the lipid uptake of pancreatic islet cells through immunofluorescence specifically comprises the following steps:

step 1, culturing islet cells to 3 × 10⁶～5×10⁶Secondly, subculturing the islet cells, sucking a certain amount of cell suspension after subculturing, inoculating the islet cells to a confocal dish, performing starvation treatment after the cells adhere to the wall, and interfering the cells with single dose of statins;

step 2, incubating the cells obtained in the step 1 by using Dil-LDL, washing the cells for three times by using PBS, fixing the cells by using paraformaldehyde, discarding liquid, and washing the cells by using deionized water;

step 3, dyeing the cells washed in the step 2 by using DAPI, discarding liquid after dyeing, washing for 3 times by using deionized water, and washing for 3min each time;

step 4, drying the cells obtained in the step 3;

and 5, observing the condition that the cells obtained in the step 4 take in the low-density lipoprotein cholesterol under a fluorescence microscope.

The present invention is also characterized in that,

in the step 1, subculture is first-second-fourth, the volume of the cell suspension inoculated into the confocal dish is 500 mu L, the starvation treatment time is 12h, and the cell intervention time is 48 h.

In the step 1, the islet cells are islet β cell line MIN6 cells.

In the step 2, the time for incubating the cells by the Dil-LDL is 4-6 h, the concentration of the paraformaldehyde is 4%, the volume of the cells fixed by the paraformaldehyde is 1-2 mL, the time for fixing the cells by the paraformaldehyde is 20-30 min, and the cells are washed by deionized water for 3-5 times for 5s each time.

The volume of the DAPI staining in the step 3 is 1-2 mL, and the time of the DAPI staining is 5 min.

In the step 4, the drying temperature is 40-80 ℃, and the drying time is 2-5 min.

And 5, when the fluorescence detection is carried out in the step 5, dividing a nine-square grid area on the cover of the confocal dish for fluorescence photographing.

In the step 5, the multiple of fluorescence detection is 20 times of a mirror, and the light sensitivity is ISO 800.

The invention has the beneficial effects that: the invention judges the change of the low-density lipoprotein cholesterol ingested by the islet cells through immunofluorescence detection, and can draw the conclusion that: the islet cells take up low-density lipoprotein cholesterol and are increased, the immunofluorescence intensity is obviously increased, the sampling in the experimental process is uniform, and the result reliability is high. The method provides a new method for detecting the function of the pancreatic islet, and has a great application prospect in aspects of assisting scientific research experiments, detecting medicine and the like.

Drawings

FIG. 1 is a schematic view of the present invention taken by microscopic examination;

FIG. 2 is a graph showing the results of fluorescence detection in example 1 of the present invention;

FIG. 3 is a graph showing the results of fluorescence detection in example 2 of the present invention;

FIG. 4 is a graph showing the results of fluorescence detection in example 3 of the present invention.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

The invention relates to a method for detecting the influence of a medicament on the lipid uptake of islet cells through immunofluorescence, which specifically comprises the following steps:

step 1, culturing islet cells in a T25 flask to grow to 3 × 10⁶～5×10⁶Respectively, carrying out passage on islet cells, culturing for one fourth, sucking 500 mu L of cell suspension, inoculating to a confocal dish, after the inoculated cells adhere to the wall, carrying out starvation treatment for 12h, and carrying out statinsThe single dose intervenes in the cells for 48 h.

The islet cells are islet β cell line MIN6 cells.

And 2, incubating the cells obtained in the step 1 for 4-6 hours by using fluorescence-labeled low-density lipoprotein (Dil-LDL), cleaning the cells for three times by using sterile phosphate Physiological Buffer Solution (PBS), fixing the cells for 1-2 min by using 1-2 mL of 4% paraformaldehyde, discarding liquid, and washing the cells for three times by using deionized water, wherein each time lasts for 5 seconds.

And 3, dyeing the cells obtained in the step 2 by using 1-3 mL of DAPI, discarding the liquid after dyeing, and washing with deionized water for 3 times, wherein each time lasts for 3 min.

And 4, drying the cells obtained in the step 3 at 60 ℃ for 2-5 min.

And 5, observing the condition that the cells obtained in the step 4 take in the low-density lipoprotein cholesterol under a fluorescence microscope.

The specific operation process is as follows: closing a fluorescence channel, turning on a power supply of a fluorescence exciter, preheating for 15min, putting the cells obtained in the step 4 into the fluorescence exciter, turning on a microscope light source during preheating, finding the cells to be observed, selecting a proper objective lens and adjusting the focal length, turning off the microscope light source after preheating is finished, turning on the fluorescence channel, exciting red light by green light to detect the low-density lipoprotein under the conditions of 20 times of a lens and ISO800, slightly scribing by a fine pen to divide the cells into 9 grids when detecting by manually adjusting the exposure time, and as shown in figure 1, each grid is one, and the part with large area can be one more than one according to the needs.