阅读说明：本技术 构建动脉粥样硬化过程中体外泡沫细胞形成模型的方法 (Method for constructing in-vitro foam cell forming model in atherosclerosis process ) 是由 穆玉明 袁晨 关丽娜 刘丽云 拜合提亚·塔依尔 吴治胜 于 2021-06-21 设计创作，主要内容包括：本发明涉及细胞模型构建方法技术领域,是一种构建动脉粥样硬化过程中体外泡沫细胞形成模型的方法,其在Transwell小室培养内皮细胞和巨噬细胞得到两种细胞共培养体系,向这两种细胞共培养体系添加TNF-α和ox-LDL得到泡沫细胞诱导体系并培养得到含有泡沫细胞的细胞培养体系,即为泡沫细胞形成模型。以该泡沫细胞形成模型能够模拟体外动脉粥样硬化早期形成过程,该构建泡沫细胞形成模型的方法相较于现有模型构建方法,可明显减少造模时间及经费消耗,为动脉粥样硬化诊治过程提供一种新的可供选择的体外细胞筛选模型。(The invention relates to the technical field of cell model construction methods, in particular to a method for constructing an in-vitro foam cell formation model in an atherosclerosis process. Compared with the existing model construction method, the method for constructing the foam cell formation model can obviously reduce the molding time and the expenditure consumption, and provides a new optional in-vitro cell screening model for the atherosclerosis diagnosis and treatment process.)

1. A method for constructing an in-vitro foam cell formation model in an atherosclerosis process is characterized by comprising the steps of respectively culturing endothelial cells and macrophages in an upper chamber and a lower chamber of a Transwell chamber to obtain two cell co-culture systems, adding TNF-alpha and ox-LDL to the two cell co-culture systems to obtain a foam cell induction system, culturing the foam cell induction system to obtain a cell culture system containing foam cells, wherein the cell culture system containing the foam cells is the foam cell formation model.

2. The method for constructing a model for foam cell formation in vitro during atherosclerosis according to claim 1, wherein the endothelial cells are human umbilical vein endothelial cells; or/and the macrophage is a macrophage lineage.

3. The method for constructing a model for foam cell formation in vitro during atherosclerosis according to claim 2, wherein the human umbilical vein endothelial cells are human umbilical vein endothelial cells HUVEC; or/and the macrophage is RAW264.7 macrophage line.

4. A method according to claim 3 for modeling foam cell formation in vitro during atherosclerosis, comprising the steps of: culturing the human umbilical vein endothelial cells in an upper chamber of a Transwell chamber, culturing the RAW264.7 macrophage in a lower chamber of the Transwell chamber, wherein the upper chamber and the lower chamber of the Transwell chamber are communicated through the bottom of the upper chamber, culturing the human umbilical vein endothelial cells for 48 hours, culturing the RAW264.7 macrophage for 24 hours, replacing culture mediums of the two with an M199 culture medium containing TNF-alpha and ox-LDL to obtain a foam cell induction system, and culturing the foam cell induction system for 24 hours to obtain a cell culture system containing foam cells, namely a foam cell formation model.

5. The method of claim 4, wherein the culture medium of human umbilical vein endothelial cells is replaced with M199 medium containing TNF- α and ox-LDL after culturing for 24 hr and then culturing with RAW264.7 macrophage cells for 24 hr.

6. Method for the modeling of foam cell formation in vitro during atherosclerosis according to any of claims 1 to 5, characterized in that the concentration of ox-LDL in the foam cell induction system is between 10mg/L and 100 mg/L.

7. The method for modeling foam cell formation in vitro during atherosclerosis according to claim 6, characterized in that the concentration of ox-LDL in the foam cell induction system is 50mg/L to 100 mg/L.

8. The method for constructing a foam cell formation model in vitro during atherosclerosis according to claim 1 or 2 or 4 or 5 or 7, wherein the concentration of TNF- α in the foam cell inducing system is 10ng/mL to 20 ng/mL.

9. The method for constructing a model of foam cell formation in vitro during atherosclerosis according to claim 3, wherein the concentration of TNF- α in the foam cell induction system is 10ng/mL to 20 ng/mL.

10. The method for constructing a model of foam cell formation in vitro during atherosclerosis according to claim 6, wherein the concentration of TNF- α in the foam cell inducing system is 10ng/mL to 20 ng/mL.

Technical Field

The invention relates to the technical field of cell model construction methods, in particular to a method for constructing an in-vitro foam cell forming model in an atherosclerosis process.

Background

Atherosclerosis (AS) seriously harms human health and life, and cardiovascular and cerebrovascular diseases caused by AS are still serious public health problems at present, and the morbidity and mortality of the cardiovascular and cerebrovascular diseases are the first of all diseases. AS is a multifactorial, involved chronic inflammatory process of the vessel wall that has been produced and progressed for decades before overt clinical symptoms appear. Because of the concealment and the potential hazard, the early diagnosis of AS lesion is convenient for taking timely and effective prevention, control and treatment measures, and the early diagnosis has important clinical significance for improving the life quality and prognosis of patients.

Recent studies have demonstrated that chronic inflammatory responses are a very important factor in the development and progression of atherosclerosis. Vascular endothelial cell damage and endothelial dysfunction are the initiating links in the development of atherosclerosis, the damage occurring before a significant pathological change. After the injury, a plurality of adhesion molecules, chemotactic factors and tissue factors are secreted to induce and promote the inflammatory reaction of the vascular intima. Monocytes subsequently recruited to the lesion differentiate into macrophages that begin to phagocytose ox-LDL deposited under the intima, with the gradual increase of ox-LDL accumulated in macrophages, causing intracellular cholesterol crystal nucleation, destruction of lysosome stability, resulting in cholesterol efflux disorder, cholesterol metabolic imbalance, macrophage foaming, and impaired cell function.

The existing animal model preparation method can generate spontaneous AS, but because the purchase and feeding costs of experimental animals are high, and the tissue specimens available for research are too few, the time cost and the expenditure budget are high, so that the animal model selection for experimental research has certain limitations. At present, scholars culture smooth muscle cells, endothelial cells and mononuclear cells sequentially from bottom to top in a self-made mechanically stretchable cell culture device to obtain a multi-cell co-culture system, but the main principle of foam cell formation at present is that the content of cholesterol ester in macrophage RAW264.7 (M phi) exceeds 60 percent.

Disclosure of Invention

The invention provides a method for constructing an in-vitro foam cell forming model in an atherosclerosis process, which overcomes the defects of the prior art, and prepares a foam cell forming model capable of inducing foam cell formation by culturing an injured endothelial-macrophage in-vitro co-culture system so as to simulate an in-vitro atherosclerosis early-stage forming process.

The technical scheme of the invention is realized by the following measures: a method for constructing an in-vitro foam cell formation model in an atherosclerosis process comprises the steps of respectively culturing endothelial cells and macrophages in an upper chamber and a lower chamber of a Transwell chamber to obtain two cell co-culture systems, adding TNF-alpha (tumor necrosis factor) and ox-LDL (oxidized low density lipoprotein) into the two cell co-culture systems to obtain a foam cell induction system, culturing the foam cell induction system to obtain a cell culture system containing foam cells, wherein the cell culture system containing the foam cells is the foam cell formation model.

The following is further optimization or/and improvement of the technical scheme of the invention:

the endothelial cells can be human umbilical vein endothelial cells, and the endothelial cells can be human umbilical vein endothelial cells HUVEC or other human vascular endothelial cells.

The macrophage may be a macrophage line, and the macrophage may specifically be a RAW264.7 macrophage line.

The method for constructing the in-vitro foam cell forming model in the atherosclerosis process is specifically carried out according to the following method: culturing the Human Umbilical Vein Endothelial Cells (HUVEC) in an upper chamber of a Transwell chamber, culturing RAW264.7 macrophages (M phi) in a lower chamber of the Transwell chamber, wherein the upper chamber and the lower chamber of the Transwell chamber are communicated through the bottom of the upper chamber, culturing the human umbilical vein endothelial cells for 48 hours, culturing the RAW264.7 macrophages for 24 hours, replacing a culture medium of the two with an M199 culture medium containing recombinant tumor necrosis factor-alpha (TNF-alpha) and ox-LDL to obtain a foam cell induction system, and culturing the foam cell induction system for 24 hours to obtain a cell culture system containing foam cells, namely a foam cell formation model.

The human umbilical vein endothelial cells are cultured for 24 hours firstly, then are cultured together with RAW264.7 macrophages for 24 hours, and then culture mediums of the human umbilical vein endothelial cells and the RAW264.7 macrophages are replaced by M199 culture mediums containing TNF-alpha and ox-LDL for continuous culture.

The seeding density of the human umbilical vein endothelial cells can be 5.0 multiplied by 10⁴The seeding density of the RAW264.7 macrophage can be 2.0 x10 per ml⁴One per ml.

The seeding density of human umbilical vein endothelial cells and RAW264.7 macrophages reaches 70 to 80 percent as much as possible.

The Transwell chamber comprises two parts, and the components which are sequentially overlapped from top to bottom are as follows: an upper chamber covered with a permeable membrane at the bottom and a lower chamber of the same material as a normal orifice plate.

The upper chamber of the Transwell chamber can be provided with cell culture cells with the same number as the lower chamber, the bottom of the upper chamber is a polycarbonate membrane (polycarbonate membrane) with micropores and the pore size of the membrane is 0.4-8.0 μm.

The diameter of the upper chamber culture pond of the Transwell chamber is 12mm, and the area is 1.1cm²(ii) a The diameter of the lower chamber culture pond is 24mm, and the area is 4.5cm²The cells are cultured in the corresponding cell culture wells.

The concentration of ox-LDL in the foam cell induction system may be 10mg/L to 100mg/L, and further may be 30mg/L to 80 mg/L. Still further, the concentration of ox-LDL in the foam cell induction system may be from 50mg/L to 80 mg/L.

In the foam cell induction system, the concentration of TNF-alpha is 10ng/mL to 20 ng/mL.

The invention relates to a method for constructing an in-vitro foam cell forming model in the atherosclerosis process, which comprises the steps of culturing endothelial cells and macrophages by utilizing upper and lower chambers which are mutually permeable in a Transwell chamber to form two cell co-culture systems, then inducing the cell co-culture systems to prepare a foam cell forming model capable of inducing the foam cells to form, and simulating the early formation process of the in-vitro atherosclerosis by using the foam cell forming model. Compared with the existing model construction method, the method for constructing the foam cell forming model can obviously reduce the molding time and the expenditure consumption, and provides a new optional in-vitro cell screening model for the atherosclerosis diagnosis and treatment process. In addition, the construction method has the following advantages:

(1) in the invention, a Transwell chamber is used for simulating the in-vitro AS forming process, and the model construction method is simple and convenient to manufacture;

(2) the preparation of the foam cell forming model in the invention takes short time, the construction can be successful in 72h, and the final 24h can form atherosclerosis early-stage marker event, namely foam cell formation;

(3) the foam cell forming model (namely a cell culture system containing foam cells) is stable in forming and good in repeatability, and results of different groups can be conveniently compared;

(4) the foam cell forming model system is economical and effective, and the time and labor cost are greatly saved;

(5) the forming result of the foam cell forming model can be determined in a short time by a flow cytometer, oil red O staining and a cholesterol kit, and the operation is convenient.

Drawings

FIG. 1 is the establishment of a lesion endothelial-macrophage co-culture system.

In FIG. 1, A is a schematic diagram of a co-culture system; b: upper human umbilical vein endothelial cells HUVEC; c: lower macrophage RAW264.7, D: electron micrograph of macrophage in blank control group; e, taking an electron microscope picture after macrophage intervention by 50mg/L ox-LDL; f, 80mg/L ox-LDL intervention macrophage electron microscope picture.

FIG. 2 shows the flow cytometry results of TNF- α and ox-LDL induced expression of the inflammatory factor CD44 by RAW264.7 cells.

In FIG. 2, A: treated with 10ng/mL TNF- α and ox-LDL; b: treatment with 20ng/mL TNF- α and ox-LDL; c: untreated control, D: and (5) quantifying the result.

FIG. 3 is a graph showing that ox-LDL induced RAW264.7 cells to foam cells when using 10ng/mL TNF-. alpha.for intervention by oil-Red-O staining.

In FIG. 3, A: no ox-LDL treatment; b: 20mg/L ox-LDL treatment; c: 40mg/L ox-LDL treatment, D: 60mg/L ox-LDL treatment, E: 80mg/L ox-LDL treatment, F: quantitative results,: p <0.05 in comparison with the 20mg/L group and P <0.05 in comparison with the 40mg group.

FIG. 4 shows the quantification of cholesterol in macrophages.

In FIG. 4, A is a total cholesterol fluorescence absorption standard curve, B is: a free cholesterol fluorescence absorption standard curve; c: a standard curve concentration dilution table; d: macrophage activity following ox-LDL intervention.

FIG. 5 shows the specific gravity of cholesterol esters in lipid-bearing cells at different levels.

Detailed Description

The present invention is not limited by the following examples, and specific embodiments may be determined according to the technical solutions and practical situations of the present invention.

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The endothelial cells in the examples below were HUVEC-T1 cells, human umbilical vein endothelial cells from Wuhan Ponciri Life technologies, Inc.; the macrophage is RAW264.7-T1 cell of Wuhan Punuoist Life technologies, Inc.; ox-LDL is a product of Guangzhou Yiyuan biotechnology limited; the TNF-alpha is a product of Beijing Boaosen biotechnology limited; the Transwell cell is specifically manufactured by corning corporation of America.

The culture medium used for culturing macrophages in the following examples was 90% high-sugar DMEM (zhongqiao new boat, china) supplemented with the following amounts of substances: 10% fetal bovine serum (Biological Industries/Israel), 1% double antibody (Gbico/USA). The cell culture conditions are 95% air and 5% CO in a constant temperature cell culture box₂，37 ℃。

The medium used for culturing endothelial cells was 93% of a basic medium ECM (science, USA) supplemented with the following amounts of substances: 5% fetal bovine serum (scienll, USA), 1% endothelial growth factor (scienll, USA), 1% double antibody (scienll, USA). The cell culture conditions are 95% air and 5% CO in a constant temperature cell culture box₂，37 ℃。

The invention is further described below with reference to the following examples:

example (b): the method for constructing the in-vitro foam cell forming model in the atherosclerosis process is carried out according to the following method: human Umbilical Vein Endothelial Cells (HUVEC) were seeded onto gelatin-coated polycarbonate filters in Transwell chambers at a seeding density of 5.0X 10⁴Per ml, endothelial monolayer can be formed after culturing for 48 hours; RAW264.7 macrophage cell line (M.phi.) was plated in the Transwell lower chamber at a seeding density of 2.0X 10 for RAW264.7 macrophages⁴And culturing the cells per mL together with the HUVEC in the upper chamber for 24h, and then replacing the culture medium with M199 culture medium containing TNF-alpha 10 ng/mL-20 ng/mL and ox-LDL 10 mg/100 mg/L for culturing for 24h to obtain a cell culture system containing foam cells, namely a foam cell forming model.

Establishment of a damaged endothelial-macrophage co-culture system as shown in figure 1, panel A, B, C; the picture of the electron microscope after the 50mg/L ox-LDL intervenes in the macrophages is shown as the E picture in figure 1; the image of the electron microscope after the 80mg/L ox-LDL intervenes in macrophages is shown as F in figure 1.

The E, F plot of FIG. 1 shows a significant change in macrophage morphology compared to the D plot of FIG. 1, with the cells in E, F plots being more or less foamy.

The research process of the method for constructing the in-vitro foam cell forming model in the atherosclerosis process comprises the following steps:

(1) under the condition of different concentrations of TNF-alpha, HUVEC is induced to express an inflammatory factor CD44, so that endothelial cell injury opens the initiation link of atherosclerosis.

The process is to simulate damaged blood vessel in vivo, firstly, cleaning Human Umbilical Vein Endothelial Cells (HUVEC) growing well in a culture dish with PBS, adding pancreatin for digestion and collection, centrifuging, re-suspending with fresh culture medium, counting the cells, diluting the cells to 5.0 × 10⁴And (2) inoculating the cells to a gelatin-coated Transwell polycarbonate filter, culturing for 2 days to form an endothelial monolayer, and then changing the endothelial cell specific medium to M199 medium containing 10ng/mL of recombinant tumor necrosis factor-alpha (TNF-alpha) and 20ng/mL of recombinant tumor necrosis factor-alpha (TNF-alpha), respectively, for 24 h.

After cell inoculation, the HUVEC gradually changes from round to spindle shape by taking pictures with a microscope every day, and the density can reach 70 to 80 percent after 2 days of culture. Note that: firstly, before taking a reagent by a liquid shifter, balancing a gun head by the reagent (slowly sucking liquid and repeatedly blowing and beating for three times). ② the liquid on the outer wall of the lance head can not be added into the reaction system.

After the reaction is finished, absorbing and removing the supernatant of the culture medium, and rinsing the supernatant once by PBS containing no calcium ions and magnesium ions; taking 25mL of the medium as an example, 1mL of a digestion solution (0.1% trypsin) is added and the mixture is digested at room temperature (25 ℃) or 37 ℃ for 2min to 5 min; observing under an inverted microscope, finding that cytoplasm retracts and gaps are enlarged, immediately absorbing and removing digestive juice, and adding a culture medium containing serum with equal volume to terminate digestion; the liquid in the bottle is sucked by a suction pipe, and the cells on the wall of the bottle are blown repeatedly. The blowing and beating actions need to be gentle and foam is avoided as much as possible; transferring to a centrifuge tube, centrifuging for 5min at 1000r, and removing the supernatant; washing with cell staining buffer centrifugation 1-2 times; cell concentration was adjusted to 1 × 10 using cell staining buffer⁶The volume is/mL for standby.

Add 5ul of antibody to 3.0X10 as per the instructions⁵Cells were mixed well and incubated at 4 ℃ for 30 minutes in the dark. Adding appropriate amount of cell for dyeingThe buffer resuspended the cells, the cell suspension centrifuged at 1000r for 5 minutes and the supernatant discarded. 0.4 mL of cell stain buffer (or PBS containing 0.1% BSA) was added to resuspend the cells, and detected and analyzed by flow cytometry, as shown in FIG. 2.

Fig. 2D shows: the quantitative result shows that the model expresses the CD44 antibody obviously, the model effect is not different statistically after being treated by 10ng/mL TNF-alpha and 20ng/mL TNF-alpha, so that the subsequent experiments use 10ng/mL TNF-alpha for treatment.

(2) Foam cell formation was induced under conditions of varying concentrations of oxidized low density lipoprotein.

The process is to simulate damaged blood vessel in vivo, firstly, cleaning Human Umbilical Vein Endothelial Cells (HUVEC) growing well in a culture dish with PBS, adding pancreatin for digestion and collection, centrifuging, re-suspending with fresh culture medium, counting the cells, diluting the cells to 3.0 × 10⁵One/ml, inoculated onto a gelatin coated Transwell polycarbonate filter. Then, the well-grown RAW264.7 macrophages were washed with PBS, trypsinized, centrifuged, resuspended in fresh medium, counted, and diluted to 2.0 × 10⁴one/mL, plated in the lower chamber of Transwell, after 24h of co-culture with HUVEC in the upper chamber, the medium was replaced with M199 medium containing 10ng/mL recombinant tumor necrosis factor-alpha (TNF-alpha) and 0mg/L, 10mg/L, 20mg/L, 40mg/L, 60mg/L, 80mg/L L, 100mg/L of ox-LDL, respectively, for 24h of culture.

(3) Effect of ox-LDL concentration on foam cell formation.

And (3) adding culture media containing different concentrations of ox-LDL into the endothelial cell-macrophage co-culture system obtained in the step (2) and then culturing, so that foam cell formation results under the conditions of different concentrations of ox-LDL can be obtained. The concentration of ox-LDL in the experiment was set to 0mg/L, 10mg/L, 20mg/L, 40mg/L, 60mg/L, 80mg/L, 100mg/L, respectively.

(4) And (4) identifying foam cells.

For the co-culture system of two cells at the end of the experiment, washing with PBS solution, 1. the cultured Cell sample is fixed on Cell ORO Fixative for 15-25 min. And 2, washing the mixture with PBS buffer solution or normal saline slightly and drying the mixture slightly. 3.60% isopropanol was washed dropwise for 20s to 30 s. And 4, dropwise adding oil red O staining solution, and sealing and staining for 10-15 min. Color separation, adding 60% isopropanol and washing slightly to remove the dye liquor. A slight wash with PBS buffer or saline. And 7, adding Mayer hematoxylin staining solution, and counterstaining the nuclei for 1min to 2 min. ORO Buffer is added for 1 min. Air dried, then observed in bright field using an inverted microscope and photographed. The results of the experiment are shown in FIG. 3.

And quantitatively analyzing and counting the red area of the lipid drop by using the color distinguishing function in the Image processing software Image J so as to obtain quantitative information.

After the co-culture system of the two cells was treated with ox-LDL of different concentrations, the foam cells generated in the co-culture system were visualized using oil red staining, which caused lipid droplets to appear red, and cells with lipid droplets aggregated were confirmed as foam cells, and the degree of foam cell formation was indicated by observing the amount of oil red.

As can be seen from fig. 3, there are no red cells in panel a, and B, C, D, E shows a concentration-dependently increased number of red cells (in the figure, a part of the cells are darker and actually red), i.e., the foam cells are observed in panel B, C, D, E in fig. 3, and the result shows that the foam cell model is successfully established.

Furthermore, we observed changes in macrophage cell activity (as shown in FIG. 4) and in the amount of cholesterol in macrophages after co-incubation of various concentrations of OX-LDL with macrophages. Generally, the specific gravity of cholesterol ester in macrophage is less than 50%, after cells are induced by ox-LDL, the experimental result shows that when the concentration is 0mg/L, 10mg/L, 20mg/L, 40mg/L, 60mg/L, 80mg/L and 100mg/L, the total cholesterol, the cholesterol ester content and the specific gravity in the cells are increased in a concentration-dependent manner along with the increase of the concentration, and the foam cell formation degree is gradually increased, as shown in figure 5. In FIG. 5, when the concentration of ox-LDL exceeds 60mg/L, the specific gravity of cholesterol ester in the cells exceeds 60%, respectively, indicating that a large amount of cholesterol is accumulated in the cells and the cells become foam cells. In the absence of ox-LDL, no foam cells were formed even with TNF- α in the medium. Therefore, the concentration of ox-LDL plays a critical role in the formation of foam cells.

The technical characteristics form an embodiment of the invention, which has strong adaptability and implementation effect, and unnecessary technical characteristics can be increased or decreased according to actual needs to meet the requirements of different situations.