CN117004556A - Method for extracting and purifying primary glomerular mesangial cells of mice - Google Patents
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Abstract
The invention provides a method for extracting and purifying primary glomerular mesangial cells of a mouse, which comprises the following steps: extraction of glomeruli, culture of mesangial cells, identification of glomeruli and mesangial cells. According to the invention, in the process of extracting cells from the tissue, the mixed solution is subjected to a series of extraction and purification processes such as full digestion, gradient cell sieving, secondary adhesion, gradient centrifugation and the like, so that glomerular loss and other tissue and impurity residues of the kidney are effectively reduced, the extraction purity of the glomerulus is further improved, and finally, the multi-cell marker comprehensive detection is carried out through immunofluorescence identification markers of different parts of the kidney tissue, so that the primary glomerular mesangial cells of the mice with better identification effect and higher purity are obtained.
Description
Technical Field
The invention belongs to the technical field of mesangial cells, and particularly relates to an extraction and purification method of primary mesangial cells of mice.
Background
Important pathophysiological changes in Chronic Kidney Disease (CKD) are the sustained decline in renal function and the progressive production of urinary proteins, both of which are closely related to the glomerular filtration membrane system. The glomerular filtration membrane system is relatively complex in structure and comprises a basement membrane, glomerular mesangial cells and podocytes with podophy at the periphery. Therefore, the extraction of the high-purity primary glomerular mesangial cells and podocytes has important application prospects in the aspects of explaining and researching the occurrence and the development of CKD, carrying out basic research of clinical drug screening and the like.
The kidney is one of the most differentiated organs of the human body, the kidney parenchyma wrapped by the kidney envelope (Capsule) is divided into the renal Cortex (Cortex) and the renal Medulla (Medulla, divided into the external Medulla and the internal Medulla), the Medulla is composed of the renal cone (Pyramid) and the renal column, several renal cones synthesize one renal nipple (papilide), then gather into the small renal calves (Minor calves), synthesize the large renal calves (Major calves), and finally pass out of the phylum of the kidney through the renal Pelvis (Pelvis) to act as the Ureter (Ureter). The functional units of the kidney are called nephrons and include Bao Manshi capsule, glomeruli, proximal tubule, loops (divided into descending, ascending thin and thick segments), distal tubule and collecting duct. The glomerulus has a complex structure and mainly consists of a filtration membrane system, including basement membrane, endothelial cells, glomerular mesangial cells and podocytes with podophy at the periphery. These cells are arranged in a certain position on Bao Manshi capsule, and can be used as highly specialized glomerular filtration membrane system with unique functional structure, and participate in the filtration process of kidney macromolecular proteins, so that their anatomical structure determines that extraction is difficult.
Glomerular disease is the leading cause of end stage renal disease. Glomerular injury can be caused by systemic disease (e.g., diabetic kidney disease) or primary glomerular injury (e.g., igA nephropathy). In most human glomerulopathies, activation and proliferation of mesangial cells and injury and loss of podocytes play a key role. To study the molecular mechanisms behind human glomerular disease, mice are widely used to simulate human glomerular disease. However, transformation studies based thereon have been challenging.
Mesangial cells are considered to be endothelial-derived mesenchymal stromal cells without specifically expressed cell markers. Although genes such as platelet-derived growth factor receptor α, platelet-derived growth factor receptor β, desmin and α8 integrin are strongly expressed in mesangial cells, these genes are also highly expressed in other stromal cells without specificity, and thus identification and authentication are difficult.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an extraction and purification method for the primary glomerular mesangial cells of the mice aiming at the defects of the prior art, and the method can extract and purify the primary glomerular mesangial cells of the mice with better identification effect and higher purity.
In order to solve the technical problems, the invention adopts the following technical scheme: a method for extracting and purifying primary glomerular mesangial cells of a mouse is shown in fig. 1, and specifically comprises the following steps:
s1, extracting glomeruli:
s101, separating and digesting: taking kidneys of 6-8-week-old male mice without kidney Capsule removal, placing the kidneys wrapped by kidney Capsule (Capsule) into precooled Hank's balanced salt solution containing 1% of streptomycin by mass, substantially dividing the kidneys into kidney Cortex (Cortex) and kidney Medulla (Medulla, divided into external marrow and internal marrow), wherein the Medulla consists of kidney cones (Pyramid) and kidney columns, synthesizing one kidney nipple (Papilane) by several kidney cones, converging into kidney calves (Minor calves), synthesizing kidney calves (Major calves), finally taking out kidney gate migration as ureters (urea) through renal Pelvis (Pelvis), separating kidney Cortex and external marrow parts, placing the kidney Cortex and the external marrow parts into Hank's balanced salt solution containing collagenase IV at a concentration of 0.75mg/mL and trypsin inhibitor, shearing into block-shaped tissues, placing the block-shaped tissues into preheated digestive juice at 37 ℃, adding the water bath with shaking volume, and the like substances to obtain precooled culture medium;
s102, gradient filtration: placing a 70-mu m cell sieve on a 50-mL centrifuge tube a, pumping the digestion mixture obtained in the step S101 into the 50-mL centrifuge tube a through the 70-mu m cell sieve by using a suction pipe, grinding the digestion mixture by using a sterilized injection plug of a 5-mL syringe, flushing the digestion mixture by using Hank' S balanced salt solution, and collecting a filtered solution; placing a 40 μm cell sieve on a 50mL centrifuge tube b, pumping the filtered solution into the 50mL centrifuge tube b through the 40 μm cell sieve by using a suction tube, retaining the substances above the 40 μm cell sieve, turning over the 40 μm cell sieve and placing the 40 μm cell sieve into a 10cm culture dish a, and flushing the substances above the 40 μm cell sieve into the 10cm culture dish a by using Hank's balanced salt solution;
s103, shaking and suspending: shaking the 10cm dish a in S102, and collecting the suspension liquid a into a 50mL centrifuge tube c;
s104, secondary adhesion: placing a 40 μm cell sieve on a 50mL centrifuge tube d, pumping the suspension liquid a obtained in the step S103 into the 50mL centrifuge tube e through the 40 μm cell sieve by using a suction tube, retaining the substance above the 40 μm cell sieve, turning over the 40 μm cell sieve and placing the 40 μm cell sieve into a 10cm culture dish b, and flushing the substance above the 40 μm cell sieve into the 10cm culture dish b by using Hank' S balanced salt solution; shaking the 10cm culture dish b, collecting the suspension liquid b into a 50mL centrifuge tube f, centrifuging, discarding supernatant, and re-suspending with serum-free Ham's F-12 culture medium to obtain a re-suspension mixture;
s105, gradient centrifugation of polyvinylpyrrolidone: the method comprises the steps of (1) filling a 50% polyvinylpyrrolidone layering liquid into a 15mL centrifuge tube, dropwise adding a resuspension mixed liquid obtained in S104 from the side wall to the position above the liquid level of the 50% polyvinylpyrrolidone layering liquid, centrifuging, discarding a supernatant, and reserving a bottom sediment, namely a glomerular sediment;
s2, culturing mesangial cells: re-suspending the glomerular sediment obtained in S105 with a mesangial cell culture medium, and inoculating and culturing, climbing out cells for 3-5 days, changing liquid every day, and carrying out passage every other day, and taking the cells of the 3 rd-5 th generation to obtain the mesangial cells;
the membranous cell culture medium is prepared from the following raw materials in percentage by mass: 70.6% of DMEM culture solution, 23.4% of Ham's F-12 culture solution, 5% of fetal bovine serum and 1% of green streptomycin;
s3, glomerular identification: identifying glomerular precipitate obtained in S105 by using podocyte marker nephrotic protein (Nephrin), and determining that the glomerulus precipitate is positive;
s4, identifying mesangial cells: the positive detection of the mesangial cell positive marker alpha-smooth muscle actin (alpha-SMA) is adopted, and the negative detection of the epithelial cells, namely podocyte marker nephrosis protein (Nephrin) and the negative detection of the endothelial cell marker close-coupled protein-1 (Zo-1) are adopted.
Preferably, the digestion solution in S101 is a DMEM medium containing collagenase IV at a concentration of 0.75mg/mL and trypsin inhibitor at a concentration of 0.75 mg/mL.
Preferably, the precooling medium in S101 is DMEM medium with 10% fetal bovine serum by mass fraction.
Preferably, the shaking speed of shaking in S101 is 120 rpm/min.
Preferably, the centrifugation conditions in S104 are: centrifuge at 4℃and 500 rpm/min for 3 min.
Preferably, the centrifugation conditions in S105 are: centrifugation was carried out at 12000 rpm/min at 4℃for 60 min.
Compared with the prior art, the invention has the following advantages:
the primary glomerular mesangial cells of the mice extracted and purified by the method have the advantages that the primary cells are obtained by multiple gradient purification, the purity is good, the activity is high, the impurities are few, only a small amount of mice kidneys are needed, and enough primary glomerular mesangial cells can be obtained for culture and identification by one-time extraction. The whole process of separation is aseptic operation, and the pollution rate is small. In the process of extracting cells through tissues, the mixed solution is subjected to a series of extraction and purification processes such as full digestion, gradient cell sieving, secondary adhesion, gradient centrifugation and the like, so that glomerular loss and other tissue and impurity residues of the kidney are effectively reduced, the extraction purity of the glomerulus is further improved, and finally, the multi-cell marker comprehensive detection is carried out through immunofluorescence identification markers at different parts of the kidney tissue, so that the primary glomerular mesangial cells of the mice with better identification effect and higher purity are obtained.
The invention is described in further detail below with reference to the drawings and examples.
Drawings
FIG. 1 is a drawing showing the extraction process separated in example 1 of the present invention.
FIG. 2 is a graph showing glomerular extraction and identification in example 1 of the present invention.
FIG. 3 is a graph showing the extraction and identification of mesangial cells in example 1 of the present invention.
Detailed Description
Example 1
Male mice of 6-8 weeks old, C57BL/6J background, from Beijing Fukang Biotech Co., ltd, were housed in Standard Pathogen Free (SPF) laboratories, 4 mice per cage, were free to acquire food and water, room temperature 20.+ -. 2 ℃, humidity 55.+ -. 5%, light/dark cycle 12 hours. All experiments were performed at the same time per day to avoid the effects of circadian rhythms. The study met the ethical criteria of the committee for animal care and use (IACUC).
The method for extracting and purifying primary mesangial cells of the mouse of this example is shown in fig. 1, and specifically comprises the following steps:
s1, extracting glomeruli:
s101, separating and digesting: the method comprises the steps of removing the neck from 4-6 male mice with age of 6-8 weeks, putting the mice into 75% alcohol for soaking for 1-5 minutes, taking kidneys without kidney Capsule, immediately putting the kidneys into a culture dish with the diameter of 10cm containing a precooled Hank's balanced salt solution containing 1% of green streptomycin, wherein the kidney structure is shown in the upper left part of fig. 1, kidney essence wrapped by kidney Capsule (Capsule) is divided into kidney Cortex (Cortex) and kidney Medulla (Medulla, divided into external marrow and internal marrow), the Medulla consists of a kidney cone (Pyramid) and a kidney column, several kidney cones synthesize a kidney nipple (Papilane), then gather into small kidney calyx (Minor calves), synthesize large kidney calves (Major calves), finally taking out of the kidney phylum via a renal Pelvis (Pelvis) to act as Ureter (Urter), separating the kidney Cortex, the external marrow and the internal marrow according to the anatomical structure, putting the kidney Cortex and the external marrow into a kidney essence (Medulla) with the concentration of 0.75mg/mL and a collagen-containing balance salt solution with the concentration of 0.75mg/mL, and cutting the kidney essence into pieces with the concentration of 1mm, and the balance salt solution is formed by the method 3 Left and right squares, the tissue was then placed in a preheated digest (5% CO) in a water bath at 37℃ 2 Environment), shaking at 120 rpm/min for 45-60 min, adding an equal volume of pre-chilled medium to the digested material to terminate the reaction to obtain a digested mixture; all reactions were started in this step and were carried out on ice;
the digestion solution is a DMEM culture solution containing collagenase IV with the concentration of 0.75mg/mL and trypsin inhibitor with the concentration of 0.75 mg/mL;
the precooling culture medium is DMEM culture solution with the mass fraction of 10% of fetal calf serum;
the precooled Hank's balanced salt solution is purchased commercially and is purchased from HyClone company;
the DMEM culture solution is purchased from the company of Semer Feier science and technology Co., ltd;
s102, gradient filtration: placing a 70-mu m cell sieve on a 50-mL centrifuge tube a by using disinfection forceps, pumping the digestion mixture obtained in the step S101 into the 50-mL centrifuge tube a through the 70-mu m cell sieve by using a suction tube, grinding substances on the 70-mu m cell sieve by using a sterilized injection plug of a 5-mL syringe, flushing the substances with Hank' S balanced salt solution for 2-3 times, collecting filtrate, and finally keeping the glomeruli in the filtrate below the cell sieve; placing a 40 μm cell sieve on another 50mL centrifuge tube b with disinfection forceps, pumping the filtered solution into the 50mL centrifuge tube b through the 40 μm cell sieve by using a suction tube, retaining the substance above the 40 μm cell sieve, and finally leaving glomeruli above the cell sieve, at the moment, turning over the 40 μm cell sieve and placing the 40 μm cell sieve into a 10cm culture dish a, and flushing the substance above the 40 μm cell sieve into the 10cm culture dish a with Hank's balanced salt solution;
s103, shaking and suspending: shaking the 10cm dish a in S102, clearly observing under a microscope that the kidney tubules are more easily and firmly adhered to the surface of the substrate of the dish, while most of the kidney glomeruli float with shaking, collecting the suspension liquid a into a 50mL centrifuge tube c;
s104, secondary adhesion: placing a 40 μm cell sieve on a 50mL centrifuge tube d with disinfection forceps, pumping the suspension liquid a obtained in the step S103 into the 50mL centrifuge tube e through the 40 μm cell sieve by using a suction tube, retaining substances above the 40 μm cell sieve, finally retaining glomeruli above the cell sieve, turning over the 40 μm cell sieve, placing the 40 μm cell sieve into a 10cm culture dish b, and flushing the substances above the 40 μm cell sieve into the 10cm culture dish b by using Hank' S balanced salt solution; gently shaking the 10cm culture dish b, collecting the suspension liquid b into a 50mL centrifuge tube f, centrifuging for 3 minutes at 4 ℃ and the rotating speed of 500 rpm/min, discarding the supernatant after centrifuging, and re-suspending with 5mL of serum-free Ham's F-12 culture medium to obtain a re-suspension mixture;
the serum-free Ham's F-12 medium is commercially available from Semer Feier technologies Co., ltd;
s105, gradient centrifugation of polyvinylpyrrolidone: loading 5mL of polyvinylpyrrolidone layering liquid with the mass fraction of 50% into a 15mL centrifuge tube, slowly dripping the resuspension mixed liquid obtained in S104 from the side wall to the position above the liquid level of the 50% polyvinylpyrrolidone layering liquid, centrifuging for 60 minutes at the temperature of 4 ℃ and the rotating speed of 12000 rpm/min, slightly taking out the centrifuge tube after centrifuging, and obtaining that the polyvinylpyrrolidone layering liquid is divided into 3-4 layers, namely the success, wherein the uppermost layer is a proximal tubular cell, the lower layer is a distal tubular cell, the middle layer is the polyvinylpyrrolidone layering liquid, and the bottommost layer is a glomerular sediment with the maximum density; discarding the supernatant, and reserving the bottom sediment, namely glomerular sediment;
the 50% polyvinylpyrrolidone layering liquid is Hank's balanced salt solution containing 50% polyvinylpyrrolidone; polyvinylpyrrolidone is a biocompatible surfactant that reduces the physical adhesion of glomeruli to the surface of the centrifuge tube; polyvinylpyrrolidone is commercially available from GE Healthcare (Pharmacia);
cell counting was performed by dropping the extracted and purified cell mixture using a cell counting plate or a cell counter, and the result showed that (8.+ -. 1). Times.10 could be obtained on average in two kidneys of one mouse 3 The extraction efficiency is high; as shown in figure 2, the identification of glomerular immunofluorescence cells shows that the development of podocyte marker nephrosis protein (Nephrin) is positive, and the glomerular immunofluorescence cells have complete spherical structure, high cell activity, no pollution and good extraction effect;
s2, culturing mesangial cells: re-suspending the glomerular sediment obtained in S105 with a mesangial cell culture medium, and inoculating and culturing, climbing out cells for 3-5 days, changing liquid every day, and carrying out passage every other day, and taking the cells of the 3 rd-5 th generation to obtain the mesangial cells;
the membranous cell culture medium is prepared from the following raw materials in percentage by mass: 70.6% of DMEM culture solution, 23.4% of Ham's F-12 culture solution, 5% of fetal bovine serum and 1% of green streptomycin;
the Ham's F-12 culture solution is purchased from the company Semer Feier science and technology Co., ltd;
FIG. 1 shows an extraction process of separating and digesting kidneys according to different kidney tissue structures, extracting high-purity glomeruli through purification operations such as full digestion, gradient cell sieving, secondary adhesion, gradient centrifugation and the like, and culturing mesangial cells.
Immunofluorescent cell identification of mesangial cells the glomeruli were observed under 4-fold, 10-fold and 40-fold microscopes, respectively, from left to right in the figure as shown in fig. 2. From left to right, there are respectively a glomerular fusion immunofluorescence image, a glomerular nuclei DAPI staining positive control image and a glomerular nephritic protein (Nephrin) staining image. The positive marker of the mesangial cells is positive in color development of alpha-smooth muscle actin (alpha-SMA), has complete mesangial cell structure, high cell activity, no pollution and good extraction effect; the kidney disease protein (Nephrin) and the endothelial cell marker close-coupled protein-1 (Zo-1) are negative, which shows that the glomerular mesangial cells extracted and cultured by the invention are almost free of other kidney tissue structure impurities such as epithelial cells, podocytes and endothelial cells;
s3, glomerular identification: identifying glomerular precipitate obtained in S105 by using podocyte marker nephrotic protein (Nephrin), and determining that the glomerulus precipitate is positive; as shown in fig. 2, the upper three figures show the morphology under the microscope after glomerular extraction, from left to right, at 4-fold, 10-fold and 40-fold, respectively, of the glomerulus observed under the microscope; clear globose glomerular structure can be seen under a high-power microscope, and the glomerular shape is full and bright, so that the activity is higher; clear view under a microscope, few impurities and no pollution; the lower three images are glomerular immunofluorescence identification, namely a glomerular fusion immunofluorescence image, a glomerular cell nucleus DAPI staining positive control image and a glomerular nephrosis protein (Nephrin) staining image from left to right, so that the glomerulus is stained by the nephrosis protein (Nephrin) to emit green fluorescence, and the glomerular fusion immunofluorescence image and the glomerular fusion positive control image can show a complete glomerular morphological structure;
the podocyte marker kidney disease protein (Nephrin) is purchased commercially and purchased from Shanghai ai Ebola anti-trade company;
s4, identifying mesangial cells: the mesangial cells were obtained by co-detection of positive markers of mesangial cells, namely alpha-smooth muscle actin (alpha-SMA), negative of epithelial cells, namely podocyte markers, namely Nephrin (Nephrin), and negative of endothelial cell markers, namely closely connected protein-1 (Zo-1), as shown in FIG. 3:
the first column on the left side shows the form under a microscope after the culture of the mesangial cells, and the mesangial cells are observed under the microscope from top to bottom at 4 times, 10 times and 40 times respectively; the spindle-shaped glomerular mesangial cell structure which is clearly stretched can be seen under a high-power microscope, the glomerular mesangial cells are arranged in a folded mode around the glomeruli, and the spindle-shaped mesangial cell structure has the advantages of unfolding form, clear color, higher activity, clear visual field under the microscope, less impurities and no pollution;
the second column on the left side is stained with a mesangial cell positive marker alpha-smooth muscle actin (alpha-SMA), and a mesangial cell fusion immunofluorescence image, a mesangial cell nucleus DAPI staining positive control image and a mesangial cell alpha-smooth muscle actin (alpha-SMA) staining image are respectively stained from top to bottom, so that the mesangial cells stained with the alpha-smooth muscle actin (alpha-SMA) emit green fluorescence, and can show complete and stretched glomerular morphology structures after being fused with the positive control staining of the cell nucleus DAPI;
the third column on the left side is that mesangial cells are stained with epithelial cell/podocyte marker nephrotic protein (Nephrin), and a mesangial cell fusion immunofluorescence image, a mesangial cell nucleus DAPI staining positive control image and a mesangial cell nephrotic protein (Nephrin) staining image are respectively obtained from top to bottom, so that the mesangial cells cannot be stained with the nephrotic protein (Nephrin), green fluorescence is not generated in the images, and therefore, no impurity components of the epithelial cells/podocytes exist in mesangial cell culture;
the first column on the right side is that the mesangial cells are stained by endothelial cell marker-tight junction protein-1 (Zo-1), and a mesangial cell fusion immunofluorescence image, a mesangial cell nucleus DAPI staining positive control image and a tight junction protein-1 (Zo-1) staining image are respectively stained from top to bottom, so that the mesangial cells can not be stained by the endothelial cell marker-tight junction protein-1 (Zo-1), green fluorescence does not exist in the image, and therefore, no impurity component of the endothelial cells exists in mesangial cell culture;
the alpha-smooth muscle actin (alpha-SMA) and podocyte marker nephropathy protein (Nephrin) are purchased from Shanghai Ebolan anti-trade company; the tight junction protein-1 (Zo-1) is commercially available from Shanghai Seikovia biological reagent Co., ltd;
cell counting was performed by dropping the extracted and purified cell mixture using a cell counting plate or a cell counter, and the result showed that (8.+ -. 1). Times.10 could be obtained on average in two kidneys of one mouse 3 The extraction efficiency is high; as shown in figure 1, the identification of glomerular immunofluorescence cells shows that the development of podocyte marker nephrosis protein (Nephrin) is positive, and the glomerular immunofluorescence cells have complete spherical structure, high cell activity, no pollution and good extraction effect; immunofluorescence cell identification of mesangial cells is shown in figure 2, and the mesangial cell positive marker alpha-smooth muscle actin (alpha-SMA) is positive in color development, has complete mesangial cell structure, high cell activity, no pollution and good extraction effect; the epithelial cells/podocyte marker nephropathy protein (Nephrin) and the endothelial cells marker zona compacta protein-1 (Zo-1) are shown to be negative, which indicates that the glomerular mesangial cells extracted and cultured according to the invention are almost free of other renal tissue structure impurities such as epithelial cells/podocytes and endothelial cells.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the present invention. Any simple modification, variation and equivalent variation of the above embodiments according to the technical substance of the invention still fall within the scope of the technical solution of the invention.
Claims (6)
1. A method for extracting and purifying primary glomerular mesangial cells of a mouse, which is characterized by comprising the following steps:
s1, extracting glomeruli:
s101, separating and digesting: taking kidneys of 6-8 week old male mice without kidney capsule removal, placing the kidneys in precooled Hank's balanced salt solution containing 1% of streptomycin by mass fraction, separating kidney cortex and outer marrow parts, placing the kidneys into DMEM culture solution containing collagenase IV with concentration of 0.75mg/mL and trypsin inhibitor with concentration of 0.75mg/mL, shearing into block tissues, placing the block tissues into digestive juice preheated by a water bath at 37 ℃ for shaking, and adding an equal volume of precooled culture medium into digested substances to obtain a digestion mixture;
s102, gradient filtration: placing a 70-mu m cell sieve on a 50-mL centrifuge tube a, pumping the digestion mixture obtained in the step S101 into the 50-mL centrifuge tube a through the 70-mu m cell sieve by using a suction pipe, grinding the digestion mixture by using a sterilized injection plug of a 5-mL syringe, flushing the digestion mixture by using Hank' S balanced salt solution, and collecting a filtered solution; placing a 40 μm cell sieve on a 50mL centrifuge tube b, pumping the filtered solution into the 50mL centrifuge tube b through the 40 μm cell sieve by using a suction tube, retaining the substances above the 40 μm cell sieve, turning over the 40 μm cell sieve and placing the 40 μm cell sieve into a 10cm culture dish a, and flushing the substances above the 40 μm cell sieve into the 10cm culture dish a by using Hank's balanced salt solution;
s103, shaking and suspending: shaking the 10cm dish a in S102, and collecting the suspension liquid a into a 50mL centrifuge tube c;
s104, secondary adhesion: placing a 40 μm cell sieve on a 50mL centrifuge tube d, pumping the suspension liquid a obtained in the step S103 into the 50mL centrifuge tube e through the 40 μm cell sieve by using a suction tube, retaining the substance above the 40 μm cell sieve, turning over the 40 μm cell sieve and placing the 40 μm cell sieve into a 10cm culture dish b, and flushing the substance above the 40 μm cell sieve into the 10cm culture dish b by using Hank' S balanced salt solution; shaking the 10cm culture dish b, collecting the suspension liquid b into a 50mL centrifuge tube f, centrifuging, discarding supernatant, and re-suspending with serum-free Ham's F-12 culture medium to obtain a re-suspension mixture;
s105, gradient centrifugation of polyvinylpyrrolidone: the method comprises the steps of (1) filling a 50% polyvinylpyrrolidone layering liquid into a 15mL centrifuge tube, dropwise adding a resuspension mixed liquid obtained in S104 from the side wall to the position above the liquid level of the 50% polyvinylpyrrolidone layering liquid, centrifuging, discarding a supernatant, and reserving a bottom sediment, namely a glomerular sediment;
s2, culturing mesangial cells: re-suspending the glomerular sediment obtained in S105 with a mesangial cell culture medium, and inoculating and culturing, climbing out cells for 3-5 days, changing liquid every day, and carrying out passage every other day, and taking the cells of the 3 rd-5 th generation to obtain the mesangial cells;
the membranous cell culture medium is prepared from the following raw materials in percentage by mass: 70.6% of DMEM culture solution, 23.4% of Ham's F-12 culture solution, 5% of fetal bovine serum and 1% of green streptomycin;
s3, glomerular identification: identifying glomerular sediment obtained in the step S105 by using podocyte marker nephrotic protein, wherein the glomerulus sediment is positive;
s4, identifying mesangial cells: adopts the common detection of mesangial cell positive marker alpha-smooth muscle actin positive, epithelial cell, namely podocyte marker nephrosis protein negative and endothelial cell marker closely connected protein-1 negative.
2. The method for extracting and purifying primary mesangial cells of mice according to claim 1, wherein the digestive juice in S101 is DMEM culture solution containing collagenase IV at a concentration of 0.75mg/mL and trypsin inhibitor at a concentration of 0.75 mg/mL.
3. The method for extracting and purifying primary mesangial cells of mice according to claim 1, wherein the pre-chilled medium in S101 is DMEM medium containing 10% fetal bovine serum by mass.
4. The method for extracting and purifying primary mesangial cells of mice according to claim 1, wherein the shaking speed of shaking in S101 is 120 rpm/min.
5. The method for extracting and purifying primary mesangial cells of mice according to claim 1, wherein the centrifugation conditions in S104 are: centrifuge at 4℃and 500 rpm/min for 3 min.
6. The method for extracting and purifying primary mesangial cells of mice according to claim 1, wherein the centrifugation conditions in S105 are: centrifugation was carried out at 12000 rpm/min at 4℃for 60 min.
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