Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
The experimental animals are Kunming mice, 8-10 weeks old, free of mouse specific pathogens, purchased and fed to Shanshi university medical school. All animals were well nourished and had normal mental and neurological status. The Shanzhi university college of medicine, the ethical committee for animal experiments approved the use of Kunming mice as subjects (No. SUMC 2018-064).
Phenol red-free high-sugar DMEM medium was purchased from the bio-tech company of the wunpro, with the product number PM150316.
Example 1
A method for obtaining mesenchymal stem cells with multidirectional differentiation potential and reproductive regulation function by carrying out induced banding culture on a mouse testis comprises the following specific steps.
(1) The male mice born for 0-3 days are killed by soaking in ethanol water solution with the volume fraction of 75% for 15min, the mice are soaked in iodophor solution for 15min, soaked and disinfected in ultra-clean bench by ethanol water solution with the volume fraction of 75% for 5min, meanwhile, the iodophor attached to the surfaces of the mice is removed, and the disinfected mice are put into physiological saline for cleaning.
(2) The mice were placed in sterile dishes with sterile gauze for dissection, the lower abdominal skin was cut transversely, then longitudinally to the anus, and the skin was spread to expose the bladder and testes on both sides of the bladder. The testis was gently lifted with micro forceps and the testis, epididymis and the tape were cut off together.
(3) The removed tissue was placed in 15% by mass sterile drops of phosphate buffered saline (PBS, available from Lonza), and the tissue fluid and blood were washed 3 times with 15% by mass sterile PBS until washed. Separating out the ribbon tissue with microscopic forceps, soaking the separated ribbon tissue in penicillin/streptomycin mixed solution (purchased from Lonza) for 3min, washing with PBS with 15% sterile mass fraction for 3 times,
(4) The prepared tape tissue was placed in a 15mL sterile centrifuge tube, about 3mLI collagenase (1 mg/mL concentration) was added (from Worthington) (10 tape tissues from 5 mice were added with 3mLI collagenase), and the tape tissue was loosened by digesting fibrin around the tape tissue with a constant temperature shaker at 37℃at 250r/min, and the interstitial cells of the tape were allowed to climb out of the tissue mass.
(5) An equal volume of phenol red-free high-sugar DMEM medium (composition by volume: 15% fetal bovine serum, 1% penicillin/streptomycin mixture, balance phenol red-free high-sugar DMEM medium) containing 15% Fetal Bovine Serum (FBS) was added to terminate the digestion of type I collagenase, 1200rmp was centrifuged for 5min, the supernatant was discarded, and 5mL of phenol red-free high-sugar DMEM medium containing 15% fetal bovine serum was added to fully resuspend the obtained mesenchymal stem cells and transferred into a sterile culture flask.
(6) Cells were placed in a 5% CO2, 37℃saturated humidity incubator for stationary culture and medium was changed after the cells had attached (about 3 days).
(7) The cell growth was observed with an inverted microscope. And (5) carrying out passage after the cell fusion rate reaches about 80%. Cell colonies were punctuated with trypsin (Gibco, life technologies) and passaged, and repeatedly subcultured to passage 3 to establish a cell bank to obtain testicular-introduced mesenchymal stem cells (GT-MSCs). The punctiform digestion method comprises adding 1 drop of pancreatin to the local part of the cells in the form of agglomerate, and then adding 1mL pancreatin to completely disperse and digest for 3min.
10 Pieces of the zona pellucida tissues are extracted to obtain one dish (25 cm2) of primary cells, and the cell quantity is 100 ten thousand. After 20 days of culture, the cell fusion rate reaches 80%, and subculture can be performed. The morphology and cell size of the second generation GT-MSCs are shown in FIG. 1. As can be seen in FIG. 1, GT-MSCs are typically fibroblast-like, in elongated, adherent, and fusiform morphology.
Example 2
This example is a study of the growth rate of the cells obtained in example 1, and the growth curve of the cells was examined by CCK-8 (Cell Counting Kit-8), and the specific procedure and results are as follows.
(1) Collecting cells (third generation) grown in 2-3 days (logarithmic growth phase), preparing single cell suspension, regulating concentration of single cell suspension after cell counting, adding 100 μl single cell suspension into 96-well plate, and regulating density of cells to be tested to 4000-5000 cells/well.
(2) The culture was performed with a cell culture medium, which was a high-sugar DMEM medium (15% fbs, 1% penicillin/streptomycin mixed solution) containing no phenol red, and the 96-well plate was placed in a 5% CO2, 37 ℃ incubator for 1-7 days. Once per day at the same time point.
(3) The cell culture medium was discarded, and the floating dead cells were removed by washing 1 time with PBS, and 100. Mu.L of fresh phenol red-free high-sugar DMEM medium and 10. Mu.L of CCK-8 solution (Abbe 50003) were added to each well.
(4) After further incubation in a cell incubator for 1 hour, the absorbance (OD value) at a wavelength of 450nm was detected by an enzyme-labeled instrument, and the detection was repeated 3 times to obtain an average value. Daily test results were recorded.
(5) The time axis is used as the horizontal axis, the absorbance (OD value) is used as the vertical axis, the proliferation curve of the cells is plotted, and the exponential growth phase of the cells can be seen from the proliferation curve of the cells.
As can be seen from FIG. 2, GT-MSCs grew to log phase for 2-4 days. 10 pieces of the tissue with the leading tape are taken and cultured according to the culture method, and after subculturing to the third generation of cells, the cell count is carried out, and the cell quantity is 2.72 multiplied by 107.
Example 3
This example demonstrates the characterization of the cell phenotype of the mesenchymal stem cells obtained in example 1 by flow cytometry and immunofluorescence, with the following procedure and results.
1. Flow cytometry
(1) Article preparation, the antibodies FITC anti-mouse CD90.2, FITC anti-mouse CD71, APC anti-mouse CD34, APC anti-mouse CD45 (Biolegend), fetal bovine serum FBS (purchased from Propoxel) were removed and placed on ice in the dark;
(2) Taking the 3 rd generation GT-MSCs cells in the logarithmic growth phase, slightly blowing the cells to avoid cell disruption before the digestion method, centrifuging the digested cells at 1500rpm for 5min, and collecting the cells;
(3) Preparing a 1.5mL EP tube, grouping according to the tested antibodies to prepare antibody marks, and leaving a blank control tube;
(4) Discarding the supernatant of the collected cells, adding 1mL of 4 ℃ precooled PBS (Biyun Tian), and re-suspending the cells into single-cell suspension;
(5) 1mL of cell suspension is sucked and added into a 1.5mL EP tube, and the cells are washed twice by precooled 1mL of PBS, and the centrifugal machine is precooled at 4 ℃ in advance, and then the cells are collected by centrifugation for 5min under the condition of 4500 rpm;
(6) Preparing a sealing liquid, namely preparing the sealing liquid according to the volume ratio of goat serum to PBS=1:9, and adding 100 mu L of sealing liquid into each tube;
(7) Discarding PBS, adding the prepared sealing solution into cells, re-suspending into cell suspension, adding 100 mu L of the sealing solution into each EP tube, and incubating for 10min;
(8) After the incubation is completed, antibody (FITC anti-mouse CD71(2.5μL/100μL)、APC anti-mouse CD34(2.5μL/100μL)、APC anti-mouse CD45(2.5μL/100μL) or FITC anti-mouse CD90.2 (4. Mu.L/100. Mu.L) is added to each tube in proportion, and the mixture is placed on ice and incubated for 15min in a dark place;
(9) Placing the incubated cells in a 4 ℃ precooling centrifuge, centrifuging for 5min at 4500rpm, and discarding the supernatant;
(10) Add 500. Mu.L PBS per tube, resuspend the cell suspension and filter with cell filtration mesh;
(11) And (5) detecting in a light-shielding machine.
As can be seen from fig. 3 and 4, the cell phenotype was established by flow cytometry using a panel of surface markers, showing positive cells for clusters CD71, CD90 and CD44 of MSCs markers and their negative cells for CD45, CD34, with CD90 and CD44 expression of up to 97.7% and 99.1%, CD 44-0.302% and CD 34-2.06%. The GT-MSCs are indeed shown to have a MSCs phenotype, which meets the International Society of Cytotherapy (ISCT) standard.
2. Immunofluorescent staining
Sterilizing the super clean bench, placing the prepared articles on the super clean bench, sterilizing by ultraviolet irradiation for 30min, taking out the reagent, and re-heating at room temperature.
(1) The preparation of the cell climbing sheet comprises the steps of taking a 24-hole culture plate, adding a small amount of high-sugar DMEM culture medium without phenol red at the bottom of the culture plate, uniformly spreading the culture plate bottom, putting the cell climbing sheet at the bottom of the 24-hole culture plate, and tightly attaching the cell climbing sheet to the plate bottom;
(2) Taking second generation (P2) cells in an exponential growth phase, and carrying out passage on a 24-pore plate according to a proper density, and growing on a climbing plate, wherein the cell density is not excessive, and preferably, the cells do not have stacking adhesion growth, so that the fluorescence of the photographed cells is clear and complete;
(3) Discarding the high-sugar DMEM culture medium without phenol red when the cell is attached to the wall and grown and fused to about 40%, if the cell needs to be added with medicine treatment, the cell density can reach 30%, and then the DES treatment is carried out for 24 hours;
(4) Discarding the culture medium, slowly blowing with precooled PBS, cleaning away suspended and detached dead cells, repeating for 3 times for 3min each time;
(5) Adding 200 mu L of paraformaldehyde with the mass fraction of 4% into each cell, and fixing the cells for 15min;
(6) Washing with PBS again for 3 times and 5min each time;
(7) 200 mu L of Triton X-100 aqueous solution with the volume fraction of 0.5% is added into each cell at room temperature to puncture the cell membrane, so that the primary antibody binding solution can easily pass through the cell membrane to enter cytoplasm;
(8) Repeating step (5);
(9) Dripping 200 mu L of goat serum with the volume fraction of 10% on each hole of cells for sealing, and standing at room temperature for incubation for 45-60 min;
(10) Preparing primary antibodies, namely diluting the primary antibodies according to the specification ratio of PDGFR-alpha (purchased from Abcam (U.S.), ab97355, dilution ratio of 1:200), NKX2.5 (purchased from CST Co (U.S.), 3868, dilution ratio of 1:200);
(11) Discarding the serum sealing liquid, not washing, adding 200 mu L of primary antibody into each hole, and placing the mixture in a refrigerator at a temperature of 4 ℃ for incubation overnight;
(12) Discarding the primary antibody, washing with PBS for 10min, and washing for 3 times;
(13) Dripping secondary antibody (Alexa Fluor 488 marked goat anti-rabbit IgG (H+L) (1:500) to incubate for 60min at room temperature, and taking care of light-shielding operation;
(14) Repeating step (5);
(15) Dripping anti-fluorescence quenching agent containing DAPI (1 mg/mL) to fully spread the climbing sheet;
(16) Lightly prying the climbing sheet by forceps, reversely buckling the climbing sheet on a glass slide, sealing the climbing sheet by transparent nail polish and fixing the climbing sheet on the glass slide to prevent the climbing sheet from moving;
(17) And placing the prepared climbing sheet under a confocal microscope, observing and shooting, and paying attention to light-shielding operation.
As can be seen from FIG. 5, TG-MSCs expressed mesodermal markers NKX2.5 and PDGFR-alpha, further demonstrating the potential of TG-MSCs for multipotent differentiation.
Example 4
This example a pluripotency study was performed on the testicular traction mesenchymal stem cells obtained in example 1 by an experiment of inducing multidirectional differentiation, with the following specific procedures and results.
1.1 Adipogenic Induction of differentiation
(1) Inoculating 40 ten thousand third-generation cells onto a six-hole plate according to a plating method to uniformly distribute the cells, discarding the high-sugar DMEM culture medium without phenol red when the fusion density reaches 80% -90% after the cells are subjected to wall-attached culture for 2 days, rinsing 3 times by PBS, discarding the PBS, and adding 2mL of fat-forming induced differentiation culture medium A (purchased OriCell, MUXMX-90031/200 mL);
(2) Continuous culture for 3 days, removing old solution with a pipette, and adding 2mL of lipid-forming induced differentiation medium B (OriCell, MUXMX-90031/200 mL);
(3) After culturing for 24 hours, removing the lipid-forming induced differentiation culture medium B solution, adding 2mL of the lipid-forming induced differentiation culture medium A solution again, and continuing culturing for 3 days;
(4) According to the time sequence, the lipid-forming induction differentiation culture medium A liquid and the lipid-forming induction differentiation culture medium B liquid are used for 3 to 5 times in turn, and then the lipid-forming induction differentiation culture medium B liquid is used for continuous culture for 4 to 7 days, and the liquid is changed every 2 to 3 days;
(5) After 3 weeks of induction culture, oil red O staining was performed until the lipid droplets became round and large enough (lipid droplet volume was greater than 40% of the cell volume).
1.2 Oil red O dyeing method
(1) Taking out the induced differentiated cells, and washing with PBS for 3 times for 5min each time;
(2) 200 mu L of paraformaldehyde solution with the mass fraction of 4% is added, and the mixture is fixed for 30min in a refrigerator at the temperature of 4 ℃;
(3) Discarding 4% paraformaldehyde solution, and washing with PBS for 3 times for 5min each time;
(4) Discarding PBS, adding 1mL of oil red O dye solution working solution (purchased from OriCell, OILR-10001) (storage solution according to volume ratio: distilled water=3:2, mixing well, filtering with neutral filter paper), and incubating at room temperature for 30min;
(5) Discarding the working solution of the oil red O dye liquor, and flushing with PBS for 3 times, each time for 5min;
(6) Rinsed once with distilled water, observed in an inverted phase contrast microscope and photographed.
2.1 Chondrogenic induced differentiation
(1) The OriCell mouse bone marrow mesenchymal stem cells are selected to be cartilage induced and differentiated kit (purchased from OriCell, MUXMX-90041/200 mL) and the kit is prepared2ML of mouse bone marrow mesenchymal stem cell chondrogenic differentiation additive I (hereinafter referred to as additive I) is added in totalA premix solution was prepared in 97mL of a cell basal medium (hereinafter referred to as basal medium);
(2) Taking 40 ten thousand third-generation cells, digesting and centrifuging, transferring the cells into a 15mL centrifuge tube, and centrifuging at 800rpm for 5min;
(3) Discarding the supernatant, adding 0.5mL of premix, re-suspending the precipitate obtained by the previous step of centrifugation, washing cells, and centrifuging at 560rpm for 5min at room temperature;
(4) Repeating the step (3), and cleaning the cells again;
(5) Adding a cartilage-forming differentiation complete medium of the mesenchymal stem cells of the mice (hereinafter referred to as an additive II) according to a proportion (10 mu L of the additive II is added into 1mL of premix), sucking the additive II with a dosage required by an experiment, adding the additive II into the premix with a corresponding volume, and preparing the complete medium of the cartilage-forming differentiation, and gently blowing and uniformly mixing;
(6) Re-suspending the precipitate obtained in the step (4) by using 0.5mL of cartilage-forming complete culture medium, and centrifuging at 560rpm for 5min at room temperature again;
(7) The centrifuge tube lid was unscrewed for gas exchange and placed in an incubator at 37 ℃ with 5% CO2 for incubation (centrifuge tube was not shaken for 24 h).
(8) Starting from inoculation, replacing cells with fresh chondrogenic complete medium every 2-3d, and taking about 0.5mL chondrogenic differentiation complete medium per tube, taking care not to suck out cartilage balls;
(9) After the cartilage induction complete culture medium is replaced, the bottom of the centrifugal tube is flicked to separate the cartilage balls from the bottom of the tube and suspend the cartilage balls in the liquid. Slightly unscrewing the cover of the centrifugal tube, and placing the tube into an incubator with the temperature of 37 ℃ and the concentration of 5% CO2 for continuous induction culture;
(10) After a typical duration of 21-28 days of induction, the cartilage spheres may be formalin-fixed and paraffin-embedded sectioned and finally stained with alisxin blue.
2.2 Allinnet blue staining experiments
2.2.1 Paraffin section was made
2.2.1.1 Fixing, namely cleaning the induced cartilage balls by PBS, soaking in 4% paraformaldehyde solution (purchased from Biyun Tian, P0099) and fixing for more than 30min;
2.2.1.2 dehydration, namely dehydration of the fixed cartilage ball by using gradient concentration alcohol in the following steps of 50% alcohol-70% alcohol-80% alcohol-95% alcohol-absolute alcohol, wherein each stage is 30min;
2.2.1.3, because alcohol and paraffin are not compatible, the dehydration needs to be transited by dimethylbenzene in the following way:
(1) Uniformly mixing dimethylbenzene and absolute alcohol according to a volume ratio of 1:1, and soaking cartilage balls in the mixture for 2 hours;
(2) Soaking cartilage balls in pure xylene for 1.5h;
(3) Changing new dimethylbenzene, and continuously soaking for 1h;
2.2.1.4 wax impregnation to remove the transparencies from the cartilage balls, wax impregnation treatment is required to allow paraffin to penetrate into the interior for embedding:
(1) Uniformly mixing dimethylbenzene and paraffin according to a volume ratio of 1:1, soaking cartilage balls in the mixture, and placing the mixture in a 40 ℃ oven for 40min;
(2) Soaking the cartilage balls treated in the step (2) in pure paraffin, and placing the pure paraffin in a 55 ℃ oven for 30min;
2.2.1.5 embedding, namely taking out the cartilage ball, placing the cartilage ball in a mould, pouring paraffin, standing and cooling, taking out the cartilage ball after the paraffin is fully cooled and formed, and trimming wax blocks.
2.2.1.6 Slicing, namely continuous slicing, wherein the thickness of each slice is 3 mu m;
2.2.1.7 sticking the cartilage ball slice to the glass slide by using an adhesive, and drying in a 35 ℃ oven;
2.2.1.8 dewaxing:
(1) Xylene soaked sections for 15min, and new xylene soaked sections for 10min;
(2) Mixing dimethylbenzene and absolute alcohol uniformly according to a volume ratio of 1:1, and soaking and slicing for 10min;
(3) Sequentially soaking the slices in 95%, 85%, 70% and 50% alcohol for 10min, and air drying.
2.2.2 Alinixin blue staining
(1) Dripping alixin blue staining solution on the paraffin section, completely covering the specimen, and staining for 1h in a 37 ℃ water bath kettle;
(2) Washing the glass slide with tap water for 5min;
(3) Dehydrating the slice with gradient ethanol (50% ethanol-70% ethanol-80% ethanol-95% ethanol-anhydrous ethanol) for 5min at each concentration for 1 time, and air drying in a fume hood while transparent with xylene;
(4) After the neutral resin is sealed, the neutral resin is observed and photographed under an inverted microscope.
3.1 Osteogenic induced differentiation
(1) Formulated as osteoinductive differentiation complete medium (available from OriCell, MUXMX-90021/200 mL) to be usedThe high-quality fetal bovine serum (20 mL) and the osteogenic differentiation additive (3 mL) in the osteogenic differentiation kit of the mesenchymal stem cells of the mice are all addedBasal medium (177 mL);
(2) Taking 40 ten thousand second generation GT-MSCs cells, inoculating the cells onto a six-hole plate according to a plating method, uniformly distributing the cells, discarding a common culture medium (a high-sugar DMEM culture medium without phenol red) when the fusion density reaches 80% -90% after the cells are subjected to wall-attached culture for 2 days, washing the cells 3 times by using PBS, adding 2mL of an osteogenic differentiation complete culture medium for induction, changing a liquid every three days (the osteogenic differentiation complete culture medium), inducing morphological change and growth condition of the cells after 4 weeks, and carrying out staining detection by alizarin red after calcium nodules appear. To prevent osteoblast shedding, it is suggested that after a large number of calcium nodules appear during the osteogenesis, the replacement form is changed to a half replacement every two days (osteogenesis induced differentiation complete medium).
3.2 Alizarin Red staining
(1) Taking out the cells after the osteogenesis differentiation, discarding the complete osteogenesis differentiation culture medium, and flushing for 3 times with PBS for 5min each time;
(2) 200 mu L of paraformaldehyde solution with the mass fraction of 4% is added, and the mixture is fixed for 30min in a refrigerator at the temperature of 4 ℃;
(3) Discarding 4% paraformaldehyde solution, and washing with PBS for 3 times for 5min each time;
(4) 1mL alizarin red dye solution (purchased from OriCell, ALIR-10001) is added into each hole, and the dyeing treatment is carried out for 3-5min;
(5) Discarding alizarin red dye liquor, and repeating the step (3);
(6) Osteoinductive differentiation staining results in the plates were observed under an inverted phase contrast microscope and recorded.
As can be seen from FIG. 6, a large number of lipid droplets were seen in the cytoplasm after 3 weeks of adipogenesis, about 85% of cells were positive (B in FIG. 6) after oil red O staining, alizarin red staining results showed that cells were able to exhibit a large amount of calcium salt deposition after 4 weeks of osteogenic differentiation, about 80% of cells were positive (A in FIG. 6), and in chondrogenic differentiation, a Li Xinlan primitive immunohistochemical staining showed that the induced group of aggregated chondrocytes (C in FIG. 6) after 4 weeks of induction, the above results showed that TG-MSCs had adipogenic, osteogenic, chondrogenic multipotential differentiation potential, and mesodermal differentiation direction, thereby demonstrating that TG-MSCs had the potential to induce differentiation into mesoderm.
Example 5
In this example, the hormone regulating function of the testicular interstitial cells was identified by qualitative analysis of the G protein-coupled receptor gene RXFP2 (receptor for the primary product INSL3 of testicular interstitial cells) by nucleic acid gel electrophoresis technique, and the specific procedures and results were as follows.
(1) Preparation of running buffer
The nucleic acid electrophoresis buffer was prepared as a TAE buffer, and the solution was diluted to 1 XTAE buffer. The 50×TAE buffer formulation is shown in Table 1.
TABLE 150 xTAE Buffer formulation (50 times dilution for use)
| Reagent(s) | Weighing value | Method of |
| Tris (trometamol) | 242g | Placing the mixture in a 1L beaker, adding about 600mL of deionized water, and fully and uniformly stirring; |
| Na2EDTA·2H2O | 37.2g | |
| CH3 COOH (glacial acetic acid) | 57.1mL | After sufficient dissolution, the pH was adjusted to 8.3 with NaOH and deionized water was added to a volume of 1L. |
(2) Preparation of agarose gel
Washing the gel-making mould and comb with distilled water, placing on a gel-making flat plate, closing the edge of the mould, setting up the comb, preparing 1% agarose gel (100 mL 1 xTAE buffer+1g agarose), heating to dissolve, adding 10 mu LYEAGREEN nucleic acid dye (10000 x, purchased from the following holothurian, 10204 ES), fully mixing, pouring into the mould, and waiting for cooling;
(3) After 30-45min at room temperature, the gel is completely coagulated, the comb is carefully pulled out, and the gel is placed in an electrophoresis tank;
(4) Pouring electrophoresis buffer solution into the electrophoresis tank, preferably 1mm beyond the gel surface, if bubbles exist in the sample hole, and removing;
(5) Adding 10 Xvolume of loading buffer (10213 ES from the living organism of the next year) into the DNA sample, mixing, and slowly adding the sample mixture into the immersed gel sample adding hole by using a gun;
(6) And (3) switching on a power supply, wherein red is a positive electrode, black is a negative electrode, and the DNA sample is cut and recorded to swim from the negative electrode to the positive electrode (one end close to the sample adding hole is negative). The voltage is generally 60-100V, and the electrophoresis is carried out for 20-40 min;
(7) Judging whether electrophoresis is stopped or not according to the electrophoresis position of the indicator;
(8) After electrophoresis, the power supply is turned off, the electrophoresis band and the position thereof are observed on a gel imager, and the size of the amplified product is compared with a standard Marker of nucleic acid molecular weight.
The results in FIG. 7 show that the normal control group, the solvent control group and the different administration experimental groups all express RXFP2 positive, and GT-MSCs have hormone regulating function.
Example 6
In this example, the hormone regulating function of the testicular mesenchymal cells was identified by qualitative analysis of the G protein-coupled receptor gene RXFP2 (receptor of the primary product INSL3 of the testicular mesenchymal cells) of GMSCs by Western blotting (Western Blot), and the specific procedures and results are as follows.
1. Cellular protein extraction and concentration determination
1.1 Total cellular protein extraction
(1) Spreading the cell plates into a 60mm culture dish, and extracting cell proteins after the cells are treated by the medicine;
(2) Preparing RIPA lysate (purchased from Panera, AAPR, 51-100) and PMSF (purchased from 100mM, purchased from Biotol, B14001) on ice for dissolving, placing PBS on a refrigerator for precooling at 4 ℃, preparing a 1.5mL EP tube on ice for use, cleaning a cell scraper, and drying in a drying box for later use;
(3) Preparing a cell lysate working solution according to the cell quantity and the cell volume (1 mL/1×107 cells), namely preparing the working solution by (calculated according to the volume ratio, RIPA lysate: PMSF=100:1), shaking and uniformly mixing, and then placing on ice for standby, and paying attention to the preparation for use at present;
(5) Taking out the cells, discarding the high-sugar DMEM culture medium without phenol red in the culture dish, and gently washing the culture medium with PBS for 3 times to wash off the fetal bovine serum in the culture medium, thereby avoiding influencing protease inhibitors;
(4) Adding a proper amount of lysate into each dish of cells (80 mu L of cell lysate is added into each dish with the diameter of 60 mm), rapidly scraping cells at the bottom of the dish by using a cell scraper, scraping the cells as completely as possible, scraping the cells sufficiently, transferring the lysate containing cell fragments into a 1.5mL EP tube, and performing lysis on ice for 30min sufficiently, wherein the cell scraper needs to be replaced between different cells, and the whole process is performed on ice to avoid protein degradation;
(5) Ultrasonically crushing each group of cell lysate for 4 times by using a homogenizing ultrasonic instrument, wherein each time is 5s, and the interval is 3 s;
(6) Pre-cooling the centrifuge to 4 ℃ in advance, placing 1.5mL of EP tube in the centrifuge and balancing, and centrifuging at 13500rpm for 20min at 4 ℃;
(7) Taking supernatant and total cell proteins after centrifugation, subpackaging into 1.5mL EP tubes, simultaneously subpackaging 6 mu L for measuring protein concentration, adding 5 Xloading buffer solution according to the ratio of 5 Xloading buffer solution to protein=1:4, heating the EP tubes on a 100 ℃ dry constant temperature heater for 10min after uniform mixing, placing the EP tubes on ice for cooling, identifying groups, and storing in a-20 ℃ refrigerator after the extraction date.
1.2 Protein concentration determination
(1) Preparation of 1.5mL EP tube, PBS, BSA standard protein (from BCA kit, purchased from Panera, AAPR 161-30);
(2) Preparing a BSA protein standard sample with the concentration of 0.5 mug/mL by using a protein standard substance preparation solution for preparing a standard curve;
(3) Preparing BCA working solution, namely preparing an appropriate amount of BCA working solution according to the ratio of reagent A to reagent B=50:1 according to the required amount of 200 mu L added to each hole, and fully and uniformly mixing;
(4) Adding standard substances into a 96-well plate according to the sequence shown in Table 2, taking an average value by dividing 3 complex wells, and setting a concentration gradient to prepare a protein standard curve;
TABLE 2 protein standard curve
| Sequence number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Standard substance concentration (μg/mL) | 0 | 0.025 | 0.05 | 0.1 | 0.2 | 0.3 | 0.5 | 0.5 |
| PBS(μL) | 20 | 19 | 18 | 16 | 12 | 8 | 4 | 0 |
| Standard substance after dilution (MuL) | 0 | 1 | 2 | 4 | 8 | 12 | 18 | 20 |
(5) Diluting a protein sample to be detected by using PBS 10 times, namely adding 18 mu L of PBS and 2 mu L of protein sample into each hole of a 96-well plate, and setting an average value of 3 multi-hole spheres for each sample to reduce errors;
(6) 200 mu L of BCA working solution is added into each hole, the holes are covered with a cover and then are placed in a constant-temperature oscillator with the temperature of 37 ℃ of an ELISA plate to shake for 30min, and the result is prevented from being influenced by bubbles in the sample loading process;
(7) Taking out the 96-well plate, cooling to room temperature, opening the plate cover, placing on an enzyme-labeled instrument, measuring the absorbance (OD) with the wavelength of 562nm, setting the standard protein concentration as the abscissa (mg/mL), drawing a standard protein curve (as shown in figure 9), displaying a unitary once equation corresponding to R2 and the straight line, if R2 is more than 0.995, proving that the model is reliable, substituting the measured absorbance of each sample into the equation to obtain the corresponding protein concentration, dividing the obtained protein concentration by the dilution factor, and obtaining the protein concentration of the sample.
Western Blot detection of target protein expression level
2.1 Preparation of Polyacrylamide gel (SDS-PAGE)
(1) Cleaning a BIO-RAD glass plate and a comb with distilled water in advance, placing the BIO-RAD glass plate and the comb in a drying box for drying, clamping the glass plate with a glue making clamp, aligning the bottom, clamping the glass plate on a glue making frame, detecting leakage with distilled water, and after no leakage is confirmed, using filter paper to distill water and suck the distilled water to dry, so that glue making can be performed;
(2) Preparing concentrated glue and separating glue, namely selecting separating glue and concentrated glue with proper concentrations according to requirements, preparing reagents according to the table 3, preparing the separating glue and the concentrated glue, preparing the separating glue firstly, and preparing the concentrated glue after the separating glue is solidified, wherein the solidification time of the separating glue and the separating glue is about 30 minutes.
TABLE 3 preparation of SDS-PAGE separating gel and concentrating gel
(3) After the preparation of the separating gel is completed, the separating gel solution is reversely and uniformly mixed up and down, the gap of the glass plate is firstly injected into the uniformly mixed separating gel solution, the height is about 3/5-4/5 of that of the glass plate, then 75% alcohol liquid is used for sealing, air is isolated to enable the separation to be quickly solidified, air bubbles are emptied at the same time, the boundary of the separating gel is kept horizontal, 75% alcohol is poured out after the separating gel is solidified, filter paper is used for sucking dry, the separating gel is not touched, then concentrated gel is injected until the glass plate is filled, then a comb is quickly and flatly inserted, air bubbles are avoided in the process, the action of inserting the comb is quick, and the concentrated gel is prevented from being solidified in advance;
(4) Concentrating the gel at room temperature for about 30min to solidify, placing the prepared gel plate in ddH2 O storage solution for moisturizing, and storing in a refrigerator at 4deg.C for use after 1 week.
3. Western immunoblotting (Western Blot)
(1) Taking out gel prepared in advance within 1 week from a refrigerator at the temperature of 4 ℃, correctly clamping a gel plate in a vertical electrophoresis tank, adding enough electrophoresis liquid into the tank to be flush with a glass plate, and slowly and vertically pulling out a comb, wherein the aim of avoiding damaging a sample loading hole is fulfilled;
(2) Loading, namely loading the protein liquid according to the same mass, wherein the loading amount of each sample is 20 mug, converting the loading volume according to the protein concentration measured by a BCA detection method, and sequentially adding the corresponding protein sample and 5 mug of pre-dyed protein Marker (purchased from Thermo Siamer);
(3) Electrophoresis, namely placing the inner tank into the outer tank, adding a proper amount of electrophoresis liquid into the outer tank to enable the electrophoresis liquid to permeate through the metal wires at the bottom of the inner tank, and covering the tank cover of the electrophoresis tank. The positive electrode and the negative electrode of the power supply are correctly connected, the power supply is turned on, the parameters are set to be 60V constant pressure running concentrated glue, after the Marker strips of the pre-dyed protein are obviously separated, the pressure can be increased to 120V constant pressure running separation glue until the Marker strips corresponding to the molecular weight of the target protein are sufficiently clear and separated, namely, the tube wall power supply is realized, and the electrophoresis is finished;
(4) Electric conversion:
a. cutting a proper PVDF film according to the size of the gel, marking the left and right sides and the front and back sides, soaking the PVDF film in methanol solution for 5min, and activating the PVDF film;
b. and (3) cutting gel, namely taking out a glass plate, tilting the glass plate by using a warping plate, cutting a required gel strip at a relative molecular weight position of gel by referring to the Marker strip position, and measuring the molecular weight of protein in the experiment as shown in Table 4.
TABLE 4 relative molecular weights for the desired proteins of interest
| Protein to be detected | Molecular weight (kDa) |
| Anti-LC3 Rabbit mAb | 14-16 |
| Anti-P62Rabbit mAb | 60 |
| Anti-Nanog Rabbit mAb | 29-42 |
| Anti-Oct4 Rabbit mAb | 45 |
| Anti-GAPDH Rabbit mAb | 36 |
| Anti-β-actin Rabbit mAb | 42 |
| Anti-RXFP2 Rabbit mAb | 86 |
C. Preparing a tray for transferring films, filling enough electro-transfer buffer solution prepared in advance, putting the film transfer clips, a foam-rubber cushion and filter paper into the tray for pre-wetting, enabling the electro-transfer buffer solution to pass through the devices, placing the foam-rubber cushion and 3 layers of cut filter paper on a negative plate of the film transfer clips according to the order, carefully transferring the cut rubber to the filter paper, then reversely buckling a PVDF film on the gel, paying attention to the left-right order, lightly evacuating bubbles, avoiding touching a protein surface of the PVDF film by hands, pasting another layer of foam-rubber cushion and the filter paper on a positive plate, and tightly closing the film transfer clips after only pressing the positive plate;
d. The method comprises the steps of electrotransfer, namely placing a transfer film clamp into a transfer film groove, adding enough electrotransfer buffer solution and an ice plate, covering an electrotransfer groove cover, transferring the transfer film groove into an ice box, filling ice blocks with ice, connecting a power supply, adjusting parameters, starting transfer film, wherein the transfer film parameters of the experiment are constant current of 200mA, and the electrotransfer time is about the molecular weight plus 30min;
e. Sealing, namely, sealing liquid (5% skimmed milk) can be prepared in advance at the beginning of film transfer, and the sealing liquid is placed on a shaking table for uniform mixing so as to prevent the follow-up luminous background from being affected incompletely by the dissolution of the skimmed milk powder, and after the electric transfer is finished, placing the PVDF film into the sealing liquid, and slowly sealing the shaking table for 1h at normal temperature;
f. Transferring the corresponding PVDF membrane into the corresponding primary anti-dilution liquid according to the Marker strip position phase, and slowly shaking overnight on a shaking table at 4 ℃ for more than 8 hours;
g. washing PVDF membrane with TBST the next day, placing it on a room temperature shaking table for rapid washing for 10min, repeating for 3 times;
h. Transferring the PVDF membrane into secondary antibody diluent prepared in advance, and slowly incubating on a room-temperature shaking table for 1h;
i. Washing PVDF film with TBST, placing it on a room temperature shaking table, rapidly washing for 10min, repeating for 3 times, and washing off secondary antibody diluent;
j. Preparing chemiluminescent liquid according to the proportion of the luminescent liquid A to B=1:1, fully and uniformly mixing, and performing light-shielding operation in the operation process, placing PVDF film protein surface upwards in a chemiluminescent instrument after uniformly attaching the luminescent liquid to the strip, opening Image Lab 3.0.1 software, and performing light emission after setting parameters and exposure time;
k. The method comprises the steps of strip analysis, namely, storing and deriving images, comparing gray values of proteins to be detected among different groups by using Image Lab 3.0.1 software, aligning the internal references of each group to obtain a ratio by taking the gray value of the internal reference of the control group as a reference, comparing the gray value of the target protein value of each sample with the ratio of the internal reference to obtain the relative gray value of each experimental group, and obtaining the relative multiples of the target protein expression of the other treatment groups by taking the target proteins of the control group as a reference. Each protein of interest was repeatedly tested 3 times and statistically analyzed using SPASS software.
In this example, living mice were subcutaneously injected with DES at different concentrations (DES concentrations of 0.1. Mu.g/kg.d, 1. Mu.g/kg.d, 10. Mu.g/kg.d, 100. Mu.g/kg.d), treated with no treatment (NC) and solvent (DM), and after the mice were bred, the testis tape of the mice was extracted, and GT-MSCs were cultured.
As shown in fig. 10, RXFP2 protein levels were down-regulated with increasing DES concentration, dose-dependent, and differences were statistically significant compared to the no-treatment control group (NC), indicating that hormonal regulation of DES-induced pediatric cryptorchism was affected.
Example 7
In this example, the effect of Western Blot (Western Blot) on the pluripotency of GT-MSCs by Nanog and Oct4 (Nanog and Oct4 are regulatory factors necessary for mesenchymal stem cells to maintain stem cells self-renewal and pluripotency) in subcultured 2 nd generation GT-MSCs was quantitatively analyzed by Western Blot to identify the pluripotency of testicular mesenchymal cells and the effect of DES on GT-MSCs pluripotency, and the method was carried out in the same manner as in example 6.
In this example, GT-MSCs were subcultured cells of passage 2, the group without any treatment was set as no-treatment control group (NC), the group treated with solvent was set as solvent control group (DM), chloroquine drug treatment 4H was set as CQ group (CQ), diethylstilbestrol drug treatment 24H was set as DES24H, DES treatment 24H and chloroquine treatment 1H was set as D+C24H.
As shown in FIG. 8, the experimental results demonstrate that the mechanism of DES-induced pediatric cryptorchidism is to inhibit Nanog and Oct4 protein expression by blocking autophagic flow, thereby affecting the pluripotency of GT-MSCs cells.
Example 8
In this example, LC3 (LC 3 is an autophagy-based marker) and P62 (autophagy degradation substrate marker) protein levels in subcultured generation 2 GT-MSCs were analyzed by Western blotting (Western Blot), and studies on the disease mechanism of DES-induced pediatric cryptorchism were explored, and the specific implementation method was the same as in example 6. The increase in the relative expression level of LC3-II/LC3-I reflects autophagosome formation, and promotes autophagy flow. The treatment groups DES treatment 12H (DES 12H), 24H (DES 24H), 48H (DES 48H) and des+cq combined treatment 12H (d+c12h), 24H (d+c24h), 48H (d+c48h) were set with the group without any treatment as the no-treatment control group (NC) and the group with solvent treatment as the solvent control group (DM).
As shown in FIG. 11, the experimental results indicate that DES inhibits autophagy of GT-MSCs, while chloroquine blocks autophagy flow (i.e., blocks degradation of LC 3), causing accumulation of LC3, and thus CQ and DES act synergistically to inhibit autophagy. Further demonstrated that DES induced autophagy of GT-MSCs was involved in mouse cryptorches and inhibited autophagy.