CN116103223A - Culture method for mouse supporting cells - Google Patents
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0683—Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells
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Abstract
The invention relates to a culture method for a mouse supporting cell, which relates to the technical field of biology, and can be used for separating and purifying to obtain the mouse supporting cell, detecting the release condition of cytochrome C and the expression change of Bcl-2 family proteins (Bax, bcl-2) by a laser confocal microscope, discussing the molecular mechanism of a testis supporting cell regulated by environmental estrogen, and providing a research basis for promoting the growth of the mouse supporting cell by soybean isoflavone.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a culture method for a mouse supporting cell.
Background
Phytoestrogen is a plant component that is structurally and functionally similar to estradiol and has the ability to bind to estrogen receptors. At present, more domestic researches are about methods for extracting and measuring the extract, and the aspects of resisting tumors, osteoporosis, climacteric syndrome and the like. Soy isoflavones are structurally similar to estrogens and therefore bind to estrogen receptors and thus exhibit estrogenic and antiestrogenic activities, which are important characteristics of phytoestrogens depending on their local concentration, endogenous estrogen content and estrogen receptor levels in tissues and organs. Soy isoflavones have a weaker estrogenic activity and the antiestrogenic activity is mainly exhibited in disease states where body estrogen levels exceed the normal range. The regulation of hormone-related disorders by soy isoflavones is based on its planar biphenol structure similar to that of the endogenous hormones 17β estradiol and diethylstilbestrol, a synthetic product, which is also similar to tamoxifen (tamoxifen). It is generally believed that nonsteroidal estrogens are bound to 17βE 2 The same site of occupied Estrogen Receptor (ER) acts.
The number of support cells plays a very important role in the spermatogenesis process and is closely related to the number of spermatogenesis, and a certain number of support cells can only maintain a certain number of spermatogenic cells, so that the lower the number of support cells, the lower the upper limit of spermatogenesis. Meanwhile, the change of the support cells in the juvenile period can influence the size of the testis and the sperm physiology after the adult period, and possibly influence the sperm quality. Early exposure of soy isoflavones can cause structural and functional changes in the male reproductive system, and low doses of soy isoflavones can promote growth of mouse supporting cells.
Mitochondrial cytochrome C is a valve for apoptosis to occur and can determine the manner in which cells die by regulating their energy metabolism. Whether cells undergo apoptosis or necrosis after stimulation depends largely on intracellular ATP levels. Intracellular ATP can still be maintained at appropriate levels after cytochrome C release, and cytochrome C released into the cytosol can mediate apoptosis by activating caspases. Bcl-2 family members can be divided into two major classes, differing in structure and function: family members that inhibit apoptosis (including Bcl-2, bcl-xl, etc.) and family members that promote apoptosis (including Bax, bak, bid, bad, etc.). The two substances are combined with each other and inhibited each other, and whether apoptosis occurs or not is often determined by the relative quantity of the two substances.
Disclosure of Invention
The invention aims to provide a culture method for mouse supporting cells.
The invention relates to a culture method for a mouse supporting cell, which comprises the following steps:
(1) Isolation of support cells: killing the mice, aseptically collecting testes at two sides, and placing the testes in a dish containing PBS; the whole testis collection process is completed within 30min; removing fat pad, epididymis and testis tunica albuginea of each testis, adding PBS, blowing, and blowing off the seminiferous tubules; adding PBS containing collagenase 0.5-1.5 g/L which is 8-12 times of tissue volume, and placing into an incubator at 37deg.C with 5% CO 2 Culturing for 10-15 min under the condition; then transferring into a centrifuge tube, blowing for 1-2 times, standing until the fine tube section of the yeast is settled, and sucking out the supernatant; repeating the above steps for 1 time, adding PBS containing digestive enzyme, placing into an incubator, and heating at 37deg.C and 5% CO 2 Culturing for 5-10 min under the condition, adding cell culture solution to terminate digestion, transferring into a centrifuge tube, centrifuging or standing, collecting precipitate, adding DMEM culture medium, blowing and mixing to obtain single cell suspension;
(2) Cell count: taking out the prepared single cell suspension, adding trypan blue, mixing, calculating the number and total number of living cells, dead cells and cell clusters by using a red cell counting plate, and regulating the cell density to 2-4 multiplied by 10 5 /mL；
(3) Isolation and purification of support cells: inoculating the single-cell suspension with the cell density adjusted in the step (2) into CO 2 Culturing in incubator, discarding non-adherent sperm cells together with culture medium, adding culture medium, culturing for 24 hr, discarding culture medium, adding Tris-HCl solution, hypotonic treating for 3min, washing with PBS for 3 times, adding fresh culture medium, and placing CO 2 At 37℃in an incubator with 5% CO 2 Culturing for 24-48 h under the conditions of 95% air and saturated humidity to obtain a support cell;
(4) Support cell culture: inoculating the support cells into a culture flask, and adding a culture solution to maintain the density of the support cells at 0.5-1.5X10 6 /ml, put in CO 2 In an incubator at 5% CO 2 Culturing in 95% air at 34 deg.c and saturated humidity, selecting 75-85% support cell for passage to cover glass-paved bottle, and culturing in CO 2 Culturing is carried out continuously in the incubator, and the support cells for the mice are obtained after culturing.
Further, the cell culture solution in the step (1) consists of 10% of NBS by mass and 1% of DMEM culture solution containing penicillin and streptomycin by mass, wherein the sum of the mass percentages of the penicillin and the streptomycin in the DMEM culture solution is 1%.
Further, in the step (1), the digestive enzyme is composed of 1.5g/L hyaluronidase and 0.25% trypsin by mass. .
Further, the trypan blue in the step (2) is 0.4% in mass percent.
Further, the concentration of Tris-HCl solution in step (3) was 20mmol.
Further, the cell culture liquid in the step (4) is prepared from 2-3 mL of DMEM culture liquid, 0.1-0.5 mL of serum and 10 -9 mol/L soybean isoflavone.
The invention has the following beneficial effects.
The method can separate and purify the mouse supporting cells, detect the release condition of cytochrome C and the expression change of Bcl-2 family proteins (Bax and Bcl-2) by a laser confocal microscope, discuss the molecular mechanism of the testis supporting cells regulated by environmental estrogens, and provide a research basis for the soybean isoflavone to promote the growth of the mouse supporting cells.
Drawings
FIG. 1 is a graph showing testis support cell culture for 12 hours;
FIG. 2 is a graph showing the average fluorescence optical density values of cytochrome C in the soybean isoflavone treated group; in the figure, 1.DMSO control, 2.10 -9 mol/L soybean isoflavone treatment group, 3.10 -7 mol/L soybean isoflavone treatment group, 4.10 -5 A mol/L soybean isoflavone treatment group;
FIG. 3 is a graph showing the average fluorescence optical density values of Bax in the soybean isoflavone treated group, in which 1.DMSO control, 2.10 - 9 mol/L soybean isoflavone treatment group, 3.10 -7 mol/L soybean isoflavone treatment group, 4.10 -5 A mol/L soybean isoflavone treatment group;
FIG. 4 is a graph of average fluorescence optical density values of soybean isoflavone treated Bcl-2; in the figure, 1.DMSO control, 2.10 -9 mol/L soybean isoflavone treatment group, 3.10 -7 mol/L soybean isoflavone treatment group, 4.10 -5 A mol/L soybean isoflavone treatment group;
FIG. 5 is a fluorescent expression pattern of soybean isoflavone group cytochrome C; in the figure, a green cytochrome C positive signal diagram, and B is a red mitochondrial positive signal; 1DMSO control group, 2.10 -9 mol/L soybean isoflavone treatment group, 3.10 -7 mol/L soybean isoflavone treatment group, 4.10 -5 A mol/L soybean isoflavone treatment group;
FIG. 6 is a fluorescence expression pattern of soybean isoflavone Bax; in the figure, A is a green Bax positive signal graph, and B is a red mitochondrial positive signal; 1DMSO control group, 2.10 -9 mol/L soybean isoflavone treatment group, 3.10 -7 mol/L soybean isoflavone treatment group, 4.10 -5 A mol/L soybean isoflavone treatment group;
FIG. 7 is a fluorescent expression pattern of soybean isoflavone Bcl-2; in the figure, A is a green Bcl-2 positive signal graph, and B is a red mitochondrial positive signal; 1DMSO control group, 2.10 -9 mol/L soybean isoflavone treatment group, 3.10 -7 mol/L soybean isoflavone treatment group, 4.10 -5 mol/L soybean isoflavone treatment group.
Detailed Description
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
For the purposes of clarity, technical solutions and advantages of embodiments of the present invention, the following detailed description will clearly illustrate the spirit of the present disclosure, and any person skilled in the art, after having knowledge of the embodiments of the present disclosure, may make alterations and modifications to the technology taught by the present disclosure without departing from the spirit and scope of the present disclosure.
The exemplary embodiments of the present invention and the descriptions thereof are intended to illustrate the present invention, but not to limit the present invention.
Example 1
The culture method for the mouse supporting cells of the present embodiment is performed according to the following:
(1) Preparing solution and sterilizing equipment reagent: 10 -9 mol/L、10 -7 mol/L、10 -5 The mol/L soybean isoflavone, the glass instrument used in the experiment requires to be sterilized for 2-4 hours at 180 ℃ in a dry heat sterilization box after being cleaned; the plastic article was steam sterilized at 121℃for 15 minutes at 15 lbs.
(2) Isolation of support cells: 12-15 male mice of 7-8 days are taken and killed by cervical dislocation. The testes on both sides were collected aseptically and placed in a small dish with PBS. The whole testis collection process was completed within 30 min. Carefully removing fat pad, epididymis and leucoma of testis by using pointed forceps, adding a proper amount of PBS (2-3 ml), and blowing off the seminiferous tubules by using a suction tube. Adding PBS containing 1g/L collagenase 10 times of tissue volume, placing into incubator, heating at 37deg.C, and adding 5% CO 2 The reaction is carried out for 15min (shaking for several times). Then transferring into 5ml centrifuge tube, gently blowing for 1-2 times, standing, gently sucking the supernatant after the fine tube segment of the yeast is settled, repeating the above stepsThe process was 1 time. Adding PBS containing 1.5g/L hyaluronidase and 0.25% trypsin, placing into an incubator, and allowing to act for 5-10 min under the same conditions, and standing under an inverted microscope to obtain soft powder of the fine tube segment of the yeast, wherein some of the soft powder is dissipated into single cells or small cell clusters. Adding fresh DMEM culture solution containing 10% NBS, 1% cyan and streptomycin, stopping digestion, transferring into a centrifuge tube, centrifuging at 1000r/min for 3min or standing for 5min, and gently sucking the supernatant after settling the soft scattered seminiferous tubules and dissociated spermatogenic cells. 1.5ml DMEM culture medium is added again, and the mixture is blown 8 to 10 times and blown several times to prepare single cell suspension.
(3) Cell count: taking out 1 drop of the prepared cell suspension, adding 10ul of 0.4% trypan blue, mixing, counting the number of living cells, dead cells, cell mass and total number of cells (cell mass connected by more than 2 cells without blowing is recorded only 1 time) by using a red blood cell counting plate, and adjusting the cell density to 3×10 5 /ml。
(4) Isolation and purification of support cells: on the testicular seminiferous tubule epithelium of 7-8 d mice, only 2 types of cells exist, namely type a spermatogonial stem cells and supporting cells. Inoculating the prepared single cell suspension into CO 2 Culturing in an incubator, according to the difference of the adherence speed of the supporting cells and the spermatogonia, using a selective adherence method, discarding the non-adherent spermatogonia along with the culture medium when the supporting cells start to adhere to polarization and the spermatogonia are still suspended (in vitro culture for about 6-7 h), adding the culture medium to continue culturing for 24 hours, discarding the culture medium, adding 3m L20 mmol Tris-HCl solution, performing hypotonic treatment for 3 minutes, flushing with PBS solution for 3 times, adding fresh culture medium, and placing CO 2 At 37℃in an incubator with 5% CO 2 Culturing for 24-48 hours under the conditions of 95% air and saturated humidity, and further purifying the support cells. The liquid is changed for 1 time every 2-3 days later.
(5) Support cell culture: the support cells were inoculated into 50ml flasks (3 ml/flask of broth: serum=9:1) and the final cell density was maintained at 1×10 6 /ml, put in CO 2 Culturing in incubator under 5% CO 2 95% air, 34 ℃ saturated humidity. Cell climbing tablet: selecting 80% growth supporting cells for passage to spreadIn bottle dish with cover glass, CO 2 Culturing in an incubator.
(6) The detection method comprises the following steps: and a laser confocal microscope is used for detecting the release condition of the cytochromes C and the expression change of Bcl-2 family proteins (Bax and Bcl-2), so that the influence of the soybean isoflavone on the supporting cells of the mice can be promoted by comprehensively evaluating the soybean isoflavone.
(7) Immunofluorescence cytochemistry procedure for cytochrome C: the treated cell climbing sheet is moved into a DMEM solution of 400n MMito Tracker Red and incubated for 30min at 37 ℃ in a dark place; transferring into DMEM liquid drops, and cleaning for 2 times at 37 ℃ in a dark place for 5min each time; transferring into 4% paraformaldehyde solution, and fixing at 37deg.C for 30min in dark place; washing the cleaning solution at 37 ℃ for 3 times in a dark place for 5min each time; incubating the membrane permeation solution for 20min in a dark place to increase the permeability of the cell membrane; the cleaning liquid is fully cleaned for 3 times in a dark place for 5min each time; blocking in 1% BSA in the dark for 1h to block the non-specific binding sites of the antibodies; and (3) moving into 1: incubation in 200 diluted murine cytochrome C primary antibody, protected from light at 4 ℃ overnight; washing the washing liquid for 3 times in a dark place to sufficiently remove unbound antibody, wherein each time is 5min; and (3) moving into 1:200 dilution of secondary antibody (FITC-labeled goat anti-mouse Ig G), incubated at room temperature for 40min in the dark; washing the washing liquid for 3 times in a dark place to remove unbound secondary antibodies, wherein each time is 5min; and (5) sealing the sheet, and observing under a laser confocal microscope.
(8) Immunofluorescence cytochemistry procedure for Bcl-2 family proteins: the treated cell climbing sheet is moved into DMEM liquid of 400n MMito Tracker Red to be incubated for 30min at 37 ℃ in a dark place; transferring into DMEM liquid drops, and cleaning for 2 times at 37 ℃ in a dark place for 5min each time; transferring into 4% paraformaldehyde liquid drop for fixing for 30min; washing with cleaning solution for 3 times, each time for 5min; incubating in the membrane permeation solution for 30min to increase the permeability of the cell membrane; cleaning with cleaning solution for 3 times, each time for 5min; blocking for 1h in 1% BSA to block non-specific binding sites of the antibody; and (3) moving into 1: incubation in 200 diluted primary antibodies (rabbit Bax, bcl-2 antibodies) overnight at 4 ℃; washing the solution for 3 times to sufficiently remove unbound antibody for 5min each time; and (3) moving into 1:200 dilution of secondary antibody (FITC labeled goat anti-rabbit Ig G), incubated at room temperature for 40min in the dark; washing the washing liquid for 3 times in a dark place to remove unbound secondary antibodies, wherein each time is 5min; and (5) sealing the sheet, and observing under a laser confocal microscope.
(9) Preparation of epidemic fluorescent cell chemical main solution: phosphate Buffered Saline (PBS) comprising KCl 0.2g, KH 2 PO 4 0.2g、Na Cl 8g、Na 2 HPO 4 1.1407g, pH 7.4, adding distilled water to 1000ml; the fixing solution comprises paraformaldehyde 0.4 g, distilled water 9ml and 10×PBS 1ml, and the pH value is adjusted to 7.4; the membrane permeation solution comprises Triton X100500 μl, mgCL.6H 2 O0.061 g, naCl 0.292g, hepes 0.477g, sucrose 10.269g and NaN 3 0.02g of PBS with the pH value adjusted to 7.4 is fixed to 100ml; the wash solution included 9ml of triple distilled water, 10. Mu.l of Tween20, 10. Mu.l of Triton X100, and 1ml of 10 XPBS; the blocking solution contained 1ml of the washing solution and 0.01g of Bovine Serum Albumin (BSA).
The detection results of this embodiment are as follows:
(1) Morphological observation and purity of support cells: the isolated cultured support cells were continuously observed using an inverted phase contrast microscope. The morphological characteristics of the support cell in-vitro culture are that after the support cell is cultured for 4 hours, the support cell starts to adhere, the support cell is observed under an inverted phase contrast microscope, the support cell becomes flat, cytoplasm is elongated, and a vacuole structure with different sizes is visible, and adjacent cells are connected in an interweaving way; cells enter the proliferation phase after 12 hours of culture, the cell gap becomes smaller, and the cell connection is very firm (as in FIG. 1, testis support cell culture for 12 hours, phase contrast microscope 200X).
(2) The purity of the separated supporting cells can reach more than 90% by adopting a combined enzyme digestion and selective adherence method and the statistical result of cell count (Table 1)
(3) Confocal microscopy showed that we detected cytochrome C green positive signal expression in both the control and each of the soy isoflavone treated groups (see figure 2). As can be seen from fig. 2, 10 -9 The mol/L soybean isoflavone treated group has stronger signal of cytochrome C compared with the control group, and the signal of cytochrome C gradually weakens along with the increase of the concentration of the soybean isoflavone. Indicating that low concentration of soy isoflavone promotes cellsProliferation.
FIG. 2 average fluorescence optical density values of cytochrome C in soybean isoflavone treated group:
dmso control 2.10 -9 mol/L soybean isoflavone treatment group 3.10 -7 mol/L soybean isoflavone treatment group 4.10 -5 mol/L soybean isoflavone treatment group
(4) Bcl-2 family protein expression profile: confocal microscopy showed that we detected Bax, bcl-2 green positive signal expression in both the control and each soy isoflavone treated group (see fig. 3, 4). As can be seen from fig. 3, at 10 -9 The signal of Bax in the mol/L soybean isoflavone treated group was weaker than that in the control group, and the signal of Bax was gradually increased as the concentration of soybean isoflavone was increased. As can be seen from fig. 4, at 10 -9 The Bcl-2 signal in the mol/L soybean isoflavone treated group was stronger than that in the control group, and the Bcl-2 signal was gradually decreased as the soybean isoflavone concentration was increased.
Claims (6)
1. A method for culturing a mouse supporting cell, comprising the steps of:
(1) Isolation of support cells: killing the mice, aseptically collecting testes at two sides, and placing the testes in a dish containing PBS; the whole testis collection process is completed within 30min; removing fat pad, epididymis and testis tunica albuginea of each testis, adding PBS, blowing, and blowing off the seminiferous tubules; adding PBS containing collagenase 0.5-1.5 g/L which is 8-12 times of tissue volume, and placing into an incubator at 37deg.C with 5% CO 2 Culturing for 10-15 min under the condition; then transferring into a centrifuge tube, blowing for 1-2 times, standing until the fine tube section of the yeast is settled, and sucking out the supernatant; repeating the above steps for 1 time, adding PBS containing digestive enzyme, placing into an incubator, and heating at 37deg.C and 5% CO 2 Culturing for 5-10 min under the condition, adding cell culture solution to terminate digestion, transferring into a centrifuge tube, centrifuging or standing, collecting precipitate, adding DMEM culture medium, blowing and mixing to obtain single cell suspension;
(2) Cell count: taking out the prepared single cell suspension, adding trypan blue, mixing, and counting the number of living cells, dead cells and cell clusters by using a red cell counting plateAnd total number of cells, and adjusting cell density to 2-4×10 5 /mL；
(3) Isolation and purification of support cells: inoculating the single-cell suspension with the cell density adjusted in the step (2) into CO 2 Culturing in incubator, discarding non-adherent sperm cells together with culture medium, adding culture medium, culturing for 24 hr, discarding culture medium, adding Tris-HCl solution, hypotonic treating for 3min, washing with PBS for 3 times, adding fresh culture medium, and placing CO 2 At 37℃in an incubator with 5% CO 2 Culturing for 24-48 h under the conditions of 95% air and saturated humidity to obtain a support cell;
(4) Support cell culture: inoculating the support cells into a culture flask, and adding a culture solution to maintain the density of the support cells at 0.5-1.5X10 6 /ml, put in CO 2 In an incubator at 5% CO 2 Culturing in 95% air at 34 deg.c and saturated humidity, selecting 75-85% support cell for passage to cover glass-paved bottle, and culturing in CO 2 Culturing is carried out continuously in the incubator, and the support cells for the mice are obtained after culturing.
2. The method according to claim 1, wherein the cell culture solution in step (1) is composed of 10% by mass of NBS, 1% by mass of a DMEM medium containing penicillin and streptomycin, and the sum of the mass percentages of penicillin and streptomycin in the DMEM medium is 1%.
3. The method according to claim 1, wherein in the step (1), the digestive enzyme is composed of hyaluronidase at a concentration of 1.5g/L and trypsin at a mass percentage of 0.25%. .
4. The method of claim 1, wherein the trypan blue content in step (2) is 0.4% by mass.
5. The method for culturing mouse supporting cells according to claim 1, wherein the concentration of Tris-HCl solution in step (3) is 20mmol.
6. The method according to claim 1, wherein the cell culture solution in the step (4) is composed of 2 to 3mL of DMEM culture solution, 0.1 to 0.5mL of serum and 10 -9 mol/L soybean isoflavone.
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| CN108384745A (en) * | 2018-01-26 | 2018-08-10 | 安徽科技学院 | A kind of method that improved two steps enzyme is separately cultured sustentacular cell of testis |
| CN114181891A (en) * | 2021-12-03 | 2022-03-15 | 中国农业大学 | Efficient culture method of mouse testicular organoid |
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| CN108384745A (en) * | 2018-01-26 | 2018-08-10 | 安徽科技学院 | A kind of method that improved two steps enzyme is separately cultured sustentacular cell of testis |
| CN114181891A (en) * | 2021-12-03 | 2022-03-15 | 中国农业大学 | Efficient culture method of mouse testicular organoid |
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