CN114672450B - Method for separating and purifying theca cells - Google Patents
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- CN114672450B CN114672450B CN202210091170.XA CN202210091170A CN114672450B CN 114672450 B CN114672450 B CN 114672450B CN 202210091170 A CN202210091170 A CN 202210091170A CN 114672450 B CN114672450 B CN 114672450B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a method for separating and purifying theca cells, belonging to the field of cell culture. The method comprises the following steps: collecting sterile follicular fluid discarded on an ovum taking day, and separating to obtain ovarian tissues; digesting the obtained ovarian tissue by using a PBS solution containing type I collagenase, collecting suspended cells, removing undigested theca tissue in the suspended cells by using a cell sieve, and collecting the ovarian cells; and (3) resuspending the obtained ovarian cells, centrifuging at a low speed for a short time, collecting precipitates to obtain primarily purified theca cells, then paving the primarily purified theca cells on an uncoated culture dish, and further purifying the theca cells to obtain the separated and purified theca cells. The method can overcome the defect of extracting the theca cells from the conventional ovarian tissues, is simple and easy to operate, can quickly purify to obtain the theca cells, provides a new thought and technical route for the sources of the theca cells, and brings a new breakthrough for the research bottlenecks of the theca cells and the ovarian tissues.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a method for separating and purifying theca cells.
Background
Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases of women of childbearing age, the incidence rate reaches 5-10%, and the main clinical manifestations are menstruation or amenorrhea, abnormal uterine bleeding, infertility, hirsutism and acne. The PCOS patients have gestational diabetes and gestational hypertension with high risk. Long-term complications of PCOS include insulin resistance, impaired glucose tolerance, type 2 diabetes, obesity, dyslipidemia, heart disease, hypertension, depression, sleep apnea syndrome, endometrial cancer, and the like. PCOS is harmful to the health of sick women for a long time, but a radical treatment method is not available due to unclear etiology and pathogenesis, and patients need lifelong follow-up and lifelong treatment. Androgen augmentation plays a key role in the pathogenesis of PCOS. Androgens are synthesized in women primarily in the follicular cells of the ovary. Therefore, the isolated culture of human follicular membrane cells is of great significance for studying the pathogenesis of PCOS.
The prior human theca cells are mainly obtained from human ovarian specimens in the gynecological operation process: first, the follicle is removed from the ovumCutting the nidus matrix; cutting the follicle in half in a culture medium, scraping granular cells in the follicle, and tearing off a follicle membrane layer; the follicular film layer was digested with 0.05% collagenase I,0.05% collagenase IA and 0.01% dnase; the digested theca cells are treated with a cell culture medium containing 0 in 5%O 2 ，5％CO 2 ，90％N 2 Culturing under the condition that a fibronectin coated culture dish is adopted. With the progress of medical technology, the protection of the ovarian function in the gynecological operation process of women is more and more emphasized, so the sources of ovarian specimens are more and more in short supply. This trend has led to slow progress in the research related to human follicular membrane cells.
Disclosure of Invention
The invention aims to provide a method for separating and purifying theca cells, which aims to solve the problems in the prior art, and provides a new thought and technical route for sources of the theca cells by separating and purifying the theca cells from the discarded follicular fluid in the ovum taking process, so that a new breakthrough is brought to the bottleneck of the research of the theca cells.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for separating and purifying theca cells, which comprises the following steps:
step 1: collecting sterile follicular fluid discarded on an egg taking day, and separating to obtain ovarian tissue;
step 2: digesting the obtained ovarian tissue by using a PBS solution containing type I collagenase, collecting suspended cells, removing undigested theca tissue in the suspended cells by using a cell sieve, and collecting the ovarian cells;
and step 3: and (3) after the ovarian cells are re-suspended, centrifuging at a low speed for a short time, and collecting precipitates to obtain primarily purified theca cells.
And 4, step 4: and (3) spreading the primarily purified theca cells on an uncoated culture dish, performing cell culture by adopting a DMEM/F12 culture medium containing antibiotics and fetal calf serum, and further purifying the theca cells to obtain the separated and purified theca cells.
Preferably, in step 1, the step of isolating and obtaining ovarian tissue specifically comprises the following steps: filtering the collected sterile follicular fluid by using a 100-micron cell sieve, flushing ovarian tissue by using a PBS (phosphate buffer solution) solution, preliminarily removing granular cells and granular cell masses, and collecting the ovarian tissue staying on the surface of the cell sieve.
Preferably, in step 2, the digestion treatment conditions are: digesting the ovarian tissue by PBS containing 5mg/mL type I collagenase for 90min at 37 ℃; wherein the volume ratio of the ovarian tissue to the PBS solution containing the type I collagenase is 1:10.
preferably, the cell sieve used in step 2 is 40 μm.
Preferably, in step 3, the low-speed short-time centrifugation is specifically: after the ovarian cells are resuspended by 10 percent Percoll solution, centrifuging for 30s at 100g, collecting 1/4 layer precipitated cells close to the bottom, resuspending in PBS solution, after transferring the upper layer liquid, blowing, centrifuging for 30s at 100g, discarding the upper layer liquid, taking 1/4 layer precipitated cells close to the bottom, resuspending in PBS solution, combining the precipitates collected twice, cleaning with PBS, and resuspending for later use.
Preferably, in step 4, the uncoated culture dish is used for culturing the cells, and the culture solution is changed at 24h to remove the granular cells with slow adherence.
Preferably, in step 4, the cells are cultured in DMEM/F12 medium containing 10% fetal bovine serum, 100IU/mL penicillin and 100. Mu.g/mL streptomycin.
The invention also provides application of the theca cells separated and purified by the method in preparation of an ovarian specimen.
The invention discloses the following technical effects:
the invention extracts the theca cells from the follicular fluid obtained in the ovum taking process, overcomes the defects of the traditional method for extracting the theca cells from the ovarian tissues, and has the main difficulty that the theca cells are mixed with a plurality of granular cells and the size and the quality of the two kinds of cells are relatively close. The invention optimizes the separation and purification conditions, and removes granular cells and granular cell clusters through preliminary filtration of a 100-micron cell sieve; by using gradient density centrifugate, centrifuging at low speed for a short time, collecting quickly sinking theca cells, and purifying the theca cells; the method comprises the steps of culturing cells by using a culture dish which is not pre-coated, and removing granular cells with slow adherence by methods such as changing liquid at 24h of culture, thereby achieving the purpose of further purifying the theca cells.
Immunohistochemical analysis is carried out on the theca cells obtained by the method, and the CYP17A1 specifically expressed by the theca cells in the ovarian tissues separated from the follicular fluid obtained in the ovum taking operation process is found to be positive; the immunofluorescence method detects that 97.9 +/-3.6% of the separated cells express CYP17A1, and the expression of the granulosa cell specific marker FSHR is not detected; the real-time quantitative fluorescent PCR method shows that CYP17A1 mRNA can be detected in the separated cells; it was found by chemiluminescence analysis that androstenedione (3.43. + -. 1.76 pmol/10) was detectable in the culture medium of the isolated cells 5 Cells), testosterone (2.50. + -. 1.65ng/10 5 A cell). The method can obtain the theca cells with high purification degree, can be used for related research of ovarian tissues, provides a new thought and technical route for sources of the theca cells, and brings a new breakthrough for bottleneck of the research of the theca cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a graph showing immunohistochemical detection of ovarian tissue isolated and collected from follicular fluid according to the present invention; a: immunohistochemical staining detection of the positive of the marker of the follicular membrane cells, androgen synthesis key enzyme CYP17A1, B: normal rabbit IgG was used as a negative control for immunohistochemical staining;
FIG. 2 is the observed white light of the microscope of the present invention for culturing the oocyst membrane cells which have been grown adherent in vitro;
FIG. 3 shows the immunofluorescence assay results of isolated theca cells of the present invention; respectively detecting a follicular membrane cell marker CYP17A1 and a granular cell marker FSHR (follicle stimulating hormone receptor) by an immunofluorescence method, wherein a negative control adopts PBS; the length of the ruler is 100 mu m;
FIG. 4 shows that CYP17A1 mRNA (3 duplicate wells) was detected in the isolated cells, and the reference gene GAPDH (2 duplicate wells) was detected by the quantitative fluorescent PCR assay of the present invention.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
Example 1
(1) Isolating ovarian tissue, and removing granulosa cells by primary filtration
Collecting the waste follicular fluid with sterile container, and standing for more than 30 min.
Sucking the sediment at the bottom layer of the follicular fluid, placing the sediment in a 50mL sterile centrifugal tube, centrifuging for 5min at 500g, discarding the supernatant, adding a proper amount of PBS into the centrifugal tube, and blowing and beating the tube to form a suspension.
Placing a sterilized 100-micron cell sieve in a 6cm culture dish, sucking the suspension into the cell sieve by a suction pipe in a grading manner, and finally sucking PBS (phosphate buffer solution) to repeatedly purge ovarian tissues in the cell sieve, so as to primarily filter and remove granular cells and granular cell masses.
(2) Isolation of ovarian cells
Transferring the ovarian tissues on the cell sieve into a 15mL sterile centrifugal tube by using an ophthalmic forceps, adding PBS (phosphate buffer solution) containing 5mg/mL type I collagen protease prepared in advance, wherein the volume ratio of the ovarian tissues to the PBS containing the type I collagen protease is 1:10, digestion at 37 ℃ for 90min.
Blowing PBS solution containing ovary tissue and type I collagenase every 15min to promote digestion. After pipetting, the mixture was allowed to stand for 1min, and the upper cell suspension was aspirated by a pipette and transferred to a new 15mL sterile centrifuge tube. PBS containing 5mg/mL collagenase I was added to the tubes containing the undigested ovarian tissue.
The cell suspension was centrifuged at 500g for 5min, the supernatant was discarded, PBS was added to resuspend the cells, and the process was repeated twice.
Placing a sterilized 40-micron cell sieve in a 6cm culture dish, sucking the cell suspension into the cell sieve by using a suction tube, sucking the sieved cell suspension, placing the cell suspension in a new 15mL sterile centrifuge tube, centrifuging for 5min at 500g, removing supernatant, and collecting ovarian cells.
(3) Further purifying the theca cells
Ovarian cells were resuspended in 10% Percoll solution (200. Mu.L 100%Percoll + 1800. Mu.L PBS), centrifuged at 100g for 30 seconds, the lower 1/4 layer of the pelleted cells collected was resuspended in PBS, the upper layer was transferred to a new centrifuge tube, after pipetting, centrifuged at 100g for 30 seconds, the upper layer was discarded, and the lower 1/4 layer of pelleted cells collected was resuspended in PBS. The twice pelleted cells were washed twice with PBS and resuspended in PBS for further characterization and culture.
(4) Culture of thecal cells
Adding DMEM/F12 culture solution containing 10% fetal bovine serum, 100IU/mL penicillin and 100. Mu.g/mL streptomycin to the pellet, counting with a cell counting plate, adjusting cell density according to experiment requirements, seeding the cells on non-precoated culture dishes, and adjusting the content of 5% CO at 37% 2 And (5) culturing in a humidifying incubator. The medium was changed 24h after the first culture to remove nonadherent cells. After that, the solution was changed every 24 hours.
(5) Identification of thecal cells
(1) And (3) dehydrating, embedding paraffin, and slicing the separated ovarian tissue, and detecting an alveolar membrane cell specific marker, namely androgen synthesis key enzyme CYP17A1 by an immunohistochemical method. Reference Harpelund Poulsen K et al Dysregistration of FGFR signaling by a selective inhibitor recovery cell survivals in human total carbohydrates of booth segments and alcohols the synthetic amino in total repeat 34.
As shown in FIG. 1, it was found by immunohistochemical analysis that CYP17A1 expressed specifically in theca cells in ovarian tissue isolated from follicular fluid obtained during the ovariectomy procedure was positive in the present invention.
(2) The number of theca cells is 1 × 10 5 Density of/mL on sterile cover slips and placed in 24-well culture dishes, 1mL of DMEM/F12 medium containing 10% fetal bovine serum, 100IU/mL penicillin and 100. Mu.g/mL streptomycin was added, and the content of 5% CO was determined at 37% 2 Culturing in a humidified incubator for 48 hr, detecting the expression of the follicular membrane cell marker CYP17A1 and the granulosa cell marker FSHR (follicle stimulating hormone receptor) by immunofluorescence method, and referring toThe literature Xu D et al SIRT2 plants a novel roll on progresterone, estroplastic and testesterone synthesis via PPARs/LXR pathways in a bone overview granular cells J Steroid Biochem Mol Biol 185 (2019).
As shown in fig. 2, the oocyst membrane cells cultured in vitro were observed under a microscope under white light; as shown in FIG. 3, 97.9. + -. 3.6% of the isolated cells were found to express CYP17A1 by immunofluorescence, and no expression of the granulosa cell-specific marker FSHR was detected. The length of the scale is 100 μm.
(3) After 48 hours of in vitro culture of the oocyst membrane cells, the cells were replaced with serum-free medium (DMEM/F12 medium containing 100IU/mL penicillin and 100. Mu.g/mL streptomycin), and cultured for 24 hours, and then subjected to real-time quantitative fluorescent PCR experiments using the GeneJET RNA Purification Kit (Thermo Fisher Scientific, USA), revertAID First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA), taqman Gene Expression Assays (Thermo Fisher Scientific, USA) kits to detect the Expression of CYP17A1 in the cells, and the levels of Testosterone and Androstenedione in the culture using the immunochemiluminometry Kit Testosterone Assay Kit (R & D Systems, USA), respectively.
As shown in FIG. 4, it was found that CYP17A1 mRNA was detectable in the isolated cells by the quantitative fluorescent PCR analysis. It was found by chemiluminescence analysis that androstenedione (3.43. + -. 1.76 pmol/10) could be detected in the culture medium of the isolated test cells 5 Cells), testosterone (2.50. + -. 1.65ng/10 5 A cell).
The invention extracts the theca cells from the follicular fluid obtained in the ovum taking process, and the main difficulty is that a plurality of granular cells are mixed in the theca cells, and the size and the quality of the two cells are relatively close. The invention (1) uses a 100 mu m cell sieve to filter and remove most granular cells and granular cell clusters, (2) uses gradient density centrifugate to carry out low-speed short-time centrifugation and collect quickly sinking theca cells, (3) adopts a culture dish without pre-coating to carry out cell culture, and (4) changes the liquid at the 24 th hour of the primary culture to remove the granular cells with slow adherent growth, thereby achieving the purpose of purifying the theca cells. The invention extracts the theca cells from the follicular fluid discarded in the ovum taking operation, thereby greatly expanding the sources of the theca cells and breaking through the related research bottleneck of the theca cells.
On the basis of the prior art, the invention simplifies the conditions of tissue digestion and cell culture, reduces the steps of liquid preparation, improves the efficiency of separation and culture of the theca cells, saves the experimental cost and achieves good cell culture effect (as shown in figure 2): (1) The components of digestive enzymes are simplified, 3 digestive enzymes such as collagenase I, collagenase IA, deoxyribonuclease and the like are used for digesting follicular membrane tissues in the prior art, and only collagen I protease is used for digesting ovarian tissues, so that a good digestion effect is achieved; (2) The components of the culture medium are simplified, the culture medium of the theca of the prior art comprises 8 components (fetal bovine serum, horse serum, ulrosER G (serum substitute), insulin, selenium, vitamin E, antibiotics and DMEM/F12 culture medium), the culture medium adopted by the invention only contains fetal bovine serum, antibiotics and DMEM/F12 culture medium, the theca cell grows well adherent to the wall, and the function of secreting androgen is still kept; (3) In the previous research, a culture dish pre-coated with fibronectin is used for carrying out the culture of the theca cells, and the culture dish which is not pre-coated is used for achieving a good cell culture effect and further effectively removing the granulocytes with slow adherence.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (5)
1. A method for separating and purifying theca cells is characterized by comprising the following steps:
step 1: collecting sterile follicular fluid discarded on an ovum taking day, and separating to obtain ovarian tissues;
step 2: digesting the obtained ovarian tissue by using a PBS solution containing type I collagenase, collecting suspended cells, removing undigested theca tissue in the suspended cells by using a cell sieve, and collecting the ovarian cells;
and step 3: after the obtained ovarian cells are resuspended, centrifuging at a low speed for a short time, and collecting precipitates to obtain primarily purified theca cells;
and 4, step 4: the primarily purified theca cells are laid on an uncoated culture dish, a DMEM/F12 culture medium containing antibiotics and fetal calf serum is adopted for cell culture, the theca cells are further purified, and the separated and purified theca cells are obtained;
in the step 1, the separation and acquisition of the ovarian tissue specifically comprises the following steps: filtering the collected sterile follicular fluid by using a 100-micron cell sieve, flushing ovarian tissues by using a PBS (phosphate buffer solution) solution, preliminarily removing granular cells and granular cell masses, and collecting the ovarian tissues staying on the surface of the cell sieve;
in step 2, the digestion conditions are as follows: digesting the ovarian tissue for 90min at 37 ℃ by using PBS containing 5mg/mL type I collagenase; wherein the volume ratio of the ovarian tissue to the PBS solution containing type I collagenase is 1:10;
in the step 3, the low-speed short-time centrifugation is specifically as follows: after the ovarian cells are resuspended by 10 percent Percoll liquid, centrifuging for 30s at 100g, collecting 1/4 layer of precipitated cells close to the bottom to be resuspended in PBS solution, after transferring the upper layer liquid, blowing and beating, centrifuging for 30s at 100g, discarding the upper layer liquid, taking 1/4 layer of precipitated cells close to the bottom to be resuspended in PBS solution, combining the precipitates collected twice, cleaning with PBS, and resuspending for later use.
2. The method for separating and purifying theca cells as claimed in claim 1, wherein the cell sieve used in the step 2 is 40 μm.
3. The method for separating and purifying the theca cells as claimed in claim 1, wherein in the step 4, the uncoated culture dish is used for culturing the cells, and the culture medium is changed at the 24 th hour to remove the granular cells with slower adherence.
4. The method for separating and purifying theca cells as claimed in claim 1, wherein in the step 4, the cell culture is performed by using DMEM/F12 culture solution containing 10% fetal bovine serum, 100IU/mL penicillin and 100. Mu.g/mL streptomycin.
5. Use of the isolated and purified theca cells of any one of claims 1-4 in the preparation of an ovarian specimen.
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