CN107034177B - Bovine vacuolar membrane cell in-vitro culture regulator and application thereof - Google Patents
Bovine vacuolar membrane cell in-vitro culture regulator and application thereof Download PDFInfo
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- C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
Abstract
The invention provides a regulator for in-vitro culture of bovine theca cells, which takes in-vitro culture solution of the bovine theca cells as a matrix, and the matrix contains melatonin. When the bovine theca cells are cultured by using the in-vitro culture solution added with the melatonin, the melatonin can inhibit the synthesis of progesterone of the bovine theca cells and inhibit StAR and CASP3 expression induced by LH + IGF1, but has no influence on CYP11A1 and CYP17A1 expression. However, melatonin can promote the proliferation of bovine theca cells, and as an endocrine regulatory factor of bovine ovarian function, melatonin can inhibit the effects of LH and IGF1 by down-regulating the synthesis of steroid hormones, thereby delaying the differentiation of the membrane cells. The melatonin is applied to a preparation for promoting the membrane cell proliferation or inhibiting the synthesis of steroids of bovine theca cells, and has good application value.
Description
Technical Field
The invention relates to the technical field of livestock gamete embryo engineering, in particular to a bovine vacuolar membrane cell in-vitro regulator and application thereof.
Background
The theca cell, one of the 3 major cells that make up the follicle in the ovary, plays a role in various functions of the ovary, such as steroid synthesis, follicular development, and follicular atresia. The theca cells originate in the ovary as fibroblast-like stromal cells, which secrete androgens, interact with Follicle Stimulating Hormone (FSH) to promote granulosa cell proliferation and follicle development.
Melatonin, N-acetyl-5 Methoxytryptamine (MT), is secreted mainly from the pineal gland, and has diverse biological functions including regulation of biological cycle rhythms, sleep function, reproduction, antioxidant stress function, and the like. It has been shown that in mammals, melatonin affects sexual development and reproductive function by binding to binding sites on the hypothalamic-pituitary-gonadal axis and activating its receptors (Clemens et al, Life Sciences, 2001, 69: 27-35; Hu et al, General and Comparative Endocrinology, 2017, 242: 101-. In male animals, melatonin can influence reproductive function in both seasonal and non-seasonal reproductive animals by regulating secretion of GnRH, LH, testosterone synthesis and maturation of the testes (Tian et al, Journal of Pineal Research, 2014, 57: 239-. Melatonin is capable of promoting the development of ovine, porcine, bovine, mouse and human oocytes and embryos (Mayo et al, cellular and Molecular Life Sciences, 2002, 59: 1706-1713; He et al, Animal Reproduction Science, 2016, 172: 164-172), on the other hand, melatonin exhibits very strong antioxidant and anti-apoptotic capabilities and can be added to a culture medium to increase the fertilization rate and early embryo development rate of oocytes cultured in vitro (Ishizuka et al, Journal of Pineal Research, 2000, 28: 48-51; Voiculescu et al, Journal of Medicine and Life, 2014, 7: 488-492).
Melatonin has been studied less in The synthesis of Steroid hormones in ovarian cells, and in rat granulosa cells, melatonin promotes progesterone synthesis (Fiske et al, Endocrinology, 1984, 114: 407) and has been studied to show no effect on progesterone synthesis (Nakamura et al, The Journal of Steroid Biochemistry and Molecular Biology, 2014, 143: 233) 239. The effect of melatonin on membrane cell proliferation is not reported in the literature. Recently, melatonin was considered a growth factor in human endometrium (Arjmand et al, Molecular Biotechnology, 2016, 58: 684-694). At present, no literature reports the influence of melatonin on bovine theca cell gene expression, and only reports indicate that melatonin suppresses synthesis of androgen stimulated by immature testicular mesenchymal cells LH, due to down-regulation of CYP11A1 and CYP17A1 genes (Qin et al, regenerative Biomedicine one, 2015, 31: 638-. Melatonin is capable of promoting the synthesis of granulocytic steroid hormones (Webley and Luck, Journal of Reproduction and Fertility, 1986, 78: 711-. To date, there have been no reports of the effects of melatonin on bovine theca cells.
Disclosure of Invention
The invention aims to provide a regulator for in-vitro culture of bovine theca cells.
The invention also aims to provide a method for promoting the in vitro culture and proliferation of bovine theca cells.
The invention further aims to provide application of the melatonin in inhibiting synthesis of bovine theca cell steroid hormone and application of the melatonin in preparing medicaments for promoting proliferation of bovine theca cell somatotheca cells/inhibiting synthesis of the membrane cell steroid hormone.
An in-vitro culture regulator for bovine theca cells takes in-vitro culture solution of bovine theca cells as a matrix, and the matrix contains melatonin.
The regulator for in vitro culture of bovine theca cells as described above preferably has a melatonin concentration of 0.023 μ g/mL to 2.3 μ g/mL, more preferably has a melatonin concentration of 0.23 μ g/mL to 2.3 μ g/mL.
The regulator for in vitro culture of bovine theca cells is preferably a culture medium prepared by mixing Ham's F-12 and DMEM at a volume ratio of 1:1, wherein the culture medium contains 10% by volume of fetal bovine serum, 2.0mmol/L glutamine, 0.12mmol/L gentamicin and 38.5mmol/L sodium bicarbonate, and 30ng/ml Luteinizing Hormone (LH).
The regulator for in vitro culture of bovine theca cells is preferably a culture medium prepared by mixing Ham's F-12 and DMEM at a volume ratio of 1:1, wherein the culture medium contains 10% by volume of fetal bovine serum, 2.0mmol/L glutamine, 0.12mmol/L gentamicin and 38.5mmol/L sodium bicarbonate, and 30ng/ml Luteinizing Hormone (LH) and 30ng/ml insulin-like growth factor (IGF 1).
The regulator for in vitro culture of bovine theca cells is preferably a culture medium prepared by mixing Ham's F-12 and DMEM at a volume ratio of 1:1, wherein the culture medium contains 10% by volume of fetal bovine serum, glutamine at a concentration of 2.0mmol/L, gentamicin at a concentration of 0.12mmol/L and sodium bicarbonate at a concentration of 38.5mmol/L, and insulin-like growth factor I (IGF1) at a concentration of 30 ng/ml.
A method for promoting the in vitro culture proliferation of bovine theca cells is to culture the bovine theca cells in the in vitro culture regulator of the bovine theca cells.
Preferably, the bovine theca cells are isolated from large follicles of 8-22 mm.
Further, the invention provides application of the melatonin in promoting bovine theca cell proliferation.
Further, the invention provides the application of the melatonin in inhibiting the synthesis of bovine theca cell steroid hormone.
Furthermore, the invention provides the application of the melatonin in preparing synthetic drugs for promoting the in vitro proliferation of bovine theca cells.
Further, the invention provides an application of the melatonin in preparing a medicament for inhibiting the synthesis of bovine theca cell steroid hormone.
Further, the steroid hormone is progesterone.
The invention has the beneficial effects that:
in the invention, when bovine theca cells are cultured in vitro, melatonin has no influence on synthesis of progesterone of the membrane cells under induction of Luteinizing Hormone (LH), the Luteinizing Hormone (LH) has a cell proliferation effect alone, the melatonin can stimulate bovine membrane cells to proliferate in a dose-dependent manner under the condition of auxiliary existence of IGF1, and IGF1 can increase sensitivity of the membrane cells to the melatonin. Melatonin can be induced by LH + insulin-like growth factor (IGF1) to inhibit the synthesis of steroid hormones such as progesterone in membrane cells in a dose-dependent manner. Melatonin suppresses LH + IGF 1-induced expression of StAR and CASP3, but has no effect on CYP11a1 and CYP17a1 expression. However, melatonin can promote bovine follicular membrane cell proliferation, and as an endocrine regulator of bovine ovarian function, melatonin can inhibit the effects of LH and IGF1 by down-regulating steroid hormone synthesis, thereby delaying membrane cell differentiation.
The melatonin is applied to a preparation for promoting the membrane cell proliferation or inhibiting the synthesis of steroids of bovine theca cells, and has good application value. The melatonin can be used as a novel pharmaceutical preparation for promoting the membrane cell proliferation of the bovine theca cells and popularized and applied to the livestock embryo engineering.
Drawings
FIG. 1 is a graph comparing the proliferation of membrane cells with different concentrations of melatonin in the presence of LH with the addition of LH and IGF 1.
Fig. 2 is a graph comparing the synthesis of progesterone with varying concentrations of melatonin in the presence of LH and IGF 1.
FIG. 3 is a graph showing the expression of membrane cell StAR genes in the absence and addition of melatonin when LH and IGF1 were added to all cells.
FIG. 4 is a graph showing the expression of CASP3 gene in membrane cells treated with no addition of melatonin, when LH and IGF1 were added to the cells in total.
FIG. 5 is a graph showing the expression results of CYP11A1 gene in membrane cells without addition of melatonin treatment when LH and IGF1 were added to all cells.
FIG. 6 is a graph showing the expression of CYP17A1 gene in membrane cells without addition of melatonin treatment when LH and IGF1 were added to all of them.
Wherein, the different lower case letters in the figure indicate that there is a difference (P <0.05)
Detailed Description
The inventor has found through extensive research that in the presence of IGF1, melatonin can inhibit progesterone synthesis in a dose-dependent manner and can stimulate bovine follicular membrane cell proliferation, the stimulation capacity of the melatonin on the cell proliferation is limited, and IGF1 can increase the sensitivity of membrane cells to the melatonin. Melatonin significantly inhibits the synthesis of progesterone by membrane cells. The study also found that melatonin treatment of bovine membranous cells down-regulates CASP3 expression. Melatonin has effects in inhibiting synthesis of membrane cell steroid hormone and resisting apoptosis; the melatonin can regulate the functions of bovine ovarian membrane cells, inhibit the synthesis of steroid hormones by down-regulating the expression of a steroid synthesis related enzyme gene (StAR), and inhibit apoptosis by down-regulating the expression of an apoptosis related gene (CASP3), thereby stimulating the proliferation of the membrane cells. Understanding the mechanism of action of melatonin helps to decipher how this anti-differentiation factor regulates normal and abnormal ovarian function (e.g., follicular cysts).
The following description of the present invention is provided in connection with specific examples and should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The sources of reagents and hormones used may be as follows: ham's F-12(F12), DMEM medium, gentamicin, sodium bicarbonate, trypan blue, protease, collagenase, hyaluronidase, deoxyribonuclease, LH from Sigma (beijing), Fetal Bovine Serum (FBS) from Gibco (shanghai), recombinant human IGF-1 from Sciencell (San Diego, CA).
ELISA determination reagent: bovine progesterone (BIM, San Francisco, CA).
RNA detection reagent: one-chain synthesis kit, fluorescence quantitative kit (Tiangen Biochemical technology Co., Ltd., Beijing).
Example 1 Effect of melatonin on the Synthesis of bovine blastocyst cell steroids
1. Cell culture
The ovaries of non-pregnant cows were collected from a large slaughterhouse in the suburb of Beijing, collected, stored in 0.9% physiological saline containing 1% streptomycin, and transported on ice to a laboratory within 1 hour. The membrane cells are separated from large follicles with the diameter of 8-22 mm, and the follicular blood vessels are clear and follicular fluid is transparent. After the large follicle is extracted with follicular fluid, the large follicle is longitudinally incised with a scalpel, and the remaining granulosa cells are scraped off by a blunt separation method. Membrane cells were isolated by microdissection and digested for 1 hour at 37 ℃ in a shaker. Undigested membrane cells were filtered through a 149 μm filter. The membrane cells were centrifuged at 50g for 7 min at 4 ℃, washed 2 times with medium (DMEM and F12 in a volume ratio of 1:1 containing 2.0mmol/L glutamine, 0.12mmol/L gentamicin and 38.5mmol/L sodium bicarbonate) and resuspended in serum free medium (containing 1.25mg/ml collagenase and 0.5mg/ml DNase) to prevent cell aggregation prior to plating. The purity of the membrane cells should be greater than 90%.
The viability of the membrane cells was evaluated by trypan blue method and averaged 92%. About 4.0X 105One cell/well, 2ml of medium containing 10% by volume Fetal Bovine Serum (FBS) in 5% CO2And cultured under 95% air condition for 48 hours, and membrane cells were obtained after washing 2 times with 1ml of serum-free medium per well.
Treatment of bovine megavesicular membrane cells with IGF1 and melatonin
After culturing the membrane cells obtained in step 1 in a medium containing 10% FBS by volume (DMEM and F12 containing 2.0mmol/L glutamine, 0.12mmol/L gentamicin and 38.5mmol/L sodium bicarbonate at a volume ratio of 1: 1) for 48 hours, the membrane cells were treated with 0, 0.023 (0.1. mu. mol/L), 0.23 (1.0. mu. mol/L) or 2.3. mu.g/ml (10. mu. mol/L) of 10% FBS-containing medium containing melatonin for 48 hours, with or without 30ng/ml of IGF 1. The medium was changed every 24 hours. After the culture was completed, the medium was collected for measurement of progesterone content, and cells were collected for cell counting. Since progesterone synthesis cannot be induced by IGF1 in the absence of LH, all treatments contained 30ng/ml LH. This experiment was designed for 3 different replicates. Each replicate membrane cell was derived from 5-7 large follicles on 4-6 bovine ovaries.
3. Detection method
Progesterone was assayed using the method recommended by the ELISA kit (BIM) 2 times per sample. The coefficient of variation within the assay batch was 9.6% and 8.7%, respectively.
TZ20 for cell countingTMCounter (BIO-RAD, Shanghai, China). The details are as follows: wash 2 times with 0.5ml PBS before trypsinization (0.25%, 0.5ml), digest for 5 minutes, aspirate 10 μ l after pipetting.
4. The result of the detection
Cell counts and progesterone hormone secretion were analyzed by 2 x 4 factor ANOVA in SPSS software. The different means were compared by Tukey and the data are presented as means. + -. standard error. The results showed that melatonin treated alone, in amounts of 0.23. mu.g/ml and 2.3. mu.g/ml, stimulated the proliferation of membrane cells 1.12-fold and 1.13-fold (P)<0.05), but melatonin had no effect on cell proliferation when added at 0.023 μ g/ml. When the addition amount of melatonin is 0.023 mu g/ml, 0.23 mu g/ml and 2.3 mu g/ml in the presence of IGF1, the cell number can be increased by 1.12, 1.16 and 1.14 times (P)<0.05). The results are shown in FIG. 1. Progesterone is expressed as pM/105cells. The results of the measurement of progesterone content are shown in FIG. 2, which illustrates that melatonin dose-dependently decreases LH and IGF1 induced progesterone synthesis, e.g., 0.23 μ g/ml and 2.3 μ g/ml melatonin inhibits progesterone synthesis by 25.3% and 32.2% (P)<0.05). Melatonin had no effect on progesterone production in the absence of IGF1 in the presence of LH (P)>0.05)。
Example 2 Effect of melatonin on CYP11A1, CYP17A1, CASP3 and StAR mRNA expression
1. Cell culture
Membrane cells obtained by cell culture as in example 1 were cultured in a medium containing 10% FBS for 24 hours, and then treated with 3 treatments: controls, IGF1(30ng/ml) and melatonin (2.3. mu.g/ml) + IGF1(30ng/ml), all treatments included 30ng/ml LH. After the 24-hour culture, the medium was aspirated and the cells lysed for RNA extraction. The experiment was designed for 3 different replicates. Each replicate membrane cell was derived from 5-7 large follicles on 4-6 bovine ovaries.
RNA extraction and RT-PCR quantitative determination of expression level
After the culture is finished, sucking the culture medium in each hole; 0.5ml of TRNzol reagent (Tiangen Biochemical technology Co., Ltd., Beijing) was added to the wells, and RNA was extracted. Each treatment included 2 RNA samples. RNA samples were measured for absorbance at 260nm using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) and the RNA samples were diluted with DEPC water and stored at-80 ℃.
CYP11A1, CYP17A1, and StAR gene primers are described in published literature (Spicer et al, Biol logy of Reproduction, 2008, 78: 243-. The primer sequence of the Caspase 3(CASP3) gene is as follows: an upstream primer: CTTCCACGAAAATACTGGCATG, downstream primer: TGAA TGTTTCCCTGAGGTTTGC, probe: TCGATCTGGTACAGACGTG are provided. For all RT-PCR, a control without template and without reverse transcription was set to ensure that the PCR reaction was free of DNA or other contamination. After the PCR reaction, the size of the target gene fragment was determined by agarose gel. The expression of the target gene was normalized with the housekeeping gene 18S ribosomal RNA (Spicer et al, B biology of Reproduction, 2008, 78: 243--△△CTAnd (4) calculating.
3. The result of the detection
Gene expression was analyzed by ANOVA in SPSS software, the different averages were compared by Tukey's method, and data are expressed as mean. + -. standard error.
The results are shown in FIGS. 3-6, in which StAR gene encodes steroid hormone acute regulatory protein, which acts on the outer mitochondrial membrane, mediates and promotes the transport of cholesterol, a substrate of steroid, to the inner membrane via the outer mitochondrial membrane, and gradually synthesizes steroid hormone under the action of cholesterol side chain lyase P450scc and the like. The CYP11A1 gene codes P450scc, is the first step in the process of catalyzing estrogen synthesis, namely converting cholesterol into pregnenestrone, and is a key rate-limiting enzyme for androgen and estrogen biosynthesis. The CYP17A1 gene encodes cytochrome enzyme P450c17, which converts pregnenestrone and progesterone into 17-hydroxypregnenestrone and progesterone. StAR, CYP11A1 and CYP17A1 are rate-limiting enzymes in the synthesis of progesterone. Caspase 3 expressed by CASP3 gene is a major downstream effector of apoptosis and a marker of granular apoptosis.
The results indicate that LH and IGF1 treated membrane cells, CYP11a1 and CYP17a1 gene expression were not affected by melatonin. However, 2.3 μ g/ml melatonin inhibited StAR and CASP3 gene expression (P < 0.05). Therefore, the melatonin can regulate the functions of bovine ovarian membrane cells, inhibit the synthesis of steroid hormones by down-regulating the expression of a steroid synthesis related enzyme gene (StAR), inhibit apoptosis by down-regulating the expression of an apoptosis related gene (CASP3), and further stimulate the proliferation of the membrane cells.
Claims (6)
1. Application of melatonin in promoting bovine theca cell proliferation is disclosed, wherein the melatonin is used for down-regulating expression of apoptosis related gene CASP3 to inhibit apoptosis so as to stimulate the membrane cell proliferation.
2. The use according to claim 1, wherein the in vitro culture medium for bovine theca cell proliferation comprises melatonin.
3. The use of claim 1, wherein the melatonin concentration is from 0.023 μ g/mL to 2.3 μ g/mL.
4. Use according to claim 1 or 2, wherein the melatonin concentration is from 0.23 μ g/ml to 2.3 μ g/ml.
5. The use according to claim 2, wherein the in vitro culture medium is a culture medium of Ham's F-12 and DMEM mixed at a volume ratio of 1:1, wherein the culture medium comprises 10% by volume fetal bovine serum, 2.0mmol/L glutamine, 0.12mmol/L gentamicin and 38.5mmol/L sodium bicarbonate, and 30ng/ml luteinizing hormone and 30ng/ml insulin-like-one growth factor.
6. The use of claim 1, wherein the bovine theca cells are isolated from large follicles ranging from 8 to 22 mm.
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