CN114015642A - Bovine follicular granulosa cell in-vitro culture regulator and application thereof - Google Patents
Bovine follicular granulosa cell in-vitro culture regulator and application thereof Download PDFInfo
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/85—Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
- C12N2501/855—Corticotropin [ACTH]
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Abstract
The invention provides a regulator for in vitro culture of bovine follicular granulosa cells and application thereof, wherein the regulator contains adrenocorticotropic hormone (ACTH). When the bovine follicular granular cells are cultured by using the in-vitro culture solution added with the adrenocorticotropic hormone, the adrenocorticotropic hormone can promote the synthesis of progesterone of the bovine follicular granular cells, the adrenocorticotropic hormone can down-regulate the expression of a P450arom gene and up-regulate the expression of a P450scc gene, but the adrenocorticotropic hormone has no influence on the expression of a StAR gene and a 3 beta-HSD gene. Adrenocorticotropic hormone is used as an endocrine regulatory factor of the bovine ovarian function, and the synthesis of steroid hormone is up-regulated to promote the differentiation of granular cells. The adrenocorticotropic hormone is applied to a preparation for enhancing the synthesis of the steroid of the bovine follicular granular cells, and has good application value.
Description
Technical Field
The invention belongs to the technical field of livestock gamete embryo engineering, and particularly relates to a bovine follicular granular cell in-vitro culture regulator and application thereof.
Background
The follicular granulosa cell is the most important somatic cell in the follicle and plays an important role in local microenvironment regulation of the follicle. Early granulosa cells are differentiated from follicular epithelial cells. Granulosa cells, as important steroid hormone synthetic cells, can promote ovary synthesis and secretion of multiple hormones and growth factors such as estrogen and progestogen, regulate and control the growth, differentiation and maturation of theca cells and oocytes through gap connection, and further regulate and control the processes of follicular development, maturation, ovulation, follicular atresia and the like.
Cows are capable of inducing adrenocortical hormone secretion under various stress conditions by activating the hypothalamic-pituitary-adrenal axis (Dobson et al, 2000). Corticosteroids decrease the pulse frequency of LH secretion and eventually the preovulatory LH surge does not occur, leading to ovulation failure and induction of ovarian cysts (Dobson et al, 2006; Ribadu et al, 2000; Colitti et al, 2007; Paredes et al, 2011). Ovarian cyst is one of the most common reasons for causing the infertility of dairy cows, seriously influences the breeding efficiency of the dairy cows and restricts the development of dairy farms and the economic benefits of breeding industries. According to foreign research reports, the incidence of ovarian cyst is 6.7% -13.1% (Wiltbank et al, 2002). Ovarian cyst can reduce conception rate, prolong calving interval, increase breeding times and elimination rate, thereby affecting the fertility of dairy cows, finally reducing the economic benefit of dairy farms and causing serious economic loss to the dairy industry.
Exogenous adrenocorticotropic hormone (ACTH) can induce cow to generate follicular cyst, and changes the pulse frequency of LH secretion mainly by activating hypothalamus-pituitary-adrenal axis, so that the pre-ovulation LH peak cannot appear but cannot ovulate, and then ovarian cyst is formed, and the mechanism of the ovarian cyst is consistent with that of ovarian cyst caused by stress (Amweg et al, 2013; Biran et al, 2015; Velazquez et al, 2013). The former research mostly develops from the change of follicular follicle in milk cow, hormone and receptor gene expression, and although some progress is made, in the production practice, when the follicular cyst is confirmed, the hormone and gene expression change shown in the research cannot completely explain the abnormal change state of the body when the follicular follicle cannot normally ovulate in time, and the unpredictability of follicular cyst formation causes certain difficulty for a researcher to analyze the molecular mechanism formed by the follicular cyst. At present, no report is available on the effect of corticotropin on bovine follicular granulosa cells.
Disclosure of Invention
The invention aims to provide an in vitro culture regulator for bovine follicular granular cells, which is used for promoting the in vitro culture and synthesis of progesterone by the bovine follicular granular cells.
The invention also aims to provide a new application of the adrenocorticotropic hormone, in particular to an application of the adrenocorticotropic hormone in promoting the synthesis of the progesterone of the bovine follicular granular cell.
In order to achieve the purpose, the invention adopts the following technical scheme that:
an in vitro culture regulator of bovine follicular granulosa cell for promoting in vitro culture of bovine follicular granulosa cell to synthesize progesterone, which comprises a progesterone content of 10%-10～10-6A basal culture solution of adrenocorticotropic hormone in mol/L.
The regulator for in vitro culture of bovine follicular granular cells as described above, preferably, the basal medium is a mixture of DMEM and Ham's F-12 at a volume ratio of 1: 1.
The regulator for in vitro culture of bovine follicular granulosa cells as described above, wherein the basic culture medium further comprises 10% by volume of fetal bovine serum, penicillin at a concentration of 100U/mL and streptomycin at a concentration of 0.1 mg/mL.
As described abovePreferably, the concentration of the adrenocorticotropic hormone is 10-7mol/L。
The regulator for in vitro culture of bovine follicular granular cells as described above, preferably wherein the corticotropin is a recombinant murine corticotropin protein.
A culture method for promoting the in vitro differentiation of bovine follicular granulosa cells, comprising culturing bovine follicular granulosa cells in the in vitro culture regulator for bovine follicular granulosa cells as described above.
Application of adrenocorticotropic hormone in promoting in-vitro synthesis of steroid hormone by bovine follicular granular cells.
Application of adrenocorticotropic hormone in preparing medicine for promoting bovine follicular granular cell differentiation in vitro.
Application of adrenocorticotropic hormone in promoting in-vitro synthesis of progesterone by bovine follicular granular cells.
Preferably, the corticotropin is used in the concentration of 10 in the in vitro culture of bovine follicular granulosa cells-10～10-6mol/L。
Further, preferably, the adrenocorticotropic hormone is used at a concentration of 10 in the in vitro culture of bovine follicular granulosa cells-7mol/L。
As mentioned above, preferably, the culture medium used for the in vitro culture of the bovine follicular granular cells is a basal medium containing DMEM and Ham's F-12 culture solution at a volume ratio of 1:1, and fetal bovine serum at a volume ratio of 10%, penicillin at a concentration of 100U/mL and streptomycin at a concentration of 0.1mg/mL are added.
The invention has the beneficial effects that:
the invention provides a regulator for in-vitro culture of bovine follicular granulosa cells, which is used for promoting the expression of P450scc and inhibiting the expression of P450arom by adrenocorticotropic hormone when the bovine follicular granulosa cells are cultured in vitro, but has no influence on StAR and 3 beta-HSD expression. Adrenocorticotropic hormone is used as an endocrine regulatory factor of the bovine ovarian function, and the differentiation of granulosa cells is accelerated by up-regulating the synthesis of steroid hormone.
The invention provides a new application of corticotropin, which is used for promoting the synthesis of progesterone in the in vitro culture of bovine follicle-stimulating cells. Furthermore, the adrenocorticotropic hormone is applied to a preparation for promoting the synthesis of the steroid of the bovine follicular granular cells, so that the application value is good. The adrenocorticotropic hormone can be used as a novel pharmaceutical preparation for promoting the synthesis of steroid hormone by cow follicle granular cells and popularized and applied to livestock embryo engineering.
Drawings
FIG. 1 is a graph comparing the synthesis of progesterone by granulosa cells treated with IGF1 at different concentrations of corticotropin.
FIG. 2 is a graph showing the results of P450scc gene expression in granulosa cells in the absence and presence of corticotropin when IGF1 was added in total.
FIG. 3 is a graph showing the results of the expression of granulosa cell P450arom gene in the absence and presence of corticotropin when IGF1 was added in total.
FIG. 4 is a graph showing the results of StAR gene expression in granulosa cells in the absence and addition of corticotropin when IGF1 was added in total.
FIG. 5 is a graph showing the results of the expression of the granulosa cell 3 β -HSD gene in the absence and presence of corticotropin when IGF1 was added in total.
In the above figures, the difference (P <0.05) is indicated by mol/L abbreviated as M.
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Modifications and substitutions may be made thereto 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 ovary used in the embodiment of the invention is collected from slaughterhouses in big factories and counties of Hebei province, and the adrenocorticotropic hormone is synthesized by Nanjing Laong biotechnology limited. The reagent sources used were as follows: ham's F-12(F12), DMEM medium, Fetal Bovine Serum (FBS) from Gibco (shanghai), recombinant human IGF-1 from Science 11(San Diego, CA), bovine progesterone Radioimmunoassay (RIA): the research institute of biotechnology in Beijing northern, RNA detection reagent: one-chain synthesis kit and fluorescent quantitative kit (Tiangen Biochemical technology Co., Ltd., Beijing) are used, and the adrenocorticotropic hormone used is recombinant murine adrenocorticotropic hormone protein.
Example 1 Effect of adrenocorticotropic hormone on the Synthesis of steroid hormones in bovine follicular granulosa cells
1. Cell culture
Ovaries were collected at slaughter, placed in 0.9% saline containing penicillin and streptomycin at concentrations of 100IU/mL, kept at 37 ℃ with a vacuum flask and transported back to the laboratory over 2 hours. A10 mL syringe is used for extracting liquid in a follicle with the length of 3-8 mm from the surface of a cow ovary obtained from a slaughterhouse, the blood vessel of the follicle is required to be clear, and the follicular liquid is required to be transparent. The follicular fluid was collected in a 15mL centrifuge tube and then centrifuged at 500g for 5min, the cells were pelleted at the bottom of the tube and the supernatant removed. Granulosa cells were isolated from medium sized follicles 3-8 mm in diameter. Resuspend cells in PBS buffer, and repeat this centrifugation 4 times. The cells were resuspended in DMEM medium containing 10% fetal bovine serum and filtered through a 200 mesh filter screen to remove cell clumps, yielding granulocytes with a purity greater than 90%.
The granulocytes were counted with a cell counter and approximately 2X 10-fold plated with 5mL of a culture medium containing 10% fetal bovine serum (10% FBS)5cells/mL, in 5% CO2And (3) standing and culturing in an incubator at 37 ℃, wherein the granular cells can adhere to the wall after 12 hours generally, and changing the culture solution after the adherence of the cells is observed. The culture medium was changed every 24 hours.
When cell growth fused to more than 80%, the culture broth was discarded, followed by 2 rinses with PBS. Adding 1mL of 0.25% trypsin digestion solution, digesting at 37 ℃ for 3min, observing cell contraction and rounding under a microscope, and adding 1mL of culture solution containing 10% fetal bovine serum (10% FBS) to terminate digestion; collecting cells in a centrifuge tube, centrifuging at 1500rpm for 5min, collecting precipitate, washing with PBS 3 times, re-suspending the collected cells with culture solution, counting, and performing 1 × 105The cells/mL were plated in 12-well plates at 37 ℃ with 5% CO2Culturing in an incubator.
Wherein the 10% FBS culture solution is DMEM and Ham's F-12 at a volume ratio of 1:1 as basal medium, and is added with 10% fetal bovine serum, penicillin at a concentration of 100U/ml and streptomycin at a concentration of 0.1 mg/ml.
2. Treatment of bovine follicular granulosa cells with corticotropin
Culturing the granular cells obtained in the step 1 in a culture solution containing 10% FBS by volume for 24 hours, discarding the culture solution, washing with PBS for 2 times, and respectively using the PBS containing the granular cells with the concentration of 0 and 10 percent-6、10-7、10-8、10-9、10-10And (3) treating the culture solution for 48 hours in serum-free culture solution containing mol/L adrenocorticotropic hormone (ACTH). After the culture was completed, the culture solution was collected for measurement of progesterone content. This experiment was designed for 3 different replicates. Wherein the serum-free culture solution is a mixture of DMEM and Ham's F-12 at a volume ratio of 1:1, does not contain fetal bovine serum, and contains 100U/ml penicillin and 0.1mg/ml streptomycin.
3. Detection assay
Progesterone was measured by radioimmunoassay.
Progesterone hormone secretion levels were analyzed by one-way factor ANOVA in SPSS software. Data are expressed as mean ± sem. Progesterone hormone is expressed as ng/mL. The results of the assay are shown in FIG. 1 and demonstrate that corticotropin enhances progesterone synthesis in granulosa cells.
Example 2 Effect of corticotropin on P450scc, P450arom, StAR, 3 β -HSD mRNA expression
1. Cell culture
Granular cells obtained by cell culture according to example 1 were cultured in a culture medium containing 10% FBS by volume for 24 hours, and then the culture medium was discarded and washed 2 times with PBS at concentrations of 0 and 10-6、10-7、10-8、10-9、10-10Treating with mol/L adrenocorticotropic hormone in serum-free culture solution for 48h, collecting the culture solution, and splitting cellsAfter being resolved, the RNA is used for extraction. Experimental design 3 different replicates were averaged.
RNA extraction and RT-PCR quantitative determination of expression level
After the culture is finished, sucking the culture solution of each hole; 0.5mL of TRIzol reagent (Tiangen Biochemical technology Co., Ltd., Beijing) was added to the wells, and RNA was extracted. The RNA samples were measured for absorbance at 260nm using a TECAN microplate detector (Tecan Group Ltd., Mannedorf, Switzerland) and diluted with DEPC water and stored at-80 ℃.
P450scc, P450arom, StAR and 3 β -HSD gene primers are described in published literature (Hilda M et al, 2017; the Su et al, 2017). The primers were synthesized by Shanghai bioengineering, Inc. Three replicates per sample. Housekeeping gene GAPDH was used as the reference gene. Adopts a relative quantitative method 2-△△CtThe formula compares the expression levels of the mRNA of the genes of interest.
3. The result of the detection
Analysis of gene expression data was performed using ANOVA in SPSS software and data are presented as mean. + -. standard error.
The results are shown in FIGS. 2-5, where cytochrome P450 aromatase (P450arom), a member of the P450 cytochrome enzyme superfamily, is a key enzyme in the steroid hormone production pathway, which catalyzes the biosynthesis of estrogen from androgen. The regulation of this gene directly affects the efficiency of androgen to estrogen conversion. The StAR gene encodes steroid hormone acute regulatory protein, which can act on the outer mitochondrial membrane, mediate and promote the transfer of cholesterol substrate from the outer mitochondrial membrane to the inner membrane, then is converted into pregnenolone under the action of cholesterol side chain lyase P450scc, enters cytoplasm after being transferred out of mitochondria, and is converted into progestogen under the action of 3 beta-HSD. The synthesis of progestagen is under multiple regulation by StAR, P450scc, 3 beta-HSD, etc.
The results show that the expression of StAR and 3 beta-HSD genes is not changed when the adrenocorticotropic hormone is used for treating the bovine follicular granulosa cells. Corticotropin can inhibit P450arom gene expression and promote P450scc gene expression (P < 0.05). Thus, it can be seen that corticotropin can regulate bovine ovarian granulosa cell function, promote progesterone synthesis by up-regulating steroid synthesis related gene P450scc, and possibly inhibit estrogen synthesis by down-regulating expression of P450arom gene.
Claims (10)
1. An in vitro culture regulator for bovine follicular granulosa cells, which is used for promoting in vitro culture of bovine follicular granulosa cells to synthesize progesterone and comprises a progesterone content with a concentration of 10%-10～10-6A basal culture solution of adrenocorticotropic hormone in mol/L.
2. The regulator of claim 1, wherein the basal medium is a mixture of DMEM and Ham's F-12 at a volume ratio of 1: 1.
3. The regulator of claim 2, wherein the basic culture medium further comprises 10% by volume of fetal bovine serum, penicillin at a concentration of 100U/mL and streptomycin at a concentration of 0.1mg/mL, and the adrenocorticotropic hormone is recombinant murine adrenocorticotropic hormone protein.
4. A culture method for promoting the in vitro differentiation of bovine follicular granulosa cells, comprising culturing bovine follicular granulosa cells in the in vitro culture regulator of bovine follicular granulosa cells according to claims 1-4.
5. Application of adrenocorticotropic hormone in promoting in-vitro synthesis of steroid hormone by bovine follicular granular cells.
6. Application of adrenocorticotropic hormone in preparing medicine for promoting bovine follicular granular cell differentiation in vitro.
7. Application of adrenocorticotropic hormone in promoting in-vitro synthesis of progesterone by bovine follicular granular cells.
8. Use according to claim 7Wherein said corticotropin is used in a concentration of 10 in the in vitro culture of bovine follicular granulosa cells-10～10-6mol/L。
9. The use according to claim 7, wherein the corticotropin is used in a concentration of 10 in the in vitro culture of bovine follicular granulosa cells-7mol/L。
10. The use according to claim 7, wherein the culture medium used for the in vitro culture of bovine follicular granulosa cells is a basal medium comprising DMEM and Ham's F-12 in a volume ratio of 1:1, supplemented with 10% by volume fetal bovine serum and penicillin at a concentration of 100U/mL and streptomycin at a concentration of 0.1 mg/mL.
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| US20070292427A1 (en) * | 2006-05-09 | 2007-12-20 | Khan Shafiq A | Substantially pure steroidogenesis inducing peptide and uses thereof |
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| US20160145579A1 (en) * | 2006-03-08 | 2016-05-26 | Kwalata Trading Limited | Regulating stem cells |
| US20070292427A1 (en) * | 2006-05-09 | 2007-12-20 | Khan Shafiq A | Substantially pure steroidogenesis inducing peptide and uses thereof |
| CN108342352A (en) * | 2018-04-20 | 2018-07-31 | 北京市农林科学院 | Application of the N- carbamylglutamic acids in pig follicle granular cell in vitro culture |
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