CN108342352A - Application of the N- carbamylglutamic acids in pig follicle granular cell in vitro culture - Google Patents

Application of the N- carbamylglutamic acids in pig follicle granular cell in vitro culture Download PDF

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CN108342352A
CN108342352A CN201810359826.5A CN201810359826A CN108342352A CN 108342352 A CN108342352 A CN 108342352A CN 201810359826 A CN201810359826 A CN 201810359826A CN 108342352 A CN108342352 A CN 108342352A
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vitro
cell
carbamylglutamic acids
vitro culture
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CN108342352B (en
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冯涛
刘彦
白佳桦
许晓玲
宋玉清
肖霖力
肖银霞
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Beijing Academy of Agriculture and Forestry Sciences
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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
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins

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Abstract

The present invention provides a kind of new purposes of N carbamylglutamic acids, specifically, N carbamylglutamic acids can be used for promoting pig follicle granular cell in-vitro multiplication, and inhibit the secretion of progesterone and estradiol for the application in preparing pig follicle granular cell in vitro culture agent.

Description

Application of the N- carbamylglutamic acids in pig follicle granular cell in vitro culture
Technical field
The present invention relates to livestock culturing technical fields, and in particular to N- carbamylglutamic acids are external in pig follicle granular cell Application in culture.
Background technology
Granular cell is maximum cell mass in ovary, have powerful secreting function, egg mother cell can by with its Between gap connection obtain needed for nutriment, to be developed to maturation.Functioning cell of the granular cell as ovarian follicle, The steroid hormone of secretion takes part in ovarian follicular growth and starts, is proliferated, differentiation, locking, ovulating to corpus luteum generation whole process, during which Quantity, form and the function of granular cell also occur to change accordingly.Granulosa cell proliferation and apoptosis are not only hung down by hypothalamus- The control of body-ovary shafting, is also regulated and controled by ovary itself autocrine and paracrine factor.Recent studies suggest that some are sought The secreting function of proliferation and steroid hormone of granular cell can also be regulated and controled by supporting the factor such as green-tea extract.Granulosa cell proliferation and Differentiation is the main path that it functions, and proliferation is mainly shown as increasing for cell quantity, and breaks up and then show as steroids The variation of hormone secretory activity.
N- carbamylglutamic acids (N-carbamylglutamate, NCG) are the analogues of N-acetylglutamat, are The rate-limiting enzyme of arginine (Arginine, ARG) synthesis, addition N- carbamylglutamic acids can promote arginic endogenous in daily ration Property synthesis.Arginine is the limiting amino acid of piglet growth, and adding arginine in daily ration can be to piglet development and health State plays a variety of regulating and controlling effects.Breeding can be improved research shows that adding arginine in Suffolk sheep daily rations in Chevez etc. Power, but do not improve ovulation rate.Recent studies suggest that blood plasma can be promoted by adding N- carbamylglutamic acids in pregnant sow daily ration Middle arginine concentrations promote angiogenesis.On high yield cow, the addition of N- Glucosamines can improve protein utilization rate, increase Milk performance and the content for reducing ammonia nitrogen in milk, blood plasma and urine, and on reproductive performance generate it is potential influence, but it is specific how shadow It rings and does not know.
The influence research that N- carbamylglutamic acids and arginine influence gonad granulocyte proliferation is less.On people, smart ammonia Acid is considered as an endometrial cell growth factor, mainly by inhibiting Epithelial Cell Apoptosis to play a role.D- asparagus ferns Propylhomoserin can stimulate lizard follicular epithelium cell Proliferation in vitro.So far, without related N- carbamylglutamic acids to the big ovum of pig Steep the report that granulosa cell proliferation influences.
Invention content
The purpose of the present invention is to provide a kind of new purposes of N- carbamylglutamic acids, for preparing pig follicle particle Application in cell injuring model agent.
To achieve the above object, the present invention use technical solution below for：
N- carbamylglutamic acids are for the application in preparing pig follicle granular cell in vitro culture agent.
Application as described above, it is preferable that the N- carbamylglutamic acids, which are used to prepare, promotes pig follicle granular cell body The accelerating agent of outer proliferation.
Application as described above, it is preferable that the N- carbamylglutamic acids are used to prepare with IGF-1 and/or FSH promotes pig Follicular cell in-vitro multiplication and the reagent or drug for inhibiting progesterone and estradiol production.
Application as described above, it is preferable that dosage of the N- carbamylglutamic acids in vitro in nutrient chemical is 19 μ g/ ml-190μg/ml。
Application as described above, it is preferable that the in vitro culture agent is the DMEM containing para-insulin No.1 growth factor Culture medium.
Application as described above, it is preferable that the DMEM culture mediums further include follicular stimulating hormone.
Application as described above, it is preferable that the DMEM culture mediums are FBS, 0.12mmol/L containing 5% PS, 5% The sodium bicarbonate of gentamicin and 38.5mmol/L.
The present invention also provides a kind of cell injuring model agent, N- carbamylglutamic acids are contained in the in vitro culture agent.
In-vitro culture medium as described above, it is preferable that the in vitro culture agent is containing described in 19 μ g/ml-190 μ g/ml The DMEM culture mediums of N- carbamylglutamic acids.
In-vitro culture medium as described above, it is preferable that also contain PS, FBS, gentamicin and carbon in the in vitro culture agent Sour hydrogen sodium.
The beneficial effects of the present invention are：
The present invention provides N- carbamylglutamic acids for the application in preparing pig follicle granular cell in vitro culture agent, tool It says to body, N- carbamylglutamic acids can be used for preparing the culture solution that pig follicle granular cell cultivates required energy fast breeding in vitro Or accelerating agent, it is used for the proliferation of pig follicle granular cell.It is relevant for promoting pig follicle to can be additionally used in preparation clinical research Granulocyte is proliferated, but inhibits the reagent or drug of progesterone and estradiol.
Specific implementation mode
Following embodiment should not be construed as limiting the invention for further illustrating the present invention.Without departing substantially from this Under the premise of spirit and essence, modification or replacement made for the present invention belong to scope of the invention.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art. The source of agents useful for same and hormone can be as follows：DMEM culture mediums, gentamicin, sodium bicarbonate, trypan blue, clostridiopetidase A, deoxidation Ribalgilase, follicular stimulating hormone (FSH) are purchased from Sigma (Beijing), and Swine serum (PS) and fetal calf serum (FBS) are purchased from Gibco (Shanghai), recombinant human insulin No.1 growth factor (IGF-1) are purchased from Sciencell (San Diego, CA), and testosterone is purchased from Steraloids (Wilton, NH).
ELISA measure reagents：Pig progesterone ELISA kit, pig estradiol ELISA kit (BIM, San Francisco, CA).
Embodiment 1
1. cell culture
Nogestational replacement gilt ovary acquisition is stored in after acquisition from the large-scale slaughterhouse in Beijing outer suburbs containing 1% green chain In 0.9% physiological saline of mycin, transports in 1 hour reach laboratory on ice.Large follicle granular cell is taken out with asepsis injector It is derived from the ovarian follicle that diameter is more than 5mm.After centrifugation with the DMEM culture mediums of serum-free (gentamicin containing 0.12mmol/L, The sodium bicarbonate of 38.5mmol/L, 1.25mg/ml clostridiopetidase As and 0.5mg/ml DNase) it is resuspended, prevent cell before bed board solidifying Collection.
Granular cell survival ability is assessed with Trypan Blue, average out to 37%.About spread 2.0 × 105A cells/well is used 1ml is containing the culture medium that volume ratio is 5% Swine serum (PS) and 5% fetal calf serum (FBS) in 5%CO2Under 95% air conditions Training base is replaced in culture 48 hours for during which every 24 hours, and each hole obtains particle after washing 2 times with the culture medium of 0.5ml serum-frees Cell.
Processing of 2.IGF-1, FSH and N- carbamylglutamic acid (NCG) to pig large follicle granular cell
The granular cell that step 1 obtains is respectively with containing IGF-1 (30ng/mL), IGF-1 (30ng/mL)+NCG (1.9 μ g/ ML, 19 μ g/mL, 190 μ g/mL, 1900 μ g/mL), IGF-1 (30ng/mL)+FSH (30ng/mL) and IGF-1 (30ng/mL)+ FSH (30ng/mL)+NCG (1.9 μ g/mL, 19 μ g/mL, 190 μ g/mL, 1900 μ g/mL) DMEM culture mediums (containing 5% PS, The gentamicin of 5% FBS, 0.12mmol/L and the sodium bicarbonate of 38.5mmol/L) in culture 48 hours, culture medium every 24 is small Shi Genghuan is primary.Simultaneously using DMEM culture mediums (containing 5% PS, 5% FBS, 0.12mmol/L gentamicin and The sodium bicarbonate of 38.5mmol/L) as a control group.After terminating culture, collection culture medium is used to measure progesterone and estrogen contains Amount collects cell and is used for cell count.Because testosterone is the precursor of synthetic estrogen, therefore all processing all include 500ng/ The testosterone of mL.3 different repetitions of this experimental design, each granular cell repeated is on 4-10 pig ovaries 30-50 large follicle.
3. detection method
Cell count TZ20TMCounter (BIO-RAD, Shanghai, China) carries out.It is specific as follows：In trypsase It is washed 2 times, is digested 5 minutes with PBS (0.5mL) before (0.25%, 0.5mL) digestion, 10 μ l are inhaled after piping and druming and are measured.
What progesterone (Progesterone, P4) and estradiol (Estradiol, E2) were recommended using ELISA kit (BIM) Method is measured, and each sample measures 2 times.The coefficient of variation measured in batch is respectively 7.1% and 8.6%.
4. testing result
Cell count and progesterone, estradiol hormone secretion are analyzed with the one-way ANOVA in SPSS softwares.Different averages Compared with Tukey methods, data are indicated with average ± standard error (SEM).
Cell counts are as shown in table 1, and independent IGF-1 has no significant effect pig large follicle granulosa cell proliferation, and right According to group, individually processing is compared with IGF-1, and the NCG additions of doses can promote granulosa cell proliferation, as NCG concentration increases are in Now first promote the effect inhibited afterwards, wherein the NCG of 190 μ g/mL concentration remarkably promotes granulosa cell proliferation (P<0.05)；With compare Group and IGF-1, which are individually handled, to be compared, and IGF-1 and FSH, which are jointly processed by, can remarkably promote large follicle granulosa cell proliferation (P<0.05)； Compared with IGF-1 and FSH are jointly processed by, NCG additions can further promote granulosa cell proliferation, be presented as NCG concentration increases First promote the effect inhibited afterwards, wherein 19 μ g/mL and the NCG of 190 μ g/mL concentration remarkably promote granulosa cell proliferation (P< 0.05)。
Influences of 1 NCG of table to pig large follicle granulosa cell proliferation
Note：Same row difference lowercase letter has differences (P<0.05).
Steroid hormone measurement result is as shown in table 2, and independent IGF-1 processing can dramatically increase progesterone and estradiol production 20% and 46% (P<0.05), at the same add NCG (190 μ g/mL) can significantly inhibit IGF-1 induction progesterone and estradiol production (P<0.05)；Compared with control group and IGF-1 individually processing, IGF-1 and FSH, which are jointly processed by, can increase separately progesterone and estradiol Secrete 1.4 times, 1.2 times and 2.5 times, 1.7 times of (P<0.05), it is total to significantly suppress IGF-1 and FSH for NCG (190 μ g/mL) additions The progesterone and estradiol production (P of isogeneous induction<0.05), the level of progesterone and estradiol is less than or maintains an equal level with control group.
These results suggest that in the presence of IGF-1, no matter FSH presence or absence, NCG can promote pig large follicle Granulocyte is proliferated, and inhibits progesterone and estradiol production.
Influences of 2 NCG of table to pig large follicle granular cell steroid-secreting
Note：Same row difference lowercase letter has differences (P<0.05).
It should be noted that the IGF-1 and FSH in the present invention are routinely added in the basal medium for cultivate granular cell Ingredient, in order to allow cell to be proliferated as early as possible to a certain extent, to complete some factor pairs before cell ageing and apoptosis The experiment that cell Proliferation influences.It is equipped in the present embodiment and is not added with IGF-1 plus IGF-1 and IGF-1 and FSH is added to compare for totally 3 kinds, Purpose is further to promote the proliferation of granular cell to highlight NCG on the basis of basal medium.Compare different NCG additions Dosage the result shows that, can promote cell Proliferation when the additive amount of NCG is 19 μ g/mL-190 μ g/mL, and can be shown in 190 μ g/mL It writes and inhibits to be increased by the steroid-secreting that IGF-1 or IGF-1 and FSH is induced.

Claims (10)

1.N- carbamylglutamic acids are for the application in preparing pig follicle granular cell in vitro culture agent.
2. application according to claim 1, which is characterized in that the N- carbamylglutamic acids are used to prepare promotion pig follicle The accelerating agent of granular cell in-vitro multiplication.
3. application according to claim 1, which is characterized in that the N- carbamylglutamic acids are used with IGF-1 and/or FSH Promote pig follicle granular cell in-vitro multiplication in preparation and inhibits the reagent or drug of progesterone and estradiol production.
4. application according to claim 1, which is characterized in that the N- carbamylglutamic acids use in nutrient chemical in vitro Amount is 19 μ g/ml-190 μ g/ml.
5. according to the application described in any one of claim 1-4, which is characterized in that the in vitro culture agent is to contain class pancreas islet The DMEM culture mediums of plain No.1 growth factor.
6. application according to claim 5, which is characterized in that the DMEM culture mediums further include follicular stimulating hormone.
7. application according to claim 5, which is characterized in that the DMEM culture mediums be containing 5% PS, 5% FBS, The gentamicin of 0.12mmol/L and the sodium bicarbonate of 38.5mmol/L.
8. a kind of cell injuring model agent, which is characterized in that contain N- carbamylglutamic acids in the in vitro culture agent.
9. in-vitro culture medium according to claim 8, which is characterized in that the in vitro culture agent is to contain 19 μ g/ml- The DMEM culture mediums of N- carbamylglutamic acids described in 190 μ g/ml.
10. in-vitro culture medium according to claim 9, which is characterized in that in the in vitro culture agent also contain PS, FBS, Gentamicin and sodium bicarbonate.
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Cited By (1)

* Cited by examiner, † Cited by third party
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Publication number Priority date Publication date Assignee Title
CN114015642A (en) * 2021-10-27 2022-02-08 北京市农林科学院 Bovine follicular granulosa cell in-vitro culture regulator and application thereof

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