CN109852680A - Application of the FGFR1 gene in pig ovary granular cell - Google Patents
Application of the FGFR1 gene in pig ovary granular cell Download PDFInfo
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- CN109852680A CN109852680A CN201811588890.7A CN201811588890A CN109852680A CN 109852680 A CN109852680 A CN 109852680A CN 201811588890 A CN201811588890 A CN 201811588890A CN 109852680 A CN109852680 A CN 109852680A
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Abstract
The present invention discloses a kind of application of FGFR1 gene in pig ovary granular cell, belongs to cell engineering and gene engineering technology field.The present invention has detected in 180 ages in days (ovary prematurity) and 200 ages in days (ovary maturity) pig ovary tissue, in different size follicular cell FGFR1 mRNA relative expression quantity, and after overexpression/interference FGFR1 granular cell proliferation, apoptosis situation, be sow folliculus ovarii growth course in molecular mechanism accumulate material.Meanwhile the gene of FGFR1 as the result is shown of the invention participates in promoting the proliferation of granular cell, inhibits the apoptosis of granular cell, further illustrates that FGFR1 gene may participate in the development and maturation of sow folliculus ovarii.
Description
Technical field
The invention belongs to cell engineerings and gene engineering technology field, and in particular to a kind of FGFR1 gene is in pig ovary
Application in granulocyte.
Background technique
Mammal is after puberty, as the growth of hypothalamic pituitary gonadal axis and Secretion are perfect, ovary machine
It can be mature on the whole, folliculus ovarii gradually mature ovulation under the stimulation of reproductive hormone, and form a cycle process.According to ovum
Be soaked the Morphology educated, and ovarian follicle can be roughly divided into three primordial follicle, growing follicle and graaffian follicle growth phases.
Ovarian follicle is mainly made of egg mother cell, granular cell and theca cell.In Ovarian Follicles granular cell first with
Simple squamous shape mode surrounds an egg mother cell and forms primordial follicle, and gradually by flat in subsequent Ovarian Follicles
Shape becomes cubic or columnar cell, and continues to multiply to several layers, forms parietal layer granular cell and wraps up the ovarian cumulus of egg mother cell
Granular cell, granular cell can produce growth factor in this course, and interacts and generate with thecal cells
Estrogen provides necessary condition for the maturation of egg mother cell and ovulation.
Fibroblast growth factor acceptor 1 (Fibroblast growth factor receptor 1, FGFR1) is
A kind of transmembrane protein, belongs to receptor tyrosine kinase.Studies have shown that PLC can be passed through after FGFR1 is in conjunction with its ligand FGFs
The signal transduction paths such as γ, MAPK, PI3K-AKT generate important function to the proliferation of cell, differentiation, survival and transfer process.
The expression about FGFR1 there is no to change the relevant report to pig ovary granular cell function point analysis both at home and abroad at present.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a kind of FGFR1 gene pig not
Application in maturation/mature ovarian and different size follicular cell.
Another object of the present invention is to provide a kind of application of FGFR1 gene in pig ovary granular cell.
Change expression quantity of the FGFR1 gene in granular cell by technique for gene engineering, determines it in pig ovary particle
Application in cell Proliferation, apoptosis.
A further object of the present invention is to provide the siRNA segments (siRNA) for inhibiting FGFR1 gene expression.
The purpose of the invention is achieved by the following technical solution:
The present invention provides a kind of FGFR1 gene in pig prematurity/mature ovarian and different size follicular cell
Using.
Under vitro, in prematurity and mature ovary tissue, the mRNA phase of FGFR1 gene in mature ovarian tissue
It is extremely significant to expression quantity to be higher than prematurity ovary.
Under vitro, in different size follicular cell, FGFR1 gene in large follicle (>=5mm) granular cell
MRNA relative expression quantity it is extremely significant be higher than small ovarian follicle (≤3mm).
The present invention also provides a kind of application of FGFR1 gene in pig ovary granular cell.
Under vitro, FGFR1 gene promotes the proliferation of gonad granulocyte, inhibits the apoptosis of gonad granulocyte.
The present invention provides the siRNA (si-FGFR1) for inhibiting FGFR1 gene expression, and sequence is as follows:
Si-FGFR1:5 '-CCACCTACTTCTCCGTCAA-3 '.
Verification result of the invention is as follows:
1, the present invention detects FGFR1 mRNA relative expression in 200 age in days sow ovary tissues by qRT-PCR method
It measures extremely significant higher than (P < 0.01) 180 age in days sow (Fig. 1).These results illustrate that the expression of FGFR1 may participate in sow ovary
It reaches maturity.
2, the present invention detects FGFR1 gene mRNA phase in pig large follicle (>=5mm) granular cell by qRT-PCR method
It is extremely significant to expression quantity to be higher than (P < 0.01) small ovarian follicle (≤3mm) (Fig. 2).These results illustrate that the expression of FGFR1 may participate in
The development of folliculus ovarii.
3, the present invention is detected by EdU method, after overexpressing FGFR1 gene in granular cell, with control group
(pcDNA3.1) (P < 0.01) (Fig. 3) is increased compared to the proliferation rate of granular cell, after interfering FGFR1 gene expression, with control group
(NC) (P < 0.01) (Fig. 4) is reduced compared to the proliferation rate of granular cell.
4, the present invention is detected by Annexin V-FITC/PI technology, and FGFR1 gene is overexpressed in granular cell
Afterwards, the apoptosis rate of granular cell reduces (P < 0.05) (Fig. 5) compared with control group (pcDNA3.1), interferes FGFR1 gene expression
Afterwards, the apoptosis rate of granular cell increases (P < 0.01) (Fig. 6) compared with control group (NC).
The present invention for research object, uses molecular cytobiology side with FGFR1 gene (Gene ID:100153248)
Method studies its application in pig ovary granular cell, confirms FGFR1 gene answering in pig ovary granular cell for the first time
With, and pass through and overexpress and interfere FGFR1 gene to confirm that FGFR1 can participate in promoting pig ovary granulosa cell proliferation, inhibition
Granulocyte apoptosis further illustrates that FGFR1 gene may participate in promoting the maturation of folliculus ovarii.
The present invention has the following advantages and effects with respect to the prior art:
The present invention has detected in 180 ages in days and 200 age in days pig ovary tissues, FGFR1 in different size follicular cell
Proliferation, the apoptosis situation of granular cell after relative expression quantity and overexpression/interference FGFR1 of mRNA are sow folliculus ovarii
Molecular mechanism in growth course accumulates material.Meanwhile the gene of FGFR1 as the result is shown of the invention participates in promoting granular cell
Proliferation inhibits the apoptosis of granular cell, further illustrates that FGFR1 gene may participate in the development and maturation of sow folliculus ovarii.
Detailed description of the invention
Fig. 1 is the relative expression quantity of FGFR1 mRNA in 180 ages in days and 200 age in days pig ovary tissues；
Fig. 2 is the relative expression quantity of FGFR1 mRNA in large and small follicular cell；
Fig. 3 is the result figure of granulosa cell proliferation after EdU method detection overexpression FGFR1；
Fig. 4 is the result figure of granulosa cell proliferation after the detection interference FGFR1 expression of EdU method；
Fig. 5 is the result figure of apoptosis of granulosa cell after Annexin V-FITC method detection overexpression FGFR1；
Fig. 6 is the result figure of regulation apoptosis of granulosa cell after the detection interference FGFR1 expression of Annexin V-FITC method.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
It should be understood that embodiment described in this specification is not intended to limit just for the sake of explaining the present invention
The present invention, parameter, ratio of embodiment etc. can adaptation to local conditions make a choice and substantial effect had no to result.It is removed in embodiment
It is this field conventional reagent and method and step outside specified otherwise.
Embodiment 1 constructs FGFR1 gene overexpression vector
BioEdit software analysis finds FGFR1 gene C DS region sequence without two kinds of restriction enzymes of BamH I and Xba I
Restriction enzyme site, and there are BamH I and Xba I digestion positions for pcDNA3.1 carrier (being purchased from Invitrogen company, article No. V79020)
Point.5.0 area software design FGFR1 gene C DS primer of primer premier, upstream and downstream primer add respectively BamH I and
Xba I restriction enzyme site sequence.Using pig ovary granular cell cDNA as template, PCR amplification target fragment, purified recycling, double enzymes
It cuts, connect pcDNA3.1 carrier, conversion, screening, (endotoxin-free plasmid is a small amount of for the correct rear extracting endotoxin-free plasmid of sequencing identification
Extracts kit is purchased from Magen company), it is named as pcDNA3.1-FGFR1.
The culture of 2 gonad granulocyte of embodiment
(1) ovary tissue for taking slaughterhouse to acquire is put into rapid on ice containing being placed in 1% dual anti-PBS or physiological saline
Take back laboratory；
(2) ovary of acquisition is turned rapidly after Sterile culture room cleans 3 times with PBS or physiological saline (dual anti-containing 1%)
Enter superclean bench, and is shallowly inserted into ovary antral follicles with the sterile disposable syringe of 1mL and draws liquor folliculi；
(3) liquor folliculi drawn is placed in the centrifuge tube containing appropriate DMEM, and 800rpm room temperature is centrifuged 5min；
(4) it discards supernatant, then is resuspended with DMEM, centrifugation, repeated washing cell 2 times；Preparation DMEM complete medium: 89%
DMEM in high glucose+10%FBS+1% is dual anti-；
(5) cell is resuspended with complete medium, is inoculated in 75mL culture bottle；37 DEG C are placed in, 5%CO2It is stood in incubator
Culture.
Described is dual anti-for penicillin and streptomysin.
The inoculation and transfection of 3 gonad granulocyte of embodiment
(1) when granular cell convergence degree reaches 90% or so, culture medium is abandoned, is cleaned cell 3 times with the PBS of preheating；
(2) 0.25% trypsin digestion is added, is put into incubator 3min or so, microscopically observation to most cells
Equivalent terminate liquid (complete medium) is added immediately and terminates digestion for levitating；
(3) PBS is cleaned 2 times, and period 800rpm is centrifuged 5min；
(4) cell precipitation is gently resuspended with complete medium, uniformly assigns in each hole, supplement body with complete medium
Product, gently shakes up, puts in incubator and cultivate；
(5) left and right for 24 hours, observes granular cell state, prepares transfection when cell confluency degree is up to 70~90% or so；
(6) transfection method is by Invitrogen company3000 kit specifications carry out；Every group
3 repetitions are set；
(7) cell after transfecting is placed in 37 DEG C, 5%CO2Continue to cultivate in incubator；
(8) according to experiment purpose 1~3 day collection cell after transfection.
4 RNA of embodiment extracting and reverse transcription
Cell total rna is extracted referring to Takara company's T RIzol operational manual, specific steps are as follows:
(1) after ovary tissue liquid nitrogen grinding, 1mL TRIzol is added by the tissue mass of every 50~100mg, and blow repeatedly
It beats several times；When extracting total serum IgE from adherent granular cell, by every 10cm2Tissue culture plate floor space be directly added into 1mL
TRIzol；
(2) 10min is stood on ice sufficiently to crack tissue/cell, 12000rpm is centrifuged 5min, abandons precipitating and sucts clear Yu Xin
In 1.5mL RNase-free pipe；
(3) 200 μ L chloroforms (every 1mL TRIzol) is added and acutely shakes 15~30s, stand 15min on ice, 4 DEG C
12000rpm is centrifuged 15min；
(4) upper strata aqueous phase is drawn to be placed in new 1.5mL RNase-free EP pipe；
(5) 500uL isopropanol (every 1mL TRIzol) is added, stands 10min on ice after mixing of lightly turning upside down,
4 DEG C of 12000rpm are centrifuged 10min；
(6) it abandons supernatant and is placed on room temperature, 75% ethyl alcohol-DEPC of 1mL is added to wash RNA, 4 DEG C of 12000rpm along tube wall
Supernatant is abandoned after centrifugation 5min；
(7) it is dried in vacuo 5~10min, pays attention to avoiding RNA precipitate dry excessive；
(8) DEPC water is added to dissolve RNA precipitate.
PrimeScriptTM RT Master Mix (Perfect Real of the mRNA reverse transcription referring to TaKaRa company
Time) cDNA reverse transcription reagent box carries out.
5 qRT-PCR of embodiment
The detection of gene relative expression quantity uses Maxima SYBR Green qPCR Master Mix (2X) in the present invention
Kit (Thermo Scientific company) carries out.Experimental result is using the content for comparing Ct value method test sample gene, tool
Body calculation formula is as follows:
Gene relative expression quantity=2{ < ﹙ experimental group target gene Ct Zhi ﹚-﹙ experimental group reference gene Ct Zhi ﹚ >-< ﹙ control group target gene Ct Zhi ﹚-﹙ control group reference gene Ct Zhi ﹚ > }
Wherein GAPDH is as reference gene, qRT-PCR primer used in the present invention are as follows:
QRT-PCR-FGFR1 Forward:5 '-GGCTACAAGGTCCGTTATG-3 '；
Reverse:5 '-CAATCTTACTCCCATTCACC-3 '；
QRT-PCR-GAPDH Forward:5 '-TCGGAGTGAACGGATTTG-3 '；
Reverse:5 '-TCACCCCATTTGATGTTGG-3 '.
The detection of 6 granulosa cell proliferation of embodiment
The Cell- of EdU method detection cell proliferation experiment reference Guangzhou Rui Bo Biotechnology Co., Ltd in the present invention
LightTM567 In vitro Kit of EdU Apollo is carried out.Specific step is as follows (by taking 96 orifice plates as an example):
(1) the dilution proportion Edu solution that 1000:1 is pressed with cell culture medium, prepares appropriate 50 μM of Edu culture mediums；
(2) every hole is added 100 μ L, 50 μM of Edu culture mediums and is incubated for 2 hours, abandons culture medium；
(3) PBS is cleaned cell 1~2 time, every time 5 minutes；
(4) every hole is added 50 μ L cell fixers (PBS containing 4% paraformaldehyde) and is incubated at room temperature 30 minutes, abandons fixer；
(5) 50 μ L 2mg/mL glycine are added in every hole, and decolorization swinging table is incubated for after five minutes, abandon glycine solution；
(6) 100 μ L PBS are added in every hole, and decolorization swinging table cleans 5 minutes, abandon PBS；
(7) 1 × Apollo staining reaction liquid of 100 μ L is added in every hole, is protected from light, after the incubation of room temperature, decolorization swinging table 30 minutes,
Abandon staining reaction liquid；
(8) 100 μ L bleeding agent (PBS of 0.5%Triton X) decolorization swinging tables are added to clean 2~3 times, 5 minutes every time, abandon
Bleeding agent；
(9) the dilution proportion reagent F for using deionized water bank 100:1, prepares appropriate 1 × Hoechst3342 reaction solution, is protected from light
It saves；
(10) 100 μ L 1 × Hoechst3342 reaction solutions are added in every hole, are protected from light, room temperature, decolorization swinging table are incubated for 30 minutes
Afterwards, staining reaction liquid is abandoned；
(11) every hole is added 150 μ L PBS and cleans 1~3 time；
(12) every hole is added 100 μ L PBS and saves for use；
(13) after the completion of dyeing, photo acquisition is carried out with fluorescence microscope.
The detection of 7 apoptosis of granulosa cell of embodiment
The present invention detects Apoptosis using Annexin V-FITC/PI technology, has referring to Guangzhou cortex biotechnology
Limit company FITC Annexin V Apoptosis Detection Kit with PI kit specification, concrete operation step
It is as follows:
(1) tissue culture plate is placed on room temperature, with PBS gently rinse culture plate inner cell, abandons PBS；
(2) 0.25% trypsin digestion is added, is put into incubator 3min or so, microscopically observation to most cells
Equivalent terminate liquid (complete medium) is added immediately and terminates digestion for levitating.
(3) 1000rpm is centrifuged 5min and collects cell, abandons supernatant, cleans cell twice with pre-cooling PBS.Adjust every solencyte
Number is 0.2~1.0*106It is a, 400 μ L 1*Binding Buffer are added, cell is resuspended.
(4) the every pipe of sample sequentially adds 5 μ L FITC-Annexin V, and room temperature (25 DEG C) is protected from light 15min.
(5) 10 μ L PI are sequentially added, after light mixing, 4 DEG C are protected from light 5min.
(6) it is analyzed immediately with flow cytomery after having reacted
Interpretation of result
1, there are many small ovarian follicle (diameters≤3mm for protruding from surface (before puberty) before non-full maturity for sow ovary
Left and right), it is similar to mulberries；The development of ovary is mature (when puberty) when first time heat, and there are many ovarian follicles to differ in size (to occur straight
Diameter >=5mm ovarian follicle) Ovarian surface is protruded from, and there is the sign ovulated.Therefore, the present invention with 180 ages in days (ovary not at
It is ripe) and 200 ages in days (ovary maturity) pig ovary tissue cDNA be template, 200 age in days sows are detected by qRT-PCR method
FGFR1mRNA relative expression quantity is extremely significant in ovary tissue is higher than (P < 0.01) 180 age in days sow (Fig. 1).These result explanations
FGFR1 may participate in promoting the maturation of sow ovary.
The ovary tissue of 180 ages in days and 200 ages in days derives from the Landrace×Large White Sows on the pig farm Yangjiang Bao Jun.
2, after sow ovary maturity, ovulation process is in cyclically-varying, and Ovarian surface has the ovarian follicle to differ in size to go out
It is existing.Therefore, folliculus ovarii is divided into two kinds of large follicle (>=5mm) and small ovarian follicle (≤3mm) etc. by diameter by the present invention
Grade, using pig large follicle (>=5mm), small ovarian follicle (≤3mm) granular cell cDNA as template, pass through qRT-PCR method detection pig
The variation discovery of FGFR1 gene mRNA expression, the FGFR1 gene mRNA relative expression quantity in pig large follicle (>=5mm) granular cell
It is extremely significant to be higher than (P < 0.01) small ovarian follicle (≤3mm) (Fig. 2).These results illustrate that FGFR1 may participate in promoting the development of ovarian follicle.
Tissue-derived commodity sow (the Ternary Pig three way cross in Guangzhou Jiahe slaughterhouse Wang Gang of the folliculus ovarii
Pig).
3, after acquiring pig ovary from slaughterhouse, it is separately cultured gonad granulocyte.The present invention passes through outside technique for gene engineering
Source synthesizes FGFR1 gene overexpression vector (pcDNA3.1-FGFR1) and siRNA segment (si-FGFR1), and is transfected
It is rear referring to EdU kit specification for 24 hours to pig ovary granular cell, the proliferative conditions of granular cell are detected.Detection
The result shows that the proliferation rate of granular cell increases (P < 0.01) compared with control group (pcDNA3.1) after overexpression FGFR1 gene
(Fig. 3), after interfering FGFR1 gene expression, the proliferation rate of granular cell reduces (P < 0.01) (Fig. 4) compared with control group (NC).
Si-FGFR1:5 '-CCACCTACTTCTCCGTCAA-3 '.
Above-mentioned siRNA segment is synthesized by Guangzhou Ribo Bio Co., Ltd.；Control group NC comes from Guangzhou
Rui Bo Biotechnology Co., Ltd.
4, after acquiring pig ovary from slaughterhouse, it is separately cultured gonad granulocyte.The present invention passes through outside technique for gene engineering
Source synthesizes FGFR1 gene overexpression vector (pcDNA3.1-FGFR1) and small interference fragment (si-FGFR1), and transfected to
In pig ovary granular cell, referring to FITC Annexin V Apoptosis Detection Kit with PI explanation after 48h
Book detects the apoptosis situation of granular cell.Testing result shows after overexpressing FGFR1 gene, with control group
(pcDNA3.1) (P < 0.05) (Fig. 5) is reduced compared to the apoptosis rate of granular cell, after interfering FGFR1 gene expression, with control group
(NC) (P < 0.01) (Fig. 6) is increased compared to the apoptosis rate of granular cell.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Agricultural University Of South China
<120>application of the FGFR1 gene in pig ovary granular cell
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-FGFR1 Forward
<400> 1
ggctacaagg tccgttatg 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-FGFR1 Reverse
<400> 2
caatcttact cccattcacc 20
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GAPDH Forward
<400> 3
tcggagtgaa cggatttg 18
<210> 4
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GAPDH Reverse
<400> 4
tcaccccatt tgatgttgg 19
<210> 5
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> si-FGFR1
<400> 5
ccacctactt ctccgtcaa 19
Claims (3)
- Application of the 1.FGFR1 gene in pig prematurity/mature ovarian and different size follicular cell, it is characterised in that:Under vitro, in prematurity and mature ovary tissue, the mRNA of FGFR1 gene is with respect to table in mature ovarian tissue It is extremely significant higher than prematurity ovary up to measuring；Under vitro, in different size follicular cell, FGFR1 base in the large follicle granular cell more than or equal to 5mm The extremely significant small ovarian follicle being higher than less than or equal to 3mm of the mRNA relative expression quantity of cause.
- Application of the 2.FGFR1 gene in pig ovary granular cell, it is characterised in that:Under vitro, FGFR1 gene promotes the proliferation of gonad granulocyte, inhibits the apoptosis of gonad granulocyte.
- 3. inhibiting application of the siRNA of FGFR1 gene expression in pig ovary granular cell, it is characterised in that: the application Environment is vitro；The siRNA of the inhibition FGFR1 gene expression, sequence are as follows:Si-FGFR1:5 '-CCACCTACTTCTCCGTCAA-3 '.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110373416A (en) * | 2019-06-18 | 2019-10-25 | 华南农业大学 | Application of the RBP1 gene in sow gonad granulocyte |
| CN111334475A (en) * | 2020-03-13 | 2020-06-26 | 吉林省农业科学院 | Application of INSRR gene, and product and method for regulating and controlling granulosa cells |
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| CN105695463A (en) * | 2016-01-19 | 2016-06-22 | 华南农业大学 | Application of PIK3R2 in pig ovarian granular cells |
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| US20080254002A1 (en) * | 2004-09-03 | 2008-10-16 | Edelberg Jay M | Bone Marrow Derived Oct3/4+ Stem Cells |
| CN105695463A (en) * | 2016-01-19 | 2016-06-22 | 华南农业大学 | Application of PIK3R2 in pig ovarian granular cells |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373416A (en) * | 2019-06-18 | 2019-10-25 | 华南农业大学 | Application of the RBP1 gene in sow gonad granulocyte |
| CN110373416B (en) * | 2019-06-18 | 2022-09-20 | 华南农业大学 | Application of RBP1 gene in sow ovarian granulosa cells |
| CN111334475A (en) * | 2020-03-13 | 2020-06-26 | 吉林省农业科学院 | Application of INSRR gene, and product and method for regulating and controlling granulosa cells |
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