阅读说明：本技术 利用miR-212及其靶基因调控卵巢颗粒细胞的方法 (Method for regulating and controlling ovarian granulosa cells by using miR-212 and target gene thereof ) 是由 张锫文 朱砺 张顺华 沈林園 牛丽莉 赵叶 陈蕾 郭鑫宇 吴霜 李欣荣 肖茜 于 2021-04-02 设计创作，主要内容包括：本发明提供了一种利用miR-212及其靶基因调控卵巢颗粒细胞的方法,属于细胞工程和生物工程的技术领域,本发明以miR-212为研究对象,首次实现利用miR-212在猪卵巢颗粒细胞增值方面的方法,该方法包括步骤：将miR-212转染入卵巢颗粒细胞中。本发明提供了一种显著的调控卵巢颗粒细胞的增值,本发明方法简单、易于实施推广。本发明的研究表明,通过对猪卵巢颗粒细胞进行分离、培养,转染miR-212后显著促进颗粒细胞的增殖能力,为后续卵泡发育相关研究提供理论基础。(The invention provides a method for regulating and controlling ovarian granulosa cells by using miR-212 and a target gene thereof, belonging to the technical field of cell engineering and bioengineering, and the method takes miR-212 as a research object and realizes the method for increasing the value of the porcine ovarian granulosa cells by using miR-212 for the first time, and the method comprises the following steps: miR-212 is transfected into ovarian granule cells. The invention provides a method for remarkably regulating and controlling the proliferation of ovarian granulosa cells, and the method is simple and easy to implement and popularize. The research of the invention shows that the pig ovarian granulosa cells are separated and cultured, the proliferation capacity of the granulosa cells is remarkably promoted after miR-212 is transfected, and a theoretical basis is provided for the subsequent research related to follicle development.)

1. A method for regulating and controlling ovarian granulosa cells by using miR-212 and a target gene thereof, wherein the method comprises the following steps: miR-212 is transfected into ovarian granule cells.

2. The method of claim 1, further comprising isolating, seeding, subculturing, and harvesting operations prior to performing the transfection.

3. The method of claim 1, wherein the nidulans granulosa cells are porcine ovarian granulosa cells.

4. The method of claim 2, wherein the separating operation is performed as follows:

1) taking out ovaries after slaughtering sows, putting the ovaries into PBS (phosphate buffer solution), washing, and then preserving the ovaries in physiological saline at 38 ℃ to separate ovarian granulosa cells within 1 hour;

2) after the ovary is put into preheated physiological saline with the temperature of 37 ℃ and cleaned, the surface of the ovary is evenly and lightly scratched by a scalpel every 1 mm, follicular fluid is collected, the follicular fluid after being sucked is put into a 15ml centrifuge tube, centrifuged for 8 minutes at 1000rpm, supernatant is discarded, and the ovary is lightly blown by preheated PBS to be evenly cleaned and centrifuged for twice;

3) the ovarian granulosa cell culture fluid was prepared with 20% FBS plus DMEM and 1% antibiotics.

5. The method of claim 2, wherein before inoculation, after the ovarian granulosa cells proliferate to 85%, the original culture medium is discarded, washed twice with PBS, 1ml pancreatin is added by a Pasteur pipette, the mixture is placed in a 37-degree cell incubator, after standing for 2min, fresh culture medium is added to stop digestion, the cells are blown down, and the suspension is sucked into a 15ml centrifuge tube; finally, the cells are evenly washed by the culture solution and inoculated into a 12-well plate for culture.

6. The method according to claim 2, wherein the cells are seeded into 12-well culture plates one day before cell transfection, and transfection is performed when the density of proliferating cells reaches 35 to 45%, and harvesting is performed after 48 hours.

7. The method of claim 2, wherein the transfection is performed according to the instructions of the lipofectamine3000 kit.

8. The method of claim 3, wherein the antibiotic is penicillin, streptomycin, or amphotericin.

9. The method of claim 5, wherein said harvesting is performed by adding RNAiSoPlus to each well, repeatedly pipetting after one minute of lysis, and storing the sample at-80 ℃.

10. The method according to claim 1, wherein in step (2), the cells are cultured at a density of 40%.

Technical Field

The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to a method for regulating and controlling ovarian granulosa cells by using miR-212 and a target gene thereof.

Background

The follicular granulosa cell is an important condition for follicular development and maintenance of normal function, and plays an extremely important role in maintaining ovarian reproductive function. It has been found by researchers that the process of follicular atresia degeneration is triggered by apoptosis of granulosa cells in the follicle, which eventually leads to follicular atresia. In follicular granulosa cells, hormones such as steroid hormone, follitropin and luteinizing hormone can be secreted, and cytokines (progesterone, stem cell growth factor, epidermal growth factor and the like) can be produced to regulate the growth and development of follicles and oocytes.

The birth rate of piglets in the production of live pigs is obviously and positively correlated with the economic benefit of production, and how to improve the number of piglets plays a crucial role in the genetic breeding work of modern pigs. At present, the method mainly depends on genetic auxiliary marker selection for improving the number of the sows yielding seeds, and is a temporary and non-polar remarkable improvement method for screening marker gene homozygous or heterozygous individuals and crossbreeding with pig species with better property of yielding seeds such as Taihu pigs and the like. However, the number of born sows is also related to the development of follicles, and ovarian granulosa cells directly influence the development of the follicles, so that the exploration of conditions influencing the proliferation of the ovarian granulosa cells has important significance for promoting the development and ovulation of the follicles in production practice.

miRNA is a small molecular RNA with the length of about 22nt, most of which have no coding capacity, and mainly depends on the combination of a seed sequence at 2-8 sites and a 3' end untranslated region of a coding gene, so that a silencing compound target gene plays a biological function. A great deal of research shows that miRNA plays an important role in regulating biological processes such as follicular development, atresia and the like, and has a great deal of reports in related fields such as ovarian cancer and the like. However, how to regulate the proliferation of ovarian granulosa cells using miRNA is still under study.

Disclosure of Invention

In view of the shortcomings and needs of the prior art, it is an object of the present invention to provide a method for modulating ovarian granulosa cells using miR-212 and its target gene.

In order to achieve the above object, the present invention provides the following solutions:

miR-212 is transfected into ovarian granule cells.

As a preferred technical scheme of the invention, before the transfection, the method also comprises the operations of separation, inoculation, subculture and sample collection.

As one embodiment of the present invention, the nidulans granulosa cells are porcine ovarian granulosa cells.

In one embodiment of the present invention, the separation is carried out according to the following method:

1) taking out ovaries after slaughtering sows, putting the ovaries into PBS (phosphate buffer solution), cleaning, and then preserving the ovaries in physiological saline at 38 ℃ to separate ovary granular cells within 1 hour;

2) placing the ovary into preheated 37-degree normal saline, cleaning, uniformly and slightly scratching the surface of the ovary at intervals of 1 mm by using a scalpel, collecting follicular fluid, placing the aspirated follicular fluid into a 15ml centrifuge tube, centrifuging at 1000rpm for 8 minutes, removing supernatant, slightly blowing and uniformly cleaning by using preheated PBS, and centrifuging for twice;

3) the ovarian granulosa cell culture fluid was prepared with 20% FBS plus DMEM and 1% antibiotics.

As an embodiment of the invention, before inoculation, after the ovary granular cells are proliferated to 85%, the original culture medium is discarded, the ovary granular cells are washed twice by PBS, 1ml of pancreatin is added by a Pasteur pipette, the ovary granular cells are placed in a 37-DEG cell culture box, the ovary granular cells are placed for 2min, then fresh culture medium is added to stop digestion, the cells are blown down, and the suspension is sucked into a 15ml centrifuge tube; finally, the cells are evenly washed by the culture solution and inoculated into a 12-well plate for culture.

As an embodiment of the invention, the day before cell transfection, the cells are inoculated into a 12-well culture plate, transfection is carried out when the density of the proliferating cells reaches 35-45%, and sampling is carried out after 48 hours.

As a preferred embodiment of the present invention, transfection is carried out according to the instructions of the lipofectamine3000 kit.

As a preferred embodiment of the invention, the antibiotics are penicillin, streptomycin and amphotericin.

As a preferred embodiment of the invention, the harvesting is performed by adding RNAiosolplus to each well, repeatedly pipetting after one minute of lysis and storing the sample at-80 ℃.

As a preferred embodiment of the present invention, in step (2), the cells are cultured at a density of up to 40%.

The invention has the beneficial effects that:

the invention provides a method for remarkably regulating and controlling the proliferation of ovarian granulosa cells, and the method is simple and easy to implement and popularize. The research of the invention shows that the pig ovarian granulosa cells are separated and cultured, the proliferation capacity of the granulosa cells is remarkably promoted after miR-212 is transfected, and a theoretical basis is provided for the subsequent research related to follicle development.

Drawings

FIG. 1 shows the secondary structure of miR-212

FIG. 2 is a graph showing the results of the transfection efficiency experiment according to the present invention;

FIG. 3 is a graph showing experimental results of transfection of an ovarian granule cell with a mix inhibitor NC, which is added with a CCK reagent to detect cell proliferation;

FIG. 4 is a graph showing the results of experiments for transfecting the ovary granular cells with the mimics oligonucleotides NC, and detecting the expression quantity of the CyclinD gene by the oligonucleotides NC;

FIG. 5 is a graph showing the results of experiments for detecting the expression level of cyclin gene by transfecting the ovarian granulosa cells with the mimics NC;

FIG. 6 is a graph of the relationship of targets for detection of miR-212 and FGF16 using a dual-luciferase reporter system;

FIG. 7 is a graph showing the results of experiments for detecting the expression level of cyclin D gene after ovarian granulosa cells are transfected with FGF16 siRNA to interfere the expression;

FIG. 8 is a graph showing the results of experiments for detecting the expression level of P53 protein after ovarian granulosa cells are transfected with FGF16 siRNA to interfere the expression of the siRNA.

Detailed Description

The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

Example 1

First, experimental scheme

1. Separation and culture of swine ovary granular cells

After the sows are slaughtered, the ovaries are taken out, washed in PBS, stored in physiological saline at 38 ℃ and taken back to the laboratory within 1 hour to separate ovarian granulosa cells.

After the ovary is put into preheated physiological saline with the temperature of 37 ℃ and cleaned, the surface of the ovary is evenly and lightly scratched by a scalpel every 1 mm, follicular fluid is collected, the follicular fluid after being sucked is put into a 15ml centrifuge tube, the centrifuge is carried out for 8 minutes at 1000rpm, the supernatant is discarded, and the process is repeated twice after the ovary is evenly cleaned by lightly blowing and beating with preheated PBS.

The prepared ovarian granulosa cell culture solution is 20% FBS plus DMEM and 1% triantibody (penicillin, streptomycin and amphotericin)

2. Cell inoculation, passage, transfection and sample collection

And (3) discarding the original culture medium when the ovary granular cells proliferate to 85%, washing twice by using PBS, adding 1ml of pancreatin by using a Pasteur pipette, putting the cells into a 37-degree cell culture box, adding a fresh culture medium to stop digestion after 2min, blowing the cells, and sucking the suspension into a 15ml centrifuge tube. Finally, the cells are evenly washed by the culture solution and inoculated into a 12-well plate for culture. One day before cell transfection, cells are inoculated into a 12-well culture plate (the corresponding density is inoculated according to the experiment requirement), when the density of the proliferated cells reaches 40%, miR-212 can be transfected for the first time, and samples are collected in 48 hours. Samples were collected, rnalsoplus was added to each well, lysed for one minute and repeatedly aspirated off by pipetting, and samples were stored at-80 ℃.

3. Cell transfection

Transfection was started when the cell density was 40% and was performed according to the instructions of the lipofectamine3000 kit, and the collection treatment or the subsequent detection was performed after 48 hours.

Second, detection scheme

RNA extraction and reverse transcription

The TRIpure kit is used for extracting total RNA of tissues and cells, and comprises the following specific steps:

(1) tissue homogenization: 50-100mg of tissue was ground to powder (while adding liquid nitrogen, the ground tissue was ground) and 1ml of TRIpure was added to the ground tissue. The 12-well C2C12 cell sample was lysed by adding 1ml of TRIPURE directly to the well of the cell culture plate and removing the cell lysate to the EP tube via a pipette.

(2) protein/RNA/DNA isolation stage: the homogenate with the TRIpure added thereto was incubated at 15 to 30 ℃ for 5 minutes and then 200. mu.l of chloroform was added to 1ml of TRIpure. And (3) performing violent vortex oscillation by using a vortex instrument to fully crack the mixture, and then incubating the mixture for 2-3 minutes at normal temperature. Centrifugal force of 12,000 Xg for 15 minutes (2-8 ℃ C.).

(3) After the solution is centrifuged, three layers of water sample solutions are obtained, and the uppermost water sample layer is gently transferred to a clean EP tube by using a pipette. To this, isopropyl alcohol was then added in an amount half that of the above lysate. The mixed solution was gently inverted upside down and mixed, incubated at room temperature for 10 minutes, and centrifuged for 10 minutes (12,000 Xg). This step is used for precipitation of RNA.

(4) The supernatant was decanted from the centrifuged solution and washed once with 75% ethanol (1 ml). Centrifuge at 500 Xg for 5 minutes. This step is used for RNA rinsing.

(5) The supernatant was decanted off the tube and allowed to air dry for a few minutes. 30 ml of RNase-free water-soluble RNA was aspirated with a pipette, RNA quality and concentration were determined with a NanoDrop ND-2000 spectrophotometer, and RNA (-80 ℃) was finally stored.

2. Reverse transcription PCR

2.1 reverse transcription of mRNA

The 1st Strand cDNA Synthesis kit is used for reverse transcription of mRNA and comprises the following specific steps:

(1) reactions for removing genomic DNA

To ensure the quality of the experiment, the reaction was performed on ice by adding the reagents to the PCR tube, mixing them instantaneously and reacting them at 42 ℃ for 2 minutes.

(2) Preparation of cDNA

And continuously adding reaction system reagents into the reaction PCR tube. Instantly separated and mixed evenly. The reaction program is 37 ℃, 15 min; diluting to 100ul after the reaction is finished at 85 ℃ for 5s, mixing uniformly and storing at-20 ℃.

2.2 reverse transcription of miRNA

And performing miRNA reverse transcription according to the instruction of the miRNA RT-QPCR kit. Reacting the reaction solution at 37 ℃ for 60 min; the reaction was carried out at 85 ℃ for 5 min. After the reaction is finished, diluting to 100ul, mixing uniformly and packaging, and storing at-20 ℃ for later use.

CCK-8 experiment

The granular cells were inoculated into 96-well cell plates, treated for 9 replicates each, and transfected to a cell density of about 40%, and after 48 hours the absorbance of the cell supernatant was detected using the franchise CCK-8 kit.

EDU experiment

(1) Ovarian granulosa cells were seeded in 96-well cell plates, and 100. mu.L of EdU diluent was added to the well to be stained after transfection of the cells for reaction. EdU dilutions can be made in a ratio of EdU solution (reagent a) to cell culture medium of 1: 1000, final concentration of 50 μ M;

(2) preheating PBS in a water bath at 37 ℃, washing cells for 1-2 times by using PBS, and washing cells for 1-3 times by using a decolorizing shaker;

(3) adding 50-100 μ L of 4% paraformaldehyde into each well, incubating at room temperature, discarding formaldehyde after 30min, washing with PBS for 3-5 times, and decolorizing and shaking for 2-3 times;

(4) adding 100 mu L of currently prepared Apollo liquid into each hole for dyeing (the fuel needs to be currently prepared, the preparation sequence of 500 mu L of Apollo dyeing liquid of a system is 469 mu L of deionized water, 25 mu L of reagent B, 5 mu L of reagent C, 1.5 mu L of reagent D and 5mg of reagent E), processing in dark, incubating for 30min at room temperature, discarding the dyeing liquid, adding 0.5 percent TritonX-100100 mu L into each hole, and cleaning for 3-4 times on a decolorization shaking bed after 15 min;

(5) the nuclear fuel was prepared by mixing Hochest (reagent F) with deionized water at a ratio of 1: 100, adding 50 mu L of nuclear dye solution into each hole, processing in the dark, incubating at room temperature for 30min, discarding the dye solution, and adding 100 mu L of PBS into each hole for washing for 2-3 times;

(6) observed under a fluorescent microscope and photographed.

5.qRT-PCR

5.1 fluorescent quantitation of mRNA

And preparing a PCR reaction solution. The mixed reaction system was placed on a CFX96 Real-Time PCR detection system instrument. The PCR reaction procedure is divided into two steps. Firstly: the pre-denaturation reaction is carried out at 95 ℃ for 3 min; secondly, the method comprises the following steps: the PCR reaction is carried out for 40 cycles at 95 ℃ for 30s, wherein the annealing temperature is the optimal annealing temperature according to the temperature gradient reaction result. The PCR result is corrected by taking beta-actin as an internal reference. The relative expression amount of mRNA was calculated by the 2-. DELTA.Ct method.

5.2 fluorescent quantitation of miRNA

The mixed reaction system is placed on a CFX96 Real-Time PCR instrument for completion. Meanwhile, U6 is used as an internal reference for miRNA quantification, and a 2-delta Ct method is adopted to calculate the relative expression quantity of miRNA. Three technical replicates were made for each sample. The upstream primer of the miRNA is the mature sequence of the miRNA itself, while the downstream primer is a universal primer provided in the kit.

6.WesternBlot

6.1 protein extraction

1) Washing the cells with PBS for 2-3 times, and after the last washing, pouring out the PBS and sucking residual liquid as much as possible by using a pipettor;

2) add the appropriate volume of RIPA lysate (protease inhibitor added within minutes before use) to the plate/flask for 3-5 min. Repeatedly shaking the culture plate/bottle to make the reagent fully contact with the cells;

3) scraping the cells with a cell scraper, and transferring the cells into a 1.5ml centrifuge tube;

4) lysis on ice for 30min, during which time the pipette was repeatedly blown to ensure complete lysis of the cells. Then, the mixture is centrifuged at 12000rpm and 4 ℃ for 10min, and the supernatant is collected, namely the total protein solution.

6.2 protein concentration determination

Taking the undenatured protein solution, and detecting by using a BCA protein concentration determination kit.

6.3 denaturation of proteins

Adding the protein solution into 5-reduced protein loading buffer solution according to the ratio of 4:1, performing boiling water bath denaturation for 15min, and storing in a refrigerator at-20 ℃ for later use;

6.4SDS-PAGE electrophoresis

1) Cleaning the glass plate;

2) preparing glue and loading;

2.1) after the glass plate is naturally dried, combining a concave glass plate and a flat glass plate into a pair, putting the pair into a glue maker, inserting an inclined inserting plate to fix the glass plate, checking whether the bottoms are aligned or not, and avoiding glue leakage;

2.2) preparing the separation gel with different concentrations according to experimental requirements, adding TEMED, immediately mixing uniformly, filling the separation gel to a proper height, and before filling, trying by using a comb, wherein the distance between the comb teeth and the liquid level of the separation gel is about 5-8 mm. Then, pure water was slowly and uniformly added to the upper layer of the separation gel until the gel was filled. After about 30min, pouring out the water on the upper layer of the separation gel after the gel is solidified and sucking the rest water with absorbent paper;

2.3) preparing 5 percent of concentrated glue according to the formula, adding TEMED, immediately mixing uniformly, and filling glue. The remaining space was filled with the gel concentrate and a comb was inserted into the gel concentrate, noting that no air bubbles were present below the comb.

2.4) after the concentrated gel is solidified, taking down the gel making device, and carefully pulling out the comb to prepare for starting electrophoresis;

2.5) putting the gel maker into an electrophoresis tank, adding enough electrophoresis liquid, and then loading the sample for electrophoresis. The voltage of the concentrated gel is 75V, and the voltage of the separation gel is 120V. Electrophoresis is finished until the bottom of the column is about 1cm in bromophenol blue, and membrane transfer is carried out.

6.5 transfer film

1) Preparing 6 pieces of filter paper of 7 x 9cm and a piece of PVDF (0.45um) membrane with a proper size, wherein the PVDF membrane is activated for 2min by methanol before being used;

2) placing a clamp for transferring the membrane, two sponge pads, a glass rod, filter paper and an activated PVDF membrane in a basin with transfer liquid;

3) open the clamp, white on the left and black on the right, add a piece of sponge and three layers of filter paper on both sides.

4) Carefully peel off the separation gel onto filter paper, place PVDF membrane on the gel without air bubbles, cover the membrane with three pieces of filter paper and remove air bubbles. Finally, covering another spongy cushion;

5) and in the membrane rotating condition (wet rotation), 300mA constant current is used for membrane rotating for half an hour, and the membrane rotating equipment is placed in ice water for cooling in the membrane rotating process.

6.6 immune response

1) Putting the transferred membrane into an incubation groove filled with TBST, quickly rinsing once, adding the skimmed milk, placing on a decolorization shaking table, and sealing for 30min at room temperature;

2) diluting the primary antibody according to the antibody specification, pouring the confining liquid in the incubation groove after the preparation, adding the prepared primary antibody, and incubating the shaking table at 4 ℃ overnight (shaking table is slowly shaken);

3. recovering primary antibody, rapidly rinsing the membrane with TBST for three times, adding TBST, placing on a decolorizing shaker, and rapidly eluting for three times for 5min each time;

4) diluting the secondary antibody with TBST according to the proportion of 1:5000, then adding the secondary antibody into an incubation tank, placing the secondary antibody on a shaker for slow shaking, and incubating the secondary antibody for 30min at room temperature;

5) the membrane was rapidly rinsed three times with TBST, then TBST was added, and the membrane was rapidly eluted 5min at a time three times on a decolorizing shaker.

6.7 chemiluminescence

Mixing ECLA solution and ECLB solution in centrifuge tube in the same volume, sticking double-layer PE gloves or other transparent film on the exposure box, putting PVDF film with protein surface facing upwards between the two films of the exposure box, adding mixed ECL solution to react thoroughly, removing residual liquid after 1-2min, covering with upper film, and pressing film. The pressed film is developed and fixed with a developing and fixing agent. The exposure conditions are adjusted according to the different luminous intensities.

6.8 gel image analysis

And scanning and archiving the film, finishing and decoloring by Photoshop, and analyzing the optical density value of a target zone by an Alpha software processing system.

7. Data statistics

The experimental data were analyzed using SPSS (version 22.0). All data are expressed as mean ± Standard Error (SEM). Differences between groups were measured using one-way analysis of variance or t-test. A significant average of differences between groups was determined to be P < 0.05.

8 results of the experiment

8.1 miR-212 transfection efficiency

In order to deeply explore the effect of miR-212 in the fat development process, the influence of an overexpression mimics (mimics) and an inhibitor (inhibitor) of miR-212 transfected ovarian granulosa cells and negative contrast on the proliferation condition of the ovarian granulosa cells is observed. Transfection of overexpression mimics and inhibitors significantly improves or reduces the expression level of miR-212 in ovarian granulosa cells.

8.2 Effect of miR-212 on ovarian granulosa cell proliferation

The CCK-8 cell proliferation detection kit is used for detecting the influence of overexpression and miR-212 inhibition on ovarian granule cell proliferation, compared with a control group, the transfection miR-212 inhibitor remarkably promotes cell proliferation, and the overexpression treatment group is opposite. The EdU results are consistent with CCK results, with significantly greater numbers of novacells when cells were treated with miR-212 inhibitor than in the control group, and significantly lower numbers of novacells in the over-expression treated group.

8.3 Effect of miR-212 on proliferation marker Gene

Consistent with the results, the expression levels of several cell cycle-related kinases, cyclinD and cyclinE, also show a trend of significant up-regulation along with the reduction of the expression level of miR-212, and the results show that miR-212 can inhibit the proliferation of ovarian granulosa cells by regulating and controlling cyclin kinase-related genes.

8.4 screening of miR-212 target Gene

miR-212 is found to be highly conserved in mammals such as human, mouse, rat, macaque and the like through sequence alignment among different species. In order to further understand the molecular regulation mechanism of miR-212 on ovarian granulosa cell differentiation, the target gene of miR-212 is predicted through three common target gene prediction databases of pictar, miRBase and TargetScan, and the prediction result shows that the miR-212 seed sequence can be completely targeted to the 3' UTR region combined with FGF 16.

8.5 validation of the relationship between miR-212 and FGF16 targets

Further verifying the binding relationship between FGF16 and miR-212, a luciferase report experiment was performed. The binding of the mimic of miR-212 to WT-FGF16 shows that the luciferase activity is reduced remarkably, while the luciferase activity is not changed remarkably in the Mut-FGF16 group, and the target relation between miR-212 and FGF16 is further proved. These results indicate that miR-212 promotes ovarian granulosa cell differentiation by targeted modulation of FGF 16.

8.6 Effect of FGF16 on ovarian granulosa cell proliferation

In the experiment, siRNA is used for interfering the expression quantity of a target gene, and a fluorescence quantification method is used for detecting the influence on the proliferation of the ovarian granular cells after the target gene is interfered.