CN111334475B

CN111334475B - Application of INSRR gene, and product and method for regulating and controlling granulosa cells

Info

Publication number: CN111334475B
Application number: CN202010177694.1A
Authority: CN
Inventors: 王春昕; 张明新; 赵云辉; 翟博; 苑志宇; 赵�卓; 徐菁; 吴翠玲
Original assignee: Jilin Academy of Agricultural Sciences
Current assignee: Jilin Academy of Agricultural Sciences
Priority date: 2020-03-13
Filing date: 2020-03-13
Publication date: 2022-03-01
Anticipated expiration: 2040-03-13
Also published as: CN111334475A

Abstract

The invention providesAn INSRR gene application, a product for regulating and controlling granular cells and a method, which relate to the technical field of biology. The application of the INSRR gene in the preparation of products for regulating granulosa cells and products and methods for regulating granulosa cells include regulating granulosa cell apoptosis, regulating the expression of at least one of the granulosa cell LHR gene, FSHR gene, ER β gene and IGF1R, and regulating at least one of the granulosa cell secreted hormones; wherein the hormone comprises E₂、P₄At least one of ACT, INH, and FS.

Description

Application of INSRR gene, and product and method for regulating and controlling granulosa cells

Technical Field

The invention relates to the technical field of biology, in particular to application of an INSRR gene, a product for regulating and controlling granulosa cells and a method.

Background

Follicular development is mainly regulated by reproductive hormones, gonadotropins are the main signals for ovarian development, and they act on the ovaries through the blood circulation system to produce gonadal hormones. A large number of primordial follicles accumulate in the ovaries after formation, and only a few dominant follicles can develop to preovulatory large follicles under the action of Follicle Stimulating Hormone (FSH). The preovulatory large follicles produce large amounts of E₂And in the absence of the corpus luteum, causes a Luteinizing Hormone (LH) spike to occur, resulting in ovulation.

Primordial follicles are formed by the transformation of oocytes to approximately 20-35mm in diameter by the cessation of mitosis and entry into meiosis. Each primordial follicle is composed of a primary oocyte and several granulosa cells that surround the oocyte in polar form and suppress the primordial follicle recruitment process by secreting anti-mullerian hormone.

After primordial follicle is initially recruited, the primordial granulosa cells change from flat to cubic or high columnar shape, then the oocyte genome is activated, the granulosa cells express follicle stimulating hormone receptors, and the oocytes grow and differentiate. In mice, it was found that kit ligands produced by granulosa cells can promote oocyte growth. After follicle recruitment, the oocyte will express Connexin 37(Connexin 37, Cx37) to form gap links for completing the transfer of substances between granulocytes and oocyte through the link, providing the necessary nutritional and regulatory signals for oocyte maturation.

Secondary follicular development begins with the appearance of a second layer of granulosa cells. The oocyte regulates the proliferation of cumulus cells and granulosa cells, the synthesis and secretion of steroid hormones, and thus inhibits the luteinization of the oocyte. At the secondary follicular stage, follicular cells emerge, which begin to express receptors for gonadotropins with granulosa cells. Gap junction protein 43(Connexin 43, Cx43) which plays an essential role in regulating secondary follicular formation is also expressed on granulosa cells. As granulosa cells differentiate, the diameter of the follicle gradually increases, with decreasing levels of Activin (ACT) and increasing levels of Inhibin (Inhibin, INH) in the follicle. In the glaff follicle, granulosa cells differentiate into parietal granulosa cells, periluminal granulosa cells, cumulus cells and radiocorona cells.

At follicle maturation, the volume reaches a maximum, the follicular fluid is at a maximum, and the follicular wall also thins, the follicle at this time being called mature follicle. The number of follicles developing side by side to mature follicles is not constant, may be multiple, or may be only one, depending mainly on whether the animal is a multiparous animal or a single-born animal. The changes in the follicles during this period are absolutely dependent on gonadotropin secretion.

It can be seen that granulosa cells and their associated hormone secretion provide sufficient physical and chemical conditions for oocyte development, and are crucial for supporting oocyte developmental maturation, the transmission of endocrine, i.e. other environmental signals. Therefore, the development of a regulation method for regulating granulosa cells and products thereof has an important role in understanding the physiological and biochemical functions of granulosa cells and follicles.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide the application of INSRR gene in preparing products for regulating and controlling granulosa cells, so that the prepared products for regulating and controlling granulosa cells have at least one function of regulating and controlling granulosa cell apoptosis, hormone secretion and gene expression.

It is a second object of the invention to provide a product for the modulation of granulosa cells.

The third purpose of the invention is to provide a regulation method of granular cells.

The fourth purpose of the invention is to provide the product for regulating the granular cells or the application of the regulation method of the granular cells.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the invention, there is provided the use of the INSRR gene in the manufacture of a product for the regulation of granulosa cells; the regulatory particle cells include: regulating at least one of granulosa cell apoptosis, regulating expression of granulosa cell genes, and regulating secretion of hormones by granulosa cells;

the regulating the expression of granulosa cell genes comprises regulating at least one of LHR gene, FSHR gene, ER β gene and IGF 1R; the hormone comprises E₂、P₄At least one of ACT, INH, and FS.

According to another aspect of the invention there is also provided a product for the regulation of granulosa cells comprising a substance for over-expressing the INSRR gene and/or a substance for inhibiting the expression of the INSRR gene.

According to another aspect of the invention, there is also provided a method of modulating granulosa cells, the method comprising up-regulating or down-regulating the expression of the INSRR gene in the granulosa cells.

According to another aspect of the present invention, the present invention also provides the use of the product for regulating granulosa cells or the regulation method of granulosa cells in at least one of the following (x1) to (x 4);

(x1) regulating follicular development;

(x2) regulating ovarian development;

(x3) preparing a product for regulating follicular development;

(x4) preparing a product for regulating ovarian development.

Compared with the prior art, the invention has the following beneficial effects:

based on the regulation effect of the INSRR gene on the granulosa cells found in the experiment, the invention provides application of the INSRR gene in preparation of products for regulating the granulosa cells, a regulation method of the granulosa cells and application of the INSRR gene and the products.

Experiments show that the INSRR gene can promote apoptosis of granulosa cells; the INSRR gene is also related to the expression of LHR gene, FSHR gene, ER beta gene and IGF1R gene in granulosa cells, and can inhibit the expression of LHR gene, FSHR gene and ER beta gene and promote the expression of IGF1R gene. Experiments show that the INSRR gene can regulate and control the secretion of hormones of granulocytes, wherein the hormones mainly comprise estradiol (E)₂) Progesterone (P)₄) Activin (ACT), Inhibin (INH) and Follistatin (FS), the INSRR gene promotes E₂、P₄And secretion of ACT, inhibiting secretion of INH and FS.

Based on the regulation effect of the INSRR gene on the granulosa cells, the regulation method of the granulosa cells and the product for regulating the granulosa cells can effectively regulate apoptosis of the granulosa cells and regulate secretion of reproductive hormone related to follicular development. Meanwhile, based on the regulation and control effect of the granulosa cells on the development of the follicle and the ovary, the regulation and control method of the granulosa cells and the product for regulating and controlling the granulosa cells can be further applied to the regulation and control of the development of the follicle and the ovary or used for preparing the product for regulating and controlling the development of the follicle and the ovary, the regulation and control effect on the follicle and/or the ovary in vivo or in vitro is realized, and the method and the guarantee of the product are provided for the research on the development and physiological and biochemical functions of the granulosa cells, the follicle and the ovary.

The method for regulating and controlling the granulosa cells, provided by the invention, is used for regulating and controlling the products of the granulosa cells and the application of the products, can deepen the understanding of the maturation mechanism of the oocytes at the later stage of follicular development, further excavate factors related to follicular development, perfect signal paths related to follicular development, disclose the mechanism of follicular development and optimize an oocyte in-vitro culture system. The method has important significance in further carrying out superovulation of mammals, embryo in-vitro production, multi-fetus variety breeding and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the relative expression level of INSRR gene following transfection of granulosa cells with siRNA in example 1 of the present invention;

FIG. 2 shows the detection result of apoptosis in flow cells after INSRR gene interference in example 2 of the present invention;

FIG. 3 is a graph showing the rate of granulosa cell apoptosis following INSRR gene interference in example 2 of the present invention;

FIG. 4 is a graph showing the proliferation rate of granulosa cells after interference of the INSRR gene in example 2 of the present invention;

FIG. 5 is a graph showing the effect of INSRR gene interference on sheep granulosa cell gene expression in example 3 of the present invention;

FIG. 6 shows the effect of INSRR gene on ovine granulosa cell E after disruption in example 4 of the present invention₂The effect of secretion;

FIG. 7 shows the interaction of INSRR gene on ovine granulosa cells P in example 4 of the present invention₄The effect of secretion;

FIG. 8 is a graph showing the effect of interference of the INSRR gene on the secretion of ACT by sheep granulocytes in example 4 of the present invention;

FIG. 9 is a graph showing the effect of interference of the INSRR gene on INH secretion from sheep granulocytes in example 4 of the present invention;

FIG. 10 is a graph showing the effect of interference of the INSRR gene on FS secretion in sheep granulocytes in example 4 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to one aspect of the invention there is provided the use of an INSRR gene in the manufacture of a product for regulating granulosa cells, wherein regulating granulosa cells comprises regulating at least one of granulosa cell apoptosis, regulating expression of a granulosa cell gene, and regulating secretion of hormones by granulosa cells. The regulating the expression of granulosa cell genes comprises regulating at least one of LHR gene, FSHR gene, ER β gene and IGF 1R; the hormone comprises E₂、P₄At least one of ACT, INH, and FS.

Insulin Receptor-Related Receptor (INSRR) genes encode Insulin Receptor-Related receptors (IRRs), which belong to the Insulin Receptor family, which belongs to the RTK type ii subfamily of the Receptor tyrosine kinase family (RTKs), which further comprises Insulin Receptors (IR), Insulin-like growth factor receptors (IGF-R), which activate intracellular tyrosine kinases upon binding to respective ligands, such as Insulin, Insulin-like growth factors (IGF-1 or IGF-2), initiating intracellular signaling to exert physiological functions in organisms. At present, the function research of insulin receptor related receptor (IRR) is less, and experiments show that INSRR gene is related to apoptosis of granulosa cells and can promote the granulosaApoptosis of the cell; the INSRR gene is also related to the expression of LHR, FSHR and ER β genes related to oocyte development in granulocytes, and can inhibit the expression of LHR, FSHR and ER β genes; the INSRR gene is also involved in the expression of the IGF1R (insulin-like growth factor 1 receptor) gene in granulosa cells, and is capable of promoting the expression of the IGF1R gene. Experiments show that the INSRR gene can regulate and control the secretion of hormones of granulocytes, wherein the hormones mainly comprise estradiol (E)₂) Progesterone (P)₄) Activin (ACT), Inhibin (INH) and Follistatin (FS), the INSRR gene promotes E₂、P₄And secretion of ACT, inhibiting secretion of INH and FS.

Granulosa cells of mature follicles are the largest group of cells that make up follicles, and during follicular development, the proliferation, differentiation, and apoptosis of granulosa cells play an important role in the development and maturation of oocytes. Studies have shown that if follicular apoptosis of granulosa cells during development leads to follicular atresia, the pregnancy rate after embryo transfer is higher with a low rate of granulosa cell apoptosis in vitro fertilization.

The hormone secreted by granulosa cells also plays an important role in the growth and development of follicles and ovaries. For example, Inhibin (INH) secreted by granulosa cells has the functions of regulating follitropin and stimulating androgen secretion from ovarian follicular membranes; the Activin (ACT) secreted by the granulosa cells is involved in the development of early follicles, is related to the atresia of mature follicles, and has the effects of promoting the maturation of the follicles and enhancing the functions of the granulosa cells; after the Follistatin (FS) secreted by granulosa cells is combined with ACT, the follicular atresia of ACT can be prevented, and the follicle can be promoted to continue to grow and mature. Meanwhile, the roles played by INH and ACT in follicular development are opposite, with the level of ACT in the follicles decreasing as granulosa cells differentiate, while INH gradually increases. Estradiol secreted by granulosa cells (E)₂) And progesterone (P)₄) It is also a reproduction-related hormone with multiple effects, and has multiple effects on ovarian development.

Each primordial follicle is composed of a primary oocyte and several granulosa cells. The follicular development is mainly regulated by reproductive hormone, gonadotropin is a main signal for ovarian development, a large number of primordial follicles are formed and gathered on ovaries, and develop to preovulatory large follicles under the regulation of Follicle Stimulating Hormone (FSH), and the preovulatory large follicles generate a large amount of E₂And in the absence of the corpus luteum, promotes the appearance of an LH surge, resulting in ovulation. IGF1R promotes the continued development of luminal follicles, which has an effect on FSHR expression and estradiol synthesis, leading to impaired granulosa cell proliferation and arrest in follicular development. It can be seen that in organisms, the generation and development of follicles are completed by the cooperation of a plurality of factors, and the expression of LHR, FSHR, ER beta and IGF1R on granulosa cells is regulated, so that a good basis is provided for further research on the development mechanism of follicles and control of the development of follicles and ovaries.

Based on the functions of the INSRR gene provided by the invention, the INSRR gene provided by the invention is applied to preparation of a product for regulating granulosa cells, and the product at least adopts up-regulation or down-regulation of the expression level of the INSRR gene to realize the regulation effect on the granulosa cells.

In some alternative embodiments, the product achieves at least one of the following regulatory effects by up-regulating the expression of the INSRR gene: inhibiting granular cell apoptosis; up-regulating IGF1R gene expression level; down-regulating the expression level of at least one of LHR gene, FSHR gene and ER beta gene; promotion of E₂、P₄And secretion of at least one of ACT; and, inhibiting secretion of at least one of INH and FS.

In some alternative embodiments, the product down-regulates the expression of the INSRR gene to achieve at least one of the following regulatory effects: promoting granular cell apoptosis; down-regulating the expression level of IGF1R gene; up-regulating the expression level of at least one of LHR gene, FSHR gene and ER beta gene; inhibition of E₂、P₄And secretion of at least one of ACT; and, promoting secretion of at least one of INH and FS.

It will be appreciated that the product is not limited to effecting up-regulation or down-regulation of the INSRR gene, but also up-regulation and/or down-regulation of the expression of other genes to effect modulation of other hormones, gene expression or other physiological and biochemical activities in the granulosa cells. The product regulates and controls granular cells, and can be regulated and controlled in vivo or in vitro.

In the above examples, when the product is used for regulating the expression of genes in granulosa cells, the product can simultaneously regulate the expression of the HR gene, FSHR gene, ER β gene and IGF 1R; it is also possible to regulate the expression of only one, two or three of the genes: for example, but not limited to, the product contains a substance for overexpressing the INSRR gene and a substance for overexpressing at least one of the LHR gene, the FSHR gene and the ER beta gene, and the product can realize the function of up-regulating the expression level of IGF1R gene and simultaneously generating no inhibition effect on at least one of the LHR gene, the FSHR gene and the ER beta gene.

In the above examples, when the product is used to promote or inhibit E₂、P₄Secretion of at least one of ACT, INH and FS, and the product can regulate the five hormones at the same time or only one, two, three or four of the hormones. For example, the product contains a substance which inhibits the expression of the INSRR gene, together with a substance which promotes E₂、P₄And ACT, which can promote secretion of INH and FS while inhibiting E alone₂、P₄And some hormones in ACT, or not E₂、P₄And a function of inhibiting secretion of ACT.

Use of the INSRR gene in the preparation of a product for regulating granulosa cells may, in some alternative embodiments, be achieved by including in the product a substance capable of up-regulating and/or down-regulating the INSRR gene. For example, but not limited to, the INSRR gene may be used by at least one of the following ways: down-regulating expression of a gene by containing an siRNA capable of interfering with expression of the gene, or expressing DNA of the siRNA; overexpression of the gene is achieved by a recombinant vector containing the gene expressing INSRR; regulating and controlling a gene by a protein or a compound drug or the like capable of inhibiting the expression of a target gene; the gene is regulated by containing cells or microorganisms capable of expressing the gene having the function of regulating the expression of the gene.

In some alternative embodiments, the INSRR gene used in the preparation of a product for regulating granulosa cells comprises the sequence shown in SEQ ID No. 1; or a sequence having at least 90% homology with the sequence shown in SEQ ID NO. 1. Wherein a sequence having more than 90% homology refers to a sequence which is not completely identical to a target sequence due to deletion, insertion or change of nucleotides, but has at least 90% similarity and has the same function as the target sequence. Sequences with more than 90% homology to the rest of the text are likewise understood.

In the above embodiments, "comprising" means that the gene may contain, in addition to a sequence having a sequence as shown in SEQ ID NO.1 or having 90% or more homology with SEQ ID NO.1, other functional units such as one or more of a promoter, a terminator, an enhancer and a transcription factor binding region; may also contain sequences encoding one or more of markers, tags and resistance genes; vector sequences and the like can also be included; or cDNA of the gene with the sequence shown in SEQ ID NO.1 or with more than 90 percent of homology with SEQ ID NO. 1. The other genes "comprising" as shown in the sequence listing are also understood in the same way and are not described in detail.

Based on the inventive concept of the application of the INSRR gene in the preparation of products for regulating granulosa cells, the invention also provides a product for regulating granulosa cells, wherein the product comprises a substance for over-expressing the INSRR gene and/or a substance for inhibiting the expression of the INSRR gene, and the regulation of granulosa cells is realized by regulating the expression of the INSRR gene.

The product for regulating and controlling the granular cells provided by the invention comprises but is not limited to reagents, kits, medicaments or culture media and the like. The product may achieve regulation of the INSRR gene by comprising a substance capable of up-regulating the INSRR gene and/or down-regulating the INSRR gene. The substances in the product for up-regulating the INSRR gene and down-regulating the INSRR gene respectively and independently comprise at least one of DNA, RNA, protein, polypeptide, compound drugs, cells and microorganisms. Examples may include, but are not limited to: siRNA, in order to interfere INSRR gene expression, play the role in lowering INSRR gene expression, said siRNA preferably contains as shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, more preferably contains as shown in SEQ ID NO.4 sequence; a plasmid expressing the siRNA; a cell or recombinant microorganism containing said siRNA or said plasmid; a vector, such as a DNA plasmid or viral vector, capable of expressing the INSRR gene to achieve overexpression of the INSRR gene; a polypeptide or protein capable of promoting or inhibiting the INSRR gene, such as a transcription factor or an inhibitor of a transcription factor; compounds that promote or inhibit the INSRR gene, such as inhibitors or agonists that interfere with the signaling pathway of INSRR gene expression.

Based on the regulatory effect of the INSRR gene on granulosa cells found in the present invention, the present invention also provides a method for regulating granulosa cells, comprising up-regulating or down-regulating the expression of the INSRR gene in granulosa cells. The regulation of granulosa cells according to the present invention includes the following aspects: regulating granular cell apoptosis; regulating expression of granulosa cell genes, the regulated genes including at least one of IGF1R gene, LHR gene, FSHR gene, and ER β; regulating secretion of hormones by granulosa cells, said hormones including E₂、P₄At least one of ACT, INH and FS. The regulation method of the granular cells can be realized by at least one of the following embodiments:

in some alternative embodiments, the expression level of the INSRR gene is up-regulated to achieve at least one of the following regulatory effects: inhibiting granular cell apoptosis; up-regulating IGF1R gene expression level; down-regulating the expression level of at least one of LHR gene, FSHR gene and ER beta gene; promotion of E₂、P₄And secretion of at least one of ACT; and, inhibiting secretion of at least one of INH and FS.

In some alternative embodiments, the expression level of the INSRR gene is downregulated such that at least one of the following regulatory effects is achieved: promoting granular cell apoptosis; down-regulating the expression level of IGF1R gene; up-regulating the expression level of at least one of LHR gene, FSHR gene and ER beta gene;inhibition of E₂、P₄And secretion of at least one of ACT; and, promoting secretion of at least one of INH and FS.

It should be noted that the present invention is not limited to regulating the expression of the INSRR gene in the granulosa cell and simultaneously regulating the expression of other genes in the granulosa cell, so as to achieve regulation of other genes, hormones, or other physiological and biochemical activities in the granulosa cell.

In the above examples, the regulation and control method can simultaneously regulate and control the expression of the HR gene, FSHR gene, ER β gene and IGF1R when regulating and controlling the expression of the genes in the granulosa cells; it is also possible to regulate the expression of only one, two or three of the genes: for example, but not limited to, the method may be a method for down-regulating the expression of the INSRR gene and simultaneously down-regulating at least one of the LHR gene, FSHR gene and ER β gene, so as to achieve down-regulation of the expression level of IGF1R gene without changing the expression level of one or more of the LHR gene, FSHR gene and ER β gene.

In the above examples, the modulation method is used to promote or inhibit E₂、P₄Secretion of at least one of ACT, INH and FS may be regulated simultaneously or only one, two, three or four of these hormones. For example, but not limited to, it may be desirable to up-regulate the expression of the INSRR gene to promote E₂、P₄And secretion of ACT, while up-regulating genes that promote secretion of INH and FS, to avoid inhibition of secretion of INH and FS.

In some alternative embodiments, the INSRR gene used in the preparation of a product for regulating granulosa cells comprises the sequence shown in SEQ ID No. 1; or a sequence having at least 90% homology with the sequence shown in SEQ ID NO. 1.

Based on the proliferation, differentiation and apoptosis of granulosa cells and the development effect of hormones on follicles and ovaries, the regulation method of granulosa cells provided by the invention or the product for regulating granulosa cells can also be applied to the following aspects: (x1) regulating follicular development; (x2) regulating ovarian development; (x3) preparing a product for regulating follicular development; (x4) preparing a product for regulating ovarian development. By obtaining the method or the product for regulating and controlling the granulosa cells, the follicles and the ovaries, the understanding of the maturation mechanism of the oocytes at the later stage of follicular development is deepened, for example, the method for regulating and controlling the granulosa cells provided by the application interferes with the development of the granulosa cells, the follicular development can be indirectly controlled, and a technical guarantee is provided for the maturation matrix of the follicles; by regulating the expression of genes or hormones in granulosa cells through the regulating method or the product, factors related to follicular development can be further excavated, a signal path related to follicular development is perfected, and a mechanism of follicular development is disclosed. As the granular cells play a key role in the development of follicles, the regulation and control of the granular cells can optimize an oocyte in-vitro culture system, have important significance in the development of superovulation of mammals, embryo in-vitro production, multi-fetus variety breeding and the like, and provide technical and material guarantee for livestock breeding and the screening of livestock with excellent fertility.

The invention is not limited to the use of the INSRR gene in the preparation of products for the regulation of granulosa cells, methods of regulation of granulosa cells and the sources of granulosa cells, follicles and ovaries involved in their use, examples of sources including, but not limited to, one or more of cattle, horses, cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink and humans.

In some alternative embodiments the INSRR gene comprises the sequence shown in SEQ ID NO. 1; or, the sequence containing at least more than 90% homology with the sequence shown in SEQ ID NO.1 is derived from sheep, so that the sources of granulosa cells, follicles and ovaries are also corresponding to the optimized sheep, which has important significance for further carrying out sheep superovulation, lamb superovulation, sheep embryo in-vitro production, multiparous sheep variety breeding and the like, and is beneficial to the development of sheep breeding industry.

The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments. The experimental methods used in the examples of the present invention are all conventional experimental methods in molecular biology, and can be performed according to conventional molecular biology experimental manuals or kit instructions.

Example 1

INSRR gene siRNA interference:

3 pairs of interference fragments are designed aiming at the INSRR gene of sheep, the three pairs of fragments respectively and independently contain sequences shown in SEQ ID NO. 2-4 in sequence, and 3' ends respectively and independently overhang unpaired TT is shown as follows:

INSRR siRNA-1(968)：5’-CCAACCACAUUGUGGGCAAtt-3’(SEQ ID NO.5)；

INSRR siRNA-2(1988)：5’-GCAUCAACUUGGAGUACUUtt-3’(SEQ ID NO.6)；

INSRR siRNA-3(3720)：5’-CCUUGCUGCCAACAAGUUUtt-3’(SEQ ID NO.7)。

the relative INSRR expression levels after 3 pairs of interfering fragments were 65.82. + -. 3.57%, 69.87. + -. 3.56% and 46.44. + -. 3.70% of the blank, respectively, as shown in FIG. 1, and therefore, the 3 rd pair of interfering fragments were selected for subsequent cell transfection experiments.

"control" in FIG. 1 represents an untreated control group; "INSRR" represents the interfered group of INSRR genes. "siRNA-1" represents the INSRR gene siRNA-1 treated group; "siRNA-2" represents the INSRR gene siRNA-2 treated group; "siRNA-3" represents the INSRR gene siRNA-3 treated group. Data are presented as mean ± SEM, statistical analysis using t-test. Indicates significant difference, p < 0.05.

Example 2

Effect of INSRR gene on apoptosis and proliferation of sheep granulosa cells:

the apoptosis condition of sheep granulosa cells after INSRR gene interference is detected through flow apoptosis detection, and the result is shown in figure 2, wherein 'siINSRR' represents an INSRR gene siRNA interference treatment group, and 'control' represents an untreated control group, and the INSRR gene interference group granulosa cell apoptosis rate is 16.03 +/-0.54% and is remarkably higher than that of a blank control group (6.98 +/-0.07%).

The MTT method is used for detecting the sheep granulosa cell proliferation rate after INSRR gene interference, and the results are shown in fig. 3 and 4, wherein the cell proliferation rate after INSRR gene interference is not obviously changed. "control" in FIGS. 3 and 4 represents an untreated control group; "INSRR" represents the interfered group of INSRR genes. Data are presented as mean ± SEM, statistical analysis using t-test. Indicates significant difference, p < 0.05.

Example 3

And (3) detecting the gene expression quantity of LHR gene, FSHR gene and ER beta gene related to oocyte development in the sheep granulosa cell after INSRR gene interference. The INSRR gene was used as a receptor gene related to the insulin receptor, and the expression level of IGF1R gene was detected.

After INSRR gene interference is found, the expression level of IGF1R gene in granulosa cells is obviously reduced, and the expression levels of LHR gene, FSHR gene and ER beta gene related to follicular development are all obviously increased. It is shown that INSRR gene exerts inhibitory effect on expression of LHR gene, FSHR gene and ER beta gene in sheep granulosa cells, and expression level of IGF1R gene is also affected by interference of INSRR gene, as shown in FIG. 5.

"control" in FIG. 5 represents an untreated control group; "si-INSRR" represents the interference handling group. "IGF 1R" represents the expression level of IGF1R gene; "LHR" represents the expression level of LHR gene; "FSHR" represents the amount of FSHR gene expression; "ER β" represents the expression level of ER β gene; data are presented as mean ± SEM, statistical analysis using t-test. Indicates significant difference, p < 0.05.

Example 4

INSRR gene on sheep granulosa cell E₂、P₄Regulation of ACT, INH and FS secretion:

the supernatant in the pellet cell culture dish after the interference is subjected to E through an ELISA method₂、P₄ACT, INH and FS detection, as shown in fig. 6 to 10. Finding granulosa cells E₂、P₄And secretion of ACT decreased significantly, and secretion of INH and FS increased significantly. Indicates that the INSRR gene can play a function in the sheep ovarian granulosa cells and is used for E in the cells₂、P₄And ACT secretion, and has inhibitory effect on INH and FS secretion in sheep granulosa cells. "control" in FIGS. 6 to 10 represents an untreated control group; "INSRR" represents the interference handling group. Data are expressed as mean. + -. SEM, using t-testAnd (5) carrying out statistical analysis. Indicates significant difference, p < 0.05.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Jilin province academy of agricultural sciences

<120> application of INSRR gene, and product and method for regulating and controlling granulosa cells

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 3906

<212> DNA

<213> sheep (Ovis aries)

<400> 1

atggccgtgc ccagcctgtg gccctgggtg gcttgcctgc tggccgtgct gctgagcctg 60

ggctttgggc tggacacccg ggaggtgtgc ccctctctgg acatccgttc cgaagtggcc 120

gagctgcgca ggctcgagaa ctgttctgtg gtggaaggcc atctgcagat cctgctgatg 180

tttaccgcca ccggcgaaga ctttaggggc ctgagcttcc ccaggctgac ccaggtgacc 240

gactacctgc tgctgttccg cgtgtatggt ctggagtccc tgcgcgacct gttccccaac 300

ctggccgtga tccgcggcgc cagactgttc ctgggctacg ccctcgtgat ctttgagatg 360

ccccatatga gggacgtggg tctgcccgct ctgggcgctg tgctgcgtgg tgctgtgagg 420

gtggagaaga accaggagct gtgccatctc agcaccatcg actggggact gctgcagccc 480

agccccggag ctaaccatat cgtggggaac aaactggggg aggaatgtgc cgacgtgtgc 540

cccggactgc tcggcgctac cggagagcca tgcgccagga ccacctttag cggccatacc 600

gactaccgat gctggacctc tagccactgc cagaaggtgt gcccctgccc caggggtctc 660

gcctgtaccg tgaggggaga atgctgccat accgagtgcc tgggcggatg tagccgccct 720

gaggacccta gggcctgcgt ggcttgtagg catctgtact tccagggcgc atgtcataga 780

gcctgccctc caggcaccta ccagcacgaa agctggaggt gcgtgaccgc cgagaggtgt 840

gccagcctga gaagcgtgcc cggcagggct tccaccttcg gaatccatca gggcagctgt 900

ctggctcagt gtcctcccgg cttcacccgg aacgacagca gcatcttttg tcataagtgc 960

gagggcctgt gccctaaaga gtgcaaagtg ggcaccaaga ccatcgacag cgtgcaggcc 1020

gcccaggacc tggtgggatg tacccatgtg gagggaagcc tgatcctgaa cctgaggcag 1080

ggatataacc tcgagctgga gctgcagagg agcctgggcc tggtggaaac catcaccgga 1140

ttcctgaaaa tcaagcatag ctttgctctg gtgagtctcg gctttttcaa aaacctcaag 1200

ctgatcaggg gggacaccat ggtggacgga aactataccc tgtacgtgct ggacaaccag 1260

aacctgcagc agctcggcag ttgggtggct gccggcctga ccatccccgt gggaaaaatc 1320

tacttcgctt tcaaccccag gctgtgcctg gagcatatct accgcctcga ggaggtgacc 1380

ggcaccaaag gcaggcagaa caaggccgaa atcaaccccc gcaccaacgg cgaccgcgcc 1440

gcttgccaga ccagaaccct caggttcgtg agcaacgtga ccgaagccaa caggatcctg 1500

ctgaggtggg aaaggtacga acccctggag gccagggacc tgctgagttt tatcgtgtat 1560

tacaaggaga gccccttcca gaacgccacc gagtatgtgg ggcccgacgc ctgcggcgct 1620

cagagctgga acctgctgga cgtggagctg ccactgtcca ggacccagga gcccggcgtg 1680

accctggcac ctctgaaacc ctggacccag tacgccgtgt tcgtgagggc catcaccctc 1740

accaccgccg aagacagccc tcatcaggga gcccagagcc ccatcgtgta cctgcgcacc 1800

ctgcccgccg cccctaccgt gcctcaggac gtgatcagca ccagcaacag cagcagccac 1860

ctgctggtga gatggaagcc ccccatccag aggaacggca acatcaccta ctacctcgtg 1920

ctctggcagc gcctggctga ggacagcgac ctctacctga acgactactg ccacagaggc 1980

ctccgcctgc ccacctccaa caacgacccc cgcttcgacg gcgaagacgg cgaactggaa 2040

gccgacaggg aacccggatg ctgcccctgc cagcatccac cccccggaca ggtgctgccc 2100

cctctggaag cccaggaggc cagcttccag aaaaagttcg agaacttcct gcataacgcc 2160

atcaccatcc ccaagagccc ctggaaggtg accagcatca acaagagccc ccagcgcgac 2220

agcggaagga gcaggcgagc cgctgaggcc ctgaggctgg gaggtaacag cagcgacttc 2280

aaaatccagg aagacaaggt gccaagggaa agagcagtgc tgagcggcct gaggcatttc 2340

accgagtaca ggatcgacat ccatgcctgc aaccatgctg cacacaccgt gggatgcagc 2400

gccgccacct tcgtgtttgc caggaccatg ccccataggg aagctgacgg aatccccggc 2460

aaggtggcct gggaagcagc cagtaagagc tccgtgctgc tccgctggct ggagccccct 2520

gaccctaacg gtctgatcct gaagtacgag atcaaataca ggaggctggg cgaggaggca 2580

accgtgctgt gcgtgagtag gctgaggtac gctaaattcg gcggcgtgca tctggcactg 2640

ctgccacccg gcaactacag cgccagggtg agggccacca gcctggctgg aaacggaagc 2700

tggaccgaca gtgtggcatt ctacatcccc ggccccgagg aggaggactc cggaggactg 2760

catgtgctgc tgaccgtgac ccccgtggga ctgatgctgc tgatcatcct ggccgccctg 2820

ggctttttct attccaagaa gcgcaactcc accctgtatg ccagcgtgaa ccccgaatac 2880

ttctccgcct cccatatgta catccccgac gagtgggaag tgccccgcga acagatcagc 2940

atcatccgcg aactgggaca gggcagcttc ggcatggtgt acgagggcct ggcccagggc 3000

ctggaagtgg gagaggagcc cacccccgtg gctctgaaga ccgtgaacga gctggccagc 3060

cccagggaga ggatcgaatt tctgaaggaa gccagtgtga tgaaagcctt taaatgtcat 3120

catgtggtga ggctgctggg cgtggtgagt cagggacagc ccaccctggt gatcatggaa 3180

ctgatggcaa ggggagacct gaagagccat ctgaggagcc tgaggcccga ggccgaaaac 3240

aaccccggac tccccaggcc cgccctggga gacatgatcc agatggctgg cgagatcgct 3300

gacggaatgg cctacctggc cgccaacaaa ttcgtgcata gggacctggc cgcccgaaac 3360

tgcatggtga gccaggactt taccgtgaaa atcggcgact tcggaatgac cagggacgtg 3420

tatgagaccg actactaccg caaaggcggc aaaggactgc tgcccgtgag gtggatggct 3480

cccgagagcc tcaaggacgg catcttcacc acccatagcg acgtgtggag cttcggagtg 3540

gtgctgtggg agatcgtgac cctggctgag cagccctatc agggactgag caacgaacag 3600

gtgctgaaat tcgtgatgga cggcggagtg ctggaggagc tggagagctg tcctgtgcag 3660

ctgcaggagc tgatgcagag gtgttggcag cagaacccaa ggctgaggcc taccttcacc 3720

catatcctgg acagcatcca ggaggagctg aggcccagct ttaggctgct gagtttctac 3780

cattcccccg gctgcagagg tgctagggca tccctccccc ccaccgacgc tgagcctgac 3840

agccctagaa cccccaaaga ggccagcagc gactttagcc cccagaacgg cggccccggc 3900

cattag 3906

<210> 2

<211> 19

<212> RNA

<213> Artificial sequence

<400> 2

ccaaccacau ugugggcaa 19

<210> 3

<211> 19

<212> RNA

<213> Artificial sequence

<400> 3

gcaucaacuu ggaguacuu 19

<210> 4

<211> 19

<212> RNA

<213> Artificial sequence

<400> 4

ccuugcugcc aacaaguuu 19

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<400> 5

ccaaccacau ugugggcaat t 21

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence

<400> 6

gcaucaacuu ggaguacuut t 21

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<400> 7

ccuugcugcc aacaaguuut t 21

Claims

1. Regulation and controlINSRRApplication of gene expressed substance in preparing product for regulating and controlling sheep granule cell apoptosis.

2. Use according to claim 1, characterized in that down-regulation is carried outINSRRThe expression quantity of the gene promotes the apoptosis of the sheep granulosa cells.

3. Use according to claim 1 or 2, characterized in that saidINSRRThe gene has a sequence shown as SEQ ID NO. 1.

4. A method of modulating apoptosis in a non-therapeutic sheep granulosa cell comprising down-regulating apoptosis in a sheep granulosa cellINSRRExpression of the gene.

5. The method of claim 4, wherein the down-regulation isINSRRThe expression quantity of the gene promotes the apoptosis of the sheep granulosa cells.

6. The method of claim 5, wherein the downregulating comprises downregulating using siRNAINSRRAnd (4) expressing the gene.

7. The method of claim 6, for down-regulationINSRRThe siRNA expressed by the gene has a sequence shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4.

8. The method of claim 7, for down-regulationINSRRThe siRNA expressed by the gene has a sequence shown in SEQ ID NO. 4.

9. The method of any one of claims 4 to 8, wherein the method is performed in a single cycleINSRRThe gene has a sequence shown as SEQ ID NO. 1.