Application of trichostatin A (TSA) to preparation of medicament for inhibiting activities of swine ovary granular cells

A technology of trichostatin and granulosa cells, used in drug combinations, sexual diseases, etc.

Inactive Publication Date: 2012-02-01
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no relevant reports on the effect of TSA on granulosa cells

Method used

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  • Application of trichostatin A (TSA) to preparation of medicament for inhibiting activities of swine ovary granular cells
  • Application of trichostatin A (TSA) to preparation of medicament for inhibiting activities of swine ovary granular cells
  • Application of trichostatin A (TSA) to preparation of medicament for inhibiting activities of swine ovary granular cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] experimental animals

[0025] The experimental animals of the present invention are general commercial sows provided by Nanjing Tianhuan Pig Slaughterhouse.

[0026] Reagent

[0027] DMEM / F12 culture dry powder: GIBCO Company; newborn fetal bovine serum: Lanzhou Minhai Company;

[0028] MTT Cell Proliferation and Cytotoxicity Detection Kit: KGI; Antibiotics: Sigma;

[0029] KCl, K 2 HPO 4 , NaCl, Na 2 HPO 4 Other chemical reagents are analytically pure: Sinopharm Chemical Reagent Co., Ltd.

[0030] 1.1 Collection of porcine ovaries and selection of follicles

[0031] Pig ovaries were collected at Nanjing Tianhuan Slaughterhouse, placed in 37°C normal saline, brought back to the laboratory within 2 hours, and washed three times with preheated PBS supplemented with penicillin and streptomycin (Sigma). Granulosa cells in follicles were selected as experimental materials.

[0032] 1.2 Obtaining porcine ovary granulosa cells

[0033] Granulosa cells were obtained f...

Embodiment 2

[0050] reagent

[0051] Trizol, RNase-free glycogen: Invitrogen; M-MLV Reverse Transcriptase: Promega; DEPC: Sigma; 2×Taq PCR Master Mix: Nanjing Tiangen Biotechnology Co., Ltd.; Oligo dT, dNTP MixtureRNase inhibitor, SYBR Premix Ex TaqTM: Takara company; DNA marker: Takara company; Boric acid, agarose, Tris, bromophenol blue: Nanjing Dazhi Biological Co., Ltd.; chloroform, isopropanol, anhydrous ethanol are all Analytical grade: Sinopharm Chemical Reagent Co., Ltd.

[0052] Solution preparation

[0053] (1) 10×TBE buffer storage solution: Tris 108.99g, boric acid 55.62g, EDTA 29.23g dissolved in 1000mL double-distilled water, pH 8.0-8.2, autoclaved, and stored at 4°C or room temperature.

[0054] (2) Bromophenol blue solution: dissolve 0.25 g of bromophenol blue in 100 mL of sterile double-distilled water, and store at 4°C or room temperature.

[0055] (3) 2× loading buffer: Tris-HCl 100mmol / L pH6.8, SDS 4.0%(W / V), bromophenol blue 0.2%(W / V), glycerol 20%(V / V), DTT 200mM....

Embodiment 3

[0083] Reagents and Kits

[0084] DMEM / F12 medium: Gibco BRL Company; Fetal Bovine Serum: Lanzhou Minhai Serum Co., Ltd.; Trypsin, EDTA·Na 2, Penicillin G, streptomycin sulfate: Sigma; Trizol, RNase-free glycogen: Invitrogen; M-MLV Reverse Transcriptase: Promega; Oligo dT, dNTP MixtureRNase inhibitor, SYBR Premix Ex TaqTM: from Takara; 2× Taq PCR MasterMix: Nanjing Tiangen Biotechnology Co., Ltd.; boric acid, agarose, Tris, bromophenol blue: Nanjing Dazhi Biological Co., Ltd.; DEPC: SIGMA Company; NaCl, KCl, NaHCO 3 、KH 2 PO4, Na 2 HPO4, DMSO, chloroform, isopropanol, absolute ethanol: Sinopharm Chemical Reagent Co., Ltd. Cell cycle detection kit; Nanjing KGI Biotechnology Co., Ltd.

[0085] 3.1 The effect of TSA on the cycle of ovarian granulosa cells detected by flow cytometry

[0086] (1) Inoculate the granulosa cells in the logarithmic growth phase in a culture flask, and treat the porcine ovary granulosa cells with 0ng / mL, 10ng / mL and 200ng / mL TSA when the cells grow...

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Abstract

The invention belongs to the field of medicaments and veterinary medicines and discloses application of trichostatin A (TSA) to preparation of a medicament for inhibiting activity of swine ovary granular cells. In the invention, the influence of the TSA to the activities of the swine ovary granular cells is systemically studied, and a result shows that the TSA has no influence to the growth of the swine ovary granular cells at low concentration (below 10ng / mL), with the increase of the concentration, the growth of the swine ovary granular cells is inhibited (P<0.05), and the inhibition effect is dose-dependent. After the granular cells are treated by using 200ng / mL TSA, the cell cycle is retardant within a period G0 / G1, and the marked decrease of gene expression of Cyclin D2 and CDK4 (Cyclin-Dependent Kinase 4) is possible to be an important reason for the cycle retardance. Accordingly, the TSA can be applied to preparation of the medicament for inhibiting the activities of the swine ovary granular cells.

Description

technical field [0001] The invention belongs to the field of drugs and veterinary drugs, and relates to the application of trichostatin A in the preparation of drugs for inhibiting the activity of porcine ovary granulosa cells. technical background [0002] Granulosa cells are very important somatic cells in the ovary, which synthesize and secrete hormones and various growth factors at different stages of development, and exert their effects through autocrine / paracrine pathways. The growth and differentiation of granulosa cells play a vital role in the growth and development of mammalian follicles, especially in the maturation of oocytes. Follicle development starts from primordial follicles, passes through primary follicles, secondary follicles and antral follicles and finally becomes mature follicles. [0003] In each estrous cycle, a large number of follicles start to grow in the ovary of mammals, but most of them fail to mature, and gradually degenerate during each deve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/165A61P15/08
Inventor 徐银学陈侠赵永艳张宝乐
Owner NANJING AGRICULTURAL UNIVERSITY
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