Effect Of GABRP On The Proliferation Of Buffalo Mammary Epithelial Cells And Their Expression Of Genes Related To Lactation

Gamma-amminobutyric acid is an important components of inhibitory neurotransmitters in the brain, which plays an important role in the transmission of nerve signals. Besides brain tissue, gamma-amino acid and its receptors are also present in many kinds of peripheral tissues. There are three types of GABA receptors （GABARs）:GABAARs/GABABRs/GABAcRs. The GABAaRs and GABAcRs are Cl--selective heteropentameric channels and GABAbRs are G-protein coupling metabotropic receptors. GABRP was a new type of GABAA receptor subunit. Recently, there were many studies about GABRP in cancer tissues, which could regulate the proliferation and migration of tumor cells, but few studies on the role of the peripheral tissues. In the breast tissue, GABRP was mainly related to the migration of breast cancer cells while no research was on the proliferation and differentiation of mammary epithelial cells. Our lab found that GABRP was a target gene of Stat5a and the expression level of GABRP was significantly increased during late pregnancy of mouse. So it was supposed that π subunit may play an important role in the development of breast and lactation. This study aimed to observe the effect of GABRP on mammary epithelial cell proliferation and expression of genes associated with lactation, which was a preliminary understanding of function of GABRP at cellular level, laid a foundation for the further understanding of buffalo mammary cell growth regularity and lactation mechanism.1. Cloning of buffalo GABRP gene and its expression in buffalo mammary tissue. The specific primers was designed and synthesised referred to bovine gabrp sequence （NM_<sub>001015618.1） in the GenBank database. buffalo mammary tissue total RNA as a template, The cDNA of GABRP gene was amplified by RT-PCR. Results showed that the obtained fragment shared 99.4% with that of river buffalo, which showed that the clone was buffalo GABRP gene; QRT-PCR technique was applied to examine the expression of GABRP gene in different periods of buffalo mammary tissue. Results showed the mRNA of GABRP gene, which in non-lactation was significantly higher than that of lactation （P<0.05）, existed in both lactation and non-lactation of buffalo mammary tissue; Immunohistochemistry was to locate of GABRP protein in lactation of buffalo mammary tissue. Results showed that GABRP protein was mostly in the alveolar epithelial cells of buffalo mammary.2. Isolation and culture of buffalo mammary epithelial cells in vitro. Fresh mammary tissue was obtained from a lactating buffalo. Tissue explant and trypsin digestion methods were used to obtain and purify mammary epithelial cells, and the specific cytoskeletal protein of breast epithelial cell, keratin 18 was identified by immunofluorescence technique. Results showed that the mammary epithelial cells were isolated from mammary explants and buffalo mammary epithelial cells had keratin 18, normal proliferation capacity.3. The effect of GAB A and GABAA receptor agonist muscimol on the proliferation of buffalo mammary epithelial cells and their expression of genes related to lactation.50μm/L, 100μm/L,500μm/L GABA,0.5μm/L,5μm/L, 50μm/L muscimol was added to medium of buffalo mammary epithelial cell. Mammary epithelial cell proliferation was analyzed by CCK8 kits. The effects of GABA and muscimol on buffalo epithelial cell cycle distribution was tested by flow cytometry; The mRNA expression level of CyclinDl, GABRP, mTOR, FABP3, GLUT of buffalo epithelial cell with GABA and muscimol treatment was analyzed by QRT-PCR. Results showed that GABA and muscimol could significantly inhibit cell proliferation significantly （P<0.05） and the inhibition efficiency was related to the concentration of muscimol and GABA and the treatment time. The flow cytometry results showed that treatment of GABA and muscimol increased the cell proportion of G1 but reduced the cell proportion of S, G2. The QRT-PCR results showed GABA and muscimol significantly reduced the expression level of CyclinD1 and GABRP and had different effects on the expression of mTOR, FABP3 and GLUT 1 gene during the lactation.In conclusion, GABA was involved in mammary development and lactation regulation process by pi subunits of GAB AA receptor.