| In this study, Xinong Saanen goat mammary gland tissue was collected for primary epithelial cell culture. With the method of tissue explants culture and high density cultivation, we isolated and cultured the diary goat mammary gland epithelial cells. Then we studied the functional characteristics of cultured cells via immunofluorescence,oil red staining and RT-PCR. We also compared several transfection methods and figured out the suitable one for the cultured cells. The cells were used as material to extract RNA for the 3'UTR cloning of H-FABP gene. The CDs and 3'UTR sequence of H-FABP gene were analysed via bioinformation method. Finally we constructed the pEGFP-N1-HFABP vector and studied the function of H-FABP gene on the cultured diary goat mammary gland epithelial cells. We believe these research work will contribute to further explaining the function and molecular regulation mechanism of H-FABP in lipid metabolism on mammary gland. To be concrete, our studies were taken based on the following aspects:1.Based on the in vitro culturing system developed for epithelial cells in mammary gland of Xinong Saanen dairy goats using tissue explants culture, high density cultivation and continuous passaging, the cultured epithelial cells were successfully grown to passage 96. The cells with the model number of chromosome of 60 demonstrated a typical'S'shape curve; the positive gene expression of keratin, EMA, vimentin andβ-casein was detected; the cytoplasmic lipid droplets were observed following the oil red staining; the cultured cells expressed the mRNA ofβ-casein. In conclusion, the current in vitro culture system can obtain the normal mammary gland epithelial cells with the function of secretion. We transfected the isolated cells with pEGFP-N1 via Lipofectamine2000 for one time, twice, respectively and also via electropotation under different parameter. Finally, we developed a better transfection system for dairy goat mammary gland epithelial cell: electropotation under 750V/cm for 1ms and 3 times. 40-50 precent cells were survival after electropotation and showed excellent growth ability; 30 precent cells could be observed strong green fluorescent.2.The complete 3'UTR of H-FABP gene was successfully cloned, which was 248bp. The homologies of CDs nucleotide sequence of H-FABP gene with the counterparts of bovine, human and mouse were 97%, 88% and 83%,and the homologies of 3'UTR nucleotide sequence were 96%, 84% and 70%, respectively. H-FABP is non transmembrane protein and has no signal peptide. The 3-dementional structure of H-FABP shows that it consists of 10 antiparallelβ-strands and 2 short helices, which forms a closed protein structure with a large cavity inside.3.We successfully generated the pEGFP-N1-HFABP eukaryotic expression vector and transfected it into the mammary epithelial cells via electroporation. After transfection, we used real time PCR to analyze the change of mRNA expression of other related genes. The results showed that after transfection, the expression of H-FABP mRNA was increased to 29.97 fold. The mRNA of AFABP and LXR gene were both up-regulated to 1.22 fold and the mRNA of TIP47 gene were up-regulated to 1.32 fold while the HSL gene was unaffected. This result suggested there is relationship between the mRNA level of H-FABP gene and AFABP, LXR or TIP47 gene.
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