Isolation,Culture And Immortalization Of Chicken Oviduct Epithelial Cells

Chicken oviduct epithelium can produce a large amount of albumin protein in the daily cycle,which provides an effective model for the production of pharmaceutical proteins,making it a hot spot for research.At present,the research progress on the fallopian oviduct bioreactor model is relatively slow,which results from the short of stable immortalized chicken oviduct epithelial cells(c OECs),being used to study the secretion mechanism of the fallopian oviduct epithelial cells.The purpose of this study was to isolate and culture c OECs firstly,then to transform human telomerase reverse transcriptase(human telomerase reverse transcriptase,h TERT)into c OECs to obtain immortalized cell lines,and subsequently to identify their biological characteristics.In addition,the effects of estrogen and progesterone on the secretion of ovalbumin were evaluated in the immortalized c OECs.1.Isolation,culture and identification of chicken oviduct epithelial cells.Primary cOECs were isolated by using 1%dithiothreitol(DTT)to dissolve mucus,0.25%Dispase enzyme to digest the minced fallopian oviduct membrane tissue,and differential adherent culture to purify c OECs.Immunofluorescence(IF)staining of c OECs markers,which include ovalbumin(OVA),Estrogen Receptor alpha 1(ESR1),cytokeratin 18(CK-18),progesterone receptor(PGR),was conducted,and other cOECs markers including OVA,lysozyme(LYZ),follicular mucus(OVM),and avidin(AVD)were also detected with RT-PCR.The results showed that(1)Using 1%DTT to dissolve the mucus on the surface of the fallopian oviduct membrane,0.25%Dispase to digest the minced oviduct membrane tissues,and differential adherent culture to purify c OECs,is an effective method for isolating c OECs.(2)The c OECs are not only polygonal,cobblestone-like cells,representing a typical epithelial cell-like morphology,but also adherent ones with contact inhibition.(3)The c OECs were positive to IF staining of OVA,ESR1,keratin 18 and PGR.(4)The m RNA expression of other c OECs markers,or OVA,LYZ,AVD and OVM,was detected in the c OECs by means of RT-PCR.2.Retrovirus-mediated immortalization of chicken oviduct epithelial cells.The retrovirus plasmids pBABE-puro-hTERT and pCMV-VSV-G were co-transfected PLAT-GP cells,the cell culture medium was collected,the retrovirus was concentrated by ultracentrifugation,and the titer was determined by conventional methods.Retroviral infection of primary c OECs was conducted,48 hours of infection,cell screening was done with 1μg/m L puromycin for 8days,and then expansion and subculture of monoclonal c OECs was performed,and the cells were cryopreservated.The h TERT expression was detected in the immortalized c OECs using RT-PCR,Western blot and IF staining.Using the same methods as in the previous chapter,the expression of c OECs markers was determined in the immortalized c OECs.The growth curve of immortalized c OECs was graphed,and its proliferation activity was determined using EDU labeling method.Finally,the soft agar cloning test was conducted to verify the tumorigenesis of the immortalized c OECs.The results show that(1)obtained by random selection,one immortalized c OECs line could be continuously subcultured for more than 50 passages;(2)the h TERT expression was verified in the immortalized c OECs;(3)the immortalized c OECs are epithelial cell-like in morphology;(4)the immortalized c OECs are positive not only to IF staining of the surface markers including ESR1,OVA,keratin 18 and PGR,and RT-PCR analysis indicated that other c OEC markers,including OVA,LYZ,AVD and OVM;(5)the proliferative activity of the immortalized c OECs is consistent with that of the primary c OECs,maintaining a normal level of division;(6)the soft agar cloning test illustrated that the immortalized c OECs are suspending in the soft agar,not dividing and proliferating,which was oppositive to the normal c OECs,indicating that immortalized c OECs are not tumorigenic.3.Preparation of chicken ovalbumin polyclonal antibodies and the effects of estrogen and progesterone on the secretion of fallopian oviduct epithelial cells.Separated from the eggs,Ovalbumin was mixed with the immune adjuvants and emulsified,and then injected subcutaneously into rabbits for 3 times at an interval of 2 weeks.One week after the third immunization,blood was collected to separate anti-serum,or ovalbumin polyclonal antibodiy,which preliminarily purified by ammonium sulfate precipitation.With addition of estrogen or progesterone alone,or together to the culture medium of the immortalized c OECs,the ovalbumin content in the culture medium was monitored by ELISA at time points of 24 and 48 hours of culture,evaluating effects of estrogen and progesterone on the secretion of ovalbumin.The results show that(1)the titer of ovalbumin polyclonal antibody is 1:2.56×10~4;(2)Both estrogen and progesterone can stimulate ovalbumin secretion,the effect of estrogen is stronger than that of progesterone,and the effect of the two hormens is better than that of a single alone.In conclution,this study established an effective method for the isolation and cultivation of chicken c OECs,and successfully obtained immortalized c OECs with expression of h TERT,which maintain the characteristics of c OECs.In addition,a chicken ovalbumin polyclonal antibody was prepared,and ELISA analysis proved that both estrogen and progesterone could promote the immortalized cOECs to secrete ovalbumin.