Establishment Of Cow Endometrial Immortalized Cell Lines

The endometrium as the wall layer of the uterus has the functions of promoting the cycle of estrous cycle,gestation of the fetus,endocrine,etc.When the pathogenic microorganism invades the uterus,the endometrium not only acts as a physical barrier against microbial invasion,but also activates the local innate immune system of the uterus to recover self-steady state.The endometrial structure is complex and the function is staggered,which makes it difficult to systematically elucidate the mechanisms of physiological and pathological processes such as pregnancy and endometritis.Although the study of primary cells as the model can be the most realistic approach to the in vivo,the primary cells still have shortcomings such as time consuming,laborious,and large individual differences.Therefore,the establishment of stable passage of immortalized cow endometrial cell lines is of great significance for the study of the complex function of the endometrium.In this experiment,the human telomerase reverse transcriptase(hTERT)was transferred into cow endometrial stromal cells(ESCs)and epithelial cells(EECs),and the immortalized cow endometrial stromal cell line and epithelial cell line were successfully screened.The function of the establishment cell line was tested by detecting keratin,vimentin,cell growth curve analysis,determination of telomere length,karyotype analysis,estrogen and progesterone stimulation.The results are as follows:(1)Cow ESCs and EECs were obtained by 0.25% trypsinization and tissue block culture.The morphology and cell type of the two isolated cells were identified by microscopic morphology observation and cell immunofluorescence.The results showed that the ESCs were fusiform and adherent.EECs showed typical paving stones and agglomerated adherent growth;vimentin expression in cow ESCs was positive,and keratin expression in EECs was positive.Proven to successfully obtain high purity ESCs and EECs.(2)The pCI-neo-hTERT plasmid was transferred into cow endometrial stromal cells,and the positive clones of endometrial stromal cells were obtained after screening for 14 days at a concentration of 500 μg/mL neomycin(G418).The positive clones were named TERT-ESCs after expanded culture;The expression of hTERT in TERT-ESCs was positive by detection of the method of cell immunofluorescence;The morphology of TERT-ESCs was fusiform and adherent growth during the passage;The results of CCK 8 analysis showed that the of TERT-ESCs was normal;The results of Serum dependence analysis showed that serum-promoted TERT-ESCs proliferation in a concentration-dependent manner;RT-PCR results showed that TERT-ESCs normally expressed normal steroid hormone receptors compared with primary ESCs.Estrogen was concentration-dependent on proliferation of TERT-ESCs;The low concentration of progesterone promoted the proliferation of TERT-ESCs,and the high concentration of progesterone inhibited the proliferation;The results of apoptosis analysis showed that the apoptosis rate of TERT-ESCs was parallel to that of primary cells;Karyotype analysis showed that the number of chromosomes remained normal during the process of infinite passage.TERT-ESCs have been passaged to the 50 th generation,indicating that the hTERT gene can maintain cells telomeres to promote immortalization.(3)The pCI-neo-hTERT plasmid was transferred into endometrial cow epithelial cells,and the positive clones of endometrial epithelial cells were obtained after 14 days of screening with G418 at a concentration of 400 μg/mL.The positive clones were named TERT-EECs after expanded culture;The expression of hTERT in TERT-EECs was positive by detection of the method of cell immunofluorescence;During the passaged,TERT-EECs vimentin was still positive;TERT-EECs still maintained good proliferative activity;serum concentration was dependent;The results of RT-PCR showed that TERT-EECs can also express steroid hormone receptors normally compared with primary EECs;Estrogen and progesterone can promote cell proliferation and inhibit cell proliferation respectively;Karyotype analysis showed that the number of chromosomes remained normal during the passage of EECs transfected with hTERT.In summary,we successfully acquired immortalized cow endometrial stromal cell lines and epithelial cell lines by transferred into pCI-neo-hTERT plasmids.A series of assays showed that the two cell lines could be infinitely passaged in vitro and functioned normally,which proved that the obtained cell lines could be used in later experiments.