Preliminary Study On The Establishment Of Immortalized Cell Line Of Mongolian Horse Fetal Fibroblasts

The life span of most cultured cells in vitro is limited.When the survival state of cells is not good,it will affect the progress of research,and some cells are difficult to obtain due to sampling and purification.Therefore,the establishment of immortalized cells has become a means to provide stable cell lines for scientific research.Horse is a difficult animal model for sampling,and with the development of our team ’s horse breeding and developmental biology related topics,obtaining immortalized horse cell lines has become a research demand.Therefore,in this study,we overexpressed human telomerase reverse transcriptase(hTERT)in primary cultured Mongolian horse fetal fibroblast(EFF),which was proved effective by multiple species.The possibility of establishing immortalized Mongolian Horse Fibroblast Cell Line to lay the foundation for subsequent research was explored.This study is mainly composed of the following three parts :1.The 34-day-old horse fetus was obtained by the technology of hedging embryo.The Mongolian horse EFFs with irregular triangle,spindle-shaped,oval nucleus,clear edge,vigorous growth,vortex-shaped or parallel arrangement were purified by collagenase IV digestion and separation method.It was proved by in vitro culture that the Mongolian horse EFFs could be normally passaged for 17 generations.At the same time,it paved the way for subsequent experiments.We used the purified Mongolian horse EFFs to determine the optimal screening concentration of hygromycin B on this cell was 50 μg / m L.2.The pLV-hTERT-IRES-hygro lentivirus expressing hTERT and hygromycin resistance was used to infect Mongolian horse EFFs,and the positive cells hTERT-EFFs were obtained after screening with 50 μg / m L hygromycin B.Microscopic observation showed that the cell morphology was normal,and RT-PCR detection showed that the infection was successful,and exogenous hTERT m RNA could be stably expressed in cells.The cells can be passaged for more than 30 generations,effectively extending the number of cell passages in the existing laboratory in vitro culture system.However,the growth curve measured by CCK-8 method showed that the proliferation ability of hTERT-EFFs was significantly lower than that of primary EFFs(P < 0.05),and the cell apoptosis detected by flow cytometry also showed that the apoptosis rate of hTERT-EFFs was significantly higher than that of primary EFFs(P < 0.05).Similarly,the detection of cell senescence by β-galactosidase staining also showed that the positive rate of hTERT-EFFs β-galactosidase staining was high.3.By referring to the literature,we speculated that although the number of in vitro passages of hTERT-EFFs increased,the reason for the decline in cell quality might be that human hTERT was incompatible with horse telomerase gene(TERC).To further determine this hypothesis,we compared the TERT nucleotide,protein and TERC sequences of multiple species,respectively.The results showed that the TERT nucleotide sequence was highly similar to the protein sequence in multiple species,and there were differences in the key sites of TERC between species.We believe that this may be the fundamental reason for the incompatibility of human TERT and horse TERC in hTERT-EFFs,thereby affecting cell quality.Taken together,this study demonstrates that overexpression of hTERT alone is not sufficient to obtain equine immortalized cells,and points to the necessity of overexpressing equine-derived TERT or co-expressing hTERT and hTERC as a follow-up strategy.This study lays the necessary laboratory methods and data foundation for the real establishment of equine fibroblast immortalized cell lines.