| The expression of NSE in corpus luteum of the rat was detected by RT-PCR, in situhybridization and immunochemistry at the level of mRNA and protein respectively, and theeffects of NSE in proliferaton and degradation of luteal cells were investigated to reveal therelation between NSE and the apoptosis of luteal cells.Meanwhile, the coding sequence (CDS)of NSE gene of dairy goats was cloned and its bioinformatics was analyzed. The main resultsare as follows:1. The results of RT-PCR, in situ hybridization and immunochemistry revealed that themRNA and protein of NSE all existed in both the corpus luteum tissues and the cultured lutealcells in vitro.2. By adding NSE of different concentrations and its antibody into cell-culture mediumof rat luteal cells in vitro, analyzing the relative expression content of bax, bcl-2, NSE and theratio of bcl-2/bax in different experimental groups by grey value half quantitative method, wefound that when there were NSE in cell-culture medium, the ratio of bcl-2/bax were higherthan the control group and gradually increased with the increasing concentration of NSE;when there were NSE antibody in cell-culture medium, the ratio of bcl-2/bax were lower thanthe control group.Meanwhile, the relative expression content of NSE in luteal cells graduallyincreased with the increasing concentration of NSE; in contrast, when there were NSEantibody in cell-culture medium, the relative expression content of NSE in luteal cells wasobviously lower than the control group. All these results showed that NSE can increase theratio of bcl-2/bax in the cultured luteal cells, so it may play a role in promoting cellulargrowth and preventing apoptosis of luteal cells.3. The coding sequence of NSE of dairy goats was identified (GenBank accession No.JN887466) by molecular cloning and bioinformatic analysis was carried on on the sequence.Its nucleotide sequence homology was found to be97.9%,89.3%,90%and92.6%,respectively, compared with that of Bos taurus, Rattus norvegicus, Mus musculus and Homosapiens, while the amino acid sequence homology was99.1%,97%,97.2%and98.2%respectively. These results first showed that the functional amino acids coded by the NSE genewere highly conserved in Caprine, Bos taurus, Rattus norvegicus, Mus musculus and Homosapiens. It was implied that the gene NSE in dairy goats had close homology to that of NSE of other species.In conclusion, the experiment demonstrated that the mRNA and protein of NSE allexisted in both the corpus luteum tissues and the cultured luteal cells in vitro, and NSE canincrease the ratio of bcl-2/bax in the cultured luteal cells, so it may play a role in promotingcellular growth and preventing apoptosis of luteal cells, preliminarily revealed the relationbetween NSE and the apoptosis of luteal cells.Meanwhile, the coding sequence (CDS) of NSEgene of dairy goats was successfully cloned and its bioinformatics was analyzed,provided atheoretical basis for future study on the function of NSE and research on the relation betweenNSE and the apoptosis of luteal cells by silencing the NSE gene with RNAi technology, italso enriched the genetic information of dairy goats. |
