大鼠周期黄体中NSE的表达及山羊NSE基因CDS的克隆
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摘要
为了研究大鼠黄体中神经元特异性烯醇化酶（NSE）的表达情况，揭示其与黄体细胞生长凋亡的关系，本实验用RT-PCR、免疫组化、免疫细胞化学、原位杂交等方法同时从mRNA水平和蛋白水平对大鼠黄体组织和体外培养的黄体细胞中NSE的表达进行了研究，并对NSE对体外培养的大鼠黄体细胞生长的影响进行了初步研究。同时，本实验还对山羊NSE基因的CDS区进行了克隆并对其序列进行了分析。主要结果如下：
1. RT-PCR、免疫组化、免疫细胞化学、原位杂交等结果表明，无论是在黄体组织还是在体外培养的大鼠黄体细胞中都存在NSE mRNA和蛋白的阳性表达。
2.通过在大鼠黄体细胞培养液中加入不同浓度的NSE及其抗体，利用灰度值半定量方法分析不同实验组中Bcl-2和Bax的相对表达量和Bcl-2/Bax的比值及NSE的相对表达量，我们发现，黄体细胞培养液中含NSE时，bcl-2/bax的比值要大于空白对照组，且随着NSE浓度的升高，bcl-2/bax的比值逐渐增大；培养液中含NSE抗体时，bcl-2/bax的比值要小于空白对照组。同时，随着培养液中NSE浓度的升高，细胞NSE的相对表达量逐渐升高；相反，培养液中含NSE抗体时，细胞NSE的相对表达量明显低于对照组。综上所述，NSE能促进黄体细胞中bcl-2/bax比值的增大，可能具有促进黄体细胞生长抑制细胞凋亡的作用。
3.通过克隆测序，本实验确定了山羊NSE基因的CDS区序列（GenBank登陆号:JN887466）并对其进行了生物信息学分析。奶山羊NSE基因CDS区核苷酸序列与牛、大鼠、小鼠和人的同源性分别为97.9%,89.3%,90%和92.6%,其氨基酸序列与牛、大鼠、小鼠和人的同源性分别为99.1%,97%,97.2%和98.2%。这些结果首次表明NSE基因编码的功能氨基酸在山羊、牛、大鼠、小鼠和人上高度保守，山羊NSE基因与其他物种的NSE基因具有很高的同源性。
综上所述，本实验证明大鼠黄体组织和细胞中均存在NSE mRNA和蛋白的表达，且NSE能促进黄体细胞中bcl-2/bax比值的增大，可能具有促进黄体细胞生长抑制细胞凋亡的作用，初步揭示了NSE与黄体细胞生长凋亡的关系；本实验还成功克隆了山羊NSE基因的CDS，并对其生物信息学进行了分析，为以后通过RNAi技术敲除NSE基因深入研究其功能及其与黄体细胞凋亡的关系提供理论依据，同时，也丰富了山羊的遗传学信息。
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.
1. RT-PCR、免疫组化、免疫细胞化学、原位杂交等结果表明，无论是在黄体组织还是在体外培养的大鼠黄体细胞中都存在NSE mRNA和蛋白的阳性表达。
2.通过在大鼠黄体细胞培养液中加入不同浓度的NSE及其抗体，利用灰度值半定量方法分析不同实验组中Bcl-2和Bax的相对表达量和Bcl-2/Bax的比值及NSE的相对表达量，我们发现，黄体细胞培养液中含NSE时，bcl-2/bax的比值要大于空白对照组，且随着NSE浓度的升高，bcl-2/bax的比值逐渐增大；培养液中含NSE抗体时，bcl-2/bax的比值要小于空白对照组。同时，随着培养液中NSE浓度的升高，细胞NSE的相对表达量逐渐升高；相反，培养液中含NSE抗体时，细胞NSE的相对表达量明显低于对照组。综上所述，NSE能促进黄体细胞中bcl-2/bax比值的增大，可能具有促进黄体细胞生长抑制细胞凋亡的作用。
3.通过克隆测序，本实验确定了山羊NSE基因的CDS区序列（GenBank登陆号:JN887466）并对其进行了生物信息学分析。奶山羊NSE基因CDS区核苷酸序列与牛、大鼠、小鼠和人的同源性分别为97.9%,89.3%,90%和92.6%,其氨基酸序列与牛、大鼠、小鼠和人的同源性分别为99.1%,97%,97.2%和98.2%。这些结果首次表明NSE基因编码的功能氨基酸在山羊、牛、大鼠、小鼠和人上高度保守，山羊NSE基因与其他物种的NSE基因具有很高的同源性。
综上所述，本实验证明大鼠黄体组织和细胞中均存在NSE mRNA和蛋白的表达，且NSE能促进黄体细胞中bcl-2/bax比值的增大，可能具有促进黄体细胞生长抑制细胞凋亡的作用，初步揭示了NSE与黄体细胞生长凋亡的关系；本实验还成功克隆了山羊NSE基因的CDS，并对其生物信息学进行了分析，为以后通过RNAi技术敲除NSE基因深入研究其功能及其与黄体细胞凋亡的关系提供理论依据，同时，也丰富了山羊的遗传学信息。
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.
引文
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