【作者】 张宇；

【导师】 白素英；

【作者基本信息】 东北林业大学 ， 动物遗传育种与繁殖， 2018， 硕士

【摘要】 麝鼠(Ondatra zibethicus Linnaeus)又称为青根貂,是田鼠亚科麝鼠属中唯一物种。雄性麝鼠有一对泌香腺,繁殖时可分泌具有明显芳香气味的乳白色麝鼠香。麝鼠香的主要药用成分为麝香酮,与天然麝香相近,所以具有成为天然麝香替代品的巨大潜力。麝鼠相较于其它鼠类,前列腺组织非常发达。研究发现腹腔注射麝鼠香可使小鼠的前列腺组织增大,所以麝鼠香除了具有外激素的作用还可能具有调节麝鼠自身发育的作用。本研究中通过RNA-seq测序技术,对麝鼠泌香期和非泌香期的前列腺组织进行了转录组测序,并通过生物信息学从整体水平上对麝鼠前列腺的发育进行了研究。同时,本研究中建立了麝鼠前列腺成纤维样细胞的原代培养方法,构建了麝鼠前列腺成纤维样细胞的细胞模型,证明了该细胞体外培养的可行性。结合RNA-seq测序结果和原代培养的细胞模型,对原代细胞进行了麝鼠香干预试验,将传代至第3代的原代培养麝鼠前列腺成纤维样细胞分为2组,10μg/ml麝鼠香组记为A1组,0.1%DMSO对照组记为B1组。实验结果显示,A1组的Bcl-2转录水平高于B1组约4倍,Bax基因转录水平较B1组低。麝鼠香可以提高细胞Bcl2基因的转录水平,降低Bax基因的转录水平,提高Bcl2/Bax的比值,起到抗细胞凋亡的作用。为初步研究麝鼠香调节Bc12/Bax比值的作用机制,本实验使用HEK293T细胞,并利用麝鼠香的主要成分麝香酮进行干预,将复苏后传代至第3代的HEK293T细胞分为5组,3μg/ml麝香酮组记为A2组,5μg/ml麝香酮组记为B2组,7μg/ml麝香酮组记为C2组,9μg/ml麝香酮组记为D2组,0.1%DMSO对照组记为E2组。每组设置3个生物学重复。相较于E2组,A2、B2、C2和D2组NF-κB基因的转录水平分别为E2组的1.39倍、1.53倍、1.72倍和1.78倍;PI3K基因的转录水平分别为E2组的1.41倍、1.58倍、1.71倍和1.79倍;ERK基因的转录水平分别为E2组的1.27倍、1.39倍、1.53倍和1.77倍;MEK基因的转录水平分别为E2组的1.2倍、1.29倍、1.32倍和1.38倍;p53基因的转录水平分别为E2组的1.08倍、1.11倍、0.99倍和1.1倍。结果发现:麝香酮会提升NF-κB、PI3K、MEK和ERK基因的转录水平,且具有明显的量效关系。对于p53基因的影响并无明显规律。因此推测,麝香酮调节细胞Bcl-2、Bax基因转录水平比值的改变可能是通过调节上述信号通路来完成的。通过RNA-seq测序部分和药物干预细胞的实验结果进行推测,麝鼠香对于麝鼠本身不只可以起到外激素的作用,还可以调节自身的生长发育情况,如可以促进麝鼠前列腺的发育。麝鼠香中的麝香酮等亲脂性气味分子随血液循环到达前列腺组织,c75134_g2转录本所编码的蛋白在这一过程中起到亲脂性气味分子载体的作用,并帮助其与受体结合,通过调节Bcl-2家族基因的转录水平,起到抗细胞凋亡的作用,调节前列腺组织的发育过程。更多还原

【Abstract】 Muskrat(Ondatra zibethicus Linnaeus)also called aone mink.It is the only species of muskrat subfamily of voles.Male muskrat have a pair of musk glands.Musk glands can secrete aromatic white musk on breeding season.The main medicinal components of muskrat musk is muscone,and it is similar to natural musk.So it has great potential to be a substitute for natural musk.Muskrat compared to other murine,prostate tissue is very developed,and the experiments have proved that intraperitoneal injection of muskrat musk can result in prostate tissue increased.So it not only has the effect of pheromones,may also have to adjust their development role.Through RNA-seq sequencing technology in this experiment,the transcriptional library of muskrat secreting stage and non-secreting stage of prostate tissue was sequenced.Through the bioinformatics from the overall level of muskrat prostate development are discussed.At the same time,this experiment established into primary muskrat prostate fibroblast like cells culture method,successfully constructed a cell model of muskrat prostate fibroblast like cells,and has been proved the feasibility of the cell culture in vitro.Combined with the results of RNA-seq sequencing and the cell model of primary culture,conducted the muskrat intervention trial of primary cells.The third generations of muskrat cultured prostate fibroblast like cells were divided into 2 groups,10 μg/ml of muskrat musk group denoted as A1 group,0.1%DMSO control group was recorded as group B1.Experimental results show that the transcriptional level of Bcl-2 in group A1 was about 4 times higher than that in group B1.The transcriptional level of Bax gene was lower than that in B1.Muskrat musk can increase the transcription level of Bcl2 gene,reduce the transcription level of Bax gene,enhancing the ratio of Bcl2/Bax to anti-apoptosis.For a preliminary study on the mechanism for adjusting the Bcl2/Bax ratio of muskrat musk,this experiment using HEK293T cells,and the intervention using muscone.The HEK293T cells were divided into 5 groups,the group of 3μg/ml muscone was set as group A2;the group of 5 μg/ml muscone was set as group B2;the group of 7 μg/ml muscone was set as group C2;the group of 9 μg/ml muscone was set as group D2;the group of 0.1%DMSO was recorded as group E2.Set three biological replicates in each group.Compared with group E2,the transcription level of NF-κB gene in A2,B2,C2 and D2 groups were 1.39 times,1.53 times,1.72 times and 1.78 times of E2,respectively;the transcriptional level of the PI3K gene was 1.41 times,1.58 times,1.71 times,and 1.79 times of the E2 group;the transcriptional level of the ERK gene was 1.27 times,1.39 times,1.53 times and 1.77 times of the E2 group;the transcriptional level of the MEK gene was 1.2 times,1.29 times,1.32 times and 1.38 times of group E2;the transcriptional level of the p53 gene was 1.08 times,1.11 times,0.99 times and 1.1 times of group E2.The results showed that Muscone increased the transcriptional level of NF-κB,PI3K,MEK and ERK genes,and they have a significant dose-effect relationship with the concentration of Muscone.But there was no obvious effect on the p53 gene.Therefore,it is presumed that the change of the Bcl-2 and Bax gene ratio of the Muscone regulated cells may be accomplished by regulating these signal pathway.Predicted by RNA-seq sequencing and drug intervention cell experimental results,muskrat musk itself not only plays the role of pheromones,also may regulate the growth and development of their own,for example,can promote the development of the prostate.The muscone and other lipophilic odorant molecules with the blood circulation to the prostate tissue.In this process,the protein encoded by the c75134-g2 transcript acts as a lipophilic molecular carrier to help it bind to the receptor.By regulating the transcriptional level of the Bcl-2 gene,it plays a role in anti-apoptosis and regulates the development of the prostate tissue.更多还原