【作者】 姚雅馨；

【导师】 李和平； 关伟军；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2012， 博士

【摘要】 我国的野生梅花鹿数量十分稀少,早在1978年就被列为我国的国家Ⅰ级重点保护野生动物,中国濒危动物红皮书也将其列为濒危(Endangered)等级。当人类对于这样的珍稀物种,如果在其濒危灭绝甚至灭绝之前,未曾利用任何方式将其种质资源保存下来的话,那么当它一旦遭遇灭绝,就意味着野生梅花鹿的整个细胞生物学和分子生物学以及对其可能展开研究的基本材料就会永远丧失。因此,如果能够将濒危动物的遗传资源以体细胞库的形式保存下来,并找到适合的技术手段将体细胞的多潜能性挖掘出来,那么使之再现的愿望将逐步得以实现。由于从克隆青蛙到诱导多能干(iPS)细胞,科研工作者一直在为解决细胞重编程中的安全问题和效率问题进行不屑的努力,希望通过添加小分子化合物这一安全而直接的方式使体细胞完成重编程过程,Reversine是一种小分子化合物,利用它处理成体细胞后能够明显将其转变为一种与干细胞类似的“原始状态”,这种经过处理的细胞具备了分化的潜能性,并且能够提高核移植胚胎的发育水平。因此,本研究以梅花鹿的耳缘组织为研究材料,利用组织块贴壁法培养原代梅花鹿细胞并成功建立起该组织的成纤维细胞系,利用胰酶消化法对其进行传代,并根据细胞的生长状态绘制细胞生长曲线；当细胞生长至100%接触抑制时,对其进行冻存,并统计冻存细胞的样本含量；后进行复苏,检测复苏前后的细胞活力；利用吉姆萨染色法对梅花鹿成纤维细胞的核型进行分析。结果显示,利用组织块贴壁法能够培养出梅花鹿的原代细胞并成功建立起该组织的成纤维细胞系,细胞样本含量为137,其中每份细胞数量为(1-3)×106个/ml；核型分析后确定该样本来源于东北梅花鹿,染色体数目为2n=66。在梅花鹿成纤维细胞库构建成功后,本研究开始着眼于挖掘体细胞的多潜能性上首先利用5μM的reversine处理复苏后48小时的梅花鹿成纤维细胞4d,观察处理前后的细胞形态变化,并利用流式细胞仪检测细胞周期的分布情况和凋亡现象,之后利用间接免疫荧光法检测reversine处理前后细胞的增殖情况和细胞骨架的变化趋势；为了体现reversine具有使成体细胞去分化的能力,本研究将处理后的成纤维细胞分别向成骨、成脂和成肝样细胞进行定向诱导分化；后利用共聚焦显微技术定性,流式细胞检测技术定量分析了诱发细胞去分化潜能的组蛋白修饰情况。结果显示,经reversine处理的细胞的BrdU的表达量明显降低,细胞处于分裂期的数量明显降低。说明经过reversine处理的细胞增殖能力降低并获得了多核细胞；经过定向诱导分化,成功将经过reversine处理后的细胞诱导分化成为成骨、成脂和成肝样细胞,通过了免疫组织化学和RT-PCR的检测,证明了诱导的成功；共聚焦的定性和流式细胞仪的定量检测后发现,经过reversine处理后的成纤维细胞乙酰化水平明显升高,而甲基化和磷酸化水平均有一定程度的降低,这在表观遗传修饰水平上证明了reversine能够通过重编程使梅花鹿的成纤维细胞获得去分化的能力。能够使濒危灭绝动物复原的有效手段当属构建异种核移植胚胎,本研究以梅花鹿的成纤维细胞作为供体细胞,以成熟的牛MⅡ期卵母细胞作为核受体进行异种核移植,比较异种核移植、同种核移植与体外受精胚胎的发育水平,并利用reversine处理核移植胚胎,以期能够达到提高重构胚发育水平的目的,并利用间接免疫荧光法检测连接组蛋白H1的表达特点。结果显示,在本研究中成功构建了牛-鹿异种核移植胚胎,并经过培养获得了异种核移植胚胎的囊胚,对其发育水平进行比较后发现牛-鹿异种核移植胚胎的发育时间并没有滞后于牛-牛同种核移植胚胎,但是发育到囊胚期的效率却明显低于同种核移植胚胎(16±8.8%vs7士3.9%,P<0.05),而经过reversine处理的牛-牛同种核移植胚胎与牛-鹿异种核移植胚胎的发育水平都有明显的提高(16±8.8%vs28.2±5.9%,P<0.05；3.7±3.9%vs16.1±5.3,P<0.05)；并且通过间接免疫荧光法检测到在卵母细胞中特异表达的连接性组蛋白H1foo与体细胞类型H1组蛋白之间存在着一种此消彼长的变化趋势,正常受精胚胎的卵母细胞特异性的连接组蛋白H1Foo与体细胞类型连接组蛋白H1的角色转换发生在16-细胞期甚至更晚,而同种核移植胚胎在2-细胞期的时候就要进行这样的转换,这从连接组蛋白的角度上解释了核移植胚胎的重编程过程不完整的原因。而经过Reversine的处理后,核移植胚胎的连接组蛋白的表达模式得到了一定程度的纠正,其表达模式与体外受精胚胎的表达模式较为接近,这种组蛋白表达模式的纠正解释了经过reversine处理的异种核移植胚胎发育水平明显升高的原因。更多还原

【Abstract】 The sika deer (Cervus nippon) was listed as the national level I protected wild animals of China, and endangered rating in China Red Data Book of Endangered Animals.If the wild sika deers have not been preserved in any way before extinction, not only the genetic resources will lost evermore, but also the research on biological mechanisms of various unknown cells and molecules of the extinct spaces will not be realized as well as the wish to regenerate the animals through somatic cell cloning. Therefore, there is a very urgent need to commence conservation of endangered species. Moreover, with the future development of science and technology, the roles of cell lines will become increasingly prominent and they may be useful in currently unforeseen applications.Then we should find a way to dig the potentiality of the somatic cell, we wish this species will regenerate by this way.From the cloned frogs to induced pluripotent stem cell(iPS), the researchers are struggling in the problems of safety and effectivity in process of reprogramming, they try to add different kinds of small compounds to figure it out, reversine is a small compound, the mouse muscle cells cells were transformed as a stem-like cell to an original state after treated with reversine, moreover, the development of nuclear transfer embryos could increased by using this small compound.Therefore, in the present study, the sika deer’s ear marginal was used as the research material, the primary cells were cultured by using the method of tissue piece, then the cells was passaged by Trypsin digesting. According to the cell growth state, the cell growth curve was described. The cells were frozen when100%contact inhibiting. Count the number of cryopreserved cell, then recovery the cells and detected cell viability of the recovery. The fibroblast karyotype was analyzed by Giemsa staining. The results showed that, tissues from sika deer’s ear marginal were isolated to culture fibroblasts and to develop a fibroblast cell line, the number of sample is137, the cell number of each sample is (1-3)×106/ml.Chromosome analysis showed that the chromosome number is2n=66,we can definite the sample was from the sika deer.After the fibroblast cell bank was established successfully, the present research was began to focus on the totipotency of somatic cell.5μM reversine were used to treat the recovered fibroblast for4d. At first, the cell morphologic changed were observed before and after treatment, then the flow cytometry were use to detect the cell cycle distribution and apoptosis, then the indirect immunofluorescence assay was used to detected the reversine treated cells proliferation and cytoskeletal changes. In order to reflect the dedifferentiate ability of reversine treated adult cells, we induced these cells to the osteogenic, adipogenic and liver cells before and after reversine treatment. Then the confocal microscopy and flow cytometry were used to analysis the histone modifications of the dedifferentiation cells. The results showed the reversine-treated fibroblast become the multinucleated cells by decreased the expression of the BrdU,and this kind of cell could be induced to differentiate into osteogenic, adipogenic and liver cells, then immunohistochemistry and RT-PCR were used to detect. After confocal qualitative and flow cytometry quantitative detection, we found the level of acetylation was incresed after reversine treatment, the level of methylation and phosphorylation was decresed, which indicated that reversine could induce the Sika Deer fibroblast into more multipotent progenitor-type cells by dedifferentiation.The most useful way to regenerate the endangered animals is to built the interspecies somatic cell nuclear transfer(ISCNT) embryos. In this study, the sika deer fibroblasts were used as the donor cells, the bovine MⅡ stage of oocytes were used as the receptor to built the ISCNT embryos.Then we compare the developmental level of ISCNT embryos, in vitro fertilization (IVF)embryos and normal NT embryo. In order to improve the developmental level of reconstructed embryo, we use reversine to deal with these NT embryos, then the indirect immunofluorescence assay was used to detect the expressed characteristics of the linker histone. The results showed that the cattle-deer ISCNT embryos was successfully constructed in this study, and ISCNT embryonic blastocyst were obtained successfully. Then compare the developmental time of cattle-deer species ISCNT embryo was not hysteresis to the normal NT embryos, but the efficiency of development to the blastocyst stage was much lower than the normal NT embryos (16±8.8%vs7±3.9%>P<0.05). It’s surprise that, the cattle-cattle normal NT embryos and cattle-deer interspecies NT embryos’ developmental level has been improved significantly after reversine treatment(16±8.8%vs28.2±5.9%, P<0.05;3.7±3.9%vs16.1±5.3, P<0.05);After analyze the expressed pattern between oocyte-specific linker histone Hlfoo and the somatic cell types H1protein, a shift trend were existed. The shift trend expressed pattern of H1Foo and H1was started in the16-cell stage or even later in the normal fertilized embryos,however, this kind of shift trend expression in normal NT embryos was happened in2-cell stage, which explains the phenomenon of incomplete reprogramming in the NT embryos. After reversine treatment, the expressed patterns of linker histone in NT embryos was corrected similar with the expression pattern of IVF embryo.更多还原