三黄鸡体细胞重编程及iPSC培养体系的优化
发布时间：2018-10-12 16:06
【摘要】：诱导多能干细胞(Induced pluripotent stem cell, iPSC)是通过将外源多能因子导入到已分化的细胞中,促进多能基因表达,使细胞重编程而获得多能性的一类细胞。iPSC与ESC (Embryonic Stem Cell)类似,具有分化成各种类型细胞和三个胚层的潜能。相比分离胚胎干细胞牺牲胚胎的局限性,iPS技术可以诱导多种来源的细胞,给研究人员提供了更多的研究材料,并规避了破坏胚胎等伦理问题。iPSC技术应用在禽类物种上,可以促进珍稀鸟类和优良种禽的保种育种。本论文研究主要从广西三黄鸡体细胞出发诱导iPSC,并优化iPSC的培养系统,为建立一套以干细胞为基础的家禽保种育种平台奠定基础。研究主要分为三部分,分别为分离三黄鸡体细胞、诱导重编程三黄鸡胚成纤维细胞、鸡iPSC的培养条件的优化,结果如下：(1)三黄鸡体细胞分离。本实验分离三黄鸡幼鸡羽毛细胞,采用不同种类培养液、胰蛋白酶不同时间消化、不同浓度血清培养液分离培养羽毛细胞。发现胰蛋白酶消化10分钟,DMEM/F12培养液培养可以分离到羽毛细胞,但羽毛细胞传代后增殖速度减慢趋向死亡。分离并得到三黄鸡幼鸡心、肝、肌肉组织、鸡胚成纤维细胞。(2)三黄鸡胚成纤维细胞诱导重编程。采用含有Oct4、Sox2、Klf4 Lin28、c-Myc多能转录因子Piggybac转座子脂质体法转染CEF细胞,转染培养10天后没有出现细胞克隆。使用含有不同Sox2、Lin28、Oct4、Nanog、c-Myc和Klf-4多能因子组合的慢病毒载体感染CEF细胞,发现Sox2、Lin28、 Oct4、Nanog组合,Sox2、Oct4、c-Myc、Klf-4组合均可实现细胞重编程,诱导出碱性磷酸酶阳性克隆。(3)鸡iPSC的培养系统的优化。通过配制不同营养成分的培养液,改变培养液中添加BRL条件培养液比例、FGF浓度、LIF浓度,对比BRL、STO、MEF饲养层,对比昆明小鼠和ICR小鼠MEF饲养层,对比丝裂霉素C处理和物理辐射处理MEF饲养层,培养并观察鸡iPSC的生长状态。实验结果发现ICR小鼠MEF细胞经丝裂霉素C处理制备饲养层适合鸡iPSC生长,ICR小鼠MEF细胞经Y射线辐射处理制备饲养层需要在培养液中添加4-10ng/ml LIF才能维持iPSC的生长,KM小鼠MEF细胞经丝裂霉素C处理和Y射线辐射处理制备饲养层不适合在本实验培养体系中培养iPSC。综上作述,本研究分离获得了三黄鸡体细胞并实现诱导重编程,优化了鸡iPSC的培养条件,保持细胞良好的生长状态,为利用iPSC技术开展地方优质珍稀家禽品种的保种育种奠定了基础。。
[Abstract]:Inducible pluripotent stem cell (Induced pluripotent stem cell, iPSC) is a kind of cells that obtain pluripotency by introducing exogenous pluripotent factors into differentiated cells, promoting the expression of pluripotent genes and reprogramming cells. IPSC is similar to ESC (Embryonic Stem Cell). It has the potential to differentiate into various types of cells and three embryonic layers. Compared with the limitations of isolating embryonic stem cells at the expense of embryos, iPS technology can induce cells from a variety of sources, provide researchers with more research materials and avoid ethical issues such as embryo destruction. IPSC technology is used in poultry species. It can promote the conservation breeding of rare birds and fine breed birds. In this paper, iPSC, was induced from somatic cells of Guangxi Sanhuang chicken and the culture system of iPSC was optimized, which laid a foundation for the establishment of a set of breeding platform based on stem cells for poultry breeding. The study was mainly divided into three parts: isolation of three yellow chicken somatic cells, induction of three yellow chicken embryo fibroblasts, and optimization of the culture conditions of chicken iPSC. The results were as follows: (1) three yellow chicken somatic cells were isolated. In this experiment, feather cells were isolated and cultured in different kinds of culture medium, trypsin digestion at different time and different concentration of serum in Sanhuang chicken feather cells. It was found that after 10 minutes of trypsin digestion, feather cells could be isolated in DMEM/F12 culture medium, but the proliferation rate of feather cells slowed down to death after passage. The chicken heart, liver, muscle tissue and embryo fibroblasts were isolated and obtained. (2) Tri-yellow chicken embryo fibroblasts were induced to reprogram. CEF cells were transfected with Piggybac transposon liposome containing Oct4,Sox2,Klf4 Lin28,c-Myc multifunctional transcription factor. After 10 days of transfection, no cell clones were found. Lentivirus vectors containing different combinations of Sox2,Lin28,Oct4,Nanog,c-Myc and Klf-4 multipotent factors were used to infect CEF cells. It was found that Sox2,Lin28, Oct4,Nanog combination and Sox2,Oct4,c-Myc,Klf-4 combination could reprogram cells and induce alkaline phosphatase positive clones. (3) Optimization of chicken iPSC culture system. The proportion of BRL conditioned medium, FGF concentration, LIF concentration, BRL,STO,MEF feeder layer, MEF feeder layer of Kunming mice and ICR mice were changed by preparing different nutrient components. Compared with mitomycin C treatment and physical radiation treatment MEF feeder layer was cultured and observed the growth state of chicken iPSC. The results showed that the feeder layer of MEF cells of ICR mice treated with mitomycin C was suitable for chicken iPSC growth, and that of MEF cells of ICR mice prepared by Y ray irradiation needed to add 4-10ng/ml LIF to the culture medium to maintain the growth of iPSC. KM The preparation of feeder layer by mitomycin C and Y-ray irradiation on mouse MEF cells is not suitable for iPSC. culture in this experimental culture system. In this study, the somatic cells of Sanhuang chicken were isolated and reprogrammed, the culture conditions of chicken iPSC were optimized, and the cells were kept in a good growth state. It laid a foundation for the breeding of local high-quality and rare poultry varieties by using iPSC technology.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831
，
本文编号：2266752
[Abstract]:Inducible pluripotent stem cell (Induced pluripotent stem cell, iPSC) is a kind of cells that obtain pluripotency by introducing exogenous pluripotent factors into differentiated cells, promoting the expression of pluripotent genes and reprogramming cells. IPSC is similar to ESC (Embryonic Stem Cell). It has the potential to differentiate into various types of cells and three embryonic layers. Compared with the limitations of isolating embryonic stem cells at the expense of embryos, iPS technology can induce cells from a variety of sources, provide researchers with more research materials and avoid ethical issues such as embryo destruction. IPSC technology is used in poultry species. It can promote the conservation breeding of rare birds and fine breed birds. In this paper, iPSC, was induced from somatic cells of Guangxi Sanhuang chicken and the culture system of iPSC was optimized, which laid a foundation for the establishment of a set of breeding platform based on stem cells for poultry breeding. The study was mainly divided into three parts: isolation of three yellow chicken somatic cells, induction of three yellow chicken embryo fibroblasts, and optimization of the culture conditions of chicken iPSC. The results were as follows: (1) three yellow chicken somatic cells were isolated. In this experiment, feather cells were isolated and cultured in different kinds of culture medium, trypsin digestion at different time and different concentration of serum in Sanhuang chicken feather cells. It was found that after 10 minutes of trypsin digestion, feather cells could be isolated in DMEM/F12 culture medium, but the proliferation rate of feather cells slowed down to death after passage. The chicken heart, liver, muscle tissue and embryo fibroblasts were isolated and obtained. (2) Tri-yellow chicken embryo fibroblasts were induced to reprogram. CEF cells were transfected with Piggybac transposon liposome containing Oct4,Sox2,Klf4 Lin28,c-Myc multifunctional transcription factor. After 10 days of transfection, no cell clones were found. Lentivirus vectors containing different combinations of Sox2,Lin28,Oct4,Nanog,c-Myc and Klf-4 multipotent factors were used to infect CEF cells. It was found that Sox2,Lin28, Oct4,Nanog combination and Sox2,Oct4,c-Myc,Klf-4 combination could reprogram cells and induce alkaline phosphatase positive clones. (3) Optimization of chicken iPSC culture system. The proportion of BRL conditioned medium, FGF concentration, LIF concentration, BRL,STO,MEF feeder layer, MEF feeder layer of Kunming mice and ICR mice were changed by preparing different nutrient components. Compared with mitomycin C treatment and physical radiation treatment MEF feeder layer was cultured and observed the growth state of chicken iPSC. The results showed that the feeder layer of MEF cells of ICR mice treated with mitomycin C was suitable for chicken iPSC growth, and that of MEF cells of ICR mice prepared by Y ray irradiation needed to add 4-10ng/ml LIF to the culture medium to maintain the growth of iPSC. KM The preparation of feeder layer by mitomycin C and Y-ray irradiation on mouse MEF cells is not suitable for iPSC. culture in this experimental culture system. In this study, the somatic cells of Sanhuang chicken were isolated and reprogrammed, the culture conditions of chicken iPSC were optimized, and the cells were kept in a good growth state. It laid a foundation for the breeding of local high-quality and rare poultry varieties by using iPSC technology.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831
，
本文编号：2266752
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