【摘要】：誘導多能干細胞(Induced pluripotent stem cell, iPSC)是通過將外源多能因子導入到已分化的細胞中,促進多能基因表達,使細胞重編程而獲得多能性的一類細胞。iPSC與ESC (Embryonic Stem Cell)類似,具有分化成各種類型細胞和三個胚層的潛能。相比分離胚胎干細胞犧牲胚胎的局限性,iPS技術(shù)可以誘導多種來源的細胞,給研究人員提供了更多的研究材料,并規(guī)避了破壞胚胎等倫理問題。iPSC技術(shù)應用在禽類物種上,可以促進珍稀鳥類和優(yōu)良種禽的保種育種。本論文研究主要從廣西三黃雞體細胞出發(fā)誘導iPSC,并優(yōu)化iPSC的培養(yǎng)系統(tǒng),為建立一套以干細胞為基礎(chǔ)的家禽保種育種平臺奠定基礎(chǔ)。研究主要分為三部分,分別為分離三黃雞體細胞、誘導重編程三黃雞胚成纖維細胞、雞iPSC的培養(yǎng)條件的優(yōu)化,結(jié)果如下：(1)三黃雞體細胞分離。本實驗分離三黃雞幼雞羽毛細胞,采用不同種類培養(yǎng)液、胰蛋白酶不同時間消化、不同濃度血清培養(yǎng)液分離培養(yǎng)羽毛細胞。發(fā)現(xiàn)胰蛋白酶消化10分鐘,DMEM/F12培養(yǎng)液培養(yǎng)可以分離到羽毛細胞,但羽毛細胞傳代后增殖速度減慢趨向死亡。分離并得到三黃雞幼雞心、肝、肌肉組織、雞胚成纖維細胞。(2)三黃雞胚成纖維細胞誘導重編程。采用含有Oct4、Sox2、Klf4 Lin28、c-Myc多能轉(zhuǎn)錄因子Piggybac轉(zhuǎn)座子脂質(zhì)體法轉(zhuǎn)染CEF細胞,轉(zhuǎn)染培養(yǎng)10天后沒有出現(xiàn)細胞克隆。使用含有不同Sox2、Lin28、Oct4、Nanog、c-Myc和Klf-4多能因子組合的慢病毒載體感染CEF細胞,發(fā)現(xiàn)Sox2、Lin28、 Oct4、Nanog組合,Sox2、Oct4、c-Myc、Klf-4組合均可實現(xiàn)細胞重編程,誘導出堿性磷酸酶陽性克隆。(3)雞iPSC的培養(yǎng)系統(tǒng)的優(yōu)化。通過配制不同營養(yǎng)成分的培養(yǎng)液,改變培養(yǎng)液中添加BRL條件培養(yǎng)液比例、FGF濃度、LIF濃度,對比BRL、STO、MEF飼養(yǎng)層,對比昆明小鼠和ICR小鼠MEF飼養(yǎng)層,對比絲裂霉素C處理和物理輻射處理MEF飼養(yǎng)層,培養(yǎng)并觀察雞iPSC的生長狀態(tài)。實驗結(jié)果發(fā)現(xiàn)ICR小鼠MEF細胞經(jīng)絲裂霉素C處理制備飼養(yǎng)層適合雞iPSC生長,ICR小鼠MEF細胞經(jīng)Y射線輻射處理制備飼養(yǎng)層需要在培養(yǎng)液中添加4-10ng/ml LIF才能維持iPSC的生長,KM小鼠MEF細胞經(jīng)絲裂霉素C處理和Y射線輻射處理制備飼養(yǎng)層不適合在本實驗培養(yǎng)體系中培養(yǎng)iPSC。綜上作述,本研究分離獲得了三黃雞體細胞并實現(xiàn)誘導重編程,優(yōu)化了雞iPSC的培養(yǎng)條件,保持細胞良好的生長狀態(tài),為利用iPSC技術(shù)開展地方優(yōu)質(zhì)珍稀家禽品種的保種育種奠定了基礎(chǔ)。。
[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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831
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本文編號：2266752