小鼠诱导性多能干细胞的建立及其分化研究

发布时间：2018-01-04 05:34

本文关键词：小鼠诱导性多能干细胞的建立及其分化研究　出处：《郑州大学》2013年硕士论文　论文类型：学位论文

【摘要】：树突状细胞(dendritic cells, DC)属于专职抗原递呈细胞,其能摄取、加工、处理和递呈抗原,并将后者携带的信息呈递给T淋巴细胞,进而引发一系列免疫应答,其效率是所有该类细胞中最强的。DC参与多种病理生理过程,并具有强大的免疫调节功能,因此以DC为基础的免疫治疗已成为肿瘤治疗的一种新模式。然而DC获取困难,通过患者自体骨髓间充质干细胞分离培养和扩增后获取自体内源性DC,技术操作困难,且患者难以承受,其疗效目前尚有争议,不适合临床广泛应用。通过胚胎干细胞(embryonic stem cell, ESC)定向分化制备DC,则具有伦理学限制,而且所得DC并非自体细胞,存在免疫排斥的可能。诱导性多能干细胞(induced pluripotent stem cells, iPSCs)技术的出现则为临床上DC的肿瘤免疫治疗所需的种子细胞的制备提供了另一种新途径。iPSCs是由体细胞诱导而成的干细胞,具有和ESC类似的发育多潜能性,在形态学、基因表达状况和表观遗传修饰方面都与ESC十分相似。iPSCs的应用价值在于获得方法相对简单和稳定,在技术上和伦理上都比其他方法更有优势,在再生医学中起着非常重要的作用。鉴于此,本研究拟通过多因子重编程技术,诱导小鼠成熟体细胞为iPSCs,并利用免疫组织化学、荧光、拟胚体(EB)实验、RT-PCR及Western blot等多项形态学和分子生物学技术对诱导而来的iPSCs进行分析鉴定。最后通过在iPSCs培养基中分别单独或联合添加不同的促使DC生长的细胞营养因子,将iPSCs在体外定向诱导分化为DC。目的利用逆转录病毒载体系统,通过病毒包装技术,将小鼠成纤维细胞重编程为iPSCs并对其进行鉴定。并在此基础上,将iPSCs在体外定向诱导分化为DC。材料与方法第一部分是iPSCs的诱导与鉴定。方法如下：采用逆转录病毒转染的方法将4种外源多能因子Oct4、Sox2、c-Myc、Klf4质粒转染入病毒包装Plat-E细胞,搜集病毒上清感染原代培养的小鼠鼠尾成纤维细胞,观察记录多能干细胞诱导生成的过程,并对其进行鉴定分析。主要采取以下鉴定方法,1)采用碱性磷酸酶(AP)染色观察未分化的iPSCs是否具备ESC的特征,再结合形态学和免疫细胞化学的观察,鉴定iPSCs是否具有全能性；2)采用免疫荧光的方法鉴定干细胞标志物Oct、Sox2、SSEA-1在诱导而来的iPSCs表面的表达；3)拟胚体(EB)实验检测是否iPSCs能够分化发育成胚体,并用免疫荧光的方法检测胚体中是否存在3个胚层来源细胞；4) Western blot检测多能基因的蛋白表达：为进一步验证感染小鼠成纤维细胞的多能基因蛋白表达情况,利用Western blot方法检测已诱导成功的iPSCs多能因子的蛋白表达情况。所用Western抗体同免疫组化抗体；5)基因表达分析(RT-PCR):采用RT-PCR分别检测Oct4、Sox2、c-Myc、Klf4多能基因在成功诱导的iPSCs、ESC M1及小鼠成纤维细胞中的表达水平,并作对比分析。第二部分为iPSCs分化为DC的研究。方法如下：将诱导成功的iPSCs基础培养体系内分别单独或联合添加不同的细胞因子,如白细胞介素IL-3(10ng/mL), IL-7(10ng/mL), IL-15(20ng/mL)。每2-3d半量换液,添加新鲜细胞因子。培养12～14d后,继续添加细胞因子粒细胞-巨噬细胞集落刺激因子(GM-CSF)(4ng/mL)(?)肿瘤坏死因子(TNF)(50ng/mL),继续培养3～5d,倒置显微镜下观察细胞形态。结果 1鼠尾成纤维细胞的培养及iPSCs的诱导采用胰酶消化法原代培养鼠尾成纤维细胞,培养次日,倒置相差显微镜下观察即可见典型的成纤维细胞特征；之后进行iPSCs的诱导,多能干细胞因子病毒感染第2d即可观察到成纤维细胞形态开始变化,细胞变平,较圆,同时出现小集落状的增殖群体。两周后可出现大量的致密而突起的克隆,细胞排列紧密,界限较清,集落边缘折光强。采用机械法挑取克隆并进行传代。 2iPSCs的鉴定我们分别利用AP染色法、EB实验、Western blot及免疫荧光染色法对诱导成的iPSCs进行了鉴定,鉴定结果均符合ESC的特征；采用RT-PCR的方法检测多能基因mRNA水平,可见所建立的iPSCs细胞系与ESC相同,均表达内源性的Oct4、Sox2、c-Myc、Klf4,而分化后的成纤维细胞不表达干细胞全能性基因Oct4；由于外源基因的导入,所建立的iPSCs细胞系外源基因RNA水平较高,而小鼠ESC和小鼠成纤维细胞外源性基因几乎完全沉默。 3体外诱导iPSCs向DC分化体外诱导iPSCs向DC分化主要是采取在iPSCs培养基中添加几种能使干细胞向DC分化的营养因子,如IL、GM-CSF、TNF、干细胞生长因子以及DC促生长因子FMS样酪氨酸激酶3配体(FIt3L)、脂多糖等。结果可见典型的DC形态,提示iPSCs在特定因子的诱导下是可以分化为DC的。结论通过4种外源多能因子的转导,小鼠鼠尾成纤维细胞成功诱导为iPSCs,并具有iPSCs的功能和形态特征； iPSCs在特定因子的诱导下,可以成功分化为DC,具备DC的表型特征；不但为临床上DC的肿瘤免疫治疗所需的种子细胞提供了一种新的来源,同时也有助于我们了解胚胎时期DC的发育及其免疫调节作用,为临床疾病防治和药物研究提供非常有价值的实验平台。
[Abstract]:Dendritic cells (dendritic, cells, DC) are professional antigen presenting cells, the uptake, processing, processing and presenting antigens, and the latter will carry the information presentation to T cells, triggering a series of immune response, its efficiency is the strongest of all the cells in the.DC involved in many physiological and pathological processes, and has strong immune regulating function, so DC based immunotherapy has become a new model for tumor therapy. However, DC are difficult to obtain, through autologous bone marrow mesenchymal stem were isolated and amplified in DC cells for autologous source technology, difficult operation, and the patient is difficult to bear, its efficacy is controversial that is not suitable for clinical application. The embryonic stem cells (embryonic stem cell, ESC) differentiation of DC was prepared with ethical restriction, and the DC is not stored in the autologous cells, immune rejection may be induced. Pluripotent stem cells (induced pluripotent stem cells iPSCs) technology is the emergence of the seed cells for tumor immunotherapy clinical DC required for the preparation of a new way of.IPSCs is induced by somatic cells into stem cells, has the development potential, and ESC similar in morphology that gene expression and epigenetic modifications are very similar with ESC value of.IPSCs is to obtain the method is relatively simple and stable in technology and ethics have more advantages than other methods, plays a very important role in regenerative medicine.
In view of this, this study proposed by multi factor reprogramming, mature somatic cells in mice induced by iPSCs, and the use of immunohistochemistry, fluorescence, embryoid body (EB) experiment, while RT-PCR and Western to blot and a number of morphological and molecular biology techniques to analyze the identification of induced iPSCs. Finally, in the iPSCs culture medium respectively alone or in combination with added neurotrophic factor promotes DC growth of different iPSCs in the differentiation of DC.
objective
The mouse fibroblast was reprogrammed into iPSCs and identified by retrovirus vector system. Based on that, iPSCs was induced to differentiate into DC. in vitro.
Materials and methods
The first part is the induction and identification of iPSCs. The method is as follows: using the method of retroviral transfection of 4 kinds of exogenous multi factor Oct4, Sox2, c-Myc, Klf4 plasmid was transfected into Plat-E cells the virus packaging, collect virus supernatant of mice infected rat primary cultured tail fibroblasts, observe the recording process induced pluripotent stem cell generation, and carries on the appraisal analysis. Mainly take the following identification methods, 1) using alkaline phosphatase (AP) staining was observed in undifferentiated iPSCs have the characteristics of ESC, combined with the observation of morphology and immunocytochemistry, identification of totipotency is iPSCs; 2) were identified by immunofluorescence method of stem cell marker Oct, Sox2, SSEA-1 and iPSCs in the induction of surface expression; 3) embryoid bodies (EB) test whether iPSCs can differentiate into embryo development, and detected by immunofluorescence in the embryo of the existence of 3 Ectoderm derived cells; 4) expression of Western blot gene can detect protein: to further validate the infection of mouse fibroblasts to gene expression, the expression is detected by Western blot method has been successfully induced pluripotent iPSCs protein. The factor Western antibody with immunohistochemistry antibody; 5) gene expression analysis (RT-PCR) were determined with RT-PCR Oct4, Sox2, c-Myc, Klf4 can be successfully induced gene in iPSCs, ESC and M1 in the expression level of fiber cells, and make comparative analysis.
The second part is the differentiation of iPSCs into DC. The method is as follows: the basis of successful induced iPSCs culture system were added alone or in combination with different cytokines, such as interleukin IL-3 (10ng/mL), IL-7 (10ng/mL), IL-15 (20ng/mL). Each 2-3D was half changed, adding fresh cytokines training. After 12 ~ 14d, continue to add the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) (4ng/mL) (?) tumor necrosis factor (TNF) (50ng/mL), cultured for 3 ~ 5D, the cells were observed under inverted microscope.
Result
The culture of 1 rat tail fibroblasts and the induction of iPSCs
Using trypsin digestion method cultured rat tail fibroblasts cultured on the next day, observed under inverted phase contrast microscope which showed typical fibroblast characteristics; then iPSCs induced pluripotent stem cell factor 2D virus infection can be observed the morphology of fibroblasts cells began to change, flattened, rounded and small colony like proliferation group. Two weeks after the emergence of a large number of dense and protruding cloned cells arranged closely, well-defined, colony edge. Positive clones and strong refraction were passaged by mechanical method.
Identification of 2iPSCs
We use AP staining method, EB experiments on induction into the iPSCs blot and Western were identified by immunofluorescence staining, the identification results are consistent with the characteristics of ESC; using RT-PCR method to detect multiple gene mRNA levels, visible established cell line iPSCs and ESC were the same, the endogenous expression of Oct4, Sox2. C-Myc, Klf4, and the differentiation of fibroblasts did not express stem cell pluripotency gene Oct4; due to the introduction of exogenous gene, iPSCs cell line RNA gene high level of ESC and mouse fibroblasts of exogenous gene almost completely silent.
3 differentiation of iPSCs into DC in vitro
In vitro differentiation of DC to iPSCs is mainly adopted in the iPSCs culture medium can add several stem cells to the differentiation of DC nutritional factors, such as IL, GM-CSF, TNF, stem cell growth factor and DC growth factor FMS like tyrosine kinase 3 ligand (FIt3L) and lipopolysaccharide. The results showed typical morphology of DC, suggesting that iPSCs in the induction of specific factors can be differentiated into DC.
conclusion
Through the transduction of 4 exogenous pluripotent factors, the mouse tail fibroblasts were successfully induced to be iPSCs, and had the functional and morphological characteristics of iPSCs.
Under the induction of specific factors, iPSCs can successfully differentiate into DC and possess the phenotypic characteristics of DC.
It not only provides a new source of seed cells for tumor immunotherapy in DC, but also helps us to understand the development of DC and its immunomodulatory effect in embryo, providing a valuable experimental platform for clinical disease prevention and drug research.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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