脂肪基质细胞的生物学特性研究及体细胞的重编程载体制备

发布时间：2018-07-11 16:41

本文选题：脂肪基质细胞(ADSCs) + 生物学特性　；参考：《西北农林科技大学》2010年硕士论文

【摘要】： 干细胞研究与应用在疾病治疗、新药研发、药效和毒性评估、克隆动物、转基因动物生产、发育生物学等多个领域产生了极其重要的影响。理论上,胚胎干细胞(embryonic stem cell,ESCs)具有无限增殖和分化为所有类型细胞的能力,然而由于存在伦理争议和免疫排斥等问题,其应用受到限制。近年的研究倾向于寻找可替代ESCs的细胞类型,如对成体干细胞、通过核移植或重编程的方法获得的与ESCs相似的亚全能干细胞等的研究为干细胞的应用提供了更广阔的前景。脂肪基质细胞(adipose-derived stromal cells, ADSCs)由于来源广泛,取材方便,干细胞含量高等特点,已成为成体干细胞研究的热点,然而ADSCs的自我更新和多向分化潜能的分子基础尚不明确,且体外增殖和分化能力受到一定的限制。在ESCs亚全能分化特性维持机制中,Oct3/4、Sox2和Nanog起到了核心作用,因此,本研究着眼于ADSCs中多潜能转录因子的表达情况以及构建体细胞重编程载体的研究,以期为干细胞的研究提供更好的实验材料。本研究以15日龄SD大鼠为实验动物,采集腹股沟皮下脂肪组织,通过胶原酶消化法获得ADCs,培养在DMEM生长培养基中。传代培养后利用RT-PCR、免疫细胞化学染色、流式细胞仪分析和特异性染色等细胞生物学和分子生物学实验技术鉴定了大鼠ADCs的基本生物学特性和多潜能转录因子的表达情况；通过特定因子诱导其向脂肪细胞、成骨细胞和神经细胞分化,并利用上述实验技术对其分化能力进行了评估；获得了包含多潜能转录因子Oct3/4、Sox2、cMyc和Klf4的慢病毒表达载体,并对其进行了鉴定、包装、浓缩及侵染能力检测,为ADSCs的自我更新和多向分化潜能的分子机理及重编程为iPS细胞的研究奠定了基础。 1、大鼠ADSCs的分离培养从大鼠腹股沟皮下脂肪组织分离获得ADSCs,培养在DMEM完全培养基中,并进行传代培养。实验证明,新鲜接种的ADSCs是一种混合的细胞群,呈三角形、梭形、球形或多角形,传代培养后,球形、三角形和多角形细胞不断减少,细胞形态趋于一致,多呈梭形,并且形成了成纤维样的细胞集落。 2、大鼠ADSCs生物学特性的检测 RT-PCR检测结果证明大鼠ADSCs不仅表达多潜能转录因子Oct3/4、Sox2和Nanog,而且其下游的靶基因如cMyc、Fgf4、Gal等在ADSCs中也有表达；免疫细胞化学分析表明ADSCs表达间充质干细胞表面标志CD105,而不表达造血干细胞表面标志CD34和内皮细胞表面标志CD31,并且多潜能转录因子Oct3/4、Sox2、Nanog以及cMyc和端粒酶在ADSCs的细胞核和细胞质内都有表达,而胚胎阶段性抗原SSEA1主要在细胞质内表达；流式细胞仪检测显示第2代大鼠ADSCs中Oct3／4,Sox2,Nanog和SSEA1的表达水平分别为2.0%,3.7%,2.4%和2.6%；并且碱性磷酸酶(alkaline phosphatase,AP)染色呈阳性。 3、大鼠ADSCs诱导分化为脂肪细胞体外通过胰岛素、氢化可的松和转铁蛋白诱导第2代大鼠ADSCs,发现诱导1天即有个别细胞中出现了脂滴,随着诱导时间的延长,脂滴不断积累并融合形成大脂滴,油红O染色呈阳性,诱导11d后,RT-PCR检测表达脂肪细胞标志基因PPARγ、FAS和LPL,并且与对照组相比差异显著。 4、大鼠ADSCs诱导分化为成骨细胞体外可通过氯化锂诱导ADSCs分化为成骨细胞。氯化锂诱导1天后,细胞形态由成纤维样转变成方块状,随着诱导时间的延长,AP活性不断提高,并且形成了广泛的钙化结节,茜素红染色呈阳性,免疫细胞化学染色结果显示表达成骨细胞特异性标志蛋白OSX和OPN。 5、大鼠ADSCs诱导分化为神经细胞通过在无血清培养基中添加β-巯基乙醇可诱导ADSCs分化为神经样细胞,诱导20min后,细胞内缩并伸出突触,1h后,形成了具有多级突触的神经元样细胞,并连接成网,免疫细胞化学染色检测表达神经细胞特异性蛋白Nestin、Tau和NF。 6、多潜能转录因子载体的鉴定、包装及转染通过PCR和酶切的方法鉴定四个慢病毒转移载体(中国科学院上海生命科学研究院肖磊博士馈赠),结果显示四个慢病毒载体中分别包含了人的多潜能转录因子Oct3／4、Sox2、cMyc和Klf4的ORF序列,然后在293T细胞中包装获得病毒上清液,高速离心浓缩,并转染大鼠ADSCs测定转染效率,发现获得的病毒液转染效率太低,不适合进行后面的研究。综上所述,本实验成功的分离了大鼠ADSCs,并对其进行了生物学特性研究,结果证明ADSCs表达间充质干细胞表面标志CD 105,不表达造血干细胞表面标志CD34和表皮细胞表面标志CD31,并且表达多种胚胎干细胞特异性的多潜能转录因子,在本实验建立的诱导体系下具有向脂肪细胞、成骨细胞和神经细胞分化的潜能。此外,本实验成功制备了体细胞重编程载体,但其感染效率仍需提高。
[Abstract]:Stem cell research and applications have been extremely important in the fields of disease treatment, new drug development, drug efficiency and toxicity assessment, cloning animals, transgenic animal production, developmental biology and many other fields. In theory, embryonic stem cell (ESCs) has the ability to proliferate and differentiate infinitely to all types of cells, however, because of its existence The application of ethical disputes and immune rejection is limited. In recent years, research tends to find the cell types that can be replaced by ESCs, for example, adult stem cells, which are similar to ESCs by nuclear transplantation or reprogramming, provide a broader prospect for the use of stem cells. Adipose-derived stromal cells, ADSCs) has become a hot spot in adult stem cell research because of its wide source, convenient extraction and high stem cell content. However, the molecular basis of self-renewal and multidirectional differentiation of ADSCs is not clear, and the ability of proliferation and differentiation in vitro is limited. The differentiation characteristics of ESCs subtotal energy are maintained. In the mechanism, Oct3/4, Sox2 and Nanog have played a key role. Therefore, this study focuses on the expression of multipotential transcription factors in ADSCs and the research on the construction of somatic cell reprogramming carriers in order to provide better experimental materials for the research of stem cells.
In this study, 15 day old SD rats were used as experimental animals to collect subcutaneous fat tissues of the groin and obtain ADCs by collagenase digestion. They were cultured in the DMEM growth medium. After subculture, RT-PCR, immunocytochemical staining, flow cytometer analysis and specific staining were used to identify the large cell biology and molecular biology experiments. The basic biological characteristics of rat ADCs and the expression of multipotential transcriptional factors; induced by specific factors to differentiate into adipocytes, osteoblasts and nerve cells, and evaluate their differentiation using the above experimental techniques, and obtain the lentivirus expression vector containing the multipotential transcription factors, Oct3/4, Sox2, cMyc and Klf4. The identification, packaging, concentration and detection of infection ability were carried out, which laid the foundation for the molecular mechanism of ADSCs's self renewal and multidirectional differentiation potential and the research of reprogramming for iPS cells.
1, isolation and culture of ADSCs in rats
ADSCs was obtained from the subcutaneous fat tissue of the rat groin and cultured in the DMEM complete medium and carried out. The experiment showed that the fresh ADSCs was a mixed cell group, which was triangular, spindle, spherical or polygonal. After the culture, the cells were decreasing, the shape of the cells tended to be consistent, and the cell morphology tended to be consistent. It was spindle shaped and formed fibroblast like cell colonies.
2, detection of biological characteristics of ADSCs in rats
The results of RT-PCR test showed that ADSCs not only expressed the multipotential transcription factors Oct3/4, Sox2 and Nanog, but also the target genes in the downstream, such as cMyc, Fgf4, Gal, etc., were also expressed in ADSCs, and the immunocytochemical analysis showed that the ADSCs expression of mesenchymal stem cells was CD105, but did not express the surface marker CD34 and endothelial cell surface of the hematopoietic stem cells. CD31, and multipotential transcription factors Oct3/4, Sox2, Nanog, cMyc and telomerase are expressed in the nucleus and cytoplasm of ADSCs, while the embryonic stage antigen SSEA1 is mainly expressed in the cytoplasm, and the flow cytometry shows that the expression levels of Oct3 / 4, Sox2, Nanog and SSEA1 are 2%, 3.7%, 2.4 respectively in the ADSCs of the second generation rats. % and 2.6%; and alkaline phosphatase (AP) staining was positive.
3, rat ADSCs induced to differentiate into adipocytes
Second generation ADSCs rats were induced by insulin, hydrocortisone and transferrin in vitro. It was found that lipid droplets appeared in a few cells for 1 days. With the prolongation of the induction time, lipid droplets accumulated and fused to form large fat drops. The oil red O staining was positive. After the induction of 11d, RT-PCR was used to detect and express the fat cell marker gene PPAR gamma, FAS and LPL. The difference was significant compared with the control group.
4, ADSCs induced differentiation into osteoblasts in rats
In vitro, ADSCs can be induced to differentiate into osteoblasts through lithium chloride. After 1 days of induction of lithium chloride, the cell morphology is transformed from fibroid to square. With the prolongation of the induction time, the activity of AP continues to increase and forms a broad calcified nodule. Alizarin red staining is positive. The results of immunocytochemical staining show that the expression of osteoblast is specific. Sex marker protein OSX and OPN.
5, rat ADSCs induced differentiation into neural cells
By adding beta mercaptoethanol into the serum-free medium, ADSCs can be induced to differentiate into nerve like cells. After induction of 20min, the cells constriction and protruding synapses. After 1h, the neuron like cells with multilevel synapses are formed and connected to the net. Immunocytochemical staining is used to detect and express the specific protein Nestin, Tau and NF..
6, identification, packaging and transfection of multipotential transcription factor vectors.
Four lentivirus transfer vectors were identified by PCR and enzyme digestion. The results showed that the four lentivirus carriers included human multipotential transcription factors Oct3 / 4, Sox2, cMyc and Klf4 ORF sequences, and then packaged in 293T cells to obtain the supernatant of the virus, at high speed. After concentrating and transfecting rat ADSCs, the transfection efficiency was detected. It was found that the transfection efficiency of the obtained virus solution was too low to be suitable for subsequent studies.
To sum up, the experiment successfully separated the rat ADSCs and studied its biological characteristics. The results showed that ADSCs expressed CD 105 on the surface of mesenchymal stem cells, did not express the surface marker CD34 of the hematopoietic stem cells and the surface marker CD31 of the epidermal cells, and expressed the specific pluripotent transcription factors of a variety of embryonic embryonic stem cells. The induced system has the potential to differentiate into adipocytes, osteoblasts and nerve cells under the induced system. In addition, the somatic cell reprogramming carrier is successfully prepared in this experiment, but the efficiency of the infection still needs to be improved.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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