本文選題：脂肪基質(zhì)細(xì)胞(ADSCs) + 生物學(xué)特性��； 參考：《西北農(nóng)林科技大學(xué)》2010年碩士論文

【摘要】： 干細(xì)胞研究與應(yīng)用在疾病治療、新藥研發(fā)、藥效和毒性評(píng)估、克隆動(dòng)物、轉(zhuǎn)基因動(dòng)物生產(chǎn)、發(fā)育生物學(xué)等多個(gè)領(lǐng)域產(chǎn)生了極其重要的影響。理論上,胚胎干細(xì)胞(embryonic stem cell,ESCs)具有無限增殖和分化為所有類型細(xì)胞的能力,然而由于存在倫理爭(zhēng)議和免疫排斥等問題,其應(yīng)用受到限制。近年的研究?jī)A向于尋找可替代ESCs的細(xì)胞類型,如對(duì)成體干細(xì)胞、通過核移植或重編程的方法獲得的與ESCs相似的亞全能干細(xì)胞等的研究為干細(xì)胞的應(yīng)用提供了更廣闊的前景。脂肪基質(zhì)細(xì)胞(adipose-derived stromal cells, ADSCs)由于來源廣泛,取材方便,干細(xì)胞含量高等特點(diǎn),已成為成體干細(xì)胞研究的熱點(diǎn),然而ADSCs的自我更新和多向分化潛能的分子基礎(chǔ)尚不明確,且體外增殖和分化能力受到一定的限制。在ESCs亞全能分化特性維持機(jī)制中,Oct3/4、Sox2和Nanog起到了核心作用,因此,本研究著眼于ADSCs中多潛能轉(zhuǎn)錄因子的表達(dá)情況以及構(gòu)建體細(xì)胞重編程載體的研究,以期為干細(xì)胞的研究提供更好的實(shí)驗(yàn)材料。 本研究以15日齡SD大鼠為實(shí)驗(yàn)動(dòng)物,采集腹股溝皮下脂肪組織,通過膠原酶消化法獲得ADCs,培養(yǎng)在DMEM生長(zhǎng)培養(yǎng)基中。傳代培養(yǎng)后利用RT-PCR、免疫細(xì)胞化學(xué)染色、流式細(xì)胞儀分析和特異性染色等細(xì)胞生物學(xué)和分子生物學(xué)實(shí)驗(yàn)技術(shù)鑒定了大鼠ADCs的基本生物學(xué)特性和多潛能轉(zhuǎn)錄因子的表達(dá)情況；通過特定因子誘導(dǎo)其向脂肪細(xì)胞、成骨細(xì)胞和神經(jīng)細(xì)胞分化,并利用上述實(shí)驗(yàn)技術(shù)對(duì)其分化能力進(jìn)行了評(píng)估；獲得了包含多潛能轉(zhuǎn)錄因子Oct3/4、Sox2、cMyc和Klf4的慢病毒表達(dá)載體,并對(duì)其進(jìn)行了鑒定、包裝、濃縮及侵染能力檢測(cè),為ADSCs的自我更新和多向分化潛能的分子機(jī)理及重編程為iPS細(xì)胞的研究奠定了基礎(chǔ)。 1、大鼠ADSCs的分離培養(yǎng) 從大鼠腹股溝皮下脂肪組織分離獲得ADSCs,培養(yǎng)在DMEM完全培養(yǎng)基中,并進(jìn)行傳代培養(yǎng)。實(shí)驗(yàn)證明,新鮮接種的ADSCs是一種混合的細(xì)胞群,呈三角形、梭形、球形或多角形,傳代培養(yǎng)后,球形、三角形和多角形細(xì)胞不斷減少,細(xì)胞形態(tài)趨于一致,多呈梭形,并且形成了成纖維樣的細(xì)胞集落。 2、大鼠ADSCs生物學(xué)特性的檢測(cè) RT-PCR檢測(cè)結(jié)果證明大鼠ADSCs不僅表達(dá)多潛能轉(zhuǎn)錄因子Oct3/4、Sox2和Nanog,而且其下游的靶基因如cMyc、Fgf4、Gal等在ADSCs中也有表達(dá)；免疫細(xì)胞化學(xué)分析表明ADSCs表達(dá)間充質(zhì)干細(xì)胞表面標(biāo)志CD105,而不表達(dá)造血干細(xì)胞表面標(biāo)志CD34和內(nèi)皮細(xì)胞表面標(biāo)志CD31,并且多潛能轉(zhuǎn)錄因子Oct3/4、Sox2、Nanog以及cMyc和端粒酶在ADSCs的細(xì)胞核和細(xì)胞質(zhì)內(nèi)都有表達(dá),而胚胎階段性抗原SSEA1主要在細(xì)胞質(zhì)內(nèi)表達(dá)；流式細(xì)胞儀檢測(cè)顯示第2代大鼠ADSCs中Oct3／4,Sox2,Nanog和SSEA1的表達(dá)水平分別為2.0%,3.7%,2.4%和2.6%；并且堿性磷酸酶(alkaline phosphatase,AP)染色呈陽(yáng)性。 3、大鼠ADSCs誘導(dǎo)分化為脂肪細(xì)胞 體外通過胰島素、氫化可的松和轉(zhuǎn)鐵蛋白誘導(dǎo)第2代大鼠ADSCs,發(fā)現(xiàn)誘導(dǎo)1天即有個(gè)別細(xì)胞中出現(xiàn)了脂滴,隨著誘導(dǎo)時(shí)間的延長(zhǎng),脂滴不斷積累并融合形成大脂滴,油紅O染色呈陽(yáng)性,誘導(dǎo)11d后,RT-PCR檢測(cè)表達(dá)脂肪細(xì)胞標(biāo)志基因PPARγ、FAS和LPL,并且與對(duì)照組相比差異顯著。 4、大鼠ADSCs誘導(dǎo)分化為成骨細(xì)胞 體外可通過氯化鋰誘導(dǎo)ADSCs分化為成骨細(xì)胞。氯化鋰誘導(dǎo)1天后,細(xì)胞形態(tài)由成纖維樣轉(zhuǎn)變成方塊狀,隨著誘導(dǎo)時(shí)間的延長(zhǎng),AP活性不斷提高,并且形成了廣泛的鈣化結(jié)節(jié),茜素紅染色呈陽(yáng)性,免疫細(xì)胞化學(xué)染色結(jié)果顯示表達(dá)成骨細(xì)胞特異性標(biāo)志蛋白OSX和OPN。 5、大鼠ADSCs誘導(dǎo)分化為神經(jīng)細(xì)胞 通過在無血清培養(yǎng)基中添加β-巰基乙醇可誘導(dǎo)ADSCs分化為神經(jīng)樣細(xì)胞,誘導(dǎo)20min后,細(xì)胞內(nèi)縮并伸出突觸,1h后,形成了具有多級(jí)突觸的神經(jīng)元樣細(xì)胞,并連接成網(wǎng),免疫細(xì)胞化學(xué)染色檢測(cè)表達(dá)神經(jīng)細(xì)胞特異性蛋白Nestin、Tau和NF。 6、多潛能轉(zhuǎn)錄因子載體的鑒定、包裝及轉(zhuǎn)染 通過PCR和酶切的方法鑒定四個(gè)慢病毒轉(zhuǎn)移載體(中國(guó)科學(xué)院上海生命科學(xué)研究院肖磊博士饋贈(zèng)),結(jié)果顯示四個(gè)慢病毒載體中分別包含了人的多潛能轉(zhuǎn)錄因子Oct3／4、Sox2、cMyc和Klf4的ORF序列,然后在293T細(xì)胞中包裝獲得病毒上清液,高速離心濃縮,并轉(zhuǎn)染大鼠ADSCs測(cè)定轉(zhuǎn)染效率,發(fā)現(xiàn)獲得的病毒液轉(zhuǎn)染效率太低,不適合進(jìn)行后面的研究。 綜上所述,本實(shí)驗(yàn)成功的分離了大鼠ADSCs,并對(duì)其進(jìn)行了生物學(xué)特性研究,結(jié)果證明ADSCs表達(dá)間充質(zhì)干細(xì)胞表面標(biāo)志CD 105,不表達(dá)造血干細(xì)胞表面標(biāo)志CD34和表皮細(xì)胞表面標(biāo)志CD31,并且表達(dá)多種胚胎干細(xì)胞特異性的多潛能轉(zhuǎn)錄因子,在本實(shí)驗(yàn)建立的誘導(dǎo)體系下具有向脂肪細(xì)胞、成骨細(xì)胞和神經(jīng)細(xì)胞分化的潛能。此外,本實(shí)驗(yàn)成功制備了體細(xì)胞重編程載體,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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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