免疫磁珠体外分离纯化大鼠毛囊干细胞的实验研究

发布时间：2018-01-01 04:10

本文关键词：免疫磁珠体外分离纯化大鼠毛囊干细胞的实验研究　出处：《重庆大学》2008年硕士论文　论文类型：学位论文

【摘要】： 在毛囊的形态发生和毛发的周期性生长过程中,毛囊干细胞(Hair follicle stem cells, HSCs)起着至关重要的作用,它们能自我更新,并呈现出多能性,能形成一系列表皮、皮脂腺以及毛囊的细胞[1-5]。目前研究表明,毛囊干细胞位于毛囊外根鞘的隆突区,即bulge区域[6-8],它们不但对毛发再生及促进皮肤损伤后功能与结构的完全修复有重要意义,而且在干细胞生物学的核心问题,如基因及蛋白图谱、干细胞可塑性及干细胞微环境的研究中,毛囊干细胞也是一个十分有益的模型[9]。但由于组织中干细胞的数量相对稀少,且与它们的子代细胞细胞无显著差异,故很难区分它们。这极大地障碍了对毛囊干细胞的深入研究。如何获得足量高纯度、活性好、保持低分化状态的毛囊干细胞,是当前研究急需解决的问题。目前,毛囊干细胞的筛选纯化主要有两类方法:差速贴壁法和免疫法。差速贴壁法分选精度低,实验重复性差,并非理想的分选方法。免疫法是利用干细胞表面表达的特殊抗原,即干细胞标记物来实现分选目的。目前常用的鼠类毛囊干细胞阳性标记物有Keratin 15,β1-integrin,α6-integrin和CD34 [2、4、8、10],阴性标记物主要有CD71[11]。免疫法主要包括流式细胞仪分选法和免疫磁珠分选法。免疫磁珠分选(Magnetic activated cell sorting,MACS)技术近年来发展起来的细胞分选的新方法。与其他分选方法相比,免疫磁珠法具有分离效率高,细胞活性好,操作简便等优点。目前,MACS技术已经在分选血液系统细胞研究领域中得到广泛应用[12],但在上皮类干细胞中尚未成功开展。本研究尝试使用Vario MACS法分选原位组织里的毛囊干细胞并获得了理想的纯化富集效果。主要研究方法和结果如下: ①大鼠毛囊干细胞的组织定位为了确定毛囊干细胞在大鼠触须部皮肤中的组织定位,我们采用免疫组织化学和免疫荧光技术,分别检测目前公认的毛囊干细胞阳性标记CD34,α6-integrin,β1-integrin,以及分化指标CD71在大鼠触须毛囊中的表达。结果表明,CD34、α6-integrin、β1-integrin的表达位于毛囊外根鞘细胞的胞浆和胞膜,而毛球部细胞未见表达。CD71在表皮、毛球有强表达,在毛囊外根鞘弱表达。我们的研究确认毛囊干细胞位于毛囊的外根鞘。分别采用组织块培养法和改良消化法,用无血清的KSFM培养基培养了毛囊原代bulge细胞。细胞生长良好,具有良好的克隆形成能力,且CD34,α6-integrin,β1-integrin阳性表达。表明我们的实验方法能成功地培养毛囊bulge区细胞。 ②Vario MACS高效富集大鼠毛囊干细胞技术的建立本部分实验选用目前公认最好的三种大鼠毛囊干细胞表面标记物制定四种分选策略:CD34单标阳性分选,α6单标阳性分选,CD34和CD71双标复合分选以及α6和CD71双标复合分选。我们用MACS技术从大鼠触须毛囊bulge区细胞悬液中纯化富集毛囊干细胞并进行荧光标记,通过流式检测细胞荧光强度鉴定目标细胞并评价分选效率,以筛选出最为理想的分选方案。流式检测结果显示四种分选策略均可获得理想的分选效率,目标细胞群中干细胞的阳性率分别为:CD34bri细胞阳性率为81.24%,α6bri细胞阳性率为100.00%,CD34briCD71dim细胞阳性率为82.54%,α6briCD71dim细胞阳性率为89.24%。双标复合分选策略可以区分干细胞和它的子代细胞,比单标阳性分选获得的干细胞纯度更高。而CD34抗体分选操作步骤比α6更为简单快捷,在分选效率差别不大的情况下,我们认为CD34和CD71双标复合分选是最理想的分选策略。 ③CD34bri毛囊干细胞生物学特性的研究本部分实验以Vario MACS分选获得的CD34bri细胞和CD34dim细胞为实验对象,研究毛囊干细胞的生物学特性,并对Vario MACS分选技术进行进一步的评估。我们检测分选前后细胞活性,用荧光显微镜观察抗体在细胞中的标记情况,分别培养CD34bri细胞和CD34dim细胞并绘制生长曲线,免疫细胞化学检测细胞中CD34,α6-integrin和β1-integrin的表达,扫描电镜和透射电镜分别观察这两种细胞的表面形态和超微结构形态。研究结果表明,Vario MACS分选的大鼠毛囊干细胞生长较缓慢,细胞活力受到一定影响。体外用KSFM培养基培养CD34bri和CD34dim细胞。细胞生长曲线显示,与CD34dim细胞相比,CD34bri细胞增殖能力更强,具有更好的克隆形成能力。CD34,α6-integrin和β1-integrin在这两种细胞中的表达集中于胞质和胞膜,并且在CD34bri细胞中表达明显强于CD34dim细胞。CD34在CD34dim细胞中只有很弱的表达。扫描电镜可见分选出来的CD34bri细胞呈圆形,形状规则,细胞表面黏附有数量不等的圆形磁珠,而CD34dim细胞、培养7d和直接消化获得的毛囊bulge细胞的表面均未见有磁珠。透射电镜下可见CD34bri细胞细胞核较大,核仁明显,胞浆小,核浆比例大,细胞器少,呈幼稚细胞的典型形态。本研究的意义首先是成功地利用Vario MACS法高效地纯化富集大鼠毛囊干细胞,获得了该干细胞的电镜形态学图象,而且更重要的是分离的毛囊干细胞具有良好的活性状态,并首次在体外培养成功,这为下一步建立毛囊干细胞系奠定了重要基础。
[Abstract]:In the period of hair follicle morphogenesis and growth of hair follicle stem cells (Hair follicle stem cells, HSCs) plays a vital role, they can self renew and exhibit pluripotency, can form a series of epidermis, sebaceous gland and hair follicle cells [1-5. The present study showed that hair follicle stem cells located in the outer root sheath of hair follicle area, bulge area [6-8], they are not only to promote the regeneration and repair after injury has important significance to fully function and structure of the skin and hair, the core issues in stem cell biology, such as gene and protein profiles, studies of stem cell plasticity and microenvironment of stem cells in hair follicles. Stem cell is a useful model for [9]. but due to a relatively small number of stem cells in tissue, and no significant difference with their offspring cells, so it is difficult to distinguish between them. This greatly hindered the hair follicle Further research on stem cells. How to obtain the hair follicle stem cells with high purity, good activity and low differentiation is an urgent problem to be solved at present.
At present, the purified cells there are two main methods: hair follicle stem differentialadherence method and immune method. Differentialadherence method separation and low accuracy, poor reproducibility experiments, the sorting method is not ideal. The immune method is the use of stem cell surface expression of the special antigen, namely stem cell markers to achieve separation purpose at present commonly used rodent hair follicle stem cells positive markers Keratin 15, beta 1-integrin, alpha 6-integrin and CD34 [2,4,8,10], negative markers are mainly CD71[11]. immunoassay including flow cytometry sorting method and immunomagnetic separation method.
Immunomagnetic separation (Magnetic activated cell sorting, MACS) a new method of cell separation technology developed in recent years. Compared with other separation methods, immunomagnetic beads method has high separation efficiency, good cell activity, easy operation etc.. At present, the MACS technology has been widely used in the research field of [12] cell sorting in the blood system. But in the epithelial stem cells has not been successfully carried out.
In this study, the Vario MACS method was used to select the hair follicle stem cells in the tissue in situ and to obtain the ideal purification and enrichment effect. The main research methods and results are as follows:
Tissue localization of rat hair follicle stem cells
In order to determine the hair follicle stem cells in the tissue localization of whisker skin in rats, we used immunohistochemistry and immunofluorescence technique, were used to detect the currently accepted hair follicle stem cells labeled CD34, 6-integrin alpha, 1-integrin beta, and CD71 expression of differentiation markers in rat whisker hair follicle. The results showed that CD34, alpha 6-integrin, cytoplasm and membrane 1-integrin expression in the hair follicle outer root sheath cells, and hair bulb cells no expression of.CD71 in epidermis, hair ball has strong expression in the outer root sheath weak expression. Our study confirmed that hair follicle stem cells located in the outer root sheath of the hair follicle.
Using tissue culture method and modified digestion method, culture medium of primary hair follicle cells with bulge serum free KSFM medium. The cells grow well, with good clone formation ability and CD34, 6-integrin alpha, 1-integrin beta expression. Experiments show that our method can successfully cultured hair follicle cells bulge.
Establishment of Vario MACS high efficiency enrichment of rat hair follicle stem cells
This part of experiment three currently recognized the best rat follicle stem cell surface markers to develop four kinds of sorting strategies: CD34 single labeled positive sorting, alpha 6 single standard positive sorting, CD34 and CD71 double labeled compound separation and alpha 6 and CD71 double composite separation. Enrichment of hair follicle stem cells were labeled and purified to our hair follicle bulge cells from rat contact with MACS technology in suspension, the fluorescence intensity was detected by flow cytometry identification of target cells and estimate the sorting efficiency, in order to screen the ideal separation scheme.
Flow cytometry results showed that the separation efficiency of four kinds of sorting strategy can obtain ideal stem cells, the positive rate was the target cell population: the positive rate of CD34bri cells was 81.24%, the positive rate of alpha 6bri cells was 100%, the positive rate of CD34briCD71dim cells was 82.54%, alpha 6briCD71dim cell positive rate was 89.24%.
Double composite separation strategy can distinguish between stem cells and their progeny cells higher than single labeled stem cell sorting. The purity of positive CD34 antibody and separation steps than alpha 6 is more simple, a little difference in separation efficiency, we believe that the CD34 and CD71 double composite separation is sorting strategy the ideal.
Study on the biological characteristics of CD34bri hair follicle stem cells
CD34bri cells and CD34dim cells in this part of the experiment by Vario MACS separation obtained as the experimental object, the biological characteristics of hair follicle stem cell research, and further assessment of Vario MACS separation technology. We detected cell activity before and after sorting, labeling with fluorescence microscopy antibody in cells, CD34bri cells and CD34dim cells were cultured and draw the growth curve, CD34 were detected by immunocytochemistry, the expression of 6-integrin alpha and beta 1-integrin, scanning electron microscope and transmission electron microscope were used to observe the surface morphology and ultrastructural structure of the two kinds of cell morphology.
The results show that the Vario MACS separation of rat follicle stem cell growth is slow, the cell viability affected. In vitro cultured in KSFM CD34bri and CD34dim cells. Cell growth curve showed that, compared with CD34dim cells, CD34bri cell proliferation ability, cloning has better forming ability of.CD34, the expression of 6-integrin alpha and beta 1-integrin in these two types of cells concentrated in the cytoplasm and membrane, and the expression of.CD34 was stronger than that of CD34dim cells in CD34dim cells was weakly expressed in CD34bri cells. Scanning electron microscopy showed that the separated CD34bri cells were round, irregular shape, cell surface adhesion to different number of circular beads, and CD34dim cells 7d, surface culture and direct digestion obtained hair follicle bulge cells there was no beads. Transmission electron microscopy showed CD34bri cells large nucleus, obvious nucleolus, cytoplasm, nucleus The proportion of the pulp is large and the organelle is few, which is typical of the infantile cells.
The significance of this study is the first successful use of Vario MACS method for efficient purification of rat hair follicle stem cell enrichment, the morphology image of the stem cells, and the more important is the separation of the hair follicle stem cells with activity in good condition, and for the first time successfully cultured in vitro, the next step for the establishment of hair follicle stem cells the Department has laid an important foundation.

【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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