本文關(guān)鍵詞：免疫磁珠體外分離純化大鼠毛囊干細(xì)胞的實(shí)驗(yàn)研究　出處：《重慶大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 毛囊 毛囊干細(xì)胞 免疫磁珠 細(xì)胞分離純化

【摘要】： 在毛囊的形態(tài)發(fā)生和毛發(fā)的周期性生長(zhǎng)過程中,毛囊干細(xì)胞(Hair follicle stem cells, HSCs)起著至關(guān)重要的作用,它們能自我更新,并呈現(xiàn)出多能性,能形成一系列表皮、皮脂腺以及毛囊的細(xì)胞[1-5]。目前研究表明,毛囊干細(xì)胞位于毛囊外根鞘的隆突區(qū),即bulge區(qū)域[6-8],它們不但對(duì)毛發(fā)再生及促進(jìn)皮膚損傷后功能與結(jié)構(gòu)的完全修復(fù)有重要意義,而且在干細(xì)胞生物學(xué)的核心問題,如基因及蛋白圖譜、干細(xì)胞可塑性及干細(xì)胞微環(huán)境的研究中,毛囊干細(xì)胞也是一個(gè)十分有益的模型[9]。但由于組織中干細(xì)胞的數(shù)量相對(duì)稀少,且與它們的子代細(xì)胞細(xì)胞無顯著差異,故很難區(qū)分它們。這極大地障礙了對(duì)毛囊干細(xì)胞的深入研究。如何獲得足量高純度、活性好、保持低分化狀態(tài)的毛囊干細(xì)胞,是當(dāng)前研究急需解決的問題。 目前,毛囊干細(xì)胞的篩選純化主要有兩類方法:差速貼壁法和免疫法。差速貼壁法分選精度低,實(shí)驗(yàn)重復(fù)性差,并非理想的分選方法。免疫法是利用干細(xì)胞表面表達(dá)的特殊抗原,即干細(xì)胞標(biāo)記物來實(shí)現(xiàn)分選目的。目前常用的鼠類毛囊干細(xì)胞陽(yáng)性標(biāo)記物有Keratin 15,β1-integrin,α6-integrin和CD34 [2、4、8、10],陰性標(biāo)記物主要有CD71[11]。免疫法主要包括流式細(xì)胞儀分選法和免疫磁珠分選法。 免疫磁珠分選(Magnetic activated cell sorting,MACS)技術(shù)近年來發(fā)展起來的細(xì)胞分選的新方法。與其他分選方法相比,免疫磁珠法具有分離效率高,細(xì)胞活性好,操作簡(jiǎn)便等優(yōu)點(diǎn)。目前,MACS技術(shù)已經(jīng)在分選血液系統(tǒng)細(xì)胞研究領(lǐng)域中得到廣泛應(yīng)用[12],但在上皮類干細(xì)胞中尚未成功開展。 本研究嘗試使用Vario MACS法分選原位組織里的毛囊干細(xì)胞并獲得了理想的純化富集效果。主要研究方法和結(jié)果如下: ①大鼠毛囊干細(xì)胞的組織定位 為了確定毛囊干細(xì)胞在大鼠觸須部皮膚中的組織定位,我們采用免疫組織化學(xué)和免疫熒光技術(shù),分別檢測(cè)目前公認(rèn)的毛囊干細(xì)胞陽(yáng)性標(biāo)記CD34,α6-integrin,β1-integrin,以及分化指標(biāo)CD71在大鼠觸須毛囊中的表達(dá)。結(jié)果表明,CD34、α6-integrin、β1-integrin的表達(dá)位于毛囊外根鞘細(xì)胞的胞漿和胞膜,而毛球部細(xì)胞未見表達(dá)。CD71在表皮、毛球有強(qiáng)表達(dá),在毛囊外根鞘弱表達(dá)。我們的研究確認(rèn)毛囊干細(xì)胞位于毛囊的外根鞘。 分別采用組織塊培養(yǎng)法和改良消化法,用無血清的KSFM培養(yǎng)基培養(yǎng)了毛囊原代bulge細(xì)胞。細(xì)胞生長(zhǎng)良好,具有良好的克隆形成能力,且CD34,α6-integrin,β1-integrin陽(yáng)性表達(dá)。表明我們的實(shí)驗(yàn)方法能成功地培養(yǎng)毛囊bulge區(qū)細(xì)胞。 ②Vario MACS高效富集大鼠毛囊干細(xì)胞技術(shù)的建立 本部分實(shí)驗(yàn)選用目前公認(rèn)最好的三種大鼠毛囊干細(xì)胞表面標(biāo)記物制定四種分選策略:CD34單標(biāo)陽(yáng)性分選,α6單標(biāo)陽(yáng)性分選,CD34和CD71雙標(biāo)復(fù)合分選以及α6和CD71雙標(biāo)復(fù)合分選。我們用MACS技術(shù)從大鼠觸須毛囊bulge區(qū)細(xì)胞懸液中純化富集毛囊干細(xì)胞并進(jìn)行熒光標(biāo)記,通過流式檢測(cè)細(xì)胞熒光強(qiáng)度鑒定目標(biāo)細(xì)胞并評(píng)價(jià)分選效率,以篩選出最為理想的分選方案。 流式檢測(cè)結(jié)果顯示四種分選策略均可獲得理想的分選效率,目標(biāo)細(xì)胞群中干細(xì)胞的陽(yáng)性率分別為:CD34bri細(xì)胞陽(yáng)性率為81.24%,α6bri細(xì)胞陽(yáng)性率為100.00%,CD34briCD71dim細(xì)胞陽(yáng)性率為82.54%,α6briCD71dim細(xì)胞陽(yáng)性率為89.24%。 雙標(biāo)復(fù)合分選策略可以區(qū)分干細(xì)胞和它的子代細(xì)胞,比單標(biāo)陽(yáng)性分選獲得的干細(xì)胞純度更高。而CD34抗體分選操作步驟比α6更為簡(jiǎn)單快捷,在分選效率差別不大的情況下,我們認(rèn)為CD34和CD71雙標(biāo)復(fù)合分選是最理想的分選策略。 ③CD34bri毛囊干細(xì)胞生物學(xué)特性的研究 本部分實(shí)驗(yàn)以Vario MACS分選獲得的CD34bri細(xì)胞和CD34dim細(xì)胞為實(shí)驗(yàn)對(duì)象,研究毛囊干細(xì)胞的生物學(xué)特性,并對(duì)Vario MACS分選技術(shù)進(jìn)行進(jìn)一步的評(píng)估。我們檢測(cè)分選前后細(xì)胞活性,用熒光顯微鏡觀察抗體在細(xì)胞中的標(biāo)記情況,分別培養(yǎng)CD34bri細(xì)胞和CD34dim細(xì)胞并繪制生長(zhǎng)曲線,免疫細(xì)胞化學(xué)檢測(cè)細(xì)胞中CD34,α6-integrin和β1-integrin的表達(dá),掃描電鏡和透射電鏡分別觀察這兩種細(xì)胞的表面形態(tài)和超微結(jié)構(gòu)形態(tài)。 研究結(jié)果表明,Vario MACS分選的大鼠毛囊干細(xì)胞生長(zhǎng)較緩慢,細(xì)胞活力受到一定影響。體外用KSFM培養(yǎng)基培養(yǎng)CD34bri和CD34dim細(xì)胞。細(xì)胞生長(zhǎng)曲線顯示,與CD34dim細(xì)胞相比,CD34bri細(xì)胞增殖能力更強(qiáng),具有更好的克隆形成能力。CD34,α6-integrin和β1-integrin在這兩種細(xì)胞中的表達(dá)集中于胞質(zhì)和胞膜,并且在CD34bri細(xì)胞中表達(dá)明顯強(qiáng)于CD34dim細(xì)胞。CD34在CD34dim細(xì)胞中只有很弱的表達(dá)。掃描電鏡可見分選出來的CD34bri細(xì)胞呈圓形,形狀規(guī)則,細(xì)胞表面黏附有數(shù)量不等的圓形磁珠,而CD34dim細(xì)胞、培養(yǎng)7d和直接消化獲得的毛囊bulge細(xì)胞的表面均未見有磁珠。透射電鏡下可見CD34bri細(xì)胞細(xì)胞核較大,核仁明顯,胞漿小,核漿比例大,細(xì)胞器少,呈幼稚細(xì)胞的典型形態(tài)。 本研究的意義首先是成功地利用Vario MACS法高效地純化富集大鼠毛囊干細(xì)胞,獲得了該干細(xì)胞的電鏡形態(tài)學(xué)圖象,而且更重要的是分離的毛囊干細(xì)胞具有良好的活性狀態(tài),并首次在體外培養(yǎng)成功,這為下一步建立毛囊干細(xì)胞系奠定了重要基礎(chǔ)。
[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.

【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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