本文選題：細胞培養(yǎng) + 外根鞘細胞　； 參考：《昆明醫(yī)學院》2011年碩士論文

【摘要】：目的：分離和培養(yǎng)出毛囊來源的種子細胞,體外和體內(nèi)誘導毛囊形成,并探討毛囊的生物學特征和種子細胞在毛囊形成中的作用。 方法：(1)用酶消化法結合組織塊培養(yǎng)法從新生兒包皮獲取皮膚成纖維細胞(Fb)；(2)購買人的毛囊外根鞘細胞株(HHFORSC)和人毛囊毛乳頭細胞株(HHDPC),并行復蘇、培養(yǎng)和傳代；(3)將毛囊外根鞘細胞(ORSC)、毛乳頭細胞(DPC)和絲裂霉素干預后的成纖維細胞按一定組合制成細胞懸液,并按不同的接種順序接種于海藻酸鈉3D細胞支架上,構建毛囊的體外三維模型,培養(yǎng)8周,行HE染色光鏡觀察毛囊形成的情況；同時將該三維模型移植入bal/bcl裸鼠皮下,飼養(yǎng)裸鼠8周,移植部位取材后分別行HE染色、免疫組化和電鏡觀察毛囊形成的情況。 結果：(1)成功獲得人Fb；(2)復蘇、培養(yǎng)和傳代的HHFORSC、HHDPC和自行分離、培養(yǎng)的Fb生長正常；(3)體外構建的毛囊三維模型,HE染色可見支架中有均勻散在分布的毛囊混合細胞,未見到角化物質(zhì)及毛囊結構；(4)裸鼠皮下移植物HE染色可見細胞聚集成團,有呈環(huán)狀排列的毛囊樣結構,CK14,CK15、β1整合素和波形絲蛋白染色陽性；電鏡下模型中可見貼附在支架上的毛囊細胞和紅細胞。(5)先接種用絲裂霉素干預的成纖維細胞1×103于海藻酸鈉3D細胞支架上培養(yǎng)一周后再接種DPC:ORSC (1:5)細胞懸液,裸鼠體內(nèi)移植物HE染色不僅可見明顯的毛囊樣結構形成,而且形成毛囊樣結構的數(shù)目較多；(6)MTT顯示濃度為10ng/ml的骨形成蛋白4(BMP-4)對毛乳頭細胞和外根鞘細胞均具有明顯的促增值作用；(7)種子細胞的體外培養(yǎng)基用KGM組毛囊細胞聚集成細胞團塊,部分呈環(huán)狀排列,而用MSCM組則有細胞團樣結構,未見環(huán)狀結構。 結論： 本試驗使用的酶消化法和組織塊培養(yǎng)法獲得的Fb保持了良好的生長特性；體外毛囊三維模型培養(yǎng)未能誘導出毛囊樣結構,但在bal/bcl裸鼠體內(nèi)誘導出了毛囊樣結構,且該模型中DPC保持了誘導毛囊形成的能力,ORSC保持了毛囊干細胞的特點。此外,本試驗探索出了誘導毛囊形成的最佳細胞組合、接種順序和比例是先接種用絲裂霉素干預的成纖維細胞1×103于海藻酸鈉3D細胞支架上,培養(yǎng)一周后再接種DPC:ORSC(1:5)細胞懸液；還證實了濃度為10ng/ml的BMP-4能明顯促進DPC和ORSC的增殖；種子細胞的體外培養(yǎng)基用KGM明顯優(yōu)于MSCM。本試驗為成功重建毛囊指明了方向。
[Abstract]:Aim: to isolate and culture the seed cells from hair follicles and induce hair follicle formation in vitro and in vivo, and to investigate the biological characteristics of hair follicles and the role of seed cells in hair follicle formation. Methods: (1) Human hair follicle outer root sheath cell line (HHFORSC) and human hair follicle hair papilla cell line (HHDPC) were obtained from the skin fibroblasts (FB); (2) of newborn by enzyme digestion and tissue mass culture, and were resuscitated, cultured and subcultured. (3) hair follicle outer root sheath cells (ORSC), dermal papilla cells (DPC) and fibroblasts treated with mitomycin were combined to make cell suspension and inoculated on sodium alginate 3D cell scaffold according to different inoculation order to construct the three-dimensional model of hair follicle in vitro. After 8 weeks of culture, the hair follicle formation was observed by HE staining, and the three dimensional model was transplanted into bal/bcl nude mice subcutaneously and fed for 8 weeks. The hair follicle formation was observed by HE staining, immunohistochemistry and electron microscope. Results: (1) Human Fb was successfully obtained; (2) HHFORSC HHDPC was resuscitated, cultured and isolated, and the cultured Fb grew normally; (3) the hair follicle 3D model was constructed in vitro and stained with HE, and there were hair follicle mixed cells scattered in the scaffold. No keratocystis and hair follicles were found. (4) the cells gathered into clusters in HE staining of subcutaneous grafts in nude mice, and the hair follicle-like structures, such as CK14, CK15, 尾 1 integrin and vimentin, were positive for the expression of CK14, CK15, 尾 1 integrin and vimentin, respectively. The hair follicle cells and erythrocytes attached to the scaffold were observed under electron microscope. (5) fibroblasts treated with mitomycin were first cultured on sodium alginate 3D scaffold for one week and then inoculated with DPC: ORSC (1:5) cell suspension. He staining of grafts in nude mice not only showed the formation of hair follicle-like structure, but also formed a large number of hair follicle-like structures. (6) Bone morphogenetic protein 4 (BMP-4) at the concentration of 10ng/ml significantly promoted the proliferation of both dermal papilla cells and outer root sheath cells. In MSCM group, there were cell clusters like structure, no ring structure. Conclusion: the Fb obtained by enzyme digestion and tissue mass culture has good growth characteristics, and hair follicle-like structure can not be induced by three-dimensional model culture of hair follicles in vitro. But hair follicle-like structure was induced in nude mice. In this model, the ability of bal/bcl to induce hair follicle formation was maintained. ORSC maintained the characteristics of hair follicle stem cells. In addition, the optimal cell combination for hair follicle formation was found in this experiment. The inoculation sequence and proportion were as follows: 1 脳 103 fibroblasts treated with mitomycin were first inoculated on sodium alginate 3D scaffold, and then inoculated with DPC: ORSC (1:5) cell suspension for one week. It was also confirmed that BMP-4 at the concentration of 10ng/ml could significantly promote the proliferation of 10ng/ml and the seed cell culture medium in vitro was better than that of 10ng/ml. This experiment pointed out the direction of successful hair follicle reconstruction.
【學位授予單位】：昆明醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

【參考文獻】

相關期刊論文 前10條

1 鮑衛(wèi)漢,徐少駿;病理性瘢痕成纖維細胞的增殖和生物合成特性[J];杭州醫(yī)學高等專科學校學報;2001年04期

2 馮潔瑩;范瑞強;;廣州市1561名中學生痤瘡流行病學調(diào)查[J];嶺南皮膚性病科雜志;2008年01期

3 王文嶺;楊蓉婭;蘇有明;;種痘樣水皰病4例[J];中國麻風皮膚病雜志;2008年02期

4 陳艷清;李世榮;曹川;馮智;夏珊;李丹;;血管緊張素對增生性瘢痕成纖維細胞纖維連接蛋白合成的影響[J];中國美容醫(yī)學;2007年04期

5 王鋒;王啟鳳;趙雪萍;;皮膚干細胞的研究進展及其應用[J];中國臨床康復;2006年21期

6 楊擎宇;汪樊;馮愛平;;尋常性痤瘡嚴重度的影響因素分析[J];中國皮膚性病學雜志;2009年07期

7 付小兵,程飚;創(chuàng)傷修復和組織再生幾個重要領域研究的進展與展望[J];中華創(chuàng)傷雜志;2005年01期

8 肖仕初,夏照帆,楊s，

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