本文選題：毛囊干細(xì)胞 + 脫細(xì)胞支架��； 參考：《新疆醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的:(1)比較不同毛囊干細(xì)胞的培養(yǎng)方法,建立毛囊干細(xì)胞(Hair follicle stem cells,HFSCs)的體外培養(yǎng)方法,用目前公認(rèn)的標(biāo)記物和其他手段對(duì)其進(jìn)行鑒定;(2)尋找標(biāo)記毛囊干細(xì)胞的方法,并用CM-Dil標(biāo)記毛囊干細(xì)胞,探討CM-Dil標(biāo)記毛囊干細(xì)胞的可行性,并觀察標(biāo)記后的毛囊干細(xì)胞與膀胱脫細(xì)胞基質(zhì)共培養(yǎng)形成的細(xì)胞-支架復(fù)合物的熒光表達(dá)情況,保證移植細(xì)胞的質(zhì)量及標(biāo)記細(xì)胞的示蹤效果;(3)誘導(dǎo)毛囊干細(xì)胞向平滑肌細(xì)胞分化并鑒定,觀察熒光標(biāo)記細(xì)胞-支架復(fù)合物后細(xì)胞生長(zhǎng)情況;(4)評(píng)估細(xì)胞-支架復(fù)合物對(duì)于兔膀胱缺損模型的修補(bǔ)作用,觀察并鑒定細(xì)胞-支架復(fù)合物修補(bǔ)處特異性標(biāo)記物的表達(dá)。方法:(1)選用出生2月的新西蘭兔,經(jīng)過(guò)嚴(yán)格消毒擬切除觸須部位后,切取含毛囊的組織,在無(wú)菌環(huán)境下使用顯微外科技術(shù)分離出毛囊組織和毛囊外根鞘部。用中性蛋白酶和胰酶消化毛囊外根鞘部;利用差速貼壁法篩選貼壁快的細(xì)胞后,培養(yǎng)的細(xì)胞可供進(jìn)一步傳代或冷凍處理。培養(yǎng)出來(lái)的細(xì)胞應(yīng)用倒置顯微鏡、電子顯微鏡、Giemsa染色、免疫熒光染色、流式細(xì)胞儀等鑒定,將β1整合素、CK15、CD34,CK7等細(xì)胞表面標(biāo)志物作為鑒定依據(jù);(2)分離培養(yǎng)兔毛囊干細(xì)胞并鑒定,取CM-Dil儲(chǔ)存液5ul,用PBS稀釋成1ml的5ug/m1(即5u M)CM-Dil工作液。將貼壁生長(zhǎng)、狀態(tài)良好P3代毛囊干細(xì)胞(細(xì)胞總量約為5~6×106)終止培養(yǎng),將培養(yǎng)液吸出,用PBS輕洗貼壁細(xì)胞2次,加入5ug/m1(即5u M)CM-Dil工作液,37℃孵育15min,-4℃孵育5min,PBS洗滌兩遍,加入體積為25ml的培養(yǎng)瓶中繼續(xù)培養(yǎng),第2天換液。倒置相差熒光顯微鏡綠色熒光下進(jìn)行觀察。將第三代MFSCs在轉(zhuǎn)化生長(zhǎng)因子β1(TGF-β1)和骨形態(tài)蛋白4(BMP4)的作用下向平滑肌細(xì)胞誘導(dǎo)分化,用細(xì)胞免疫組化的方法檢測(cè)平滑肌細(xì)胞特異性標(biāo)記物的表達(dá);(3)使用CM-Dil標(biāo)記HFSCs并與兔膀胱脫細(xì)胞基質(zhì)共培養(yǎng)成細(xì)胞-支架復(fù)合物,利用熒光倒置顯微鏡觀察標(biāo)記后細(xì)胞及細(xì)胞-支架復(fù)合物,確定熒光標(biāo)記情況;手術(shù)回植細(xì)胞-支架復(fù)合物至兔膀胱缺損模型觀察生長(zhǎng)情況;取得實(shí)驗(yàn)動(dòng)物膀胱,選取修補(bǔ)位置,制成石蠟切片,使用熒光倒置顯微鏡觀察組織切片的熒光表達(dá),利用免疫組化的方法檢測(cè)組織結(jié)構(gòu)成分和平滑肌細(xì)胞特異性標(biāo)記物的表達(dá)。結(jié)果:(1)第三代HFSCs在倒置顯微鏡下表現(xiàn)為幼圓鋪路石樣形態(tài),二步酶法、顯微分離技術(shù)聯(lián)合免疫磁珠法、差速貼壁法能很好的培養(yǎng)出毛囊干細(xì)胞;(2)CM-Dil標(biāo)記的第三代HFSCs熒光表達(dá)穩(wěn)定,與兔膀胱脫細(xì)胞基質(zhì)共培養(yǎng)得到的細(xì)胞-支架復(fù)合物仍有較強(qiáng)的熒光表達(dá);TGF-β1和BMP4誘導(dǎo)鑒定后確定細(xì)胞的干細(xì)胞功能;HFSCs向平滑肌細(xì)胞誘導(dǎo)7天后,具有平滑肌細(xì)胞特有的“波峰-波谷”樣生長(zhǎng)特點(diǎn)。表達(dá)平滑肌細(xì)胞特異性標(biāo)記物:a-SMA;(3)實(shí)驗(yàn)兔分別于術(shù)后7d、16d各死亡一只,死因?yàn)槁┠?組織切片熒光表達(dá)有所減弱但仍存在,與未修補(bǔ)位置界限較清,Masson染色發(fā)現(xiàn)組織結(jié)構(gòu)齊全,肌纖維表達(dá)明顯,免疫組化表達(dá)平滑肌細(xì)胞特異性標(biāo)記物:a-SMA。結(jié)論:(1)HFSCs原代獲取后,在體外培養(yǎng)條件下生長(zhǎng)良好,并能維持干細(xì)胞的特性,能作為組織工程研究及應(yīng)用的首選種子細(xì)胞;二步酶法、顯微分離技術(shù)聯(lián)合免疫磁珠法、差速貼壁法能很好的培養(yǎng)出毛囊干細(xì)胞;(2)熒光標(biāo)記的HFSCs在體內(nèi)能夠穩(wěn)定表達(dá);TGF-β1和BMP4能使HFSCs向平滑肌細(xì)胞分化;(3)膀胱脫細(xì)胞基質(zhì)BAMG在實(shí)驗(yàn)兔體內(nèi)8周后可基本吸收降解,是一種良好的生物支架材料,HFSCs在體內(nèi)能夠分化為平滑肌細(xì)胞。HFSCs和BAMG采用靜態(tài)接種的方法可在體外構(gòu)建組織工程膀胱,修補(bǔ)缺損的兔膀胱效果良好,是一種理想的膀胱修補(bǔ)重建材料。
[Abstract]:Objective: (1) to compare the culture methods of different hair follicle stem cells, to establish the culture method of Hair follicle stem cells (HFSCs) in vitro, and to identify it with the commonly recognized markers and other means. (2) to find a method to mark hair follicle stem cells, and to mark hair follicle stem cells by CM-Dil, and to explore the CM-Dil labeling of hair follicle stem cells. The fluorescent expression of the cell scaffold complex formed by the co culture of the hair follicle stem cells and the vesical acellular matrix was observed, and the quality of the transplanted cells and the tracer effect of the labeled cells were ensured. (3) the differentiation and identification of the hair follicle stem cells to the smooth muscle cells were induced and the fluorescent labeled cell scaffold complex was observed. Growth situation; (4) evaluate the repair effect of cell scaffold complex on the rabbit bladder defect model, observe and identify the expression of specific markers at the repair of cell scaffold complex. Methods: (1) the New Zealand rabbits born in February were selected and the hair follicle containing tissues were cut after strict disinfection of the tentacles, and the use of the tissue in a sterile environment was used in a sterile environment. The microsurgical technique was used to isolate the hair follicle tissue and the outer root sheath of the hair follicle. The outer root sheath of the hair follicle was digested with neutral protease and trypsin; the cultured cells could be further passaged or frozen after screening the fast cells with differential adherence. The cultured cells were used in inverted microscopy, electron microscopy, Giemsa staining, and immunofluorescence staining. Color, flow cytometry, etc. identified the surface markers of beta 1 integrin, CK15, CD34, CK7 and other cell surface markers. (2) isolation and culture of rabbit hair follicle stem cells and identification of CM-Dil storage liquid 5ul, PBS diluted into 1ml 5ug/m1 (5u M) CM-Dil working fluid. Culture, the culture solution was sucked out, 2 times with PBS light washing adherent cells, adding 5ug/m1 (5u M) CM-Dil working fluid, incubating 15min at 37 degrees C, incubating 5min at -4 C, PBS washing for two times, adding in the culture bottle of 25ml, and changing liquid in second days. The third generation MFSCs in the transforming growth factor was observed. The differentiation of beta 1 (TGF- beta 1) and bone Morphin 4 (BMP4) was induced to smooth muscle cells. The expression of specific markers of smooth muscle cells was detected by cellular immunofluorescence. (3) CM-Dil labeled HFSCs was used to co culture with the rabbit bladder acellular matrix to form a cell branch complex, and the labeled cells were observed by fluorescence inverted microscope. And the cell scaffold complex was used to determine the fluorescence labeling; the growth of the replanted cell scaffold complex to the rabbit bladder defect model was observed. The experimental animal bladder was obtained, the repair position was selected and the paraffin section was made. The fluorescence expression of the tissue section was observed by the fluorescence inversion microscope, and the tissue structure was detected by immunohistochemical method. Results: (1) the third generation of HFSCs in the inverted microscope showed the shape of the paving stone under the inverted microscope, the two step enzyme method, the microseparation technique combined with the immunomagnetic bead method, the differential adherence method could produce the hair follicle stem cells well; (2) the fluorescence expression of the third generation HFSCs marked by the CM-Dil was stable, and the rabbit bladder The cell scaffold complex produced by the acellular matrix still had strong fluorescent expression, and TGF- beta 1 and BMP4 were induced to determine the stem cell function of the cells. The specific "wave peak valley" like growth characteristics of smooth muscle cells were characterized by HFSCs to smooth muscle cells for 7 days. The specific marker of smooth muscle cells: a-SMA; (3). The rabbits died in 7d and 16d after the operation, each died because of leakage of urine; the fluorescence expression of the tissue section was weakened but still existed, and the limit was clear with the unrepaired position. Masson staining found that the tissue structure was complete, the expression of muscle fibers was obvious, and the specific markers of smooth muscle cells were expressed by immunohistochemistry: (1) after the original HFSCs was obtained, it was cultured in vitro. Under the condition of cultivation, it can grow well and can maintain the characteristics of stem cells. It can be used as the preferred seed cell for tissue engineering research and application. Two step enzyme method, microseparation technique combined with immunomagnetic bead method, differential adherence method can produce hair follicle stem cells well; (2) the fluorescent labeled HFSCs can be expressed steadily in the body; TGF- beta 1 and BMP4 can make HFSCs direction The cell differentiation of smooth muscle cells (3) the bladder acellular matrix BAMG can be basically absorbed and degraded after 8 weeks in the experimental rabbit. It is a good scaffold material. HFSCs can differentiate into smooth muscle cells.HFSCs and BAMG by static inoculation in vitro, which can construct tissue worker Cheng Bangguang in vitro, and the effect of repairing the defect of rabbit's bladder is good. Ideal reconstructive materials for bladder repair.

【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R694

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張麗萍,袁卓庭,馬曉兵,聶青;化學(xué)藥物瘤內(nèi)注射合并加溫放射綜合治療中晚期腫瘤的初步探討[J];海軍總醫(yī)院學(xué)報(bào);1998年04期

2 林常敏;β-連環(huán)蛋白在毛囊形態(tài)發(fā)生及毛囊干細(xì)胞增生分化的作用[J];醫(yī)學(xué)研究生學(xué)報(bào);2004年04期

3 曹慶雋;孫曼妮;;毛囊干細(xì)胞的研究進(jìn)展[J];基層醫(yī)學(xué)論壇;2007年11期

4 耿松梅;王劍利;王萬(wàn)卷;譚升順;彭振輝;;毛囊干細(xì)胞定位和體外向表皮分化[J];中國(guó)醫(yī)學(xué)科學(xué)院學(xué)報(bào);2006年03期

5 譚挺;胡志奇;周洪軍;;顯微分離培養(yǎng)與免疫磁珠法分離純化人毛囊干細(xì)胞[J];中國(guó)修復(fù)重建外科雜志;2008年02期

6 劉宏偉;程飚;付小兵;;局部組織腎素-血管緊張素系統(tǒng)在皮膚損傷修復(fù)和再生中的作用[J];中國(guó)病理生理雜志;2011年01期

7 王振顯;蔡文清;龐國(guó)勛;宋永周;陳康寧;孫福振;楊濤;;人脫細(xì)胞羊膜負(fù)載大鼠骨髓間充質(zhì)干細(xì)胞構(gòu)建組織工程膀胱的研究[J];中國(guó)老年學(xué)雜志;2010年02期

8 趙奎;賈宗菲;弓慧敏;王振華;王恩峰;龐全海;;毛囊干細(xì)胞的分離方法及其應(yīng)用[J];中國(guó)畜牧獸醫(yī);2010年12期

9 彭文標(biāo);劉春曉;謝群;朱臂琳;呂騰榮;;富集兔毛囊干細(xì)胞構(gòu)建組織工程化尿道的研究[J];上海交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2013年03期

10 柯錦城;程波;;毛囊干細(xì)胞對(duì)坐骨神經(jīng)損傷修復(fù)的影響[J];中國(guó)組織工程研究;2012年32期

，

本文編號(hào)：1775366