毛囊干细胞脱细胞支架复合物修复兔膀胱缺损的研究

发布时间：2018-04-20 00:13

本文选题：毛囊干细胞 + 脱细胞支架　；参考：《新疆医科大学》2016年博士论文

【摘要】：目的:(1)比较不同毛囊干细胞的培养方法,建立毛囊干细胞(Hair follicle stem cells,HFSCs)的体外培养方法,用目前公认的标记物和其他手段对其进行鉴定;(2)寻找标记毛囊干细胞的方法,并用CM-Dil标记毛囊干细胞,探讨CM-Dil标记毛囊干细胞的可行性,并观察标记后的毛囊干细胞与膀胱脱细胞基质共培养形成的细胞-支架复合物的荧光表达情况,保证移植细胞的质量及标记细胞的示踪效果;(3)诱导毛囊干细胞向平滑肌细胞分化并鉴定,观察荧光标记细胞-支架复合物后细胞生长情况;(4)评估细胞-支架复合物对于兔膀胱缺损模型的修补作用,观察并鉴定细胞-支架复合物修补处特异性标记物的表达。方法:(1)选用出生2月的新西兰兔,经过严格消毒拟切除触须部位后,切取含毛囊的组织,在无菌环境下使用显微外科技术分离出毛囊组织和毛囊外根鞘部。用中性蛋白酶和胰酶消化毛囊外根鞘部;利用差速贴壁法筛选贴壁快的细胞后,培养的细胞可供进一步传代或冷冻处理。培养出来的细胞应用倒置显微镜、电子显微镜、Giemsa染色、免疫荧光染色、流式细胞仪等鉴定,将β1整合素、CK15、CD34,CK7等细胞表面标志物作为鉴定依据;(2)分离培养兔毛囊干细胞并鉴定,取CM-Dil储存液5ul,用PBS稀释成1ml的5ug/m1(即5u M)CM-Dil工作液。将贴壁生长、状态良好P3代毛囊干细胞(细胞总量约为5~6×106)终止培养,将培养液吸出,用PBS轻洗贴壁细胞2次,加入5ug/m1(即5u M)CM-Dil工作液,37℃孵育15min,-4℃孵育5min,PBS洗涤两遍,加入体积为25ml的培养瓶中继续培养,第2天换液。倒置相差荧光显微镜绿色荧光下进行观察。将第三代MFSCs在转化生长因子β1(TGF-β1)和骨形态蛋白4(BMP4)的作用下向平滑肌细胞诱导分化,用细胞免疫组化的方法检测平滑肌细胞特异性标记物的表达;(3)使用CM-Dil标记HFSCs并与兔膀胱脱细胞基质共培养成细胞-支架复合物,利用荧光倒置显微镜观察标记后细胞及细胞-支架复合物,确定荧光标记情况;手术回植细胞-支架复合物至兔膀胱缺损模型观察生长情况;取得实验动物膀胱,选取修补位置,制成石蜡切片,使用荧光倒置显微镜观察组织切片的荧光表达,利用免疫组化的方法检测组织结构成分和平滑肌细胞特异性标记物的表达。结果:(1)第三代HFSCs在倒置显微镜下表现为幼圆铺路石样形态,二步酶法、显微分离技术联合免疫磁珠法、差速贴壁法能很好的培养出毛囊干细胞;(2)CM-Dil标记的第三代HFSCs荧光表达稳定,与兔膀胱脱细胞基质共培养得到的细胞-支架复合物仍有较强的荧光表达;TGF-β1和BMP4诱导鉴定后确定细胞的干细胞功能;HFSCs向平滑肌细胞诱导7天后,具有平滑肌细胞特有的“波峰-波谷”样生长特点。表达平滑肌细胞特异性标记物:a-SMA;(3)实验兔分别于术后7d、16d各死亡一只,死因为漏尿;组织切片荧光表达有所减弱但仍存在,与未修补位置界限较清,Masson染色发现组织结构齐全,肌纤维表达明显,免疫组化表达平滑肌细胞特异性标记物:a-SMA。结论:(1)HFSCs原代获取后,在体外培养条件下生长良好,并能维持干细胞的特性,能作为组织工程研究及应用的首选种子细胞;二步酶法、显微分离技术联合免疫磁珠法、差速贴壁法能很好的培养出毛囊干细胞;(2)荧光标记的HFSCs在体内能够稳定表达;TGF-β1和BMP4能使HFSCs向平滑肌细胞分化;(3)膀胱脱细胞基质BAMG在实验兔体内8周后可基本吸收降解,是一种良好的生物支架材料,HFSCs在体内能够分化为平滑肌细胞。HFSCs和BAMG采用静态接种的方法可在体外构建组织工程膀胱,修补缺损的兔膀胱效果良好,是一种理想的膀胱修补重建材料。
[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.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R694

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