体外诱导人脂肪间充质干细胞向表皮细胞和成纤维细胞分化

发布时间：2018-04-24 09:50

本文选题：脂肪间充质干细胞 + 诱导分化　；参考：《大连医科大学》2011年硕士论文

【摘要】：背景:临床上常见的慢性溃疡、烧伤、外伤等造成的皮肤损伤,需要及时覆盖创面,以促进创面愈合及功能重塑。近年来,采用自体干细胞和生长因子的再生医学,为组织损伤的修复提供了新的思路,同时也为以细胞为基础的治疗提供了新的途径。研究表明,成体干细胞治疗,为组织损伤的修复提供了一种安全、有效的临床治疗方法。目的:采用人脂肪间充质干细胞(ADSCs)与EGF、bFGF、IGF-1、TGFβ1等生长因子进行体外诱导培养,从ADSCs的分离培养,到对其增殖能力和表面分子表达的检测,并从转录水平和蛋白水平进行系统分析。拟建立ADSCs向表皮细胞和成纤维细胞诱导分化的规范实验体系。方法:从脂肪组织中分离ADSCs,并检测第3代和第10代ADSCs的表面分子表达及第3、5、10代ADSCs的细胞增殖情况。将ADSCs在含有EGF、bFGF、IGF-1等生长因子的诱导培养基中进行体外诱导培养,蛋白水平,免疫荧光检测CK 19的抗原表达,同时透射电镜下观察细胞超微结构。将ADSCs在含有TGFβ1、bFGF等生长因子的诱导培养基中进行体外诱导培养,转录水平,检测I型胶原的mRNA含量;蛋白水平,免疫荧光检测I型胶原的抗原表达,同时透射电镜下观察细胞超微结构。结果:(1)第3、10代ADSCs的CD90表达强阳性,CD34表达弱阳性,CD106表达阴性,第3、10代细胞均一性分别为94.2%,97.5%。第3代和第10代的ADSCs在细胞形态学、增殖能力和表面分子的表达方面无明显改变。(2)ADSCs在含有EGF、bFGF、IGF-1等生长因子的诱导培养基中进行体外诱导培养,第3天,部分细胞由梭形变成不规则形,第5天开始,细胞逐渐融合成铺石路样改变。免疫荧光结果显示,CK 19表达阳性,阳性率为15.6%。诱导15天后,透射电镜下,可见黑素小体、透明角质颗粒。(3)ADSCs在含有TGFβ1、bFGF等生长因子的诱导培养基中进行体外诱导培养,ADSCs诱导7天后,转录水平上,I型胶原的mRNA含量为对照组的两倍多;免疫荧光结果显示,与阴性对照组比,诱导组I型胶原蛋白的表达增加。诱导15天后,透射电镜下,可见胶原微纤维。结论:联合人脂肪间充质干细胞与EGF、bFGF、IGF-1、TGFβ1等生长因子进行体外诱导培养, ADSCs在一定条件下,可以向表皮细胞和成纤维细胞分化。从而,为皮肤组织再生提供了种子细胞的来源,同时也为皮肤损伤修复的临床应用提供了理论基础。
[Abstract]:Background: skin injury caused by chronic ulcer, burn and trauma in clinic needs to cover the wound in time to promote wound healing and functional remodeling. In recent years, regenerative medicine with autologous stem cells and growth factors has provided new ideas for the repair of tissue injury and a new approach to cell-based therapy. Studies have shown that adult stem cell therapy provides a safe and effective method for the repair of tissue injury. Objective: to study the effects of human adipose mesenchymal stem cells (ADSCs) and EGFFGFGFIGF-1TGF- 尾 1 on the proliferation and surface molecular expression of human adipose mesenchymal stem cells (ADSCs) in vitro. To establish a standard experimental system for the differentiation of ADSCs into epidermal cells and fibroblasts. Methods: ADSCs were isolated from adipose tissue and the expression of surface molecules in the 3rd and 10th generation of ADSCs and the proliferation of ADSCs in the 3rd and 10th generation were detected. The expression of CK19 antigen was detected by immunofluorescence, and the expression of CK19 antigen was detected by immunofluorescence. The ultrastructure of the cells was observed by transmission electron microscope (TEM). ADSCs was cultured in culture medium containing TGF 尾 1bFGF in vitro, transcription level was measured to detect the mRNA content of type I collagen, protein level, antigen expression of type I collagen was detected by immunofluorescence. At the same time, the ultrastructure of cells was observed under transmission electron microscope. Results the strong positive expression of CD90 and the weak positive expression of CD106 in ADSCs in the 3rd and 10th generation of ADSCs were negative, and the homogeneity of the cells in the 10th generation of the 3rd generation were 94.2and 97.5g, respectively. The third and tenth passages of ADSCs had no obvious changes in cell morphology, proliferation ability and surface molecule expression. They were induced in vitro in the medium containing EGFFbFGFIGF-1, and cultured in vitro on the 3rd day. Some of the cells changed from fusiform to irregular, and gradually fused into paved road-like changes on the 5th day. Immunofluorescence showed positive expression of CK19, and the positive rate was 15.6. After 15 days of induction, melanosome and hyaline keratin granules were observed in the culture medium containing TGF 尾 1bFGF for 7 days. The mRNA content of type I collagen was more than twice as high as that of the control group at the transcriptional level, and the expression of type I collagen protein in the induced group was higher than that in the negative control group. After 15 days of induction, collagen microfibers were observed under transmission electron microscope. Conclusion: the combination of human adipose mesenchymal stem cells and EGFFGFF-1TGF- 尾 1 growth factors can differentiate ADSCs into epidermal cells and fibroblasts under certain conditions. Thus, it provides the source of seed cells for skin tissue regeneration, and also provides a theoretical basis for the clinical application of skin injury repair.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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