利用羊膜上皮干细胞抑制皮肤增生性瘢痕形成的研究

发布时间：2018-06-28 23:33

本文选题：羊膜 + 羊膜上皮细胞　；参考：《第四军医大学》2012年硕士论文

【摘要】：病理性瘢痕是皮肤创伤修复过程中异常增生所致，主要包括增生性瘢痕（Hypertrophic Scar，HS）和瘢痕疙瘩（Keloid，K）。两者组织学特点都是成纤维细胞增值，细胞外基质过度分泌、胶原过度沉积。临床表现为瘙痒、疼痛、严重可导致关节的功能障碍等。目前增生性瘢痕的发生机制尚不明确，且临床治疗复杂及困难。所以对抑制皮肤瘢痕形成的研究就尤为重要。研究表明瘢痕的形成因素之一是多种炎性因子参与的病理过程，使正常的创面愈合结果变为瘢痕的形成。文献报道，Gordon等利用小鼠伤口组织，采用腺病毒介导的IL-10转染的方法，证实过表达的IL-10可以明显的抑制伤口炎症反应，从而减少瘢痕的形成，进一步验证了抗炎症策略在防止瘢痕的形成，，以及治疗中具有重要作用。羊膜是胎膜的一部分，包括来源于胚胎外胚层的上皮细胞（Humanamniotic epithelial cells,hAECs）和胚胎中胚层的间质细胞（Human amnioticmesenchymal cells,hAMCs），羊膜上皮细胞具有部分干细胞特性，可分化为三个胚层不同类型的细胞。现临床以成功的应用在眼科，利用羊膜上皮细胞的干细胞特性，通过抑制炎症反应治疗角膜溃疡和翼状胬肉等疾病。本实验通过构建兔耳瘢痕模型，利用羊膜上皮干细胞抑制炎症因子、免疫源性极低等优点，在创面周围定期注射羊膜上皮干细胞悬液，研究羊膜上皮干细胞抑制瘢痕形成中所起的作用。为增生性瘢痕的防治和治疗开辟一条新的道路。第一部分实验：羊膜上皮细胞的分离、培养及鉴定 1.主要方法从胎膜组织中分离羊膜组织，采用胰蛋白消化法、LG-DMEM培养基培养。取生长状态良好的羊膜上皮细胞分别进行流式细胞术、免疫荧光染色、成骨成脂多向分化等实验进行干细胞特性的鉴定。 2.主要结果 FCM结果显示不同程度的阳性表达CD29、CD90、CD105，CD34、CD45为阴性。其中阳性率最高的是CD29，说明羊膜上皮细胞兼有间充质干细胞的表型特征。免疫荧光染色显示SSEA-4，Oct-4均有阳性表达。hAECs成功定向诱导成脂成骨。 3.主要结论羊膜上皮细胞具有一般干细胞的特性。第二部分实验：瘢痕成纤维细胞的分离、培养 1.主要方法取外科手术中废弃的增生性瘢痕组织，采用组织快培养法、DMEM培养基培养。 2.主要结果原代培养时间较长，大约1周时间有少许细胞从组织块周围爬出，生长较快，镜下观察，细胞为长梭形，较正常皮肤成纤维细胞略短。 3.主要结论成功的培养出瘢痕的成纤维细胞。第三部分实验：羊膜上皮细胞与瘢痕成纤维细胞的共培养及鉴定 1.主要方法将羊膜上皮细胞种于6孔板中，待完全贴壁时，3个孔放入Transwell,另外3个孔为对照，将瘢痕成纤维细胞种于Transwell中,加入IL-6炎性因子刺激下，让两种细胞共同培养，分别在共培养的1、2、3天提取RNA，进行细胞周期、细胞凋亡及PCR检测。 2.主要结果细胞周期结果显示1d，3d的实验组中瘢痕成纤维细胞增殖要低于对照组，说明羊膜上皮细胞对瘢痕成纤维细胞的增殖有抑制作用。细胞凋亡结果显示1d、3d实验组瘢痕成纤维细胞凋亡的比对照组数值略高。PCR检测显示，实验组的OCT-4、Ι型胶原较对照组高，而TIMP-1低于对照组。 3.主要结论两种细胞共培养后，羊膜上皮细胞对瘢痕成纤维细胞的增殖和凋亡有作用，PCR检测TIMP-1、OCT-4、Ι型胶原等指标随着时间不同各自的表达不同。第四部分实验：利用动物模型检测羊膜上皮细胞在瘢痕防治中的作用 1.主要方法手术切除双侧兔耳内侧面，形成直径为1×1cm的4个方形全层皮肤缺损；在右耳创面周围注射羊膜上皮细胞悬液，左耳创面周围注射PBS缓冲液。每周注射一次，定期观察。4周后实验组创面完全上皮化，肿块较小。对照组肿块明显。取瘢痕组织进行HE染色及激光共聚焦观察. 2.主要结果 HE染色结果显示实验组上皮化完全、略显增生状态，基底层细胞排列明显，其下的真皮组织中未见炎性细胞分布，成纤维细胞分布与对照组相比较为分散。共聚焦可观察到移植的羊膜上皮细胞。对照组HE染色显示成纤维细胞分布集中，增生明显。共聚焦未见羊膜上皮细胞。 3.主要结论通过肉眼观察、HE染色及共聚焦检测，说明羊膜上皮干细胞有效的抑制瘢痕的形成。综上所述，本实验利用羊膜上皮细胞的干细胞特性抑制增生性瘢痕形成为目的，通过抑制炎症反应,减少创面愈合时间为切入点，分别在体内和体外实验中证明这一推测的可靠性。为此，羊膜上皮干细胞的应用有可能成为瘢痕预防和治疗的新手段和切入点。
[Abstract]:Pathological scar is caused by abnormal hyperplasia in the process of skin wound repair, mainly including Hypertrophic Scar (HS) and keloid (Keloid, K). Both histologic features are fibroblast increment, excessive extracellular matrix secretion, and excessive collagen deposition. The clinical manifestation is pruritus, pain, and severe joint functional barrier. At present, the mechanism of hypertrophic scars is not clear, and its clinical treatment is complex and difficult. Therefore, the study of suppressing skin scar formation is particularly important.
The research shows that one of the factors of scar formation is the pathological process of various inflammatory factors involved in the pathological process, which makes the result of normal wound healing into scar formation. It is reported that Gordon and so on use the method of adenovirus mediated IL-10 transfection in the wound tissue of mice and confirm that the overexpressed IL-10 can obviously inhibit the wound inflammation reaction and thus reduce the wound inflammation. The formation of less scarring further confirms that the anti-inflammatory strategy plays an important role in preventing scar formation and treatment.
Amniotic membrane is part of the fetal membrane, including the epithelial cells (Humanamniotic epithelial cells, hAECs) derived from the embryonic ectoderm (hAECs) and the interstitial cells of the embryonic mesoderm (Human amnioticmesenchymal cells, hAMCs). The amniotic epithelial cells have some stem cell characteristics and can be divided into three different types of cells. In the ophthalmology department, we use the characteristics of stem cells of amniotic epithelial cells to treat corneal ulcer and pterygium by inhibiting inflammatory reaction.
In this experiment, the rabbit ear scar model was constructed and the amniotic epithelial stem cells were used to inhibit the inflammatory factors and the extremely low immunogenic advantages. The amniotic epithelial stem cell suspension was injected around the wound surface regularly to study the effect of amniotic epithelial stem cells on the formation of scar formation, which opened a new way for the prevention and treatment of hypertrophic scars.
Part one experiment: isolation, culture and identification of amniotic epithelial cells
1. main methods
The amniotic membrane tissue was separated from the fetal membrane, the trypsin digestion method and the LG-DMEM culture medium were used. The amniotic epithelial cells with good growth state were carried out by flow cytometry, immunofluorescence staining, and osteogenic and multidifferentiation experiments to identify the characteristics of stem cells.
2. main results
The FCM results showed that the positive expression of CD29, CD90, CD105, CD34 and CD45 was negative. The positive rate was CD29, indicating that the amniotic epithelial cells had both the phenotypic characteristics of mesenchymal stem cells. The immunofluorescence staining showed that SSEA-4, Oct-4 showed that.HAECs was successfully directed to induce osteogenesis.
3. main conclusions
Amniotic epithelial cells have the characteristics of general stem cells.
The second part experiment: isolation and culture of scar fibroblasts
1. main methods
Tissue culture and DMEM culture were used to remove the hypertrophic scar tissue from surgical operation.
Base culture.
2. main results
The primary culture time was longer, and some cells were crawling out of the tissue around 1 weeks. The growth was faster. The cells were observed under the microscope. The cells were long spindle shaped, and the cells were slightly shorter than those of normal skin fibroblasts.
3. main conclusions
The cicatricial fibroblasts were successfully developed.
The third part experiment: co culture and identification of amniotic epithelial cells and scar fibroblasts.
1. main methods
The amniotic epithelial cells were planted in the 6 orifice plate. When they were completely adhered to the wall, 3 holes were put into Transwell and the other 3 holes were used as control. The scar fibroblasts were planted in Transwell. Under the stimulation of IL-6 inflammatory factors, two kinds of cells were co cultured and RNA was extracted from the co cultured 1,2,3 days, and cell cycle, apoptosis and PCR detection were carried out.
2. main results
The cell cycle results showed that the proliferation of cicatricial fibroblasts in the experimental group of 1D was lower than that in the control group, indicating that the amniotic epithelial cells could inhibit the proliferation of cicatricial fibroblasts. The apoptosis results showed that the apoptosis of the scar fibroblasts in the 3D experimental group was slightly higher than that of the control group,.PCR, and the OCT-4 of the experimental group. The results showed that the apoptosis of cicatricial fibroblasts in the experimental group of 3D was higher than that of the control group. The former was higher than the control group, but the TIMP-1 was lower than that of the control group.
3. main conclusions
After co culture of two kinds of cells, amniotic epithelial cells have a effect on the proliferation and apoptosis of scar fibroblasts. PCR detection of TIMP-1, OCT-4, collagen type and other indexes vary with time.
The fourth part of the experiment: using animal models to detect the role of amniotic epithelial cells in the prevention and treatment of scar.
1. main methods
4 square full layer skin defects with diameter of 1 x 1cm were formed by surgical excision of the inner side of the bilateral rabbit ear. The amniotic epithelial cell suspension was injected around the right ear wound, and the PBS buffer was injected around the left ear wound. Once a week, the wound was completely epithelialization and the mass was smaller after.4 weeks. The scar tissue was obvious. HE staining and laser confocal observation were carried out.
2. main results
The results of HE staining showed that the experimental group was completely epitheliized and slightly proliferated. The cells in the basal layer were arranged obviously. There was no distribution of inflammatory cells in the lower dermal tissue. The distribution of fibroblasts was scattered with the control group. The confocal coke can observe the transplanted amniotic epithelial cells. The HE staining showed that the fibroblasts were concentrated and proliferated. It was obvious that amniotic epithelial cells were not found in confocal.
3. main conclusions
HE staining and confocal microscopy showed that amniotic epithelial stem cells effectively inhibited scar formation.
To sum up, this experiment uses the characteristics of the stem cells of amniotic epithelial cells to inhibit the formation of hypertrophic scar. By inhibiting the inflammatory response and reducing the healing time of the wound as a breakthrough point, the reliability of this inference is proved in both in vivo and in vitro. Therefore, the application of the amniotic membrane stem cells may be the prevention and treatment of scar. The novice section and the entry point of the treatment.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【参考文献】