發(fā)布時(shí)間：2018-06-28 23:33

  本文選題：羊膜 + 羊膜上皮細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：病理性瘢痕是皮膚創(chuàng)傷修復(fù)過程中異常增生所致，主要包括增生性瘢痕（Hypertrophic Scar，HS）和瘢痕疙瘩（Keloid，K）。兩者組織學(xué)特點(diǎn)都是成纖維細(xì)胞增值，細(xì)胞外基質(zhì)過度分泌、膠原過度沉積。臨床表現(xiàn)為瘙癢、疼痛、嚴(yán)重可導(dǎo)致關(guān)節(jié)的功能障礙等。目前增生性瘢痕的發(fā)生機(jī)制尚不明確，且臨床治療復(fù)雜及困難。所以對(duì)抑制皮膚瘢痕形成的研究就尤為重要。 研究表明瘢痕的形成因素之一是多種炎性因子參與的病理過程，使正常的創(chuàng)面愈合結(jié)果變?yōu)轳：鄣男纬�。文獻(xiàn)報(bào)道，Gordon等利用小鼠傷口組織，采用腺病毒介導(dǎo)的IL-10轉(zhuǎn)染的方法，證實(shí)過表達(dá)的IL-10可以明顯的抑制傷口炎癥反應(yīng)，從而減少瘢痕的形成，進(jìn)一步驗(yàn)證了抗炎癥策略在防止瘢痕的形成，，以及治療中具有重要作用。 羊膜是胎膜的一部分，包括來源于胚胎外胚層的上皮細(xì)胞（Humanamniotic epithelial cells,hAECs）和胚胎中胚層的間質(zhì)細(xì)胞（Human amnioticmesenchymal cells,hAMCs），羊膜上皮細(xì)胞具有部分干細(xì)胞特性，可分化為三個(gè)胚層不同類型的細(xì)胞。現(xiàn)臨床以成功的應(yīng)用在眼科，利用羊膜上皮細(xì)胞的干細(xì)胞特性，通過抑制炎癥反應(yīng)治療角膜潰瘍和翼狀胬肉等疾病。 本實(shí)驗(yàn)通過構(gòu)建兔耳瘢痕模型，利用羊膜上皮干細(xì)胞抑制炎癥因子、免疫源性極低等優(yōu)點(diǎn)，在創(chuàng)面周圍定期注射羊膜上皮干細(xì)胞懸液，研究羊膜上皮干細(xì)胞抑制瘢痕形成中所起的作用。為增生性瘢痕的防治和治療開辟一條新的道路。 第一部分實(shí)驗(yàn)：羊膜上皮細(xì)胞的分離、培養(yǎng)及鑒定 1.主要方法 從胎膜組織中分離羊膜組織，采用胰蛋白消化法、LG-DMEM培養(yǎng)基培養(yǎng)。取生長狀態(tài)良好的羊膜上皮細(xì)胞分別進(jìn)行流式細(xì)胞術(shù)、免疫熒光染色、成骨成脂多向分化等實(shí)驗(yàn)進(jìn)行干細(xì)胞特性的鑒定。 2.主要結(jié)果 FCM結(jié)果顯示不同程度的陽性表達(dá)CD29、CD90、CD105，CD34、CD45為陰性。其中陽性率最高的是CD29，說明羊膜上皮細(xì)胞兼有間充質(zhì)干細(xì)胞的表型特征。免疫熒光染色顯示SSEA-4，Oct-4均有陽性表達(dá)。hAECs成功定向誘導(dǎo)成脂成骨。 3.主要結(jié)論 羊膜上皮細(xì)胞具有一般干細(xì)胞的特性。 第二部分實(shí)驗(yàn)：瘢痕成纖維細(xì)胞的分離、培養(yǎng) 1.主要方法 取外科手術(shù)中廢棄的增生性瘢痕組織，采用組織快培養(yǎng)法、DMEM培養(yǎng) 基培養(yǎng)。 2.主要結(jié)果 原代培養(yǎng)時(shí)間較長，大約1周時(shí)間有少許細(xì)胞從組織塊周圍爬出，生長較快，鏡下觀察，細(xì)胞為長梭形，較正常皮膚成纖維細(xì)胞略短。 3.主要結(jié)論 成功的培養(yǎng)出瘢痕的成纖維細(xì)胞。 第三部分實(shí)驗(yàn)：羊膜上皮細(xì)胞與瘢痕成纖維細(xì)胞的共培養(yǎng)及鑒定 1.主要方法 將羊膜上皮細(xì)胞種于6孔板中，待完全貼壁時(shí)，3個(gè)孔放入Transwell,另外3個(gè)孔為對(duì)照，將瘢痕成纖維細(xì)胞種于Transwell中,加入IL-6炎性因子刺激下，讓兩種細(xì)胞共同培養(yǎng)，分別在共培養(yǎng)的1、2、3天提取RNA，進(jìn)行細(xì)胞周期、細(xì)胞凋亡及PCR檢測。 2.主要結(jié)果 細(xì)胞周期結(jié)果顯示1d，3d的實(shí)驗(yàn)組中瘢痕成纖維細(xì)胞增殖要低于對(duì)照組，說明羊膜上皮細(xì)胞對(duì)瘢痕成纖維細(xì)胞的增殖有抑制作用。細(xì)胞凋亡結(jié)果顯示1d、3d實(shí)驗(yàn)組瘢痕成纖維細(xì)胞凋亡的比對(duì)照組數(shù)值略高。PCR檢測顯示，實(shí)驗(yàn)組的OCT-4、Ι型膠原較對(duì)照組高，而TIMP-1低于對(duì)照組。 3.主要結(jié)論 兩種細(xì)胞共培養(yǎng)后，羊膜上皮細(xì)胞對(duì)瘢痕成纖維細(xì)胞的增殖和凋亡有作用，PCR檢測TIMP-1、OCT-4、Ι型膠原等指標(biāo)隨著時(shí)間不同各自的表達(dá)不同。 第四部分實(shí)驗(yàn)：利用動(dòng)物模型檢測羊膜上皮細(xì)胞在瘢痕防治中的作用 1.主要方法 手術(shù)切除雙側(cè)兔耳內(nèi)側(cè)面，形成直徑為1×1cm的4個(gè)方形全層皮膚缺損；在右耳創(chuàng)面周圍注射羊膜上皮細(xì)胞懸液，左耳創(chuàng)面周圍注射PBS緩沖液。每周注射一次，定期觀察。4周后實(shí)驗(yàn)組創(chuàng)面完全上皮化，腫塊較小。對(duì)照組腫塊明顯。取瘢痕組織進(jìn)行HE染色及激光共聚焦觀察. 2.主要結(jié)果 HE染色結(jié)果顯示實(shí)驗(yàn)組上皮化完全、略顯增生狀態(tài)，基底層細(xì)胞排列明顯，其下的真皮組織中未見炎性細(xì)胞分布，成纖維細(xì)胞分布與對(duì)照組相比較為分散。共聚焦可觀察到移植的羊膜上皮細(xì)胞。對(duì)照組HE染色顯示成纖維細(xì)胞分布集中，增生明顯。共聚焦未見羊膜上皮細(xì)胞。 3.主要結(jié)論 通過肉眼觀察、HE染色及共聚焦檢測，說明羊膜上皮干細(xì)胞有效的抑制瘢痕的形成。 綜上所述，本實(shí)驗(yàn)利用羊膜上皮細(xì)胞的干細(xì)胞特性抑制增生性瘢痕形成為目的，通過抑制炎癥反應(yīng),減少創(chuàng)面愈合時(shí)間為切入點(diǎn)，分別在體內(nèi)和體外實(shí)驗(yàn)中證明這一推測的可靠性。為此，羊膜上皮干細(xì)胞的應(yīng)用有可能成為瘢痕預(yù)防和治療的新手段和切入點(diǎn)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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