人表皮細胞去分化的實驗研究
發(fā)布時間：2018-08-18 16:01
【摘要】： 皮膚是人體最大的器官，位于體表，易于受傷，在創(chuàng)傷愈合過程中，表皮細胞進行增殖、游走、覆蓋創(chuàng)面、分化從而完成創(chuàng)面的修復。表皮細胞在創(chuàng)面修復過程中發(fā)生一系列的表型變化，從而完成創(chuàng)傷修復。研究中發(fā)現(xiàn)表皮干細胞在維持表皮的自我更新，保持皮膚正常的表皮結(jié)構(gòu)與功能方面起著重要作用。如何調(diào)動機體內(nèi)的表皮干細胞來促進創(chuàng)面的愈合以及皮膚的功能性修復是人們普遍關(guān)注的研究熱點。研究發(fā)現(xiàn)激活組織干細胞或通過誘導損傷附近細胞去分化，再次獲得干細胞特性以誘導內(nèi)源性再生能力是生物界普遍存在的現(xiàn)象。去分化的細胞重新獲得干細胞形態(tài)和細胞分裂潛能，形成新的單元來修復損傷。近年來，在創(chuàng)傷修復領(lǐng)域里的去分化研究成為研究熱點。已有研究發(fā)現(xiàn)在特殊條件下皮膚創(chuàng)面修復過程中表皮細胞可以發(fā)生去分化，從而促進創(chuàng)面的修復。 目前，研究表皮細胞的方法主要有：(1)人體的皮膚組織活體取材；(2)對培養(yǎng)的表皮細胞進行科學研究；(3)對動物皮膚及表皮細胞進行研究，從而推測人表皮細胞在各種干預(yù)因素作用下發(fā)生的變化及其機制。 本研究首先篩選一種合適的人表皮在體研究的動物模型，然后利用此動物模型對移植后的人表皮進行形態(tài)學研究。在此基礎(chǔ)上，進一步設(shè)計去表皮基底層細胞的表皮移植的動物模型，從而對移植的表皮細胞去分化現(xiàn)象進行研究，為皮膚創(chuàng)傷修復研究提供理論基礎(chǔ)。 1、動物模型的建立 應(yīng)用中性蛋白酶消化后機械分離獲得包皮表皮皮片，再把表皮皮片移植到裸鼠體內(nèi)。設(shè)計兩種動物實驗?zāi)Ｐ停?1)在裸鼠背部創(chuàng)面游離移植表皮皮片；(2)在裸鼠背部皮下移植表皮皮片。分別于移植后3d、5d、7d取材。對標本進HE染色和CK10、CK14、p63的免疫組織化學染色進行形態(tài)學研究。 2、研究表皮皮片移植到裸鼠皮下后表皮細胞的表型改變 把表皮皮片移植到裸鼠皮下后于移植后3d、5d、7d取材，對標本進行HE染色和CK10、CK14、CK19、β1整合素、PCNA、p63、c-Myc的免疫組織化學染色進行形態(tài)學研究。 3、設(shè)計去基底層表皮皮片移植的動物模型，對移植的去基底層表皮進行去分化的研究 用Ⅳ型膠原反復粘貼并沖洗表皮皮片，以去除包括表皮干細胞在內(nèi)的表皮基底層細胞。用免疫組織化學方法從形態(tài)學角度來驗證此方法去除基底層細胞的可行性。 把去基底層細胞的表皮皮片用DAPI標記后移植到裸鼠皮下，術(shù)后7d取材，對標本進行HE染色和CK10、CK14、CK19、β1整合素、PCNA、p63、的免疫組織化學染色進行移植后表皮細胞表型改變的形態(tài)學研究。以α6整合素和CD71分子為表皮干細胞標志物應(yīng)用流式細胞儀法檢測移植后存活表皮細胞的表型改變。 1、表皮皮片移植到裸鼠皮下后皮片成活，成活率較高，達60％。移植后表皮細胞增殖、生長、分化，與在體皮膚創(chuàng)傷后的表皮細胞的變化一致。CK14、p63在基底層及附基底層陽性表達。而移植表皮皮片到裸鼠背部創(chuàng)面的方法表皮成活不理想，裸鼠自身表皮逐漸生長至移植表皮下方，并最終達到創(chuàng)面愈合，移植表皮皮片脫落。 2、表皮皮片移植到裸鼠皮下后基底層細胞增殖，p63、PCNA和CK14在基底層和副基底層陽性表達。CK10陽性表達于棘層和顆粒層，表達強度弱于移植前。表皮干細胞的標志物CK19和β1整合素陽性細胞數(shù)量明顯多于移植前表皮。c-Myc在成活的表皮基底層和副基底層細胞中陽性表達。實驗中發(fā)現(xiàn)，在成活的表皮皮片的棘層出現(xiàn)與基底層和副基底層細胞表型一致的散在分布的細胞或細胞島。 3、Ⅳ型膠原處理前后的表皮片行HE和免疫組織化學檢測顯示：處理前的表皮片可見明顯的表皮角，而處理后的皮片表皮角消失，皮片的最下層無排列緊密的細胞存在。處理前的表皮片基底層的細胞CK14陽性，基底層以上CK10染色陽性，處理后CK14陰性而皮片全層CK10染色陽性。移植的去基底層表皮細胞的皮片呈間斷成活，以移植皮片有成活表皮細胞為成活標準，皮片存活率為33.3％。去基底層移植后7d的表皮皮片細胞排列分層不明顯，無整齊排列的基底層細胞，與未去基底層的表皮皮片移植7d的HE結(jié)果有明顯差別。去基底層表皮移植7d取材標本CK19和β1整合素染色陽性細胞呈多層散在分布，表皮中PCNA陽性細胞大量出現(xiàn)，細胞排列散在，分層不明顯，p63陽性細胞也大量出現(xiàn)在成活的表皮片各個層次中，有的成團樣聚集，分布不均。流式細胞儀檢測結(jié)果：去基底層表皮皮片α6~+CD71~-細胞占0.016％，α6~+CD71~+占0.021％；移植后7dα6~+CD71~-細胞占1.366％，α6~+CD71~+占3.528％，，移植前與移植后α6~+CD71~-和α6~+CD71~+細胞比例有明顯差異(P＜0.05)。 1、將分離的表皮皮片移植到裸鼠皮下可以成功建立人表皮在體研究實驗動物模型。這為研究人表皮細胞在皮膚創(chuàng)傷修復中的作用提供了研究平臺。 2、移植成活的表皮細胞與人體皮膚創(chuàng)面愈合過程中表皮細胞發(fā)生的表型變化相似，基底層細胞增生活躍，CK14、p63、PCNA陽性細胞增多，多層分布。成活的表皮皮片中表皮干細胞標志物β1整合素和CK19陽性細胞增多，提示表皮皮片的異種移植促進了表皮干細胞的分裂、增殖。 3、移植的表皮棘層出現(xiàn)散在分布的島狀細胞團，免疫組化染色證實為短暫擴增細胞或表皮干細胞。在皮膚創(chuàng)傷修復過程中，一定條件下表皮細胞可能發(fā)生去分化，從而促進創(chuàng)傷修復。 4、將分離的表皮用Ⅳ型膠原反復粘連并沖洗來可以成功去除表皮基底層細胞。 5、在去表皮基底層表皮皮片移植到裸鼠的動物實驗?zāi)Ｐ脱芯恐羞M一步證實了表皮細胞去分化這一生物學現(xiàn)象存在的可靠性。皮膚創(chuàng)傷修復過程中除了有靜止干細胞的激活外，在一定條件下終未分化的表皮細胞去分化也參與皮膚創(chuàng)傷的修復。
[Abstract]:Skin is the largest organ in the human body, located on the body surface, easy to be injured. In the process of wound healing, epidermal cells proliferate, migrate, cover the wound, and differentiate to complete the wound repair. Epidermal cells undergo a series of phenotypic changes in the process of wound repair, thus completing the wound repair. Self-renewal plays an important role in maintaining the normal structure and function of the epidermis. How to mobilize the epidermal stem cells in vivo to promote wound healing and functional repair of the skin is a hot research topic. It has been found that activating tissue stem cells or inducing the cells near the injury to dedifferentiate can be obtained again. Dedifferentiated cells regain the morphological and mitotic potential of stem cells and form new cells to repair injuries. In recent years, the study of dedifferentiation in the field of wound repair has become a hot topic. It has been found that skin wounds under special conditions are damaged. During the process of surface repair, epidermal cells can dedifferentiate, thus promoting wound repair.
At present, the main methods to study the epidermal cells are: (1) human skin tissue in vivo; (2) scientific research on the cultured epidermal cells; (3) animal skin and epidermal cells were studied to speculate the changes and mechanisms of human epidermal cells under various intervention factors.
In this study, we first selected a suitable animal model for in vivo study of human epidermis, and then used this animal model to study the morphology of human epidermis after transplantation. It provides a theoretical basis for wound healing research.
1, the establishment of animal models.
Two animal models were designed: (1) epidermal grafts were transplanted freely on the wounds of the back of the nude mice; (2) epidermal grafts were transplanted subcutaneously on the back of the nude mice. P63 immunohistochemical staining was used for morphological study.
2, we studied the phenotypic changes of epidermal cells after skin grafting in nude mice.
The epidermal skin grafts were transplanted into nude mice subcutaneously and taken 3, 5 and 7 days after transplantation. The specimens were stained with HE and immunohistochemical staining of CK10, CK14, CK19, beta 1 integrin, PCNA, p63 and c-Myc.
3. Designing an animal model of dermal skin graft to dedifferentiate the transplanted dermal skin.
The epidermal basal layer cells, including epidermal stem cells, were removed by repeated adherence of collagen type IV and rinsing of epidermal skin grafts.
DAPI-labeled epidermal grafts were transplanted subcutaneously into nude mice. The specimens were stained with HE and immunohistochemical staining with CK10, CK14, CK19, beta-1 integrin, PCNA, p63. The morphological changes of epidermal cell phenotype were studied after transplantation. The phenotypic changes of viable epidermal cells after transplantation were detected by flow cytometry.
1. The survival rate of epidermal skin graft in nude mice was 60%. The proliferation, growth and differentiation of epidermal cells were consistent with the changes of epidermal cells after skin trauma in vivo. The epidermis gradually grew below the grafted epidermis and eventually healed and the grafted epidermis flaked off.
2. After subcutaneous skin grafting, the basal layer cells proliferated and p63, PCNA and CK14 were positively expressed in the basal layer and parabasal layer. CK10 was positively expressed in the spinous layer and granular layer, and the expression intensity was weaker than that before transplantation. Positive expression was found in the cells of the basal and parabasal layers. In the surviving epidermis, scattered cells or cell islands were found in the spinous layer, which was consistent with the phenotype of the cells in the basal and parabasal layers.
The results of HE and immunohistochemistry showed that the corners of the epidermis before and after treatment disappeared, and there were no close-packed cells in the bottom layer of the skin. The survival rate of the grafted skin was 33.3%. On the 7th day after the basal layer was removed, the cells in the epidermis were not obviously stratified, and the cells in the basal layer were not well arranged. After 7 days of epidermal grafting, the positive cells of CK19 and beta 1 integrin staining were scattered in many layers. PCNA positive cells were found in a large number of epidermis, the cells were scattered in many layers, and the layers were not obvious. P63 positive cells also appeared in many layers of the surviving epidermal grafts, and some of them were clustered. Flow cytometry showed that the percentage of alpha 6 ~ + CD71 ~ - cells was 0.016%, that of alpha 6 ~ + CD71 ~ + cells was 0.021%, that of alpha 6 ~ + CD71 ~ - cells was 1.366% and that of alpha 6 ~ + CD71 ~ + cells was 3.528% on the 7th day after transplantation, and that of alpha 6 ~ + CD71 ~ - and alpha 6 ~ + CD71 ~ + cells were significantly different before and after transplantation (P < 0.05).
1. Human epidermis can be successfully transplanted into nude mice to establish an animal model in vivo, which provides a platform for the study of the role of human epidermal cells in skin wound healing.
2. The phenotypic changes of epidermal cells during wound healing were similar to those of human skin. The basal layer cells proliferated and the positive cells of CK14, p63 and PCNA increased. The epidermal stem cell markers beta 1 integrin and CK19 positive cells increased in the surviving epidermal skin grafts, suggesting that xenotransplantation of epidermal skin grafts was possible. It promotes the division and proliferation of epidermal stem cells.
3. Scattered islands of cells appeared in the transplanted epidermal spinous layer. Immunohistochemical staining confirmed the transient expansion of cells or epidermal stem cells.
4, we can successfully remove epidermal basal layer cells by separating the epidermis with repeated adhesion and washing with type IV collagen.
5. In addition to the activation of stationary stem cells, the dedifferentiation of undifferentiated epidermal cells is also involved in skin wound repair under certain conditions. Repair.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R329
本文編號：2189968
[Abstract]:Skin is the largest organ in the human body, located on the body surface, easy to be injured. In the process of wound healing, epidermal cells proliferate, migrate, cover the wound, and differentiate to complete the wound repair. Epidermal cells undergo a series of phenotypic changes in the process of wound repair, thus completing the wound repair. Self-renewal plays an important role in maintaining the normal structure and function of the epidermis. How to mobilize the epidermal stem cells in vivo to promote wound healing and functional repair of the skin is a hot research topic. It has been found that activating tissue stem cells or inducing the cells near the injury to dedifferentiate can be obtained again. Dedifferentiated cells regain the morphological and mitotic potential of stem cells and form new cells to repair injuries. In recent years, the study of dedifferentiation in the field of wound repair has become a hot topic. It has been found that skin wounds under special conditions are damaged. During the process of surface repair, epidermal cells can dedifferentiate, thus promoting wound repair.
At present, the main methods to study the epidermal cells are: (1) human skin tissue in vivo; (2) scientific research on the cultured epidermal cells; (3) animal skin and epidermal cells were studied to speculate the changes and mechanisms of human epidermal cells under various intervention factors.
In this study, we first selected a suitable animal model for in vivo study of human epidermis, and then used this animal model to study the morphology of human epidermis after transplantation. It provides a theoretical basis for wound healing research.
1, the establishment of animal models.
Two animal models were designed: (1) epidermal grafts were transplanted freely on the wounds of the back of the nude mice; (2) epidermal grafts were transplanted subcutaneously on the back of the nude mice. P63 immunohistochemical staining was used for morphological study.
2, we studied the phenotypic changes of epidermal cells after skin grafting in nude mice.
The epidermal skin grafts were transplanted into nude mice subcutaneously and taken 3, 5 and 7 days after transplantation. The specimens were stained with HE and immunohistochemical staining of CK10, CK14, CK19, beta 1 integrin, PCNA, p63 and c-Myc.
3. Designing an animal model of dermal skin graft to dedifferentiate the transplanted dermal skin.
The epidermal basal layer cells, including epidermal stem cells, were removed by repeated adherence of collagen type IV and rinsing of epidermal skin grafts.
DAPI-labeled epidermal grafts were transplanted subcutaneously into nude mice. The specimens were stained with HE and immunohistochemical staining with CK10, CK14, CK19, beta-1 integrin, PCNA, p63. The morphological changes of epidermal cell phenotype were studied after transplantation. The phenotypic changes of viable epidermal cells after transplantation were detected by flow cytometry.
1. The survival rate of epidermal skin graft in nude mice was 60%. The proliferation, growth and differentiation of epidermal cells were consistent with the changes of epidermal cells after skin trauma in vivo. The epidermis gradually grew below the grafted epidermis and eventually healed and the grafted epidermis flaked off.
2. After subcutaneous skin grafting, the basal layer cells proliferated and p63, PCNA and CK14 were positively expressed in the basal layer and parabasal layer. CK10 was positively expressed in the spinous layer and granular layer, and the expression intensity was weaker than that before transplantation. Positive expression was found in the cells of the basal and parabasal layers. In the surviving epidermis, scattered cells or cell islands were found in the spinous layer, which was consistent with the phenotype of the cells in the basal and parabasal layers.
The results of HE and immunohistochemistry showed that the corners of the epidermis before and after treatment disappeared, and there were no close-packed cells in the bottom layer of the skin. The survival rate of the grafted skin was 33.3%. On the 7th day after the basal layer was removed, the cells in the epidermis were not obviously stratified, and the cells in the basal layer were not well arranged. After 7 days of epidermal grafting, the positive cells of CK19 and beta 1 integrin staining were scattered in many layers. PCNA positive cells were found in a large number of epidermis, the cells were scattered in many layers, and the layers were not obvious. P63 positive cells also appeared in many layers of the surviving epidermal grafts, and some of them were clustered. Flow cytometry showed that the percentage of alpha 6 ~ + CD71 ~ - cells was 0.016%, that of alpha 6 ~ + CD71 ~ + cells was 0.021%, that of alpha 6 ~ + CD71 ~ - cells was 1.366% and that of alpha 6 ~ + CD71 ~ + cells was 3.528% on the 7th day after transplantation, and that of alpha 6 ~ + CD71 ~ - and alpha 6 ~ + CD71 ~ + cells were significantly different before and after transplantation (P < 0.05).
1. Human epidermis can be successfully transplanted into nude mice to establish an animal model in vivo, which provides a platform for the study of the role of human epidermal cells in skin wound healing.
2. The phenotypic changes of epidermal cells during wound healing were similar to those of human skin. The basal layer cells proliferated and the positive cells of CK14, p63 and PCNA increased. The epidermal stem cell markers beta 1 integrin and CK19 positive cells increased in the surviving epidermal skin grafts, suggesting that xenotransplantation of epidermal skin grafts was possible. It promotes the division and proliferation of epidermal stem cells.
3. Scattered islands of cells appeared in the transplanted epidermal spinous layer. Immunohistochemical staining confirmed the transient expansion of cells or epidermal stem cells.
4, we can successfully remove epidermal basal layer cells by separating the epidermis with repeated adhesion and washing with type IV collagen.
5. In addition to the activation of stationary stem cells, the dedifferentiation of undifferentiated epidermal cells is also involved in skin wound repair under certain conditions. Repair.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R329
【參考文獻】
相關(guān)期刊論文 前4條
1 付小兵 ,孫同柱 ,孫曉慶 ,盛志勇;表皮細胞生長因子治療創(chuàng)面出現(xiàn)干細胞島現(xiàn)象的方法學鑒別研究[J];解放軍醫(yī)學雜志;2002年05期
2 付小兵!100037北京,孫曉慶!100037北京,孫同柱!100037北京,董永洪!100037北京,顧小曼!100037北京,陳偉!100037北京,盛志勇!100037北京;表皮細胞生長因子治療創(chuàng)面出現(xiàn)的干細胞島現(xiàn)象[J];中華醫(yī)學雜志;2001年12期
3 趙志力,付小兵,孫同柱,陳偉,孫曉慶;成人正常皮膚和瘢痕組織表皮干細胞定位與表達特征的研究[J];中華燒傷雜志;2003年01期
4 付小兵,李建福,盛志勇;表皮干細胞:實現(xiàn)創(chuàng)面由解剖修復到功能修復飛躍的新策略[J];中華燒傷雜志;2003年01期
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