毛囊细胞与组织工程皮肤的构建
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摘要
近年来,随着组织工程技术的迅速发展,组织工程皮肤已开始在临床上广泛使用。然而目前的组织工程皮肤的种子细胞来源主要是皮肤真皮成纤维细胞和表皮细胞,由此构建的组织工程皮肤具有和正常皮肤相似的真皮和表皮双层结构但没有毛囊、皮脂腺等皮肤附属器官,在组织结构上与正常皮肤组织仍有较大差异。
毛囊细胞在皮肤创伤修复中具有重要作用,可以作为种子细胞来源参与皮肤创伤后修复,而目前国内外利用毛囊细胞构建组织工程皮肤的文献报道较少。本研究在分离培养毛囊外根鞘细胞、毛乳头细胞、真皮成纤维细胞以及表皮细胞的基础上,对毛乳头细胞进行体外诱导,以证明其干细胞特性,为其作为毛囊化组织工程皮肤种子细胞提供依据;利用组织工程技术,以毛乳头细胞作为真皮细胞,以外根鞘细胞和表皮细胞成功制备组织工程皮肤;同时,将毛乳头细胞和外根鞘细胞重组并在肾被膜下培养,试图寻找新的制备含毛囊结构组织工程皮肤的方法。具体实验如下:
1、以简便快捷的培养方法获得大量生长状态良好的毛囊外根鞘细胞和毛乳头细胞:以新生SD大鼠为细胞来源,应用机械法拔取完整生长初期毛囊,组织块法培养毛乳头细胞和外根鞘细胞。
2、利用常规的体外诱导方法对毛乳头细胞进行体外诱导,分别得到类成骨细胞和类脂质细胞。
3、用实验室设计的组织工程皮肤培养系统,分别用毛乳头细胞和外根鞘细胞,毛乳头细胞和表皮细胞构建组织工程皮肤,结果均构建出含真皮和表皮的组织工程皮肤,表明毛乳头细胞可作为组织工程皮肤的种子细胞。
4、将毛乳头细胞与外根鞘细胞重组离心,并将离心所得细胞团放入SD大鼠的肾被膜下培养。两周后取出,经HE染色观察毛囊形成情况。结果移植物中有多个毛囊样结构出现。
本实验成功构建了以毛乳头细胞为真皮细胞、以表皮细胞和外根鞘细胞为表皮细胞的组织工程双层皮肤,并证明了毛乳头细胞的干细胞特性。结果表明毛囊细胞作为种子细胞,不但增加了组织工程皮肤种子细胞的来源范围,也为构建含毛囊结构的组织工程皮肤提供新的思路与方法,为组织工程皮肤的进一步完善奠定了新的理论基础,也为皮肤缺损的修复提供了新的治疗方法。
With the rapid advancement in the technology of tissue engineering (TE) over the years, TE skin has seen extensive application in clinic. At present, the main origin of seed cells of tissue engineering skin is usually dermal fibroblasts and keratinocytes. In consequence, there exist profound differences between normal skin and tissue engineering skin, with the later having no skin adnexa, such as hair follicle, sebaceous gland and sweat gland and so on.
Hair follicles play a very important role in skin wound healing, serving as origins of seed cells. However, until now, documents or reports on constructing tissue engineering skin with hair follicle cells are scarcely found in China as well as in the world. In this study, the researcher firstly separates in vitro outer root sheath cells, dermal papilla cells of hair follicle, fibroblast and epidermal cells, and then uses dermal papilla cells and fibroblast as hypodermal cells, and outer root sheath cells and epidermal cells as epidermal cells to construct tissue engineering skin. In this study, the dermal papilla cells was inducted in vitro, and the characteristics of stem cell was identified, this work provides the accordance for the dermal papilla cells used as seed cells in tissue engineering skin with hair follicle. At the same time, the dermal papilla cells were reorganized with outer root sheath cells, and the cell clumps was cultured under the renal capsule, and by this way, we tried to search for the new method of constructing tissue engineering skin with hair follicle. Experiment is specified as follows:
1、Vibrissas in anagen stage of newly born SD rats serve as cell sources. Outer root sheath cells are cultured by tissue culturing, and dermal papilla cells are obtained by culturing the intact dermal papillas. In these studies, large numbers of well-cultured outer root sheath cells and dermal papilla cells are cultured in easy and fast ways.
2、Dermal papilla cells were induced in vitro by conventional methods, and the osteogenic and adipogenous cells were obtained.
3、Using the technology of full-thickness tissue engineering skin culture in the laboratory, tissue engineering skins were constructed with the dermal papilla cells、the outer root sheath cells were cultured firstly and epidermal cells, and the dermal papilla cells were used for dermis seed cells, and the outer root sheath cells and the epidermal cells were used for epidermis seed cells. As the result, the tissue engineering skin was constructed successfully, and this indicates that the dermal papilla cells can be used as seed cell in tissue engineering skin construction.
4、The dermal papilla cells and the outer root sheath cell were reorganized and centrifugated, and the cell clumps were cultured under the renal capsule of SD rat . Two weeks later, the clumps were taken out and stained by hematoxylin-eosin (HE) and many hair follicle structures were found .
In this study, we constructed full-thick tissue engineering skin successfully with dermal papilla cells as hypodermal cells and epidermal cells and outer root sheath cells as epidermal cells, and proved the dermal papilla cells’characteristic as stem cell. It turns out that the employment of hair follicle cells as seed cells not only enlarges the range of seed cells for TE skin, but also provide new method for construction of tissue engineering skin with hair follicle, and contributes to the research on tissue engineering skin and provides a new clinical method of skin defects restoration.
毛囊细胞在皮肤创伤修复中具有重要作用,可以作为种子细胞来源参与皮肤创伤后修复,而目前国内外利用毛囊细胞构建组织工程皮肤的文献报道较少。本研究在分离培养毛囊外根鞘细胞、毛乳头细胞、真皮成纤维细胞以及表皮细胞的基础上,对毛乳头细胞进行体外诱导,以证明其干细胞特性,为其作为毛囊化组织工程皮肤种子细胞提供依据;利用组织工程技术,以毛乳头细胞作为真皮细胞,以外根鞘细胞和表皮细胞成功制备组织工程皮肤;同时,将毛乳头细胞和外根鞘细胞重组并在肾被膜下培养,试图寻找新的制备含毛囊结构组织工程皮肤的方法。具体实验如下:
1、以简便快捷的培养方法获得大量生长状态良好的毛囊外根鞘细胞和毛乳头细胞:以新生SD大鼠为细胞来源,应用机械法拔取完整生长初期毛囊,组织块法培养毛乳头细胞和外根鞘细胞。
2、利用常规的体外诱导方法对毛乳头细胞进行体外诱导,分别得到类成骨细胞和类脂质细胞。
3、用实验室设计的组织工程皮肤培养系统,分别用毛乳头细胞和外根鞘细胞,毛乳头细胞和表皮细胞构建组织工程皮肤,结果均构建出含真皮和表皮的组织工程皮肤,表明毛乳头细胞可作为组织工程皮肤的种子细胞。
4、将毛乳头细胞与外根鞘细胞重组离心,并将离心所得细胞团放入SD大鼠的肾被膜下培养。两周后取出,经HE染色观察毛囊形成情况。结果移植物中有多个毛囊样结构出现。
本实验成功构建了以毛乳头细胞为真皮细胞、以表皮细胞和外根鞘细胞为表皮细胞的组织工程双层皮肤,并证明了毛乳头细胞的干细胞特性。结果表明毛囊细胞作为种子细胞,不但增加了组织工程皮肤种子细胞的来源范围,也为构建含毛囊结构的组织工程皮肤提供新的思路与方法,为组织工程皮肤的进一步完善奠定了新的理论基础,也为皮肤缺损的修复提供了新的治疗方法。
With the rapid advancement in the technology of tissue engineering (TE) over the years, TE skin has seen extensive application in clinic. At present, the main origin of seed cells of tissue engineering skin is usually dermal fibroblasts and keratinocytes. In consequence, there exist profound differences between normal skin and tissue engineering skin, with the later having no skin adnexa, such as hair follicle, sebaceous gland and sweat gland and so on.
Hair follicles play a very important role in skin wound healing, serving as origins of seed cells. However, until now, documents or reports on constructing tissue engineering skin with hair follicle cells are scarcely found in China as well as in the world. In this study, the researcher firstly separates in vitro outer root sheath cells, dermal papilla cells of hair follicle, fibroblast and epidermal cells, and then uses dermal papilla cells and fibroblast as hypodermal cells, and outer root sheath cells and epidermal cells as epidermal cells to construct tissue engineering skin. In this study, the dermal papilla cells was inducted in vitro, and the characteristics of stem cell was identified, this work provides the accordance for the dermal papilla cells used as seed cells in tissue engineering skin with hair follicle. At the same time, the dermal papilla cells were reorganized with outer root sheath cells, and the cell clumps was cultured under the renal capsule, and by this way, we tried to search for the new method of constructing tissue engineering skin with hair follicle. Experiment is specified as follows:
1、Vibrissas in anagen stage of newly born SD rats serve as cell sources. Outer root sheath cells are cultured by tissue culturing, and dermal papilla cells are obtained by culturing the intact dermal papillas. In these studies, large numbers of well-cultured outer root sheath cells and dermal papilla cells are cultured in easy and fast ways.
2、Dermal papilla cells were induced in vitro by conventional methods, and the osteogenic and adipogenous cells were obtained.
3、Using the technology of full-thickness tissue engineering skin culture in the laboratory, tissue engineering skins were constructed with the dermal papilla cells、the outer root sheath cells were cultured firstly and epidermal cells, and the dermal papilla cells were used for dermis seed cells, and the outer root sheath cells and the epidermal cells were used for epidermis seed cells. As the result, the tissue engineering skin was constructed successfully, and this indicates that the dermal papilla cells can be used as seed cell in tissue engineering skin construction.
4、The dermal papilla cells and the outer root sheath cell were reorganized and centrifugated, and the cell clumps were cultured under the renal capsule of SD rat . Two weeks later, the clumps were taken out and stained by hematoxylin-eosin (HE) and many hair follicle structures were found .
In this study, we constructed full-thick tissue engineering skin successfully with dermal papilla cells as hypodermal cells and epidermal cells and outer root sheath cells as epidermal cells, and proved the dermal papilla cells’characteristic as stem cell. It turns out that the employment of hair follicle cells as seed cells not only enlarges the range of seed cells for TE skin, but also provide new method for construction of tissue engineering skin with hair follicle, and contributes to the research on tissue engineering skin and provides a new clinical method of skin defects restoration.
引文
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