人表皮干细胞复合裸鼠脱细胞真皮支架构建组织工程皮肤及移植的实验研究
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摘要
目的:用组织工程学原理,通过体外分离扩增人表皮干细胞作为种子细胞,复合自己制备的异种(裸鼠)脱细胞真皮支架材料,探讨体外构建活性组织工程皮肤的可行性。将该组织工程皮肤移植于实验动物创面,检测和了解该组织工程皮肤的结构和功能。
方法:用中性蛋白酶消化法,分离小儿包皮的表皮,制成单细胞悬液, IV型胶原快速粘附10-15min (370C),分别取快速粘附细胞和未黏附的细胞培养。用K-SFM培养基,补充10%的干细胞培养专用胎牛血清(FBS)。用免疫组织化学测定角蛋白15(CK15)、角蛋白19(CK19)和β1整合素(β1-integrin)和用流式细胞仪检测CD71等指标,对进表皮干细胞行鉴定。以胰蛋白酶法制备(裸鼠)脱细胞真皮支架,并进行组织学观察。将分离培养传代的表皮干细胞接种于铺好IV型胶原的脱细胞真皮表面,置入干细胞培养体系培养后,再行气液界面培养构建组织工程皮肤。HE染色切片观察组织工程皮肤结构。将组织工程皮肤移植于实验动物创面,以单纯脱细胞真皮移植作对照。肉眼观察移植后的创面情况,对移植后的皮肤取材行组织学观察及免疫组织化学鼠抗人广谱角蛋白染色确定组织来源。
结果:1.表皮干细胞能被IV型胶原快速黏附;2.黏附的细胞在培养皿中后很快贴壁,第3-4d形成一些小集落,其后细胞逐渐增殖,至第7d左右逐渐有大集落形成。而未黏附的细胞集落形成早1-2d,集落形成数明显少于实验组。黏附细胞免疫组织化学染色阳性率CK15为50.80%+1.81、CK19为51.40%+2.22、和β1-integrin为51.40%+1.90,CD71阳性率约1.91%+0.21。黏附细胞具有表皮干细胞特性,阳性率与文献报道的差别不大。3、制备的脱细胞真皮支架呈乳白色,柔软有弹性,光镜下见表皮去除,真皮内无其他细胞成分,胶原纤维结构保存完好,排列有序。4、表皮干细胞能快速黏附到由IV型胶原包被的脱细胞真皮支架上,迅速贴壁生长,无排斥反应。联合培养约两周后,可融合形成含1-2层细胞的复合皮。5、活性复合皮移植于实验动物的创面后,创面愈合良好,未见排斥反应,创面收缩不明显; 4周后,移植皮肤取材切片HE染色,光镜下见创面愈合,皮层含2-3层细胞,胶原排列整齐,未见皮肤附属结构。组织工程皮肤鼠抗人广谱角蛋白染色为阳性反应,对照组染色阴性,鼠皮广谱角蛋白染色阴性,说明移植皮肤的皮肤结构来源于实验用的人体种子细胞,表皮干细胞向表皮细胞分化。脱细胞真皮组无表皮结构形成,表面逐渐干燥、变硬,创面收缩,3周后创面愈合。组织取材HE染色未见表皮层结构形成,为红染的疤痕样组织,鼠抗人广谱角蛋白染色为阴性反应。
结论:1、表皮干细胞能被IV型胶原快速黏附,利用这一特性,可以用来筛选表皮干细胞,并在K-SFM培养体系中分裂增殖,产生大量的子代细胞,可作为皮肤组织工程的种子细胞。
2、胰蛋白酶法制备的异体脱细胞真皮,能去除表皮层及真皮内各种细胞成分,胶原纤维结构保存完好,可作为组织工程用脱细胞真皮支架材料。
3、扩增、传代的第二代表皮干细胞能快速黏附在预铺好IV型胶原的脱细胞真皮上,在表皮干细胞培养体系中生长,联合气液界面培养,能构建活性复层结构的人工皮肤。
4、构建的活性组织工程皮肤能修复实验动物创面,4周后的组织学检测显示,表皮干细胞分化为表皮细胞,组织内未见有皮肤附属结构形成。
Auto-skin grafting will be fist used when extensive skin loss occurs, when suffer a deficient of donor sites available for skin graft, other methods always used in clinic such as small stamps of autografting, mesh skin grafting, intermingle transplantation of auto-allografting, micro-skin-grafting and cultured epidermal Auto-grafting, because lack of dermis, can't resolve the problems regarding the cosmesis and function satisfactorily. Tissue-engineering skin can help resolving this problem, but there are some trouble in all kinds of tissue-engineering skin now, some are just dermis scaffold, some may lead to reject reaction because of the variaton of epidermal cells. Therefore, it will have clinical importance to composite a permanent skin substitute with ideal functions and no reject reaction.
Objective: Investigate the feasibility constructing living composite skin of tissue-engineering. To construct tissue-engineered skin with cultured epidermal stem cells and acellular dermal scaffold and investigate the feasibility of repairing animal skin defection with the tissue-engineered skin. To research the construction and function of the tissue-engineered skin.
Methods: Epidermis was separated from dermis of child's forskin,and dissociated into single cells. epidermal cells were incubated for 10-15min at 370C on dishes coated with collagen typeⅣ,then the adherent and nonadherent cells were cultured respectively in the K-SFM medium, supplemented with 100g/L fetal calf serum (FBS, for stem cell). The cells were examined for phenotypes under microscope, the expressions of Cytokeratine 15 (CK15) , Cytokeratine 19 (CK19) andβ1-integrin were detected with immunohistochemical method, andCD71 were detected with flow cytometry. Acellular dermal scaffolds were prepared by using of trypsin .Well-growing human epidermal stem cells which isolated were seeded onto the surface of acellular dermal scaffold which covered by collagen typeⅣto construct living tissue-engineered skin. This kinds of skin were transplanted to full skin wound in SD rats, compared with transplatation of acellular dermal scaffold only. The wounds were oberserved after operation. Tissue samples were harvested and examined by means of histology and immunohistochemistry method.
Results:1、The epiderm stem cells can be adhered rapidly by collagen typeⅣ; 2、the adhered cells formed more and bigger colonies than that of nonadhered cells. The positive expressions by immunohistochemical method of CK15 are50.80%+1.81, CK19 are 51.40%+2.22,β1-integrin are51.40%+1.90, and the positive expressions by flow cytometry method of CD71 is1.91%+0.21. 3. The acellular xeno-dermis prepared firstly has been removed of the epidermis and all cellular components of the dermis. but the basement membrane complex and the three-dimensional structure of the collagen scaffold appeared intact. 4, ESCs can soon adhered to the surface and seed on the acellular dermal scaffold which voverd by collagen typeⅣ. A living tissue-engineered skin include 1-2 layers of epithelium was constructed in vitro after 2 weeks Co-cultivation. 5, After the tissue-engineered skin being transplated onto the wounds, it healed well with no rejiection and only a little contraction. The tissue histology structure also demonstrated that the wounds had healed. The neoepithelium was 1-2 layers thick consisting of epidermal cells, but there is no auxiliary structure of skin can be seen . The positive expression of broad-spectrum cytokeratine demonstrated that the neoskin was regenerated from grafted human cells. But in control group, the acellular dermal scaffolds transplated become dry because of no epithelium. In the end, the acellular dermal scaffolds seperated from the wounds and the wound contraction , 3 weeks later, the wounds heal with scar, tissue histology show that the expression of broad-spectrum cytokeratine is negative.
Conclusion:1、The character of epiderm stem cell can be adherent by collagen typeⅣcan be used to separate ESCs, ESCs can grow rapidly in K-SFM medium, and produce massive daughter cells, so can be used as the seeds of tissue-engineered skin.
2、The acellular dermal scaffolds prepared by method of Trypsin had been demonstrated that it had been removed of the epidermis and all cellular components of the dermis. but its basement membrane complex and the three-dimensional structure of the collagen scaffold appeared intact. Can be used as scaffold of TE.
3、The ESCs can be seeded onto the surface of scaffold after amplification and passage, after 2 weeks’growth and Co-cultivation in K-SFM medium, can construct a living tissue-engineered skin with human epidermal stem cells and acellular dermal scaffold in vitro .
4、After transplantation onto animal wounds,the composite skin was demonstrated to be a good skin substitut to repair skin defection, tissue history show that the ESCs can differentiate to epithelium, duration the observation there is no auxiliary structure of skin can be seen.
方法:用中性蛋白酶消化法,分离小儿包皮的表皮,制成单细胞悬液, IV型胶原快速粘附10-15min (370C),分别取快速粘附细胞和未黏附的细胞培养。用K-SFM培养基,补充10%的干细胞培养专用胎牛血清(FBS)。用免疫组织化学测定角蛋白15(CK15)、角蛋白19(CK19)和β1整合素(β1-integrin)和用流式细胞仪检测CD71等指标,对进表皮干细胞行鉴定。以胰蛋白酶法制备(裸鼠)脱细胞真皮支架,并进行组织学观察。将分离培养传代的表皮干细胞接种于铺好IV型胶原的脱细胞真皮表面,置入干细胞培养体系培养后,再行气液界面培养构建组织工程皮肤。HE染色切片观察组织工程皮肤结构。将组织工程皮肤移植于实验动物创面,以单纯脱细胞真皮移植作对照。肉眼观察移植后的创面情况,对移植后的皮肤取材行组织学观察及免疫组织化学鼠抗人广谱角蛋白染色确定组织来源。
结果:1.表皮干细胞能被IV型胶原快速黏附;2.黏附的细胞在培养皿中后很快贴壁,第3-4d形成一些小集落,其后细胞逐渐增殖,至第7d左右逐渐有大集落形成。而未黏附的细胞集落形成早1-2d,集落形成数明显少于实验组。黏附细胞免疫组织化学染色阳性率CK15为50.80%+1.81、CK19为51.40%+2.22、和β1-integrin为51.40%+1.90,CD71阳性率约1.91%+0.21。黏附细胞具有表皮干细胞特性,阳性率与文献报道的差别不大。3、制备的脱细胞真皮支架呈乳白色,柔软有弹性,光镜下见表皮去除,真皮内无其他细胞成分,胶原纤维结构保存完好,排列有序。4、表皮干细胞能快速黏附到由IV型胶原包被的脱细胞真皮支架上,迅速贴壁生长,无排斥反应。联合培养约两周后,可融合形成含1-2层细胞的复合皮。5、活性复合皮移植于实验动物的创面后,创面愈合良好,未见排斥反应,创面收缩不明显; 4周后,移植皮肤取材切片HE染色,光镜下见创面愈合,皮层含2-3层细胞,胶原排列整齐,未见皮肤附属结构。组织工程皮肤鼠抗人广谱角蛋白染色为阳性反应,对照组染色阴性,鼠皮广谱角蛋白染色阴性,说明移植皮肤的皮肤结构来源于实验用的人体种子细胞,表皮干细胞向表皮细胞分化。脱细胞真皮组无表皮结构形成,表面逐渐干燥、变硬,创面收缩,3周后创面愈合。组织取材HE染色未见表皮层结构形成,为红染的疤痕样组织,鼠抗人广谱角蛋白染色为阴性反应。
结论:1、表皮干细胞能被IV型胶原快速黏附,利用这一特性,可以用来筛选表皮干细胞,并在K-SFM培养体系中分裂增殖,产生大量的子代细胞,可作为皮肤组织工程的种子细胞。
2、胰蛋白酶法制备的异体脱细胞真皮,能去除表皮层及真皮内各种细胞成分,胶原纤维结构保存完好,可作为组织工程用脱细胞真皮支架材料。
3、扩增、传代的第二代表皮干细胞能快速黏附在预铺好IV型胶原的脱细胞真皮上,在表皮干细胞培养体系中生长,联合气液界面培养,能构建活性复层结构的人工皮肤。
4、构建的活性组织工程皮肤能修复实验动物创面,4周后的组织学检测显示,表皮干细胞分化为表皮细胞,组织内未见有皮肤附属结构形成。
Auto-skin grafting will be fist used when extensive skin loss occurs, when suffer a deficient of donor sites available for skin graft, other methods always used in clinic such as small stamps of autografting, mesh skin grafting, intermingle transplantation of auto-allografting, micro-skin-grafting and cultured epidermal Auto-grafting, because lack of dermis, can't resolve the problems regarding the cosmesis and function satisfactorily. Tissue-engineering skin can help resolving this problem, but there are some trouble in all kinds of tissue-engineering skin now, some are just dermis scaffold, some may lead to reject reaction because of the variaton of epidermal cells. Therefore, it will have clinical importance to composite a permanent skin substitute with ideal functions and no reject reaction.
Objective: Investigate the feasibility constructing living composite skin of tissue-engineering. To construct tissue-engineered skin with cultured epidermal stem cells and acellular dermal scaffold and investigate the feasibility of repairing animal skin defection with the tissue-engineered skin. To research the construction and function of the tissue-engineered skin.
Methods: Epidermis was separated from dermis of child's forskin,and dissociated into single cells. epidermal cells were incubated for 10-15min at 370C on dishes coated with collagen typeⅣ,then the adherent and nonadherent cells were cultured respectively in the K-SFM medium, supplemented with 100g/L fetal calf serum (FBS, for stem cell). The cells were examined for phenotypes under microscope, the expressions of Cytokeratine 15 (CK15) , Cytokeratine 19 (CK19) andβ1-integrin were detected with immunohistochemical method, andCD71 were detected with flow cytometry. Acellular dermal scaffolds were prepared by using of trypsin .Well-growing human epidermal stem cells which isolated were seeded onto the surface of acellular dermal scaffold which covered by collagen typeⅣto construct living tissue-engineered skin. This kinds of skin were transplanted to full skin wound in SD rats, compared with transplatation of acellular dermal scaffold only. The wounds were oberserved after operation. Tissue samples were harvested and examined by means of histology and immunohistochemistry method.
Results:1、The epiderm stem cells can be adhered rapidly by collagen typeⅣ; 2、the adhered cells formed more and bigger colonies than that of nonadhered cells. The positive expressions by immunohistochemical method of CK15 are50.80%+1.81, CK19 are 51.40%+2.22,β1-integrin are51.40%+1.90, and the positive expressions by flow cytometry method of CD71 is1.91%+0.21. 3. The acellular xeno-dermis prepared firstly has been removed of the epidermis and all cellular components of the dermis. but the basement membrane complex and the three-dimensional structure of the collagen scaffold appeared intact. 4, ESCs can soon adhered to the surface and seed on the acellular dermal scaffold which voverd by collagen typeⅣ. A living tissue-engineered skin include 1-2 layers of epithelium was constructed in vitro after 2 weeks Co-cultivation. 5, After the tissue-engineered skin being transplated onto the wounds, it healed well with no rejiection and only a little contraction. The tissue histology structure also demonstrated that the wounds had healed. The neoepithelium was 1-2 layers thick consisting of epidermal cells, but there is no auxiliary structure of skin can be seen . The positive expression of broad-spectrum cytokeratine demonstrated that the neoskin was regenerated from grafted human cells. But in control group, the acellular dermal scaffolds transplated become dry because of no epithelium. In the end, the acellular dermal scaffolds seperated from the wounds and the wound contraction , 3 weeks later, the wounds heal with scar, tissue histology show that the expression of broad-spectrum cytokeratine is negative.
Conclusion:1、The character of epiderm stem cell can be adherent by collagen typeⅣcan be used to separate ESCs, ESCs can grow rapidly in K-SFM medium, and produce massive daughter cells, so can be used as the seeds of tissue-engineered skin.
2、The acellular dermal scaffolds prepared by method of Trypsin had been demonstrated that it had been removed of the epidermis and all cellular components of the dermis. but its basement membrane complex and the three-dimensional structure of the collagen scaffold appeared intact. Can be used as scaffold of TE.
3、The ESCs can be seeded onto the surface of scaffold after amplification and passage, after 2 weeks’growth and Co-cultivation in K-SFM medium, can construct a living tissue-engineered skin with human epidermal stem cells and acellular dermal scaffold in vitro .
4、After transplantation onto animal wounds,the composite skin was demonstrated to be a good skin substitut to repair skin defection, tissue history show that the ESCs can differentiate to epithelium, duration the observation there is no auxiliary structure of skin can be seen.
引文
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