真皮多能干细胞复合血浆构建真皮再生模版及移植观察

Construction and transplantation of dermal template using dermal multipotent stem cells seeded into human plasma

背景:要实现组织工程皮肤生物学功能重建,应从重建功能正常的真皮入手,使之成为汗腺、血管、神经和脂肪再生的场所。目的:以真皮多能干细胞为种子细胞构建真皮再生模版,观察其移植后的成活状态。设计、时间及地点:单因素设计,重复观察,于2006-01/06在中山大学眼科中心国家重点实验室和中山大学动物中心进行。材料:6周龄BALB/c裸鼠10只,由中山大学动物中心提供。人皮肤多能干细胞来源于接受瘢痕整形手术患者的正常皮肤,由汕头大学医学院附属第二医院烧伤整形科提供。人O型血浆为大连制药有限公司产品。方法:取10mL人O型血浆,与生理盐水、氨甲环酸混匀后,添加1×105个经5-Brdu标记的人皮肤多能干细胞克隆,振荡致细胞团均匀分布在血浆中,再滴加氯化钙,放置37℃培养箱30min后成凝胶状,然后加入含胎牛血清、B27的DMEM/F12培养液,培养四五天细胞融合成片,吸干凝胶表面水分,使之成膜状,即为真皮模版。裸鼠麻醉后,背部制作全层皮肤缺损创面,然后植入真皮模版,覆盖创面。主要观察指标:真皮模版移植后观察血管化程度,创面上皮化后免疫组织化学检测模版中5-Brdu标记干细胞的成活状态。结果:人皮肤多能干细胞在血浆中增殖良好,真皮模版为白色生物膜,厚(500±254)μm,真皮模版中部分细胞仍表达nestin。真皮模版植入后,第3天模版下有少量的血管生成,第9天大量细胞迁入模版中。创面上皮化后第1,3周,皮肤组织的表皮和真皮中均可见大量5-BrdU阳性细胞分布。结论:人皮肤多能干细胞接种于血浆中构建的真皮模版移植后,能较快血管化,且真皮模版干细胞在受体组织中能成活下来。

BACKGROUND： In order to realize the biological function reconstruction of tissue-engineered skin, reconstruction of dermal template is necessary to benefit regeneration of sweat gland, vessel, nerve, and lipid tissue. OBJECTIVE： To construct dermal template with dermal multipotent stem cells and to observe the survival state of grafts after transplantation. DESIGN, TIME AND SETTING： Single factor, repetitive observation. The study was performed at the Animal Center and State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University from January to June 2006. MATERIALS： Ten 6-week-old BALB/c naked mice were provided by the Animal Center of Sun Yat-sen University. Human dermal multipotent stem cells were from normal skin of people undergoing scar plastic operation, provided by Department of Burn and Plastic Surgery, Second Affiliated Hospital of Medical School of Shantou University. Human O-type plasma was purchased from Dalian. METHODS： 10 mL O-type plasma was mixed with normal saline and tranexamic acid. Dermal multipotent stem cells marked by 5-BrdU were placed in the plasma, and incubated in calcium chloride at 37℃ for 30 minutes, followed by incubation in DMEM/F12 supplemented by fetal bovine serum and B27. The cells were confluent and formed slices. The water was extracted, and dermal template was obtained. Full-thickness defect skin wounds were made on the back of animals, and dermal template was transplanted. MAIN OUTCOME MEASURES： NeovascularizaUon of dermal template was observed and 5-BrdU expression cells in the skin tissues after wound healing was detected by immunofluorescence techniques. RESULTS： Human dermal multipotent stem cells proliferated well in the plasma; constructed dermal template was white biofilm, （500±254） μ m thickness. Some cells in the dermal template still expressed nestin. New vessel formation was seen under the dermal template on day 3 after transplantation; on day 9, many cells grew into the template. Large numbers of 5-BrdU expressed cells were detected in the dermis and epidermis on healed wounds. CONCLUSION： Dermal template could be well neovascularized after transplantation and cells in the template could survive in recipient skin tissues.