ES细胞源性表皮干细胞与类真皮构成皮肤类似物的分化

【目的】以ES细胞源性表皮干细胞为种子细胞构建皮肤类似物,探讨其在皮下的分化潜能。【方法】Hoechst33342标记E14-ES细胞,经羊膜诱导4d后,形成表皮干细胞克隆,并以其作为种子细胞,取129胎鼠成纤维细胞与复合凝胶-明胶海绵复合,形成皮肤类似物,埋植于129小鼠皮下组织。术后2周、4周、6周、8周取材,做HE染色观察,β1整合素、CK15、CK19、CEA、CK18免疫组化荧光双标和免疫组化观察。【结果】术后2周和4周,植块内可见单层立方或柱状上皮构成的管状和泡状结构,6周和8周后,可见角化复层上皮、毛囊样、汗腺样、皮脂腺样等结构,免疫双标结果显示,植入2周和4周,带有蓝色核标记的细胞形成的管状或泡状结构呈β1整合素、CK15、CK19、CEA和CK18阳性,6周和8周后,HE染色中汗腺样结构呈CEA、CK18阳性。【结论】ES细胞源性表皮干细胞与类真皮构建的皮肤类似物,在同种小鼠皮下有分为角化复层上皮、汗腺样、毛囊样、皮脂腺样等结构的潜能。

MSCs构建的类真皮对ES细胞源性表皮干细胞分化潜势的影响

目的探讨以MSCs构建类真皮对ES细胞源性表皮干细胞分化潜能的影响。方法大鼠骨髓MSCs与复合凝胶-明胶海绵组成类真皮,Hoechst33342标记E14-ES细胞,经羊膜诱导4 d后,形成表皮干细胞克隆,将二者构成皮肤类似物,埋植于129小鼠皮下。术后2、4、6和8周取材,做HE染色,β1整合素、CK15、CK19、CEA、CK18免疫荧光双标和免疫组化观察。结果术后2和4周,植块中以形成单层立方或柱状上皮构成的管状和泡状结构为主,6和8周后,可见角化复层上皮、毛囊样、汗腺样、皮脂腺样等结构,皮脂腺样结构多见。免疫双标结果显示,植入2和4周,带有蓝色核标记的细胞形成的管状或泡状结构,呈β1整合素、CK15、CK19、CEA和CK18阳性,6和8周后,HE染色汗腺样结构呈CEA、CK18阳性。结论ES细胞源性表皮干细胞与骨髓MSCs构建的类真皮,在同种小鼠皮下可分化出角化复层上皮、汗腺样、毛囊样、皮脂腺样等结构。

小鼠皮肤成纤维细胞的体细胞核移植

取成年小鼠唇部皮肤进行培养, 分离成纤维细胞并血清饥饿培养1 周, 用作核供体。对成年小鼠进行超排, 取卵母细胞用作核受体,核移植重构胚经SrCl2 激活处理6 h后, 同mM16培养液和小鼠输卵管上皮细胞共培养, 把发育到早期囊胚的重构胚转移至小鼠胎儿成纤维细胞饲养层上, 添加ES细胞条件培养液,消化分离ICM,然后接种培养, 对孵出的ES细胞样集落进行鉴定培养。结果显示, 小鼠唇部皮肤成纤维细胞为核供体, 核移植重构胚2 细胞率为54 05%, 桑椹胚率17 14%, 囊胚率6 90 %, 对照组卵丘细胞的核移植重构胚2 细胞率为60 00%, 桑椹胚率21 85%, 囊胚率11 69 %, 但2种供体细胞在支持核移植重构胚发育能力上差异不显著。成纤维细胞重构囊胚中6个囊胚分离出ES细胞样集落, 3个ES细胞样集落可稳定传代; 对照组卵丘细胞重构囊胚中9个囊胚中分离出ES细胞样集落, 5个ES细胞样集落可稳定传代。从核移植重构胚中分离出的ES细胞样集落具有岛状或巢状群体生长形态,生长旺盛的集落可自发分化成单个散在或片状存在的上皮样或梭形细胞, 碱性磷酸酶检测为阳性, 常规冻存复苏, 仍显示ES细胞特征。

Fabrication of a Flexible Electric Skin Using a Bionic Cell and Study of Its Sensing Ability

Imitating the structure and function of the Pacinian corpuscle in the human body,a Bionic Cell(BC)with a liquid core was designed and made with polyvinylidene fluoride(PVDF),and an Electronic Skin(ES)sensor was successfully obtained by embedding the BCs into silicone rubber.The passive sensing ability of the ES for surface vibration pressure was measured.The results showed that the ES could detect waveform,frequency,amplitude and other parameters of the surface,and it exhibited not only a high sensitivity of 0.46563 fc/Pa,but also a large pressure measurement range of 0.6 Pa–6014 Pa.A falling real object impact experiment was carried out,and the results indicated that the ES output charge was linearly related to the falling height of the object and logarithmically related to the mass of the object.Then,a crawling insect experiment and human pulse experiment were performed to study the perception ability of the ES to irregular pressure.Due to its special structure,simple fabrication process,and high sensitivity wide measurement range,the ES fabricated in this paper is expected to be used as robot skin with tactile perception ability in the near future.