摘要
目的毛乳头细胞被广泛用于组织工程毛囊和皮肤构建。探讨用含血清的角质形成细胞培养基(keratinocyte medium,KM)和普通培养基(normal medium,NM)培养毛乳头细胞的生物学特性差异,以及KM培养的毛乳头细胞是否可用于组织工程毛囊研究。方法采用两步酶消化法从除皱术时切下的头皮标本中分离培养毛乳头,接种后分别采用KM和NM培养毛乳头细胞并传代。记录毛乳头最早贴壁时间及细胞迁出时间,接种5d时统计毛乳头贴壁率。倒置显微镜下观察两组细胞形态学变化,计数第3代细胞每皿聚集体个数;记录细胞最大传代次数;细胞融合成膜后对消化的细胞膜片行HE染色观察。取第3代细胞行MTT法检测细胞增殖;免疫荧光染色和ALP染色观察α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和ALP在两组细胞中的表达,并统计ALP蓝色阳性区域面积。结果KM组毛乳头最早贴壁时间及细胞迁出时间分别为24h及64h,早于NM组的48h及80h。5d时KM组及NM组毛乳头贴壁率分别为54.17%及36.78%。NM组细胞体积较KM组大。NM组第3代细胞每皿形成(3.06±1.12)个聚集体,KM组每皿形成(9.25±1.73)个聚集体,两组比较差异有统计学意义(P<0.05)。KM组细胞融合后细胞膜片行HE染色可见细胞呈多层性;NM组细胞不能多层性生长,未行HE染色观察。NM组细胞最多可传7代;KM组最多可传15代。MTT检测显示KM组细胞增殖迅速,7d后KM组吸光度值明显高于NM组,差异有统计学意义(P<0.05)。免疫荧光染色显示,两组第3代细胞α-SMA均为阳性。ALP染色显示,NM组第6代细胞中仅少量细胞表达ALP,阳性区域面积为(987±146)μm2;KM组第14代细胞ALP染色仍为阳性,阳性区域面积为(8757±558)μm2;两组比较差异有统计学意义(P<0.05)。结论毛乳头细胞在含血清的KM中增殖迅速,聚集生长明显,传代次数增多;用含血清KM培养毛乳头细胞用于组织工程毛囊构建是可行的。
Objective Dermal papillae cells are widely applied to reconstruction of tissue engineered hair follicle and skin.To investigate the difference of the biological characteristics of dermal papillae cells cultured with keratinocyte medium（KM） and normal medium（NM）,and to determin whether it is feasible for the reconstruction of tissue engineered hair follicle using dermal papillae cells cultured in KM.Methods Scalp samples were obtained in rhytidectomy procedure.Dermal papillaes were isolated by two steps digestive treatment,then cultured with KM and NM in two groups.The time of dermal papillae adherence and cell outgrowth was recorded and the rate of dermal papillae adherence was determined after 5 days.As well as,the difference of cell morphology was observed through inverted phase contrast microscope.The maximum generations were determined in two groups and the cell sheets were observed by HE staining.In third-generation cells,the number of aggregates in every dish and the proliferation by MTT were compared between two groups.Meanwhile,the expression ofα-smooth muscle actin（α-SMA） and ALP were detected by immunofluorescence and specific staining in two groups.Results Dermal papillaes of KM group had a higher rate of adherence and fast outgrowth.The rates of adherence were 54.17%and 36.78%in KM group and in NM group,respectively.In KM group,cells adhered after 24 hours and outgrew after 64 hours.While,cells adhered after 48 hours and outgrew after 80 hours in NM group.The cells were bigger in NM group than in KM group.In third-generation cells,3.06±1.12 and 9.25±1.73 aggregates formed in NM group and KM group,respectively,the difference was significant（P0.05）.In addition,cells could form cell sheets which were muti-layers in KM group.Mostly 7 and 15 generations could been subcultured in NM group and KM group,respectively.The result of MTT indicated that cells proliferated more actively in KM group;absorbance value of KM group was significantly higher than that of NM group after 7 days（P0.05�
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2010年第2期138-142,共5页
Chinese Journal of Reparative and Reconstructive Surgery
