【摘要】 目的:采用单纯胰酶和中性蛋白酶联合胰酶两种不同的消化方式,观察表皮细胞表面抗原α6整合素和转铁蛋白受体CD71表达的变化,为荧光激活细胞分选术分选表皮干细胞选择合适的消化方法。方法:选取中山大学附属第一医院外科门诊包皮环切手术患者的皮肤标本10份,患者均知情同意,年龄14～22岁。每份标本均分为2份,分别用胰酶、中性蛋白酶联合胰酶进行消化,共20份,分为胰酶组、中性蛋白酶联合胰酶组,10份/组。胰酶组将皮条放入质量浓度为2.5g/L的胰酶中,4℃消化过夜。次日撕除表皮角质层,以弯镊轻刮皮肤上皮面,获得表皮基底层细胞;中性蛋白酶联合胰酶组将皮条放入质量浓度为2.5g/L的中性蛋白酶中,4℃消化过夜。次日轻撕下皮肤表皮层,将其剪成1mm×1mm大小的碎块,以质量浓度为2.5g/L的胰酶消化5min,获得表皮基底层细胞。分别取两组细胞悬液300μL,加入碘化丙啶标记死亡细胞,流式细胞仪检测死亡细胞百分率,计算细胞活力。以荧光标记的α6整合素和CD71抗体标记细胞表面抗原,通过流式细胞术检测抗原表达。剩余细胞以表皮细胞培养液重悬,移入预先铺有100mg/LⅣ型胶原的培养瓶中,接种密度为5×104/cm2,3d换液1次,观察两组细胞的生长情况。结果:实验纳入包皮环切手术患者的皮肤标本10份,每份标本分别用胰酶、中性蛋白酶联合胰酶进行消化,共20份,全部进入结果分析。①不同酶消化后两组细胞活力的比较:中性蛋白酶联合胰酶组细胞活力明显高于胰酶组[(86±1.81)%,(82±1.48)%,P<0.01]。②不同酶消化后两组细胞抗原的表达:胰酶组α6briCD71dim细胞占(8.00±1.69)%,α6briCD71bri细胞占(36.00±18.03)%,α6dim细胞占(40.00±10.08)%;中性蛋白酶联合胰酶组3种细胞所占比例依次为(30.00±8.59),(8.00±1.63),(38.00±8.72)%,两组各抗原的表达基本相似(P>0.05)。③不同酶消化后两组细胞形态学观察结果:胰酶组细胞贴壁及伸展较中性蛋白酶联合胰酶组稍迟0.5～1.0h,此后培养过程中生长增殖情况无明显差异,一般7d左右接近融合,传代。培养第7天,表皮细胞体积小,核浆比例大,可见夹杂有成纤维细胞。中性蛋白酶联合胰酶组细胞30min内基本贴壁,并有少量细胞伸展,60min后大部分细胞伸出短小伪足,胞质展开,折光率减低。培养第7天,表皮细胞体积小,核浆比例大,未见夹杂有成纤维细胞。结论:从对表面抗原表达影响的角度来看,单纯胰酶消化与中性蛋白酶联合胰酶消化后α6briCD71dim,α6briCD71bri,α6dim这3种表皮细胞的检出率基本相似,两种消化方法可根据习惯选用。但中性蛋白酶联合胰酶消化对细胞活力影响小,消化时机容易掌握,对细胞损伤相对小,适用于荧光激活细胞分选术分选表皮干细胞。更多还原

【Abstract】 AIM: To observe the effects of different digestion methods (purepancreatin and neutral protease matching with pancreatin) on theexpression of surface antigen α6 integrin and transferrin receptor CD71 inepidermic cells, so as to choose a suitable digestion method to separatehuman epidermal stem cells by fluorescence-activated cell sorting (FACS)techniques.METHODS: Ten human foreskins from routine circumcisions werecollected from the out-patient clinic of surgery, First Affiliated Hospital,Sun Yat-Sen University. The patients aged 14- 22 years were all agreed tojoin the experiment. Each specimen was divided into two parts, anddigested with pancreatin and neutral protease matching with pancreatin,respectively, totally 20 portions, including pancreatin group and neutralprotease matching with pancreatin group with 10 portions in each group.Those strips in the pancreatin group were digested with 0.25% trypsogen at4 ℃overnight. On the next morning, the cuticular layer was striped off andthe epithelial layer was scratched with curved scissors. Primary basalkeratinocytes were collected from the cell suspension. Those in the neutralprotease matching with pancreatin group were incubated in 2.5 mg dispaseper ml at 4 ℃. At the following day, cuticle was striped off, and was cutinto 1 mm×1 mm broken bits. Sheet of epidermis were digested for 5minutes into pancreatin to gain basal lamina of epidermis cells. Cellsuspension 300 μL was gained in the two groups, respectively, adding intopropichium iodide for accounting the cell viability of death cells with flowcytometer to calculate cell activity. Labeling epidermic cell with anti-α6integrin mAb G0H3 and mAb MA712 against the human transferrinreceptor CD71 for flow cytometric analysis, and others were resuspensedwith epidermic cell medium, and moved into 100 mg/L flask coated withcollagen IV at a final seeding cell density of 5×104 viable cells per squarecentimeter. The growth medium was changed every 3 days. The growthsituation of cells was observed.RESULTS: Ten human foreskins in patients after routine circumcisions,totally 20 portions of samples that were digested with pancreatin andneutral protease matching with pancreatin, were all involved in the resultanalysis. ① Comparison of cell viability of the two groups after digesting indifferent enzyme: It was higher in the neutral protease matching withpancreatin group than that in the pancreatin group [(86±1.81) %, (82±1.48)%, P < 0.01]. ② Expression of cell antigen in the two groups afterdigesting in different enzyme: The cells of α6briCD71dimin the pancreatingroup was (8.00±1.69)%\ The cells of α6briCD71bri occupied (36.00±18.03)%\The cells of α6dim accounted for (40.00±10.08)%. The 3 types of cells in theneutral protease matching with pancreatin group occupied (30.00±8.59),(8.00±1.63), (38.00±8.72)%, in order. Most of the expression of antigenwere similar (P > 0.05). ③ Results of cell morphological observation afterdigesting in different enzyme: Adherence and extension in the pancreatingroup were late slightly for 0.5- 1.0 hours as compared with the neutralprotease matching with pancreatin group. The growth and proliferationduring cultured process had insignificant difference, about 7 daysapproached to confluence and transfer of culture. At the 7th day, volume ofepidermic cells was small, and karyoplasms were big, showing fibroblast. Inthe neutral protease matching with pancreatin group, the cells adheredwithin 30 minutes, small amount of cell extended. After 60 minutes,exhibited small cell size, cytoplasm outspread, refracting rate decreased. Atthe 7th day after culture, the volume of epidermic cells was small with a bignuclear and there were no fibroblast.CONCLUSION: From the angle of effects on expression of superficialantigen, checking rates of α6briCD71dim, α6briCD71bri, α6dim after pancreatin andneutral protease matching with pancreatin digestion are similar generally,so the two digestion methods can be used according to habit. But theneutral protease matching with pancreatin digestion has little effect on thecell viability. It is easy to master the time of digestion. This method hasslight effect on cell injury, and is suitable for separating epidermal stemcells by FACS techniques.更多还原