摘要
目的:评估Ⅳ型胶原黏附法分选表皮干细胞的分选效果及对细胞生物学特性的影响。方法:实验于2004-11/2005-05在中山大学附属第一医院外科实验室完成。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5份(患者知情同意),进行表皮细胞的分离培养。取培养的二三代表皮细胞,消化后加入铺有100mg/LⅣ型胶原的培养瓶中分选。取分选细胞悬液于培养箱中过夜培养,第2天取出爬片,磷酸缓冲液冲洗3min×2次,去除残留培养液,4%多聚甲醛室温下固定10min,磷酸缓冲液冲洗3min×2次,以含0.1%Triton-100的3%山羊血清封闭、透膜20min,滴加稀释好的角蛋白K19/β1整合素抗体进行双标,4℃过夜孵育,磷酸缓冲液冲洗3min×2次,加入配置好的双标用二抗,37℃下孵育20min,磷酸缓冲液冲洗3min×2次,加入5mg/L的Hochest33258,标记细胞核,37℃下孵育5min,磷酸缓冲液冲洗3min×2次,滴加含40%甘油的碳酸氢钠缓冲液,盖玻片封片,以上步骤均在暗室中操作,同时以磷酸缓冲液代替一抗作为阴性对照。激光共聚焦显微镜下观察,拍照。同时表达角蛋白K19、β1整合素认为是表皮干细胞。分别取分选前及筛选后细胞各4×106个,检测表皮干细胞(α6briCD71dim细胞)、短暂扩增细胞(α6briCD71bri细胞)及终末分化细胞(α6dim细胞)的百分率及分选前后的细胞周期。结果:①分选后细胞体积小,均匀一致,核浆比例大,免疫荧光检测同时表达K19、β1整合素。②与分选前细胞比较,分选后细胞中表皮干细胞α6briCD71dim细胞、短暂扩增细胞α6briCD71bri细胞的百分率明显增高,差异显著[(9.41±0.31)%,(38.74±1.09)%;(43.66±1.12)%,(48.92±2.49)%,P<0.01]。③与分选前细胞比较,分选后细胞G0/G1期所占比例明显增高,差异显著[(80.80±1.69)%,(94.37±1.32)%,P<0.01]。提示分选后细胞处于相对静止状态的表皮干细胞所占比例高。结论:Ⅳ型胶原黏附分选出的细胞含有高纯度的表皮干细胞,此分选方法对细胞活力无明显影响。能够为组织工程皮肤构建提供理想的种子细胞。
AIM： To isolate human epidermal stem calls by type Ⅳ collagen, and explore its effect on the cellular biological characteristics. METHODS： The experiment was conducted in the Laboratory of Surgery, First Hospital Affiliated to Sun Yat-Sen University from November 2004 to May 2005. Five human foreskins from routine circumcisions ware collected from the Clinic of Surgery, First Hospital Affiliated to Sun Yat-Sen University to isolate and culture. Epidermal stem calls of the second and third passages ware cultured in cultural bottles with type Ⅳ collagen （100 mg/L） after digestion. Cell suspension was cultured in the incubator ovemight. On the next rooming, the cuticular layer was washed with phosphate buffer twice for 3 minutes to get rid of the residual cultural fluid, and then fixed for 10 minutes at the room temperature with 4% of polyformat dehyde, washed with phosphate buffer twice for 3 minutes, sealed with 3% of goat serum containing 0.1% Triton-100; diluted keratin K19/131 integrin antibody was added for double labeling and incubated at 4 ~C overnight, washed with phosphate buffer twice for 3 minutes each time, and then the prepared double-labeling was added with second antibody, incubated at 37 ℃ for 20 minutes, washed with phosphate buffer twice for 3 minutes each time, and 5 mg/L Hochest 33258 was added to label the nuclear, incubated at 37 ℃ for 5 minutes, washed with phosphate buffer twice for 3 minutes each time, and sodium bicarbonate solution, containing 40% of glycerine, was added, and the sample was sealed with cover glass. All above-mentioned procedures ware conducted in dark room, and phosphate buffer was taken as the negative control instead of first antibody. Laser confocal microscopy was adopted for observation and photo taking. Meanwhile, keratin K19 and 131 integrin ware taken as epidermal stem calls. 4×106 calls before and after the sorting ware selected to detect the ratios of epidermal stem calls （α6briCD71dim calls）, short-term proliferated calls （α6briCD71 bri calls） and terminally differentiated calls （α6dim calls） as wall as the call cycle. RFSULTS. （1） The experimental group calls showed a small call size, blastlike morphology with a highnuclear-to-cytoplasmic ratio, expressed Keratin 19 and β1 integdn at the same time. （2） The ratios of α6briCD71dim and α6briCD71 bri calls ware significantly enhanced with remarkable differences [（9.41 ±0.31）%, （38.74±1.09）% ; （43.66±1.12）%, （48.92±2.49）% ,P 〈 0.01]. （3） Compared with calls before sorting, the ratio of calls at G0/G1 phase after the sorting was markedly increased with significant differences [（80.80±1.69）%,（94.37±1.32）%,P 〈 0.01], which indicated that the ratio of epidermal stem calls in comparatively quiescent condition was higher after sorting. CONCLUSION; Those adhesion calls by type Ⅳ collagen are epidermal stem calls. Selective adhesion of kerstinocytes to type Ⅳ collagen is a feasible approach to isolate epidermal stem calls, and could provide ideal seeded calls for skin tissue engineering.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第7期1201-1204,I0001,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
广东省自然科学基金资助项目(31685)
广东省科技计划项目基金资助(2004B35001002)
广州市科学技术局资助项目(2006Z3E5131)~~
