中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2011年
2期
117-120
,共4页
黑素细胞%细胞培养技术%白癜风
黑素細胞%細胞培養技術%白癜風
흑소세포%세포배양기술%백전풍
Melanocytes%Cell culture techniques%Vitiligo
目的 从吸引疱疱顶微小皮片建立人表皮黑素细胞(MC)原代培养,以人羊膜为支架培养MC,观察羊膜对MC黏附、增殖和树突发育的影响.方法 用负压吸疱法分离疱顶微小皮片,多巴染色后对取自同一个体下腹部和前臂屈侧皮片的MC进行计数.皮片经胰酶消化后自基底面轻刮表皮细胞建立原代MC培养,将培养的第2～4代MC接种至新鲜或冻存羊膜上共孵育不同时间后,倒置显微镜下观察MC形态和树突发育.行石蜡包埋组织切片,HE染色后观察MC对羊膜的黏附.用MTT比色法测定接种在羊膜上的MC存活率.结果 前臂屈侧吸引疱疱顶皮片多巴染色阳性细胞个数为(1543.1±113.3)个/mm2,腹部皮片为(857.4±101.7)个/mm2.在4周内建立MC原代培养至少需要从2个疱顶(面积约25.1 mm2)的前臂皮片获取的表皮细胞.倒置显微镜下观察,与单纯接种细胞培养皿的MC比较,接种至新鲜或冻存羊膜上继续培养4、8、12 d的MC形态多为两极,树突细长延伸.HE染色示MC能黏附并均匀分布在羊膜的基底膜层表面.MTT法测定结果示,羊膜对MC的分裂增殖有抑制,但冻存羊膜与新鲜羊膜对MC增殖抑制差异无统计学意义(P＞0.05).结论 人前臂屈侧皮肤富含活性MC,是获取吸引疱疱顶微小皮片建立MC原代培养的较好部位.人羊膜支持MC黏附生长和树突发育,有望成为一种能荷载体外培养扩增MC的人源生物支架.
目的 從吸引皰皰頂微小皮片建立人錶皮黑素細胞(MC)原代培養,以人羊膜為支架培養MC,觀察羊膜對MC黏附、增殖和樹突髮育的影響.方法 用負壓吸皰法分離皰頂微小皮片,多巴染色後對取自同一箇體下腹部和前臂屈側皮片的MC進行計數.皮片經胰酶消化後自基底麵輕颳錶皮細胞建立原代MC培養,將培養的第2～4代MC接種至新鮮或凍存羊膜上共孵育不同時間後,倒置顯微鏡下觀察MC形態和樹突髮育.行石蠟包埋組織切片,HE染色後觀察MC對羊膜的黏附.用MTT比色法測定接種在羊膜上的MC存活率.結果 前臂屈側吸引皰皰頂皮片多巴染色暘性細胞箇數為(1543.1±113.3)箇/mm2,腹部皮片為(857.4±101.7)箇/mm2.在4週內建立MC原代培養至少需要從2箇皰頂(麵積約25.1 mm2)的前臂皮片穫取的錶皮細胞.倒置顯微鏡下觀察,與單純接種細胞培養皿的MC比較,接種至新鮮或凍存羊膜上繼續培養4、8、12 d的MC形態多為兩極,樹突細長延伸.HE染色示MC能黏附併均勻分佈在羊膜的基底膜層錶麵.MTT法測定結果示,羊膜對MC的分裂增殖有抑製,但凍存羊膜與新鮮羊膜對MC增殖抑製差異無統計學意義(P＞0.05).結論 人前臂屈側皮膚富含活性MC,是穫取吸引皰皰頂微小皮片建立MC原代培養的較好部位.人羊膜支持MC黏附生長和樹突髮育,有望成為一種能荷載體外培養擴增MC的人源生物支架.
목적 종흡인포포정미소피편건립인표피흑소세포(MC)원대배양,이인양막위지가배양MC,관찰양막대MC점부、증식화수돌발육적영향.방법 용부압흡포법분리포정미소피편,다파염색후대취자동일개체하복부화전비굴측피편적MC진행계수.피편경이매소화후자기저면경괄표피세포건립원대MC배양,장배양적제2～4대MC접충지신선혹동존양막상공부육불동시간후,도치현미경하관찰MC형태화수돌발육.행석사포매조직절편,HE염색후관찰MC대양막적점부.용MTT비색법측정접충재양막상적MC존활솔.결과 전비굴측흡인포포정피편다파염색양성세포개수위(1543.1±113.3)개/mm2,복부피편위(857.4±101.7)개/mm2.재4주내건립MC원대배양지소수요종2개포정(면적약25.1 mm2)적전비피편획취적표피세포.도치현미경하관찰,여단순접충세포배양명적MC비교,접충지신선혹동존양막상계속배양4、8、12 d적MC형태다위량겁,수돌세장연신.HE염색시MC능점부병균균분포재양막적기저막층표면.MTT법측정결과시,양막대MC적분렬증식유억제,단동존양막여신선양막대MC증식억제차이무통계학의의(P＞0.05).결론 인전비굴측피부부함활성MC,시획취흡인포포정미소피편건립MC원대배양적교호부위.인양막지지MC점부생장화수돌발육,유망성위일충능하재체외배양확증MC적인원생물지가.
Objective To establish a primary culture of human melanocytes from tiny skin sheets harvested by using a suction blister method, to carry out a serial subcultivation of the melanocytes with human amniotic membrane (AM) as a scaffold, and to observe the influence of AM on the adhesion, proliferation and dendrite development of melanocytes. Methods Tiny skin sheets were collected from the flexual forearm or lower abdomen of a healthy male volunteer by a suction blister method and melanocytes in the skin sheet were counted following Dopa staining under a microscope. The trypsinized skin sheets were scraped with a scalpel to harvest melanocytes which were subjected to a primary culture. Then, the melanocytes were inoculated onto fresh or cryopreserved AM followed by a culture for various durations (4, 8 and 12 days). The morphology and dendrite development of melanocytes were visualized under an inverted microscope after dopa-staining, cell viability evaluated by MTT assay, the adhesion to AM examined by hematoxylin and eosin (HE) staining protocol. Results The density of melanocytes was 1543.1±13.3 cells per mm2 and 857.4±101.7 cells per mm2 in skin sheets obtained from the forearm flexure and lower abdomen of the volunteer, respectively. A skin sheet of about 25.1 mm2 from approximately two blister roof was required to ensure the success of primary culture of melanocytes within 1 month. After culture on fresh or cryopreserved AM for 4, 8, and 12 days, most melanocytes were bi-polar with extended slender dendrites compared with those cultured in common cell culture medium. HE staining showed that melancytes adhered and were evenly distributed on the basement membrane of AM. MTT assay showed that the AM inhibited the proliferation of melanocytes, and no statistical difference was observed in the inhibitory effect between fresh AM and cryopreserved AM (P＞ 0.05). Conclusions Enriched with melanocyes, flexural forearm is a preferable donor site to offer skin sheets for primary culture of melanocytes. Human AM could improve the adhesive growth and dendrite development of melanocytes, and may serve as a promising bioscaffold for in vitro expansion of melanocytes.
