CAJ | 학술논문

背景:通过体外培养人角膜缘干细胞并进行传代、建系研究可为角膜缘干细胞移植的基础与临床研究提供足够的细胞储备.目的:探讨一种体外培养人角膜缘干细胞传代、建系的方法.设计:随机对照观察.单位:赣南医学院.材料:实验于2003-06/2004-04在赣南医学院科研中心与中山大学医学院眼科医院国家重点实验室完成.新鲜人角膜缘组织块分别取自2名健康人角膜组织供体,均经过供体者知情同意.RPMI-1640(Sigma R8755,含L-谷氨酰胺),200 g/L胎牛血清(Gibco 16140-071).DMEM培养基、硫酸软骨素、人表皮生长因子购自美国Sigma公司,HEPES、DMSO购自美国Gibco公司,100%甘油购自上海运佳黄浦制药有限公司,戊二醛购自德国E.Merk公司,乙醇、盐酸、丙酮、甲醛等购自北京化学试剂公司,0.25%胰蛋白酶液购自上海新华制药厂,上述试剂均为分析纯级.方法:人角膜缘深部色素区组织块经消化后,分别在含有RPMI-1640、200 g/L胎牛血清的培养瓶与以羊膜细胞外基质为培养载体的培养皿中进行体外培养.光镜及扫描电镜观察原代及传代培养细胞的生长情况;采用台盼蓝排斥实验计算冻存细胞逐代冻存处理后的复苏率.主要观察指标:①人角膜缘干细胞体外原代及传代培养观察结果.②细胞冻存复苏率.结果:①原代培养结果:经PRMI-1640培养基中培养1 d后,倒置相差显微镜下可见培养瓶中细胞多已贴壁,均匀稀疏排列成单层并贴于培养瓶底部.②传代培养结果:传第2代时添加人表皮生长因子后,细胞分散成单层,贴壁生长旺盛.所有细胞传至第30代后形态的变异性增加明显,细胞体积明显增大,形态呈圆形或不规则圆形,密集成群.在培养液中传33代后,细胞仍然保持旺盛的分化、增殖能力,并且更适宜在羊膜细胞外基质上生长.③细胞冻存复苏率:冻存细胞的复苏率为82.2%.结论:将人角膜缘干细胞经体外培养33代并逐代冻存后初步建立了人角膜缘干细胞系,人角膜缘干细胞更适合在含有羊膜细胞外基质为底物的培养基中生长. 揹景:通過體外培養人角膜緣榦細胞併進行傳代、建繫研究可為角膜緣榦細胞移植的基礎與臨床研究提供足夠的細胞儲備.目的:探討一種體外培養人角膜緣榦細胞傳代、建繫的方法.設計:隨機對照觀察.單位:贛南醫學院.材料:實驗于2003-06/2004-04在贛南醫學院科研中心與中山大學醫學院眼科醫院國傢重點實驗室完成.新鮮人角膜緣組織塊分彆取自2名健康人角膜組織供體,均經過供體者知情同意.RPMI-1640(Sigma R8755,含L-穀氨酰胺),200 g/L胎牛血清(Gibco 16140-071).DMEM培養基、硫痠軟骨素、人錶皮生長因子購自美國Sigma公司,HEPES、DMSO購自美國Gibco公司,100%甘油購自上海運佳黃浦製藥有限公司,戊二醛購自德國E.Merk公司,乙醇、鹽痠、丙酮、甲醛等購自北京化學試劑公司,0.25%胰蛋白酶液購自上海新華製藥廠,上述試劑均為分析純級.方法:人角膜緣深部色素區組織塊經消化後,分彆在含有RPMI-1640、200 g/L胎牛血清的培養瓶與以羊膜細胞外基質為培養載體的培養皿中進行體外培養.光鏡及掃描電鏡觀察原代及傳代培養細胞的生長情況;採用檯盼藍排斥實驗計算凍存細胞逐代凍存處理後的複囌率.主要觀察指標:①人角膜緣榦細胞體外原代及傳代培養觀察結果.②細胞凍存複囌率.結果:①原代培養結果:經PRMI-1640培養基中培養1 d後,倒置相差顯微鏡下可見培養瓶中細胞多已貼壁,均勻稀疏排列成單層併貼于培養瓶底部.②傳代培養結果:傳第2代時添加人錶皮生長因子後,細胞分散成單層,貼壁生長旺盛.所有細胞傳至第30代後形態的變異性增加明顯,細胞體積明顯增大,形態呈圓形或不規則圓形,密集成群.在培養液中傳33代後,細胞仍然保持旺盛的分化、增殖能力,併且更適宜在羊膜細胞外基質上生長.③細胞凍存複囌率:凍存細胞的複囌率為82.2%.結論:將人角膜緣榦細胞經體外培養33代併逐代凍存後初步建立瞭人角膜緣榦細胞繫,人角膜緣榦細胞更適閤在含有羊膜細胞外基質為底物的培養基中生長.
배경:통과체외배양인각막연간세포병진행전대、건계연구가위각막연간세포이식적기출여림상연구제공족구적세포저비.목적:탐토일충체외배양인각막연간세포전대、건계적방법.설계:수궤대조관찰.단위:공남의학원.재료:실험우2003-06/2004-04재공남의학원과연중심여중산대학의학원안과의원국가중점실험실완성.신선인각막연조직괴분별취자2명건강인각막조직공체,균경과공체자지정동의.RPMI-1640(Sigma R8755,함L-곡안선알),200 g/L태우혈청(Gibco 16140-071).DMEM배양기、류산연골소、인표피생장인자구자미국Sigma공사,HEPES、DMSO구자미국Gibco공사,100%감유구자상해운가황포제약유한공사,무이철구자덕국E.Merk공사,을순、염산、병동、갑철등구자북경화학시제공사,0.25%이단백매액구자상해신화제약엄,상술시제균위분석순급.방법:인각막연심부색소구조직괴경소화후,분별재함유RPMI-1640、200 g/L태우혈청적배양병여이양막세포외기질위배양재체적배양명중진행체외배양.광경급소묘전경관찰원대급전대배양세포적생장정황;채용태반람배척실험계산동존세포축대동존처리후적복소솔.주요관찰지표:①인각막연간세포체외원대급전대배양관찰결과.②세포동존복소솔.결과:①원대배양결과:경PRMI-1640배양기중배양1 d후,도치상차현미경하가견배양병중세포다이첩벽,균균희소배렬성단층병첩우배양병저부.②전대배양결과:전제2대시첨가인표피생장인자후,세포분산성단층,첩벽생장왕성.소유세포전지제30대후형태적변이성증가명현,세포체적명현증대,형태정원형혹불규칙원형,밀집성군.재배양액중전33대후,세포잉연보지왕성적분화、증식능력,병차경괄의재양막세포외기질상생장.③세포동존복소솔:동존세포적복소솔위82.2%.결론:장인각막연간세포경체외배양33대병축대동존후초보건립료인각막연간세포계,인각막연간세포경괄합재함유양막세포외기질위저물적배양기중생장.
BACKGROUND: The investigation of culturing, passaging and establishing human limbal stem cells can strengthen the recognition of the stem cells and provide the enough cellular reserve for the basic and clinical research of limbal stem cell transplantation.OBJECTIVE: To explore a method of pessaging and establishing cell line of human limbal stem cells cultured in vitro.DESIGN: Randomized controlled observation.SETTING: Gannan Medical College.MATERIALS: The experiment was performed at Scientific Center of Gannan Medical College and the National Key Laboratory of Ophthalmology Hospital Affiliated to Sun Yat-Sen University from June 2003 to April 2004. Fresh human limbus corneae were isolated from two healthy donors. Procedures were performed according to the informed consent of the donors. Main reagents contained RPMI-1640 (Sigma R8755, containing L-glutamine) and 200 g/L fetal calf serum (FCS) (Gibco 16140-071). DMEM medium, chondroitin sulfatase and human epidermal growth factor (hEGF) were purchased from Sigma Co. USA; HEPES and DMSO were bought from Gibco, USA; 100% glycerinum was purchased from Yunjia Huangpu Pharmaceutical Product Limited Company, PRC; glutaraldehyde was bought from E.Merk, Germany; Alcohol, chlorhydric acid, acetone and methyl aldehyde were purchased from Beijing Chemical Agent Company, PRC; 0.25% parenzyme was bought from Shanghai Xinhua Pharmaceutical Factory, PRC.Above-mentioned reagents were analytical pure grade.METHODS: After digestion, human limbal tissues in limbal basilar part with an abundant pigment were cultured in the culture flask containing RPMI-1640 and 200 g/L FCS and in culture dish containing amniotic extracellular matrix (AECM) as the cultural supporter. Primary and passage cells were observed under light microscope and scanning electron microscope (SEM). The revival ratio of stem cell refrigeration of every generation was calculated by the trypanblau exclusion experiment.MAIN OUTCOME MEASURES: ① Observational results of limbal stem cells during the primary culture and serial subcultivation in vitro, and ② revival ratio of stem cell refrigeration.RESULTS: ①Findings of primary culture: Most limbal stem cells in the culture flask had the adherence and were arrayed uniformly sparsely to form monolayer and adhered to the bottom of culture flask under the inverted phase contrast microscope after 1-day culture. ② Findings of serial subcultivation: After human epidermal growth factor (hEGF) was added into the second passage, cells were scattered into the monolayer and adhered to grow quickly.Morphological variability of all the cells increased obviously when passage the 30th generation. The cellular volume was obviously increasing, and the round or irregular round cells gathered together. The 33rd generation human limbal stem cells still could vigorously differentiate, proliferate and grow in ACEM. ③ The revival ratio of stem cell refrigeration was 82.2%.CONCLUSION: The human limbal stem cell lines were preliminarily established by culturing and freezing the cells of 33 generations in vitro. The human limbal stem cell lines preferred to grow in the culture dish containing AECM as the cultural supporter.