中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
Chinese Journal of Experimental Ophthalmology
2015年
9期
781-786
,共6页
组织工程角膜%细胞培养%脂肪源干细胞%口腔黏膜上皮细胞%生物标志物/代谢%细胞层片技术%眼表重建%兔
組織工程角膜%細胞培養%脂肪源榦細胞%口腔黏膜上皮細胞%生物標誌物/代謝%細胞層片技術%眼錶重建%兔
조직공정각막%세포배양%지방원간세포%구강점막상피세포%생물표지물/대사%세포층편기술%안표중건%토
Tissue engineering,corneal%Cells,cultured%Adipose tissue-derived stem cells/cytology%Mouth mucosa/cytology%Biological markers/metabolism%Cell sheet technology%Ocular surface reconstruction%Rabbits
背景 组织工程角膜学的发展为角膜盲的治疗提供了新的选择,但目前暂未发现培养方法简单、可行性好的角膜上皮种子细胞和理想的支架材料.研究表明,脂肪源性干细胞(ADSCs)具有自我更新能力同时也具有类上皮的特质,而温度敏感性材料(TRSs)作为支架进行干细胞培养也为细胞层片技术的进步提供了技术支撑. 目的 研究兔ADSCs在TRSs上的培养特性,并与经典的口腔黏膜上皮细胞(OMECs)特性进行比较,探讨ADSCs作为眼表重建种子细胞的可行性.方法 异丙基丙烯酰胺溶于二丙醇后均匀涂于直径35 mm的聚苯乙烯培养皿表面,利用电子束照射制备TRSs,兔颈背部皮下脂肪组织2～3g经消化培养后获得ADSCs,同时取出兔口腔黏膜组织进行消化培养,获得OMECs.将2种干细胞均接种于TRSs上继续培养,比较2种细胞形态、生长速度、脱附时间和成活细胞的总计数,将ADSCs层片和OMECs层片行组织病理学检查,观察2种细胞的形态特征;采用免疫组织化学法检测2种细胞中干细胞标志物和上皮细胞标志物的表达;应用扫描电子显微镜检查2种细胞层片的表面超微结构.结果 自制TRSs透明性和光滑度与普通培养皿接近,任意观察的5个样品中有4个水接触角＞10°,成功率为80％.TRSs上培养的ADSCs呈长梭形,OMECs呈不规则圆形.ADSCs生长周期在TRSs上为12～ 14 d,脱附时间为(46.0±9.6) min,细胞总计数为(7.9±1.1)×105/片,而TRSs培养的OMECs生长周期为14～16 d,脱附时间为(91.9±10.9) min,细胞计数为(45.8±26.5) ×105/片,2种细胞间层片脱附时间和细胞计数的差异均有统计学意义(P=0.002、0.028).组织病理学检查显示,TRSs上的ADSCs呈1～3层排列,而OMECs呈4～5层覆层结构.免疫组织化学染色显示,ADSCs和OMECs细胞层片细胞角蛋白12(CK12)及干细胞标志物p63、ATP结合转运蛋白G超家族成员2(ABCG2)均呈阳性表达.扫描电子显微镜下观察可见ADSCs和OMECs表面均有致密的上皮微绒毛结构,细胞间连接紧密.结论 自制的TRSs可作为脂肪干细胞的培养支架,ADSCs取材广泛,TRSs上培养的ADSCs层片细胞活力好,可操作性强,可作为眼表重建新的种子来源.
揹景 組織工程角膜學的髮展為角膜盲的治療提供瞭新的選擇,但目前暫未髮現培養方法簡單、可行性好的角膜上皮種子細胞和理想的支架材料.研究錶明,脂肪源性榦細胞(ADSCs)具有自我更新能力同時也具有類上皮的特質,而溫度敏感性材料(TRSs)作為支架進行榦細胞培養也為細胞層片技術的進步提供瞭技術支撐. 目的 研究兔ADSCs在TRSs上的培養特性,併與經典的口腔黏膜上皮細胞(OMECs)特性進行比較,探討ADSCs作為眼錶重建種子細胞的可行性.方法 異丙基丙烯酰胺溶于二丙醇後均勻塗于直徑35 mm的聚苯乙烯培養皿錶麵,利用電子束照射製備TRSs,兔頸揹部皮下脂肪組織2～3g經消化培養後穫得ADSCs,同時取齣兔口腔黏膜組織進行消化培養,穫得OMECs.將2種榦細胞均接種于TRSs上繼續培養,比較2種細胞形態、生長速度、脫附時間和成活細胞的總計數,將ADSCs層片和OMECs層片行組織病理學檢查,觀察2種細胞的形態特徵;採用免疫組織化學法檢測2種細胞中榦細胞標誌物和上皮細胞標誌物的錶達;應用掃描電子顯微鏡檢查2種細胞層片的錶麵超微結構.結果 自製TRSs透明性和光滑度與普通培養皿接近,任意觀察的5箇樣品中有4箇水接觸角＞10°,成功率為80％.TRSs上培養的ADSCs呈長梭形,OMECs呈不規則圓形.ADSCs生長週期在TRSs上為12～ 14 d,脫附時間為(46.0±9.6) min,細胞總計數為(7.9±1.1)×105/片,而TRSs培養的OMECs生長週期為14～16 d,脫附時間為(91.9±10.9) min,細胞計數為(45.8±26.5) ×105/片,2種細胞間層片脫附時間和細胞計數的差異均有統計學意義(P=0.002、0.028).組織病理學檢查顯示,TRSs上的ADSCs呈1～3層排列,而OMECs呈4～5層覆層結構.免疫組織化學染色顯示,ADSCs和OMECs細胞層片細胞角蛋白12(CK12)及榦細胞標誌物p63、ATP結閤轉運蛋白G超傢族成員2(ABCG2)均呈暘性錶達.掃描電子顯微鏡下觀察可見ADSCs和OMECs錶麵均有緻密的上皮微絨毛結構,細胞間連接緊密.結論 自製的TRSs可作為脂肪榦細胞的培養支架,ADSCs取材廣汎,TRSs上培養的ADSCs層片細胞活力好,可操作性彊,可作為眼錶重建新的種子來源.
배경 조직공정각막학적발전위각막맹적치료제공료신적선택,단목전잠미발현배양방법간단、가행성호적각막상피충자세포화이상적지가재료.연구표명,지방원성간세포(ADSCs)구유자아경신능력동시야구유류상피적특질,이온도민감성재료(TRSs)작위지가진행간세포배양야위세포층편기술적진보제공료기술지탱. 목적 연구토ADSCs재TRSs상적배양특성,병여경전적구강점막상피세포(OMECs)특성진행비교,탐토ADSCs작위안표중건충자세포적가행성.방법 이병기병희선알용우이병순후균균도우직경35 mm적취분을희배양명표면,이용전자속조사제비TRSs,토경배부피하지방조직2～3g경소화배양후획득ADSCs,동시취출토구강점막조직진행소화배양,획득OMECs.장2충간세포균접충우TRSs상계속배양,비교2충세포형태、생장속도、탈부시간화성활세포적총계수,장ADSCs층편화OMECs층편행조직병이학검사,관찰2충세포적형태특정;채용면역조직화학법검측2충세포중간세포표지물화상피세포표지물적표체;응용소묘전자현미경검사2충세포층편적표면초미결구.결과 자제TRSs투명성화광활도여보통배양명접근,임의관찰적5개양품중유4개수접촉각＞10°,성공솔위80％.TRSs상배양적ADSCs정장사형,OMECs정불규칙원형.ADSCs생장주기재TRSs상위12～ 14 d,탈부시간위(46.0±9.6) min,세포총계수위(7.9±1.1)×105/편,이TRSs배양적OMECs생장주기위14～16 d,탈부시간위(91.9±10.9) min,세포계수위(45.8±26.5) ×105/편,2충세포간층편탈부시간화세포계수적차이균유통계학의의(P=0.002、0.028).조직병이학검사현시,TRSs상적ADSCs정1～3층배렬,이OMECs정4～5층복층결구.면역조직화학염색현시,ADSCs화OMECs세포층편세포각단백12(CK12)급간세포표지물p63、ATP결합전운단백G초가족성원2(ABCG2)균정양성표체.소묘전자현미경하관찰가견ADSCs화OMECs표면균유치밀적상피미융모결구,세포간련접긴밀.결론 자제적TRSs가작위지방간세포적배양지가,ADSCs취재엄범,TRSs상배양적ADSCs층편세포활력호,가조작성강,가작위안표중건신적충자래원.
Background Development of corneal tissue engineering creates a new therapeutic method for severe corneal diseases.However,ideal seed cells and scaffold for corneal surface reconstruction have not yet been investigated well.Adipose-derived stem cells (ADSCs) are varified to have a self-renewal ability and epithelioid features,and temperature-responsive scaffolds (TRSs) can offer technical support for stem cell sheet.Objective This study was to investigate the characteristics of ADSCs cultured on TRSs and compare these features to typical oral mucosal epithelial cells (OMECs),and therefore to explore the feasibility of reconstruction of ocular surface with ADSCs as seed cells.Methods Self-made TRSs were prepared by adding isopropyl alcohol dissolved poly-Nisopropylacrylamide (PNIPAAm) to each polystyrene tissue culture dish and then irradiating using an election beam.Subcutaneious fatty tissue of rabbit neck was obtained to culture ADSCs,and 4 pieces of oral cavity mucosal tissue were digested and cultured to obtain OMECs.Then the ADSCs and OMECs were incubated on TRSs,and cell morphology,growth rate,detached duration and survival counts were compared between ADSCs and OMECs.The ADSCs sheet and OMECs sheet were stained with hematoxylin and eosin for morphological examination.Immunochemistry was used to observe the expressions of stem-cell biomakers and epithelioid-cell biomakers in the cells.The ultrastructure of cell surface was observed under the scanning electron microscope.Results Self-made TRSs were similar to ordinary culture dish in the transparancy and smoothness.The water contact angle of 4 in 5 samples were ＞10° with the effective rate upto 80％.A DSCs showed the elongated fusiform in shape,while OMECs showed a cobblestone appearance.The growth cycle,detached duration and cell number of ADSCs were 12-14 days,(46.0 ±9.6) minutes and (7.9 ±1.1)×105/sheet,and those of OMECs were 14-16 days,(91.9 ±10.9) minutes and (45.8 ±26.5)×105/sheet,respectively,showing statistically significant differences in the detached duration and cell counts between ADSCs and OMECs (P=0.002,0.028).Hematoxylin and eosin staining showed that ADSCs sheet comprised only 1-3 layer cells,while OMECs showed 4-5 layer cells.ATP-binding cassette superfamily G member 2 (ABCG2),p63 and cytokeratin 12 (CK12) were positively expressed in both ADSCs sheet and OMECs sheet.Closely packed cells and typical eithelial microvilli in the cell surface were exhibited in both ADSCs sheet and OMECs sheet under the scanning electron microscope.Conclusions Self-made TRSs can be used as scaffold of ADSCs.The ADSCs sheet on the TRSs appears to have a good cell vitality and therefore is a new seed source of ocular surface reconstruction.
