中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
6期
511-516
,共6页
鲍慧婧%邹俊%尹烁%崔磊
鮑慧婧%鄒俊%尹爍%崔磊
포혜청%추준%윤삭%최뢰
脂肪干细胞%生物相容性%组织工程%聚羟基乙醇
脂肪榦細胞%生物相容性%組織工程%聚羥基乙醇
지방간세포%생물상용성%조직공정%취간기을순
Adipose derived stem cells%Biocompatibility%Tissue engineering%Polylactic-co-glycolic acid
背景 种子细胞和支架材料是角膜组织工程研究的主要课题.脂肪干细胞具有来源广泛、增生和分化能力强的特点而成为目前组织工程种子细胞的研究热点,聚羟基乙醇(PLGA)作为高分子可降解支架材料已成功用于构建多种组织器官.目的 探讨兔脂肪干细胞的生物学特性及其复合多孔支架材料聚羟基乙醇(PLGA)的生物相容性,为进一步构建脂肪干细胞组织工程化角膜基质提供实验基础.方法 取雌性新西兰大白兔颈背部脂肪组织,采用消化法分离培养兔脂肪细胞,传至第4代.将传代细胞分别以3×104/cm2、3×104/cm2、3×106/cm2的密度接种于6孔板中,分别用成骨诱导培养液、成脂诱导培养液及成软骨诱导液进行诱导培养,并分别以质量分数1%茜素红-Tris-盐酸溶液、质量分数0.6%油红染液和免疫荧光法鉴定培养细胞的多向分化能力.将第4代细胞用稀释的DiO荧光染液重悬的脂肪干细胞按1×107/ml的密度接种于自制的多孔PLGA支架形成细胞-生物材料复合物,Hoechst法定量检测细胞在支架上的生长情况,并分别于接种后第1、3、7天对细胞-生物材料复合物行共焦显微镜和扫描电子显微镜检测,观察细胞在该支架上的黏附生长和基质分泌情况,评价PLGA的生物相容性.结果 原代培养的脂肪细胞7～8d后可达80% ～90%融合,呈成纤维细胞样外观.传代第4代的细胞成骨诱导2周后茜素红染色显示矿化结节及周围细胞着深红色;成脂诱导2周后油红O染色显示细胞质内布满红色脂滴颗粒;微团培养成软骨诱导2周后,免疫荧光染色结果显示Ⅱ型胶原表达阳性.细胞接种至PLGA支架材料第7天增生达到高峰期,扫描电子显微镜和共焦显微镜检测显示细胞在支架上贴附生长良好,能够在支架表面及孔隙内壁得到充分伸展和生长,细胞外基质分泌旺盛.结论 培养的兔脂肪细胞具有脂肪干细胞的多向分化功能,与多孔PLGA支架复合具有良好的生物相容性,可作为构建组织工程化角膜基质的种子细胞和支架材料.
揹景 種子細胞和支架材料是角膜組織工程研究的主要課題.脂肪榦細胞具有來源廣汎、增生和分化能力彊的特點而成為目前組織工程種子細胞的研究熱點,聚羥基乙醇(PLGA)作為高分子可降解支架材料已成功用于構建多種組織器官.目的 探討兔脂肪榦細胞的生物學特性及其複閤多孔支架材料聚羥基乙醇(PLGA)的生物相容性,為進一步構建脂肪榦細胞組織工程化角膜基質提供實驗基礎.方法 取雌性新西蘭大白兔頸揹部脂肪組織,採用消化法分離培養兔脂肪細胞,傳至第4代.將傳代細胞分彆以3×104/cm2、3×104/cm2、3×106/cm2的密度接種于6孔闆中,分彆用成骨誘導培養液、成脂誘導培養液及成軟骨誘導液進行誘導培養,併分彆以質量分數1%茜素紅-Tris-鹽痠溶液、質量分數0.6%油紅染液和免疫熒光法鑒定培養細胞的多嚮分化能力.將第4代細胞用稀釋的DiO熒光染液重懸的脂肪榦細胞按1×107/ml的密度接種于自製的多孔PLGA支架形成細胞-生物材料複閤物,Hoechst法定量檢測細胞在支架上的生長情況,併分彆于接種後第1、3、7天對細胞-生物材料複閤物行共焦顯微鏡和掃描電子顯微鏡檢測,觀察細胞在該支架上的黏附生長和基質分泌情況,評價PLGA的生物相容性.結果 原代培養的脂肪細胞7～8d後可達80% ～90%融閤,呈成纖維細胞樣外觀.傳代第4代的細胞成骨誘導2週後茜素紅染色顯示礦化結節及週圍細胞著深紅色;成脂誘導2週後油紅O染色顯示細胞質內佈滿紅色脂滴顆粒;微糰培養成軟骨誘導2週後,免疫熒光染色結果顯示Ⅱ型膠原錶達暘性.細胞接種至PLGA支架材料第7天增生達到高峰期,掃描電子顯微鏡和共焦顯微鏡檢測顯示細胞在支架上貼附生長良好,能夠在支架錶麵及孔隙內壁得到充分伸展和生長,細胞外基質分泌旺盛.結論 培養的兔脂肪細胞具有脂肪榦細胞的多嚮分化功能,與多孔PLGA支架複閤具有良好的生物相容性,可作為構建組織工程化角膜基質的種子細胞和支架材料.
배경 충자세포화지가재료시각막조직공정연구적주요과제.지방간세포구유래원엄범、증생화분화능력강적특점이성위목전조직공정충자세포적연구열점,취간기을순(PLGA)작위고분자가강해지가재료이성공용우구건다충조직기관.목적 탐토토지방간세포적생물학특성급기복합다공지가재료취간기을순(PLGA)적생물상용성,위진일보구건지방간세포조직공정화각막기질제공실험기출.방법 취자성신서란대백토경배부지방조직,채용소화법분리배양토지방세포,전지제4대.장전대세포분별이3×104/cm2、3×104/cm2、3×106/cm2적밀도접충우6공판중,분별용성골유도배양액、성지유도배양액급성연골유도액진행유도배양,병분별이질량분수1%천소홍-Tris-염산용액、질량분수0.6%유홍염액화면역형광법감정배양세포적다향분화능력.장제4대세포용희석적DiO형광염액중현적지방간세포안1×107/ml적밀도접충우자제적다공PLGA지가형성세포-생물재료복합물,Hoechst법정량검측세포재지가상적생장정황,병분별우접충후제1、3、7천대세포-생물재료복합물행공초현미경화소묘전자현미경검측,관찰세포재해지가상적점부생장화기질분비정황,평개PLGA적생물상용성.결과 원대배양적지방세포7～8d후가체80% ～90%융합,정성섬유세포양외관.전대제4대적세포성골유도2주후천소홍염색현시광화결절급주위세포착심홍색;성지유도2주후유홍O염색현시세포질내포만홍색지적과립;미단배양성연골유도2주후,면역형광염색결과현시Ⅱ형효원표체양성.세포접충지PLGA지가재료제7천증생체도고봉기,소묘전자현미경화공초현미경검측현시세포재지가상첩부생장량호,능구재지가표면급공극내벽득도충분신전화생장,세포외기질분비왕성.결론 배양적토지방세포구유지방간세포적다향분화공능,여다공PLGA지가복합구유량호적생물상용성,가작위구건조직공정화각막기질적충자세포화지가재료.
Background Seed cells and scaffold material are the important aspects of corneal tissue engineering research.Adipose-derived stem cells(ASCs) are becoming the focus of seed cells research because of their wide source,powerful proliferation and differentiation abilities.As biodegradable polymer,polylactic-co-glycolic acid(PLGA) has successfully build multiple tissues and organs.Objective Present study was to ascertain the biological characteristics of the rabbit ASCs and their biocompatibility with PLGA scaffold in vitro and to provide groundwork for further study on the reconstruction of tissue engineered corneal stroma.Methods Adipose cells were isolated from lipoaspirate of New Zealand white rabbit using collagenase Ι.The cells were cultured and passaged.The generation 4 cells were inoculated to culture plate with 6 holes at the density of 3×104/cm2,3×104/cm2,3×106/cm2 respectively and cultivated in ossification inducing medium,lipoblast inducing medium and chondroblast inducing medium to identify the characteristics of the cells.The multilineage differentiated cells were identified by alizarin red staining,oil red O staining and immunoinfluorescene technique.The generation 4 cells were re-suspended with DiO influorescence fluid at the density of 1×107/ml and seeded on PLGA scaffold to fabricate cell-PLGA constructs.Quantitative analysis of cell proliferation on PLGA was detected by Hoechst DNA assay.The attachment and growth of adipose-derived stem cells on the scaffold were observed under the scanning electron microscope(SEM) and confocal microscopy in 1 day,3,7 days after seeding for the evaluation of biocompatibility between cells and PLGA.Results Primarily cultured cells reached 80%-90% confluence after 7-8 days with the fibroblast-like appearance.Adipose-derived stem cells of rabbits differentiated into osteoblast,adipocyte and chondroblast successfully,showing the positive stain for alizarin red staining,oil red O staining and immunoinfluorescene technique respectively.Proliferation of cells on PLGA scaffold went into plateau phase at 7 days after culture.SEM and confocal microscopy revealed the well-attached,spread cells along the scaffold and abundant excellular matrix both on the surface and interior pore of scaffold.Conclusion Cultured rabbit adipose cells have the ability of potential multilineage differentiation and good biocompatibility with PLGA scaffold,which could be used to construction of tissue engineered corneal stroma.
