摘要
目的观察冷冻干燥保存方法对羊膜组织结构及生物活性因子的影响。方法取健康剖宫产产妇胎盘剥离羊膜,-40℃冻干保存,分别进行大体、光镜、扫描电镜、透射电镜观察和免疫组化分析,并与新鲜羊膜对比。结果新鲜及冻干保存的羊膜均可见5层基本组织结构,由内向外依次为上皮细胞层、基底膜、致密层、纤维母细胞和海绵层。新鲜羊膜光镜下见上皮细胞排列紧密,深面是厚而连续的基底膜;扫描电镜下,表面有突出的微绒毛,细胞间连接结构清晰;透射电镜下见上皮细胞核规则、胞质内部分线粒体空泡变性,细胞间见多桥粒连接,基底膜完整,基质中胶原微纤维结构清晰,见明暗相间的横纹。光镜下,冻干羊膜上皮细胞形态基本完好,细胞间隙加大;基底膜及致密层保持完整;纤维母细胞层、海绵层略微疏松。扫描电镜下,冻干羊膜表面略显干缩,无明显突出的微绒毛,上皮细胞完好,形态结构未发生改变。透射电镜观察,冻干羊膜表面可见微绒毛,上皮细胞完整,胞核呈灶性空化,胞浆内可见空泡,线粒体肿胀、嵴不可见;细胞间可见桥粒连接;基底膜基本正常,基质胶原纤维结构清晰。免疫组化分析,b-FGF、HGF主要分布于羊膜上皮层,ColⅣ、LN主要分布于羊膜基底膜;基质层也有b-FGF、ColⅣ、LN分布,纤维母细胞层也有HGF分布。冻干羊膜中HGF含量显著低于新鲜羊膜(P<0.05),b-FGF、ColⅣ、LN3种成分含量在新鲜和冻干羊膜中差异无显著性意义(P>0.05)。结论冷冻干燥对羊膜组织形态与生物活性因子无明显影响。
Objective To investigate the effects by freeze drying on the morphological structure and biological vitality of human amniotic membrane（AM）.Methods Fresh AM was preserved by freeze drying at-40 ℃.The structure of AM was observed by Light microscope,Scan electron microscope,Transmission electron microscope.The histological properties were analyzed by Immuno-histochemical method with image analysis system used to investigate the changes in the expression of hepatocyte growth factor（HGF）,basic fibroblast growth factor（b-FGF）,collagen type Ⅳ（Col Ⅳ） and laminin（LN）.All properties were compared with fresh AM.Results Light microscope：Five structure-layers was demonstrated in AM since fresh and preserved in freeze drying at-40 ℃.They were observed that epithelial cell,base membrane,tight layer,fibroblasts,and strata spongiosum from inner to out.The epithelial cells were arranged on the base membrane tightly in fresh AM,but loose in drying AM.Base membrane and compact layer were well-structured,but strata-fibroblast and spongy layer loose slightly.Scan electron microscope study：The links between epithelial cells in AM were kept well,the micro-hairs of the cell observed.The epithelial cells in drying AM were shrinkage,structurally-normal,but micro-hairs of the cell not observed.Transmission electron microscope study：The epithelium in AM was observed as intact structure.It is revealed which nucleus-structure kept well,vacuole-degeneration in some mitochondria and an abundant of bridge-corpuscle between cells.The structure of base membrane was complete,and displayed alternately dark and bright rings in collagenous fibers.The epithelial cells were kept complete,showed micro-hairs and spot-desmosome between cells,nucleus dissolved partly,vacuolus in cytoplast,mitochondria showed vacuolus in drying AM,while basement membrane still normal,rich of collagen fibrils in base material,being alternately dark and bright band.Analyzed by Immuno-histochemical method：b-FGF and HGF were distributed in epithelium of AM and drying AM mostly.b-FGF was distributed in stromal layer,and HGF in fibroblasts layer also.ColⅣand LN were distributed in AM basement membrane mostly,in stromal layer little.The express of HGF in the epithelial cells of AM at freeze drying storage decreased significantly compared with fresh AM（P〈0.05）.The express of b-FGF,ColⅣ and LN in drying AM was not decreased significantly（P〉0.05）.the same as that in the fresh control group（P〉0.05）.Conclusions The structure and biological vitality factors of freeze drying AM were not destroyed significantly.
出处
《南昌大学学报（医学版）》
CAS
2010年第4期43-46,F0003,共5页
Journal of Nanchang University：Medical Sciences
关键词
羊膜
冷冻干燥
组织形态
生物活性因子
amniotic membrane
freeze drying
morphology
vitality factor
