摘要
【目的】从正常兔泪腺组织及泪腺上皮细胞中分离及培养干细胞并鉴定。【方法】取4周龄左右的新西兰大白兔(0.5～1.0 kg),麻醉后取双眼主泪腺组织,右眼泪腺用于石蜡切片并行免疫组织化学检查,左眼泪腺予酶消化法分离培养并进一步纯化泪腺上皮细胞。首先,免疫组化检测兔泪腺组织切片中的ATP-binding cassette sub-family G member 2(ABCG2),巢蛋白(nestin),Tumor protein 63(P63),α-平滑肌肌动蛋白(SMA),波形蛋白(vimentin)等干细胞的标志物表达阳性的部位。其次,泪腺上皮细胞先以Peter Complete Medium(PCM)培养基培养,待出现梭形细胞后,更换Dulbecco's Modified Eagle Medium(DMEM)培养基以常规分离培养干细胞。分离出的细胞取第三代用于免疫荧光检测ABCG2,nestin,P63,α-SMA,vimentin等干细胞的标志物,同时进行成脂,成骨诱导分化。【结果】兔泪腺组织免疫组织化学检查存在ABCG2,nestin,P63,α-SMA,vimentin阳性的细胞,成功分离该细胞后,该细胞可表达ABCG2,nestin,P63,α-SMA,vimentin干细胞的标志,并可成功诱导其分化为脂肪细胞及骨细胞。【结论】兔泪腺组织中存在具有干细胞特征的细胞并能诱导分化,该细胞有望成为泪腺自我修复及重建的种子细胞,成为干眼临床治疗的新手段。
[Objective] To isolate and identify the stem cells from rabbit lacrimal gland and lacrimal gland epithelial cells in vitro.[Methods] Lacrimal gland was dissected out from about 4 weeks old New Zealand white male rabbits which weighed 0.5-1.0 kg.The right lacrimal gland was fixed and embedded in paraffin for histology,and the left one was digested for isolating the epithelial cells of lacrimal gland.Firstly,ABCG2,P63,nestin,vimentin,and α-SMA were employed to find out if there are stem cells in lacrimal gland.Secondly,Stem cells were isolated after digestion from LG and cultured in Peter Complete Medium （PCM）.After 3 days,when the spindle cells appeared,stem cells were transferred to Dulbecco＇s Modified Eagle Medium （DMEM）,then cultured and passaged these cells about 3 days per passage,the passage 3 cells were examined for the expression of stem cells markers by immuno？ uorescence （IF）,however,their ability to osteogenic and adipogenic differentiation was also tested.[Results] Stem cell markers positive cells can be isolated and proliferated from rabbit lacrimal gland.In addition,these cells successfully induced into osteoblasts and adipocytes,attesting to their stem cells property.[Conclusion] Lacrimal glands contains stem cells and these cells can be successfully induced,which might play a pivotal role in lacrimal glands repair.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2014年第3期438-444,共7页
Journal of Sun Yat-Sen University:Medical Sciences
基金
眼科学国家重点实验室自主研究基金项目(3030902113006)
关键词
泪腺
干细胞
干眼
兔
分离与鉴定
lacrimal gland
stem cell
dry eye
rabbit
isolation and identification
