摘要目的 建立玻璃体切除液组织和细胞培养的新技术.方法 实验研究.采用随机数字表法,选取30例孔源性视网膜脱离(RRD)和48例增生性糖尿病视网膜病变(PDR)患者,经睫状体平坦部玻璃体切除术后获得的玻璃体切除液标本,行0.5%荧光素钠反相标记玻璃体凝胶,经105U/L透明质酸酶和106U/L胶原酶Ⅰ联合消化,取沉淀混悬液接种于预置的0.01%多聚赖氨酸培养瓶中,倒置法培养24 h后,置入含30%胎牛血清的F12培养液中正置培养6 d,期间半量换液1次,动态观察增生膜片边缘细胞生长情况.结果 在0.01%多聚赖氨酸预置条件下,玻璃体切除液中的膜组织块经倒置培养24 h后均可半干燥贴壁,78例玻璃体切除液标本按上法贴壁,成功率100%.7d培养期间均未出现细菌、真菌及支原体污染.经0.5%荧光素钠标记,透明质酸酶联合胶原酶Ⅰ作用30 min,78例标本中玻璃体凝胶均被消化,消化率100%.部分增生膜边缘可见细胞迁移而出,呈现不同程度的生长和增生趋势.30例RRD标本中,细胞生长率43.33%(13/30);48例PDR标本中,细胞生长率37.50%(18/40),其中24例PDR-V标本中,细胞生长率41.67%(10/24).结论 酶解联合倒置半干燥细胞培养法可确保增生膜贴壁,短期培养既可避免污染,又可观察增生膜组织内细胞生长特性;该方法可用于增生膜细胞增殖活性的判断,有望成为一种预测增生性眼底疾病术后复发风险的新技术.

abstractsObjective To setup a new technique of tissue and cell culture for vitreous aspirates.Methods Experiment study.Specimens used for supporting new culture technique were selected based on random digit table.Thirty cases with rhegmatogenous retinal detachment(RRD)and forty-eight with proliferative diabetic retinopathy(PDR),which undergoing primary pars plana vitrectomy,were selected randomly and included in the study.After being antiphase stained with fluorescein-natrium(0.5%)and digested with hyaluronidase(105U/L)combined with collagenase Ⅰ(106U/L)for removing vitreous gel,sediment of vitreous fluid after centrifugation were inoculated into standard culturing bottle with which polylysine(0.01%)was pre-set.The bottle which contained F12medium with 30% fetal bovine serum was placed upside down for 24 hours and consecutively upside for 6 days.During which,F12medium was replaced once in half volume,and cell growth along the edge of sedimentary membrane was observed at time of the 3rd and the 6th day after upside culture.Results Under condition of pre-setting by polylysine(0.01%)and being placed upside down for 24 hours,pieces from vitreous fluids could adhere to the bottom of bottle in a way of semi-xeransis with adherence rate of 100%(78/78).No bacteria,fungus and mycoplasma contamination was found within 7 days.Antiphase stained with fluorescein-natrium(0.5%)and digested with hyaluronidase(105U/L)combined with collagenase Ⅰ(106U/L)for 30 minutes,vitreous gel.in 78 specimens could be digested(78/78).Cell emigration could be found in edge area of some pieces of vitreous fluid and cell growth as well as proliferation was shown.In 30 specimens of RRD,cell growth rate were 43.33%(13/30).In 48 specimens of PDR,cell growth rate were 37.50%(18/48).Concerning PDR phase V(PDR-V),cell growth rate reach 41.67%(10/24).Conclusions Enzymolysis with upside down and semi-xeransis could ensure good adherence of membrane,moreover,no contamination and obvious cell growth could be found within short-term culture.These suggested that a new technique for judging viability of cell from proliferative membrane and predicting recurrent risk after surgery of proliferative ocular fundus disease could be expected.