CAJ | 학술논문

背景原发性开角型青光眼(POAG)是一种常见致盲性眼病,其特点是房水外流阻力增加导致眼压增高.位于房水外流通道的小梁网调节房水的外流,因此研究小梁网细胞的生物学特性有着重要的意义.目的探讨POAG小梁细胞体外培养的方法及其生物学特性.方法经小梁切除术收集8例开角型青光眼患者患眼的带小梁网的深层巩膜组织块进行体外原代和传代培养,用鼠抗人层黏连蛋白(LM)单克隆抗体、兔抗人纤维连结蛋白(FN)单克隆抗体、鼠抗人神经元特异性烯醇化酶(NSE)单克隆抗体进行免疫组织化学检测以对传代细胞进行鉴定,在透射电子显微镜下对传代细胞的超微结构进行观察,并将传代小梁细胞的生物学特性与本研究组前期培养的正常小梁细胞进行比较.结果组织块培养10 d左右,可见细胞从其边缘向外生长.传代细胞在4 d内处于对数生长期,其后进入平台期,第7天细胞基本融合.第3代POAG小梁细胞及正常人眼小梁细胞中可见FN、LM和NSE均呈阳性表达,证实传代细胞为小梁细胞,而空白对照组细胞未见FN、LM和NSE表达.第3代POAG小梁细胞和正常小梁细胞中FN的A450值分别为0.35±0.06和0.26±0.01,LM的A450值分别为0.34±0.03和0.25±0.02,差异均有统计学意义(FN:t=14.446,P=0.001;LM:t=9.346,P=0.001).与正常小梁细胞比较,第3代POAG小梁细胞表面的微绒毛、细胞质的溶酶体及吞噬小泡含量减少.结论采用组织块培养法可成功在体外培养POAG小梁细胞,该研究结果为研究青光眼的发病机制提供了细胞学基础.
배경 원발성개각형청광안(POAG)시일충상견치맹성안병,기특점시방수외류조력증가도치안압증고.위우방수외류통도적소량망조절방수적외류,인차연구소량망세포적생물학특성유착중요적의의.목적 탐토POAG소량세포체외배양적방법급기생물학특성.방법 경소량절제술수집8례개각형청광안환자환안적대소량망적심층공막조직괴진행체외원대화전대배양,용서항인층점련단백(LM)단극륭항체、토항인섬유련결단백(FN)단극륭항체、서항인신경원특이성희순화매(NSE)단극륭항체진행면역조직화학검측이대전대세포진행감정,재투사전자현미경하대전대세포적초미결구진행관찰,병장전대소량세포적생물학특성여본연구조전기배양적정상소량세포진행비교.결과 조직괴배양10 d좌우,가견세포종기변연향외생장.전대세포재4 d내처우대수생장기,기후진입평태기,제7천세포기본융합.제3대POAG소량세포급정상인안소량세포중가견FN、LM화NSE균정양성표체,증실전대세포위소량세포,이공백대조조세포미견FN、LM화NSE표체.제3대POAG소량세포화정상소량세포중FN적A450치분별위0.35±0.06화0.26±0.01,LM적A450치분별위0.34±0.03화0.25±0.02,차이균유통계학의의(FN:t=14.446,P=0.001;LM:t=9.346,P=0.001).여정상소량세포비교,제3대POAG소량세포표면적미융모、세포질적용매체급탄서소포함량감소.결론 채용조직괴배양법가성공재체외배양POAG소량세포,해연구결과위연구청광안적발병궤제제공료세포학기출.
Background Primary open angle glaucoma (POAG) is a major blindness-causing disease,characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork lining the aqueous outflow pathway modulates the aqueous outflow facility. To study the biological characteristics of the trabecular meshwork cells has important significance. Objective This study was to culture the trabecular cells from primary open-angle glaucomatous eye (POAG) and study the biologic characteristics of passaged cells. Methods The deep scleral tissue with trabecular meshwork was obtained during the trabeculectomy from 8 eyes with POAG. The trabecular cells were primarily cultured and passaged in vitro. The generation 3 cells were identified by immunochemistry with the laminin (LM), fibronectin (FN) and neuron specific endolase (NSE)monoclonal antibodies. The ultrastructure was examined to observe the biological characteristics of the cells under the transmission electronic microscope. The experimental results were compared among POAG group, normal control group and blank control group. Results The primarily cultured POAG trabecular cells migrated from the edge of tissue mass about 10 days. The cells of generation 3 presented the logarithmic phase in the first 4 days and fused in the 7th day. FN,LM and NSE were positively expressed in the generated cells in POAG group and normal control group rather than blank control group. The MOD values of the generation 3 cells for FN in POAG group and normal control group were 0. 35 ± 0.06 and 0. 26 ± 0. 01, and those for LM were 0. 34 ± 0. 03 and 0. 25 ± 0. 02 respectively, showing statistically significant difference between these two groups ( FN: t = 14. 446, P＜0.001; LM: t = 9. 346, P＜0. 001 ). The microvilli, cytolysosome and phagocytic vesicle were obviously decreased in the trabcular cells of POAG group compared with normal control group under the transmission electron microscope. Conclusion The trabecular meshwork cells from POAG can be successfully cultured and passaged in vitro. It provides a cytology basis for further glaucoma research.