中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
9期
786-790
,共5页
王瑶%宫华青%杨玲玲%王茜%周庆军%王宜强
王瑤%宮華青%楊玲玲%王茜%週慶軍%王宜彊
왕요%궁화청%양령령%왕천%주경군%왕의강
羊膜%视网膜色素上皮细胞%增生%分化
羊膜%視網膜色素上皮細胞%增生%分化
양막%시망막색소상피세포%증생%분화
Amniotic membrane%Retinal pigment epithelium cell%Proliferation%Differentiation
背景 人视网膜色素上皮(RPE)细胞移植治疗视网膜变性性疾病是目前的研究热点之一,许多生物载体可制备RPE细胞单层,但多存在着细胞毒性、稳定性差及免疫反应等问题. 目的 检测羊膜对人RPE细胞增生和分化的影响,探讨其作为人RPE细胞层载体进行移植的可能性. 方法 将RPE细胞系ARPE-19细胞株用含质量分数10％胎牛血清的DMEM/F12培养基进行培养和传代,8～12代细胞用于实验.将传代细胞分为2个组,一组细胞接种于去上皮羊膜作为实验组,另一组细胞直接在培养孔内进行培养作为对照组.分别于接种后24、48、72、96 h行MTT实验,测定RPE细胞的吸光度(A492)值以评估两组细胞在增生能力方面的不同；培养3周后的细胞层行苏木精-伊红染色,检测细胞在不同培养载体上是否存在形态学方面的区别；分别收集同一孔中生长于羊膜和培养板表面的细胞,采用逆转录聚合酶链反应(RT-PCR)法检测色素上皮衍生因子(PEDF)、钙黏蛋白(N-cadherin)、β-连环蛋白(β-catenin)以及细胞连接相关蛋白的表达情况；同时收集培养3周时的细胞行透射电子显微镜和扫描电子显微镜检查,比较不同载体培养的ARPE-19细胞在超微结构方面的区别.结果 与对照组相比,实验组细胞增生的速度明显变缓,两组细胞增生情况比较差异有统计学意义(F=41.760,P=0.000).苏木精-伊红染色结果显示,同一培养孔内实验组的细胞密度明显低于对照组,说明羊膜对ARPE-19细胞增生的影响不是通过细胞分泌的可溶性因子的作用.RT-PCR结果显示,实验组细胞连接相关蛋白claudin 1、N-cadherin和PEDFmRNA在培养细胞上的表达水平均明显高于对照组,差异均有统计学意义(t=15.828,P=0.000;t=6.839,P=0.002;t=14.667,P=0.000)；Connexin 43的表达低于对照组,差异有统计学意义(t=3.358,P=0.024).超微结构分析发现羊膜载体上的细胞呈典型的多边形RPE细胞表型,分化现象明显,而普通培养板培养的RPE细胞形态主要呈梭形,细胞层厚薄不均.结论 去上皮羊膜为载体培养的RPE细胞增生速度变慢,但培养的细胞分化程度较高,提示羊膜可作为RPE细胞体外培养的良好载体,用于制备移植所需功能成熟的RPE细胞片层.
揹景 人視網膜色素上皮(RPE)細胞移植治療視網膜變性性疾病是目前的研究熱點之一,許多生物載體可製備RPE細胞單層,但多存在著細胞毒性、穩定性差及免疫反應等問題. 目的 檢測羊膜對人RPE細胞增生和分化的影響,探討其作為人RPE細胞層載體進行移植的可能性. 方法 將RPE細胞繫ARPE-19細胞株用含質量分數10％胎牛血清的DMEM/F12培養基進行培養和傳代,8～12代細胞用于實驗.將傳代細胞分為2箇組,一組細胞接種于去上皮羊膜作為實驗組,另一組細胞直接在培養孔內進行培養作為對照組.分彆于接種後24、48、72、96 h行MTT實驗,測定RPE細胞的吸光度(A492)值以評估兩組細胞在增生能力方麵的不同；培養3週後的細胞層行囌木精-伊紅染色,檢測細胞在不同培養載體上是否存在形態學方麵的區彆；分彆收集同一孔中生長于羊膜和培養闆錶麵的細胞,採用逆轉錄聚閤酶鏈反應(RT-PCR)法檢測色素上皮衍生因子(PEDF)、鈣黏蛋白(N-cadherin)、β-連環蛋白(β-catenin)以及細胞連接相關蛋白的錶達情況；同時收集培養3週時的細胞行透射電子顯微鏡和掃描電子顯微鏡檢查,比較不同載體培養的ARPE-19細胞在超微結構方麵的區彆.結果 與對照組相比,實驗組細胞增生的速度明顯變緩,兩組細胞增生情況比較差異有統計學意義(F=41.760,P=0.000).囌木精-伊紅染色結果顯示,同一培養孔內實驗組的細胞密度明顯低于對照組,說明羊膜對ARPE-19細胞增生的影響不是通過細胞分泌的可溶性因子的作用.RT-PCR結果顯示,實驗組細胞連接相關蛋白claudin 1、N-cadherin和PEDFmRNA在培養細胞上的錶達水平均明顯高于對照組,差異均有統計學意義(t=15.828,P=0.000;t=6.839,P=0.002;t=14.667,P=0.000)；Connexin 43的錶達低于對照組,差異有統計學意義(t=3.358,P=0.024).超微結構分析髮現羊膜載體上的細胞呈典型的多邊形RPE細胞錶型,分化現象明顯,而普通培養闆培養的RPE細胞形態主要呈梭形,細胞層厚薄不均.結論 去上皮羊膜為載體培養的RPE細胞增生速度變慢,但培養的細胞分化程度較高,提示羊膜可作為RPE細胞體外培養的良好載體,用于製備移植所需功能成熟的RPE細胞片層.
배경 인시망막색소상피(RPE)세포이식치료시망막변성성질병시목전적연구열점지일,허다생물재체가제비RPE세포단층,단다존재착세포독성、은정성차급면역반응등문제. 목적 검측양막대인RPE세포증생화분화적영향,탐토기작위인RPE세포층재체진행이식적가능성. 방법 장RPE세포계ARPE-19세포주용함질량분수10％태우혈청적DMEM/F12배양기진행배양화전대,8～12대세포용우실험.장전대세포분위2개조,일조세포접충우거상피양막작위실험조,령일조세포직접재배양공내진행배양작위대조조.분별우접충후24、48、72、96 h행MTT실험,측정RPE세포적흡광도(A492)치이평고량조세포재증생능력방면적불동；배양3주후적세포층행소목정-이홍염색,검측세포재불동배양재체상시부존재형태학방면적구별；분별수집동일공중생장우양막화배양판표면적세포,채용역전록취합매련반응(RT-PCR)법검측색소상피연생인자(PEDF)、개점단백(N-cadherin)、β-련배단백(β-catenin)이급세포련접상관단백적표체정황；동시수집배양3주시적세포행투사전자현미경화소묘전자현미경검사,비교불동재체배양적ARPE-19세포재초미결구방면적구별.결과 여대조조상비,실험조세포증생적속도명현변완,량조세포증생정황비교차이유통계학의의(F=41.760,P=0.000).소목정-이홍염색결과현시,동일배양공내실험조적세포밀도명현저우대조조,설명양막대ARPE-19세포증생적영향불시통과세포분비적가용성인자적작용.RT-PCR결과현시,실험조세포련접상관단백claudin 1、N-cadherin화PEDFmRNA재배양세포상적표체수평균명현고우대조조,차이균유통계학의의(t=15.828,P=0.000;t=6.839,P=0.002;t=14.667,P=0.000)；Connexin 43적표체저우대조조,차이유통계학의의(t=3.358,P=0.024).초미결구분석발현양막재체상적세포정전형적다변형RPE세포표형,분화현상명현,이보통배양판배양적RPE세포형태주요정사형,세포층후박불균.결론 거상피양막위재체배양적RPE세포증생속도변만,단배양적세포분화정도교고,제시양막가작위RPE세포체외배양적량호재체,용우제비이식소수공능성숙적RPE세포편층.
Background Human retinal pigment epithelial (RPE) cell transplantation treating retinal degenerative diseases is a researching topic,and the source of human RPE cells is a key problem.Many biological carriers can be used for the preparation of RPE cell layer.However,some advantages,such as cytotoxicity,lack of stability and immunologic reaction etc.are still existed.To study an ideal biological carrier is very important.Objective This experimental was to determine the effects of amniotic membrane on the proliferation and differentiation of human RPE cells and the possibility as a scaffold for RPE cell transplantation.Methods ARPE19 cell line cells were cultured and passaged in DMEM/F12 medium with 10％ fetal bovine serum,and 8-12generation of cells were used.The cells were divided into two groups.One group of cells were incubated on the denuded amniotic membrane,and the other group of cells were cultured in the medium (control group).MTT was performed to detect the A492 value of RPE cells for the evaluation of cell proliferation ability 24,48,72,96 hours after culture.Cell morphology was compared by histopathological examination 3 weeks after culture.The mRNA expression of pigment epithelium-derived factor (PEDF),N-cadherin,β-catenin and cell connection related proteins in the cells of both groups were assayed using reverse transcription polymerase chain reaction (RT-PCR).Ultrastructure of the cells was observed under the transmission and scan electronic microscope 3 weeks after culture.Results The number of ARPE-19 cells cultured on denuded amniotic membrane was decreased significantly in comparison with the normal culture plate(F=41.760,P =0.000).Histopatholy also showed that the cell density on amniotic membrane was lower than of normal cells on plate surface.Moreover,the expression level of claudin 1 mRNA,N-cadherin mRNA and PEDF mRNA were significantly up-regulated in denuded amniotic membrane group in comparison with control group (t=15.828,P=0.000 ;t=6.839,P=0.002 ;t=14.667,P=0.000),but the expression of Connexin 43 mRNA was down-regulated in denuded amniotic membrane group compared with control group(t=3.358,P=0.024).Ultrastructural examination revealed that ARPE-19 cells cultured on amniotic membrane exhibited a polygonal epithelial phenotype with cilium on the apical side,however,the cells cultured on normal culture plate displayed fusiform shape and uneven thickness.Conclusions Amniotic membrane plays a promoting effect on the differentiation of ARPE-19 cells and a inhibitory effect on the proliferation of ARPE-19 cells,suggesting that amniotic membrane might be an useful scaffold for the preparation of functionally mature RPE cells for clinical transplantation.
