中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
2期
127-131
,共5页
李善义%戴应%谭美华%丁勇%钟敬祥%陈建苏
李善義%戴應%譚美華%丁勇%鐘敬祥%陳建囌
리선의%대응%담미화%정용%종경상%진건소
房水%角膜内皮细胞%增生%细胞培养
房水%角膜內皮細胞%增生%細胞培養
방수%각막내피세포%증생%세포배양
Aqueous humor%Corneal endothelial cell%Proliferation%Cell culture
背景 组织工程角膜内皮层的构建需要有活性的角膜内皮细胞(CECs),因此如何在体外培养出大量有生理功能的角膜内皮种子细胞是迫切需要解决的问题. 目的 观察房水对体外培养的牛CECs生长的影响. 方法 从新鲜牛眼球前房抽取1.2 ml房水,经消毒过滤后取上清备用.从牛眼角膜组织分离牛CECs并用含质量分数10％胎牛血清的低糖DMEM培养液进行培养传代.分别在培养液中加入不同体积的房水,使房水终体积分数分别为2.5％、5.0％、10.0％、15.0％、20.0％,对照组不加房水,利用细胞计数试剂盒-8(CCK-8)比色法检测各组细胞在450 nm处的吸光度(A450)值.流式细胞仪分析10.0％房水培养对CECs细胞周期的影响.待培养细胞融合成单层后,1 ml塑料枪头尖端在培养皿底划痕并用含10％胎牛血清的DMEM培养液中加入10.0％房水培养24 h,未加入房水的培养液作为对照组,检测细胞单层的愈合情况.将细胞以6×105个/ml密度接种到培养皿中,分别用含10.0％房水的培养液和无房水的培养液孵育5d,利用DAPI荧光染色技术分析细胞密度. 结果 培养的CECs呈六角形、铺路石样的扁平状.CCK-8比色法检测表明,各培养组细胞的A450值总体比较差异有统计学意义(F=4.051,P=0.007)；与对照组比较,各房水培养组细胞的A450值均明显高于对照组,以含10.0％房水培养组CECs的A450值最高,差异有统计学意义(P＜0.01).流式细胞术分析表明,10.0％房水孵育24 h后,处于S-G2期的细胞比例为(34.80±3.13)％,对照组为(23.06±1.13)％,差异有统计学意义(t=-5.729,P=0.005).划痕愈合分析检测表明,10.0％房水组的细胞单层划痕面积为(0.116±0.019) mm2,对照组为(0.358±0.049) mm2,差异有统计学意义(t=13.842,P=0.000).DAPI荧光染色结果显示,10.0％房水组的平均细胞密度为(1439±110)个/视野,大于对照组的(1162±45)个/视野,差异有统计学意义(t=-11.020,P=0.000).结论 房水作用于培养的牛CECs后可促进CECs的生长,10.0％房水对培养的CECs有促进角膜细胞增生的作用.
揹景 組織工程角膜內皮層的構建需要有活性的角膜內皮細胞(CECs),因此如何在體外培養齣大量有生理功能的角膜內皮種子細胞是迫切需要解決的問題. 目的 觀察房水對體外培養的牛CECs生長的影響. 方法 從新鮮牛眼毬前房抽取1.2 ml房水,經消毒過濾後取上清備用.從牛眼角膜組織分離牛CECs併用含質量分數10％胎牛血清的低糖DMEM培養液進行培養傳代.分彆在培養液中加入不同體積的房水,使房水終體積分數分彆為2.5％、5.0％、10.0％、15.0％、20.0％,對照組不加房水,利用細胞計數試劑盒-8(CCK-8)比色法檢測各組細胞在450 nm處的吸光度(A450)值.流式細胞儀分析10.0％房水培養對CECs細胞週期的影響.待培養細胞融閤成單層後,1 ml塑料鎗頭尖耑在培養皿底劃痕併用含10％胎牛血清的DMEM培養液中加入10.0％房水培養24 h,未加入房水的培養液作為對照組,檢測細胞單層的愈閤情況.將細胞以6×105箇/ml密度接種到培養皿中,分彆用含10.0％房水的培養液和無房水的培養液孵育5d,利用DAPI熒光染色技術分析細胞密度. 結果 培養的CECs呈六角形、鋪路石樣的扁平狀.CCK-8比色法檢測錶明,各培養組細胞的A450值總體比較差異有統計學意義(F=4.051,P=0.007)；與對照組比較,各房水培養組細胞的A450值均明顯高于對照組,以含10.0％房水培養組CECs的A450值最高,差異有統計學意義(P＜0.01).流式細胞術分析錶明,10.0％房水孵育24 h後,處于S-G2期的細胞比例為(34.80±3.13)％,對照組為(23.06±1.13)％,差異有統計學意義(t=-5.729,P=0.005).劃痕愈閤分析檢測錶明,10.0％房水組的細胞單層劃痕麵積為(0.116±0.019) mm2,對照組為(0.358±0.049) mm2,差異有統計學意義(t=13.842,P=0.000).DAPI熒光染色結果顯示,10.0％房水組的平均細胞密度為(1439±110)箇/視野,大于對照組的(1162±45)箇/視野,差異有統計學意義(t=-11.020,P=0.000).結論 房水作用于培養的牛CECs後可促進CECs的生長,10.0％房水對培養的CECs有促進角膜細胞增生的作用.
배경 조직공정각막내피층적구건수요유활성적각막내피세포(CECs),인차여하재체외배양출대량유생리공능적각막내피충자세포시박절수요해결적문제. 목적 관찰방수대체외배양적우CECs생장적영향. 방법 종신선우안구전방추취1.2 ml방수,경소독과려후취상청비용.종우안각막조직분리우CECs병용함질량분수10％태우혈청적저당DMEM배양액진행배양전대.분별재배양액중가입불동체적적방수,사방수종체적분수분별위2.5％、5.0％、10.0％、15.0％、20.0％,대조조불가방수,이용세포계수시제합-8(CCK-8)비색법검측각조세포재450 nm처적흡광도(A450)치.류식세포의분석10.0％방수배양대CECs세포주기적영향.대배양세포융합성단층후,1 ml소료창두첨단재배양명저화흔병용함10％태우혈청적DMEM배양액중가입10.0％방수배양24 h,미가입방수적배양액작위대조조,검측세포단층적유합정황.장세포이6×105개/ml밀도접충도배양명중,분별용함10.0％방수적배양액화무방수적배양액부육5d,이용DAPI형광염색기술분석세포밀도. 결과 배양적CECs정륙각형、포로석양적편평상.CCK-8비색법검측표명,각배양조세포적A450치총체비교차이유통계학의의(F=4.051,P=0.007)；여대조조비교,각방수배양조세포적A450치균명현고우대조조,이함10.0％방수배양조CECs적A450치최고,차이유통계학의의(P＜0.01).류식세포술분석표명,10.0％방수부육24 h후,처우S-G2기적세포비례위(34.80±3.13)％,대조조위(23.06±1.13)％,차이유통계학의의(t=-5.729,P=0.005).화흔유합분석검측표명,10.0％방수조적세포단층화흔면적위(0.116±0.019) mm2,대조조위(0.358±0.049) mm2,차이유통계학의의(t=13.842,P=0.000).DAPI형광염색결과현시,10.0％방수조적평균세포밀도위(1439±110)개/시야,대우대조조적(1162±45)개/시야,차이유통계학의의(t=-11.020,P=0.000).결론 방수작용우배양적우CECs후가촉진CECs적생장,10.0％방수대배양적CECs유촉진각막세포증생적작용.
Background The construction of tissue-engineered corneal endothelium needs the functional seeding cells,so how to culture a large amount of functional corneal endothelial cells (CECs) is an urgent problem to be solved.Objective The aim of this study was to evaluate the role of aqueous humor on bovine CECs in vitro.Methods Aqueous humor of 1.2 ml was collected from the anterior chamber of bovine and sterilized,and the liquid supernatant was obtained.The bovine CECs were isolated from bovine cornea and then cultured in low glucose Dulbecco Modified Eagle Medium with 10％ fetal bovine serum (FBS) in vitro.Aqueous humor was added into the medium with the final concentration of 2.5％,5.0％,l0.0％,15.0％ and 20.0％,respectively,and no aqueous humor was added in the control group.Cell counting kit-8 (CCK-8) assay was used to detect the absorbency value of CECs for the evaluation of cell proliferation.Progression of the cell cycle was analyzed by flow cytometry (FCM).After confluence of the cells was reached,1 ml plastic spear tip was used to scratch the cell single layer,and the cells were incubated consequently in medium with 10％ FBS and with or without aqueous humor for 24 hours.Healing area of the cell single layer was measured.The cells were incubated at a density of 6 × 105 cells/ml and cultured using medium with or without 10.0％ aqueous human for 5 days,and the number of the cells was analyzed by DAPI fluorescence technique.Results Under the phase-contrast microscopy,the confluent CECs showed a slabstone-like and hexagonal appearance.CCK-8 assay revealed that the absorbance values of CECs was significantly different among the various culture groups (F=4.051,P =0.007),and the absorbance value in different concentrations of aqueous human culture groups was significantly higher than that in the control group (P ＜ 0.01).FCM showed that the percentage of the cells in S-G2 phases was (34.80-±3.13)％ in the 10.0％ aqueous humors group and (23.06±1.13)％ in the control group,showing a significant difference (t =-5.729,P=0.005).The scratch test showed that the healing area of the cell signal layer was (0.116±0.019) mm2 in the 10.0％ aqueous humors group and (0.358 ±0.049) mm2 in the control group,showing a significant difference (t =13.842,P =0.000).The density of cells in the 10.0％ aqueous humor group was (1439± 1 10)/field,which was more than (1162±45)/field in the control group (t =-11.020,P=0.000).Conclusions Aqueous humor at the concentration of 10.0％ promote the growth and proliferation of bovine CECs.The result suggests that 10.0％ aqueous humor can be used as a promoting agent during the culture of CECs.
