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
117-121
,共5页
角膜内皮细胞%衰老%p16INK4a基因%Bmi1基因
角膜內皮細胞%衰老%p16INK4a基因%Bmi1基因
각막내피세포%쇠로%p16INK4a기인%Bmi1기인
Corneal endothelial cell%Senescence%p16INK4a gene%Bmi1 gene
背景 p16INK4a基因在细胞老化和早衰过程中发挥着重要作用,是目前已知的细胞衰老的主导基因.角膜内皮细胞(CECs)周期停滞于G1期且体内缺乏增生能力,是否与细胞衰老有关尚未得知. 目的 探讨不同年龄的正常人CECs中p16INK4a基因及其抑制基因Bmi1的表达. 方法 收集临床上DX液保存的行角膜移植手术后剩余的供体周边环状角膜组织,记录供体年龄.经锥虫蓝-茜素红双染法观察CECs的活性；行苏木精-伊红染色以证实角膜材料的组织结构正常.将收集到的角膜环材料按照年龄分为＜30岁组、30～ 50岁组和＞50岁组,每组30个角膜环,其中15个用于总RNA提取,另外15个角膜环等分为3份,分别用于免疫组织化学检测、免疫荧光检测和CECs活性的观察.提取每个角膜环的总RNA,行实时荧光定量PCR检测,观察p16INK4a mRNA、Bmi1 mRNA和Ki67 mRNA在CECs中的表达.行冰冻切片免疫组织化学染色,检测p16INK4a 、Bmi1和Ki67蛋白在CECs中的表达.免疫荧光法检测p16INK4a在CECs的表达. 结果 苏木精-伊红染色结果显示,供体角膜上皮、基质和内皮结构完整,排列规则.实时荧光定量PCR检测显示随年龄增长,p16INK4a mRNA的表达增强,而Bmi1 mRNA和Ki67 mRNA的表达减弱,差异均有统计学意义(F=5.703,P=0.014;F=3.950,P=0.042;F=548.500,P=0.000),其中与＜30岁组比较,＞50岁组p16INK4a mRNA在CECs中的表达量明显升高,而Bmi1 mRNA和Ki67 mRNA的表达均明显降低,差异均有统计学意义(P=0.006、0.013、0.000).免疫组织化学结果可见,p16INK4a蛋白在各组CECs中均有表达,主要位于细胞核内,而Bmi1与Ki67在年老供体的表达强度均弱于年轻供体,与实时荧光定量PCR的结果基本一致.免疫荧光染色显示,年老供体CECs p16INK4a的表达强度强于年轻供体. 结论 细胞衰老主导基因p16INK4a的表达随年龄增加而增高,其抑制基因Bmi1的表达随年龄增加而降低.
揹景 p16INK4a基因在細胞老化和早衰過程中髮揮著重要作用,是目前已知的細胞衰老的主導基因.角膜內皮細胞(CECs)週期停滯于G1期且體內缺乏增生能力,是否與細胞衰老有關尚未得知. 目的 探討不同年齡的正常人CECs中p16INK4a基因及其抑製基因Bmi1的錶達. 方法 收集臨床上DX液保存的行角膜移植手術後剩餘的供體週邊環狀角膜組織,記錄供體年齡.經錐蟲藍-茜素紅雙染法觀察CECs的活性；行囌木精-伊紅染色以證實角膜材料的組織結構正常.將收集到的角膜環材料按照年齡分為＜30歲組、30～ 50歲組和＞50歲組,每組30箇角膜環,其中15箇用于總RNA提取,另外15箇角膜環等分為3份,分彆用于免疫組織化學檢測、免疫熒光檢測和CECs活性的觀察.提取每箇角膜環的總RNA,行實時熒光定量PCR檢測,觀察p16INK4a mRNA、Bmi1 mRNA和Ki67 mRNA在CECs中的錶達.行冰凍切片免疫組織化學染色,檢測p16INK4a 、Bmi1和Ki67蛋白在CECs中的錶達.免疫熒光法檢測p16INK4a在CECs的錶達. 結果 囌木精-伊紅染色結果顯示,供體角膜上皮、基質和內皮結構完整,排列規則.實時熒光定量PCR檢測顯示隨年齡增長,p16INK4a mRNA的錶達增彊,而Bmi1 mRNA和Ki67 mRNA的錶達減弱,差異均有統計學意義(F=5.703,P=0.014;F=3.950,P=0.042;F=548.500,P=0.000),其中與＜30歲組比較,＞50歲組p16INK4a mRNA在CECs中的錶達量明顯升高,而Bmi1 mRNA和Ki67 mRNA的錶達均明顯降低,差異均有統計學意義(P=0.006、0.013、0.000).免疫組織化學結果可見,p16INK4a蛋白在各組CECs中均有錶達,主要位于細胞覈內,而Bmi1與Ki67在年老供體的錶達彊度均弱于年輕供體,與實時熒光定量PCR的結果基本一緻.免疫熒光染色顯示,年老供體CECs p16INK4a的錶達彊度彊于年輕供體. 結論 細胞衰老主導基因p16INK4a的錶達隨年齡增加而增高,其抑製基因Bmi1的錶達隨年齡增加而降低.
배경 p16INK4a기인재세포노화화조쇠과정중발휘착중요작용,시목전이지적세포쇠로적주도기인.각막내피세포(CECs)주기정체우G1기차체내결핍증생능력,시부여세포쇠로유관상미득지. 목적 탐토불동년령적정상인CECs중p16INK4a기인급기억제기인Bmi1적표체. 방법 수집림상상DX액보존적행각막이식수술후잉여적공체주변배상각막조직,기록공체년령.경추충람-천소홍쌍염법관찰CECs적활성；행소목정-이홍염색이증실각막재료적조직결구정상.장수집도적각막배재료안조년령분위＜30세조、30～ 50세조화＞50세조,매조30개각막배,기중15개용우총RNA제취,령외15개각막배등분위3빈,분별용우면역조직화학검측、면역형광검측화CECs활성적관찰.제취매개각막배적총RNA,행실시형광정량PCR검측,관찰p16INK4a mRNA、Bmi1 mRNA화Ki67 mRNA재CECs중적표체.행빙동절편면역조직화학염색,검측p16INK4a 、Bmi1화Ki67단백재CECs중적표체.면역형광법검측p16INK4a재CECs적표체. 결과 소목정-이홍염색결과현시,공체각막상피、기질화내피결구완정,배렬규칙.실시형광정량PCR검측현시수년령증장,p16INK4a mRNA적표체증강,이Bmi1 mRNA화Ki67 mRNA적표체감약,차이균유통계학의의(F=5.703,P=0.014;F=3.950,P=0.042;F=548.500,P=0.000),기중여＜30세조비교,＞50세조p16INK4a mRNA재CECs중적표체량명현승고,이Bmi1 mRNA화Ki67 mRNA적표체균명현강저,차이균유통계학의의(P=0.006、0.013、0.000).면역조직화학결과가견,p16INK4a단백재각조CECs중균유표체,주요위우세포핵내,이Bmi1여Ki67재년로공체적표체강도균약우년경공체,여실시형광정량PCR적결과기본일치.면역형광염색현시,년로공체CECs p16INK4a적표체강도강우년경공체. 결론 세포쇠로주도기인p16INK4a적표체수년령증가이증고,기억제기인Bmi1적표체수년령증가이강저.
Background p16INK4a gene plays an important role during the aging and senility.So it was well known to be a leading gene associated with aging.Corneal endothelial cells(CECs) always get trapped in the G1 phase due to the lack of proliferative ability.Whether it is relative with cell senescence is unclear.Objective This study was to investigate the expression ofp16INK4a gene and Bmi1 gene in human CECs from different aged donors ex vivo.Methods The corneal rims,the residual of corneal tissue preserved in DX solution after penetrating keratoplasty,was used in the present study.Parameters were recorded for the donor,including the age,death to preservation interval and preservation to surgery interval.Corneal endothelium survival rate and endothelial cell density were evaluated by trypan blue-alizarin red dying immediately after penetrating keratoplasty.Routine haematoxylin and eosin staining was also performed to proof the normal structure of the cornea.Sections of corneas from different aged donors were classified into ＜30 years group,30-50 years group and ＞50 years group and were immunostained to assess the expressions of p16INK4aprotein,Bmi1 protein and Ki67 protein in CECs.Total RNA was extracted from independent corneal sample for the evaluation of p16INK4a mRNA,Bmi1 mRNA and Ki67 mRNA expression in CECs by quantitative real-time PCR(qRT-PCR).Results The endothelial cell density of each group was (3069 ±172),(2748±64),(2444 ±178)cells/mm2,respectively.Haematoxylin and eosin staining showed the normal structure of corneal epithelium,stroma and endothelium.qRT-PCR examination revealed an age-related increase in p16INK4a mRNA expression in the CECs(F =5.703,P =0.014) and a decrease in Bmi1 mRNA and Ki67 mRNA expression (F =3.950,P =0.042;F=548.500,P =0.000).The further comparison verified a significant elevation in the expression of p16INK4a mRNA in the CECs of the ＞50 years group compared with ＜30 years group and significant decline in Bmil mRNA and Ki67 mRNA the expression (P =0.006,0.013,0.000).Immunohistochemistry in situ confirmed the expression and nuclear localization of p16INK4a protein in CECs,and the expressing intensities of Bmi1 and Ki67 proteins in the elder donors were weaker than those of the younger donors.The immunofluorescence exhibited that the expressing intensity of p16INK4a protein in CECs of 58 years old donor was higher than that of 23 years old donor,showing a consistent result with that of qRT-PCR.Conclusions Expression of p16INK4a gene increases and that of Bmi1 gene decreases upon age.These results suggest that p16INK4a gene is associated with senescence of human CECs.
