中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
8期
686-690
,共5页
仝欢%尚庆丽%马景学%高建%王鑫
仝歡%尚慶麗%馬景學%高建%王鑫
동환%상경려%마경학%고건%왕흠
脉络膜新生血管%小干扰RNA%补体因子B%脐静脉内皮细胞%人%ECV-30细胞株
脈絡膜新生血管%小榦擾RNA%補體因子B%臍靜脈內皮細胞%人%ECV-30細胞株
맥락막신생혈관%소간우RNA%보체인자B%제정맥내피세포%인%ECV-30세포주
Choroidal neovascularization%Small interfering RNA%Complement factor B%Umbilical vein endothelial cell%Human%ECV-304
背景 脉络膜新生血管(CNV)是多种眼部疾病致盲的原因之一,研究发现补体系统在CNV的发病机制中起重要作用.目的 构建针对补体因子B(CFB)的小干扰RNA(siRNA)重组质粒,体外观察其对人脐静脉内皮细胞ECV-304增生的影响.方法 根据人CFB的基因序列设计引物,经PCR扩增后与质粒pRNAT-U6.1连接,得到重组质粒pRNAT-U6.1/CFB siRNA,并进行测序鉴定和PCR鉴定.ECV-304细胞株进行常规培养,用电转染技术将重组质粒或空质粒分别转染人ECV-304细胞株,分为CFB-siRNA转染组和空质粒转染组,未转染的细胞为空白对照组.细胞转染后继续培养48 h,于倒置荧光显微镜下观察绿色荧光蛋白(GFP)的表达并计算转染效率;采用半定量逆转录PCR(RT-PCR)法测定各组细胞中CFB mRNA的相对表达量;用MTT法检测各组细胞转染24、48和72 h时细胞的增生值(A)并计算生长抑制率;利用流式细胞技术检测各组细胞的生长周期变化.结果 PCR扩增的目的片段序列与CFB基因序列完全相符,ECV-304细胞转染后,倒置荧光显微镜下可见CFB-siRNA转染组和空质粒转染组的细胞中GFP呈绿色荧光.半定量RT-PCR结果显示,CFB-siRNA转染组、空质粒转染组和空白对照组的CFB mRNA相对表达量分别为0.07±0.04、0.14±0.02和0.14 ±0.03,总体比较差异有统计学意义(F=233.05,P=0.00);其中CFB-siRNA转染组CFB mRNA相对表达量明显低于空质粒转染组和空白对照组,差异均有统计学意义(均P＜0.05).MTT法检测结果显示,各组不同时间细胞增生抑制率总体比较差异有统计学意义(F分组=212.99,P=0.00);CFB-siRNA转染组细胞转染24、48、72 h后细胞增生的抑制率分别为(23.45±0.01)％、(33.48±0.02)％和(45.49±0.01)％,明显高于同时间点空质粒转染组和空白对照组,差异均有统计学意义(均P＜0.05).流式细胞仪检测结果显示,CFB-siRNA转染组、空质粒转染组和空白对照组G1期的细胞数占总细胞数的(44.4±0.5)％、(25.8±0.4)％和(27.9±0.6)％,总体比较差异有统计学意义(F=58.98,P=0.00);CFB-siRNA转染组G1期和G2期细胞所占百分比显著高于空白对照组和空质粒转染组,差异均有统计学意义(均P＜0.05).结论 重组质粒pRNAT-U6.1/CFB siRNA可通过将细胞阻滞在G1期而有效抑制人脐静脉内皮细胞的增生.
揹景 脈絡膜新生血管(CNV)是多種眼部疾病緻盲的原因之一,研究髮現補體繫統在CNV的髮病機製中起重要作用.目的 構建針對補體因子B(CFB)的小榦擾RNA(siRNA)重組質粒,體外觀察其對人臍靜脈內皮細胞ECV-304增生的影響.方法 根據人CFB的基因序列設計引物,經PCR擴增後與質粒pRNAT-U6.1連接,得到重組質粒pRNAT-U6.1/CFB siRNA,併進行測序鑒定和PCR鑒定.ECV-304細胞株進行常規培養,用電轉染技術將重組質粒或空質粒分彆轉染人ECV-304細胞株,分為CFB-siRNA轉染組和空質粒轉染組,未轉染的細胞為空白對照組.細胞轉染後繼續培養48 h,于倒置熒光顯微鏡下觀察綠色熒光蛋白(GFP)的錶達併計算轉染效率;採用半定量逆轉錄PCR(RT-PCR)法測定各組細胞中CFB mRNA的相對錶達量;用MTT法檢測各組細胞轉染24、48和72 h時細胞的增生值(A)併計算生長抑製率;利用流式細胞技術檢測各組細胞的生長週期變化.結果 PCR擴增的目的片段序列與CFB基因序列完全相符,ECV-304細胞轉染後,倒置熒光顯微鏡下可見CFB-siRNA轉染組和空質粒轉染組的細胞中GFP呈綠色熒光.半定量RT-PCR結果顯示,CFB-siRNA轉染組、空質粒轉染組和空白對照組的CFB mRNA相對錶達量分彆為0.07±0.04、0.14±0.02和0.14 ±0.03,總體比較差異有統計學意義(F=233.05,P=0.00);其中CFB-siRNA轉染組CFB mRNA相對錶達量明顯低于空質粒轉染組和空白對照組,差異均有統計學意義(均P＜0.05).MTT法檢測結果顯示,各組不同時間細胞增生抑製率總體比較差異有統計學意義(F分組=212.99,P=0.00);CFB-siRNA轉染組細胞轉染24、48、72 h後細胞增生的抑製率分彆為(23.45±0.01)％、(33.48±0.02)％和(45.49±0.01)％,明顯高于同時間點空質粒轉染組和空白對照組,差異均有統計學意義(均P＜0.05).流式細胞儀檢測結果顯示,CFB-siRNA轉染組、空質粒轉染組和空白對照組G1期的細胞數佔總細胞數的(44.4±0.5)％、(25.8±0.4)％和(27.9±0.6)％,總體比較差異有統計學意義(F=58.98,P=0.00);CFB-siRNA轉染組G1期和G2期細胞所佔百分比顯著高于空白對照組和空質粒轉染組,差異均有統計學意義(均P＜0.05).結論 重組質粒pRNAT-U6.1/CFB siRNA可通過將細胞阻滯在G1期而有效抑製人臍靜脈內皮細胞的增生.
배경 맥락막신생혈관(CNV)시다충안부질병치맹적원인지일,연구발현보체계통재CNV적발병궤제중기중요작용.목적 구건침대보체인자B(CFB)적소간우RNA(siRNA)중조질립,체외관찰기대인제정맥내피세포ECV-304증생적영향.방법 근거인CFB적기인서렬설계인물,경PCR확증후여질립pRNAT-U6.1련접,득도중조질립pRNAT-U6.1/CFB siRNA,병진행측서감정화PCR감정.ECV-304세포주진행상규배양,용전전염기술장중조질립혹공질립분별전염인ECV-304세포주,분위CFB-siRNA전염조화공질립전염조,미전염적세포위공백대조조.세포전염후계속배양48 h,우도치형광현미경하관찰록색형광단백(GFP)적표체병계산전염효솔;채용반정량역전록PCR(RT-PCR)법측정각조세포중CFB mRNA적상대표체량;용MTT법검측각조세포전염24、48화72 h시세포적증생치(A)병계산생장억제솔;이용류식세포기술검측각조세포적생장주기변화.결과 PCR확증적목적편단서렬여CFB기인서렬완전상부,ECV-304세포전염후,도치형광현미경하가견CFB-siRNA전염조화공질립전염조적세포중GFP정록색형광.반정량RT-PCR결과현시,CFB-siRNA전염조、공질립전염조화공백대조조적CFB mRNA상대표체량분별위0.07±0.04、0.14±0.02화0.14 ±0.03,총체비교차이유통계학의의(F=233.05,P=0.00);기중CFB-siRNA전염조CFB mRNA상대표체량명현저우공질립전염조화공백대조조,차이균유통계학의의(균P＜0.05).MTT법검측결과현시,각조불동시간세포증생억제솔총체비교차이유통계학의의(F분조=212.99,P=0.00);CFB-siRNA전염조세포전염24、48、72 h후세포증생적억제솔분별위(23.45±0.01)％、(33.48±0.02)％화(45.49±0.01)％,명현고우동시간점공질립전염조화공백대조조,차이균유통계학의의(균P＜0.05).류식세포의검측결과현시,CFB-siRNA전염조、공질립전염조화공백대조조G1기적세포수점총세포수적(44.4±0.5)％、(25.8±0.4)％화(27.9±0.6)％,총체비교차이유통계학의의(F=58.98,P=0.00);CFB-siRNA전염조G1기화G2기세포소점백분비현저고우공백대조조화공질립전염조,차이균유통계학의의(균P＜0.05).결론 중조질립pRNAT-U6.1/CFB siRNA가통과장세포조체재G1기이유효억제인제정맥내피세포적증생.
Background Choroidal neovascularization (CNV) is one of the causes of blindness in multiple eye diseases.Researches showed that complement system participates in the pathogenesis of CNV.Objective This study was to construct the recombinant of complement factor B-small interference RNA (CFB-siRNA) expression vector and to observe its inhibitory effect on human umbilical vein endothelial cells (ECV-304).Methods CFB gene primers were designed based on human CFB gene,and an expression vector of CFB-siRNA was constructed by inserting CFB-siRNA into pRNAT-U6.1/Neo plasmid.Recombinant plasmids were confirmed by the digestion analysis of restriction endonuclease,and all inserted sequences were verified by DNA sequencing.The recombinant pRNAT-U6.1/CFB-siRNA plasmid and the blank plasmid were transfected into ECV-304 cells in the CFB-siRNA group and blank plasmid group by electroblot,respectively,and non-transfected cells served as the normal control group.The cells were observed under the fluorescence microscope 48 hours after transfection,and the transfective efficiency was calculated.The relative expression of CFB mRNA in the cells of different groups was detected by semi-quantitative reverse transcription PCR (RT-PCR).MTT was employed to calculated the growth inhibitory rates of the cells 24,48 and 72 hours after transfection.The percentages of the cells in different cell cycles were detected by flow cytometry.Results The sequence of the target vector was identical to the designed sequence.The green fluorescence protein (GFP) was seen in both the CFB-siRNA group and the blank plasmid group.The relative expression levels of CFB mRNA were 0.07 ±0.04,0.14 ±0.02 and 0.14 ±0.03 in the CFB-siRNA group,the blank plasmid group and the normal control group,respectively,a significant difference was obtained among the three groups (F=233.05,P =0.00);the expression level of CFB mRNA in the CFB-siRNA group was significantly declined in comparison with the blank plasmid group and the normal control group (both at P＜0.05).The growth inhibitory rates of the cells were (23.45 ±0.01) ％,(33.48 ±0.02) ％ and (45.49±0.01) ％ at 24,48 and 72 hours after transfection,respectively,a significant difference was obtained among the three groups (Fgroup =212.99,P =0.00);the growth inhibitory rates in CFB-siRNA group were significantly higher than that in the blank plasmid group and normal control group (all at P＜ 0.05).The percentages of G1 phase cells were (44.4 ±0.5) ％,(25.8 ±0.4) ％ and (27.9 ± 0.6) ％ in the CFB-siRNA group,the blank plasmid group and the normal control group respectively,a significant difference was obtained among the three groups (F=58.98,P=0.00).The percentages of G1 phase and G2 phase cells in the CFB-siRNA group were significantly higher than those in the blank plasmid group and the normal control group (all at P＜0.05).Conclusions Recombinant pRNAT-U6.1/CFB siRNA inhibits the proliferation of ECV-304 cells effectively by arresting the cells in G1 intermediate phase of the growth cycle.
