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重组质粒pRNAT-U6.1/CFB siRNA的构建、鉴定及其对人脐静脉内皮细胞增生的影响
仝欢
尚庆丽
马景学
高建
王鑫
作者及单位信息
·
DOI： 10.3760/cma.j.issn.2095-0160.2015.08.003
Construction and assessment of recombinant plasmid pRNAT-U6.1/CFB siRNA and its inhibitory effect on proliferation of human umbilical vein endothelial cells
Tong Huan
Shang Qingli
Ma Jingxue
Gao Jian
Wang Xin
Authors Info & Affiliations
Tong Huan
Department of Ophthalmology, Affiliated Second Hospital of Hebei Medical College, Shijiazhuang 050000, China
Shang Qingli
Ma Jingxue
Gao Jian
Wang Xin
·
DOI： 10.3760/cma.j.issn.2095-0160.2015.08.003
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摘要

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G 1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G 1期和G 2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G 1期而有效抑制人脐静脉内皮细胞的增生。

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
ABSTRACT

BackgroundChoroidal neovascularization (CNV) is one of the causes of blindness in multiple eye diseases. Researches showed that complement system participates in the pathogenesis of CNV.

ObjectiveThis 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.

ResultsThe 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 ( F group=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 G 1 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 G 1 phase and G 2 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).

ConclusionsRecombinant pRNAT-U6. 1/CFB siRNA inhibits the proliferation of ECV-304 cells effectively by arresting the cells in G 1 intermediate phase of the growth cycle.

Choroidal neovascularization;Small interfering RNA;Complement factor B;Umbilical vein endothelial cell;Human;ECV-304
Shang Qingli, Email: nc.defanabis3102gnahsilgniq
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引用本文

仝欢,尚庆丽,马景学,等. 重组质粒pRNAT-U6.1/CFB siRNA的构建、鉴定及其对人脐静脉内皮细胞增生的影响[J]. 中华实验眼科杂志,2015,33(8)：686-690.

DOI:10.3760/cma.j.issn.2095-0160.2015.08.003

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脉络膜新生血管(choroidal neovascularization，CNV)是多种眼部疾病致盲的原因之一，其发生机制尚未完全明确，治疗效果也不尽如人意。近年来抗血管内皮生长因子(vascular endothelial growth factor，VEGF)的药物，如贝伐单抗、雷珠单抗、VEGF-Trap及康柏西普等 [ 1 , 2 , 3 , 4 ]已成为临床研究的热点，但单纯的抗VEGF治疗仍不能达到根治CNV的目的，也不能防止CNV的复发。研究发现，补体活化的旁路途径在激光诱导CNV中发挥重要作用 [ 5 , 6 ]，而作为旁路途径的关键因子，补体因子B(complement factor B，CFB)则提供了抑制CNV生成的新靶点 [ 7 ]。RNA干扰(RNA interference，RNAi)是一种外源性双链RNA抑制细胞内同源基因表达的现象，其在转录水平、转录后水平和翻译水平上阻断基因的表达，与使目标基因永久性表达沉默的基因敲除方法不同，RNAi通过降解具有同源序列靶基因的mRNA达到阻止靶基因表达的目的。RNAi技术自20世纪90年代末发现以来，已成功用于哺乳动物细胞的研究，并成为研究基因功能的有效方法，有望成为CNV特异性的基因治疗手段 [ 8 , 9 , 10 ]。本研究中构建CFB小干扰RNA(small interference RNA，siRNA)表达载体，探讨CFB在实验性CNV的形成中所产生的分子生物学效应和机制。
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备注信息
A
尚庆丽，Email： nc.defanabis3102gnahsilgniq
B
河北省应用基础研究计划重点基础研究项目 (09966111D)
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