振荡法分离小鼠视网膜色素上皮细胞提取总RNA的效果

doi:DOI:10.3760/cma.j.issn.1674-845X.2016.01.005

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摘要

目的探讨应用振荡法分离小鼠视网膜色素上皮（RPE）细胞并进行总RNA提取的效果。方法实验研究。应用机械法对4只小鼠眼制作眼杯，去除视网膜后应用RNA保护试剂及振荡的方法对小鼠RPE进行分离，将应用振荡法分离后的眼杯铺片，采用免疫细胞化学方法检测分离后RPE细胞残留情况；收集分离的细胞，提取总RNA。同时分别应用自发分离法、酶法、眼杯法各对4只小鼠眼RPE进行分离，提取总RNA，比较不同方法提取的RPE细胞总RNA量，并应用实时定量PCR方法对RPE细胞中RPE65、vWF及COL6A1 mRNA表达水平进行比较。数据采用单因素方差分析、独立样本t检验进行比较。结果采用4种方法进行RPE分离得到的总RNA量差异有统计学意义（F=8.538，P<0.01），免疫细胞化学方法证实应用振荡法可以使RPE细胞与脉络膜完全分离，得到的总RNA量为（234.47±49.13）ng，与自发分离法及酶法比较差异无统计学意义（P>0.05），但低于眼杯法（P<0.05）。4种方法得到的RPE65的mRNA表达量差异有统计学意义（F=38.453，P<0.01），通过实时定量PCR法检测振荡法RPE65的mRNA表达量与自发分离法相当（P>0.05），低于酶法（P<0.01），但要高于眼杯法（P<0.01）。体现脉络膜细胞污染程度的标志物vWF及COL6A1 mRNA相对表达量，4种方法差异有统计学意义（F=45.432、90.498，P<0.01）。振荡法vWF mRNA表达水平低于自发分离法（P<0.05），与酶法相似（P>0.05），远低于眼杯法（P<0.01）；COL6A1mRNA表达水平与自发分离法及酶法相似（P>0.05），远低于眼杯法（P<0.01）。结论应用振荡法可有效分离小鼠RPE细胞，并最大限度地降低来自脉络膜的污染，经总RNA提取后可得到较多的总RNA量及较好的质量，此方法方便快捷、稳定性较好，适合小样本量的RPE细胞的基因转录水平分析。

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	齐首楠
	王晨光
	赵亮亮
	苏冠方
	程岩
	田蕊
	杨欣悦
	王丽丽

关键词 ：视网膜色素上皮, 细胞分离, 小鼠, RNA提取

Abstract：

Objective To study the effect of a shaking method to isolate murine retinal pigment epithelium (RPE) cells for RNA extraction. Methods This was an experimental study. After an eye cup was made, an RNA-protected reagent and shaking method were used to isolate the RPE of mice (n=4). Immunohistochemistry staining was used to detect the status of the RPE residue, the detachable cells were collected and the total RNA was extracted. Other methods such as auto isolation, the enzyme method and eye-cup method were used to isolate the RPE and extract total RNA(each group n=4). The quantity of total RNA was compared among the different methods and quantitative real-time PCR was used to compare the RPE65, vWF and COL6A1 mRNA levels. The effect that the shaking method had on RPE isolation was studied. A one-way ANOVA and independent sample t-test were used in the statistical analysis of the data. Results The four methods resulted in statistically different quantities of total RNA (F=8.538, P<0.01). The shaking method resulted in complete isolation of RPE from the choroid tissue and was proven by immunohistochemistry staining. The quantity of total RNA obtained by the shaking method was 234.47±49.13 ng, which was similar to that obtained by the auto isolation and enzyme methods (P>0.05) but less than that obtained by the eye-cup method (P<0.05). A comparison of the four methods showed statistically significant differences in the expression levels of RPE65 mRNA (F=38.453, P<0.01). The expression of RPE65 mRNA was similar with the shaking and auto isolation methods (P>0.05), but was lower than the enzyme method (P<0.01) and higher than the eye-cup method (P<0.01). The expression of vWF and COL6A1 mRNA by the four methods were significantly different (F=45.432, 90.498, P<0.01). The expression of vWF mRNA by the shaking method was lower than with the auto isolation method (P<0.05), was similar to the enzyme method (P>0.05), and much lower than the eye-cup method (P<0.01). The expression of COL6A1 mRNA by the shaking method was similar to that obtained with the auto isolation and enzyme methods (P>0.05), but much lower than the eye-cup method (P<0.01). Conclusion The shaking method is effective, convenient and stable. This method can maximally decrease contamination of the choroid for total RNA extraction and allows further study of the gene transcription of RPE in small samples.

Key words： Pigment epithelium of eye Cell separation Mice RNA extraction

收稿日期: 2015-08-11

基金资助:

吉林省科技发展计划青年基金（20130522012JH）

通讯作者: 王晨光，Email：wangchenguang1977@163.com

引用本文:

齐首楠,王晨光,赵亮亮,苏冠方,程岩,田蕊,杨欣悦,王丽丽. 振荡法分离小鼠视网膜色素上皮细胞提取总RNA的效果[J]. 中华眼视光学与视觉科学杂志, 2016, 18(1): 19-24. Qi Shounan,Wang Chenguang,Zhao Liangliang,Su Guanfang,Cheng Yan,Tian Rui,Yang Xinyue,Wang Lili. A study to isolate mouse retinal pigment epithelium cells with a shaking method for RNA extraction. Chinese Journal of Optometry Ophthalmology and Visual science, 2016, 18(1): 19-24. DOI: DOI:10.3760/cma.j.issn.1674-845X.2016.01.005

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[1]	Thumann G, Dou GR, Wang YS, et al. Cell Biology of the Retinal Pigment Epithelium[M]//Ryan SJ. Retina. 5th ed. Los Angeles,2013:401-410.
[2]	McLeod DS, Taomoto M, Otsuji T, et al. Quantifying changes in RPE and choroidal vasculature in eyes with age-related macular degeneration[J]. Invest Ophthalmol Vis Sci,2002,43(6):1986-1993.
[3]	Hirami Y, Tsujikawa A, Sasahara M, et al. Alterations of retinal pigment epithelium in central serous chorioretinopathy[J]. Clin Experiment Ophthalmol,2007,35(3):225-230. DOI:10.1111/j.1442-9071.2006.01447.x.
[4]	Xin-Zhao Wang C, Zhang K, Aredo B, etal. Novel method for the rapid isolation of RPE cells specifically for RNA extraction and analysis[J]. Exp Eye Res,2012,102:1-9. DOI:10.1016/j.exer.2012.06.003.
[5]	Saari JC, Bunt AH, Futterman S, et al. Localization of cellular retinal-binding protein in bovine retina and retinal pigment epithelium, with a consideration of the pigment epithelium isolation technique[J]. Invest Ophthalmol Vis Sci,1977,16(9):797-806.
[6]	Wang N, Koutz CA, Anderson RE. A method for the isolation of retinal pigment epithelial cells from adult rats[J]. Invest Ophthalmol Vis Sci,1993,34(1):101-107.
[7]	Sakagami K, Naka H, Hayashi A, et al. A rapid method for isolation of retinal pigment epithelial cells from rat eyeballs[J]. Ophthalmic Res,1995,27(5):262-267. DOI:10.1159/000267735.
[8]	Langenfeld A, Julien S, Schraermeyer U. An improved method for the isolation and culture of retinal pigment epithelial cells from adult rats[J]. Graefes Arch Clin Exp Ophthalmol,2015,253(9):1493-1502. DOI:10.1007/s00417-015-3011-5.
[9]	Farnoodian M, Kinter JB, Yadranji Aghdam S, et al. Expression of pigment epithelium-derived factor and thrombospondin-1 regulate proliferation and migration of retinal pigment epithelial cells[J]. Physiol Rep,2015,3(1):e12266. DOI:10.14814/phy2.12266.