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After the cell growth curves was recorded, RPE cells of the 3-5th passages were utilized. 2、Three different siRNA (siRNAl,siRNA2,siRNA3) targeting against human cx43 gene and one negative control siRNA were designed and transfected into cultured human RPE cells via liposome reagent. The most effective siRNA can be determined by semi-quantitative reverse transcription PCRRT-PCR. 3、To the most effective siRNA, after transfected into human RPEs with different concentration, the cellular proliferate activities were messured by MTT colorimetry ; the percentages of RPE in different cell circle phase was assayed by FCM; the changes of phenotypical properities were observed with SCM; the protein expression of cx43 was studied through immunocytochemistry stain and Weston blot; the communication intercellular was calculated with FRAP; and the ability of recovery was assessed by using an in vitro wound healing model.4、The total proteins of siRNA1 and RPE were seperated by two-dimensional gel electrophoresis and visualized by silver staining. Proteins with significant expression alterations were selected and their peptide mass fingerprints (PMFs were obtained by matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS).The PMFs were used to search NCBInr database by Auto MS-Fit software.
实验方法:1、培养原代的人RPE细胞,经过细胞角蛋白、S-100和神经胶质原纤维酸性蛋白免疫细胞化学鉴定后,通过AO/PI染色技术确定培养细胞的存活率,描记其生长曲线,第3-5代用于以下细胞实验2、生物合成针对人cx43基因的三条小干扰RNA和一条阴性RNA通过脂质体转染RPE细胞后,通过RT-PCR的方法确定抑制效率最高的干扰片断3、将该片段以不同浓度通过阳离子脂质体转染培养的人RPE细胞后,采用MTT法观察其对细胞的增殖力的作用;通过流式细胞仪观察其对细胞周期的影响;通过扫描电镜观察其对细胞形态的影响;通过免疫细胞化学和Weston blot观察其对cx43蛋白表达的作用;采用激光共聚焦和荧光淬灭恢复技术观察荧光恢复速率平均百分率,评价其对细胞间通讯功能的影响;通过制作RPE细胞损伤模型,观察其对损伤修复能力的作用4、分离纯化转染siRNA的RPE组和正常对照组RPE细胞的全部蛋白质,应用等电聚焦电泳和SDS-PAGE双向电泳技术,银染显示分离出的蛋白质斑点,经凝胶图像分析软件对两个样本进行胶图分析,寻找差异蛋白点。
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periodograph:周期图仪
periodogram /周期图/ | periodograph /周期图仪/ | periodometer /调和分析仪/
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spectrograph:频谱仪
控制仪的主要按键和旋钮的作用与使用选择如下:频谱仪(SPECTROGRAPH) 频谱仪的表观结构如图15-6所示. 频谱仪是把从控制仪送来的代表纱条不匀的电压讯号放大,输入到55个频道中去. 然后分离出55种波谱分量,仪器内积分器把这些波谱分量在整个周期内累计.