siRNA作用后培养的人视网膜色素上皮细胞蛋白质谱分析

Protein mass spectrography for cultured human retinal pigment epithelium after treated with siRNA

目的通过抑制人视网膜色素上皮(retinal pigment epithelium,RPE)细胞缝隙连接蛋白43(connexin 43,cx43)基因的表达,寻找差异蛋白,初步探讨cx43基因对RPE细胞生命活动的意义。方法提取细胞总蛋白并定量分析,第一向电泳是用pH3～10线性IPG预制胶条进行等电聚焦,第二向电泳是用SDS-PAGE凝胶再分离蛋白。银染PAGE凝胶、干燥,Powerlook 2100XL扫描凝胶。利用Image Master 2D Elite 4.01图像分析软件进行分析,蛋白斑点相对分子质量和等电点采用二维校正法确定。随机选取图像分析中siRNA转染组与RPE细胞对照组凝胶图中volume值相差≥2倍的7个蛋白质斑点进行胶内酶切,经胶上原位酶切和质谱分析得到肽质量指纹图谱,查寻蛋白质数据库并鉴定差异蛋白质,分析其生物学意义。结果RPE细胞对照组3块凝胶共分离出蛋白质斑点861个,组间匹配率达到92%;siRNA转染组3块凝胶共分离出蛋白质斑点887个,组间匹配率达到93%。经过软件图像分析,发现volume值相差≥2倍的点共78个,其中上调的16个,下调的18个。有19个点在RPE细胞对照组存在而siRNA转染组不存在,25个在siRNA转染组存在而RPE细胞对照组不存在。随机选取7个点进行蛋白质谱分析:SP-H抗原、P27BBP、有丝分裂活性蛋白激酶、次黄嘌呤核苷酸脱氢酶和热休克蛋白70表达上调,β-actin表达下降,电压依赖阴离子通道蛋白在转染siRNA后出现表达。结论通过蛋白质谱发现cx43基因沉默后引起多种蛋白的表达失调,cx43可能参与RPE细胞的多种生命活动。

Objective To search for different proteins through inhibiting the expression of connexin 43 （cx43） at human retinal pigment epithelium（RPE） cells, and to explore the significance of cx43 on activity of human RPE cells. Methods The total proteins of siRNA and RPE cells were separated and quantitatively analyzed. The first dimensional electrophoresis was performed on immobilized pH gradient rod gels （pH 3 - 10）. Then the proteins in the rod gels were separated using SDS-PAGE gels. The silver-stained gels were dried and scanned with image scanner. The 2D image analysis was performed with image Master 2D Elite 4.01. Seven proteins with significant expression alterations（ volume value 1〉 2 ） were selected randomly and their peptide mass fingerprints（PMFs） were obtained by matrix-assisted laser desorption/ionization time of flying mass spectrometry. The PMFs were used to search protein database. The proteins were identified and their biological significance were analyzed. Results By silver staining,there were 861 protein spots in normal RPE cells group and 887 protein spots were showed in siRNA group. The average matching rate in RPE cells group and siRNA group were 92% and 93% , respectively. Compared with two groups, there were 78 different spots ,volume value of which changed more than 2 times. Among them, 16 protein spots increased in abundance and 18 spots showed lower expression. There were 19 protein spots showed in RPE cells control group but not in siRNA group, and 25 spots showed in siRNA but not in RPE cells group. Seven protein spots were randomly identified： SP-H antigen,p27BBP protein, mitogen activated protein kinase, inosine monophosphate dehydrogenase and HSP70 up regulated, while β-actin protein down regulated, and the expression of voltage-dependent anion channel was found after siRNA transfected. Conclusion The gene silencing of cx43 can cause disorder of many proteins. Cx43 may play an important role in many activities of RPE.