老龄大鼠肺损伤后肺蛋白质的双向电泳图像分析 提高电泳分辨率、重复性和一致性的技术方法

Two-dimensional gel electrophoresis analysis of protein in injured lung of the elder rats: an approach to increasing resolution, repetition and concordance of electrophoresis

目的:经气管灌注脂多糖致老龄大鼠急性肺损伤,应用蛋白质双向电泳技术,观察可提高电泳分辨率、重复性和一致性的双向电泳技术分离急性肺损伤后的老龄大鼠肺组织在蛋白质水平上发生的改变,以探讨蛋白质变化在急性肺损伤发生中可能起到的作用。方法:实验于2003-10/2004-04在全军重点实验室解放军总医院老年心血管病研究所和实验测试中心完成。①选用12只老龄SD大鼠,随机分为2组,每组6只,分别为实验组和对照组。实验组大鼠经气管灌注小剂量脂多糖造成急性肺损伤模型:用1mL无菌注射器接钝头长针在直视下经气管内注入新鲜配制的脂多糖/生理盐水混悬液,用量为0.75mg/kg;对照组动物依同法灌注等量生理盐水。②采用丙酮沉淀法提取肺组织蛋白质,在蛋白样品提取过程中,两组蛋白质组织尽量在相同条件下进行。并保证提取蛋白质的整个过程在低温状态下进行,避免在样品提取过程中发生蛋白变性、降解。其次,提取组织蛋白质时所需裂解液等试剂尽量同一批次配制。再次,应使用蛋白酶抑制剂混合物,以免在蛋白质提取和电泳过程中蛋白质遭到破坏。③对实验组和对照组动物蛋白质提取物行并联双向凝胶电泳,保证了电泳图像较高的一致性和重复性。采用双向凝胶电泳技术和计算机辅助图像分析技术,分析凝胶图像对比和找出差异表达蛋白点。电泳过程中使用预制的宽pH范围的胶条,以提高蛋白质分辨率。结果:进入结果分析老龄大鼠12只,每组6只。①对实验组和对照组大鼠肺组织蛋白质提取物行并联双向电泳,电泳图像显示有较高的一致性和可重复性。②两组肺组织采用双向凝胶电泳根据蛋白质的两个特性将其分离:等电点和分子量。并用考染方法显示了凝胶上蛋白质点的位置和丰度。结果显示这种方法对老龄大鼠肺组织蛋白质达到了很好的分离效果,经过计算机图像软件分析凝胶图像有以下结果:对照组大鼠肺组织采用双向凝胶电泳凝胶上共得到(730±52)个点,实验组大鼠肺组织电泳凝胶上得到(706±67)个点,配比率为86%,重现性较好。③经过对比分析两组凝胶,找到质和/或量有明显改善(P<0.05)的16个点。其中实验组有13个点丰度显著增高,表观分子量/等电点分别为26500/5.32,30300/5.32,30700/5.57,35000/6.16,29100/6.20,38700/6.23,30400/6.78,31600/7.30,30900/7.34,38800/6.99,27700/7.58,29700/7.78,38800/7.68;有3个点丰度显著降低,表观分子量/等电点分别为28300/4.95,23000/6.26和31700/6.26。结论:对试验组和对照组肺组织蛋白质提取物,采用宽pH范围的电泳凝胶及并联进行大鼠的双向凝胶电泳,可以有效地将脂多糖致老龄大鼠急性肺损伤时异常改变的肺脏蛋白质分离开来,这些差异表达的蛋白质可能与肺损伤后的蛋白质应激有关。

AIM: To investigate the changes of lung proteins in elder rats with acute lung injury(ALI) induced by intratracheal lipopolysaccharide(LPS) infusion with two-dimensional(2D) polypropylene gel electrophoresis based on that 2D polypropylene gel electrophoresis can promote the resolution, repetition and concordance of electrophoresis so as to probe into the possible role of changes of protein level in the attack of ALI. METHODS: The experiment was conducted in the Chinese PLA Key Laboratory,Institute of Geriatric Cardiology and Experiment Test Center, General Hospital of Chinese PLA from October 2003 to April 2004. ①Twelve SD rats were randomly assigned into the experiment and control groups with 6 in each. The rats in the experiment group were infused with low-dose LPS suspension(0.75 mg/kg, newly dispensed by saline) intratracheally by using an sterile injection syringe(1 mL) inlaid into a long blunt-end needle under a direct vision to induce ALI, and those in the control group were infused with sterile saline of the same volume as in the experiment group.②Firstly, the lung proteins were extracted by means of acetone sedimentation, and the process of extraction should be performed under the same condition in the two groups as far as possible; Also, extraction should be kept at a low temperature to avoid protein denaturation and degradation. Secondly, the reagents such as lysis solution needed were dispensed as simultaneously as possible. Thirdly, the procedures such as the addition of protease inhibitor cocktail should be supplemented to avoid the destruction of protein during extraction of protein and electrophoresis. ③The protein extracts of the two groups were analyzed by 2D polypropylene gel electrophoresis to assure high concordance and repetition of electrophoregram. 2D polypropylene gel electrophoresis and computerized image analysis software were used to analyze the comparison of electrophoregrams and find out the differential expression of protein spots. To increase the resolution of protein during the process, the electrophoresis was involved in the gel strip of the designed immobilized pH gradients with wide pH range. RESULTS: The twelve SD rats were all analyzed in the result with 6 in either group. ①After the parallel two-dimensional electrophoresis was performed to the rats in the two groups, the electrophoregrams were of high concordance and repetition. ②According to two properties of proteins, lungs in the two groups were divided into isoelectric point and molecular weight by 2D polypropylene gel electrophoresis. The protein spots and abundance on gels were shown by Coomassie brilliant blue staining. It was a good method for the separation of lung proteins in elder rats, and the results shown by computerized image analysis were as follows: The gels of rat lungs by 2D polypropylene gel electrophoresis were (730±52) protein spots in the control group, and (706±67) protein spots in the experiment group with matching rate of 86% in a good recurrence. ③By comparing these gels, there were sixteen spots significantly different in quantity and/or quality(P< 0.05). The abundance of thirteen spots was increased in the experiment group: Mr/pI of them were 26 500/5.32, 30 300/5.32, 30 700/5.57, 35 000/6.16, 29 100/6.20, 38 700/6.23, 30 400/6.78, 31 600/7.30, 30 900/7.34, 38 800/6.99, 27 700/7.58,29 700/7.78, 38 800/7.68; The abundance of three spots were decreased: Mr/pI were 28 300/4.95, 23 000/6.26 and 31 700/6.26. CONCLUSION: 2D polypropylene gel electrophoresis in parallel and with the wide pH range gel used for the lung protein extracts in the experiment and control groups can separate proteins with abnormal expression in the LPS-induced ALI of rats. These proteins with differential expression may be involved in protein stresses following ALI.