衰老对外周血淋巴细胞蛋白质组影响的研究

The effects of aging on proteome of human peripheral blood lymphocyte

目的:应用双向电泳和质谱技术,对不同年龄组外周血淋巴细胞蛋白质组进行分析,研究免疫衰老的本质,了解衰老在免疫细胞中的发生机制。方法:4组同一家族男性(祖父,父亲,儿子)血样,离心获得外周血淋巴细胞,提取细胞内全部蛋白质。用三种荧光染料标记后进行双向电泳,经不同激光扫描获得蛋白质图谱。经分析后得到差异蛋白质,挖取差异蛋白点经酶解后进行质谱测定及分析。结果:与青年组相比,老年组热休克蛋白70、波动蛋白、细丝蛋白、肌动蛋白、微管蛋白、肌球蛋白、α-烯醇化酶、Rho-二磷酸鸟苷分离抑制因子以及干扰素调节因子4表达均明显降低。而中年组未见明显差异。结论:衰老时淋巴细胞自我保护功能和抗氧化功能衰退,免疫细胞的结构发生退行性改变,糖酵解功能降低促使免疫细胞运动能力减弱,导致吞噬功能减弱,同时老年人淋巴细胞的凋亡加速,衰老个体在缺少IRF-4时,抗原提呈细胞的发育分化受到影响,机体的整体免疫能力受到影响,最终导致综合免疫衰老效应。

Objective： Two- dimensional difference gel electrophoresis （2-D DICE） and mass spectra were used to analysis the proteome of human peripheral blood lymphocytes in three different age groups for studying the nature of aging and unclosing the molecular mechanism Methods： Twelve lymphocyte protein samples in three aging generations from four families were labeled with Cy3 or Cy5 randomly;Each Cy3-1abeled sample and CyS-labeled sample were mixed on the same 2-D gel along with a Cy2-1abeled mixture of all sampies as an internal standard and run on the same gel. Using DICE technology with internal standard statisticaUy significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. MALDI-TOF-TOF MS was used to provide sensitive and accurate mass spectral data for the unique proteins. Results： Compared with that of the young group, the expression of Heat shock protein 70 （ HSP70 ）, Alpha enolase, vimentin, MYL6 protein myosin, actin, Tubulin, Filamin, Rho- GDP-inhibitorl and Interferon regulatory factor 4 （IRF-4） declined in the old group, while no prominent difference appeared in the middle group. Conclusion： The function of self-protection and antioxygen of lymphocytes have weakened,retrogressive change emerges in the structure of immunocytes. The movement ability and phagocytosis of immunocyte decline because of Glycolysis depression when the individual is aging. Simultaneously, lymphocyte apoptosis is accelerated. The development and differentiation of antigen presenting cells are affected when IRF-4 is deficient, so that the immunopoteney of organism is declined. All above-mentioned appearances may lead to the appearance of cence.