摘要
论文提要和创新点:
1.初步建立子宫内膜异位症相关蛋白质组研究技术体系,并创新性应用于子宫内膜异位症发病机制研究中,研究结果证明一维电泳和液质联用分析技术,具有重复性好鉴定正常和异位的内膜组织差异蛋白质表达谱的有用工具,在今后研究中建立了一种新的组织蛋白组学的研究方法。
2.通过建立子宫内膜异位症相关蛋白质组研究技术体系和蛋白质组分析和差异蛋白鉴定出14种唯一/成组的特异蛋白,包括胶原蛋白а-1、а-2、а-3(VI)和а-1(XIV)链,肌动蛋白、膜联蛋白A2、弹性蛋白微纤维界面定位蛋白1、铁蛋白轻链多肽变异体、墨角藻糖基转移酶10、肌球蛋白9、蛋白S100-A9、KIAA1783蛋白和两个假想蛋白。研究结果分析,认为这些特异差异蛋白功能主要涉及到子宫内膜异位症蛋白质转录水平细胞间黏附、细胞运动性和信号传导,在子宫内膜异位症发生发展中具有重要意义。还需要进一步鉴定和功能分析。
3.我们创新性对子宫内膜异位症的在位内膜和异位内膜进行差异蛋白的western blotting验证和功能分析,结果显示β-actin在异位内膜中明显上调表达。提示β-actin在子宫内膜异位完成粘附、侵袭和血管形成等病理过程中起到重要调节作用。
总之,实验结果将有助于今后纵深研究子宫内膜异位症的发病机制,并可能作为靶点,用于子宫内膜异位症病理发展过程监测指标、诊断标记物和药物治疗中靶标,具有极其重要诊断治疗意义。
With the advent of post-genomic era, functional analysis of proteins has become the major task that life science researchers are facing with. Fortunately, the newly developed proteomics technology can be used to achieve the goal of functional analysis of proteins. The proteomic methodology comprises 1) separation of complex protein mixtures, 2) quantification of separated components, and 3) identification of individual proteins and their poattranslational modifications. There are many approaches to achieving these three fundamentals. To date, the most consistently successful technology is the combination of two-dimensional gel electrophoresis (2-DE), to allow protein separation and visualization, with mass spectrometric identification. The 2DE approach had high-resolution capacity, but was labor-intensive and could not resolve very basic or acidic, very large or small proteins. For surface enhance laser desorption/ionization time-of-flight mass spectrometry only could focus on low molecular weight proteins and was difficult for protein identification. In the study we used 1DE/LC/MS approach to address above problem, which could avoid the problems of above approaches. This approach may give more useful information for the understanding of diease pathogenesis and diagnosis. Proteomic techniques, including one-dimensional electrophoresis (1DE), liquid chromatography and mass spectrometry (LC/MS), were used to screen and identify differential proteins expressing in ectopic and eutopic endometrial and western blotting were used to carry out clinical research for the experimental result to explore endometriosis-related proteins.
1.A preliminary differential proteomic analysis of endometriosi
Endometriosis is a puzzled diease. Scholars suggest that endometriosis should be genetic disease、immunologic disease、inflammation disease、disease result from bleeding、organ dependent disease、hormonal dependent disease,this indicate that we do not understand for it。In order to study this item,we first construct a new system of Proteomic techniques of endometriosis,one-dimensional electrophoresis (1DE), liquid chromatography and mass spectrometry (LC/MS),were used to screen and identify differential proteins expressing in ectopic and eutopic endometria. A total of five consenting patients(two as stageⅡ, two as stageⅢand one as stageⅣ) were selected and proven endometriosis,and five ectopic and eutopic endometrial were surgically excised from the five patients. In order to avoid individual proteome variation, the ten tissues (each for 100μg) were mixed into two samples, eutopic and ectopic endometria. the two samples (eutopic and ectopic endometria) were both run three times, 1DE/LC/MS。Most bands in the two samples were similar, and some differential bands also could be found. Three differential pair bands in the gel were selected and identified by LC/MS。The result indicate :in order to find the differential proteins, we compared the identifications from the two samples. For the first pair band, two proteins in eutopic (control) sample and fourteen in ectopic (experiment) sample. Altogether total 14 differential up-regulation proteins were identified in the three pair bands which included collagen alpha-1, -2, -3 (VI) as well as alpha-1(XIV) chain, actin, Annexin A2, EMILIN-1, Ferritin light polypeptide variant, fucosyltransferase 10, myosin-9, protein S100-A9, KIAA1783 protein and two hypothetical proteins. although we identified 14 differential proteins for endometriosis, many of them were also found in other diseases, such as tumors, inflammation, etc, therefore these proteins were lack specificity for disease diagnosis. The combination of these differential proteins should be a better choice for clinical application. Altogether, we presented a preliminary proteomic analysis of endometriosis and anticipate that above data will help us understand to nature of endometriosis and eventually develop new diagnostic and therapeutic methods of endometriosis in the future.
2. Western blotting validation
To confirm the protein identification and differential expression of up-regulation proteins in eutopic and ectopic endometrium tissues, we conducted western blot forβ-actin with a new set of tissue samples for validation (three stageⅡ, one stageⅢand one stageⅣ). The eutopic and ectopic endometrium tissues were prepared as described above. All samples for validation study were separated by SDS-PAGE with 50μg protein per lane and transferred onto Immobilon-P PVDF membrane (Millipore, Bedford, MA, USA) in CAPS transfer buffer (pH 11.0, 10 mM CAPS, 10% v/v methanol) for 45 min at 1.5 mA/cm2 on a semidry electroblotter (BioRad, CA, USA). Western blot was performed using rapid immunodetection method. After being soaked in 100% methanol for 10 s and dried in air for 15 min, the membrane was probed with mouse anti-actin mAb (Cell Signal Technology, MA, USA. 1:300 dilution) followed by horseradish peroxidase-conjugated horse anti-mouse Ig (AMS Biotechnology, Oxon, UK. 1:3000 dilution). The blot was developed using ECL detection reagent (Pierce Chemical, Rockford, IL, USA). The study showed that actin expression level in eutopic endometrium was lower than that in detected in ectopic endometrium for all five pair samples.
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