UBE3A重組蛋白片段不適合作為子宮內膜異位症的診斷標記

The recombinant protein fragment of UBE3A is not suitable as a diagnostic marker of endometriosis

張佳燕(Chia-Yen Chang)

指導教授 ： 陳凌雲

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2011年)

https://doi.org/10.6834/CSMU.2011.00063

摘要

子宮內膜異位症（endometriosis）是指子宮內膜組織出現在子宮腔以外的地方，並且進而造成發炎、疼痛、甚至不孕的問題，也是女性生殖系統中常見的良性腫瘤，雖然不是造成高死亡率的疾病，但若症狀過於嚴重，則會影響患者的生活品質甚至進而影響到患者的生育能力。此外患者即使接受治療但其復發率仍偏高，有鑑於此，如能藉由抽血檢查的方式，找到能有效偵測對子宮內膜異位症之診斷標記，將會對患者在治療追蹤及診斷上有所助益。有相關文獻指出SEREX (Serological identification of antigen by recombinant expression cloning)的實驗方法，利用癌症患者血清中的自體抗體可辨識腫瘤相關抗原 (Tumor associated antigen；TAA)來協助診斷癌症的變化或癒後追蹤的工具。本實驗室易瑜嶠醫師初步証實了Cyclin B1與IMP1可適用於作為診斷子宮內膜異位症的標記，另外日本有學者也發現anti-PDIK1L及anti-syntaxin 5此兩個autoantibody也可用來當作診斷子宮內膜異位症的新標記。有鑒於此，我們希望也能找到有特異性的TAA來當作診斷子宮內膜異位症的新標記。在本實驗中，從cancer immunome database (CIDB)去篩選與婦科疾病有關的TAA，而CIDB並無子宮的TAA，所以先篩選卵巢的TAA後，再篩選屬於男性生殖系統睪丸的TAA，然後互相交叉比對，因睪丸組織不含MHC分子不會引起免疫反應，且腫瘤特異性高，搜尋結果選擇UBE3A作研究主題。以重組蛋白的方式製備了UBE3A腫瘤相關抗原，加以純化後，先以酵素連結免疫分析法 (Enzyme-Linked Immunoassay；ELISA)篩選婦科疾病患者血清，發現結果與正常人無顯著差異性，進而以西方墨點法(western blotting) 繼續評估，先排除掉Thioredoxin融合蛋白的影響後，發現絕大部分子宮內膜異位症患者的血清都不會與UBE3A有反應，由以上結果看來UBE3A重組蛋白片段應該不適合做為診斷婦科疾病的標記。

關鍵字

UBE3A

並列摘要

Endometriosis is a condition characterized by the presence of endometrial tissue outside of the uterine cavity. This disease causes inflammation, pain, and even infertility problems. It is often seen in the female reproductive system as benign tumors. Although the disease does not have a high mortality rate, if the symptoms are too severe, it will affect the patients` quality of life and in turn affect the patients` ability to reproduce. In addition, even after the patients have received treatment, the recurrence rate is still high. Therefore, if an effective marker of endometriosis can be found through by blood tests, it would be extremely helpful in the diagnosis and treatment of the patients` condition. Relevant literature on the topic have stated that the SEREX (Serological identification of antigen by recombinant expression cloning) experimental method, can be a diagnostic and tracking tool for cancer by the use of cancer serum autoantibodies to recognize tumor-associated antigens (TAA). Yi YC, MD of our laboratory initially confirmed that Cyclin B1 and IMP1 can be applied as a diagnostic marker of endometriosis. Other Japanese scholars have also discovered the antibodies anti-PDIK1L and anti-syntaxin 5 as new tags for the diagnosis of the disease. Because of these findings, we hope to find another specific TAA to diagnose endometriosis. In this experiment, we use the cancer immunome database (CIDB) to screen gynecological diseases related TAAs. However, the CIDB does not hold information regarding TAAs of the uterus, so we must first screen ovarian TAAs, filter TAAs of the male testicles, and then cross reference the two. Because testicular tissue does not contain MHC molecules, it does not cause immune responses and is highly tumor-specific, thus we selected UBE3A as the research topic. In our experiment, we first prepared UBE3A tumor associated antigen by protein recombinant. After purification, we used Enzyme-Linked Immunoassay (ELISA) to screen the blood serum of patients with gynecological diseases, and found normal results with no significant deviation from a healthy person. We then proceded to Western Blotting for further assessment by excluding the impact of Thioredoxin fusion protein, we found that the serum of the vast majority of endometriosis patients will not react with UBE3A. Thus we concluded the recombinant protein fragment of UBE3A will not be suitable as a diagnostic marker of endometriosis.

並列關鍵字

UBE3A

參考文獻

Anderson KS, Ramachandran N, Wong J, Raphael JV, Hainsworth E, Demirkan G, Cramer D, Aronzon D, Hodi FS, Harris L, Logvinenko T, LaBaer J. Application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer. Journal of Proteome Research 2008 Apr;7(4):1490-9.

Banks L, Pim D, Thomas M. Viruses and the 26S proteasome: hacking into destruction. Trends in Biochemical Sciences 2003 Aug;28(8):452-9.

Casiano CA, Mediavilla-Varela M, Tan EM. Tumor-associated antigen arrays for the serological diagnosis of cancer. Molecular & Cellular Proteomics 2006 Oct;5(10):1745-59.

Chen YT, Gure AO, Scanlan MJ. Serological analysis of expression cDNA libraries (SEREX): an immunoscreening technique for identifying immunogenic tumor antigens. Methods in Molecular Medicine 2005;103:207-16.

Clayton-Smith J, Pembrey ME. Angelman syndrome. Journal of Medical Genetics 1992 Jun;29(6):412-5.