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標題: | 自然殺手細胞活性於前列腺癌預後的應用 Application of Natural Killer Cell Activity in Prostate Cancer Prognosis |
作者: | Yu-Chuan Lu 呂育全 |
指導教授: | 趙福杉(Fu-Shan Jaw) |
關鍵字: | 前列腺癌,自然殺手細胞,自然殺手細胞活性,外泌體,前列腺根除術, prostate cancer,natural killer cell,natural killer cell activity,exosome,radical prostatectomy, |
出版年 : | 2020 |
學位: | 博士 |
摘要: | 前列腺癌排名台灣男性十大癌症第五名,2015年有約5,000位新病人,每年約有1,200位病人不幸死於前列腺癌。在新診斷的案例中,約2/3為局部前列腺癌,也就是未轉移的前列腺癌,局部前列腺癌又可分為低度、中度與高度風險前列腺癌;另外1/3則是已轉移的前列腺癌,就是晚期前列腺癌。近年來,隨著抽血前列腺特異抗原(prostate-specific antigen, PSA)的檢查廣泛使用,局部前列腺癌的診斷比率也逐漸增加。前列腺癌的臨床診斷與追蹤治療主要以前列腺特異抗原(PSA)與影像學為主要依歸,但PSA仍有其侷限性,例如臨床上約有百分之五的前列腺癌患者PSA很低(<4 ng/ml),而腫瘤惡性度(格里森分數)卻很高,於臨床診斷上相當困難,而這群患者也較容易發展荷爾蒙抗性且生存預後也較差,因而發展新型前列腺癌的檢測與術後追蹤方式對臨床治療與追蹤十分重要。
自然殺手細胞是人體免疫中非常重要的一環,能幫助身體對抗外來的病原體與消除人體癌變的細胞,自然殺手細胞活性與腫瘤的存在與否息息相關。其具有非專一性的細胞毒殺作用,並可分泌多種細胞激素調控免疫機制,當細胞被病毒感染、腫瘤細胞沒有表現主要組織相容性複合體I (MHC-I)、或表現出異常分子被自然殺手細胞辨識出之後,自然殺手細胞會立刻釋放出穿孔素直接使目標細胞產生凋零,整個作用時間僅需要幾分鐘。而人體藉由先天的固有免疫,和後天的獲得免疫等防禦機制而達到防制或消除微生物的入侵或癌細胞的増生與存活。 本研究藉由測量自然殺手細胞活性來預測前列腺癌的預後,於前列腺癌根除手術前與術後四至六周抽取病患血液,以分析自然殺手細胞活性、自然殺手細胞數量、表面受體如NKG2A與NKG2D、PSA、白血球數量(white blood cells, WBCs)與C反應蛋白(C-Reactive Protein, CRP)等,並將分析血液中外泌體。自然殺手細胞活性是藉由NK Vue® Kit 試驗方法,檢測自然殺手細胞釋放的干擾素-γ(Interferon gamma, IFN-γ) 進而得知自然殺手細胞活性。本研究發現,前列腺癌患者的自然殺手細胞活性顯著低於健康參與者。接受前列腺根除手術的患者,術後自然殺手細胞活性顯著較術前提升,而自然殺手細胞表面受體方面,術後抑制型受體NKG2A表現量明顯低於術前。此外,手術切緣陽性患者的術後自然殺手細胞活性顯著低於切緣陰性的患者。因此,自然殺手細胞活性可搭配PSA以提高臨床治療與追蹤的準確度。 自然殺手細胞的活性與腫瘤細胞衍生的外泌體有關係,外泌體已經被證實能透過包裹的物質影響目標細胞的功能,包括mRNA, miRNA和蛋白質等,但是詳細的控制機轉仍然不清楚。這些由腫瘤細胞分泌的外泌體會在腫瘤細胞與自然殺手細胞接觸之前被分泌出來,並與自然殺手細胞上的活化接受器結合,並活化自然殺手細胞。自然殺手細胞被活化之後,這些接受器會被吞噬到細胞內並代謝掉,因而降低自然殺手細胞活性。因此,前列腺癌細胞能藉此避免自然殺手細胞攻擊而存活。而在我們的研究中也初步發現,將患者術前與術後血液中的外泌體分別與NK-92細胞共同培養24小時,絕大部分與術後外泌體共同培養的NK-92細胞表面上的活化型受體NKG2D的表現量相較於術前有上升趨勢。同時,若將健康參與者與患者術前血液中的外泌體分別與NK-92細胞共同培養,也可發現與健康參與者外泌體共同培養的NK-92細胞上的NKG2D的表現量來的較高,也就是說,患者的外泌體會影響到自然殺手細胞表面NKG2D受體的表現量,進而可能影響到自然殺手細胞的活性。後續將持續研究外泌體與NKG2A的表現量的相關性,並分析前列腺根除手術前後的外泌體上表面配體蛋白質的組成,並藉由改變外泌體上重要配體(利用抗體或抑制劑進行中和或抑制),找出影響自然殺手細胞活性最重要的配體,以尋找最適合的抑制劑。希望未來能供臨床上使用,提高病人的自體免疫,以增加前列腺癌的治癒率,提供更全面的免疫治療方法。 Prostate cancer ranks the fifth among the top ten male cancers in Taiwan. There were approximately 5,000 new patients in 2015, and nearly 1,200 patients died of prostate cancer each year. Nonetheless, about two-thirds of newly diagnosed cases are localized prostate cancer, that is, non-metastatic prostate cancer. Localized prostate cancer can be divided into low risk, intermediate risk and high risk prostate cancer, and 1/3 is metastatic prostate cancer, which is advanced prostate cancer. In recent years, blood test for prostate specific antigen (PSA) has been widely used, and the proportion of localized prostate cancer has gradually increased. The clinically indicators for diagnosis and tracking treated prostate cancer patients mainly rely on prostate specific antigen PSA and imaging. However, about 5 percent of prostate cancer patients have very low PSA (< 4 ng/ml), but with high tumor grading (Gleason score). This group of patients is more likely to develop hormonal resistance disease and the survival prognosis is much poorer. Development of new detection methods for predicting prostate cancer is important for clinical treatment and tracking. Natural killer (NK) cell plays an important role in human immune system. It can help one organism fighting against the external pathogen and internal cancerous cells. Therefore, the activity of NK cells (NKA) is believed to be associated with the existence of cancers. NK cells which were characterized with non-specific toxic effects can also secrete a variety of cytokines regulating the immune system to kill certain cancer cells or infected cells. If cells infected with a virus or tumor cells showed no MHC-I, or exhibiting abnormal molecules, NK cells release immediately perforin directly to the target cells and cause cell dying and apoptosis. The entire duration of action takes only a few minutes. By innate and acquired immunity, the human body can achieve prevention and control of tumor cells, and eliminate microbial invasion. This study was conducted by assessing the activity of NK cells to predict the prognosis of prostate cancer. Blood samples were drawn before and four to six weeks after prostatectomy to analyze the number and activity of NK cells, the surface receptors: NKG2A and NKG2D, PSA, white blood cells (WBCs), along with C-reactive protein (CRP). Analysis of exosomes in blood were also performed. The NKA was determined by the NK Vue® Kit test method, which measures the interferon-γ (IFN-γ) released by the NK cells. This study found that the activity of NK cells was evidently reduced in prostate cancer patients than in healthy participants. For patients undergoing prostatectomy, the postoperative activity of NK cells was measurably improved than before surgery. Meanwhile, with the surgical intervention, a significantly lower expression of surface receptors NKG2A (inhibitory receptor) was detected than preoperatively. In addition, postoperative NKA was significantly lower in patients with positive margins than in patients with negative margins. Therefore, NK cell activity could be combined with PSA to enhance the accuracy of clinical treatment and future follow-ups NKA is currently known to be related with small vesicles released by cancer cells, called tumor derived exosomes. It has been confirmed that exosomes can alter the functions of recipient cells via their harboring molecules such as miRNAs, mRNAs and proteins; however, the exact mechanism is not well established. The exosomes released from prostate cancer cells will bind to the receptors on NK cells to activate NKA before the direct physical contact between tumors and NK cells. After activating NK cells, the NK cell receptors will be internalized and degraded, and the NKA will be diminished. Finally, prostate cancers may survive. In our research, the exosomes in the blood of patients before and after prostatectomy were co-cultured with NK-92 cells for 24 hours. The expression of the activated receptor NKG2D on the surface of NK cell co-cultured with exosomes after prostatectomy had an upward trend compared with that before surgery. The exosomes in the blood of healthy participants and patients before prostatectomy were also co-cultured with NK-92 cells, and the expression of NKG2D on NK-92 cells co-cultured with exosomes of healthy participants was also higher. That is, the exosomes of patients will affect the expression of NKG2D receptors on the surface of NK cells, which may affect the activity of NK cells. The effort will continue to study the correlation between the expression of NKG2A and exosomes, analyze the composition of the surface ligand proteins on the exosomes before and after prostatectomy, and change the important ligands on the exosomes (using neutralizing antibodies or inhibitory agents) to find the most important ligands that affect the NKA in order to find the most appropriate inhibitor. It is hoped that it can be used clinically in the future to improve the patient's autoimmunity, increase the cure rate of prostate cancer, and provide a more comprehensive immunotherapy method. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7263 |
DOI: | 10.6342/NTU202000523 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2025-03-03 |
顯示於系所單位: | 醫學工程學研究所 |
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ntu-109-1.pdf 此日期後於網路公開 2025-03-03 | 1.49 MB | Adobe PDF |
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