本文選題：卵巢癌 + uPA/uPAR系統(tǒng)　； 參考：《吉林大學》2016年博士論文

【摘要】：卵巢癌死亡率居婦科惡性腫瘤首位。臨床上卵巢癌治療手段以腫瘤細胞減滅術為主,輔助化學治療。盡管卵巢癌治療方案明確,手術設備也日臻完善,化療藥物層出不窮,但晚期卵巢癌的五年生存率仍然僅有20%-30%。導致低存活率主要有兩點原因:(1)卵巢癌發(fā)現(xiàn)時期別已較晚,多伴有其它部位轉移,難以實施理想的腫瘤細胞減滅術;(2)化療產(chǎn)生嚴重毒、副作用,如骨髓抑制,肝、腎功能損傷等,導致患者難以按規(guī)定的療程和劑量完成化療。因此,尋找到一種有效抑制卵巢癌,同時還能對臟器功能有保護作用的藥物,成為科研人員研究的重點。尿激酶型纖溶酶原激活物(uPA)是一種絲氨酸蛋白酶,uPA通過其生長因子結構域(GFD區(qū))與細胞表面的尿激酶型纖溶酶原激活物受體(uPAR)高效結合后,激活纖溶系統(tǒng),降解細胞外基質及基底膜,促進腫瘤血管生成,激活MAPK,Src,FAK,PI3K-AKT等信號通路,在腫瘤的增殖、轉移中起重要作用。uPA與uPAR在包括卵巢癌在內的多種實體腫瘤中高表達,且其表達水平與患者的預后密切相關。Kunitz型蛋白酶抑制劑(KPI)是一類對絲氨酸蛋白酶有抑制作用的酶。其對uPA/uPAR系統(tǒng)中uPA功能可產(chǎn)生抑制作用,進而能夠抑制uPA/uPAR系統(tǒng)在腫瘤生長和轉移中的作用。另一方面,Kunitz型蛋白酶抑制劑能抑制炎性介質的釋放,抑制白細胞過度激活;抑制超氧陰離子自由基產(chǎn)生,降低脂質過氧化損傷;穩(wěn)定溶酶體膜,抑制溶酶體的降解作用;抑制Ca2+激活的K+離子通道,延遲細胞凋亡和死亡。KPI在抗腫瘤轉移和器官保護方面的研究日漸增多�；谏鲜鯧PI和uPA/uPAR系統(tǒng)的關聯(lián)以及各自的功能作用。本研究團隊前期以uPA的GFD區(qū)連接一種從人β-淀粉樣肽中獲得的KPI結構域,重組制備了新型人源Kunitz融合蛋白uPAg-KPI。擬在本研究中探索uPAg-KPI在抗卵巢癌和器官保護中的作用。目的:驗證uPAg-KPI在體內和體外實驗中對卵巢癌增殖和轉移的抑制作用,以及其對增殖、轉移相關通路的影響;驗證uPAg-KPI在肝、腎損傷模型中對肝、腎功能的保護作用,以及其對氧化平衡系統(tǒng)的影響。方法:(1)通過MTT,克隆形成試驗,流式細胞術,Transwell試驗,劃痕試驗,探討uPAg-KPI對SKOV3的生長和轉移的抑制作用。通過Western Blot初步探討其作用機制。(2)通過構建人卵巢癌SKOV3細胞的裸鼠移植瘤模型,對比裸鼠移植瘤大小和重量,比較抑瘤率,探討uPAg-KPI在體內對卵巢癌的抑制作用及在聯(lián)合應用中的輔助作用。通過免疫組化法檢測P-ERK、P-AKT、PCNA、VEGF這些與生長轉移有關蛋白,初步探討機制。(3)構建順鉑致小鼠急性腎損傷模型,通過對比小鼠生長狀態(tài),腎BUN、CRE水平,行腎臟組織病理學檢查的方法,探討uPAg-KPI在急性腎損傷中的保護作用;通過檢測腎臟勻漿組織中MDA、SOD、GSH-PX、CAT含量,初步探討作用機制。(4)構建CCl4致小鼠急性肝損傷模型,通過對比小鼠生長狀態(tài),肝ALT、AST水平,行肝組織病理學檢查的方法,探討uPAg-KPI在急性肝損傷中的保護作用;通過檢測肝臟勻漿組織中MDA、SOD、GSH-PX、CAT含量,初步探討作用機制。結果:(1)uPAg-KPI抗卵巢癌作用的體外實驗,(1)uPAg-KPI對SKOV3的抑制作用呈現(xiàn)時間和濃度依賴性,在uPAg-KPI濃度為0.5μg/μl,藥物作用48h時可抑制率可達50%。(2)uPAg-KPI組(0.5μg/μl)克隆形成數(shù)目為12%±0.76%,與對照組18%±2.18%比較,顯著降低。(3)流式細胞術檢測uPAg-KPI用藥后細胞周期的分布,加藥組G1期細胞增加,S和G2/M期細胞百分比下降,細胞周期阻滯于分裂期之前。(4)劃痕實驗中uPAg-KPI組劃痕區(qū)域細胞數(shù)量較對照組明顯降低。(5)Transwell遷移實驗中,uPAg-KPI組穿膜細胞數(shù)12.5±5.06,較對照組37.7±6.34顯著降低;Transwell侵襲實驗中,uPAg-KPI組穿膜細胞數(shù)5.5±2.46,較對照組為24.4±3.20顯著降低。(6)Western blot檢測uPAg-KPI與SKOV3細胞作用12、24、48小時后,ERK1/2,P-ERK1/2和AKT和P-AKT水平,總ERK1/2和總AKT蛋白的表達無明顯變化,p-ERK1/2和p-AKT表達水平下調。(2)uPAg-KPI抗卵巢癌作用的體內實驗中:(1)uPAg-KPI組裸鼠移植瘤的大小和重量均小于對照組。(2)比較三組用藥組抑瘤率,聯(lián)合用藥組高于順鉑組高于uPAg-KPI組(71.69%45.14%33.32%)。(3)在裸鼠終體重和生長狀態(tài)觀察對比中,順鉑組、順鉑聯(lián)合uPAg-KPI組與對照組比,體重下降明顯,以順鉑組為著;裸鼠生長狀態(tài)觀察中,順鉑及順鉑聯(lián)合uPAg-KPI組裸鼠生長狀態(tài)較差,uPAg-KPI組裸鼠一般狀態(tài)未受影響。(4)uPAg-KPI組與對照組的免疫組化結果比較,uPAg-KPI組P-ERK、P-AKT、PCNA、VEGF蛋白表達降低。(3)uPAg-KPI對順鉑所致小鼠急性腎損傷的保護作用:(1)順鉑致小鼠急性腎損傷模型中,模型組與對照組比較,血清CRE、BUN顯著升高,腎功能受損,造模成功。(2)在三組不同劑量的uPAg-KPI用藥組中,血清CRE與模型組均數(shù)明顯降低,但并無統(tǒng)計學意義。大劑量uPAg-KPI組血清BUN較模型組明顯降低,差異有統(tǒng)計學意義。(3)小鼠腎組織勻漿檢測MDA、SOD、CAT、GSH-PX水平,uPAg-KPI各劑量組MDA含量均較模型組降低;SOD檢測中,大劑量uPAg-KPI組中SOD含量較模型組升高;CAT檢測中,各劑量組uPAg-KPI組中CAT含量較模型組升高;GSH-PX含量檢測中,中劑量和大劑量uPAg-KPI組中GSH-PX含量較模型組升高。上述差異均有統(tǒng)計學意義。(4)腎臟組織HE染色后觀察,uPAg-KPI可降低造模后腎臟炎癥反應,管型減少,腎小管上皮細胞脫落、腫脹減輕。(4)uPAg-KPI對CCl4所致小鼠急性肝損傷的保護作用:(1)CCl4致小鼠急性肝損傷模型中,模型組與對照組比較,血清ALT,AST顯著升高,肝功能受損,造模成功。(2)在三組不同劑量的uPAg-KPI用藥組中,大劑量uPAg-KPI組ALT與模型組比較,明顯降低;AST結果中,中劑量和大劑量組較模型組AST降低,差異均有統(tǒng)計學意義。(3)小鼠肝組織勻漿檢測MDA、SOD、CAT、GSH-PX水平,uPAg-KPI各劑量組MDA含量均較模型組降低;在SOD檢測中,中劑量和大劑量uPAg-KPI組中SOD含量較模型組升高;CAT檢測中,中劑量和大劑量uPAg-KPI組中CAT含量較模型組升高;GSH-PX含量檢測中,中劑量和大劑量uPAg-KPI組中GSH-PX含量較模型組升高。上述差異均有統(tǒng)計學意義。(4)肝臟HE染色后觀察,uPAg-KPI可降低造模后肝臟炎癥反應,空泡變性和脂肪變減少,肝小葉結構變清晰、規(guī)則。結論:(1)uPAg-KPI對卵巢癌SKOV3細胞的增殖、侵襲、轉移有抑制作用,該作用可能與阻滯細胞周期,干擾P-ERK、P-AKT相關信號通路有關。(2)uPAg-KPI對卵巢癌的體內實驗有抑制作用,在與順鉑聯(lián)合使用時,抑制作用增強。uPAg-KPI的抑制作用可能與下調P-ERK、P-AKT、PCNA、VEGF蛋白表達有關。uPAg-KPI與順鉑聯(lián)合應用時,可能會改善順鉑毒、副作用。(3)uPAg-KPI能夠改善順鉑所致的急性腎功能損傷,降低順鉑引起的腎小管擴張,蛋白管型的形成,炎細胞浸潤和上皮細胞變性壞死。該保護功能與抗自由基損傷,降低脂質過氧化產(chǎn)物形成有關。(4)uPAg-KPI能夠改善CCl4所致的急性肝功能損傷,降低CCl4引起的肝臟炎細胞浸潤,肝細胞的變性、壞死。該保護功能與抗自由基損傷,降低脂質過氧化產(chǎn)物形成有關。
[Abstract]:The mortality of ovarian cancer is the first one in gynecologic malignant tumor. The treatment of ovarian cancer is mainly by tumor cell subtraction and adjuvant chemotherapy. Although the treatment plan of ovarian cancer is clear, the surgical equipment is becoming more and more perfect and the chemotherapy drugs emerge in endlessly, but the five year survival rate of advanced ovarian cancer is still only 20%-30%. and the low survival rate is two. Reasons: (1) ovarian cancer was found late, with other parts of the metastasis, difficult to implement the ideal tumor cell reduction; (2) chemotherapy produced severe toxic, side effects, such as bone marrow suppression, liver and kidney function damage, resulting in patients difficult to complete the prescribed course and dose of chemotherapy. Therefore, to find an effective inhibition of ovarian cancer, the same UPA is a serine protease, and uPA activates the fibrinolysis system by combining its growth factor domain (GFD region) with the urokinase type plasminogen activator receptor (uPAR) on the cell surface and activates the fibrinolysis system. Extracellular matrix and basement membrane, promoting tumor angiogenesis, activating MAPK, Src, FAK, PI3K-AKT and other signaling pathways, play an important role in the proliferation and metastasis of tumor,.UPA and uPAR are highly expressed in various solid tumors, including ovarian cancer, and the expression level is closely related to the patient's pre related.Kunitz protease inhibitor (KPI). The inhibitory activity of serine protease, which inhibits the uPA function in the uPA/uPAR system, and then inhibits the role of uPA/uPAR in tumor growth and metastasis. On the other hand, the Kunitz protease inhibitors can inhibit the release of inflammatory mediators, inhibit the overactivation of leukocytes, and inhibit the generation of superoxide anion radicals. Reducing the lipid peroxidation damage, stabilizing lysosome membrane, inhibiting the degradation of lysosome, inhibiting the K+ ion channel activated by Ca2+, delayed apoptosis and death of.KPI in anti-tumor metastasis and organ protection increasing. Based on the association of the above KPI and uPA/uPAR system and their respective functional effects, the team in the early stage of this study was uP The GFD region of A connects a KPI domain obtained from human beta amyloid peptide, and the recombinant human Kunitz fusion protein uPAg-KPI. is prepared to explore the role of uPAg-KPI in the anti ovarian cancer and organ protection in this study. Objective: to verify the inhibitory effect of uPAg-KPI on the proliferation and metastasis of oval carcinoma in vivo and in vitro, and the inhibitory effect of uPAg-KPI on the proliferation and metastasis of oocyte carcinoma in vitro and in vitro. The effect on the proliferation and metastasis related pathways; to verify the protective effect of uPAg-KPI on the liver and kidney function in the liver and kidney damage model and its effect on the oxidative balance system. Methods: (1) through MTT, clone formation test, flow cytometry, Transwell test, scratch test, and explore the inhibitory effect of uPAg-KPI on the growth and metastasis of SKOV3. Through West Ern Blot preliminarily discussed its mechanism of action. (2) by constructing a nude mouse model of human ovarian cancer SKOV3 cells, comparing the size and weight of xenografts in nude mice and comparing the tumor suppressor rate, the inhibitory effect of uPAg-KPI on ovarian cancer in the body and the assistant role in the combined application were investigated. The immunological method was used to detect P-ERK, P-AKT, PCNA, and VEGF. Long transfer related proteins, preliminary study mechanism. (3) construct cisplatin induced acute renal injury model in mice. By comparing mice growth state, kidney BUN, CRE level, and renal histopathological examination, the protective effect of uPAg-KPI in acute renal injury was explored, and the content of MDA, SOD, GSH-PX and CAT in kidney homogenate was examined, and preliminary discussion was made. Mechanism. (4) to construct a model of acute liver injury induced by CCl4 in mice, and to explore the protective effect of uPAg-KPI in acute liver injury by comparing the growth state of the mice, the level of liver ALT and AST and the pathological examination of liver tissue, and to explore the mechanism of action by detecting the content of MDA, SOD, GSH-PX and CAT in the liver homogenate. Results: (1) uPAg-KPI anti ovum. In vitro experiment of nesting cancer, (1) uPAg-KPI showed time and concentration dependence on the inhibitory effect of SKOV3. The concentration of uPAg-KPI was 0.5 g/ Mu L, and the inhibitory rate of 48h was 12% + 0.76% in the 50%. (2) uPAg-KPI group, which was significantly lower than that of the control group (18% + 2.18%). (3) the flow cytometry was used to detect uPAg-KPI. The distribution of cell cycle after the drug increased, the percentage of G1 cells in the drug group increased, the percentage of S and G2/M cells decreased and the cell cycle was blocked before the split stage. (4) the number of cells in the scratch area of the uPAg-KPI group was significantly lower than that in the control group. (5) in the Transwell migration experiment, the number of membrane cells in the uPAg-KPI group was 12.5 + 5.06, which was significantly lower than that of the control group (37.7 + 6.34). In the Transwell invasion experiment, the number of membrane cells in the uPAg-KPI group was 5.5 + 2.46, which was significantly lower than that of the control group. (6) the expression of ERK1/2, P-ERK1/2, AKT and P-AKT was not significantly changed by Western blot after the action of uPAg-KPI and SKOV3 cells for 12,24,48 hours, and the expression level and the expression level were down. (2) In vivo experiment of anti ovarian cancer effect of PI: (1) the size and weight of nude mice transplanted in group uPAg-KPI were less than that of control group. (2) the tumor inhibition rate of the three groups was compared with that of the group of uPAg-KPI (71.69%45.14%33.32%). (3) cisplatin group, cisplatin combined with uPAg-KPI group and uPAg-KPI group in the observation and comparison of the final body weight and growth state of nude mice. In the group of nude mice, the growth status of cisplatin and cisplatin combined with uPAg-KPI group was poor, and the general state of nude mice in group uPAg-KPI was not affected. (4) compared with the control group, the expression of P-ERK, P-AKT, PCNA, VEGF protein in group uPAg-KPI was lower. (3) uPAg-KPI pairs. The protective effect of platinum induced acute renal injury in mice: (1) in the model of acute renal injury in mice induced by cisplatin, the model group was compared with the control group, the serum CRE, BUN increased significantly, the renal function was damaged and the model was successful. (2) the number of serum CRE and the model group decreased significantly in the three groups of different doses of uPAg-KPI, but there was no statistical significance. Large dose uPAg- The serum BUN in KPI group was significantly lower than that in the model group. (3) the level of MDA, SOD, CAT, GSH-PX in the renal homogenate of mice was lower than that of the model group; in SOD detection, the SOD content in the large dose uPAg-KPI group was higher than that in the model group; in CAT detection, the content of each dose group was higher than that of the model group. In the GSH-PX content test, the GSH-PX content in the medium and large dose uPAg-KPI groups was higher than that of the model group. (4) after HE staining in the kidney tissue, uPAg-KPI could reduce the renal inflammation, decrease the tube type, decrease the renal tubular epithelial cells and reduce the swelling. (4) uPAg-KPI to the acute liver of mice induced by CCl4 The protective effect of injury: (1) in the model of acute liver injury induced by CCl4 in mice, the model group was compared with the control group, the serum ALT, AST increased significantly, the liver function was damaged and the model was successful. (2) in the three groups of different doses of uPAg-KPI, the large dose uPAg-KPI group ALT was significantly lower than the model group; in AST results, the medium dose and the large dose group were more A than the model group A. ST decreased, and the difference was statistically significant. (3) the level of MDA, SOD, CAT, GSH-PX in the liver homogenate of mice was lower than that of the model group; in SOD detection, the SOD content in the middle and large dose uPAg-KPI groups was higher than that in the model group; and in CAT detection, the contents of the medium and large doses of uPAg-KPI groups were higher than those in the model group. In the SH-PX content test, the GSH-PX content in the medium and large dose uPAg-KPI groups was higher than that of the model group. (4) after the liver HE staining, uPAg-KPI could reduce the liver inflammation, vacuolation and fat decrease, the liver lobule structure became clear, and the rules. Conclusion: (1) uPAg-KPI to ovarian cancer SKOV3 cells The proliferation, invasion, and metastasis have inhibitory effects, which may be related to blocking cell cycle and interfering with P-ERK, P-AKT related signaling pathways. (2) uPAg-KPI has inhibitory effect on ovarian cancer in vivo, and inhibition of the inhibitory action of.UPAg-KPI may be associated with the downregulation of P-ERK, P-AKT, PCNA, and VEGF protein expression when combined with cisplatin. The combination of Ag-KPI and cisplatin may improve cisplatin toxicity and side effects. (3) uPAg-KPI can improve the acute renal damage caused by cisplatin, reduce the dilatation of renal tubules, formation of protein tube, infiltration of inflammatory cells and degeneration and necrosis of epithelial cells caused by cisplatin. This protection work can be associated with the anti free radical damage and the formation of lipid peroxidation products. (4) (4) uPAg-KPI can improve the acute liver function damage caused by CCl4, reduce the infiltration of liver inflammatory cells, degeneration and necrosis of liver cells, and the protective function is related to the anti free radical damage and the formation of lipid peroxidation products.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.31

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