新型融合蛋白uPAg-KPI对卵巢癌的抑制作用及器官功能保护的实验研究

发布时间：2018-04-20 06:18

本文选题：卵巢癌 + uPA/uPAR系统　；参考：《吉林大学》2016年博士论文

【摘要】：卵巢癌死亡率居妇科恶性肿瘤首位。临床上卵巢癌治疗手段以肿瘤细胞减灭术为主,辅助化学治疗。尽管卵巢癌治疗方案明确,手术设备也日臻完善,化疗药物层出不穷,但晚期卵巢癌的五年生存率仍然仅有20%-30%。导致低存活率主要有两点原因:(1)卵巢癌发现时期别已较晚,多伴有其它部位转移,难以实施理想的肿瘤细胞减灭术;(2)化疗产生严重毒、副作用,如骨髓抑制,肝、肾功能损伤等,导致患者难以按规定的疗程和剂量完成化疗。因此,寻找到一种有效抑制卵巢癌,同时还能对脏器功能有保护作用的药物,成为科研人员研究的重点。尿激酶型纤溶酶原激活物(uPA)是一种丝氨酸蛋白酶,uPA通过其生长因子结构域(GFD区)与细胞表面的尿激酶型纤溶酶原激活物受体(uPAR)高效结合后,激活纤溶系统,降解细胞外基质及基底膜,促进肿瘤血管生成,激活MAPK,Src,FAK,PI3K-AKT等信号通路,在肿瘤的增殖、转移中起重要作用。uPA与uPAR在包括卵巢癌在内的多种实体肿瘤中高表达,且其表达水平与患者的预后密切相关。Kunitz型蛋白酶抑制剂(KPI)是一类对丝氨酸蛋白酶有抑制作用的酶。其对uPA/uPAR系统中uPA功能可产生抑制作用,进而能够抑制uPA/uPAR系统在肿瘤生长和转移中的作用。另一方面,Kunitz型蛋白酶抑制剂能抑制炎性介质的释放,抑制白细胞过度激活;抑制超氧阴离子自由基产生,降低脂质过氧化损伤;稳定溶酶体膜,抑制溶酶体的降解作用;抑制Ca2+激活的K+离子通道,延迟细胞凋亡和死亡。KPI在抗肿瘤转移和器官保护方面的研究日渐增多。基于上述KPI和uPA/uPAR系统的关联以及各自的功能作用。本研究团队前期以uPA的GFD区连接一种从人β-淀粉样肽中获得的KPI结构域,重组制备了新型人源Kunitz融合蛋白uPAg-KPI。拟在本研究中探索uPAg-KPI在抗卵巢癌和器官保护中的作用。目的:验证uPAg-KPI在体内和体外实验中对卵巢癌增殖和转移的抑制作用,以及其对增殖、转移相关通路的影响;验证uPAg-KPI在肝、肾损伤模型中对肝、肾功能的保护作用,以及其对氧化平衡系统的影响。方法:(1)通过MTT,克隆形成试验,流式细胞术,Transwell试验,划痕试验,探讨uPAg-KPI对SKOV3的生长和转移的抑制作用。通过Western Blot初步探讨其作用机制。(2)通过构建人卵巢癌SKOV3细胞的裸鼠移植瘤模型,对比裸鼠移植瘤大小和重量,比较抑瘤率,探讨uPAg-KPI在体内对卵巢癌的抑制作用及在联合应用中的辅助作用。通过免疫组化法检测P-ERK、P-AKT、PCNA、VEGF这些与生长转移有关蛋白,初步探讨机制。(3)构建顺铂致小鼠急性肾损伤模型,通过对比小鼠生长状态,肾BUN、CRE水平,行肾脏组织病理学检查的方法,探讨uPAg-KPI在急性肾损伤中的保护作用;通过检测肾脏匀浆组织中MDA、SOD、GSH-PX、CAT含量,初步探讨作用机制。(4)构建CCl4致小鼠急性肝损伤模型,通过对比小鼠生长状态,肝ALT、AST水平,行肝组织病理学检查的方法,探讨uPAg-KPI在急性肝损伤中的保护作用;通过检测肝脏匀浆组织中MDA、SOD、GSH-PX、CAT含量,初步探讨作用机制。结果:(1)uPAg-KPI抗卵巢癌作用的体外实验,(1)uPAg-KPI对SKOV3的抑制作用呈现时间和浓度依赖性,在uPAg-KPI浓度为0.5μg/μl,药物作用48h时可抑制率可达50%。(2)uPAg-KPI组(0.5μg/μl)克隆形成数目为12%±0.76%,与对照组18%±2.18%比较,显著降低。(3)流式细胞术检测uPAg-KPI用药后细胞周期的分布,加药组G1期细胞增加,S和G2/M期细胞百分比下降,细胞周期阻滞于分裂期之前。(4)划痕实验中uPAg-KPI组划痕区域细胞数量较对照组明显降低。(5)Transwell迁移实验中,uPAg-KPI组穿膜细胞数12.5±5.06,较对照组37.7±6.34显著降低;Transwell侵袭实验中,uPAg-KPI组穿膜细胞数5.5±2.46,较对照组为24.4±3.20显著降低。(6)Western blot检测uPAg-KPI与SKOV3细胞作用12、24、48小时后,ERK1/2,P-ERK1/2和AKT和P-AKT水平,总ERK1/2和总AKT蛋白的表达无明显变化,p-ERK1/2和p-AKT表达水平下调。(2)uPAg-KPI抗卵巢癌作用的体内实验中:(1)uPAg-KPI组裸鼠移植瘤的大小和重量均小于对照组。(2)比较三组用药组抑瘤率,联合用药组高于顺铂组高于uPAg-KPI组(71.69%45.14%33.32%)。(3)在裸鼠终体重和生长状态观察对比中,顺铂组、顺铂联合uPAg-KPI组与对照组比,体重下降明显,以顺铂组为著;裸鼠生长状态观察中,顺铂及顺铂联合uPAg-KPI组裸鼠生长状态较差,uPAg-KPI组裸鼠一般状态未受影响。(4)uPAg-KPI组与对照组的免疫组化结果比较,uPAg-KPI组P-ERK、P-AKT、PCNA、VEGF蛋白表达降低。(3)uPAg-KPI对顺铂所致小鼠急性肾损伤的保护作用:(1)顺铂致小鼠急性肾损伤模型中,模型组与对照组比较,血清CRE、BUN显著升高,肾功能受损,造模成功。(2)在三组不同剂量的uPAg-KPI用药组中,血清CRE与模型组均数明显降低,但并无统计学意义。大剂量uPAg-KPI组血清BUN较模型组明显降低,差异有统计学意义。(3)小鼠肾组织匀浆检测MDA、SOD、CAT、GSH-PX水平,uPAg-KPI各剂量组MDA含量均较模型组降低;SOD检测中,大剂量uPAg-KPI组中SOD含量较模型组升高;CAT检测中,各剂量组uPAg-KPI组中CAT含量较模型组升高;GSH-PX含量检测中,中剂量和大剂量uPAg-KPI组中GSH-PX含量较模型组升高。上述差异均有统计学意义。(4)肾脏组织HE染色后观察,uPAg-KPI可降低造模后肾脏炎症反应,管型减少,肾小管上皮细胞脱落、肿胀减轻。(4)uPAg-KPI对CCl4所致小鼠急性肝损伤的保护作用:(1)CCl4致小鼠急性肝损伤模型中,模型组与对照组比较,血清ALT,AST显著升高,肝功能受损,造模成功。(2)在三组不同剂量的uPAg-KPI用药组中,大剂量uPAg-KPI组ALT与模型组比较,明显降低;AST结果中,中剂量和大剂量组较模型组AST降低,差异均有统计学意义。(3)小鼠肝组织匀浆检测MDA、SOD、CAT、GSH-PX水平,uPAg-KPI各剂量组MDA含量均较模型组降低;在SOD检测中,中剂量和大剂量uPAg-KPI组中SOD含量较模型组升高;CAT检测中,中剂量和大剂量uPAg-KPI组中CAT含量较模型组升高;GSH-PX含量检测中,中剂量和大剂量uPAg-KPI组中GSH-PX含量较模型组升高。上述差异均有统计学意义。(4)肝脏HE染色后观察,uPAg-KPI可降低造模后肝脏炎症反应,空泡变性和脂肪变减少,肝小叶结构变清晰、规则。结论:(1)uPAg-KPI对卵巢癌SKOV3细胞的增殖、侵袭、转移有抑制作用,该作用可能与阻滞细胞周期,干扰P-ERK、P-AKT相关信号通路有关。(2)uPAg-KPI对卵巢癌的体内实验有抑制作用,在与顺铂联合使用时,抑制作用增强。uPAg-KPI的抑制作用可能与下调P-ERK、P-AKT、PCNA、VEGF蛋白表达有关。uPAg-KPI与顺铂联合应用时,可能会改善顺铂毒、副作用。(3)uPAg-KPI能够改善顺铂所致的急性肾功能损伤,降低顺铂引起的肾小管扩张,蛋白管型的形成,炎细胞浸润和上皮细胞变性坏死。该保护功能与抗自由基损伤,降低脂质过氧化产物形成有关。(4)uPAg-KPI能够改善CCl4所致的急性肝功能损伤,降低CCl4引起的肝脏炎细胞浸润,肝细胞的变性、坏死。该保护功能与抗自由基损伤,降低脂质过氧化产物形成有关。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31

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