姜黄素脂质体抑制肝癌栓塞后乏氧诱导血管生成的机制和实验研究

发布时间：2017-12-28 12:23

本文关键词：姜黄素脂质体抑制肝癌栓塞后乏氧诱导血管生成的机制和实验研究　出处：《南京中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：肝细胞肝癌栓塞前后血清HIF-lα和VEGF的表达和相关性研究目的:探讨研究肝细胞肝癌(hepatocellular carcinoma,HCC)患者经肝动脉栓塞术(transcatheter arterial embolization,TAE)前后血清乏氧诱导因子-1α(hypoxia inducible factor1 alpha,HIF-1α)和血管内皮生成因子(vascular endothelial growth factor,VEGF)的表达水平和相关性;分析血清HIF-1βα和VEGF表达水平与部分HCC临床特征的相关性。方法:收集2014年6月至2015年12月首诊确诊HCC患者42例,同期确诊肝血管瘤患者6例(对照组)。在对所有患者行临床特征分析后,行肝脏肿瘤和血管瘤的TAE治疗,栓塞时根据肿瘤部位和大小采取不同剂量的碘化油和栓塞剂。静脉采集所有患者TAE前,TAE栓塞后第3天,第7天和1个月的血清,分离血清后行酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定血清HIF-1α和VEGF表达。引入实体瘤客观评价疗效标准(RECIST1.1),根据疗效将患者分为完全疗效组(CR),部分疗效组(PR),疾病稳定组(SD)与疾病进展组(PD)。分析血清HIF-1α、VEGF与HCC各项临床特征,以及两者之间的相关性。结果:肝血管瘤TAE栓塞前血清HIF-1α和VEGF值分别为27.14±5.94 pg/mL和54.51 ± 11.38pg/mL;HCC 患者 TAE栓塞前血清 HIF-1α 和 VEGF 值为 162.19±43.03pg/mL和278.70±55.55ppg/mL,两者比较具有统计学差异(P0.05)。HCC患者TAE栓塞后第3天血清 HIF-1α 和 VEGF 达到峰值(HIF-1α:332.17±37.37 pg/mL,VEGF:398.37±69.38 pg/mL),明显高于栓塞前表达水平(P0.05)。HCC患者TAE栓塞后第7天血清HIF-1α和 VEGF 值呈下降趋势(HIF-1α:245.20±84.52pg/mL,VEGF:334.25±83.88 pg/m L),但是仍明显高于治疗前的表达水平(P0.05)。HCC患者TAE栓塞治疗后1个月复诊评估,完全疗效组(CR)7例,部分疗效(PR),疾病稳定(SD)与疾病进展组(PD)共35 例。CR 与 PR+SD+PD 两组血清 HIF-1α 和 VEGF 比较如下:(118.67±43.70 pg/mL vs 252.07±67.17pg/mL)(199.33±31.68pg/mL vs 354.75±92.82pg/mL),具有显著统计学差异(P0.05)。血清HIF-1α表达水平与血清VEGF具有明显的正相关性(r=0.67,P0.05),同时血清HIF-1α、VEGF与门静脉癌栓和远端转移临床特征具有相关性(P0.05)。结论:血清HIF-1α与VEGF在肝细胞肝癌疾病进程中具有重要意义,表明乏氧微环境诱导的血管生成在HCC侵袭转移过程中起着关键作用。同时,血清HIF-1α与VEGF表达能够有效地评估TAE栓塞治疗的疗效和预测患者生存状况。姜黄素脂质体抑制栓塞后乏氧诱导血管生成的机制研究目的:通过建立VX2兔肝癌模型,探讨栓塞后残存肿瘤组织局部乏氧诱导因子1-α(hypoxia inducible factor 1 alpha,HIF-1a)和血管内皮生成因子(vascular endothelial growth factor,VEGF)的相关性,以及姜黄素脂质体联合肝动脉栓塞(transcatheter arterial embolization,TAE)在抑制栓塞后乏氧诱导肿瘤血管生成表达中的价值。方法:(1)姜黄素脂质体:精密称取质量比为20:1:2的大豆卵磷脂、胆固醇及姜黄素置于茄形容器中,加入适量二氯甲烷使脂质完全溶解。脂质溶液形成一层均匀的薄膜,然后真空干燥过夜,使溶剂除尽。加入磷酸盐缓冲液后充分乳化,乳化温度为45℃。最后脂质体通过小孔径滤膜高压挤出保存备用。(2)VX2兔肝癌模型:将肿瘤传代模型兔猝死后取出肿瘤组织置于0.9%生理盐水中保存。将预建模型兔麻醉后行腹部正中切口,暴露肝脏左叶,取1mm×1mm× 1mm肿瘤组织种植于兔肝脏左叶,然后缝合腹部切口。所有建模成功的VX2兔常规喂养18-20天。(3)试验流程:VX2肝癌模型兔分为3组。第一组(对照组n=18):给予0.9%生理盐水假栓塞;第二组(栓塞组n=18):给予碘化油和90-180um聚乙烯醇颗粒(PVA)栓塞;第三组(姜黄素脂质体联合栓塞组n=18):给予姜黄素脂质体(20mg/kg body weight)与碘化油乳液和90-180 nm聚乙烯醇颗粒(PVA)栓塞。基于栓塞后VX2兔模型猝死时间再将每组分为3个亚组,第一亚组(n=6):栓塞后6小时组;第二亚组(n=6):栓塞后24小时组;第三亚组(n=6):栓塞后3天组。(4)介入栓塞治疗流程:所有建模成功VX2兔肝癌模型在数字血管减影机下,将微导管依次分别超选择置于腹腔干、肝固有动脉和肿瘤滋养动脉内。对照组给予0.9%生理盐水2 mL;栓塞组给予0.1 mL/kg碘化油和0.1mL 90-180 nm PVA颗粒栓塞;姜黄素脂质体联合栓塞组给予20mg/kg姜黄素脂质体和0.1 mL/kg碘化油乳化后缓慢注射,结束再给予0.1mL 90-180 nmPVA颗粒栓塞。(5)栓塞后依据不同的时间点(6小时、24小时和3天)猝死VX2兔肝癌治疗模型,取出肿瘤组织标本。通过免疫组化测定分析肿瘤组织标本内乏氧诱导细胞-1α(HIF-1(α),血管内皮生成因子(VEGF)和肿瘤微血管密度(microvesseldensity,MVD)的表达。应用聚合酶链式反应(real-time polymeranse chain reaction,RT-PCR)评估检测 VEGFmRNA 的表达水平。结果:(1)姜黄素脂质体特性姜黄素脂质体平均粒径的大小为118.2±0.91mn,脂质体表面的Zeta电位和电荷为-1.66±0.14 mV,透视电镜扫描姜黄素脂质体表面形态符合标准脂质双分子结构。(2)肿瘤体积变化对照组VX2兔在假栓塞治疗前后,肿瘤体积呈明显的上升趋势。而姜黄素脂质体联合栓塞组,其栓塞前后肿瘤体积呈现减小趋势,但栓塞后第3天与栓塞前比较数值不具备显著统计学差异(P0.05)。(3)HIF-1α表达栓塞组免疫组化染色显示:栓塞坏死肿瘤边缘残存肿瘤组织内乏氧诱导细胞-1α(HIF-1α)呈现强染色表现,而姜黄素脂质体联合栓塞组的乏氧诱导细胞-1α(HIF-1α)蛋白染色表现明显降低,两者比较具有显著地统计差异(P,0.05)。(4)VEGF表达栓塞组残存肿瘤组织内血管内皮生成因子(VEGF)呈强阳性染色表现。而姜黄素脂质体联合栓塞组,VEGF蛋白和VEGFmRNA各时间点的表达均低于栓塞组,其两者数值比较具有显著的统计学差异(P0.05)。(5)MVD表达栓塞组栓塞后6小时到3天的动态观察显示:平均微血管密度(MVD)呈现明显的上升趋势(P0.05),而姜黄素脂质联合栓塞组,各时间点的平均微血管密度(MVD)表达则呈现明显下降趋势。(6)相关性分析Spearman's相关性分析表明HIF-1α蛋白与VEGF mRNA具有显著正相关性(r=0.705,P=0.001);与VEGF蛋白的相关性为(r=0.655,P=0.003);与MVD的相关性为(r=0.521,P=0.027),同时VEGF蛋白与MVD也具有良好的相关性(r=0.519,P=0.027)。结论:乏氧诱导肿瘤血管生长在肝脏肿瘤侵袭生长和复发转移过程中具有关键的地位,而姜黄素脂质体能够明显下调HIF-1α蛋白表达水平,从而有效抑制VX2兔肝脏肿瘤栓塞后残存肿瘤组织由于乏氧诱导导致的肿瘤血管生长和复发转移。
[Abstract]:Objective to study the expression and correlation of serum HIF-l and VEGF before and after embolization of hepatic cell cancer: Study of hepatocellular carcinoma (hepatocellular, carcinoma, HCC) in patients with hepatic artery embolization (transcatheter arterial embolization, TAE) and serum hypoxia inducible factor alpha -1 (hypoxia inducible factor1 alpha, HIF-1 alpha) and vascular endothelial growth factor (vascular endothelial growth factor, VEGF) level of expression and correlation; correlation analysis of serum expression of HIF-1 alpha and VEGF levels and clinical features of part HCC. Methods: 42 patients with HCC from June 2014 to December 2015 were collected and 6 cases (control group) were diagnosed with liver hemangioma at the same time. In the analysis of clinical characteristics for all patients after TAE treatment for liver tumor and hemangioma, embolization according to tumor size and location by different dose of iodized oil and embolic agent. Serum was collected from all patients before TAE, third days, seventh days and 1 months after TAE embolism. Serum HIF-1 and VEGF expression was measured by enzyme-linked immunosorbent assay (ELISA) after serum separation. Objective to evaluate the curative effect standard (RECIST1.1) by introducing solid tumors. According to the curative effect, the patients were divided into complete response group (CR), partial curative effect group (PR), disease stabilization group (SD) and disease progression group (PD). To analyze the clinical features of serum HIF-1 alpha, VEGF and HCC, as well as the correlation between them. Results: the serum HIF-1 alpha and VEGF values of hepatic hemangioma before TAE embolization were 27.14 + 5.94 pg/mL and 54.51 + 11.38pg/mL respectively, and the serum HIF-1 alpha and VEGF values of HCC patients were 162.19 + 43.03pg/mL and 278.70 + 55.55ppg/mL before TAE embolization. On the third day after TAE embolization, the levels of serum HIF-1 alpha and VEGF reached peak in HCC patients (HIF-1 = 332.17 + 37.37 pg/mL, VEGF:398.37 + 69.38 pg/mL), which was significantly higher than that before embolization (P0.05). On the seventh day after TAE embolization, serum HIF-1 and VEGF values of HCC patients showed a downward trend (HIF-1, 245.20 + 84.52pg/mL, VEGF:334.25 + 83.88 pg/m L), but it was still significantly higher than that before treatment (P0.05). 1 months after TAE embolization for HCC patients, 7 cases (CR), partial curative effect (PR), disease stability (SD) and disease progression group (PD) were 35 cases. CR and PR+SD+PD two groups of serum HIF-1 alpha and VEGF were as follows: (118.67 + 43.70 pg/mL vs 252.07 + 67.17pg/mL) (199.33 + 31.68pg/mL vs 354.75 + 92.82pg/mL), with significant statistical difference (P0.05). There was a significant positive correlation between serum HIF-1 alpha level and serum VEGF level (r=0.67, P0.05). Meanwhile, serum HIF-1 alpha and VEGF were correlated with clinical characteristics of portal vein tumor thrombus and distal metastasis (P0.05). Conclusion: serum HIF-1 alpha and VEGF play an important role in the progression of HCC, suggesting that hypoxia induced angiogenesis plays a key role in the invasion and metastasis of HCC. At the same time, the expression of serum HIF-1 - alpha and VEGF can effectively evaluate the efficacy of TAE embolization and predict the survival of the patients. Objective to study the mechanism of curcumin liposomes inhibited hypoxia induced angiogenesis after embolization: through the establishment of rabbit VX2 liver cancer model, to investigate the residual tumor after embolization in local tissue hypoxia inducible factor alpha 1- (hypoxia inducible factor 1 alpha, HIF-1a) and vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) and curcumin lipid correlation. Combined with hepatic artery embolization (transcatheter arterial embolization, TAE) in the inhibition of embolism after hypoxia induced angiogenesis in the expression of value. Methods: (1) curcumin liposomes: the soybean lecithin, cholesterol and curcumin with mass ratio of 20:1:2 were placed in the eggshell container, and the appropriate amount of dichloromethane was added to completely dissolve the lipid. The lipid solution forms a uniform layer of thin film, then the vacuum drying for the night, so that the solvent is removed. After adding the phosphate buffer solution, the emulsification temperature is 45. At last, the liposomes are saved by high pressure extrusion of small aperture filter membrane. (2) VX2 rabbit model of liver cancer: the tumor tissue was taken out of the tumor tissue and stored in 0.9% normal saline after the sudden death of the tumor model rabbit. After the model rabbits were anesthetized, the left lobe of the liver was exposed and the 1mm x 1mm x 1mm tumor tissue was planted in the left lobe of the rabbit liver, and then the abdominal incision was sutured. All the successful VX2 rabbits were fed for 18-20 days. (3) test flow: VX2 liver cancer model rabbits were divided into 3 groups. The first group (control group n=18): given 0.9% saline sham embolization; group second (embolization group n=18): given iodized oil and 90-180um polyvinyl alcohol (PVA) embolization; third group (curcumin liposome combined with n=18 embolization group): Curcumin liposomes (20mg/kg body weight) with lipiodol emulsion and 90-180 (polyvinyl alcohol particles NM PVA) embolism. Based on the sudden death time of VX2 rabbit model after embolization, each group was divided into 3 sub groups. The first subgroup (n=6): 6 hours after embolization; second sub group (n=6): 24 hours after embolization; the Sanya group (n=6): 3 days after embolization. (4) interventional embolization treatment process: all models were successfully constructed, and the VX2 rabbit liver cancer model was placed under the digital subtraction angiography, and the microcatheter was placed in the celiac trunk, the proper hepatic artery and the tumor nutrient artery in turn. The control group was given 0.9% saline 2 mL; embolization group received 0.1 mL/kg 0.1mL 90-180 nm PVA and lipiodol embolization; curcumin liposome combined with embolization group were given 20mg/kg curcumin liposomes and 0.1 mL/kg after the end of the slow injection of lipiodol emulsion, then given 0.1mL 90-180 nmPVA particle embolization. (5) the tumor tissue specimens were taken out of the VX2 rabbit liver cancer treatment model at different time points (6 hours, 24 hours and 3 days) after embolization. The expression of -1, HIF-1 (VEGF) and microvesseldensity (MVD) in hypoxic cells of tumor tissues was detected by immunohistochemistry. The application of polymerase chain reaction (real-time polymeranse chain reaction, RT-PCR)
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

【相似文献】