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  本文關鍵詞：姜黃素脂質體抑制肝癌栓塞后乏氧誘導血管生成的機制和實驗研究　出處：《南京中醫(yī)藥大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 肝細胞肝癌 經肝動脈栓塞術 乏氧誘導因子-1α 血管內皮生長因子 乏氧誘導因子-1α 血管生成 栓塞 肝臟腫瘤

【摘要】：肝細胞肝癌栓塞前后血清HIF-lα和VEGF的表達和相關性研究目的:探討研究肝細胞肝癌(hepatocellular carcinoma,HCC)患者經肝動脈栓塞術(transcatheter arterial embolization,TAE)前后血清乏氧誘導因子-1α(hypoxia inducible factor1 alpha,HIF-1α)和血管內皮生成因子(vascular endothelial growth factor,VEGF)的表達水平和相關性;分析血清HIF-1βα和VEGF表達水平與部分HCC臨床特征的相關性。方法:收集2014年6月至2015年12月首診確診HCC患者42例,同期確診肝血管瘤患者6例(對照組)。在對所有患者行臨床特征分析后,行肝臟腫瘤和血管瘤的TAE治療,栓塞時根據腫瘤部位和大小采取不同劑量的碘化油和栓塞劑。靜脈采集所有患者TAE前,TAE栓塞后第3天,第7天和1個月的血清,分離血清后行酶聯免疫吸附試驗(enzyme-linked immunosorbent assay,ELISA)測定血清HIF-1α和VEGF表達。引入實體瘤客觀評價療效標準(RECIST1.1),根據療效將患者分為完全療效組(CR),部分療效組(PR),疾病穩(wěn)定組(SD)與疾病進展組(PD)。分析血清HIF-1α、VEGF與HCC各項臨床特征,以及兩者之間的相關性。結果:肝血管瘤TAE栓塞前血清HIF-1α和VEGF值分別為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 值為 162.19±43.03pg/mL和278.70±55.55ppg/mL,兩者比較具有統(tǒng)計學差異(P0.05)。HCC患者TAE栓塞后第3天血清 HIF-1α 和 VEGF 達到峰值(HIF-1α:332.17±37.37 pg/mL,VEGF:398.37±69.38 pg/mL),明顯高于栓塞前表達水平(P0.05)。HCC患者TAE栓塞后第7天血清HIF-1α和 VEGF 值呈下降趨勢(HIF-1α:245.20±84.52pg/mL,VEGF:334.25±83.88 pg/m L),但是仍明顯高于治療前的表達水平(P0.05)。HCC患者TAE栓塞治療后1個月復診評估,完全療效組(CR)7例,部分療效(PR),疾病穩(wěn)定(SD)與疾病進展組(PD)共35 例。CR 與 PR+SD+PD 兩組血清 HIF-1α 和 VEGF 比較如下:(118.67±43.70 pg/mL vs 252.07±67.17pg/mL)(199.33±31.68pg/mL vs 354.75±92.82pg/mL),具有顯著統(tǒng)計學差異(P0.05)。血清HIF-1α表達水平與血清VEGF具有明顯的正相關性(r=0.67,P0.05),同時血清HIF-1α、VEGF與門靜脈癌栓和遠端轉移臨床特征具有相關性(P0.05)。結論:血清HIF-1α與VEGF在肝細胞肝癌疾病進程中具有重要意義,表明乏氧微環(huán)境誘導的血管生成在HCC侵襲轉移過程中起著關鍵作用。同時,血清HIF-1α與VEGF表達能夠有效地評估TAE栓塞治療的療效和預測患者生存狀況。姜黃素脂質體抑制栓塞后乏氧誘導血管生成的機制研究目的:通過建立VX2兔肝癌模型,探討栓塞后殘存腫瘤組織局部乏氧誘導因子1-α(hypoxia inducible factor 1 alpha,HIF-1a)和血管內皮生成因子(vascular endothelial growth factor,VEGF)的相關性,以及姜黃素脂質體聯合肝動脈栓塞(transcatheter arterial embolization,TAE)在抑制栓塞后乏氧誘導腫瘤血管生成表達中的價值。方法:(1)姜黃素脂質體:精密稱取質量比為20:1:2的大豆卵磷脂、膽固醇及姜黃素置于茄形容器中,加入適量二氯甲烷使脂質完全溶解。脂質溶液形成一層均勻的薄膜,然后真空干燥過夜,使溶劑除盡。加入磷酸鹽緩沖液后充分乳化,乳化溫度為45℃。最后脂質體通過小孔徑濾膜高壓擠出保存?zhèn)溆谩?2)VX2兔肝癌模型:將腫瘤傳代模型兔猝死后取出腫瘤組織置于0.9%生理鹽水中保存。將預建模型兔麻醉后行腹部正中切口,暴露肝臟左葉,取1mm×1mm× 1mm腫瘤組織種植于兔肝臟左葉,然后縫合腹部切口。所有建模成功的VX2兔常規(guī)喂養(yǎng)18-20天。(3)試驗流程:VX2肝癌模型兔分為3組。第一組(對照組n=18):給予0.9%生理鹽水假栓塞;第二組(栓塞組n=18):給予碘化油和90-180um聚乙烯醇顆粒(PVA)栓塞;第三組(姜黃素脂質體聯合栓塞組n=18):給予姜黃素脂質體(20mg/kg body weight)與碘化油乳液和90-180 nm聚乙烯醇顆粒(PVA)栓塞�；谒ㄈ骎X2兔模型猝死時間再將每組分為3個亞組,第一亞組(n=6):栓塞后6小時組;第二亞組(n=6):栓塞后24小時組;第三亞組(n=6):栓塞后3天組。(4)介入栓塞治療流程:所有建模成功VX2兔肝癌模型在數字血管減影機下,將微導管依次分別超選擇置于腹腔干、肝固有動脈和腫瘤滋養(yǎng)動脈內。對照組給予0.9%生理鹽水2 mL;栓塞組給予0.1 mL/kg碘化油和0.1mL 90-180 nm PVA顆粒栓塞;姜黃素脂質體聯合栓塞組給予20mg/kg姜黃素脂質體和0.1 mL/kg碘化油乳化后緩慢注射,結束再給予0.1mL 90-180 nmPVA顆粒栓塞。(5)栓塞后依據不同的時間點(6小時、24小時和3天)猝死VX2兔肝癌治療模型,取出腫瘤組織標本。通過免疫組化測定分析腫瘤組織標本內乏氧誘導細胞-1α(HIF-1(α),血管內皮生成因子(VEGF)和腫瘤微血管密度(microvesseldensity,MVD)的表達。應用聚合酶鏈式反應(real-time polymeranse chain reaction,RT-PCR)評估檢測 VEGFmRNA 的表達水平。結果:(1)姜黃素脂質體特性姜黃素脂質體平均粒徑的大小為118.2±0.91mn,脂質體表面的Zeta電位和電荷為-1.66±0.14 mV,透視電鏡掃描姜黃素脂質體表面形態(tài)符合標準脂質雙分子結構。(2)腫瘤體積變化對照組VX2兔在假栓塞治療前后,腫瘤體積呈明顯的上升趨勢。而姜黃素脂質體聯合栓塞組,其栓塞前后腫瘤體積呈現減小趨勢,但栓塞后第3天與栓塞前比較數值不具備顯著統(tǒng)計學差異(P0.05)。(3)HIF-1α表達栓塞組免疫組化染色顯示:栓塞壞死腫瘤邊緣殘存腫瘤組織內乏氧誘導細胞-1α(HIF-1α)呈現強染色表現,而姜黃素脂質體聯合栓塞組的乏氧誘導細胞-1α(HIF-1α)蛋白染色表現明顯降低,兩者比較具有顯著地統(tǒng)計差異(P,0.05)。(4)VEGF表達栓塞組殘存腫瘤組織內血管內皮生成因子(VEGF)呈強陽性染色表現。而姜黃素脂質體聯合栓塞組,VEGF蛋白和VEGFmRNA各時間點的表達均低于栓塞組,其兩者數值比較具有顯著的統(tǒng)計學差異(P0.05)。(5)MVD表達栓塞組栓塞后6小時到3天的動態(tài)觀察顯示:平均微血管密度(MVD)呈現明顯的上升趨勢(P0.05),而姜黃素脂質聯合栓塞組,各時間點的平均微血管密度(MVD)表達則呈現明顯下降趨勢。(6)相關性分析Spearman's相關性分析表明HIF-1α蛋白與VEGF mRNA具有顯著正相關性(r=0.705,P=0.001);與VEGF蛋白的相關性為(r=0.655,P=0.003);與MVD的相關性為(r=0.521,P=0.027),同時VEGF蛋白與MVD也具有良好的相關性(r=0.519,P=0.027)。結論:乏氧誘導腫瘤血管生長在肝臟腫瘤侵襲生長和復發(fā)轉移過程中具有關鍵的地位,而姜黃素脂質體能夠明顯下調HIF-1α蛋白表達水平,從而有效抑制VX2兔肝臟腫瘤栓塞后殘存腫瘤組織由于乏氧誘導導致的腫瘤血管生長和復發(fā)轉移。
[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)
【學位授予單位】：南京中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.7

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