本文關(guān)鍵詞：姜黃素脂質(zhì)體抑制肝癌栓塞后乏氧誘導(dǎo)血管生成的機(jī)制和實(shí)驗(yàn)研究　出處：《南京中醫(yī)藥大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肝細(xì)胞肝癌 經(jīng)肝動(dòng)脈栓塞術(shù) 乏氧誘導(dǎo)因子-1α 血管內(nèi)皮生長(zhǎng)因子 乏氧誘導(dǎo)因子-1α 血管生成 栓塞 肝臟腫瘤

【摘要】：肝細(xì)胞肝癌栓塞前后血清HIF-lα和VEGF的表達(dá)和相關(guān)性研究目的:探討研究肝細(xì)胞肝癌(hepatocellular carcinoma,HCC)患者經(jīng)肝動(dòng)脈栓塞術(shù)(transcatheter arterial embolization,TAE)前后血清乏氧誘導(dǎo)因子-1α(hypoxia inducible factor1 alpha,HIF-1α)和血管內(nèi)皮生成因子(vascular endothelial growth factor,VEGF)的表達(dá)水平和相關(guān)性;分析血清HIF-1βα和VEGF表達(dá)水平與部分HCC臨床特征的相關(guān)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(duì)照組)。在對(duì)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(shí)根據(jù)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(gè)月的血清,分離血清后行酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immunosorbent assay,ELISA)測(cè)定血清HIF-1α和VEGF表達(dá)。引入實(shí)體瘤客觀評(píng)價(jià)療效標(biāo)準(zhǔn)(RECIST1.1),根據(jù)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(jìn)展組(PD)。分析血清HIF-1α、VEGF與HCC各項(xiàng)臨床特征,以及兩者之間的相關(guān)性。結(jié)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(jì)學(xué)差異(P0.05)。HCC患者TAE栓塞后第3天血清 HIF-1α 和 VEGF 達(dá)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(dá)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(shì)(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(dá)水平(P0.05)。HCC患者TAE栓塞治療后1個(gè)月復(fù)診評(píng)估,完全療效組(CR)7例,部分療效(PR),疾病穩(wěn)定(SD)與疾病進(jìn)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(jì)學(xué)差異(P0.05)。血清HIF-1α表達(dá)水平與血清VEGF具有明顯的正相關(guān)性(r=0.67,P0.05),同時(shí)血清HIF-1α、VEGF與門靜脈癌栓和遠(yuǎn)端轉(zhuǎn)移臨床特征具有相關(guān)性(P0.05)。結(jié)論:血清HIF-1α與VEGF在肝細(xì)胞肝癌疾病進(jìn)程中具有重要意義,表明乏氧微環(huán)境誘導(dǎo)的血管生成在HCC侵襲轉(zhuǎn)移過(guò)程中起著關(guān)鍵作用。同時(shí),血清HIF-1α與VEGF表達(dá)能夠有效地評(píng)估TAE栓塞治療的療效和預(yù)測(cè)患者生存狀況。姜黃素脂質(zhì)體抑制栓塞后乏氧誘導(dǎo)血管生成的機(jī)制研究目的:通過(guò)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(dǎo)因子1-α(hypoxia inducible factor 1 alpha,HIF-1a)和血管內(nèi)皮生成因子(vascular endothelial growth factor,VEGF)的相關(guān)性,以及姜黃素脂質(zhì)體聯(lián)合肝動(dòng)脈栓塞(transcatheter arterial embolization,TAE)在抑制栓塞后乏氧誘導(dǎo)腫瘤血管生成表達(dá)中的價(jià)值。方法:(1)姜黃素脂質(zhì)體:精密稱取質(zhì)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(zhì)完全溶解。脂質(zhì)溶液形成一層均勻的薄膜,然后真空干燥過(guò)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(zhì)體通過(guò)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(yù)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(yàn)流程:VX2肝癌模型兔分為3組。第一組(對(duì)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(zhì)體聯(lián)合栓塞組n=18):給予姜黃素脂質(zhì)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(shí)間再將每組分為3個(gè)亞組,第一亞組(n=6):栓塞后6小時(shí)組;第二亞組(n=6):栓塞后24小時(shí)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(shù)字血管減影機(jī)下,將微導(dǎo)管依次分別超選擇置于腹腔干、肝固有動(dòng)脈和腫瘤滋養(yǎng)動(dòng)脈內(nèi)。對(duì)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(zhì)體聯(lián)合栓塞組給予20mg/kg姜黃素脂質(zhì)體和0.1 mL/kg碘化油乳化后緩慢注射,結(jié)束再給予0.1mL 90-180 nmPVA顆粒栓塞。(5)栓塞后依據(jù)不同的時(shí)間點(diǎn)(6小時(shí)、24小時(shí)和3天)猝死VX2兔肝癌治療模型,取出腫瘤組織標(biāo)本。通過(guò)免疫組化測(cè)定分析腫瘤組織標(biāo)本內(nèi)乏氧誘導(dǎo)細(xì)胞-1α(HIF-1(α),血管內(nèi)皮生成因子(VEGF)和腫瘤微血管密度(microvesseldensity,MVD)的表達(dá)。應(yīng)用聚合酶鏈?zhǔn)椒磻?yīng)(real-time polymeranse chain reaction,RT-PCR)評(píng)估檢測(cè) VEGFmRNA 的表達(dá)水平。結(jié)果:(1)姜黃素脂質(zhì)體特性姜黃素脂質(zhì)體平均粒徑的大小為118.2±0.91mn,脂質(zhì)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(zhì)體表面形態(tài)符合標(biāo)準(zhǔn)脂質(zhì)雙分子結(jié)構(gòu)。(2)腫瘤體積變化對(duì)照組VX2兔在假栓塞治療前后,腫瘤體積呈明顯的上升趨勢(shì)。而姜黃素脂質(zhì)體聯(lián)合栓塞組,其栓塞前后腫瘤體積呈現(xiàn)減小趨勢(shì),但栓塞后第3天與栓塞前比較數(shù)值不具備顯著統(tǒng)計(jì)學(xué)差異(P0.05)。(3)HIF-1α表達(dá)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(nèi)乏氧誘導(dǎo)細(xì)胞-1α(HIF-1α)呈現(xiàn)強(qiáng)染色表現(xiàn),而姜黃素脂質(zhì)體聯(lián)合栓塞組的乏氧誘導(dǎo)細(xì)胞-1α(HIF-1α)蛋白染色表現(xiàn)明顯降低,兩者比較具有顯著地統(tǒng)計(jì)差異(P,0.05)。(4)VEGF表達(dá)栓塞組殘存腫瘤組織內(nèi)血管內(nèi)皮生成因子(VEGF)呈強(qiáng)陽(yáng)性染色表現(xiàn)。而姜黃素脂質(zhì)體聯(lián)合栓塞組,VEGF蛋白和VEGFmRNA各時(shí)間點(diǎn)的表達(dá)均低于栓塞組,其兩者數(shù)值比較具有顯著的統(tǒng)計(jì)學(xué)差異(P0.05)。(5)MVD表達(dá)栓塞組栓塞后6小時(shí)到3天的動(dòng)態(tài)觀察顯示:平均微血管密度(MVD)呈現(xiàn)明顯的上升趨勢(shì)(P0.05),而姜黃素脂質(zhì)聯(lián)合栓塞組,各時(shí)間點(diǎn)的平均微血管密度(MVD)表達(dá)則呈現(xiàn)明顯下降趨勢(shì)。(6)相關(guān)性分析Spearman's相關(guān)性分析表明HIF-1α蛋白與VEGF mRNA具有顯著正相關(guān)性(r=0.705,P=0.001);與VEGF蛋白的相關(guān)性為(r=0.655,P=0.003);與MVD的相關(guān)性為(r=0.521,P=0.027),同時(shí)VEGF蛋白與MVD也具有良好的相關(guān)性(r=0.519,P=0.027)。結(jié)論:乏氧誘導(dǎo)腫瘤血管生長(zhǎng)在肝臟腫瘤侵襲生長(zhǎng)和復(fù)發(fā)轉(zhuǎn)移過(guò)程中具有關(guān)鍵的地位,而姜黃素脂質(zhì)體能夠明顯下調(diào)HIF-1α蛋白表達(dá)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(dǎo)導(dǎo)致的腫瘤血管生長(zhǎng)和復(fù)發(fā)轉(zhuǎn)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(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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