【作者】 周波；

【导师】 王建华；

【作者基本信息】 复旦大学 ， 医学影像学与核医学， 2012， 博士

【摘要】 目的：研究人参皂甙Rg3对肝癌细胞McA-RH7777及血管内皮细胞的抑制作用。材料和方法：取大鼠肝癌细胞株McA-RH7777和人脐静脉内皮细胞(HUVEC),用WST-1法检测血管内皮生长因子-165(VEGF-165)、血小板衍生性生长因子-BB(PDGF-BB)和人参皂甙Rg3分别对它们的促增殖或毒性作用；PI单染法流式细胞术检测人参皂甙Rg3对各细胞株的促凋亡作用；细胞划痕试验检测人参皂甙Rg3对HUVEC的迁移抑制作用；细胞免疫荧光法(IF)和免疫蛋白杂交印迹法(WB)检测各细胞株VEGFR-2、p VEGFR-2、PDGFR-β和p PDGFR-β的表达,以及不同浓度人参皂甙Rg3对它们的抑制作用。经软件SPSS11.0作统计分析。结果：VEGF浓度在10ng/mL和PDGF浓度在l0ng/mL以上,对HUVEC有明显促增殖作用(P值均小于0.05)。(VEGF+PDGF)浓度在25ng/ml以上对大鼠肝癌细胞McA-RH7777有促增殖作用(P值小于0.05)。Rg3对HUVEC有显著的抑制作用(P<0.001)。两两比较示在0.001μM及其以上浓度,对HUVEC即有明显的抑制作用(所有P均<0.001),且与Rg3浓度存在量效关系。Rg3对大鼠肝癌细胞McA-RH7777存在显著的抑制作用(P<0.001)。两两比较示在0.001μM及其以上浓度,对McA-RH7777即有明显的抑制作用(所有P均<0.001),且与Rg3浓度存在量效关系。HUVEC空白对照、Rg3浓度0.1μM、1μM时凋亡率分别为4.05%、12.56%和20.06%。McA-RH7777在空白对照、0.1μM、1μM时凋亡率分别为2.74%、10.22%和17.53%。细胞划痕实验Rg3组划痕未缩小,至24h、48h时渐增宽。免疫细胞荧光化学法检测HUVEC和McA-RH7777的VEGFR-2受体加Rg3后磷酸化均明显受到抑制。Western Blot检测HUVEC和McA-RH7777两种细胞VEGFR-2和PDGFR-β均有表达,予生长因子VEGF. PDGF刺激后,两种受体磷酸化状态明显；分别与Rg3不同浓度共培养后,两种受体表达量(与β-actin比较)无明显变化,但磷酸化状态受到抑制,抑制程度与Rg3浓度呈正比。目的：评价动脉栓塞术(TAE)联合人参皂甙Rg3治疗大鼠肝癌的疗效,探讨各种治疗干预后肿瘤新生血管变化及分子水平变化机制。材料和方法：将大鼠肝癌细胞株McA-RH7777植入Buffalo大鼠肝脏建立大鼠肝癌模型,随机分成四组：生理盐水组(A组)、Rg3组(B组)、TAE组(C组)、TAE+Rg3组。观察大鼠生存时间、肿瘤直径、体重变化、肿瘤转移情况。各组分别于术后第1周、第2周、第4周及第8周分别作MRI平扫；同时取肿瘤组织,大体观察后作酶联免疫吸附试验(ELISA)、免疫组化及蛋白免疫印迹(WB)检测MVD (CD31标记)、HIF-1α、VEGF、VEGFR-2、p VEGFR-2、b-FGF。经软件SPSS11.0作统计分析。结果：各组间大鼠生存期差异有统计学意义(P<0.05),两两比较B组和D组生存期显著长于A组(P均<0.05)。D组比C组的相对风险度为0.32,95%CI为(0.034-3.111)(P=0.328),B组相对A组为0.11,95%CI(0.013-0.954)(P=0.045)。不同时间段各组肿瘤直径变化差异明显(P<0.001),与A组相比,B、D组术后各时间段肿瘤生长均明显减缓(P均<0.05)。使用人参皂甙Rg3的B组和D组发生肿瘤转移情况要少于A组和C组,尤其是肝内转移和远处转移差异明显(P均<0.05)。使用人参皂甙Rg3的B组和D组的大鼠术后体重恢复及增重情况好于A组和C组(P均<0.05)。ELISA检测肿瘤内VEGF各组间差异明显(P均<0.05),从术后第二周开始使用人参皂甙Rg3的B组和D组的大鼠肿瘤内VEGF明显低于A组和C组(P均<0.05)。免疫组化IHC示MVD各组间差异明显(P<0.05),使用人参皂甙Rg3的B组和D组的大鼠肿瘤内MVD计数明显低于同时间段的A组和C组(P均<0.05)。HIF-1α在术后第一周各组间差异有统计学意义(P<0.05),C、D组表达比A、B组明显增强(P<0.05),第二周到第八周各组间无明显差异。D组相对C组大鼠各时间段肿瘤内表达VEGF染色强度明显减弱(P<0.05)。B、D组肿瘤内VEGFR-2在术后四周、八周时均较A、C组有不同程度的减少,组间差异有统计意义(PP<0.05)。B组p-VEGFR-2相对于A组在术后一、二周却受到明显的抑制(PP<0.05),而D组p-VEGFR-2相对于C组在术后四周、八周抑制增强,组间差异有统计学意义(P<0.05)。WB示术后各时间段B组CD31表达低于A组(P<0.05),第二、四周D组CD31表达低于C组(P<0.05)。使用人参皂甙Rg3的B组和D组的肿瘤内b-FGF表达低于A组和C组(P均<0.05)。B组相对A组肿瘤内VEGFR-2表达和磷酸化水平各时间段受到明显抑制(P均<0.05),而D组相对C组肿瘤内VEGFR-2表达和磷酸化水平第一、二、四周受到明显抑制(P均<0.05)。结论：人参皂甙Rg3与TAE联合治疗大鼠肝癌,阻断多种促血管生成因子受体,抑制新生血管生成,提高了TAE的疗效；目的：评价人参皂甙Rg3口服胶囊联合介入动脉化疗栓塞(TACE)治疗肝癌的有效性,安全性,及抑制血管生长因子状况。材料与方法：根据自愿意向治疗原则分别纳入TACE+Rg3组141人,TACE组141人。实验方案通过伦理审查并注册,所有患者均签署知情同意书。TACE+Rg3组接受常规介入治疗同时口服人参皂甙Rg3(参一胶囊)40mg/d,至少连续口服3个月以上。对照组TACE组仅接受介入治疗。主要观察指标是生存期(0S)和无疾病进展生存期(PFS),并记录所有患者发生转移情况,不良反应情况。治疗8周后评估肿瘤反应情况,部分患者随访血清VEGF变化情况。结果：有效纳入统计TACE+Rg3组80人,TACE组82人。本研究分析时间点为2012年3月1日,共62名患者随访到死亡,其中TACE+Rg3组死亡29人,TACE组死亡33人。总OS无明显差异(24个月vs24个月,P=0.092),以Child分期分层统计发现Child B期患者TACE+Rg3治疗能显著提高中位生存期25个月(95%CI13.2-36.8)vsTACE组14个月(95%CI10.7-17.3个月),P=0.023。相对风险下降67%,95%CI(0.12-0.91),P=0.033。总中位PFS:TACE+Rg3组5个月vsTACE组4个月,P=0.064。相对风险率(Hazard rate, HR)-0.75,95%CI (0.54-1.04), P=0.089. Child分层分析后Child B期患者中位PFS为5个月(95%CI3.8-6.2个月)vs B组3个月(95%CI1.9-4.0个月),P=0.005。相对风险率(Hazard rate, HR)=0.46,95%CI (0.24-0.85),P=0.014。肿瘤反应方面TACE+Rg3组有效率(PR+SD)相对于TACE组有明显提高(72.5%vs56.1%,P=0.029)。肿瘤转移方面TACE+Rg3组发生淋巴结转移相对于TACE组有明显减少(27.5%vs43.9%,P=0.029)。首次治疗术后2个月和6个月时TACE+Rg3组血清VEGF比TACE组明显下降(P=0.000)。不良反应方面两组大部分无明显差异,TACE+Rg3组术后对白细胞恢复和胆红素恢复正常以及体重营养状况均好于TACE组(P<0.05)。结论：TACE联合人参皂甙Rg3治疗肝癌有效,安全,并能有效抑制VEGF蛋白表达。更多还原

【Abstract】 Obejective:To evaluate the suppression of HCC cells and vascular endothelial with Gensenoside Rg3. Materials and methods:Rat HCC cell lines (McA-RH7777) and human umbilical vein endothelial cell (HUVEC) were used in this part. The proliferation or toxicity of VEGF-165, PDGF-BB and Gensenoside Rg3for these cells was detected by WST-1method. The apoptosis of these cells with Gensenoside Rg3was also detected with PI staining method by flow cytometry. The inhibition of HUVEC with Gensenoside Rg3was tested by cell scratch test. The inhibition of VEGFR-2、p VEGFR-2、PDGFR-β and p PDGFR-β with Gensenoside Rg3in different concentration was detected by cell immunofluorescence and western blotting (WB). All data were processed by SPSS11.0. Results:The proliferation of HUVEC was promoted by VEGF at10ng/mL and above, or by PDGF at10ng/mL and above (P<0.05). The proliferation of HCC cell (McA-RH7777) was promoted by VEGF+PDGF at25ng/ml and above(P<0.05). The proliferation of HUVEC and McA-RH7777cells was suppressed by Gensenoside Rg3signigicantly at0.001μM and above(P<0.001). The apoptosis of HUVEC was4.05%without Gensenoside Rg3,12.56%at0.1μM of Gensenoside Rg3and20.06%at1μM Gensenoside Rg3by flow cytometry.While, the apoptosis of McA-RH7777cells was2.74%,10.22%,17.53%with OμM,0.1μM,1μM Gensenoside Rg3. HUVEC didn’t cross the scratch after incubation with Gensenoside Rg3in Scratch test. The phosphorylation of VEGFR-2of HUVEC and McA-RH7777cells was suppressed by Gensenoside Rg3in cell immunofluorescence test. The phosphorylation of VEGFR-2and PDGFR-β was found to be suppressed by Gensenoside Rg3in Western blotting test, but not in expression of VEGFR-2and PDGFR-β. Conclusions:1. Gensenoside Rg3can suppress the proliferation of HUVEC and promotes its apoptosis.2. Gensenoside Rg3can also inhibite HCC cells at high concentration. Obejective:To investigate the effects of the combination of TAE and Gensenoside Rg3for HCC in rat model, and study the mechanism of angiogenesis and molecular level. Materials and methods:Rat HCC models, established with McA-RH7777and Buffalo strait rats, were divided randomly into four groups:saline group (group A, control group), Rg3group (group B), TAE group (group C), TAE+Rg3group (group D). Rat survival time, tumor diameter, weight and tumor metastasis were evaluated. MRI plain scan were applied for five groups at1week,2weeks,4weeks and8weeks after surgery and at the same time, some rats were sacrificed for the detection of MVD(labeled by CD31), HIF-la, VEGF, VEGFR-2, p VEGFR-2and b-FGF with IHC and/or WB. All data were processed by SPSS11.0. Results:There was statistical difference in survival time among4groups (P<0.05), and group B and group D had longer survival than group A between each two groups compare (P<0.05). The hazard ratio in group D compared to group C was0.32,95%CI (0.034-3.111),(P=0.328), while group B compared to group A was0.11,95%CI (0.013-0.954),(P=0.045)。The diameter of tumor increased significantly faster than other three group(P<0.05). The occurrences of metastasis in group B and group D which Rg3was used were significantly lower than those in group A and group C(P<0.05). The weight of rats in group B and D was significantly superior to that in group A and C (P <0.05). VEGF expression of tumor was significantly different among4groups2weeks later after intervention by ELISA test (P<0.001), which was significantly lower in group B and D (P<0.05). MVD of tumor was significantly different among4groups all time after intervention by IHC (P<0.05), which was significantly lower in group B and group D (P<0.05). HIF-1α expression of tumor was not significantly different (P>0.05) except which was higher in group C and D compared with group A and B at1week after intervention by IHC (P<0.05). VEGF expression of tumor in group D was significantly lower than that in group C by IHC (P<0.05). VEGFR-2expression in group B and D was significantly lower than that in group A and C4weeks later after intervention by IHC (P<0.05). Expression of p-VEGFR-2was significantly lower in group B than it in group A at first two weeks after intervention by IHC (P<0.05), but it was until4weeks later after TAE that p-VEGFR-2in group D was lower than it in group C (P<0.05). WB test showed that CD31in group D was lower than it in group C at2and4weeks (P<0.05), while it in group B was lower than it in group A all time after intervention (P<0.05). Expression of b-FGF in group B and D was lower than it in group A and D all time by WB test (P<0.05). However, the expression and phosphorylation of VEGFR-2was inhibited significantly among4groups all time after intervention (P<0.05). Conclusions:Gensenoside Rg3, with combination of TAE for treatment of HCC, inhibits several angiogenesis-related receptors, suppresses angiogenesis, and enhances the effect of TAE. Obejective:To investigate the effects and safety of the combination of TACE and Gensenoside Rg3for HCC in patients, and study the mechanism of angiogenesis and molecular level. Materials and methods:Two haundren and eighty two patients were divided into two groups:TACE+Rg3group (n=141) and TACE group (n=141). All patients received TACE as regular and patients in TACE+Rg3group took Gensenoside Rg340mg/d at least3months. Primary outcomes were overall survival and Progression free survival. Secondary outcomes included the response rate, metastasis rate, serum VEGF level, and and safety. Results:At the end of study,80patients in TACE+Rg3group and82patients in TACE group were under analysis. And62deaths had occurred,29in TACE+Rg3group and33in TACE group. Median overall survivial was no difference in two groups(24months vs24months, P=0.092), but in all Child B patients, median survival was25months (95%CI13.2-36.8) in TACE+Rg3group vs14months in TACE group (95%CI10.7-17.3), P=0.023。 HR=0.33,95%CI(0.12-0.91), P=0.033。Median PFS was also no difference5months in TACE+Rg3group and4months in TACE group (P=0.064), HR=0.75,95%CI (0.54-1.04), P=0.089。But in all Child B patients, median PFS was5months (95%CI3.8-6.2months) in TACE+Rg3group vs3months,(95%CI1.9-4.0months) in TACE group, P=0.005。HR=0.46,95%CI (0.24-0.85), P=0.014. Response rate (PR+SD) in TACE+Rg3group was significantly better than it in TACE group (72.5%vs56.1%, P=0.029). There was less lymph nodes metastasis found in TACE+Rg3group (27.5%vs43.9%, P=0.029). The levels of serum VEGF in TACE+Rg3group was significantly lower than those in TACE group after2months and6months therapy (P=0.000). The recovery of white blood cell level,serum total bilirubin level and weight was better in TACE+Rg3group (P<0.05). There was no difference in other adverse events. Conclusion:Gensenoside Rg3is effective and safe with TACE to control HCC, and it can suppress VEGF protein expression.更多还原