射频消融联合肝动脉栓塞治疗肝癌后残余肿瘤的动物实验研究
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摘要
目的:通过动物实验研究射频消融联合肝动脉栓塞治疗肝癌后残余肿瘤生物学行为的变化,初步探讨联合治疗的机制,评价其治疗效果。
方法:健康清洁级新西兰大白兔45只在CT引导下行VX2瘤块种植法建立成肿瘤直径2.5cm～2.7cm大小的肝癌动物模型。建模成功后随机分成对照组(假性治疗组)、单纯射频治疗组和肝动脉碘油栓塞7天后射频治疗组(单极射频针裸露端长均为2cm),共3大组。每组分别于治疗结束后1天、4天、7天处死5只瘤兔。切取肿瘤中心组织及边缘组织为检测标本,全部组织经10%Formalin固定,常规石蜡包埋,4μm连续切片,对取材标本行HE染色观察治疗后病理形态学变化。肿瘤边缘组织行免疫组织化学方法检测残余肿瘤细胞凋亡、增殖细胞核抗原、血管内皮生长因子及微血管密度的表达。所有数据均用统计软件包SPSS13.0处理。计量资料用X±S表示,以P<0.05有统计学差异。
结果:1.TUNEL法及Caspase-3检测细胞凋亡结果显示:单纯射频及联合治疗组凝固性坏死区周边的肿瘤细胞凋亡指数(AI)明显高于对照组(P<0.01),以射频后1天最为明显,后逐渐减少,7天后虽明显减少但仍高于术前水平。且联合治疗组各时间点残余肿瘤细胞凋亡指数也高于单纯射频组(P<0.01)。2.单纯射频及联合治疗组残余肿瘤细胞增殖指数(PI)明显低于对照组(P<0.01),且联合治疗组残余肿瘤细胞增殖指数(PI)也低于单纯射频组(P<0.05)。3.单纯射频及联合治疗组VEGF低于对照组(P<0.05)。联合治疗组VEGF绝对值略高于单纯射频组,但差异无统计学意义(P>0.05)。4.单纯射频及联合治疗组残余肿瘤微血管密度(MVD)明显低于对照组(P<0.01)。联合治疗组残余肿瘤微血管密度(MVD)绝对值略高于单纯射频组,但差异无统计学意义(P>0.05)。5.残余肿瘤细胞VEGF与MVD表达呈正相关(P<0.01)。
结论:1.单纯射频和射频消融联合肝动脉栓塞治疗兔肝VX2肿瘤一周内均能诱导残余肿瘤细胞凋亡,同时抑制肿瘤细胞增殖。2.联合治疗与单纯射频治疗相比更能促进残余肿瘤细胞凋亡,抑制肿瘤细胞增殖。3.单纯射频和联合治疗一周内均能抑制肿瘤血管生成,且联合治疗在抑制肿瘤血管生成作用方面与单纯射频治疗作用相当。4.联合治疗后残余肿瘤内仍有VEGF表达,仍存在肿瘤血管生成。临床上如联合应用抗肿瘤血管生成药物可能会取得更好的疗效。
Objectives: To study the biological behavior of remnant tumor of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) combined with transcatheter arterial embolization (TAE); to investigate the mechanism of the combined therapy; and to assess the therapeutic efficacy of it.
Methods: Forty-five New Zealand white rabbits were implanted percutaneously with VX2 tumor cells in the left lobe of the liver under CT guidance successfully. Three weeks later, there was a focal tumor (φ2.5cm-2.7cm) in the liver of each rabbit. Then they were divided into 3 groups at random: control group (pseudo-therapy group), RFA (single electrode 2.0cm exposure) alone group and 7 days after TAE combined with RFA (single electrode 2.0cm exposure) sequentially group. Each group was divided into 3 subgroups respectively according to the killing time: 1 day, 4 days and 7 days after treatment. All the 45 rabbit-models were sacrificed after treatment and their tissues,including peripherial tumor and central tissue of the ablation area,were sectioned, embedded in paraffin, fixed in 10% Formalinand and spitted constantly with 4μm–section. They were examined pathologically by HE dying and immunohistochemical methods (Apoptosis, CD31, VEGF, PCNA). The results of immunohistochemistry were compared. All the data were analysed by SPSS 13.0 software. Quantitative data were showed by x±s, P<0.05 was defined as threshold for statistical significance .
Results: 1. Quantification assay of apoptosis measured by TUNEL and Caspase-3 staining methods indicated that apoptosis index (AI) in the margin of the tumor after RFA alone and combined therapy were both higher than that of control group significantly (P<0.01), they reached their peaks at 1 day, and decreased gradually along with the time to a low level at 7 days after treatment , but were still higher than that of pre-treatment . And AI of each subgroup after combined therapy were higher than that of RFA alone therapy significantly (P<0.01) . 2. Proliferation index (PI) of survival tumor cells after RFA alone and combined therapy were both lower than control group significantly (P<0.01) , and PI of each subgroup after combined therapy were lower than that of RFA alone therapy (P<0.05) . 3. Vascular endothelial grow factors (VEGF) of survival tumor cells after RFA alone and combined therapy were both lower than control group significantly (P<0.05). VEGF of each subgroup after combined therapy were higher than VEGF of RFA alone therapy slightly , but there was no statistical significance between them (P<0.05). 4. Microvessel density (MVD) of survival tumor cells after RFA alone and combined therapy were both lower than control group significantly (P<0.01) . MVD of each subgroup after combined therapy were higher than MVD of RFA alone therapy slightly, but there was no statistical significance between them (P<0.05) . 5. There was a positive correlation between VEGF and MVD in the combined treatment groups (P<0.01) .
Conclusions: 1. Both RFA alone and RFA combined with TAE therapy can induce the apoptosis and inhibit the cell proliferation in 1 week after treatment. 2. Compared with RFA alone , combined therapy can induce more cells to apoptosis and inhibit more cells proliferation , and can get better therapeutic efficacy . 3. Both RFA alone and RFA combined with TAE therapy can effectively inhibit tumor angiogenesis in 1 week after treatment. As for the efficacy of it, combined therapy is parallel with RFA alone. 4. Duing to tumor angiogenesis existed, combined therapy plus anti-angiogenesis drugs may obtain better therapeutic efficacy.
方法:健康清洁级新西兰大白兔45只在CT引导下行VX2瘤块种植法建立成肿瘤直径2.5cm～2.7cm大小的肝癌动物模型。建模成功后随机分成对照组(假性治疗组)、单纯射频治疗组和肝动脉碘油栓塞7天后射频治疗组(单极射频针裸露端长均为2cm),共3大组。每组分别于治疗结束后1天、4天、7天处死5只瘤兔。切取肿瘤中心组织及边缘组织为检测标本,全部组织经10%Formalin固定,常规石蜡包埋,4μm连续切片,对取材标本行HE染色观察治疗后病理形态学变化。肿瘤边缘组织行免疫组织化学方法检测残余肿瘤细胞凋亡、增殖细胞核抗原、血管内皮生长因子及微血管密度的表达。所有数据均用统计软件包SPSS13.0处理。计量资料用X±S表示,以P<0.05有统计学差异。
结果:1.TUNEL法及Caspase-3检测细胞凋亡结果显示:单纯射频及联合治疗组凝固性坏死区周边的肿瘤细胞凋亡指数(AI)明显高于对照组(P<0.01),以射频后1天最为明显,后逐渐减少,7天后虽明显减少但仍高于术前水平。且联合治疗组各时间点残余肿瘤细胞凋亡指数也高于单纯射频组(P<0.01)。2.单纯射频及联合治疗组残余肿瘤细胞增殖指数(PI)明显低于对照组(P<0.01),且联合治疗组残余肿瘤细胞增殖指数(PI)也低于单纯射频组(P<0.05)。3.单纯射频及联合治疗组VEGF低于对照组(P<0.05)。联合治疗组VEGF绝对值略高于单纯射频组,但差异无统计学意义(P>0.05)。4.单纯射频及联合治疗组残余肿瘤微血管密度(MVD)明显低于对照组(P<0.01)。联合治疗组残余肿瘤微血管密度(MVD)绝对值略高于单纯射频组,但差异无统计学意义(P>0.05)。5.残余肿瘤细胞VEGF与MVD表达呈正相关(P<0.01)。
结论:1.单纯射频和射频消融联合肝动脉栓塞治疗兔肝VX2肿瘤一周内均能诱导残余肿瘤细胞凋亡,同时抑制肿瘤细胞增殖。2.联合治疗与单纯射频治疗相比更能促进残余肿瘤细胞凋亡,抑制肿瘤细胞增殖。3.单纯射频和联合治疗一周内均能抑制肿瘤血管生成,且联合治疗在抑制肿瘤血管生成作用方面与单纯射频治疗作用相当。4.联合治疗后残余肿瘤内仍有VEGF表达,仍存在肿瘤血管生成。临床上如联合应用抗肿瘤血管生成药物可能会取得更好的疗效。
Objectives: To study the biological behavior of remnant tumor of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) combined with transcatheter arterial embolization (TAE); to investigate the mechanism of the combined therapy; and to assess the therapeutic efficacy of it.
Methods: Forty-five New Zealand white rabbits were implanted percutaneously with VX2 tumor cells in the left lobe of the liver under CT guidance successfully. Three weeks later, there was a focal tumor (φ2.5cm-2.7cm) in the liver of each rabbit. Then they were divided into 3 groups at random: control group (pseudo-therapy group), RFA (single electrode 2.0cm exposure) alone group and 7 days after TAE combined with RFA (single electrode 2.0cm exposure) sequentially group. Each group was divided into 3 subgroups respectively according to the killing time: 1 day, 4 days and 7 days after treatment. All the 45 rabbit-models were sacrificed after treatment and their tissues,including peripherial tumor and central tissue of the ablation area,were sectioned, embedded in paraffin, fixed in 10% Formalinand and spitted constantly with 4μm–section. They were examined pathologically by HE dying and immunohistochemical methods (Apoptosis, CD31, VEGF, PCNA). The results of immunohistochemistry were compared. All the data were analysed by SPSS 13.0 software. Quantitative data were showed by x±s, P<0.05 was defined as threshold for statistical significance .
Results: 1. Quantification assay of apoptosis measured by TUNEL and Caspase-3 staining methods indicated that apoptosis index (AI) in the margin of the tumor after RFA alone and combined therapy were both higher than that of control group significantly (P<0.01), they reached their peaks at 1 day, and decreased gradually along with the time to a low level at 7 days after treatment , but were still higher than that of pre-treatment . And AI of each subgroup after combined therapy were higher than that of RFA alone therapy significantly (P<0.01) . 2. Proliferation index (PI) of survival tumor cells after RFA alone and combined therapy were both lower than control group significantly (P<0.01) , and PI of each subgroup after combined therapy were lower than that of RFA alone therapy (P<0.05) . 3. Vascular endothelial grow factors (VEGF) of survival tumor cells after RFA alone and combined therapy were both lower than control group significantly (P<0.05). VEGF of each subgroup after combined therapy were higher than VEGF of RFA alone therapy slightly , but there was no statistical significance between them (P<0.05). 4. Microvessel density (MVD) of survival tumor cells after RFA alone and combined therapy were both lower than control group significantly (P<0.01) . MVD of each subgroup after combined therapy were higher than MVD of RFA alone therapy slightly, but there was no statistical significance between them (P<0.05) . 5. There was a positive correlation between VEGF and MVD in the combined treatment groups (P<0.01) .
Conclusions: 1. Both RFA alone and RFA combined with TAE therapy can induce the apoptosis and inhibit the cell proliferation in 1 week after treatment. 2. Compared with RFA alone , combined therapy can induce more cells to apoptosis and inhibit more cells proliferation , and can get better therapeutic efficacy . 3. Both RFA alone and RFA combined with TAE therapy can effectively inhibit tumor angiogenesis in 1 week after treatment. As for the efficacy of it, combined therapy is parallel with RFA alone. 4. Duing to tumor angiogenesis existed, combined therapy plus anti-angiogenesis drugs may obtain better therapeutic efficacy.
引文
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8. Zagoria RJ,Chen MY,Shen P,et al. Complications from radiofrequency ablation of liver metastases.Am Surg, 2002, 68: 204-209
9. Rhim H,Dodd GD Ⅲ,Chintapalli KN,et al.Radiofrequency thermal ablation of abdominal tumors : lessons learned from complications.Radiographics, 2004,24:41-52
10. Rhim H,Yoon K,Lee JM,et al.Major complications after radio-frequency thermal ablation of hepatic tumors:spectrum of imaging findings.Radiographics, 2003, 23:123-136
11. Livraghi T,Solbiati L,Meloni MF,Gazelle GS,Halpern EK,Goldberg SN. Treatment of focal liver tumors with percutaneous radio-frequency ablation: complications encountered in a multicenter study.Radiology 2003;226:441-451
12.Mulier S,Mulier P,Ni Y,et al.Complications of radiofrequency coagulation ofliver tumours.British Journal of Surgery,2002,89,1206-1222.
13. Livraghi T,Meloni F,Gazelle GS,et a1.Radiofrequency ablation (RF) in the treatment of hepatocellular carcinoma (HCC):experience in 259 patients.Radiology,1999,213:282_283.
14. Bowles BJ,Machi J,Limm WM,et a1. Safety and efficacy of Radiofrequency thermal ablation in advanced liver tumors.Arch Surg,2001,136:864-869.
15. Chung MH,Wood TF,Tsioulias GJ,et al.Laparoscopic radiofrequency ablation of unresectable hepatic malignancies.A phase 2 trail.Surg Endosc,2001,15:1021-1026.
16. Francica G,Marone G,Solbioti L,et al.Hemobilia,intrahepatic hematoma and acute thrombosis with cavernomatous transformation of the portal vein after percutaneous thermoablation of a liver metastasis.Eur Radiol,2000,10:926-929.
17. Choi H,Loyer EM,DuBrow RA,et al.Radiofrequency ablation of liver tumors: assessment of therapeutic response and complications.Radiographics,2001,21(SI): 541-554.
18. 刘海鹰,唐云强,崔书钟,等。原发性及转移性肝癌的射频治疗。中华普通外科杂志,2001,2:106-110.
19. Patterson EJ,Scudamore CH,Owen DA,et al.Radiofrequency ablation of porcine liver in vivo:effects of blood flow and treatment time on lesion size.Ann Surg,1998,227:559-565.
20. De Baere T,Elias D,Dromain C,et a1.Rediofrequency ablation of100 hepatic metastases with a mean follow-up of more than 1 year. AJR Am J Roentgenol,2000,175:1619—1625.
21. Dominique E,El Otmany A,Goharin A,et a1.Intraductal cooling of the main bile ducts during intraoperative radiofrequeney ablation.J Surg Oncol,2001,76:297-300.
22. Wah TM,Koenig P,Irving HC,Gervais DA,Mueller PR.Radiofrequency ablation of a central renal tumor:protection of the collecting system with a retrograde cold dextrose pyeloperfusion technique.J Vasc Interv Radiol, 2005,16:1551-1555
23. Wood TF,Rose DM,Chung M,et a1.Radiofrequency ablation of 231 unrsectable hepatic tumors:indications,limitations,and complications.Ann SurgOncol,2000,7:593-600.
24. Chopra S, Dodd GD 3rd, Chanin MP, et a1.Radiofrequency ablation of hepatic tumors adjacent to the gallbladder:feasibility and safety.AJR Am J Roentgenol,2003,180:697-701.
25. Meloni MF,GoldBerg SN,Mbser V,et al.Colonic perforation and abscess following radiofrequency ablation treatment of hepatoma.Eur J Ultrasound,2002, 15:73-76
26. Minami Y,Kudo M,Kawasaki T,et al.Percutaneous ultrasound-guided radiofrequency ablation with artificial pleural effusion for hepatocellular carcinoma in the hepatic dome. J Gastroenterol 2003;38:1066-1070
27. Machi J,Uchida S,Sumida K,et al.Ultrasound-guided radiofreqyency thermal ablation of liver tumors:percutaneous,laparoscopic,and open surgical approaches.J Gastrointest Surg, 2001,5:477-489
28. de Baere T, Risse O,Kuoch V,et a1. Adverse events during radiofrequency treatment of 582 hepatic tumors. AJR Am J Roentgenol,2003,181:695-700.
29. Goldberg SN, Gazelle GS,Dawson SL,et a1 . Tissue ablation with radiofrequency:effect of probe size,gauge,duration,and temperature on lesion volume. Acad Radiol,1995,2:399-404.
30. Machi J,Oishi AJ,Morioka WKYuM,et al.Radiofrequency thermal ablation of synchronous metastatic liver tumors can be performed safely in conjunction with colorectal cancer resection.Cancer J,2000,6(Suppl 4):S344-350.
31. Geschwind JF,Kaushik S,Ramsey DE, et al. Influence of a new prophylactic antibiotic therapy on the incidence of liver abscesses after chemoembolization treatment of liver tumors.J Vasc Interv Radiol 2002;13:1163-1166
32. Lu DS,Raman SS,Vodopich DJ,et a1.Effect of vessel size on creation of hepatic radiofrequency lesions in pigs:assessment of the“heat sink”effect. AJR Am J Roentgenol,2002.178:47-51.
33. Lim HK,Nam G,Han S,et a1. Major complications after radiofrequency thermal ablation of hepatic tumors:a report of the Korean RF study group.Radiology,2001,221 Suppl:248.
34. Goldberg SN,Solbiati L,Halpern EF,et a1.Variables affecting proper system grounding for radiofrequency ablation in an animal mode1.J Vasc Interv Radiol, 2000,11:1O69—1O75.
35. De Sio I,Castellano L,De Girolamo V,et al.Tumor dissemination after radiofrequency ablation of hepatocellular carcinoma.Hepatology, 2001,34:609-610.
36. 吴金生,褚延魁,马庆久。集束电极射频肝癌原位灭活治疗的临床研究。中华普通外科杂志,2001,4:214-215.
37. 汪毅,胡大荣,任永强,等。经皮射频毁损治疗肝脏肿瘤的近期疗效观察。中国肿瘤临床,2001,10:737-739.
38. De Baere TJ, Smayra T,Kuoch V,et al.Complications of percutaneous radiofrequency ablation of liver tumors.Radiology,2001,221(Suppl):627.
39. Solbiati L,Ierace T,Goldberg SN,et a1.Percutaneous US-guided radiofrequency tissue ablation of liver metastases:treatment and follow-up in 16 patients.Radiology,1997,202:195-2O3.
40. Di Stasi M, Buscarini L, Livraghi T,et al.Percutaneous ethanol injection in the treatment of hepatocellular carcinoma.A multicener survey of evaluation practices and complication rates.Scand J Gastroenterol,1997,32:1168-1173.
41. Llovet JM, Vilana R, Bru C, et al.Increased risk of tumor seeding after percutaneous radiofrequency ablation for single hepatocellular carcinomar Hepatology,. 2001,33:1124-1129.
42. Livraghi T,Goldberg SN,Monti F,et a1.Saline-enhanced radiofrequency tissue ablation in the treatment of liver metastases. Radiology.1997,202:205-210
43. Keltner JR, Donegan E, Hynson JM,et al.Acute renal failure after radiofrequency liver ablation of metastatic carcinoid tumor.Anesth Analg,2001, 93:587-589.
44. Curley SA, Izzo F,Delrio P,et a1.Radiofrequency ablation of unresectable primary and metastatic hepatic malignancies:results in123 patients. Ann Surg, 1999, 230:1-8.