【作者】 王永中；

【导师】 姚树坤；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 博士

【摘要】 原发性肝细胞癌(Hepatocellular Carcinoma, HCC)以下简称原发性肝癌,为世界常见难治的恶性肿瘤之一,由于其发病隐匿,确诊时常常失去了手术治疗的机会。经导管动脉内化疗栓塞术(transcather arterial chemoembolization, TACE)又称肝动脉化疗栓塞治疗,是目前公认为对不能手术切除的原发性肝癌的安全、有效、可反复的首选常规治疗方法,它近期疗效显著,可使肿瘤缩小,症状缓解,并有效杀灭肿瘤细胞,在一定时间内控制肿瘤的生长,延长患者生存时间。但由于碘油栓塞以及大量化疗药物短时间进入体内,造成肝组织缺血缺氧,导致机体整体功能的严重损害,改善TACE治疗后原发性肝癌患者的免疫功能,降低术后肿瘤的复发率,延长生存期,成为提高治疗效果的关键。中草药是天然的生物反应调节物质(Biological response modulation substance,BRMs),可以提高机体免疫力,与机体免疫系统、内分泌系统、神经系统等相互影响与协调,共同维持生命机制的稳定与平衡,具有辅助抗肿瘤,减少介入治疗的毒副作用等功能。原发性肝癌患者多有免疫功能的异常,常伴有免疫抑制因子的表达升高和/或免疫促进因子表达水平的降低。研究表明患者外周血中IL-2及肿瘤坏死因子-α(TNF-α)水平变化与原发性肝癌密切相关。IL-2为重要的细胞免疫促进因子,具有明显的抗肿瘤作用,sIL-2R为免疫抑制因子,其在血循环中与IL-2结合从而封闭阻断了IL-2的作用。TNF-α具有多种生物学功能,对多种肿瘤细胞与正常细胞具有直接的细胞毒作用,并能介导炎症与免疫反应。血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种重要的血管生长因子,对血管内皮细胞的增殖、水解基膜、迁移和血管构建的调控作用较强,且特异性高。HCC为典型的多血管肿瘤,生长迅速,且极易发生肝内血管浸润及癌栓形成,疗效欠佳。研究表明,人肝细胞癌中有较强VEGF的表达,且VEGF的表达与原发性肝癌的生长和转移密切相关。环氧合酶-2(cyclooxygenase-2, COX-2)作为前列腺素生物合成过程中的一个重要限速酶,在多种肿瘤的发生发展和转移中起重要作用,其表达受细胞内外多种因素的影响。COX - 2在肿瘤中的作用机制作主要包括①抗凋亡作用;②促进肿瘤血管生成;③增强肿瘤细胞的侵袭性;④炎性刺激及免疫抑制作用;⑤对前致癌物的氧化转化作用。有研究表明,在原发性肝癌中COX-2呈高表达,原发性肝癌患者外周血中VEGF及COX-2水平的检测对判断原发性肝癌转移和复发有着重要的价值,对指导临床治疗,改善患者预后有重要的意义。TACE治疗使得大量化疗药物短时间进入体内以及碘油栓塞,会造成肝组织缺血缺氧,导致机体整体功能的严重损害。如何改善TACE治疗后原发性肝癌患者的免疫功能,降低术后肿瘤的复发率,改善生存质量,延长生存期,是提高治疗效果的关键。目的本研究对TACE术后患者给予中药联合治疗,检测中药清肝化瘀口服液对原发性肝癌患者肝动脉化疗栓塞( transcatheter arterial chemoembolization, TACE)术后血液中CD3+, CD4+, CD4+/CD8+、自然杀伤细胞(natural killer cell, NK)及甲胎蛋白(alpha-fetoproteins,AFP)的变化,测定患者外周血中VEGF及COX-2的含量变化,观察患者综合指标,探讨中药对TACE术后机体免疫功能、肿瘤转移潜能及生存质量的影响,揭示清肝化瘀口服液对抑制肝癌细胞增殖、降低肝癌细胞COX-2、VEGF表达的作用,从免疫学和原发性肝癌转移两方面阐明清肝化瘀口服液联合介入治疗在原发性肝癌治疗中提高免疫力,减轻毒副作用,抑制肿瘤转移等方面,进一步探讨中药治疗原发性肝癌的作用以及作用靶点,从而增强病人免疫功能、提高原发性肝癌患者的生存期,为中药的临床研究提供试验依据。方法1病例选择:随机选取我院2006年2月至2007年2月间住院原发性肝癌患者60例,男38例,女22例,年龄48～65岁,平均56.3±7.7岁。所有患者均符合以下诊断标准:①术后组织学检查为原发性肝细胞肝癌;②影像学检查有明确实性占位;③甲胎蛋白(AFP)>200μg/L,持续4周以上。临床分期按2001年9月中国抗癌协会肝癌专业委员会制定标准对患者分期,Ⅰ期8例,Ⅱ期42例,Ⅲ期10例。2试验分组:随机将患者分为试验组及对照组。试验组即TACE+中药组:原发性肝癌患者30例,男20例,女10例,平均年龄55.5±8.7岁,其中Ⅰ期5例,Ⅱ期19例,Ⅲ期6例。对照组即单纯TACE组:原发性肝癌患者30例,男18例,女12例,平均年龄56.9±7.9岁,其中Ⅰ期3例,Ⅱ期23例,Ⅲ期4例。3标本采集:分别于介入术前1天和介入术后第28天早晨空腹静脉采集样血5 ml,分装不抗凝管与肝素抗凝管,不抗凝管4℃,2500 r/min离心10分钟,分离血清,-30℃保存待测;抗凝管直接送检。4外周血T细胞亚群的检测:选用三色荧光标记的免抗试剂直接荧光染色,采用流式细胞术进行测定两组患者治疗前后外周血CD3+, CD4+, CD4+/CD8+、NK细胞活性。5血清细胞因子检测:双抗体夹心酶联免疫吸附法(ELISA法)检测两组患者治疗前后血清中TNF-α和IL-2水平。6 ELISA法检测两组患者治疗前后外周血中VEGF蛋白水平表达。7 RT-PCR法检测两组患者治疗前后外周血有核细胞中COX-2基因表达。8测定TACE+中药组及单纯TACE组患者治疗前后AFP水平9检测两组患者治疗后病灶变化及肝外转移情况: CT和/或彩色多谱勒超声检查肿瘤最大直径乘以其垂直直径与治疗前比较;CT和/或超声及胸片检查,随访观察肺、骨、脑及淋巴结的转移情况。10对两组患者治疗后的疗效进行判定:按照WHO制定的实体瘤客观评价标准进行判定及分析。判定标准为:①完全缓解为病灶全部吸收;②部分缓解为病灶部分缓解;③无变化为病灶无变化;④病情进展为病灶进展。11对两组患者治疗后生活质量进行评价:根据卡氏功能状态(Karnosky performance status, KPS)评分,以治疗后较治疗前增加>10分为改善,减少>10分为下降,变化≤10分为稳定。12统计学分析:数据处理采用SPSS 13.0统计软件。计量资料以均数±标准差( X±S)表示,组间比较采用成组设计的t检验;同组治疗前后比较采用配对t检验;计数资料比较采用X2检验,以P<0.05为差异有统计学意义。结果1 TACE+中药组及单纯TACE组两组患者T细胞亚群和NK细胞比较:两组患者治疗前外周血CD4+、CD8+、CD4+/CD8+及NK测定值无显著性差异,两组具有可比性;原发性肝癌介入治疗后28日时,检测患者外周血中CD3+, CD4+, CD4+/CD8+、NK细胞测定值分别为0.23±0.18,0.18±0.04,1.12±0.46及10.68±3.01,经清肝化瘀口服液联合介入治疗后患者外周血中CD3+, CD4+, CD4+/CD8+、NK细胞测定值分别为0.55±0.11, 0.35±0.09,1.65±0.29及31.07±6.56,结果显示经清肝化瘀口服液联合介入治疗后外周血中CD3+, CD4+, CD4+/CD8+、NK细胞较单纯介入治疗组明显增高(P<0.05),提示清肝化瘀口服液可有效增强原发性肝癌介入治疗患者的细胞免疫。2原发性肝癌患者介入治疗前后血清TNF-α和IL-2水平比较:患者经介入治疗后TNF-α浓度明显下降(P<0.05),IL-2浓度增高。3 TACE+中药组及单纯TACE组介入治疗前后外周血细胞因子TNF-α及IL-2的比较:两组患者治疗前外周血细胞因子TNF-α及IL-2测定值无显著性差异,两组具有可比性;TACE+中药组介入治疗后外周血细胞因子TNF-α含量为10.85±1.67,显著低于单纯TACE组的13.56±1.55,说明清肝化瘀口服液可协助TACE有效降低TNF-α表达量(P<0.05);TACE+中药组介入治疗后外周血细胞因子IL-2含量为32.83±3.03,高于单纯TACE组的28.05±2.95,说明清肝化瘀口服液可协助TACE有效增强IL-2的表达(P<0.05)。4 TACE+中药组及单纯TACE组介入治疗前后外周血中VEGF表达的比较:两组患者治疗前,血清VEGF表达量无显著性差异,两组具有可比性;原发性肝癌患者介入治疗后28日时,检测患者外周血中VEGF表达量为456.08±175.49,经清肝化瘀口服液联合介入治疗后患者外周血中VEGF表达量为207.49±93.22,结果显示清肝化瘀口服液联合治疗可有效降低介入治疗后增高的VEGF(P<0.05)。5 TACE+中药组及单纯TACE组介入治疗前后外周血有核细胞中COX-2表达比较:治疗前,两组患者COX-2阳性表达率及表达量无显著性差异,两组具有可比性;介入治疗后28日时,检测TACE+中药组与单纯TACE治疗组患者外周血有核细胞中COX-2表达量比较发现,经清肝化瘀口服液联合介入治疗后患者外周血有核细胞中COX-2表达阳性率为47.77%,明显低于单纯TACE治疗组的55.69%(P<0.05),同时表达量也低于单纯TACE治疗组。提示清肝化瘀口服液可有效降低原发性肝癌介入治疗患者外周血有核细胞中COX-2的表达。6原发性肝癌患者中药清肝化瘀口服液联合介入治疗后患者中有6例发生肝外转移,转移率为20%,单纯TACE组发生肝外转移14例,肝外转移率为43.3%,二者差异有统计学意义(P<0.05)。7原发性肝癌患者TACE+中药组,部分缓解患者24例,有效率为80%,单纯TACE治疗组部分缓解者12例,有效率为40%,两组间差异有统计学意义(P<0.05)。8原发性肝癌患者中药清肝化瘀口服液联合介入治疗后KPS评分后判定,生活质量改善者13例,改善率为43.3%,单纯TACE组生活质量改善者6例,改善率仅20%,两组间差异有统计学意义(P<0.05)。结论1 TACE+中药组NK细胞下降的幅度低于单纯TACE治疗组,CD3+, CD4+, CD4+/CD8+的值高于单纯TACE治疗组说明清肝化瘀口服液对化疗引起的细胞免疫抑制有一定的保护作用。2 TACE+中药组外周血中VEGF及COX-2表达显著降低,说明TACE联合应用清肝化瘀口服液在抑制肿瘤内血管生成、防止肿瘤转移中具有重要作用。3 TACE+中药组在瘤体客观疗效及临床症状的缓解率上均优于单纯TACE治疗组,说明化疗介入联合应用清肝化瘀口服液治疗原发性肝癌在瘤体缩小、缓解临床症状方面优于单纯西医治疗。4治疗后两组病例生活质量的比较,TACE+中药组优于单纯TACE治疗组,提示中西医结合治疗原发性肝癌可以改善或提高患者的生活质量。5 TACE+中药组AFP值下降显著,与单纯TACE组有统计学意义,提示化疗介入配合清肝化瘀口服液可以较好地降低血中AFP水平。6从化疗的不良反应如肝功能损害出现的情况看,TACE+中药组低于单纯TACE治疗组,说明清肝化瘀口服液辅助西医化疗介入治疗原发性肝癌在减轻化疗毒副作用方面明显优于单纯西医治疗。更多还原

【Abstract】 Hepatocellular Carcinoma (HCC), called hepatama for short, is one of an incurable malignant tumor in world. Because of its hiding morbidity, the opportunity for operating therapy is often missing. Transcather arterial chemoembolization (TACE), also called hepatic arterial chemoembolization (HACE), is considered a secure, effective and repeatable first selecting conventional therapy for the inoperable hepatama. Its curative effect is significant in the near future. TACE can deflate the tumor, relieve the symptom and kill the tumor cells. It can control the growth of the tumor in a short time and prolong the live time of the patients. Because a large quantity of chemo medicine comes into body, the liver tissue will be ischemic and hypoxia and the allomeric function of organism is damaged severely. Improving the immunological function of the hepatama patients after TACE, cutting down the recurrence rate of the tumor and prolonging the live time have been the key point of the therapeutic efficacy.The traditional Chinese medicine is the natural medicine. It can improve the immunity of the organism and it can coordinate and influence each other with immune system, endocrine system and nervous system. They maintain the stabilization and balance of the life mechanism, help to resist tumor growth and decrease the side effect of the interventional therapy in common.The patients with hepatoma often have immunological function abnormality. The expression of immune suppressive factors increases and the expression of immune enhancive factors decreases. The study suggested that the change of TNF-αand IL-2 in the blood of patients with HCC had close correlation to HCC. IL-2 is an important cell immune enhancive factor and it has significant effects to resist tumor. sIL-2R is an immune suppressive factor. It can combine to the IL-2 in blood circulation and hold up the effects of IL-2. TNF-αhas many biological function and has intimate cell toxic action to many tumor cells and normal cells. It can also mediate inflammation and immune reaction.Vascular endothelial growth factor is an important vascular growth factor. It has strong effects on proliferation of vascular endothelial cell, hydrolization of basal lamina, cell immigration and vascular construction. It also has high specificness. HCC is a typical tumor with vascularity. It grows quickly and the vascular infiltration among the liver is easily to happen. The therapeutic effect is below the mark. There is study showed that the expression of VEGF was significantly in hepatoma cells and it has close relation to the growth and metastasis of HCC. Cyclooxygenase-2 is an important rate-limiting enzyme in the biosynthesis process of prostaglandin and it has essential effects in the genesis, development and metastasis of lots of tumors. There is study showed that the expression of COX-2 was high in hepatoma and the expressive level of VEGF and COX-2 in the peripheral blood had important value in judging the metastasis and recurrence of hepatoma. It also had significance in guiding clinical therapy and improving prognosis.ObjectiveIn this study, the traditional Chinese medicine had been given to the HCC patients after TACE. The change of CD3+, CD4+, CD4+/CD8+, NK cell and alpha-fetoprotein (AFP) in blood were detected after the therapy with QingGanHuaYu orally taking liquid in HCC patients after TACE. The content of VEGF and COX-2 in peripheral blood was also examined and the composite index of the patients was observed. Through the above research, the influence of traditional Chinese medicine on the immune function, the potentiality of tumor metastasis and living quality was discussed. This study revealed the effects of QingGanHuaYu orally taking liquid on the suppression of the proliferation of hepatoma cells and the decreasing of the expression of VEGF. This study illuminated the contribution of QingGanHuaYu orally taking liquid to the therapy of HCC from both immunology and HCC metastasis sides. It can improve the immunity, lessen side effects and inhibit tumor metastasis and so on. This study also discussed the effects of traditional Chinese medicine and its targets and provided experimental basis to the clinical study of the traditional Chinese medicine.Methods1 Patients selection: The patients with HCC was randomly selected from the forth hospital of Hebei Medical University from 2006.2 to 2007.2. Among the patients, there were 38 males and 22 females and the age of the patients was from 48-65 and the average age was 56.3±7.7. All the patients were consistent with the following diagnostic code: (1) the result of histology examination was HCC; (2) imaging examination identifies the position; (3) AFP>200μg/L. According to Chinese liver cancer cooperation meeting staging standard, stage I were 8 cases, stage II were 42 cases and stage III were 10 cases.2 Experimental grouping: Divide the patients randomly into experimental group and control group:The experimental group is TACE with traditional Chinese medicine therapy. Among the 30 cases of PHC patients, there were 20 males and 10 females. The average age was 55.5±8.7. The patients in stage I were 5 cases, stage II 19 cases and stage III 6 cases.The control group is merely TACE group. Among the 30 cases of PHC patients, there were 18 males and 12 females. The average age was 56.9±7.9. The patients in stage I were 3 cases, stage II 23 cases and stage III 4 cases.3 Specimen collecting: Blood sample 5ml was collected 1 day before and 28 days after the intervention in the morning on an empty stomach. The blood was stuff into non-anticoagulation and anticoagulation tubes respectively. The centrifugalization was carried out at 4oC in 2500r/min for 10min. The serum and blood plasma was divided and preserved at -30oC.4 The examination of T cell subgroup: The CD3+, CD4+, CD4+/CD8+ and NK cell activity in peripheral blood were examined through flow cytometry with the antibody labeled by triad colour fluorescent before and after the therapy in the two patients groups. 5 The examination of cytokine in serum: The serum level of TNF-αand IL-2 of the patients were detected by double antibody sandwich enzyme linked immunosorbent assay (ELISA) method.6 The VEGF level in peripheral blood of the two patients group was examined through ELISA method.7 The expression of COX-2 in gene level in blood karyocyte was detected by RT-PCR method in the two patients group before and after therapy.8 The examine of AFP in two patients group was performed.9 The detection of the tumor change and outside liver metastasis: the maximum level diameter and vertical diameter was determined by CT or multicolor ultrasonic examination by Doppler’s method. Calculated the volume of the tumor and compared them before and after therapy. The outside metastasis to lung, bone, brain and absorbent gland was detected by CT or sternite.10 The therapeutic effect assessment of the two groups after therapy: The assessment and analysis were based on the solid tumor objective evaluation criterion enacted by WHO. The assessment standard was as follow: (1) complete remission means the tumor is absorbed completely; (2) partial remission means the tumor is absorbed partly; (3) no change means the tumor has no change; (4) pathogenetic condition advancement means the tumor increases.11 The evaluation of life quality of the two groups: The evaluation was based on the Karnosky performance status (KPS). Improving means after therapy the score increases beyond 10; descending means after therapy the score decreases beyond 10; stabilizing means the score changes in 10.12 Statistical analysis: the data processing was carried out by SPSS 13.0 software. Measurement data was expressed as X±S. The compare between groups was using T test. The enumeration data was compared by X2 test. Ranked data was compared by rank-sum test. P<0.05 means the difference has statistical significance. Results1 The comparison of T cell subgroup and NK cell between two groups:the examine values of CD3+, CD4+, CD4+/CD8+ and NK cell had no significant difference before the therapy. Compared with the simple TACE group, the TACE with QingGanHuaYu orally taking liquid group had low CD3+ value (P<0.05)、CD4+ value (P<0.05) and the CD4+/CD8+ value also decreased (P<0.05).2 The comparison of TNF-αand IL-2 level in serum between prior and circa of the interventional therapy: compared with prior therapy, the TNF-αlevel decreased (P<0.05) and the IL-2 level (P<0.05) increased significantly after therapy.3 The comparison of TNF-αand IL-2 level in serum between the two groups: compared with the simple TACE group(13.56±1.55), the TNF-αlevel(10.85±1.67) decreased (P<0.05); and the IL-2 level (P<0.05) increased significantly in the TACE with QingGanHuaYu orally taking liquid group(32.83±3.03) compared with the simple group(28.05±2.95).4 After the interventional therapy, the VEGF level in serum and the expression of COX-2 in the blood karyocyte Compared with before the interventional therapy, the difference had statistical significance (P<0.05). The comparison of VEGF expression between the two groups: the VEGF level in TACE with QingGanHuaYu orally taking liquid group (207.49±93.22) was significant lower than that of simple TACE group (456.08±175.49) (P<0.05).5 The comparison of COX-2 in peripheral blood karyocyte between two groups: the value of COX-2 in TACE with QingGanHuaYu orally taking liquid group was 47.77%, significant lower than that of simple TACE group 55.69%. The difference had statistical significance (P<0.05).6 In the TACE with QingGanHuaYu orally taking liquid group, there were 6 patients had metastasis outside liver and the metastasis rate was 20%. In the simple TACE group, there were 14 patients had metastasis outside liver and the metastasis rate was 43.3%. The difference between the two group had statistical significance (P<0.05).7 In the TACE with QingGanHuaYu orally taking liquid group, there were 24 patients had partial remission and the effective rate was 80%. In the simple TACE group, there were 12 patients had partial remission and the effective rate was 40%. The difference between the two group had statistical significance (P<0.05).8 According to the KPS evaluation standard, there were 13 patients had life quality improvement and the improving rate was 43.3% in the TACE with QingGanHuaYu orally taking liquid group. There were 6 patients had life quality improvement and the improving rate was only 20% in the simple TACE group. The difference between the two group had statistical significance (P<0.05).Conclusion1 In the TACE with QingGanHuaYu orally taking liquid therapy group, the NK cell decreasing extent of NK cell is lower than that of simple TACE therapy group and the value of CD3+, CD4+, CD4+/CD8+ were higher than that of simple TACE therapy group. So, QingGanHuaYu orally taking liquid has protection to the cell immunity of the HCC patients after chemo.2 The expression of VEGF and COX-2 in peripheral blood degraded significantly in the TACE with QingGanHuaYu orally taking liquid therapy group. Using QingGanHuaYu orally taking liquid after TACE therapy can inhibit the angiogenesis in tumor.3 In the TACE with QingGanHuaYu orally taking liquid therapy group, the objective tumor curative effect and the relief rate of clinical symptom were better than the simple TACE group. The TACE therapy combined with QingGanHuaYu orally taking liquid can deflate the volume of the tumor and relieve the clinical symptom.4 When it comes to the comparison of the life quality, the patients accepted TACE with QingGanHuaYu orally taking liquid therapy were living better than those of accepting the simple TACE therapy. So, combined treatment of traditional Chinese medicine and western medicine toHCC can improve the patients’life quality significantly.5 In the TACE with QingGanHuaYu orally taking liquid therapy group, the value of AFP in peripheral blood descended largely. When compared with the simple TACE therapy group, the difference had statistical significance. The TACE therapy combined the QingGanHuaYu orally taking liquid therapy can degrade the level of AFP in peripheral blood of the patients with HCC.6 On the adverse effect of the TACE therapy such as liver function lesion, in the TACE with QingGanHuaYu orally taking liquid therapy group, it is less than that of the simple TACE therapy group. QingGanHuaYu orally taking liquid can reduce and lessen the side effects after the TACE therapy in the patients with HCC.更多还原