【作者】 王琨；

【导师】 刘鲁明；

【作者基本信息】 复旦大学 ， 中西医结合临床， 2011， 博士

【摘要】 概述胰腺癌是一种高度恶性的消化道肿瘤,其的死亡率几乎是100%,在我国胰腺癌的发病率也呈上升趋势,2005年上海市就有新增胰腺癌患者1746例,它已经成为癌症死亡的第5位主要诱因。高强度聚焦超声(HIFU)技术是通过聚焦超声的热消融作用来达到靶向破坏瘤组织的目的,其优点是无需采用侵入手段,即可用于治疗体内深部肿瘤,缺点是作为局部治疗手段存在一定的局限性。中医药治疗胰腺癌因其改善症状、延长患者生存而有其独到之处,临床上采用清胰化积(QYHJ)方联合HIFU治疗晚期胰腺癌的综合治疗模式,取得一定的疗效,但相关的临床研究为小样本分析,且缺乏相应的实验研究。因此迫切需要进行系统和深入的临床和实验研究,来揭示QYHJ方联合HIFU治疗晚期胰腺癌的价值。目的本研究目的在于通过临床分析和实验研究,了解QYHJ方联合HIFU治疗晚期胰腺癌的疗效,确定QYHJ方与HIFU联合治疗晚期胰腺癌的临床价值。方法1.清胰化积方联合高强度聚焦超声治疗晚期胰腺癌的临床分析从2007年12月到2010年7月共有86例晚期胰腺癌患者入选此研究,其中男性55例,女31例,平均年龄58.2岁(30-81岁)。TNM分期：III期21例,Ⅳ期65例,伴肝转移53例。所有患者中有69患者曾接受以吉西他滨为主化疗或介入治疗方案失败,包括15患者曾接受过胰腺病灶放射治疗进展的,余17例患者均为不能耐受化疗或拒绝放化疗的。所有病例中有18例分别进行了2-3次HIFU治疗,其余68例仅接受1次HIFU治疗,伴肝转移53例患者中有8例同时进行了肝转移灶的HIFU消融治疗,有23例同时进行了肝转移灶的介入治疗。患者HIFU治疗前后和随访期间均给予QYHJ方为主的中药治疗。观察患者HIFU治疗后1周的疼痛改善情况,以及在HIFU治疗结束后1个月根据CT/MRI评价近期疗效。远期疗效观察患者生存时问,生存率和中位生存期。2.清胰化积方联合高强度聚焦超声对胰腺癌患者外周血免疫细胞的影响30例经组织病理或细胞学证实胰腺癌患者纳入研究,其中男性23例,女性7例,年龄39-85岁,中位年龄60岁。TNM分期Ⅲ期14例,Ⅳ期16例,KPS评分均≥60。所有病例在HIFU治疗后给予QYHJ为主中药煎剂连续口服28天。治疗前后均采用流式细胞术检测T细胞亚群CD3+、CD3+CD4+、CD3+CD8+、CD4+CD25+CD127low、CD8+CD28+和CD3-CD16+56+细胞及B淋巴细胞百分比,以及CD4+/ CD3+比值。3.清胰化积方联合高强度聚焦超声对Panc02瘤作用的病理学观察于C57小鼠右后肢外侧近臀部皮下接种panc02瘤株,随机分为4组,包括对照组、HIFU组、黄芪组、QYHJ组,待肿瘤直径在0.8-1.Ocm时,除对照组无功率输出假辐照外,其余各组均予瘤体HIFU消融,声功率300W,持续10秒,对照组和HIFU组于HIFU后每日生理盐水灌胃,另两组分别黄芪和QYHJ方灌胃,观察HIFU处理后不同时段各组残瘤组织病理学改变及HSP70和VEGF蛋白表达。免疫组化染色图像的分析用Image Pro Plus软件进行光密度测定分析,选择测量累积光密度(1OD)作为分析指标。4.清胰化积方联合高强度聚焦超声对荷瘤小鼠血管生成相关因子的影响于C57小鼠右后肢外侧近臀部皮下接种panc02瘤株,随机分为3组,包括对照组、HIFU组和QYHJ组,除对照组无功率输出假辐照外,其余各组均予瘤体HIFU消融,声功率300W,持续10秒,对照组和HIFU组于HIFU后每日生理盐水灌胃,QYHJ组予以QYHJ方灌胃,采用ELASA法测定HIFU处理后3天和10天不同时段小鼠血清VEGF、TGF-β、IL-6、PDGF及bFGF的值。结果1.清胰化积方联合高强度聚焦超声治疗晚期胰腺癌的临床分析近期疗效评价：完全缓解(CR)0例,部分缓解(PR)7例(8.1%),稳定(SD)48例(55.8%),进展(PD)31例(36.0%)。对HIFU辐照病灶的单独评价：完全缓解(CR)0例,部分缓解(PR)8例(9.3%),稳定(SD)64例(74.4%),进展(PD)14例(16.3%)。全组患者总的中位生存时间6个月,半年和1年生存率分别是52.0%、11.4%。73例治疗前CA19-9升高的患者,治疗后有12例下降,36例治疗前CA242升高的治疗后有15例下降。所有病例治疗后疼痛完全缓解有25例(29.1%),部分缓解36例(41.9%),缓解有效率为61/86(70.9%),单因素分析KPS评分、肝转移和9-9、CA242对生存有影响,Cox回归分析KPS评分是独立的预后因素。2.清胰化积方联合高强度聚焦超声对胰腺癌患者外周血免疫细胞的影响患者治疗后T细胞亚群CD3+、CD3+CD4+的百分比值有明显上升,与治疗前比较差异显著(P<0.01),而CD3TD8*治疗后无明显变化(P>0.05),同样CD4+/CD8+的比值治疗后亦有所上升,与治疗前比较有差异(P<0.05)。调节性T淋巴细胞(CD4+CD25+CD127low)治疗后百分比值有明显下降(P<0.01),而细胞毒T淋巴细胞(CD8+CD28+)百分比值治疗后有明显上升(P<0.01)。B淋巴细胞百分比值治疗后有明显下降,与治疗前比较差异显著(P<0.01),而CD3-CD16+56+细胞治疗后百分比值无明显变化(P>0.05)。3.清胰化积方联合高强度聚焦超声对Panc02瘤作用的病理学观察HIFU辐照后3小时的瘤体组织切片：与对照组相比HIFU组消融后H&E染色镜下见大片损伤坏死肿瘤组织,周边肿瘤细胞呈核缩、核凝的凝固性坏死表现。辐照后3天瘤体组织切片显示坏死灶周边有大量凋亡细胞,辐照后10天坏死灶基本同前,但周边凋亡细胞明显减少。HIFU辐照后10天各组HSP70表达均明显高于对照组(P<0.01),而HIFU组、QYHJ组、黄芪组各组间无差异。HIFU辐照后3天HIFU组、QYHJ组的VEGF表达均明显低于对照组(P<0.01),而对照组与黄芪组之间无明显差异(P>0.05)。HIFU辐照后10天各组VEGF表达均明显低于对照组(P<0.01), QYHJ组的VEGF表达则显著低于与HIFU组、黄芪组(P<0.01)。4.清胰化积方联合高强度聚焦超声对荷瘤小鼠血管生成相关因子的影响HIFU处理后3天及10天各组间肿瘤体积未发现有明显差异(P>0.05)。HIFU后10天,对照组与HIFU组之间血清VEGF水平无明显差异(P>0.05),而QYHJ组血清VEGF水平明显低于HIFU组和对照组(P<0.05)。HIFU处理后3天及10天对照组与HIFU组间血清IL-6水平无明显差异(P>0.05),而QYHJ组血清IL-6水平明显低于HIFU组和对照组(P<0.05)。HIFU处理后3天HIFU组血清TGF-β、bFGF明显高于对照组,而QYHJ组与对照组之间无明显差异(P>0.05)。10天后HIFU组血清TGF-β、bFGF仍明显增高,而QYHJ组与照组之间仍无明显差异。HIFU后3天及10天各组之间血清PDGF均无明显差异表达(P>0.05)。结论1.清胰化积方联合高强度聚焦超声治疗是晚期胰腺癌有效的治疗模式,部分患者甚至是伴远处转移的患者治后可以获得长期生存,KPS评分是晚期胰腺癌独立的预后影响因素。2.清胰化积方联合高强度聚焦超声治疗可以通过增加CD4+/CD8+比值、增强细胞毒T细胞以及抑制Treg来调节患者机体的细胞免疫功能,增强机体的抗瘤免疫效应。3.高强度聚焦超声辐照荷瘤小鼠可以导致胰腺癌组织发生凝固性坏死,清胰化积方可以协同高强度聚焦超声的消融作用抑制残留胰腺癌细胞的生长,进-步下调其VEGF的表达。4.清胰化积方联合高强度聚焦超声辐照荷瘤小鼠,不仅可以促进外周血VEGF和IL-6下降,还可以降低TGF-β和bFGF的水平从而消除高强度聚焦超声消融对TGF-β和bFGF的影响。更多还原

【Abstract】 IntroductionPancreatic cancer is a highly lethal disease all over the world. In 2005, there were approximately 1746 new cases of pancreatic cancer in Shanghai, China. Due to the pessimistic prognosis, the mortality of pancreatic cancer is almost 100%. It has become the fifth cause of the death of cancer disease. High Intensity Focused Ultrasound (HIFU) has been successfully used as a novel treatment for tumors in clinical practice. The primary mechanism for HIFU treatment is the thermal effect. HIFU treatment is able to lead a potential coagulative necrosis in targeted pancreatic carcinoma without damaging peripheral vital anatomy structures.Former researches indicated that traditional Chinese medicine (TCM) with this therapy was of some advantages in treating pancreatic cancer patients, especially those in advanced stages. However, most of those researches were the small scale retrospective studies or case reports. Experimental researches on TCM for pancreatic cancer were even rarely seen. So it is urgent to carry out systemic and comprehensive clinical trails as well as experimental researches to better expose the exact effects of TCM in multidisciplinary management of pancreatic cancer.Objective:We aimed to understand the way, effects and mechanism of qingyihuaji(QYHJ) herbal formula and HIFU in treating advanced pancreatic cancer through clinical researches and experimental studies, which may help to define the value of the integrative modality for the management of advanced pancreatic cancer. Material and methods:1. A clinical Research on QYHJ herbal formula and HIFU for the Treatment of Advanced Pancreatic CancerFrom July 2007 to July 2010, eighty-six advanced pancreatic cancer patients confirmed by cytology or histology were selected from patient database. Pancreatic cancer patients were treated by HIFU and QYHJ herbal formula.55 patients were male and 31 were female. Their mean age was 58.2 years with a range of 30-81 years.There were 21 patients with stage III, and 65 patients with stage IV disease.69 patients had a previous history of failure treatment with chemotherapy or radiochemotherapy.17 other patients, either refused chemotherapy and/or radiotherapy, or was not felt to be a suitable candidate for chemotherapy and/or radiotherapy. Patients were assessed CT/MRI scan performed approximately 1 month after completion of therapy. Responses were categorized by WHO criteria. Pain relief, local tumor control rate and complications were observed after HIFU treatment. Survival was estimated from the date of initiation of treatment by Kaplan-Meier method.2. The effects of QYHJ herbal formula and HIFU on imlllune cells in the peripheral blood of patients with pancreatic cancerThirty patients with pancreatic carcinoma were treated by HIFU and QYHJ herbal formula.23 patients were male and 7 were female. Their median age was 60 years with a range of 39-85 years.There were 14 patients with stage III, and 16 patients with stage IV disease. This study collected peripheral veuous blood and examine the percentage of T-lymphocyte subsets CD3+, CD3+CD4+, CD3+CD8+, CD4+CD25+CD127low, CD8+CD28+, CD3-CD16+56+and CD4+/CD8+ before and four weeks after HIFU treatment.3. Pathological observation of mice implanted with panc02 after QYHJ herbal formula and HIFU therapyC57 mice were transplanted with panc02 in the back legs. When the transplanted tumor diameter reached 0.8 to 1 cm, the mice randomized into four groups and then they were alation by HIFU:the control group (after fake-HIFU ablation given normal saline), the HIFU group (after HIFU ablation given normal saline), the HQ group (after HIFU ablation given Astragalus decoction) and the QYHJ group (after HIFU ablation given QYHJ herbal formula). Mice in each group were killed and pathologically observed in 3 hours,3 days and 10 days after operation, respectively.4. Effects of QYHJ herbal formula and HIFU on serum angiogenesis factors in mice with pancreatic cancerC57 mice were transplanted with panc02 in the back legs and randomized into three groups and then they were alation by HIFU:the control group (after fake-HIFU ablation given normal saline), the HIFU group (after HIFU ablation given normal saline) and the QYHJ group (after HIFU ablation given QYHJ herbal formula). Serum VEGF, TGF-β, IL-6, PDGF and bFGF were detected by ELASA method in 3 days and 10 days after HIFU ablation, respectively.Results:1. A clinical Study on Qiyihuaji formula and HIFU for the Treatment of Advanced Pancreatic Cancer:There were no severe complications or adverse events related to HIFU therapy in any of the patients treated. Serum level of CA19-9 decreased in 16.4% patients (12/73) and CA242 decreased in 41.7% patients (15/36). The cancer-related pain was relieved in 70.9% patients(61/86). Coagulated-necrosis of the mass by CT/MRI examination was observed. The local tumor mass change was assessed. There were no complete response (0%). And partial response (PR) were 9.3%; no change (SD) 74.4%; progressive disease (PD) 16.3%. The half year survival was 52.0% and 1-year survival 11.4% respectively, and the median survival was 6-month in this series. Karnofsky performance scale (KPS) were identified as independent predictors for overall survival by Cox proportional regression analysis.2. The effect of QYHJ herbal formula and HIFU on imlllune cells in the peripheral blood of patients with pancreatic cancerNo evidence of tumor hemorrhage, large blood vessel rupture, obstructive jaundice or gastrointestinal perforation were found in any patient. The percentage of CD3+, CD3+CD4+, CD8+CD28+and CD4+/CD8+cells were significantly elevated after treatment and the percentage of CD4+CD25+CD127low and B cells were significantly decreased. The difference was significant(P<0.05).3. Pathological observation of mice implanted with panc02 after QYHJ herbal formula and HIFU therapyAfter HIFU treatment, necrosis was observed in the centralablation area, surrounded by intermediate response belt and peripheral residual tumor tissue, was observed in the specimens from tumors. Apoptosis cells increased 3 days after HIFU and then decrease. The expression of VEGF in intermediate response belt decreased significantly, but HSP70 increased 10 days after HIFU. The expressions of VEGF 3 days after HIFU in the HIFU and QYHJ groups were lower than those in the control group (both P<0.01), while there was no significant difference in the expression of VEGF between the control and HQ group (P>0.05). The expressions of VEGF 10 days after HIFU in the QYHJ group were also lower than those in the HIFU and HQ groups (both P<0.01).4. Effect of QYHJ herbal formula and HIFU on serum angiogenesis factors inmice with pancreatic cancerThe expressions of serum VEGF in 10 days after ablation in the QYHJ group were lower than those in the control and HIFU group (both P<0.05), while there was no significant difference between the control and HIFU group (P>0.05). The expressions of serum IL-6 in 3 and 10 days after ablation in the QYHJ group were lower than those in the control and HIFU group (both P<0.05), while there was no significant difference between the control and HIFU group (P>0.05). The expressions of serum TGF-βand bFGF in 3 days after ablation in the HIFU group were higher than those in the control group (both P<0.05), while there was no significant difference between the control and QYHJ group (P>0.05). The expressions of serum TGF-βand bFGF in the HIFU group remain higher in 10 days after ablation. There was no significant difference in the expression of PDGF among all groups (P>0.05), Conclusion:1. Extracorporeal HIFU can effectively ablate pancreatic cancer and relieve cancer-related pain. The combined modality of QYHJ herbal formula and HIFU was benefit to advanced pancreatic cancer, and warrant for further prospective study. KPS was identified as independent predictors for overall survival.2. The combined modality of QYHJ herbal formula and HIFU can enhance immune function and antitumor activity for patients with pancreatic cancer.3. HIFU treatment is able to lead coagulative necrosis in targeted tumor tissue. QYHJ herbal formula and HIFU treatment can effectively down-regulate the protein expression of VEGF and inhibit the growth of remnant carcinoma.4. QYHJ herbal formula and HIFU treatment not only can reduce VEGF and IL-6 levels in peripheral blood of mice with pancreatic cancer, but also can reduce the TGF-βand bFGF levels which is triggered by HIFU ablation.更多还原