【作者】 张苗尊；

【导师】 牟一平； 汤谷平；

【作者基本信息】 浙江大学 ， 临床医学（专业学位）， 2016， 博士

【摘要】 胰腺癌是恶性度极高的消化道肿瘤,预后极差,以进展快、高发生率和高死亡率著称,目前手术是可切除胰腺癌治愈可能的唯一手段。随着腹腔镜技术的提高和腔镜器械的发展,腹腔镜远端胰腺切除术(Laparoscopic distal pancreatectomy, LDP)已成为治疗胰腺良性和低度恶性肿瘤的标准术式。至今已有大量LDP和开腹胰腺远端切除(Open Distal Pancreatetomy, ODP)对照研究,其结果表明LDP近期和远期手术效果与ODP无明显差异,在术中出血量、术后恢复方面有优势,但对于胰体尾的导管腺癌(Pancreatic ductal adenocarcinoma, PDCA), LDP应用尚存在争议,主要顾虑在于LDP能否达到与ODP一致的肿瘤根治效果及手术安全性,目前仅有少量文献报道,总体病例量较少。本研究对本中心开展的LDP和ODP治疗PDCA病例进行回顾性对照研究,比较手术疗效和肿瘤疗效,同时汇总LDP与ODP治疗PDCA对比研究的文献,进行荟萃分析,以助于更系统和客观评价LDP治疗PDAC的手术安全性和肿瘤根治性。胰腺癌的肿瘤生物学特性导致胰腺癌术后早期就容易出现局部复发和远处转移,即使手术根治性切除,术后5年生存率仍只有约25%,因此化疗在术后辅助治疗及局部进展期或晚期转移患者治疗中起重要作用,传统的化疗目前提高中晚期胰腺癌患者生存率有限,近几年随着分子靶向治疗的发展及纳米技术的提高,利用分子靶向技术及纳米载体能有效提高化疗靶向性,降低多药耐药及化疗全身毒性,为胰腺癌化疗提供了新的治疗思路。自噬在肿瘤中发挥复杂而重要的作用,是肿瘤分子靶向治疗研究的新热点,胰腺癌生长与自噬高度依赖,自噬升高也是胰腺癌抵抗化疗的策略之一。我们以普朗尼克P123为主体设计了一个新型的肿瘤靶向药物基因共输送聚阳离子纳米载体体系--P123P600iRGD (PP6iRGD)聚阳离子胶束,通过体内外研究观察PP6iRGD/DTX/Atg7 siRNA复合物释放多西紫杉醇(Docetaxel, DTX)及自噬相关蛋白7 siRNA (Atg7 siRNA),利用Atg7 siRNA促使Atg7表达下降,抑制自噬,阻止胰腺癌细胞因化疗诱导的自噬流,促进肿瘤细胞凋亡。第一部分：腹腔镜与开腹远端胰腺切除术治疗胰腺癌的病例对照研究研究目的：腹腔镜远端胰腺切除术在胰腺良性和低度恶性肿瘤应用广泛,但对于胰体尾恶性肿瘤,尤其是胰腺导管腺癌的治疗存在争议,本课题设计了病例对比研究,分析LDP和ODP治疗胰腺导管腺癌的近期及远期疗效,以助于评价LDP治疗PDAC的手术安全性和肿瘤根治性。研究方法：回顾性分析2003年4月至2015年10月开展的远端胰体尾切除术治疗胰腺导管腺癌病例,根据手术方式分为LDP和ODP组,分析患者的临床病例资料、围手术期资料、肿瘤根治性及术后随访资料。研究结果：共29例患者纳入LDP组。48例纳入ODP组。患者临床病例资料无明显统计学差异。LDP组平均手术时间显著性小于ODP组(180min vs270min,P=0.003)。术中平均出血量LDP组显著性少于ODP组(100ml vs 400ml, P=0.00)。术后肛门排气时间及术后进食流质时间LDP均少于ODP组(3d vs 3d, P=0.000; 3d vs 4d, P=0.001)。术后平均住院日LDP组显著性少于ODP组(13d vs 15d, P=0.022)。术后胰漏、术后并发症率两组间无明显统计学差异。R0切除率、肿瘤大小、淋巴结清扫数目、阳性淋巴结率、神经侵犯率LDP组和ODP组无显著性统计学差异(96.6% vs 89.6%,P=0.400:3.5cm vs 3.9cm,P=0.987；11 vs 9,P=0.072； 62.1% vs 47.9%,P=0.249；51.7% vs 43.8%,P=0.638).LDP组和ODP组术后中位生存时间分别为16月和15月。Cox多因素回归分析后,手术切缘(R0切除)、术后辅助治疗与术后生存时间正相关(P=0.000；P=0.015)。结论：1.LDP和ODP治疗胰腺胰体尾导管腺癌具有相同的手术疗效和肿瘤学根治疗效。但研究结果仍需多中心、前瞻性的随机对照研究验证。2.LDP治疗胰腺胰体尾导管腺癌安全可行,并具有创伤小、术后恢复快等微创手术优势。第二部分：腹腔镜与开腹远端胰腺切除术治疗胰腺癌病例对照研究的荟萃分析研究目的：1.荟萃分析腹腔镜与开腹胰体尾切除术治疗胰腺导管腺癌的对比研究,以助于更系统和客观评价LDP治疗PDAC的手术安全性和肿瘤根治性。研究方法：检索Pubmed.Cochame Library.Biosis Previews和Web of Science 1996年1月至2016年2月公开发表的LDP与ODP治疗PDCA临床对照研究,对R0切除率、手术时间、术中出血量、总并发症率、死亡率、术后胰漏、平均住院日、肿瘤大小、总淋巴结数、阳性淋巴结率、术后辅助化疗及总生存率等数据进行荟萃分析,数据分析采用RevMan5.2软件。研究结果：7篇文献纳入本研究,总计429例,其中LPD组205例,ODP组347例。R0切除率LDP组和ODP组无明显统计学差异(P=0.23),术中出血量LDP组明显少于ODP组,有统计学显著性差异,WMD值为-276.16(95%CI=-450.42- -101.89；P<0.01,12=76%)；平均术后住院日显示有异质性,采用随机模型合并后发现LDP组平均术后住院日少于ODP组,差异有统计学意义,WMD值为-4.12(95%CI=-5.87--2.37；P<0.01,I2=70%):LDP组和ODP组手术时间无统计学差异(P=0.51),术后胰漏、术后总并发症、死亡率无统计学差异；肿瘤大小发现异质性很高,经采用随机模型,显示LDP组肿瘤小于ODP组,差异有统计学意义WMD值为-0.48 (95%CI=-0.94--0.02; P=0.04,12=69%),经敏感性分析,倾向性配对后,LDP组和ODP组肿瘤大小无统计学差异(0.07)；总淋巴结数、阳性淋巴结率、术后辅助化疗率、术后复发率经荟萃分析后,两组无明显统计学差异。结论：LDP治疗胰体尾胰腺导管腺癌技术上是安全可行的,肿瘤根治性可以达到ODP,且有微创外科的固有优势,但研究结果仍需多中心、前瞻性的随机对照研究验证。第三部分：靶向纳米载体携带DTX和Atg7 siRNA治疗胰腺癌的研究研究目的：构建以P123为主体的具备肿瘤靶向功能的药物基因共输送胶束纳米载体体系：P123P600iRGD,评估携带DTX及Atg7 siRNA能力,探讨PP6iRGD/DTX/Atg7纳米复合物抑制自噬诱导胰腺癌凋亡的基因药物协同作用。研究方法：对合成的P123P600iRGD胶束纳米载体进行理化性质、载基因、载药物能力的研究。PP6iRGD载体体系携带Atg7 siRNA和DTX,在Panc-1体外实验中用细胞毒性实验、Western Blotting实验及qPCR实验等方法检测胰腺癌药物基因协同作用及抗肿瘤效果；在体内实验中用免疫组化、tunnel. Western Blotting实验及qPCR实验等检测PP6iRGD/DTX/Atg7纳米复合物药物基因协同作用及抗肿瘤效果。研究结果：以普朗尼克P123为主体,合成了一个新型的具有肿瘤靶向功能的药物基因共输送胶束纳米载体体系PP6iRGD。该载体在溶液中能自发组装成胶束,其疏水内核可装载药物,阳离子外壳可携带基因,可高效通过胰腺癌细胞膜,在体外低PH条件下具备药物缓慢释放能力。利用PP6iRGD载体体系携带Atg7siRNA和DTX,在Panc-1体外实验中发现DTX可诱导自噬上调,经Atg7 siRNA基因沉默后可抑制自噬,抑制Panc-1增殖,诱导细胞凋亡,体内实验中发现PP6iRGD携带Atg7 siRNA和DTX抑制自噬,抑制肿瘤生长,促进凋亡,靶向治疗组抑瘤效果更显著。结论：1、PP6iRGD胶束纳米载体系统可共输送DTX和Atg7 siRNA通过细胞膜,并发挥发挥药物基因协同治疗作用。2、PP6iRGD/DTX/Atg7体外释放DTX,诱导自噬上调,经Atg7 siRNA基因沉默后可抑制自噬,抑制Panc-1增殖,诱导细胞凋亡。3、PP6iRGD/DTX/Atg7体内实验可抑制自噬,抑制肿瘤生长,促进凋亡,靶向治疗组抑瘤效果更显著。更多还原

【Abstract】 Pancreatic cancer is one of the most challenging gastrointestinal cancer and is associated with poor prognosis. It is characterized by rapid progression, high incidence and mortality. Surgery remains the only opportunity for long-term survival for patients with resectable pancreatic cancer. With the development of laparoscopic instruments and skills, LDP has become widely accepted by surgeons for benign and low-grade tumors of the pancreas. Recent comparative studies showed that LDP has the advantage of less blood loss and fewer hospital stay days as well as fewer postoperative complications compared with open distal pancreatectomy. However, application of laparoscopic approach has been restricted for malignant pancreatic lesions, especially pancreatic ductal adenocarcinoma (PDAC), due to concerns over oncologic safety. Until now only a few studies has been reported and total cases were less. We designed a case-control study to analysis the short- and long-term outcomes of the patients undergoing either Laparoscopic distal pancreatectomy or open distal pancreatectomy for PDAC. Also we systematically review the surgical and oncologic outcomes of laparoscopic distal pancreatectomy (LDP) versus open distal pancreatectomy (ODP) for pancreatic ductal adenocarcinoma (PDAC).Tumor biology of pancreatic cancer is prone to early recurrence and metastasis postoperation. Even after curative resection, the 5-year survival remains at 25%, so chemotherapy plays an important role for patients postoperation or with local advancement or metastasis. But traditional chemotherapy has limited efficiency for pancreatic cancer. Recently with the development of molecular targeted therapy and nanotechnology, it supplies better chemotherapy effect and could reduce systemic toxicity and multidrug resistance (MDR). Autophagy acts as both tumor suppressor and tumor inducer through different mechanism, now regulation of autophagy become an important targeted therapy for cancer. Autophagy is required for tumorigenic growth of pancreaitc cancer and is one of the strategy for chemotherapy-resistant of pancreatic caner. We designed a cancer targeted micellar nanocarrier using Pluronics123 (P123) as main body which could co-delivery of Docetaxel (DTX) and Atg7 siRNA-P123P600iRGD/DTX/Atg7 siRNA(PP6iRGD/DTX/Atg7) complex. Using this PP6iRGD/DTX/Atg7 complex we studied the the release of DTX and Atg 7 siRNA in vivo and in vitro. Knockout of Atg7 by Atg7 siRNA could inhibite autophagy and induce cellular apoptosis.Part 1:Laparoscopic Versus Open Distal Pancreatectomy for Pancreatic Ductal Adenocarcinoma:A case-control studyObjective:Laparoscopic distal pancreatectomy (LDP) showed advantage of perioperation outcomes for benign and low-grade tumor of the pancreas. The application of LDP for pancreatic ductal adenocarcinoma (PDAC) didn’t gain popular acceptance and the number of LDP for PDAC remains low. We designed a case-control study to analysis the short- and long-term outcomes of the patients undergoing either Laparoscopic distal pancreatectomy or open distal pancreatectomy for PDAC.Methods:From April 2003 to October 2015,29patients were underwent LDP and 48 patients were underwent ODP for PDAC. The two groups’demographic information, perioperative outcomes and survival data were compared.Results:Baseline characteristics were comparable between the LDP and ODP groups. The mean operation time, intraoperative blood loss, first flatus, first oral intake and postoperative hospital stay were significantly less in LDP group than ODP group (180min vs270min, P=0.003; 50ml vs400ml, P=0.000; 3d vs 4d, P=0.001; 3d vs 4d, P=0.003; 13d vs 15.5d, P=0.022). The ration of R0 resection, overall postoperative morbidity and postoperative pancreatic fistula rates were similar in the two groups. There were no significant differences in tumor sizes (3.5cm vs 3.9cm, P=0.987) and number of harvested lymph nodes (11 vs 9, P=0.072). The median overall survival for LDP and ODP groups were 16.0 months and 15.0 months. Cox proportional hazards analysis showed, R1 resection and rejection of adjuvant treatment were associated with worse survival.Conclusion:The results in our study validated that LDP was technically feasible and safe for PDAC in selected patients and the short-term oncologic outcomes were not inferior to ODP in this small sample study. However the long-term oncologic safety of LDP for PDAC has to be further evaluated by multicenter or randomized controlled trials.Part 2:Laparoscopic Versus Open Distal Pancreatectomy for Pancreatic Ductal Adenocarcinoma:A Systematic Review and Meta-AnalysisObjective:To systematically review the surgical and oncologic outcomes of laparoscopic distal pancreatectomy (LDP) versus open distal pancreatectomy (ODP) for pancreatic ductal adenocarcinoma (PDAC).Methods:A systematic search of PubMed, Embase, Cochrane Library, and Web of Science was conducted from Jan 1996 to Feb 2016. All original studies comparing LDP versus ODP were included for critical appraisal. Data synthesis and statistical analysis were carried out using RevMan 5.2 software.Results:7 studies were involved in this research. The rate of R0 resection was similar in both LDP and ODP groups(P=0.23). LDP was associated with less intraoperation blood loss, postoperative hospital stay and less tumor size (P<0.01, P<0.01, P=0.04). After sensitivity analyses using propensity score analysis, tumor size was similar in both groups (P=0.07). The mean operation time, overall postoperative morbidity, mortality, number of harvested lymph nodes, ration of N1 and postoperative pancreatic fistula rates were similar in LDP and ODP. The ration of adjuvant treatment and the ration of recurrences were also similar in both groups.Conclusion:The results in this study validated that LDP was technically feasible and safe for PDAC. The oncologic outcomes were similar between LDP and ODP with less blood loss and postoperative hospital stay in LDP group. However, further randomized, controlled studies would be required to evaluate the laparoscopic approach in PDAC.Part 3:Targeted micellar nanocarriers co-delivery of DTX and Atg7 siRNA for Pancreatic cancerObjective:1. To design a tumor-targeted micellar nanocarrier using pluronic123 as main body which could co-delivery gene and drug and to evaluate the ability of drug loading and gene condense.2. To study the synergistic effect of autophagy inhibition and apoptosis induction by PP6iRGD/DTX/Atg7 nanocomplexes as well as antitumor Laparoscopic distal pancreatectomy or open distal pancreatectomy for PDAC.Methods:From April 2003 to October 2015,29patients were underwent LDP and 48 patients were underwent ODP for PDAC. The two groups’demographic information, perioperative outcomes and survival data were compared.Results:Baseline characteristics were comparable between the LDP and ODP groups. The mean operation time, intraoperative blood loss, first flatus, first oral intake and postoperative hospital stay were significantly less in LDP group than ODP group (180min vs270min, P=0.003; 50ml vs400ml, P=0.000; 3d vs 4d, P=0.001; 3d vs 4d, P=0.003; 13d vs 15.5d, P=0.022). The ration of R0 resection, overall postoperative morbidity and postoperative pancreatic fistula rates were similar in the two groups. There were no significant differences in tumor sizes (3.5cm vs 3.9cm, P=0.987) and number of harvested lymph nodes (11 vs 9, P=0.072). The median overall survival for LDP and ODP groups were 16.0 months and 15.0 months. Cox proportional hazards analysis showed, R1 resection and rejection of adjuvant treatment were associated with worse survival.Conclusion:The results in our study validated that LDP was technically feasible and safe for PDAC in selected patients and the short-term oncologic outcomes were not inferior to ODP in this small sample study. However the long-term oncologic safety of LDP for PDAC has to be further evaluated by multicenter or randomized controlled trials.Part 2:Laparoscopic Versus Open Distal Pancreatectomy for Pancreatic Ductal Adenocarcinoma:A Systematic Review and Meta-AnalysisObjective:To systematically review the surgical and oncologic outcomes of laparoscopic distal pancreatectomy (LDP) versus open distal pancreatectomy (ODP) for pancreatic ductal adenocarcinoma (PDAC).Methods:A systematic search of PubMed, Embase, Cochrane Library, and Web of Science was conducted from Jan 1996 to Feb 2016. All original studies comparing LDP versus ODP were included for critical appraisal. Data synthesis and statistical analysis were carried out using RevMan 5.2 software.Results:7 studies were involved in this research. The rate of R0 resection was similar in both LDP and ODP groups(P=0.23). LDP was associated with less intraoperation blood loss, postoperative hospital stay and less tumor size (P<0.01, P<0.01, P=0.04). After sensitivity analyses using propensity score analysis, tumor size was similar in both groups (P=0.07). The mean operation time, overall postoperative morbidity, mortality, number of harvested lymph nodes, ration of N1 and postoperative pancreatic fistula rates were similar in LDP and ODP. The ration of adjuvant treatment and the ration of recurrences were also similar in both groups.Conclusion:The results in this study validated that LDP was technically feasible and safe for PDAC. The oncologic outcomes were similar between LDP and ODP with less blood loss and postoperative hospital stay in LDP group. However, further randomized, controlled studies would be required to evaluate the laparoscopic approach in PDAC.Part 3:Targeted micellar nanocarriers co-delivery of DTX and Atg7 siRNA for Pancreatic cancerObjective:1. To design a tumor-targeted micellar nanocarrier using pluronic123 as main body which could co-delivery gene and drug and to evaluate the ability of drug loading and gene condense.2. To study the synergistic effect of autophagy inhibition and apoptosis induction by PP6iRGD/DTX/Atg7 nanocomplexes as well as antitumor effect for pancreatic cancer.Methods:PP6iRGD/DTX/Atg7 nanocomplexes was synthesized and the struture, drug loading and gene condense ability were examined. In virto and in vivo studies were carried out to evaluate the synergistic effect of autophagy inhibition and apoptosis induction by PP6iRGD/DTX/Atg7 nanocomplexes.Results:A novel tumor-targeted micellar nanocarrier was synthesized using P123 as main body and could co-delivery DTX and Atg7 siRNA through hydrophobic core and hydrophilic shell. The nanocomplexes could self-assembly in fluid.In vitro studies PP6iRGD/DTX/Atg7 nanocomplexes showed controlled release and knockout of Atg7 which inhibiting autophagy and inducing apoptosis in Panc-1 cell through MTT, Western Blotting and qPCR. In vivo studies PP6iRGD/DTX/Atg7 nanocomplexes showed inhibition of autophagy and inhibiting of the tumor growth as well as induction of apoptosis through immunohistochemical, tunned, Western Blotting and qPCR. The targeted therapy effect was obvious in vivo study.Conclusion:1. PP6iRGD/DTX/Atg7 nanocomplexes could co-delivery DTX and Atg7 siRNA and functioned the synergistic effect.2. In vitro studies PP6iRGD/DTX/Atg7 nanocomplexes showed controlled release of DTX. The release of Atg7 siRNA silenced Atg7 expression and inhibited autophagy and induced apoptosis.3. In vivo studies PP6iRGD/DTX/Atg7 nanocomplexes showed inhibition of autophagy and inhibiting of the tumor growth as well as induction of apoptosis. The targeted therapy effect was obvious in vivo study.更多还原