游离空肠移植重建下咽及颈段食管的临床应用及初步评价

自1986年9月至1988年9月,我院用游离空肠移植方法重建下咽及颈段食管癌术后缺损,共完成7例.男5例,女2例;年龄自38岁～67岁,平均57.3岁.Ⅲ期6例(T_3N_05,T_3N_11),Ⅳ期1例(T_3N_2).梨状窝癌累及喉5例,环后癌1例,颈段食管癌1例.全喉咽及颈段食管切除6例,颈段食管切除(保留喉)1例.1例发生肠段坏死,血管吻合成功率为85.7%(6/7).3、5年生存率分别为57.1%(4/7)及33.3%(1/3).介绍了手术要点,该术式的优点及局限性.     

非开胸食管内翻拔脱咽胃吻合术在下咽及颈段食管癌治疗中的应用

本文从1985～1994年5月间应用咽胃吻合术治疗下咽及颈段食管癌30例,男性24例,女性6例。其右侧梨状窝及环后区部分颈段食管8例,左侧9例。颈段食管13例。术后一侧胃壁坏死,其它均成功。     

咽胃吻合术在下咽食管缺损重建中的应用

下咽癌及颈段食管癌因其具有侵袭性的生物学特性及早期临床症状隐蔽,大多数就诊患者均为中晚期病例,因此下咽癌及颈段食管癌患者经手术切除后常常形成大范围的下咽食管缺损。对此类手术后缺损,国内外学者倾向于采用胃或肠移植一期修复,尤其咽胃吻合修复应用较广泛。2003年12月～2006年9月,我院完成5例下咽癌及颈段食管癌行全喉全下咽切除、食管内翻剥脱、咽胃吻合术,现报告如下：     

胃咽吻合术在下咽癌侵及颈段食管手术修复中的应用

目的研究胃咽吻合术在下咽癌侵及颈段食管手术修复中的应用及术后并发症的预防、处理.方法回顾我院1998-2002年采用全喉、全下咽及全食管切除、胃咽吻合术治疗下咽癌侵及颈段食管的病人3例,分析其手术方式、并发症防治体会及结果.结论胃咽吻合术对于下咽癌侵及颈段食管术后缺损具有良好的修复作用,是一值得推荐的手术方式.     

Ⅳ期喉癌、下咽癌及颈段食管癌切除后重建、保留吞咽发音功能

四川省肿瘤医院头顶科与胸外科合作，在W期喉癌、下咽癌及颈段食管癌切除后，利用带血管带组织一期重建，保留重要的吞咽及喉发音功能取得明显进展。该科利用胸大肌岛状脱皮瓣、胃代食管、结肠移植及游离空肠移植等多项技术对厂倒晚期喉癌、复发喉癌、下咽癌及预段食管癌患者成功地进行了咽食管重建。在恢复患者吞咽功能的同时，对3例患者保留了喉的发音功能。在对2例晚期下咽癌及颈段食管癌患者切除肿瘤后，分别采取游离空肠及结肠移植重建日咽、下咽及食管的同时，保留喉体组织于原位，保证了患者术后的正常唯发音功能。该研究成果于1994…     

胸大肌岛状皮瓣在下咽及颈段食管癌术后重建中的应用

目的对较晚期下咽及颈段食管癌大面积切除后的组织缺损,应用胸大肌岛状皮瓣重建恢复功能经验总结.方法自1985年5月至2000年5月期间应用该皮瓣修复下咽及颈段食管癌术后组织缺损177例.结果177例重建中1例因感染发生狭窄,12例发生咽瘘,164例一期愈合,恢复吞咽功能,一年生存率82.5%(142/172),三年生存率45.3%(24/53),五年生存率38.7%(12/31),十年生存率33.3%(4/12),十五年生存率25%(2/8).结论胸大肌岛状皮瓣能够解决较晚期下咽及颈段食管上段癌切除后组织缺损及功能重建.     

T4期下咽癌的手术治疗与修复

目的 探讨晚期下咽癌的外科治疗以及术后下咽及颈段食管缺损修复重建的方法和疗效.方法 回顾性分析25例下咽癌患者的手术方式及修复方法.其中梨状窝癌17例,下咽后壁区癌3例,环后癌5例.其中T4N0M0 9例,T4N1M0 11例,T4N2M0 5例.喉下咽食管全切除后胃代食管的咽胃吻合术7例,喉下咽颈段食管全切除游离空肠代下咽颈段食管术4例,残喉气管瓣修复下咽、颈段食管部分切除后咽部缺损8例,胸大肌肌皮瓣修复下咽、颈段食管部分切除后咽部缺损6例.所有患者颈部均行改良性和(或)择区性颈清扫.其中术前放疗8例,术后放疗17例.结果 25例患者无术中死亡,术后所有患者均恢复了正常吞咽功能,5例喉功能保留的患者不同程度的恢复了呼吸和发音功能.术后随访3～5年,无失访患者.1年内死亡3例,1、3、5年生存率分别为88.0%、48.0%和28.0%.结论 下咽及颈段食管缺损一期修复方法的应用扩大了下咽癌的手术适应证,提高了晚期下咽癌患者的生存率和生活质量.在修复方法的选择方面,肿瘤部位和手术后组织缺损的大小是选择修复方法的主要因素,同时还应结合患者的年龄和全身状况等因素,以期最大可能地减少并发症的发生.

Abstract：

Objective To explore the surgical treatment of hypopharyngeal and cervical esophageal cancers and the ways of reconstruction after hypopharyngo-oesphagectomy, and to evaluate their efficacy.Methods Twenty five patients with cancer of the laryngopharynx and cervical esophagus treated in our department between 1995 and 2007 were included in this study. Their clinical data were restrspectively analyzed. Among them, 17 cases had the tumor originated from the pyriform sinus, 3 of the posterior pharyngeal wall and 5 of the postcricoid region. Acording to the 2002 UICC criteria, all the tumors were stage T4, including 9 patients with cN0, 11 with cN1, and 5 with cN2 disease. The pharyngoesophageal defect reconstruction methods were as following: pharyngogastric anastomosis in 7 patients, free jejunal transplantion in 4, laryngotracheal flap in 8, and pectoralis major musculocutaneous flap in 6 patients. All patients were treated with modified and/or selective neck dissection. Among them, 8 cases received preoperation radiotherapy, 17 received post-operative auxiliary radiotherapy. Results There was no operation death case in this group. All patients were followed up for 3 to 5 years. Three patients died in the first year.According to Kaplan-Meier analysis, the 1 -year survival rate was 88.0%, 3-year survival rate was 48.0%,and S-year survival rate was 28.0%. Conclusions The use of primary repair of the defects of laryngopharynx and cervical esophagus expands the operative indication for cancers of the laryngopharynx and cervical esophagus, improves the survival rate and life quality of the patients. Regarding the repair method of choice, site of the tumor and size of the defect are the most important factors regarding choice of reconstruction method, while the patients' age and general condiction should also be considered to minimize the complications as more as possible.     

非开胸食管内翻拔脱术治疗下咽癌,颈段食管癌

目的：研究非开胸食管内翻拔脱术治疗下咽癌、颈段食管癌疗效及术后胃肠营养的维持。方法：自１９８５年至１９９５年间，采用非开胸食管内翻拔脱术治疗下咽癌１６例，颈段食管癌３２例，术后病理 鳞癌。其中，经食管床咽胃吻合术２５例，经胸骨后咽结肠吻合术２３例。采用高位空肠造瘘逆行插管行术后胃肠减压及维持胃肠营养。结果：术后并发症，咽胃吻合口瘘２例（８％），咽结肠吻合瘘４例（１７．４％），均治愈。喉返神经损伤３     

喉气管自体移植修复下咽癌术后缺损

探讨了喉气管自体移植修复下咽癌术后缺损的疗产。方法：对１１例下咽癌口患者术后采用喉气管自体移植修复下咽，术后随访３年。结果：６例无效力一存，天咽功能恢复了良好。１例死于肺转移。４例局部复发，其中２例已死，２例带瘤生存。结论：认为该术式简便，创伤小，吞咽功能恢复良好，但对于广泛侵及喉及食管口的下咽部病变，需选用其它更为广泛的术式或其它重建技术。     

喉气管自体移植修复下咽癌术后缺损

目的：探讨喉气管自体移植修复下咽癌术后缺损的疗效。方法：对11例下咽癌患者术后采用喉气管自体移植修复下咽，术后随访3年。结果：6例无瘤生存，吞咽功能恢复良好。1例死于肺转移。4例局部复发，其中2例已死，2例带瘤生存。结论：认为该术式简便、创伤小，吞咽功能恢复良好、但对于广泛侵及喉及食管口的下咽部病变，需选用其它更为广泛的术式或其它重建技术。     

Anxa5真核表达质粒构建及其对小鼠肝癌Hca—P细胞增殖影响观察

目的：构建pcDNA3。1-V5／HisB—Anxa5真核表达质粒,获得膜联蛋白A5（AnnexinA5,Anxa5）表达稳定上调的小鼠肝癌Hca—P细胞株,探究Anxa5上调对Hca—P增殖的影响。方法：利用RT—PCR扩增小鼠全长Anxa5编码序列,将其克隆到pcDNA3．1-V5／HisB载体中,并将构建的pcDNA3．1-V5／HisB-Anxa5表达质粒转入Hca—P细胞。采用（;418筛选及有限稀释法获得单克隆细胞株,观察细胞形态变化。蛋白质印迹法分析Anxa5表达情况,CCK-8法检测相应Hca—P细胞的增殖能力。结果：酶切及测序结果显示,pMD R 18-T—Anxa5克隆质粒和pcDNA3．1-V5／HisB-Anxa5表达质粒构建成功;获得了Anxa5表达稳定上调的单克隆细胞株;与正常Hca-P比较,Anxa5蛋白在pcDNA3．1一V5／HisB-Anxa5-Hca-P单克隆细胞中上调了59．5％,P一0．016;pcDNA3．1-V5／HisB～Anxa5-Hca—P增殖显著加快,P=0．002。结论：成功构建了Anxa5真核表达质粒并获得Anxa5表达稳定上调的小鼠肝癌Hca—P细胞株,Anxa5上调对Hca—P细胞的增殖具有促进作用,为后续研究提供实验基础。     

Construction and characterization of a PDCD5 recombinant lentivirus vector and its expression in tumor cells

The function and mechanism of the programmed cell death 5 (PDCD5) gene is not completely understood, so it is necessary to build a stable and efficient PDCD5 recombinant lentiviral expression vector. The coding region of the PDCD5 gene was PCR amplified from pCMV-SPORT6. Next, the PDCD5 fragment and the pGC-FU vector were digested with the restriction enzyme AgeI and ligated by in-Fusion technology to build the pGC-FU-PDCD5 plasmid. Competent E. coli DH5α cells were transformed and positive clones were identified by PCR. The PCR products were digested and sequenced. After sequencing, positive clones were selected to grow and propagate. The pGC-FU-PDCD5 plasmid DNA was purified and mixed with the pHelper1.0 and pHelper2.0 packaging plasmids. They were co-transfected into 293T cells. The viral titer was measured by real-time PCR. The expression of GFP and PDCD5 was detected by both Western blotting and fluorescence microscopy. Additionally, the A498, ACHN, HCT116 and LoVo tumor cell lines were transfected with the virus supernatant, and PDCD5 expression was detected in these cells. The constructed pGC-FU-PDCD5 plasmid contained the correct PDCD5 gene sequence. Strong green fluorescence in both the cytoplasm and cell membrane was observed following transfection with purified pGC-FU-PDCD5 into 293T cells. The transfection rate was greater than 95%, and the expression of the PDCD5-GFP fusion protein was confirmed by Western blotting. The titer of the recombinant PDCD5 lentiviral condensed supernatant was measured to be 2 x 10(9) Tu/ml. The transfection efficiencies of A498, ACHN and HCT116 cells were greater than 90%. However, transfection efficiency of LoVo cells was lower but still greater than 70%. The PDCD5-GFP fusion protein was stable in these transfected tumor cells, as detected by Western blotting. In conclusion, a PDCD5 recombinant lentiviral vector was successfully constructed, and high expression of the plasmid was observed in tumor cells.     

死亡受体5胞外区域的重组、表达及活性鉴定

目的: 构建死亡受体5(death receptor 5,DR5)胞外区域(eDR5)的表达载体,表达纯化重组蛋白并鉴定其生物特性.方法: 通过重叠 PCR 获得 DR5 胞外段编码序列,构建 pET-22b( )/DR5 表达载体,转化大肠杆菌 BL21(DE3),IPTG 诱导表达,Ni2 柱亲和纯化,SDS-PAGE、直接 ELISA 鉴定纯化产物的纯度和特异性,用 MTT 法检测eDR5 蛋白阻断DR5 单克隆抗体 FMU1.5 和 TRAIL 诱导人胶质瘤细胞株 U343(高表达DR5)、U373(低表达DR5 )细胞凋亡的作用.结果: 获得了 DR5 胞外段编码序列,目的蛋白在上清及包涵体中都有表达, 表达量占菌体总蛋白的 30% 以上,纯化的重组蛋白纯度达 95% 以上,蛋白产量达 9 mg/ml.ELISA 结果表明所纯化蛋白为eDR5.eDR5 蛋白可部分阻断 FMU1.5 和 TRAIL 诱导人胶质瘤细胞株 U343 细胞凋亡的作用,其阻断率与 DR5 表达相关.结论: 死亡受体 5 胞外段基因的成功重组、表达及纯化,为进一步的功能研究奠定了基础.     

Sensitivity of human, murine, and rat cells to 5-fluorouracil and 5'-deoxy-5-fluorouridine in relation to drug-metabolizing enzymes

Six cell lines differing in histological origin were studied regarding the growth inhibitory effect of fluoropyrimidines in relation to their metabolism. The human colon carcinoma cell line WiDr was most sensitive to 5-fluorouracil (FUra) (50% growth inhibitory concentration, 0.7 microM) and to its analogue 5'deoxy-5-fluorouridine (5'dFUR) (50% growth inhibitory concentration, 18 microM). The murine B16 melanoma cell line was moderately sensitive to FUra but least sensitive to 5'dFUR. The 50% growth inhibitory concentration values in the human melanoma cell lines IGR3 and M5, the transformed human intestine cell line intestine 407 and the rat hepatoma cell line H35 varied for FUra between 1.7 and 5.0 microM, and for 5'dFUR between 54 and 160 microM. Several enzymes from pyrimidine metabolism responsible for FUra metabolism were measured with FUra as a substrate. The activity of uridine phosphorylase, which catalyzes the conversion of 5'dFUR to FUra, was lowest in B16 cells correlating with the low sensitivity to 5'dFUR. When adenosine 5'-triphosphate was included in the reaction mixture for uridine phosphorylase, FUra was rapidly channeled into FUra nucleotides via its nucleoside. The rate of channeling appeared to correlate with the nucleoside phosphorylase activity in the various cell lines. In several cell lines activities of nucleotide-degrading enzymes were rather high and interfered with the measurement of orotate phosphoribosyl transferase (OPRT) with FUra as substrate. Addition of the phosphatase inhibitor glycerol-2-phosphate partly prevented breakdown of the newly formed 5-fluorouridine 5'-monophosphate and enabled measurement of OPRT. The WiDr cell line had a relatively high OPRT activity which could explain its sensitivity to FUra. The activity of thymidylate synthase was measured at a suboptimal concentration of 1 microM and at the optimal concentration of 10 microM deoxyuridine 5'-phosphate. With all cell lines the ratio between the activities at 10 and 1 microM was between 2.3 and 3.6. The activity of thymidylate synthase was lowest in WiDr and IGR3 cells and 3-4 times higher in M5 and Intestine 407 cells. The inhibition of 0.01 microM 5-fluorodeoxyuridine 5'-monophosphate was 80-90% at 1 microM deoxyuridine 5'-phosphate and 50-70% at 10 microM deoxyuridine 5'-phosphate with all cell lines. At 0.1 microM 5-fluorodeoxyuridine 5'-monophosphate enzyme activity was inhibited by 95-100%. The incorporation of FUra into RNA was relatively low in IGR3 cells and 3-5 times higher in all other cell lines.(ABSTRACT TRUNCATED AT 400 WORDS)     

载5-氟尿嘧啶两亲多糖纳米粒的制备及其对肝癌细胞HepG2的杀伤作用

背景与目的:生物可降解载药纳米微粒作为新型药物靶向传输和缓释/控释载体,可延长药物的生物半衰期,减轻药物的毒副作用,而且具有良好的生物相容性。本实验制备可生物降解的载5-氟尿嘧啶(5-fluorouracil,5-FU)葡聚糖接枝聚乳酸共聚物(5-FU/DEX-g-PLA),探讨其对人肝癌细胞HepG2的体内外杀伤作用。方法:利用分子自组装技术制备5-FU/DEX-g-PLA载药纳米微粒,透射电镜观察纳米粒形态,分光光度法计算载药率,MTT法观察对HepG2细胞的体外杀伤作用,动物实验观察其体内抑瘤效应。结果:5-FU/DEX-g-PLA纳米微粒呈球形,粒径约50nm,药物包封率约9.3%。体内药物代谢动力学数据显示,5-FU纳米制剂在血液中维持时间长于5-FU裸药;MTT结果显示,5-FU纳米组细胞生长抑制率(58.8%)与5-FU裸药组(58.0%)差异无显著性(P>0.05);体内抑瘤实验显示,5-FU纳米组肿瘤抑制率(73.1%)显著高于5-FU裸药组(57.5%)。结论:5-FU/DEX-g-PLA纳米微粒可有效抑制肝癌细胞的生长。     

Early adjuvant intraportal chemotherapy with 5-fluorouracil after hepatic resection of colorectal metastasis: a preliminary clinical and pharmacokinetic study

Intraportal continuous infusion of 5-FU (600 mg/m2/24 h during 7 days) was administered in the immediate postoperative course of 6 consecutive patients with colorectal metastases resected for cure (one segmentectomy and 5 nonanatomical local resections). One month later, a systemic continuous infusion of 5-FU was delivered at the same dose. The tolerance of intraportal chemotherapy was good despite 2 patients with mild digestive toxicity. The plasma concentrations of both unchanged 5-FU and 5,6-dihydro-5-FU (the primary metabolite of 5-FU), were determined in 2 patients using Gas Chromatography--Mass Spectrometry. The 5-FU clearance was higher after intraportal infusion than after systemic infusion (x 1.5 to 3). Hepatic extraction was variable (0.32-0.70) and lower than in reported experimental data on dogs (0.90-0.99). 5,6-dihydro-5-FU concentrations were constantly higher than 5-FU concentrations in plasma. The patient with lower hepatic extraction had the higher 5,6-dihydro-5-FU plasma concentrations. These findings suggest a predominant extrahepatic formation of plasmatic 5,6-dihydro-5-FU.     

ALK5激活型突变体在肾癌细胞中的表达及功能

目的:构建ALK5激活型真核表达质粒Flag-ALK5 T204D,证实融合蛋白在肾癌细胞内表达,并验证其对TGF-β信号通路的激活作用。方法:以野生型ALK5质粒为模板,采用重叠延伸PCR方法进行定点突变;将构建的重组质粒测序并转染到肾癌细胞ACHN中,提取细胞蛋白进行Western blot检测;利用双荧光素酶报告基因分析,检测该突变体对TGF-β信号通路典型靶基因启动子的激活作用。结果:对ALK5的表达序列进行定点突变,构建了ALK5激活型真核表达质粒Flag-ALK5 T204D;Western blot检测到融合蛋白的表达,分子量约为58kDa;Flag-ALK5 T204D能够上调TGF-β信号通路典型靶基因启动子活性。结论:成功构建了Flag-ALK5 T204D并验证了其对TGF-β信号通路的激活作用,为进一步研究ALK5的生物学特性及其功能奠定了基础。     

WNT5A 与小细胞肺癌临床特征的相关性研究及其对 细胞迁移作用的影响

  目的  检测 WNT5A 在小细胞肺癌(small cell lung cancer,SCLC) 中的表达及其促细胞迁移作用的机制。  方法  采用免疫组化的方法检测 79 例 SCLC 和 25 例正常肺组织中 WNT5A 的表达量。通过细胞划痕和 Transwell 小室检测 WNT5A 及 JNK 对 SCLC 细胞系 DMS153 迁移能力的作用,应用 Western blot 检测干扰 WNT5A 后磷酸化 JNK 含量的变化。  结果  WNT5A 在 SCLC 中表达高于正常肺组织,并且与肿瘤的临床分期、淋巴转移及远处转移相关,WNT5A 的表达与神经元特性烯醇化酶(NSE) 、胃泌素释放肽前体(Pro-GRP) 也有明显相关性。干扰 WNT5A 导致 DMS153 迁移能力下降,应用 JNK 抑制剂 SP600125 能够阻止 WNT5A 造成的细胞迁移增加。  结论  WNT5A 在 SCLC 中高表达,并且与肿瘤的转移相关,WNT5A 通过磷酸化 JNK 促进 SCLC 细胞迁移,并且可以作为 SCLC 的预测指标和治疗靶点。       

Concentration of 5-FU after hepatic artery infusion chemotherapy for liver metastases of colorectal cancer

AIM: Hepatic artery infusion chemotherapy (HAI) using 5-FU is a good method of treating patients with liver metastases from colorectal cancer. We investigated the toxicity and the response in relation to the concentration of 5-FU after HAI, and examined various factors that would have an effect on the 5-FU concentration. RESULTS: The mean 5-FU concentration was 480 ng/ml. The most frequent complication of HAI was anorexia. Three of 14 patients suffered from grade 2 or 1 anorexia. The 5-FU concentration of these patients was higher (1,010, 721, 642 ng/ml) than that of the others. The response rate of HAI was 36%. The 5-FU concentration of responders tended to be lower than that of non-responders. None of the following was related to the 5-FU concentration: whether or not the gallbladder was resected, whether or not co-lateral blood flow was recognized, or the volume of the patients' liver. CONCLUSION: 5-FU concentration after HAI has some effect on anti-tumor response and gastrointestinal toxicity.     

Cost-effectiveness analysis of first-line chemotherapies in metastatic colorectal cancer. Results of the Fédération Francophone de Cancérologie Digestive (FFCD) 9601 randomized trial

BACKGROUND: The De Gramont regimen (or high-dose LV5FU2, HD-LV5FU2) is considered a standard treatment for metastatic colorectal cancer. The aim of the study was to evaluate the efficacy and the costs of three regimens as compared to HD-LV5FU2: raltitrexed (R), LV5FU2 with a lower dose of folinic acid (LD-LV5FU2), and weekly infusional 5FU (WI-FU). METHODS: An economic analysis was performed prospectively as part of a randomized trial comparing first-line chemotherapy regimens in 294 patients with unresectable metastatic colorectal cancer. The primary endpoint was event-free survival (EFS). Direct medical costs were computed from the health system viewpoint using 2001 unit costs. RESULTS: None of the three regimens improved EFS as compared to HD-LV5FU2. R was less effective and more toxic. The mean total cost per patient was euro 15,970 for HD-LV5FU2. The cost of R (10,687 euro) was lower than that of HD-LV5FU2 (p = 0.008). The cost of LD-LV5FU2 (14,888 euro) and of WI-FU (13,760 euro) was not significantly different from that of HD-LV5FU2. CONCLUSION: The lower efficacy and increased toxicity of R made it a clinically inferior regimen despite its easy administration and lower cost. The HD-LV5FU2 protocol remains a better treatment. LD-LV5FU2 appeared a good alternative regimen because it reduced costs without jeopardizing its efficacy. The WI-FU regimen did not show a significant difference in terms of efficacy, but suggested toxicity to be slightly increased.