血管、淋巴管内皮生长因子在胰腺癌中的表达

研究血管内皮生长因子(VEGF)在胰腺癌中的表达及意义,采用:Northern blot分析法检测正常胰腺组织和胰腺癌组织中VEGF-A、c mRNA的表达;采用免疫组织化学染色法检测胰腺癌标本中VEGF-A、C蛋白的表达。结果发现胰腺癌组织中VEGF-A和VEGF-C基因的表达分别是正常胰腺组织中的3.0和3.6倍;免疫组织化学染色法显示胰腺癌组织中VEGF-A和VEGF-C的阳性率分别为50％和80％;胰腺癌细胞VEGF-A的表达与肿瘤大小呈正相关,VEGF-C的表达与淋巴结转移呈正相关,均有统计学意义(P<0.05),而VEGF-A与VEGF-C表达无关联。胰腺癌非同步联合表达血管和淋巴管内皮生长因子,前者与肿瘤大小、局部范围相关联,后者与淋巴结转移相关联。     

Suppression of growth of pancreatic cancer cell and expression of vascular endothelial growth factor by gene silencing with RNA interference

OBJECTIVE: To explore the anti‐angiogenesis and tumor cell growth suppressive effects resulted from gene silencing by RNAi in BxPC‐3 human pancreatic cancer cells. METHODS: The designation and transfection of vascular endothelial growth factor (VEGF)‐siRNA lentivirus was carried out in vitro. Real‐time PCR and western blot were conducted to measure the expression levels of VEGF mRNA and protein. Flow cytometry was employed to evaluate cell apoptosis and cell death. A lactate dehydrogenase (LDH) assay was used to assess the cytotoxicity of VEGF‐siRNA. A 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) assay was used to picture the cellular growth. For the in vivo study, BxPC‐3 cells were injected subcutaneously into nude mice to form xenografts. The mice were divided into three groups according to the intervention used. The control group, the negative control group and the knockdown group of mice were injected with saline, an empty lentivirus vehicle and lentivirus carrying VEGF‐siRNA, respectively. None of the mice died during the study. When these mice were killed, the xenografts were collected and the tumor sizes of the different groups were compared. Finally, immunohistochemistry was used to assess the VEGF expression level and microvascular density. RESULTS: After the transfection of VEGF‐siRNA lentivirus, the cellular expression of VEGF mRNA decreased to 50% of the control and the VEGF protein in the BxPC‐3 cells decreased to 30% of the control. Apoptosis and cell death increased after transfection of the VEGF‐siRNA lentivirus. The LDH assay showed high cytotoxicity induced by VEGF‐siRNA lentivirus transfection. The MTT assay showed slower cellular growth in the knockdown cells. Tumor growth suppression was observed in nude mice that had received the VEGF‐siRNA lentivirus transfection, and the tumor sizes of the xenografts in this group were clearly smaller than those in other two groups. VEGF expression and microvascular density were significantly decreased. CONCLUSION: Vascular endothelial growth factor gene silencing via VEGF‐siRNA can effectively inhibit the production of VEGF and exert an anti‐angiogenesis and tumor cell growth suppressive effect both in vitro and in vivo.     

Meta‐analysis of inoperable pancreatic cancer: gemcitabine combined with cisplatin versus gemcitabine alone

OBJECTIVES: To compare the therapeutic effects of gemcitabine (GEM) monotherapy with GEM‐cisplatin (DDP) combination chemotherapy in patients with advanced stage pancreatic cancer (APCa) through meta‐analysis. METHODS: MEDLINE and EMBASE searches were supplemented by information from trial registers of randomized controlled trials (RCTs) for GEM‐DDP combination chemotherapy and GEM alone in APCa. A quantitative meta‐analysis using updated information based on inclusion criteria from all available RCTs was carried out by two reviewers. The primary meta‐analysis involved the overall survival (OS), objective remission rate (ORR) and toxicity. RESULTS: The meta‐analysis included six RCTs. There was no significant advantage for the GEM‐DDP combination group in 6‐month survival rate (P = 0.24) or clinical benefit rate (P = 0.58). There was a marginal significant improvement for the GEM‐DDP combination group in ORR (RD = 6%, P = 0.05; RD, risk difference = risk in the GEM‐DDP combination group – risk in the GEM alone group). Moreover, there was a significant improvement for the combination group in 6‐month TTP/TTF (RD = 9%, P = 0.02). WHO grade 3–4 toxicity was higher for the GEM‐DDP combination group in terms of neutropenia (RD = 6%, P = 0.08), thrombocytopenia (RD = 8%, P = 0.17) and vomiting/nausea (RD = 11%, P = 0.07); none reached significant difference. CONCLUSION: GEM‐DDP combination should not be recommended and GEM monotherapy remains the standard treatment for patients with APCa.     

Effects of all-trans-retinoic on human gastric cancer cells BGC-823

OBJECTIVE: To determine the inhibitory effects of all‐trans‐retinoic acid (ATRA) on cell growth, cell cycle and vascular endothelial growth factor (VEGF) expression in the human gastric cancer cell line BGC‐823 in vitro. METHODS: Human gastric cancer BGC‐823 cells were treated with various concentrations of ATRA and the cell growth was then determined using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide viability assay. The cell cycle distribution was analyzed using a flow cytometer. The VEGF mRNA and protein expression were analyzed by semi‐quantitative RT‐PCR and Western blotting, respectively. RESULTS: ATRA at concentrations of 0.1–10 µmol/L inhibited the growth of BGC‐823 cells grown in culture; a time‐ and dose‐dependent inhibitory influence was found. ATRA arrested BGC‐823 cells at the G₀/G₁ phase in a dose‐dependent way. Both VEGF mRNA and protein were decreased by ATRA in a dose‐dependent way. CONCLUSION: The anti‐tumor effects of ATRA on human gastric cancer cells are associated with G₀/G₁ phase arrest and decreased VEGF expression.     

IP方案化疗对小细胞肺癌患者血清VEGF及内皮抑素的影响

目的 探讨IP方案化疗对小细胞肺癌（SCLC）患者血清血管内皮生长因子（VEGF）及内皮抑素的影响.方法 对45例SCLC患者行IP方案化疗,采用ELISA法检测其化疗前后的血清VEGF、内皮抑素,同时对20例健康对照者进行比较.结果 SCLC患者的血清VEGF、内皮抑素均高于对照者（P均＜0.01）,二者水平与患者的年龄、性别、吸烟与否无关,与临床分期有关（P＜0.05）;临床受益患者化疗后血清VEGF下降、内皮抑素升高（P均＜0.01）;血清VEGF低于平均值组的中位生存期长于高于平均值组,内皮抑素高于平均值组中位生存期长于低于平均值组（P均＜0.05）.结论 SCLC患者血清VEGF及内皮抑素的变化对判定化疗疗效及预后有一定参考价值.     

Short term chemotherapy followed by radiofrequency ablation in stage III pancreatic cancer: results from a single center

Background

Neo-adjuvant chemotherapy (CHT) has gained increasing importance in resectable and borderline resectable pancreatic cancer leading to a better performing surgery when we look at negative resection margins and selection of patients with less aggressive disease. We apply this principle to patients with Stage III (LAC) pancreatic cancer undergoing RFA and try to select patients who may benefit from a local treatment.

Methods

All patients affected by LAC were treated with RFA for a stable disease after a short CHT. Postoperative morbidity and mortality were evaluated together with overall survival (OS) and disease specific survival (DSS).

Results

We consecutively treated 57 patients affected by LAC. Median duration of CHT before RFA was 5 months. The postoperative mortality rate was zero. Overall morbidity was 14 % with RFA-related morbidity of 3.5 %. The OS and DSS were 19 months and when compared to a similar population who received RFA as up front treatment, there was no difference.

Conclusions

Our results do not support the adoption of a short CHT as a way to identify patients to treat with RFA with the most benefit. Based on this and by knowing the role of immune modulation after RFA and its specific involvement in pancreatic carcinoma, we can propose RFA as upfront treatment.     

PDGFR-β反义寡核苷酸对大鼠血管平滑肌细胞凋亡的影响

目的 :探讨血小板衍化生长因子 β受体 （PDGFR- β）反义寡核苷酸对培养的大鼠血管平滑肌细胞 （VSMC）凋亡的影响。方法 :建立 SD大鼠胸主动脉 VSMC体外增殖模型 ,取 5～ 10代细胞为实验对象 ,按实验目的分为以下几组 :1反义寡核苷酸组 ;2正义寡核苷酸组 ;3错义寡核苷酸 ;4空白对照组。利用透射电镜等观察 VSMC加药前后形态学和超微结构的变化 ,采用原位末端脱氧核苷酸转移酶介导的 d U TP缺口末端标记法 （TU NEL）和流式细胞仪观察和分析 PDGFR- β反义寡核苷酸对 VSMC凋亡的影响。结果 :1加药后 2 4h细胞形态和超微结构较加药前无明显改变 ,48h细胞形态和超微结构较加药前有明显改变。2加药后 2 4h TU NEL 染色各组未发现凋亡细胞 ,48h后反义寡核苷酸组细胞爬片染色有较多凋亡阳性细胞出现。 3加药 2 4h流式细胞仪检测未发现加药组细胞凋亡量与对照组之间有显著差异 ,48h加药组细胞凋亡量明显高于对照组。结论 :PDGFR- β反义寡核苷酸对大鼠VSMC凋亡有诱导作用。     

放射性PDGFR-β反义寡核苷酸对血管平滑肌细胞增殖和凋亡的影响

目的研究32P标记血小板衍化生长因子β受体（PDGFR-β）的反义寡核苷酸（AON）对血管平滑肌细胞（VSMC）增殖的影响,为抑制血管术后再狭窄的形成提供可能的干预。方法体外进行大鼠VSMC的培养,以510代细胞为实验对象,人工合成PDGFR-βAON,并进行32P标记,按实验目的分组。MTT法分析细胞增殖活力,以流式细胞仪测定细胞周期,用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）检测细胞凋亡。结果32P标记PDGFR-βAON作用后,VSMC的增殖强度明显弱于空白对照组（P<0.01）,而处于DNA合成前期（G0/G1）细胞百分比高于空白对照组（P<0.01）;32P标记AON组细胞凋亡率明显高于空白组（P<0.01）。结论32P-PDGFR-βAON可以诱导VSMC凋亡,抑制其增殖。     

致动脉粥样硬化不同因素对大鼠腹腔巨噬细胞和主动脉平滑肌细胞分泌表达Tenascin-C的影响

目的　观察一些致动脉粥样硬化因素对培养的大鼠腹腔巨噬细胞和主动脉平滑肌细胞分泌表达Tenascin C的影响。方法　体外分离培养大鼠腹腔巨噬细胞和主动脉平滑肌细胞 ,同步后分别加入不同刺激因子采用Westernbolt及逆转录聚合酶链反应 (RT PCR)的方法检测Tenascin C蛋白和mRNA水平表达。结果　Tenascin C在两种细胞的对照组中均不表达 ,低密度脂蛋白刺激组只有少量表达 ,其余各刺激组均呈高水平表达。加入氧化修饰低密度脂蛋白 (oxLDL) 2h后即有Tenascin C的表达 ,6h时达高峰 ,可持续到 2 4h。oxLDL与Tenascin C蛋白的表达呈明显的浓度依赖性。结论　oxLDL等致动脉粥样硬化刺激因子可在转录水平上调Tenascin C基因的表达 ,从而促进血管平滑肌细胞和巨噬细胞分泌表达高水平的Tenascin C ,这可能是动脉粥样硬化中Tenascin C沉积的主要原因     

Remodeling in the microcirculation of rat skeletal muscle during chronic ischemia

OBJECTIVE: To establish the time course and extent of remodeling of terminal microcirculation in ischemic rat skeletal muscle during prolonged low flow that does not lead to inflammation. METHODS: One common iliac artery was ligated via laparotomy in adult Sprague-Dawley rats and extensor digitorum longus (EDL) muscles removed at intervals (1, 2, and 5 weeks) postsurgery. Serial frozen EDL sections were stained to show capillaries (alkaline phosphatase), cell proliferation (antibody to proliferating cell nuclear antigen [PCNA]), terminal microvessels (antibodies to alpha-smooth muscle actin (alpha-SMA) or endothelial nitric oxide synthase [eNOS]), and macrophages (antibodies to infiltrating and resident macrophages). Total muscle eNOS protein was quantified by standard Western blotting techniques. RESULTS: Capillary proliferation was very limited in ischemic EDLs, with a modest 12% increase in the capillary/fiber ratio after 5 weeks, preceded at 2 weeks by increased numbers of PCNA-positive nuclei at capillary sites. There was no muscle necrosis or evidence of inflammation, based on macrophage staining. The number of terminal microvessels that were positive for alpha-SMA and <10 microm in diameter was fewer in ischemic EDLs at all time points, whereas the number of larger positive vessels was unchanged. eNOS-positive vessels <10 microm in diameter were stained similarly throughout ischemic muscles as the controls, and showed a similar increase in vessel/fiber ratio as the capillaries. The total eNOS protein level was similar to that in controls in ischemic EDLs after 1 and 2 weeks, but was 28% lower after 5 weeks. CONCLUSIONS: Prolonged, moderate flow reduction to skeletal muscles does not necessarily lead to inflammation or extensive capillary growth. Based on eNOS staining, the terminal microcirculation remains intact, but the loss of alpha-SMA immunoreactivity may indicate remodeling involving the "deinvestment" of microvessels by smooth muscle.     

骨骼肌卫星细胞移植后心肌梗死区血管内皮因子表达及血管新生作用

目的:研究骨骼肌卫星细胞移植后心肌梗死(MI)区血管内皮因子(VEGF)表达及血管新生作用。方法:成年Louis近交系大鼠,结扎前降支建立急性MI模型,将骨骼肌卫星细胞移植到梗死区,2W后应用免疫组化方法鉴定梗死区VEGF的表达及新生血管密度。结果:骨骼肌卫星细胞在梗死区增殖、分化良好,梗死区VEGF表达移植组明显高于对照组(P<0.05),新生血管密度移植组也高于对照组(P<0.05)。结论:移植区卫星细胞可以分泌生长因子,促进血管形成,改善梗死区细胞的生存环境。     

Results of a retrospective analysis of gemcitabine as a second-line treatment after chemoradiotherapy and maintenance chemotherapy using 5-fluorouracil in patients with locally advanced pancreatic cancer

Background  Many studies of concurrent chemoradiation therapy with 5-fluorouracil (5-FU) for locally advanced pancreatic cancer have been reported with a median survival time of approximately 10 months. Recently, gemcitabine (GEM) has been administered immediately after chemoradiation. The clinical outcome of chemoradiation therapy in conjunction with 5-FU and second-line chemotherapy with GEM after disease progression has not been clarified. Methods  Patients with locally advanced pancreatic cancer were treated with concurrent radiation therapy (1.8 Gy/fraction; total dose, 50.4 Gy) with 5-FU (200 mg/m² every day) until disease progression, followed by GEM (1000 mg/m², days 1, 8, 15, and every 4 weeks) as second-line therapy. Results  Of the 18 patients with locally advanced pancreatic cancer who received chemoradiation therapy with 5-FU, there were three partial responses, giving a response rate of 17%. The median time to progression was 170 days. The median survival time was 443 days. During chemoradiation therapy, the incidences of grade 3 or 4 anorexia, nausea, mucositis, and gastric ulcer were 33%, 22%, 17%, and 17%, respectively. Sixteen patients received second-line chemotherapy with GEM, of whom one patient had a partial response. The median time to progression from the initiation of GEM was 113 days, and median overall survival time was 231 days. Major toxicities were hematological toxicities: grade 3 or 4 leukopenia in 75% and anemia in 31%. Conclusions  The treatment strategy with concurrent chemoradiation and maintenance chemotherapy with 5-FU followed by second-line chemotherapy with GEM may be an option for locally advanced pancreatic cancer.     

Gemcitabine-based combination therapy com-pared with gemcitabine alone for advanced pancreatic cancer: a meta-analysis of nine randomized controlled trials

BACKGROUND: Pancreatic cancer is one of the most aggres-sive malignancies and chemotherapy is an effective strategy for advanced pancreatic cancer. Gemcitabine (GEM) is one of first-line agents. However, GEM-based combination therapy has shown promising efficacy in patients with advanced pancreatic cancer. This meta-analysis aimed to compare the efficacy and safety of GEM-based combination therapy versus GEM alone in the treatment of advanced pancreatic cancer.DATA SOURCES: A comprehensive search of literature was performed using PubMed, EMBASE, Web of Science and Co-chrane Central Register of Controlled Trials. A quantitative meta-analysis was performed based on the inclusion criteria from all eligible randomized controlled trials. The outcome indicators included overall survival (OS), 6-month survival, 1-year survival, progression-free survival/time-to-progression (PFS/TTP), and toxicities.RESULTS: A total of nine randomized controlled trials involv-ing 1661 patients were included in this meta-analysis. There was significant improvement in the GEM-based combination therapy with regard to the OS (HR=0.85, 95% CI: 0.76-0.95, P=0.003), PFS (HR=0.76, 95% CI: 0.65-0.90, P=0.002), 6-month survival (RR=1.09, 95% CI: 1.01-1.17, P=0.03), and the overall toxicity (RR=1.68, 95% CI: 1.52-1.86, P<0.01). However, there was no significant difference in the 1-year survival.CONCLUSIONS: GEM-based combination chemotherapy might improve the OS, 6-month survival, and PFS in advanced pan-creatic cancer. However, combined therapy also added toxicity.     

血小板衍化生长因子对血管平滑肌细胞增殖及胶原蛋白合成的影响

目的 :观察血小板衍化生长因子 （PDGF）对培养的血管平滑肌细胞 （VSMC）增殖及胶原蛋白合成的影响。方法 :采用培养的兔动脉 VSMC,应用 3H- Td R的 3H-脯氨酸掺入方法 ,观察 PDGF- BB对兔 VSMC DNA合成以及胶原蛋白合成的影响。结果 :PDGF- BB可促进处于静止状态的兔 VSMC DNA及胶原蛋白的合成 ,并呈现明显的浓度依赖关系 ,在 40 μg/ L 的浓度时 DNA及胶原蛋白的合成达到高峰 ,DNA及胶原蛋白分别处于 36 h和 48h合成最为显著。结论 :PDGF- BB可明显促进培养的 VSMC增殖及胶原蛋白的合成。     

胰腺癌组织中血管内皮生长因子mRNA与尿激酶型纤溶酶原激活物mRNA定量表达的临床意义分析

目的探讨胰腺癌组织中血管内皮生长因子(VEGF)mRNA、尿激酶型纤溶酶原激活物(uPA)mRNA定量表达的临床意义。方法自2008年1月至2011年12月于本科行胰头癌根治术的患者中筛选出经病理证实为导管腺癌的30例患者的完整资料,采用荧光定量PCR(qPCR)检测其胰腺癌组织及6例正常胰腺组织中VEGF mRNA、uPA mRNA定量表达,分析其与临床病理因素之间的关系。结果 VEGF mRNA、uPA mRNA的表达与胰腺癌的组织分化程度、神经侵犯有关。VEGF mRNA在淋巴结转移阳性组中的定量表达高于淋巴结转移阴性组,两组比较差异有统计学意义(t=20.007,P=0.000);uPA mRNA在直径≤2 cm的肿瘤组织中定量水平小于直径2 cm的肿瘤组织,两组比较差异有统计学意义(t=7.539,P=0.000)。uPA mRNA在伴有十二指肠侵犯组中的定量表达高于无十二指肠侵犯组,两组比较差异有统计学意义(t=-2.089,P=0.037)。uPA mRNA在Ⅲ期肿瘤组织中的定量表达高于Ⅰ、Ⅱ期肿瘤组织中的定量表达,两组比较差异有统计学意义(t=-9.450,P=0.000)。VEGF mRNA与uPA mRNA的表达存在正相关(r=0.334,P=0.000)。结论 VEGF mRNA、uPA mRNA在胰腺癌组织中过度表达可为肿瘤细胞的侵润创造条件。     

雌激素对PDGF诱导的血管平滑肌细胞增殖及细胞周期的影响

目的探讨17β-雌二醇（E2）对血管平滑肌细胞（VSMC）增殖及细胞周期的影响。方法分离培养VSMC,血小板源生长因子（PDGF）诱导其增殖,应用MTT法及流式细胞仪检测不同浓度E2（10和100 nmol/L）对传代VSMC增殖活性及细胞周期的影响。结果E2（10和100 nmol/L）作用下VSMC增殖活性下降,且与浓度有关;对VSMC细胞周期的分布影响主要表现为处于G0/G1期细胞数增多,G2/S期的细胞数减少（P<0.01）。结论E2具有抑制VSMC增殖的作用,其部分机制可能是通过阻滞VSMC G0/G1期向S的转化有关。     

白藜芦醇对人食管癌细胞Eca-109增殖的影响及其机制探讨

目的观察白藜芦醇（Res）对人食管癌细胞Eca-109增殖的影响,并探讨其机制。方法将Eca-109细胞分别与0、10、20、40μmol/L的Res共同培养,采用MTT法测定Res对Eca-109细胞的生长抑制率,采用RT-PCR法检测Eca-109中的VEGF mRNA。结果随着Res浓度增加、作用时间延长,Eca-109细胞生长抑制率增加（P均〈0.05）,VEGF mRNA表达量降低（P均〈0.05）。结论Res可抑制Eca-109细胞增殖,其作用可能与抑制VEGF表达有关。     

色素上皮衍生因子对缺氧/复氧血管平滑肌细胞增殖及凋亡的影响

目的:观察重组人源性色素上皮衍生因子（Pigment epithelium-derived factor,PEDF）对缺氧/复氧(H/R)后平滑肌细胞（vascular smooth muscle cells,VSMCs）增殖和凋亡的影响。方法:以组织贴块法培养VSMCs后,随机分为3组,即对照组、H/R模型组及模型+PEDF组。模型+PEDF组又按PEDF的浓度(50、100、200及400 ng/ml)分为4组。用MTT比色法检测VSMCs增殖的变化;用Hoechst/PI双染色法和流式细胞仪法(FCM)检测VSMCs的凋亡;用Western blot法测定凋亡相关蛋白Bcl-2、Bax的表达。结果:①MTT比色法检测显示,缺氧、复氧可促进VSMCs增殖;PEDF能够抑制VSMCs增殖,以200 ng/ml的PEDF作用更显著。Hoechst/PI双染色法和FCM检测显示,经100、200 ng/ml的PEDF作用后,VSMCs的凋亡率较对照组和H/R组明显增高(P<0.01)。与对照组和H/R组比较,Western blot检测显示,100、200 ng/ml的PEDF能够下调Bcl-2蛋白表达并上调Bax蛋白表达(P<0.05,P<0.01)。结论:H/R后,VSMCs增殖增多,PEDF可抑制H/R后VSMCs增殖,其作用可能与Bcl-2、Bax通路促进VSMCs凋亡有关。     

HIF-1α基因沉默对食管鳞癌细胞功能性基因的影响

目的观察食管鳞癌细胞中缺氧诱导因子-1α（HIF-1α）基因与血管内皮生长因子（VEGF）、血红素加氧酶（HO-1）、基质金属蛋白酶-2（MMP-2）基因之间的关系及其对细胞周期的影响。方法以HIF-1α基因沉默的Eca-109细胞、化学模拟缺氧细胞及未干预细胞为研究对象,通过Western blot检测VEGF、HO-1、MMP-2基因在三种细胞中的表达差异以了解其与HIF-1α基因的关系,并通过流式细胞术分析了解HIF-1α基因沉默后细胞周期的变化。结果Western blot检测发现,同空白及模拟缺氧组比较,HIF-1α基因沉默后HIF-1α蛋白表达明显下调,同时VEGF、HO-1、MMP-2的蛋白表达均出现了明显下降。流式细胞分析表明,HIF-1α基因沉默后的Eca-109细胞组同对照组相比G1、G0期细胞明显增加,G2、M期细胞变化不大,S期细胞比例明显减少。结论HIF-1α与上述三种基因存在明显相关性,进而可能影响着食管鳞癌的血管生成、侵袭、应激保护等生物学行为,HIF-1α沉默可将食管鳞癌细胞阻滞于G1、G0期。