胰腺生长因子促胰岛β细胞的生物活性研究

目的研究胰腺提取物中的胰腺生长因子对胰岛β细胞的增殖活性。方法常规培养胰岛β细胞RIN-m细胞株,按每孔1.0×104个细胞入96孔板,加药培养分别至24 h、48 h、72 h,按四甲基偶氮唑蓝比色法(methyl thiazolyltetrazolium,MTT)和5-溴脱氧尿嘧啶(5-Bromo-2′-Deoxyuridine,Brdu)法检测细胞生长增殖情况,设阴性对照,以市售百密达(BEYATTA)为阳性对照;同时采用反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)荧光定量检测RIN-m细胞内的肠胰十二指肠同源盒1(pancreatic duodenal homeobox factor-1,PDX-1)和胰岛素(insulin,INS)基因的表达情况;收集培养48 h后的细胞,以流式细胞仪检测细胞的凋亡情况。结果胰腺提取物中的胰腺生长因子能促进胰岛RIN-m细胞株的增殖,增殖率达40%以上(P<0.01),PDX-1和INS基因的同步高表达,均具有统计学差异(P<0.01)。实验组与对照组相比,细胞凋亡率降低。结论本胰腺提取物质(pancreatic extract,PE)有显著促进胰岛β细胞生长增殖、抑制其凋亡的作用,可作为糖尿病治疗新药开发重要依据。

大鼠胰腺发育不同阶段肝细胞生长因子受体编码基因的表达

目的研究肝细胞生长因子受体编码基因c-met在大鼠胰腺发育不同阶段的表达。方法采用高密度寡核苷酸芯片对孕12.5 d(E12.5)、孕15.5 d和孕18.5 d、新生、成年胰腺进行基因转录水平分析,并用RT-PCR技术验证基因在大鼠胰腺不同发育时期的表达。结果c-met基因在E15.5、E18.5较成年特异性高表达。经RT-PCR验证,c-met基因表达趋势与芯片结果相符;与芯片c-met探针所用引物不同RT-PCR,结果却在各发育阶段呈现与芯片不同的表达趋势。结论提示c-met可能在胰腺发育过程中起到调控作用,参与胚胎胰腺发育中晚期细胞功能完善的信号传导过程。并且c-met在胰腺发育中发挥作用有可能存在不同转录本。

穿心莲内酯对糖尿病大鼠血糖及胰腺胰岛素样表皮生长因子-1蛋白的影响

目的研究穿心莲内酯对糖尿病大鼠血糖及胰腺胰岛素样表皮生长因子-1(IGF-1)蛋白的影响。方法大鼠尾静脉注射链脲菌素建立1型糖尿病模型,将模型组大鼠分为糖尿病对照组、穿心莲内酯低剂量组[100mg/(kg·d)]、高剂量组[200mg/(kg·d)]3组,并设正常对照组。给药2周后,断尾采血检测各组大鼠血糖,HE染色观察大鼠胰腺形态学变化,免疫组织化学法检测各组大鼠胰腺胰岛素样表皮生长因子-1蛋白表达。结果与正常对照组比较,各组大鼠血糖明显升高(P<0.05),模型组大鼠胰腺组织胰岛明显减少,胰腺IGF-1蛋白表达比正常对照组明显减少。穿心莲内酯高、低剂量组与模型对照组比较,血糖随药物浓度升高而降低(P<0.05),胰腺组织病理损害减轻,随药物浓度升高胰腺IGF-1蛋白表达增加。结论穿心莲内酯可降低糖尿病大鼠血糖,显著改善糖尿病大鼠胰腺组织损伤,其作用机制可能与上调IGF-1蛋白表达有关。

神经鞘磷脂在人胰腺癌细胞生长中的信使作用

目的：观察人胰腺癌细胞株中SM水平：观察PACAP138对人胰腺癌细胞株（HS766T）SM水平的影响。探讨SM在信息传导中的作用：同时观察SM的分解产物SPH对细胞的增殖作用及其机制。方法：细胞培养，分别加入不同浓度的PACAP138、PACAP638和SPH。应用MTT法来观察细胞增殖程度：Fura-2/AM测定[Ca^2+]；薄层层析法测定细胞内SM含量。结果：PACAP138促进HS766T的Ca^2+和SM的生成，呈剂量依赖性，PACAP638拮抗PACAP138的上述作用。SPH促进HS766T的生长，促进HS766Ta^2+和SM的生成，呈剂量依赖性，结论：SPH能促进人胰腺癌细胞株增殖；SM的信使作用一方面是通过与PI转换，活化PKC，产生IP3，另一方面是通过刺激Ca^2+转运和Ca^2+的释放。

生长抑素联合香丹参注射液治疗重症急性胰腺炎临床研究

目的 观察生长抑素联合复方丹参注射液治疗重症急性胰腺炎（SAP）的临床疗效.方法 采用随机对照分组的临床研究方法,将2012年6月- 2016年3月收治的162例SAP患者分为对照组和观察组：对照组 （81例） 予生长抑素治疗, 治疗组 （81例） 予生长抑素联合香丹注射液治疗. 观察血清淀粉酶、 C反应蛋白、 肿瘤坏死因子和胰腺影像学变化.结果 治疗后患者临床症状、血清淀粉酶和炎症指标改善明显,治疗组和对照组的总有效率分别为98%和65%,比较差异有统计学意义（P〈0.05）.结论 生长抑素联合香丹注射液治疗重症急性胰腺炎疗效确切,临床值得推广应用.

c-met及相关基因在大鼠胰腺发育功能完善中的表达

目的：研究原癌基因c-met及其相关基因在大鼠胰腺发育及细胞功能完善过程中的表达。方法：采用高密度寡核苷酸芯片(Affymetrix芯片)对孕12．5(E12．5)、E15．5和E18．5、新生、成年胰腺进行基因转录水平分析,并用RT-PCR验证基因在大鼠胰腺不同发育时期的表达。结果：c-met基因在E15．5、E18．5较成年特异高表达。芯片中c-met转录调控基因和信号传导通路相关基因的表达趋势与c-met高度相似。RT-PCR(所用引物设计区域与芯片相同)验证,c-met表达趋势与芯片结果相符；与芯片c-met探针所用引物不同RT-PCR,结果却在各发育阶段呈现与芯片不同的表达趋势。结论：提示c-met可能在胰腺发育细胞功能完善的关键阶段起调控作用,参与胚胎胰腺发育中晚期细胞功能完善的信号传导过程。并且c-met在胰腺发育中发挥作用有可能存在不同转录本。

大鼠胰腺发育中肝细胞因子受体编码基因的表达及蛋白定位

目的研究肝细胞因子受体编码基因c-met在大鼠胰腺发育不同阶段的表达及其编码蛋白的细胞定位。方法采用RT-PCR技术检测c-met基因在大鼠胰腺不同发育时期:孕15.5 d(E15.5)和孕18.5 d(E18.5)、生后14 d(P14)、生后21 d(P21)、新生及成年胰腺的表达;并用免疫组化技术检测该基因编码的蛋白c-MET在胰腺发育不同阶段的细胞定位。结果c-met基因在E15.5、E18.5 d较成年特异性高表达,免疫组化结果显示该基因编码的蛋白c-MET在新生后的胰腺大量定位于胰岛细胞。结论c-met可能在胰腺发育过程中起到调控作用,参与胰腺发育中新生后胰岛结构重塑过程。

小鼠胰腺对梯度胆囊收缩素的应答

目的分析胆囊收缩素(CCK)对胰腺及胰腺癌发生的影响,并探讨绿茶成分表没食子儿茶素没食子酸酯(EGCG)的抗癌作用。方法将生长期小鼠随机分为4组,分别给予含大豆胰蛋白酶抑制剂(STI)0 g/L(11只)、2 g/L(11只)、4 g/L(10只)和8 g/L(11只)的饮水,2周后处死小鼠,记录胰腺湿质量。采用酶联免疫吸附试验(ELISA)检测血浆CCK水平;测定胰腺组织中蛋白质、DNA、胰蛋白酶和脂肪酶含量;免疫组织化学染色检测胰腺组织增殖细胞核抗原(PCNA)阳性细胞百分比。采用胰腺内包埋化学致癌物二甲基苯并蒽(DMBA)法建立癌发生模型。将15只小鼠随机均分为正常对照组、DMBA包埋组、DMBA+2 g/L STI组、DMBA+8 g/L STI组、DMBA+8 g/L STI+EGCG组,6周后处死小鼠,取胰腺进行组织学分析。结果2 g/L STI组CCK水平高于0 g/L STI组,8 g/L STI组高于0、2和4 g/L STI组(P<0.05)。2、4 g/L STI组胰腺湿质量高于0 g/L STI组,8 g/L STI组高于0、2 g/L STI组(P<0.05)。2、4 g/L STI组胰腺蛋白质和DNA含量高于0 g/L STI组,8 g/L STI组高于0、2和4 g/L STI组(P<0.05)。0、2、4和8 g/L STI组胰蛋白酶含量依次升高;2、4、8 g/L STI组胰脂酶含量高于0 g/L STI组(P<0.05)。0、2、4、8 g/L STI组小鼠胰腺组织PCNA阳性细胞比例依次升高(P<0.05)。胰腺癌前病变程度随饮水中的STI剂量增大而加重,EGCG可减轻此病变。结论STI可诱发梯度CCK血症,并刺激胰腺增生、胰酶生成及胰腺癌发生,而EGCG具有减轻胰腺癌前病变的作用。

AKT2短发夹状RNA抑制胰腺癌细胞生长的研究

蛋白激酶B（PKB／AKT）是一种重要的信号蛋白分子，AKT蛋白与细胞生长代谢密切相关。AKT2是AKT蛋白3种主要亚型之一，有研究证实AKT2在胰腺癌中表达增高，且AKT2对胰腺肿瘤的发生发展有重要促进作用，本实验旨在研究AKT2短发夹状RNA表达载体对胰腺癌细胞生长的影响。

针对K—ras^Asn12的短发夹RNA表达载体对胰腺癌细胞生长的抑制作用

我们利用RNAi技术达到了特异抑制胰腺癌突变K-ras^Asn12的目的，现报道如下。一、材料和方法 1．材料：AsPC-1和BxPC-3细胞株购自中科院上海细胞库。pSilenCircle试剂盒购自Allele公司。质粒抽提纯化盒购自Promrga公司。PCR引物及插入序列由上海生工生物工程公司合成。CellCounting Kit-8购自日本同仁公司。An-nexin V-FITC apoptosis detectionkit购自晶美公司。

生长激素对胰腺癌细胞株增殖活性的调控

目的探讨生长激素对胰腺癌细胞株Bxpc-3增殖活性的影响。方法MTT法检测生长激素对胰腺癌细胞株Bxpc-3增殖活性,Tunel法标记凋亡阳性细胞、免疫组化法标记增殖细胞,激光共聚焦显微镜计数免疫荧光双标Tunel阳性细胞和增殖细胞。结果10-7mol/L、10-8mol/L、10-9mol/L浓度的生长激素在第2天明显的抑制胰腺癌细胞生长,伴随作用时间的延长这种抑制作用逐渐减轻,且出现了加速胰腺癌细胞生长的作用,第4天、第5天胰腺癌细胞的生长同正常对照组相比有显著差异(P<0.01)。结论本实验研究从细胞学的角度证实生长激素在开始阶段具有抑制胰腺癌细胞生长的作用,但是这种作用伴随时间的延长逐渐消失,且出现加速了肿瘤细胞的生长的现象,为临床肿瘤病人应用生长激素提供了一定的理论依据。

血管内皮生长因子C在淋巴结转移灶和原发灶胰腺癌细胞中的表达及作用

本实验旨在观察血管内皮生长因子（VEGF）-C表达的器官差异性，以及其反义寡核苷酸（ASODN）对胰腺癌细胞生长、凋亡的影响，探讨VEGF—C在胰腺癌淋巴结转移中的机制。

蛋白磷酸酶2A抑制剂通过c—Jun氨基末端激酶／蛋白激酶B通路抑制胰腺癌细胞生长的机制

斑蝥素是蛋白磷酸酶2A（protein phosphatase 2A，PP2A）的抑制剂。PP2A可使c—Jun氨基末端激酶（c-Jun N—terminal kinase，JNK）等参与细胞生长的多种激酶去磷酸化而失活。本研究拟检测斑蝥素和经典的PP2A抑制剂冈田酸对胰腺癌细胞JNK／Akt信号通路的活性及细胞生长的影响。

酪氨酸激酶受体抑制剂STI571对胰腺癌细胞生长的抑制作用

STI571（CGP57148）是特异性酪氯酸激酶受体抑制剂，特异性阻断Abl、血小板衍化生成因子、c-kit受体。本研究旨在探讨应用STI571对胰腺癌细胞生长的影响。

Fractalkine and TGF-β1 levels reflect the severity of chronic pancreatitis in humans

MM： To clarify whether serum chemokine and cytokine levels can become useful biological and functional markers to assess the severity of chronic pancreatitis （CP）. This study aimed at clarifying whether serum chemokine and cytokine levels can become useful biological and functional markers to assess the severity of CR METHODS： Serum monocyte chemoattractant protein-1 （MCP-1）, transforming growth factor beta-1 （TGF-βI）, and soluble type fractalkine （s-fractalkine） concentrations were examined in patients with CP （n = 109） and healthy controls （n = 116）. Severity of disease was classified in patients with CP by a staging system. Relationships between stage-specific various clinical factors and serum MCP-1, TGF-β1, and s-fractalkine levels were investigated. Furthermore, 57 patients with non-alcoholic CP were similarly evaluated in order to exclude influence of alcohol intake. RESULTS： Patients with CP showed significant higher levels of serum TGF-β1 and s-fractalkine, but not MCP-1, compared to the controls. Serum TGF-β1 in the severe stage and s-fractalkine in the mild and thesevere stage of CP significantly increased compared to those of controls. However, it was observed that both TGF-β1 and s-fractalkine levels were affected by alcohol intake. In patients with non-alcoholic CP, serum TGF-β1 showed significant increase in the moderate stage of CP, and serum s-fractalkine revealed significant increase in the early stage of CP. CONCLUSION： It is suggested that the measurement of serum F-fractalkine is useful to diagnose early- stage CP. Moreover, the combined determination of both, s-fractalkine and TGF-β1, in human sera may be helpful in evaluating the severity status of CP.

MK615 inhibits pancreatic cancer cell growth by dual inhibition of Aurora A and B kinases

AIM:To investigate the anti-neoplastic effect of MK615, an anti-neoplastic compound isolated from Japanese apricot, against human pancreatic cancer cells in vitro. METHODS: Three human pancreatic cancer cell lines PANC-1, PK-1, and PK45H were cultured with MK615 at concentrations of 600, 300, 150, and 0 μg/mL. Growth inhibition was evaluated by cell proliferation assay, and killing activity was determined by lactate dehydrogenase (LDH) assay. Expression of Aurora A and B kinases was detected by real-time polymerase chain reaction (PCR) and Western blotting. Cell cycle stages were evaluated by flow cytometry. RESULTS: The growth inhibitory rates of MK615 at 150, 300, and 600 μg/mL were 2.3% ± 0.9%, 8.9% ± 3.2% and 67.1% ± 8.1% on PANC1 cells, 1.3% ± 0.3%, 8.7% ± 4.1% and 45.7 ± 7.6% on PK1 cells, and 1.2 ± 0.8%, 9.1% ± 2.1% and 52.1% ± 5.5% on PK45H cells, respectively (P <0.05). The percentage cytotoxicities of MK615 at 0, 150, 300, and 600 μg/mL were 19.6% ± 1.3%, 26.7% ± 1.8%, 25.5% ± 0.9% and 26.4% ± 0.9% in PANC1 cells, 19.7% ± 1.3%, 24.7% ± 0.8%, 25.9% ± 0.9% and 29.9% ± 1.1% in PK1 cells, and 28.0% ± 0.9%, 31.2% ± 0.9%, 30.4% ± 1.1% and 35.3 ± 1.0% in PK45H cells, respectively (P < 0.05). Real-time PCR and Western blotting showed that MK615 dually inhibited the expression of Aurora A and B kinases. Cell cycle analysis revealed that MK615 increased the population of cells in G2/M phase. CONCLUSION: MK615 exerts an anti-neoplastic effect on human pancreatic cancer cells in vitro by dual inhibition of Aurora A and B kinases.

Prognostic significance of S100A4 and vascular endothelial growth factor expression in pancreatic cancer

AIM: To investigate the expression of vascular endothelial growth factor (VEGF) and calcium-binding protein S100A4 in pancreatic cancer and their relationship to the clinicopathological parameters and prognosis of pancreatic cancer. METHODS: Expression status of VEGF and S100A4 was examined in 62 surgical specimens of primary pancreatic cancer by immunohistochemistry. Correlation between the expression of VEGF and S100A4 and clinicopathological parameters was analyzed. RESULTS: Thirty-eight of 62 (61.3%) specimens of primary pancreatic cancer were positive for S100A4. Thirty-seven (59.7%) specimens showed positive expression of VEGF. The positive correlation between S100A4 and VEGF expression was significant in cancer tissues (P < 0.001). S100A4 expression was significantly correlated with tumor size, TNM stage and poorer prognosis. VEGF expression had a significant correlation with poorer prognosis. The prognosis of 17 S100A4-and VEGF-negative cancer patients was significantly better than that of other patients (P < 0.05). Distant metastasis (P = 0.001), S100A4-(P = 0.008) and VEGF-positive expression (P = 0.016) were significantly independent prognostic predictors (P < 0.05). CONCLUSION: Over-expression of S100A4 and VEGF plays an important role in the development of pancreatic cancer. Combined examination of the two molecules might be useful in evaluating the outcome of patients with pancreatic cancer.

MATl基因沉默对胰腺癌细胞生长的影响

我们既往的研究证实，作为调控细胞周期循环的关键基因之一，人类MATl（ménage à trios1）基因在胰腺癌组织中表达增强，它参与胰腺癌的发生［1-2］。转染反义MATl基因可使胰腺癌细胞出现明显的G．期阻滞［3］。为此，本研究应用体外转录法合成靶向MATl基因的小干扰RNA（small interferenceRNA，siRNA），转染胰腺癌Capan-2细胞，观察其对细胞的生长抑制效应，探讨siRNA沉默MATl基因用于胰腺癌基因治疗的可行性。

Anti-tumor activity of erlotinib in the BxPC-3 pancreatic cancer cell line

AIM：To investigate the effect and mechanism of action of erlotinib, an epidermal growth factor receptor （EGFR） small molecule tyrosine kinase inhibitor （TKI）, in the human pancreatic cancer cell line BxPC-3 both in vitro and in vivo.METHODS： In vitro, human pancreatic cancer cell line BxPC-3 was exposed to varying concentrations of ertotinib, and its effects on proliferation, cell cycle distribution, apoptosis and the expression of proand antiapoptotic factors such as bcl-2, bcl-xl, bax and bak, and the expression of vascular endothelial cell growth factor （VEGF） were measured with 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, flow cytometric analysis, terminal deoxynucleotidyl transferase-mediated nick end labeling assay （TUNEL）, and reverse transcriptionpolymerase chain reaction （RT-PCR）. Potential effect of erlotinib on angiogenesis was examined by tube formation assay. Tumor growth suppression was observed in xenografted nude mice with pancreatic cancer in vivo. Immunohistochemical （IHC） staining for EGFR and factor VII-related antigen was undertaken to detect the microvessel density and VEGF expression in tumor tissue in xenograft nude mice.RESULTS： Erlotinib, as a single agent, repressed BxPC-3 cell growth in a dose-dependent manner, triggered G1 arrest and induced cell apoptosis, and suppressed capillary formation of endothelium in vitro. Expressions of VEGF were significantly down-regulated at a high concentration of 200 μmol/L, however, the expressions of bcl-2 and bcl-xl were decreased at 50 μmol/L. In vivo, Erlotinib-treated mice demonstrated a reduced tumor volume, weight and microvessel density as compared to the control. IHC staining showed decreased expression of EGFR and RT-PCR had lower VEGF expression in treated mice.CONCLUSION： The in vitro and in vivo findings provide evidence that BxPC-3 cells are inhibited with erlotinib treatment. Inhibition of EGFR may be a promising adjuvant chemotherapy strategy in pancreatic cancer treatment.