【作者】 汪理；

【导师】 王春友；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 目的本研究旨在检测胰腺癌中GSK-3β的表达,探讨GSK-3β的表达与胰腺癌临床病理特征的关系,进而深入探讨其在胰腺癌发生、发展中的作用。方法运用免疫组织化学SP法检测65例胰腺癌组织及10例癌旁胰腺组织中GSK-3β的表达。结果在65例胰腺癌标本中33例出现GSK-3β的阳性表达,而在癌旁胰腺组织未检测到GSK-3β的阳性表达(P<0.01)。GSK-3β蛋白的表达与胰腺癌患者的年龄、性别和肿瘤大小无关,但与胰腺癌的分化程度、转移和临床分期有关(P<0.05)。结论GSK-3β的高表达在胰腺癌的发生发展和浸润转移过程中起着重要作用。GSK-3β可能与胰腺癌患者的预后的密切相关。目的构建针对人GSK-3β基因的shRNA真核表达质粒,并筛选出基因沉默效果最佳的shRNA质粒表达载体；转染人胰腺癌细胞株PANC-1,建立稳定表达GSK-3βshRNA的细胞模型。方法针对GSK-3p基因的mRNA序列设计,分别构建3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经大肠杆菌扩增,酶切,PCR,测序鉴定,转染胰腺癌PANC-1细胞,Real-time PCR检测GSK-3βmRNA被抑制情况。选取效应最强的重组质粒和阴性对照质粒转染的PANC-1细胞,经G418筛选后,建立稳定表达GSK-3βshRNA的PANC-1细胞株(实验组)和稳定表达control shRNA的PANC-1细胞株(阴性对照组),未转染的PANC-1细胞株设为空白对照组。采用荧光显微镜和FCM观察细胞的转染情况；Real-time PCR和Western blot分析GSK-3β的表达。结果1、经测序证实,成功构建GSK-3βshRNA真核表达质粒,插入的DNA片段的序列与设计序列完全一致；2、重组质粒瞬时转染PANC-1细胞后,GSK-3βmRNA明显下调(P<0.05),其中以2号重组质粒效应最强；3、重组质粒稳定转染后检测PANC-1细胞中GSK-3β的表达,Real-time PCR和’Western blot结果均提示：与空白对照组和载体对照组比较,实验组中GSK-3βmRNA和蛋白的表达均显著降低(P<0.05)。结论本研究成功构建了携带以GSK-3β为靶向的shRNA的重组质粒。经脂质体途径稳定转染的PANC-1细胞,该shRNA能够显著抑制GSK-3β的表达。该实验为进一步研究GSK-3β的功能和以其为靶点的肿瘤的基因治疗提供了基础。目的本实验拟在体外观察基因沉默GSK-3β对胰腺癌PANC-1细胞的增殖、凋亡、周期和侵袭能力的影响,并初步探讨相关的分子机制及GSK-3β作为胰腺癌治疗靶点的可能性。方法实验分组如下：空白对照组为未转染的PANC-1细胞；载体对照组为稳定转染control shRNA的PANC-1细胞；实验组为稳定转染GSK-3βshRNA的PANC-1细胞。MTT法连续7d检测各组细胞的OD值,并绘制出细胞的生长曲线；FCM检测细胞的凋亡和周期；Transwell侵袭实验检测细胞的侵袭能力；Real-time PCR检测各组细胞中bcl-2、cyclin D1、VEGF、HIF-1αmRNA的表达；Western blot检测各组细胞中bcl-2、cyclin D1、HIF-1α蛋白的表达；ELISA检测PANC-1细胞培养上清中VEGF蛋白的浓度；EMSA检测PANC-1细胞中NF-κB的DNA结合活性。结果与对照组比较,转染GSK-3βshRNA的PANC-1细胞生长速度明显减慢；凋亡率明显增加；而且处于G0／G1期细胞比例显著升高,S期细胞比例显著降低,发生明显的G1期阻滞(P<0.05)。实验组PANC-1细胞中bcl-2、cyclin D1、VEGF基因和蛋白的表达显著下调,NF-κB的DNA结合活性降低(P<0.05)。GSK-3p沉默的PANC-1细胞中HIF-1α基因和蛋白的表达出现不一致的变化。结论GSK-3β可能在胰腺癌细胞的增殖、存活和侵袭中具有重要作用,阻断GSK-3β的表达可望成为胰腺癌分子靶向治疗的新途径。目的探讨基因沉默GSK-3β对人胰腺癌裸鼠移植瘤生长及血管生成的影响。方法人胰腺癌PANC-1细胞接种于裸鼠皮下,建立胰腺癌裸鼠移植瘤模型,分为空白对照组(种植未转染的PANC-1细胞)、载体对照组(种植转染control shRNA的PANC-1细胞)和实验组(种植转染GSK-3βshRNA的PANC-1细胞),测量各组移植瘤的重量和体积,并计算抑瘤率；免疫组化SP法检测PCNA和FⅧ蛋白的表达,并计算增殖指数(SPF)和微血管密度(MVD)；Real-time PCR和Western blot检测VEGF基因和蛋白的表达。结果实验组移植瘤的重量和体积显著低于对照组(P<0.05),抑瘤率为35.27%；实验组SPF和MVD值均小于对照组(P<0.05)；实验组移植瘤的VEGF基因和蛋白的表达较对照组显著降低(P<0.05)。结论在体内,基因干扰GSK-3β的表达可以显著抑制PANC-1细胞接种的裸鼠皮下移植瘤的生长和新生血管的形成,该效应可能与其下调VEGF的表达有关。目的探讨RNA干扰GSK-3β后,人胰腺癌PANC-1细胞对吉西他滨敏感性的改变。方法实验分组如下：空白对照组为未转染的PANC-1细胞；载体对照组为稳定转染control shRNA的PANC-1细胞；实验组为稳定转染GSK-3p shRNA的PANC-1细胞。在各组PANC-1细胞中加入不同浓度吉西他滨并作用48h；MTT检测各组细胞的增殖活性；FCM检测各组细胞的凋亡率。结果在吉西他滨作用48h后,实验组中PANC-1细胞的生长抑制率、凋亡率均显著高于对照组,而且该效应与剂量成正比,高剂量引起高抑制(P<0.05)；结论基因干扰GSK-3p的表达可显著增强吉西他滨对胰腺癌PANC-1细胞的杀伤力,并伴有NF-κB活性的降低和抗凋亡基因bcl-2的下调,这表明基因沉默GSK-3β可能通过抑制胰腺癌细胞NF-κB的活性,下调与化疗耐药相关的NF-κB的靶基因bc1-2的表达而增强胰腺癌对化疗的敏感性。因此,阻断GSK-3β的表达有望开辟胰腺癌基因治疗的新途径。更多还原

【Abstract】 Objective To investigate the relationship between the expression of GSK-3βand clinical features in pancreatic cancer, and determine the role of GSK-3βin the development of pancreatic cancer.Methods The expression of GSK-3βin pancreatic cancer tissue and adjacent normal tissue was detected by SP immunohistochemical technique.Results GSK-3βwas expressed in 33 of 65 cases. In contrast, no expression of GSK-3βin adjacent normal tissue was detectable (P<0.01). Expression of GSK-3βhad no relationship with ages, sexes and tumor sizes of pancreatic cancer patient. But the expression was significantly correlated with differentiation level, metastatic status and JPS stage (P<0.05).Conclusion High expression of GSK-3βmay play an important role in carcinogenesis and development, invasion and metastasis of pancreatic cancer. Therefore, assessment of GSK-3βexpression might be useful for predicting the prognosis of pancreatic cancer patients.Objective To construct three shRNA interference expression plasmid vectors of human GSK-3βgene, detect the expression of GSK-3βin pancreatic carcinoma PANC-1 cells after transfection with recombinant plasmids, and transfect stably the most effective recombinant plasmid into PANC-1 cells.Methods Three plasmid expression vectors coding for shRNA targeting GSK-3βand a control vector were designed. The recombinant plasmids were amplified in E. coli. DH5a, and then treated with restriction enzymes, PCR and sequencing. PANC-1 cells stably expressing GSK-3βshRNA and control shRNA were screened with G418, as the experimental group and vector control group. The cells were not transfected as the blank control group. Cells transfection state was observed by fluorescence microscope and FCM. GSK-3βexpression was assayed with Real-time PCR and Western blot.Results 1.The successful construction of recombinant plasmids was determined by DNA sequencing.2 GSK-3βexpression was notably down-regulated after transfection of shRNA plasmids in PANC-1 cells. Recombinant plasmid 2 had the strongest effect.3. Compared with the control groups, the expression of GSK-3βshow a significantly down-regulation in experimental group.Conclusion Plasmid vector expressing shRNA against GSK-3βand PANC-1 cells with stably expressing GSK-3βshRNA were constructed, which could facilitate further studies on GSK-3βfunction and its application in tumor gene therapy.Objective To investigate the effects of GSK-3βRNA interference on pancreatic cancer cell proliferation, apoptosis, cycle and invasive capability and to explore the molecular mechanism involving the procedure and the possibility of targeting GSK-3βfor pancreatic cancer therapy.Methods PANC-1 cells stably expressing GSK-3βshRNA and control shRNA were the experimental group and the vector control group. The cells were not transfected as the blank control group. The growth curves of PANC-1 cells were designed by MTT assay. The cells apoptosis and cycle were analyzed by FCM. The invasive capability of PANC-1 cells was detected by Transwell. The expression levels of bcl-2、cyclin D1、VEGF、HIF-1αwere respectively assayed with Real-time PCR, Western blot and ELISA. NF-κB DNA binding activity was detected through EMSA analysis of nuclear extracts. Results Compared with control group, the growth rate and invasive capability of PANC-1 cells in experimental group decreased notably, the apoptotic number of cells increased obviously, and cell cycle was arrest at G0/G1 phase. The expression of bcl-2, cyclin D1, VEGF, and NF-κB DNA binding activity show a significantly down-regulation in experimental group. However, in GSK-3βRNAi PANC-1 cells, the HIF-1αprotein expression was not inconsistent with the gene expression.Conclusion GSK-3βmay play an important role in proliferation, survival and invasion of human pancreatic cancer. Blocking GSK-3βin pancreatic cancer cells may offer an avenue for gene therapy.Objective To study the effects of GSK-3βRNA interference on the growth and angiogenesis of human pancreatic cancer xenografts in nude mice.Methods PANC-1 cells were incubated and were inoculated subcutaneously in athymic nude mice to establish xenograft models. The mice were divided into blank control group, vector control group and experimental group. Tumor volume and weight was measured in nude mice bearing xenografts. The inhibitory rate was calculated according to the weights of xenografts. The proteins of PCNA and FⅧwere assessed by immunohistochemical method. SPF and MVD were respectively counted according to PCNA and FⅧstaining. The expression level of VEGF was detected by Real-time PCR and Western blot.Results Compared with control group, the growth rate of human pancreatic cancer xenografts of nude mice in experimental group decreased notably (P<0.05) and the inhibitory rate was 35.17%. The SPF index, MVD count and VEGF expression of xenografts were significantly decreased in experimental group than that in control group (P<0.05).Conclusion In vivo GSK-3βRNAi can inhibit the growth and angiogenesis of human pancreatic cancer xenografts in nude mice, which may be related to the downregulation of VEGF.Objective To investigate the sensitivity of pancreatic cancer cell to gemcitabine (GE) after GSK-3βRNA interference.Methods PANC-1 cells stably expressing GSK-3βshRNA and control shRNA were the experimental group and the vector control group. The cells were not transfected as the blank control group. Different dosages of GE were added into PANC-1 cells. The growth inhibition rates and the apoptosis rates were respectively measured by MTT and FCM.Results GE caused a greater increase of the growth inhibition rate and apoptosis rates of PANC-1 cells in experimental group than in control groups in a dose-dependent manner (P<0.05)Conclusion Our data demonstrate that GSK-3βRNAi inhibits the NF-κB DNA binding activity and the expression of bcl-2 and enhances the growth inhibition and the apoptosis induced by GE. Thus GSK-3βRNAi could sensitize pancreatic cancer cells to GE. Blocking GSK-3βin pancreatic cancer cells may offer an avenue for gene therapy.更多还原