【作者】 张树华；

【导师】 王春友；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分胰腺癌组织、癌旁组织及正常组织糖代谢酶活性表达的检测目的检测胰腺正常组织、癌组织及癌旁组织中糖酵解关键酶-乳酸脱氢酶（1acatedehydrogenase，LDH）活性及其同工酶谱变化，以及琥珀酸脱氢酶（succinatedehydrogenase，SDH）活性的表达变化，分析糖酵解与胰腺癌生物学特性的关系。方法收集我院自2006年10月至2008年7月期间我院收治的12例胰腺导管腺癌患者手术切除标本和12例胰岛素瘤等良性病变及其正常胰腺组织。LDH活性检测采用LDH检测试剂盒；LDH同工酶谱检测采用法国Sebia公司的hydrasys全自动分析仪；酶组织化学染色检测癌组织、癌旁组织及胰腺正常组织中琥珀酸脱氢酶活性的变化。结果与正常胰腺组织相比，癌组织及癌旁组织中LDH活性明显升高，差异有统计学意义（P＜0．05）；癌组织及癌旁组织中LDH同工酶谱分析显示LDH4、LDH5明显升高，与正常组织相比，差异有统计学意义（P＜0．05）。酶组织化学染色显示，癌组织中琥珀酸脱氢酶表达活性较癌旁组织及正常组织活性明显降低。结论胰腺癌有氧氧化功能降低，糖酵解水平增强，其中LDH活性及其同工酶谱的变化可能和胰腺癌的发病机理相关，特异性抑制LDH可能成为胰腺癌新的治疗策略。第二部分乳酸脱氢酶特异性抑制剂体外对人胰腺癌细胞生长抑制及诱导凋亡的影响目的分析乳酸脱氢酶抑制剂-草氨酸盐（oxamate）对人胰腺癌细胞株增殖的影响及诱导凋亡的作用。方法MTT法观察终浓度为0mol／L，0．02mol／L，0．04mol／L，0．08mol／L，0．1mol／L草氨酸盐分别作用于不含血清培养基及含10％血清培养基胰腺癌PANC-1细胞株24h、48h后，检测其对细胞增殖的抑制作用，同时行Hochest33342及PI染色、流式细胞仪检测草氨酸盐对其凋亡的影响。结果草氨酸盐对人胰腺癌PANC-1细胞株的增殖有显著的抑制作用，并且随草氨酸盐浓度增加，增殖抑制明显增加，呈剂量依赖性，不含血清培养时抑制作用高于含血清组（P＜0．01），；Hochest及PI染色、流式细胞仪检测均表现细胞凋亡比例逐渐增高，且呈剂量依赖性（P＜0．01）。结论草氨酸盐能够显著抑制人胰腺癌细胞的增殖并诱导其凋亡，有可能成为治疗胰腺癌的新靶点。第三部分，LDH抑制剂-草氨酸盐体外对胰腺癌细胞细胞凋亡的可能机制的研究目的研究探讨乳酸脱氢酶抑制剂-草氨酸盐（oxamate）诱导胰腺癌细胞PANC-1凋亡的可能机制。方法体外培养人胰腺癌细胞PANC-1，不同浓度草氨酸盐（0mol／L，0．02mol／L，0．04mol／L，0．06mol／L，0．08 mol／L，0．1mol／L）作用panc-1细胞48h，流式检测细胞凋亡情况；钙离子荧光指示剂Fluo-3／AM染色，激光共聚焦下观察不同浓度草氨酸盐作用下细胞内钙含量的变化；罗丹明123（Rhodamine 123，Rh123）单染、Rh123联合PI双染联合检测细胞线粒体膜电位的变化；结果草氨酸盐干预胰腺癌细胞48h后，细胞凋亡比例逐渐增高，细胞内钙含量随着草氨酸盐浓度的升高，荧光强度也逐渐增强，呈剂量—效应关系；0．02mol／L～0．06mol／L的草氨酸盐干预时，罗丹明单染显示细胞线粒体膜电位逐渐增高，而0．08mol／L～0．1mol／L的草氨酸盐时，线粒体膜电位则出现明显下降的趋势，Rh123-／PI-细胞百分比明显增加（p＜0．05），0．1mol／L时已为（8．94±0．13）％；结论糖酵解抑制剂-草氨酸盐能够有效的诱导胰腺癌PANC-1细胞的凋亡。草氨酸盐可能通过增加胰腺癌细胞内钙离子含量，影响细胞内钙离子平衡，低浓度组主要通过抑制细胞能量代谢而诱导凋亡，而0．08mol／L和0．1mol／L的高浓度组还可能通过降低细胞线粒体膜电位，进而激活线粒体凋亡途径，诱导其凋亡。第四部分LDH-A shRNA对人胰腺癌细胞系PANC-1增殖、凋亡和LDH-A表达的影响目的肿瘤组织糖酵解增强，而乳酸脱氢酶催化丙酮酸氧化生成乳酸，是糖酵解途径的关键酶，乳酸脱氢酶A（1actate Dehydrogenase，LDH-A）与乳酸脱氢酶B（LDH-B）基因分别编码M亚基和H亚基，M、H亚基比例不同而表现为同工酶LDH_1-5。本研究通过构建LDH-A shRNA，转染PANC-1细胞，分析其对胰腺癌细胞生物学特性的影响。方法构建3条LDH-A shRNA质粒，脂质体转染质粒至PANC-1细胞中，实时荧光定量PCR法检测不同质粒转染后LDH-A mRNA的表达变化。将抑制效率最高的shRNA质粒-3转染PANC-1细胞，MTT法检测转染前后细胞增殖的变化，流式细胞仪检测细胞凋亡的变化。RT-PCR检测LDH-A表达以及酶化学染色检测转染前后LDH活性的变化。结果3种LDH-A shRNA质粒转染胰腺癌细胞后，LDH-A shRNA质粒-3的2-△△Ct值为（0．47±0．02），较正常细胞的（0．71±0．01）小，存在抑制作用，且抑制效率较最高。PANC-1细胞在转染shRNA质粒-3后12h就出现转染组吸光度值显著低于对照组，细胞出现增殖抑制，24h、36h、48h和72h，转染组的吸光度值均显著低于对照组（P＜0．01）。转染质粒组细胞凋亡也明显增高，其凋亡率可达到61．74％；转染shRNA质粒-3的胰腺癌细胞株，其LDH-A mRNA的表达明显抑制，酶化学染色显示LDH活性明显减弱。结论LDH-A shRNA通过抑制胰腺癌细胞LDH-A mRNA的表达，从而抑制其增殖及诱导细胞凋亡，可能成为治疗胰腺癌的新的策略。更多还原

【Abstract】 PartⅠAnalysis the activity of the enzyme of glycoysis in PancreaticNormal Tissue,Carcinoma and Adjacent Non-Cancerous TissueObjective To detect the activity of enzyme of glycolysis,concluding the lactatedehydrogenase （LDH） and LDH isoenzyme,and succinate dehydrogenase（SDH）,so as toresearch the relation between biological characterictic of the pancreatic cancer andglycolysis.Methods Consecutive 12 cases pancreatic ductal adenocarcinoma and 12 benign lesionssuch an insulinoma from October 2006 to July 2008 were collected,as well as normalpancreatic tissues.The total activity of the LDH was detected by the LDH Testing Kits,andthe iosenzyme pattern of LDH was inspected by the France Sebia hydrasys.The activity ofSDH was detected by the enzyme histochemical staining.Results Compared to the normal tissue,LDH activity of the pancreatic cancer andadjacent non-cancerous tissue was significantly higher （P＜0.05）.LDH iosenzyme patternin cancer tissue was also significantly different,i.e.,the percentage of LDH4 and LDH₅increased obviously,and greater than the normal tissue （P＜0.05）.SDH actvity of thepancreatic cancer was obviously higher than the adjacent non-cancerous tissur and the normal pancreatic tissues.Conclusion The alteration of SDH、LDH activity and its isoenzyme pattern are possiblyrelated to the pathogenesis of pancreatic cancer.Inhibit the LDH activity may be a newtherapeutic strategy for pancreatic cancer.PartⅡEffect of the inhibitor of LDH（oxamate） on growth andapoptosis of human pancreatic cancer cell in vitroObjective To investigate the effect of Lacate Dehydrogenase（LDH） inhibitor-oxamate onthe growth and apoptosis of human pancreatic cancer cell.Methods The inhibitional effect of oxamate was observed at 0 mol/L,0.02 mol/L,0.04mol/L,0.08 mol/L,0.1mol/L on the growth ofpanc-1 cell line in medium containing 10%fetal bovine serum and serum-free medium for 24 h and 48 h by MTT assays.Flowcytometry（FCM）,Hochest and PI staining analysis was usedto study the changes of cellapoptosis.Results Oxamate caused dose-dependent inhibition on the growth of human pancreaticcancer cell panc-1.Inhibition was much greater in serum-free medium than in medium with10% fetal bovine serum （p＜0.0 1）.Flow cytometry（FCM）,Hochest and PI staining analysisshows that the proportion of the apoptosis increased gradually,and it isdose-dependent（p＜0.01）.Conclusion LDH inhibitor-oxamate inhibits the growth of human pancreatic cancer cell and induces apoptosis.Oxamate could be a potential targent for anti-pancreatic cancer.PartⅢThe research of mechasim of pancreatic cancer cell apoptosisintroduced by inhibitor of LDH-oxamate in vitroObjective Investigate and research the possible mechasim for apoptosis of pancreaticcancer cell introduced by LDH-oxamate in vitro.Method The pancreatic cancer cell PANC-1 was treated with different concentration ofoxamate （0mol/L,0.02mol/L,0.04mol/L,0.06mol/L,0.08 mol/L,0.1mol/L） for 48h.Theapoptosis was measured by flow cytometry（Annexin V/PI）;then dyed with Fluo-3/AM.Thelight density of cells for different concentration of oxamate under confocal lasermicroscopy were investigated;mitochondrial membrane potential was detected by flowcytometry With Rodamin 123 or Rodamin 123/PI stained.Result With oxamate treated for 48h,the apoptosis of PANC-1 increased obviously.Oxamate can influenced the concentration of Ca²⁺ in PANC-1,and this effect is correspondto the concentration of oxamate.The MMP（0.02mol/L-0.06mol/L）increased,but decreasedsignificantly（0.08mol/L-0.1mol/L）.The percentage of Rh123-/PI- cells increased aftertreated with oxamate,that of 0.08mol/L-0.1mol/L increased obviously （p＜0.05）,thepercentage of 0.1 mol/L came to （8.94±0.13）%.Conclusion The glycolysis inhibitor-oxamate could induce the pancreatic cancer cellapoptosis effectively by inhibition of glycolysis.Low concentration induce the apoptosismainly by inferring the metabolism,but the 0.08mol/L-0.lmol/L could also influenced theconcentration of Ca,degrade the mitochondrial membrane potential ,lead to the apoptosis. Oxamate can induced the increase of the concentration of Ca²⁺ of PANC-1,and influencedthe following pathophysiologiacn response（mitochondrial membrane potential）.PartⅣEffect of LDH-A shRNA on the Growth,Apoptosis of Humanpancreatic cancer cell PANC-1 and the expression of LDH-AObjective:Most cancer cells exhibit increased glycolysis,Lactate dehydrogenase catalysesthe conversion of pyruvate to lactate,which is essential for the glycolytic pathway.MethodResult Conclusion.The five isoenzymes of LDH are composed of LDH-A（so-called muscletype） and LDH-B（so-called heart type） subunits.This study constructed a short hairpinRNA（shRNA） targeting LDH-A,and analyze its effects on growth,apoptosis of panc-1cells,the expression of LDH-A and the activity of the LDH.Method:Three shRNAs targeting LDH-A were combined to pGCsilencer vector,andtransfected into panc-1 cells.The expression of LDH-A after transfected by the threeshRNAs in pancreatic cancer cell lines panc-1 was detected by Quantitative Real TimePCR.After tranfected by the LDH shRNA-3 with the highest inhibited rate..cell growthwas analyzed by MTT assay,apoptosis was detected by flow cytometry.The LDH-Aexpression was detected by reverse transcription polymerase chain reaction,LDH activitywas observed with enzyme cytochemical method.Results:The 2-△△Ct of LDH-A shRNA-3 was （0.47±0.02）,less than the untransfectedpancreatic cancer cell（0.71±0.01）,the LDH-A shRNA-3 can inhibit the expression of the LDH-A most obviously.The growth of the pancreatic cancer cell was inhibitted after 12htransfected by LDH-A shRNA-3,all the Absorbance value of transfected cell in24h,36h,48h,72h discreased obviously compared to the normal pancreatic cancer cell（p＜0.01）.The apoptosis rates of the transfected cell increased to 61.74%.The inhibition ofLDH-A expression in pane-1 cells transfected by shRNA-3 was significantly and theactivity of LDH was also reduced.Conclusion:LDH-A shRNA can reduce the expression of the LDH-A,inhibit the activityof LDH,inhibit the growth and introduce the apoptosis of panc-1 cells siganificantly.Thetreatment of pancreatic cancer with the LDH-A shRNA could become the a new strategy infuture.更多还原