柠檬酸钠促胃癌MGC-803细胞凋亡的作用及机制研究

目的:探讨柠檬酸钠(SCT)促进胃癌MGC-803细胞凋亡的作用及机制。方法:胃癌MGC-803细胞分别经SCT(5、10、20 mmol/L)和5-FU(0.5 mmol/L)作用,以未处理的MGC-803为阴性对照,用流式细胞仪检测细胞周期分布及细胞凋亡率;比色法检测细胞内乳酸含量、磷酸果糖激酶1(PFK-1)活性及三磷酸腺苷(ATP)水平;Western blot检测细胞中Bcl-2、Bax、caspase-3及Cyt-c蛋白的相对表达量。结果:与阴性对照细胞比较,SCT处理的MGC-803细胞G2/M期阻滞与细胞凋亡明显增加;细胞内乳酸含量、PFK-1的活性和ATP水平均明显降低;细胞内Bcl-2的表达明显降低,而Bax、caspase-3和Cyt-c表达明显升高(均P0.05)。5-FU对MGC-803细胞乳酸含量、PFK-1的活性无明显影响(均P0.05),但其他作用与SCT相似。结论:SCT可促进MGC-803细胞凋亡,其作用可能与其抑制PFK-1的活性,降低糖酵解效率,并与线粒体凋亡通路的激活有关。     

多种人结肠癌5-氟尿嘧啶耐药细胞株的建立及耐药机制初步研究

目的:通过建立多种5-氟尿嘧啶(5-FU)耐药的人结肠癌细胞株,探讨耐药的结肠癌细胞的生物学特性与耐药机制。方法:选用人结肠癌HT-29、LoVo和SW480细胞,通过高浓度5-FU反复接触结合药物浓度递增法建立耐药株HT-29/5-FU、LoVo/5-FU和SW480/5-FU。不同浓度5-FU作用所建立的耐药细胞株及其亲本细胞后,分别用MTT法、流式细胞术、qRT-PCR、Westernblot检测细胞对5-FU的敏感性、周期分布、耐药相关分子[P-糖蛋白(P-gp)、多药耐药相关蛋白1(MRP1)、ATP结合盒超家族G成员2(ABCG2)]及第十号染色体缺失的磷酸酶和张力蛋白同源物(PTEN)与蛋白激酶B(Akt)的mRNA和蛋白的表达,并用Akt活性检测试剂盒检测细胞Akt活性。结果:与各自的亲本细胞比较,构建的HT-29/5-FU、LoVo/5-FU和SW480/5-FU对5-FU的IC50均明显升高(均P0.05),耐药指数分别为7.213、5.849和15.940。随着5-FU处理浓度的升高,亲本细胞和耐药细胞G0/G1期细胞数量均明显增加(均P0.05),但同一浓度5-FU处理下,各耐药株G0/G1期细胞数量均明显少于其对应亲本株(均P0.05)。与各自的亲本细胞比较,对应耐药株的P-gp、MRP1、ABCG2、Akt的mRNA和蛋白表达水平均明显升高,而PTEN表达均明显降低(均P0.05),且Akt活性均明显提高(均P0.05)。结论:成功建立了结肠癌5-FU耐药细胞株,其耐药机制可能与PTEN下调所致的PI3K/Akt降低PI3K/Akt通路活化有关。     

抑制Wip1磷酸酶对肝再生及肝细胞有氧糖酵解的影响

目的:探讨Wip1抑制剂GSK2830371对肝部分切除小鼠肝再生的影响及GSK2830371对肝细胞有氧糖酵解的影响。方法:小鼠2/3肝切除模型,腹腔注射GSK2830371,免疫组化Brd U染色和肝脏称重检测肝再生水平,酶学比色法检测肝脏中乳酸含量以及HK、PFK、PK和LDH的活性。GSK2830371作用于LO2肝细胞,酶学比色法检测葡萄糖和乳酸水平及关键酶活性,酶化学发光法检测ATP水平,CCK-8检测线粒体活性,Western bot和QRT-PCR检测相关机制。结果:经GSK2830371治疗,小鼠肝再生明显加快,PFK、HK和LDH的活性以及肝脏乳酸量均增高。经GSK2830371刺激,LO2消耗的葡萄糖和产生的乳酸明显增多,PFK、HK和LDH的活性增强,mTOR蛋白表达量及其磷酸化水平增高,HIF1α、PDK1、GLUT1和MCT4的mRNA表达上调。结论:GSK2830371抑制Wip1后能促进肝再生并提高肝细胞的有氧糖酵解水平。     

死亡相关蛋白激酶在结肠癌耐药中的作用

目的 探讨死亡相关蛋白激酶（DAPK）在结肠癌耐药中的作用.方法 应用免疫组织化学（免疫组化）SP法检测61例结肠癌组织及32例癌旁组织中DAPK的表达.以氟尿嘧啶（5-FU）诱导建立的结肠癌耐药细胞系HCT116/5-FU为模型.通过转染DAPK-siRNA下调DAPK的表达（DAPK-siRNA组）,转染FAM-siRNA（FAM-siRNA组）作为对照;通过过表达载体上调DAPK的表达（DAPK过表达组）.采用实时定量荧光PCR及蛋白质印迹法检测3组的DAPK、多药耐药蛋白（MRP）和P-糖蛋白（P-gp）的mRNA及蛋白表达水平;MTT法及流式细胞法分别测定3组细胞在未经5-FU处理及浓度为8 μg/ml的5-FU处理下的细胞增殖和凋亡情况.结果 DAPK在结肠癌组织中的阳性表达率明显低于癌旁组织[18.0％（11/61）比90.6％（29/32）,P＜0.05].与FAM-siRNA组比较,DAPK-siRNA组细胞中DAPK mRNA水平及蛋白表达水平均明显降低,DAPK过表达组则均显著升高（均P＜0.05）.在5-FU处理下,相比FAM-siRNA组,DAPK过表达组细胞增殖受到明显抑制,细胞凋亡率明显升高（均P＜0.05）;DAPK-siRNA组细胞增殖和细胞凋亡率均无明显变化（均P＞0.05）.与FAM-siRNA组比较,DAPK过表达组两种耐药蛋白的mRNA和蛋白表达水平均明显降低（P＜0.05）,而DAPK-siRNA组与FAM-siRNA组间差异无统计学意义（P＞0.05）.结论 DAPK能够抑制结肠癌耐药细胞的增殖,促进其凋亡,并可能通过抑制MRP和P-gp的mRNA和蛋白表达,来增强结肠癌细胞对药物的敏感性。     

巨噬细胞迁移抑制因子在大肠癌细胞LoVo中的表达及其意义

目的 检测巨噬细胞迁移抑制因子(MIF)在人大肠癌细胞LoVo中的表达,探讨其可能的意义.方法 采用实时荧光定量PCR及Western blot技术检测MIF mRNA及蛋白在人大肠癌细胞LoVo及人大肠上皮细胞FHC中的表达.结果 与FHC组相比,LoVo组中MIF在mRNA及蛋白水平的表达均显著升高.结论 MIF在大肠癌细胞中的高表达可能与其发生发展有一定的联系.     

肝X受体激动剂GW3965对人结肠癌细胞奥沙利铂耐药的逆转作用及机制

目的:探讨肝X受体(LXR)激动剂GW3965对人结肠癌细胞奥沙利铂(OXA)耐药的逆转作用及机制。方法:用OXA药物浓度持续递增法诱导人结肠癌HCT116细胞构建OXA耐药的人结肠癌HCT116/L-OHP细胞,比较HCT116/L-OHP细胞与其亲本HCT116细胞的生长情况,以及对不同浓度OXA作用的反应情况;检测HCT116/L-OHP细胞经GW3965处理48 h后OXA耐药性及自噬相关蛋白ATG-5、Beclin-1、p62、LC3的表达的变化。结果:成功构建人结肠癌耐OXA细胞HCT116/L-OHP,表现为HCT116/L-OHP细胞与亲本HCT116细胞比较,增殖能力有所减弱,但对OXA的耐药性明显增强(IC_(50):244.99μmol/L vs.10.05μmol/L,P0.05),其耐药指数(RI)为24.45。不同浓度(10、20、30μmol/L)GW3965作用后,HCT116/L-OHP细胞对OXA的IC_(50)、RI均明显降低(均P0.05),且呈浓度依赖性(IC_(50):199.49、114.71、87.32μmol/L;RI:19.89、11.40、8.69),3个浓度的逆转倍数分别为1.23、2.15、2.82;ATG-5、Beclin-1蛋白的表达明显降低,而p62、LC3-II蛋白的表达明显升高(均P0.05),且均呈浓度依赖性。结论:LXR激动剂GW3965可逆转人结肠癌细胞对OXA的耐药,其机制可能与调节自噬相关蛋白表达水平有关。     

表没食子儿茶素没食子酸酯通过PI3K/Akt通路抑制胰腺癌细胞糖酵解的实验研究

目的：探讨表没食子儿茶素没食子酸酯（EGCG）对胰腺癌细胞的增殖及糖代谢的影响。方法：用CCK8试剂盒检测不同浓度EGCG对胰腺癌PANC-1细胞增殖的影响，用葡萄糖乳酸试剂盒检测EGCG对糖酵解的影响，Western blot法检测糖酵解酶及PI3K/Akt通路蛋白的表达。结果：CCK8结果显示，EGCG可以抑制PANC-1细胞的增殖，其抑制作用具有剂量依赖性和时间依赖性（P <0.05）。葡萄糖乳酸检测实验表明，EGCG可以抑制PANC-1细胞的葡萄糖消耗和乳酸生成（P <0.05）。Western blot结果表明，EGCG可以抑制糖酵解酶己糖激酶-2(HK-2)、磷酸果糖激酶（PFK）和乳酸脱氢酶（LDH）及磷酸化PI3K和Akt的表达，且EGCG浓度越高，抑制作用越明显（P <0.05）。结论：EGCG抑制人胰腺癌细胞系PANC-1的增殖和糖酵解，其抑制作用可能与抑制PANC-1细胞中PI3K/Akt通路有关。     

靶向survivin的siRNA诱导肝癌细胞化疗敏感性的研究

目的探讨靶向survivin的siRNA对肝癌细胞化疗敏感性的影响。方法构建siRNA真核表达载体,稳定转染肝癌细胞HepG2后观察其对丝裂霉素(MMC)作用的转基因后肿瘤细胞的效应。结果测序证实siRNA真核表达载体构建成功。RT-PCR检测到siRNA在mRNA水平抑制survivin基因表达率达73%。MTT法观测siRNA作用下MMC对HepG2,HepG2/Silence(-)细胞的存活率,结果显示,仅在48 h时,加MMC组细胞的存活率(0.505±0.015)显著低于对照组(0.824±0.322)(P0.05)。当survivin的表达被有效抑制时,肝癌细胞存活率(0.520±0.017)在12 h点显著低于对照组(0.741±0.005),且48 h点达到高峰(P0.05)。免疫印迹法显示,未加MMC前survivin蛋白表达的OD值为34 273±323,加入MMC12,24,48 h survivin蛋白表达的OD值均显著降低(分别为21 415±142,16 771±122和13 672±133),均有统计学差异(均P0.05)。流式技术检测HepG2/Silence(+)加MMC组12,24,48 h细胞凋亡率与对照组比较,差异有统计学意义。Caspase-3活性检测显示HepG2/Silence(+)细胞在MMC作用下0,12,24,48 h Caspase-3活性分别为0.19±0.05,0.33±0.12,3.79±0.27和9.34±0.86;各时点间差异具显著性(均P0.05)。结论靶向survivin的siRNA不仅有效抑制肝癌细胞中survivin的表达,而且增强了细胞对MMC的敏感性,其作用机制系通过增强细胞内caspase-3活性,增加肝癌细胞的凋亡而实现的。     

PTTG对胆管癌细胞生长和5-FU敏感性影响的研究

目的探讨抑制垂体瘤转化基因(PTTG)表达后胆管癌细胞体外生长的变化及其对5-FU化疗敏感性的影响。方法将反义PTTG真核表达载体pcDNA3.1-PTTGas和空白载体pcDNA3.1(+)转染入胆管癌细胞株QBC939中,筛选获得稳定表达株tQBC939(PTTG-)和tQBC939(pcDNA3.1),并设立未转染的QBC939细胞对照组,绘制细胞生长曲线,以MTT法检测细胞增殖力的变化,流式细胞仪检测细胞周期的比例变化,并采用5-FU处理细胞后,以MTT检测3组细胞的生长抑制率,计算IC50值,流式细胞仪和Hoechst染色法检测各组细胞凋亡情况。结果 tQBC939(PTTG-)组与tQBC939(pcD-NA3.1)组和QBC939组比较,前者生长较为迅速,S期细胞比例增多,G2/M期细胞比例减少(P0.05),增殖指数较高(P0.05);经5-FU处理后,tQBC939(PTTG-)的IC50值明显低于tQBC939(pcD-NA3.1)和QBC939细胞(P0.05),细胞凋亡增多。结论转染PTTG反义DNA可增强胆管癌细胞的增殖力和对5-FU化疗的敏感性。     

甲基莲心碱逆转人结肠癌细胞奥沙利铂耐药的体外研究

目的:探讨甲基莲心碱(Nef)对人结肠癌细胞奥沙利铂(OXA)耐药的逆转作用及机制。方法:采用OXA浓度逐步递增法(2、4、8、12、24、48μmol/L)孵育人结肠癌HCT116细胞诱导构建OXA耐药株HCT116/OXA;检测Nef对HCT116/OXA细胞的细胞毒性,确定Nef的最适作用浓度和时间;分析并比较OXA(IC_(50)浓度)单独处理、Nef(最适作用浓度)单独处理、OXA(IC_(50)浓度)联合Nef(最适作用浓度)处理后,HCT116/OXA细胞的增殖,凋亡情况及凋亡相关蛋白(Bcl-2,Bax,PARP,p-PARP)的表达情况。结果:与亲本HCT116细胞比较,HCT116/OXA细胞较对OXA的耐药性明显增高(IC_(50):21.00μmol/Lvs.112.00μmol/L,P0.05),耐药指数为5.33。Nef能明显抑制HCT116/OXA的增殖有作用(P0.05),并呈浓度依赖性,其最适作用浓度、时间分别为5μmol/L、24h(细胞存活率为90%)。与OXA单独处理比较,HCT116/OXA细胞对OXA联合Nef处理的耐受性明显降低(IC_(50):112.00μmol/Lvs.45.47μmol/L,P0.05),逆转倍数为2.46;Nef单独作用对HCT116/OXA细胞的凋亡影响不明显(P0.05),但其与OXA联合作用对HCT116/OXA细胞凋亡诱导作用明显强于OXA单独作用(P0.05);与OXA或Nef单独作用比较,OXA联合Nef作用后,HCT116/OXA细胞抗凋亡蛋白Bcl-2表达明显下降,Bax、p-PARP等凋亡蛋白表达明显上升(均P0.05)。结论:Nef可逆转HCT116/OXA对OXA的耐药,机制可能与其调节Bcl-2/Bax表达水平,从而与OXA产生协同作用有关。     

Leberstoffwechsel während kalter ischämischer Inkubation in UW-Lösung am Rattenmodell

Simple cold storage of livers for transplantation activates glycolysis due to lack of oxygen. Energy derived from glycolysis may be critical for cell survival and liver cell death may occur once glycolysis is inhibited in the liver due to accumulation of end products or lack of substrates (glycogen). The relationship between cell death (lactate dehydrogenase, LDH release), anaerobic glycolysis (lactate production), and glycogen content of liver tissue was studied during cold incubation of liver slices in UW solution. Rat livers slices from male Sprague Dawley rats were incubated at 4°C in UW solution, with continuous gentle shaking, under conditions of chemical hypoxia (KCN, 5 mM). The rate of lactate production, LDH release, ATP and glycogen content were measured spectrophotometrically and by HPLC. Lactate increased nearly linearly for the first 48 h of incubation; total lactate which had accumulated after 48 h was 33.9±0.81 µmol/g and at 96 h nearly the same, 31.3±1.2 µmol/g. Glycolysis stopped, apparently, because of the depletion of liver slice glycogen which was initially 228.8±1.7 µmol/g wet wt. It decreased to 34.7±2.7 µmol/g at 48 h and to 18.7±1.1 µmol/g at 72 h and remained at this level for the next 24 h. An increased leakage of LDH occurred once glycogen metabolism (and accumulation) ceased. LDH release could be stimulated after only a few hours of cold incubation of liver tissue slices by adding glycolysis inhibitor (iodoacetic acid) to the medium. After 24 h, LDH release was 24.4±1.8% and increased to 52.8±5.2% (P<0.05, Student'st-test) with iodoacetic acid. Adding a glycolytic substrate (fructose, 10 mM) to the medium maintained lactate production for 96 h. The stimulation of glycolysis by fructose also reduced cell death: LDH release was significantly lower at 72- and 96-h incubation (P<0.001, two-way ANOVA). The ATP content was significantly higher with fructose (P<0.001). Adding glucose (20 mM) and fructose (10 mM) in combination resulted in prolonged cell survival, significantly delayed glycogen depletion and significantly higher ATP content at 48 and 72 h (two-way ANOVA). Livers from rats who had fasted for 24 h demonstrated the same LDH release at 48 h when incubated with glucose (20 mM) and fructose (10 mM). In conclusion, LDH leakage from hypoxic cold-stored liver slices is related to anaerobic glycolysis. Anaerobic glycolysis appears to continue slowly under hypothermia and provides sufficient energy for maintenance of cell viability. A stimulation of glycolysis in the cold is possible by fructose and results in prolonged cell survival under hypothermic conditions. Glycogen depletion can be slowed down by combining glucose and fructose.     

Examination of the metabolism of oedematous brain tissue

Summary We induced oedema in rabbit brains by applying cold to a defined area of the intact dura for a constant time. 24 hours later the animals were killed, and a piece of brain cortex adjacent to the region of cold application and an exactly similar piece from the other hemisphere were removed. In both pieces we determined the water content, the water uptake of the sliced brain in vitro, and the glucose metabolism of the brain slices. Respiration, carbon dioxide production in the presence of bicarbonate, consumption of glucose, and production of lactate and pyruvate were measured. The incubations were made in physiological saline with different concentrations of glucose (2.5 ml, 5 mM, and 10 mM).The mean difference of the water contents in vivo was 1.9%. The water uptake in vitro did not depend on the glucose concentration.The oedematous tissue had a slightly stimulated respiration, and its aerobic glycolysis was markedly increased.The respiration was independent of the glucose concentration. The lactate: pyruvate quotient was somewhat higher on the oedematous side, and it became greater on both sides with increasing glucose concentration.The glycolysis increased considerably between 2.5 mM and 5 mM glucose, especially in the oedematous brain. Between 5 mM and 10 mM glucose the aerobic glycolysis was accelerated only slightly by increasing glucose concentration.We think that the increased glycolysis is an expression of a preceding energy deficit. By a simple enhancement of the glucose concentration to more than 100 mg/100 ml the metabolism is not stimulated further, and the water content is not diminished.Supported by the Deutsche Forschungsgemeinschaft: H. 573/3.Part of the Dissertation, Med. Fakultät Homburg (Saar), 1972.     

VEGF-C反义核酸对结直肠癌LoVo细胞体内生长的影响

目的：探讨VEGF-C反义核酸对结直肠癌LoVo细胞体内生长的影响。方法：20只裸鼠随机均分为实验组与对照组,实验组接种转染反义VEGF-C核酸的LoVo细胞,而对照组接种转染空白质粒的LoVo细胞。观察两组肿瘤的生长情况,21 d后处死动物取移植瘤标本,用免疫组化法检测移植瘤组织中的微淋巴管密度（MLD）和微血管密度（MVD）。结果： 两组的成瘤率均为100%;实验组与对照组接种14 d后的肿瘤体积分别为（382.0±152.8）mm3和（454.2±148.7）mm3,21 d后为（745.0±250.9）mm3和（1 574.4±506.2）mm3,差异均有统计学意义（均P<0.05）;实验组肿瘤组织中MLD与MVD计数均较对照组明显减少[（11.75±2.22）/0.72 mm2 vs. （28.50±2.65）/0.72mm2,（47.75±2.99）/0.72 mm2 vs. （53.73±3.50）/0.72 mm2]（均P<0.05）。结论：转染VEGF-C反义核酸可抑制结直肠癌LoVo细胞移植瘤在裸鼠的体内生长,并抑制移植瘤淋巴管与血管的生成。

     

hTERT|CEA及CMV启动子在人结肠癌细胞株中的转录活性比较

目的：比较人端粒酶催化亚单位（hTERT）,癌胚抗原（CEA）及巨细胞病毒（CMV）启动子在人结肠癌细胞株LoVo和SW480中的转录活性。 方法：设计引物应用PCR法从人结肠癌基因组中克隆hTERT和CEA启动子;用双酶切和PCR法切除原始载体pLVX-EGFP-3FLAG中的CMV启动子后,将hTERT,CEA启动子与该载体重组,构建出重组质粒pLVX-hTERTp-EGFP-3FLAG和pLVX-CEAp-EGFP-3FLAG;将上述两种质粒及原始载体（含CMV启动子）分别瞬时转染人结肠癌细胞株LoVo和SW480后,检测两种细胞株绿色荧光蛋白表达。 结果：经PCR,酶切及测序鉴定,克隆及载体构建完全正确。CMV,hTERT及CEA启动子的转录活性（绿色荧光细胞数/总细胞数）在LoVo细胞中依次为54.7%,33.0%,9.5%;在SW480中依次为16.5%,10.1%,8.5%,差异均有统计学意义（均P<0.05）。 结论：在人结肠癌细胞株中,转录活性以CMV启动子最高,hTERT启动子次之,CEA启动子最低。该结果可为结肠癌的靶向性基因治疗研究提供参考。

     

Possible role of arachidonic acid in the regulation of lactate production in rat Sertoli cells

The aim of the study was to determine whether arachidonic acid (AA) is involved in the regulation of Sertoli cell lactate production and if this fatty acid participates in follicle-stimulating hormone (FSH) regulation of Sertoli cell function. In a first set of experiments the effect of AA and porcine pancreas phospholipase A2 (PLA2) on lactate production, glucose uptake, lactate dehydrogenase (LDH) activity and LDH A mRNA levels in Sertoli cell cultures obtained from 20-day-old rats was evaluated. In a second set of experiments the effect of two PLA2 inhibitors--quinacrine (Q) and AACOCF3--on FSH stimulation of the above-mentioned parameters of Sertoli cell function was investigated. Treatment with PLA2 and AA increased Sertoli cell lactate production. The observed action of exogenously added PLA2 involved its catalytic properties responsible for AA release. PLA2 and AA treatments also stimulated Sertoli cell glucose uptake, LDH activity and LDH A mRNA levels. In order to determine whether AA participates in FSH regulation of Sertoli cell lactate production cells were incubated with FSH in the absence or presence of the PLA2 inhibitors Q and AACOCF3. Both drugs partially inhibited the ability of FSH to stimulate lactate production, glucose uptake and LDH activity. The present investigation suggests that AA is involved in the regulation of lactate production, glucose transport, LDH activity and LDH A mRNA levels. In addition, these results suggest that cytosolic PLA2 and AA may participate in FSH-regulation of Sertoli cell energetic metabolism.     

Interaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) with glucokinase activates glucose phosphorylation and glucose metabolism in insulin-producing cells

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was recently identified as a new intracellular binding partner for glucokinase (GK). Therefore, we studied the importance of this interaction for the activity status of GK and glucose metabolism in insulin-producing cells by overexpression of the rat liver and pancreatic islet isoforms of PFK-2/FBPase-2. PFK-2/FBPase-2 overexpression in RINm5F-GK cells significantly increased the GK activity by 78% in cells expressing the islet isoform, by 130% in cells expressing the liver isoform, and by 116% in cells expressing a cAMP-insensitive liver S32A/H258A double mutant isoform. Only in cells overexpressing the wild-type liver PFK-2/FBPase-2 isoform was the increase of GK activity abolished by forskolin, apparently due to the regulatory site for phosphorylation by a cAMP-dependent protein kinase. In cells overexpressing any isoform of the PFK-2/FBPase-2, the increase of the GK enzyme activity was antagonized by treatment with anti-FBPase-2 antibody. Increasing the glucose concentration from 2 to 10 mmol/l had a significant stimulatory effect on the GK activity in RINm5F-GK cells overexpressing any isoform of PFK-2/FBPase-2. The interaction of GK with PFK-2/FBPase-2 takes place at glucose concentrations that are physiologically relevant for the activation of GK and the regulation of glucose-induced insulin secretion. This new mechanism of posttranslational GK regulation may also represent a new site for pharmacotherapeutic intervention in type 2 diabetes treatment.     

Effect of extended cold ischemia time on glucose metabolism in liver grafts: experimental study in pigs

Background/Purpose Recovery of normal carbohydrate metabolism in the liver after transplantation is highly important. The aim of the present study was to evaluate how short and long cold ischemia (CI) time followed by warm ischemia (WI) impact intrahepatic glucose metabolism in a pig liver transplantation model. Methods Twenty-six animals were divided into two transplantation groups: group I with a liver ischemia time of 5 h (n = 6), and group II with 15 h of liver ischemia (n = 7). Intrahepatic microdialysis samples were collected throughout the experiment at 20-min intervals, during the donor operation, cold preservation, liver implantation, and liver reperfusion in the recipient. Glucose, lactate, and pyruvate concentrations were analyzed and the lactate/pyruvate ratio (L/Pr) was calculated. Result There were no changes in glucose levels during CI. However, during WI, glucose and lactate increased and the increase was significantly higher in the group with longer CI (P < 0.01). The L/Pr increased at the beginning of CI but accelerated to increase during WI in both groups, with significantly prolonged and higher levels in the group with longer CI (P < 0.01). Conclusions Extended CI results in increased intrahepatic glycogenolysis, delayed restoration of aerobic glycolysis, and prolonged anaerobic glycolysis shortly after reperfusion. Improvements in glycogen protection and faster restoration of aerobic metabolism during preservation and reperfusion time seem to be necessary in order to improve liver preservation protocols per se.     

circRAD18/miR-516b/PDK1轴调节葡萄糖代谢重编程与结直肠癌增殖的关系

背景与目的 环状RNA circRAD18被发现在乳腺癌和甲状腺癌的进展中起了促进作用,但其在其他恶性肿瘤的表达及作用尚未被充分揭示。笔者前期通过生物信息学软件预测circRAD18可与miR-516b互补结合,而葡萄糖代谢关键调节酶丙酮酸脱氢酶激酶1（PDK1）可能是miR-516b的靶基因。因此,本研究初步探讨circRAD18在结直肠癌细胞中的表达及作用,及其对靶miRNA及下游靶基因的调控关系。方法 用qRT-PCR检测不同结直肠癌细胞系（SW480、SW620、HT-29）及正常结直肠上皮细胞（NCM460）中circRAD18的表达;用si-circRAD18沉默结直肠癌细胞中circRAD18的表达后,分别用CCK-8实验和相应的试剂盒检测细胞的增殖情况以及葡萄糖摄取量和乳酸产生量。用双荧光素酶报告基因实验与RNA免疫沉淀（RIP）实验分析circRAD18、miR-516b及PDK1之间的结合关系;最后,采用过表达/敲低实验进一步验证三者之间的关系。结果 与正常结直肠上皮细胞比较,circRAD18在各结直肠癌细胞系中的表达均明显上调（均P<0.05）;转染si-circRAD18后,结直肠癌细胞增殖能力、葡萄糖摄取及乳酸产生量均明显降低（均P<0.05）;双荧光素酶报告基因实验与RIP实验证实circRAD18可与miR-516b结合,而PDK1是miR-516b的下游靶基因。miR-516b模拟物及si-circRAD18的转染可明显抑制细胞葡萄糖摄取、乳酸产生及PDK1蛋白表达,且补充PDK1可逆转该抑制作用（均P<0.05）。结论 circRAD18在结直肠癌细胞中表达上调,并与结直肠癌细胞增殖能力的增强密切相关,作用机制可能与circRAD18通过海绵样吸附miR-516b后,上调PDK1表达,从而导致结直肠癌细胞葡萄糖代谢重编程有关。