缺血预处理对鼠肝细胞凋亡及调控基因（bcl—2,Fas）蛋白表达的影响

目的观察鼠肝缺血再灌注损伤对肝细胞凋亡,以及缺血预处理对缺血再灌注损伤引起的肝细胞凋亡以及对其调控基因(bcl-2,Fas)蛋白表达的影响.方法Wistar大鼠分为假手术(SO)组、缺血再灌注(IR)组、缺血预处理(IP)组,后2组中分为3个亚组(IR1,2,3,IP1,2,3).缺血均为30min.缺血预处理为缺血前采用5min缺血及5min再灌.分别于再灌注1.5,3,4.5h后处死动物采取肝脏标本,SO组于术后3.5h采取肝标本.检测细胞凋亡及bcl-2,Fas蛋白表达水平.结果IR2组和IP2组与SO组比较细胞凋亡指数(AI)显著性增加(P<0.01),IP组较IR组细胞AI显著性降低(P<0.05).电镜示凋亡细胞,但IP组较IR组的细胞器特别是线粒体损伤要轻.bcl-2蛋白表达IP组较IR组及SO组显著性增加(P<0.05),IR组与SO组无差异(P>0.05).Fas蛋白表达IR2组和IP2组较SO显著性增高(P<0.05),IP组与IR组比较无显著性差异(P>0.05).结论IR损伤可能通过激活Fas蛋白的表达而促发肝细胞凋亡;IP可能通过激活bcl-2蛋白的表达而抑制肝细胞凋亡.     

果糖在微囊化肝细胞制作过程中的细胞保护作用

目的 为了减少肝细胞在微囊化过程中的缺氧损伤,研究在缺氧状态下果糖对肝细胞的保护作用。方法 将ＳＤ大鼠分为三组;Ａ组,细胞分离的灌流液和消化液中均加果糖２０ｍｍｏｌ／Ｌ,Ｂ,Ｃ两组不加果糖;细胞分离及微囊化后,检测细胞的活率,微囊化肝细胞体外培养,Ｃ组培养液中加果糖１０ｍｍｏｌ／Ｌ,Ａ,Ｂ两组不加。     

Upregulation of GLUT2 mRNA by glucose, mannose, and fructose in isolated rat hepatocytes.

Previously, demonstrated that GLUT2 mRNA and protein are increased in liver of streptozocin-induced diabetic rats. To examine the mechanisms whereby GLUT2 mRNA is regulated, we cultured isolated hepatocytes in the absence and presence of various concentrations of glucose. Culture of hepatocytes in high glucose concentration (27.8 mM) for 20 h induced a 3.2-fold increase in GLUT2 mRNA levels compared with hepatocytes cultured without D-glucose. Interestingly, D-mannose and D-fructose could substitute for D-glucose to elevate the GLUT2 mRNA level, whereas 3-O-methyl-D-glucose, 2-deoxy-D-glucose, and sucrose, which were not metabolized or taken up by the cells, were without effect. Insulin had no significant effect on GLUT2 mRNA levels in hepatocytes in the presence or absence of D-glucose. Therefore, the regulation of the GLUT2 gene by D-glucose in hepatocytes is contrary to that reported for GLUT1 and GLUT4 genes, which are downregulated by D-glucose. These results also suggest that the elevated GLUT2 mRNA level observed in diabetic rat liver is due to the high blood glucose concentration rather than to insulin deficiency.     

Fibrocystin/polyductin modulates renal tubular formation by regulating polycystin-2 expression and function

Autosomal recessive polycystic kidney disease is caused by mutations in PKHD1, which encodes the membrane-associated receptor-like protein fibrocystin/polyductin (FPC). FPC associates with the primary cilia of epithelial cells and co-localizes with the Pkd2 gene product polycystin-2 (PC2), suggesting that these two proteins may function in a common molecular pathway. For investigation of this, a mouse model with a gene-targeted mutation in Pkhd1 that recapitulates phenotypic characteristics of human autosomal recessive polycystic kidney disease was produced. The absence of FPC is associated with aberrant ciliogenesis in the kidneys of Pkhd1-deficient mice. It was found that the COOH-terminus of FPC and the NH2-terminus of PC2 interact and that lack of FPC reduced PC2 expression but not vice versa, suggesting that PC2 may function immediately downstream of FPC in vivo. PC2-channel activities were dysregulated in cultured renal epithelial cells derived from Pkhd1 mutant mice, further supporting that both cystoproteins function in a common pathway. In addition, mice with mutations in both Pkhd1 and Pkd2 had a more severe renal cystic phenotype than mice with single mutations, suggesting that FPC acts as a genetic modifier for disease severity in autosomal dominant polycystic kidney disease that results from Pkd2 mutations. It is concluded that a functional and molecular interaction exists between FPC and PC2 in vivo.     

Dual functional role of membrane depolarization/Ca2+ influx in rat pancreatic B-cell.

Transient exposure of rat pancreatic B-cell to 50 mM K+ ([K+50]) makes exocytosis unresponsive to further depolarization, i.e., stimulation with 100 mM K+ or 1 uM glyburide, which closes the ATP-sensitive K+ (K+ATP) channel, simultaneously with [K+50] does not produce any greater insulin secretion compared with [K+50] alone. In sharp contrast, 16.7 mM glucose ([G16.7]) applied simultaneously with [K+50] elicits an insulin response markedly greater than that produced by [K+50] alone, which is not attenuated by 100 uM diazoxide, an inhibitor of K+ATP channel closure. [G16.7]-induced insulin secretion at the basal K+ concn of 4.7 mM was greatly (93%) suppressed by 100 uM diazoxide. Insulin secretion induced by [K+50] plus [G16.7] ([K+50 + G16.7]) was markedly suppressed (70%) by 1 uM nifedipine, a Ca(2+)-channel blocker and was completely abolished by 2 mM 2-cyclohexen-1-one, which reportedly decreases reduced glutathione level and blocks glucokinase. This finding indicates that insulin release induced by [K+50 + G16.7] is not due to leakage produced by toxic stimuli but to activation of exocytosis. When graded concentrations (25 and 50 mM) of K+ were applied simultaneously with [G16.7] in the presence of 100 uM diazoxide, insulin response was clearly dependent on K+ concentration, indicating that the physiological range of membrane depolarization also activates the glucose-responsive effector. Membrane depolarization/Ca2+ influx directly stimulates hormone exocytosis on one hand and activates the K+ATP channel-independent glucose-responsive effector or effectors on the other in the B-cell. The nature of the glucose-responsive effector or effectors remains to be established.     

Role of the insulin receptor substrate 1 and phosphatidylinositol 3-kinase signaling pathway in insulin-induced expression of sterol regulatory element binding protein 1c and glucokinase genes in rat hepatocytes

The mechanism by which insulin induces the expression of the sterol regulatory element binding protein 1c (SREBP-1c) and glucokinase genes was investigated in cultured rat hepatocytes. Overexpression of an NH(2)-terminal fragment of IRS-1 that contains the pleckstrin homology and phosphotyrosine binding domains (insulin receptor substrate-1 NH(2)-terminal fragment [IRS-1N]) inhibited insulin-induced tyrosine phosphorylation of IRS-1 as well as the association of IRS-1 with phosphatidylinositol (PI) 3-kinase activity, whereas the tyrosine phosphorylation of IRS-2 and its association with PI 3-kinase activity were slightly enhanced. The equivalent fragment of IRS-2 (IRS-2N) prevented insulin-induced tyrosine phosphorylation of both IRS-1 and IRS-2, although that of IRS-1 was inhibited more efficiently. The insulin-induced increases in the abundance of SREBP-1c and glucokinase mRNAs, both of which were sensitive to a dominant-negative mutant of PI 3-kinase, were blocked in cells in which the insulin-induced tyrosine phosphorylation of IRS-1 was inhibited by IRS-1N or IRS-2N. A dominant-negative mutant of Akt enhanced insulin-induced tyrosine phosphorylation of IRS-1 (but not that of IRS-2) and its association with PI 3-kinase activity, suggesting that Akt contributes to negative feedback regulation of IRS-1. The Akt mutant also promoted the effects of insulin on the accumulation of SREBP-1c and glucokinase mRNAs. These results suggest that the IRS-1-PI 3-kinase pathway is essential for insulin-induced expression of SREBP-1c and glucokinase genes.     

Rxa对过氧化氢诱导人类肝细胞系Fas mRNA表达及Ca2+流变化影响的单细胞分析

目的　探讨Rxa对过氧化物所致肝细胞凋亡时细胞保护作用的可能分子机制。方法应用全细胞膜片钳单细胞逆转录聚合酶链反应 (RT PCR)技术进行Rxa对过氧化氢 (H2 O2 )诱导人类肝细胞系 (L0 2细胞 )FasmRNA表达的单细胞分析 ,应用全细胞膜片钳显微荧光单细胞浆游离钙浓度 ([Ca2 ]i)测量技术进行同期瞬时Ca2 流变化观察。用流式细胞术观察早期凋亡细胞指数。结果　H2 O2 作用于L0 2细胞 2h电泳图分析可见FasmRNA表达 ,[Ca2 ]i (1115 .2 8± 2 2 7.16 )nmol/L、早期凋亡细胞指数骤升为 16 .18± 0 .6 5 ;而同期Rxa处理组电泳图分析未见FasmRNA特异性扩增条带出现 ;[Ca2 ]i、早期凋亡细胞指数较低 (P <0 .0 1、P <0 .0 1)。结论　Rxa可能通过抗氧化和钙阻滞作用阻抑L0 2细胞胞浆段Fas信号传导途径的活化 ,发挥细胞保护作用。     

十二指肠空肠旁路术和袖状胃切除术对糖尿病大鼠肝脏葡萄糖激酶表达的影响

目的探讨十二指肠空肠旁路手术（DJB）和袖状胃切除术（SG）对糖尿病大鼠肝脏葡萄激酶（GCK）表达的影响。方法制作GK大鼠（非肥胖糖尿病大鼠）和正常SD大鼠DJB和SG两种术式的动物模型．动态观察术后空腹血糖和胰岛素的变化。术后8周取肝组织标本,分别采用实时定量RT—PCR和Western blot检测术后肝脏GCK mRNA和蛋白表达水平。结果GK大鼠DJB组和SG组术后各时间点空腹血糖均显著低于术前水平（均P〈0．01）,假手术组1周内空腹血糖降低,2周后逐渐恢复到术前高水平：SD大鼠各种手术后血糖水平无明显改变（均P〉0．05）。GK大鼠DJB术后肝脏GCK mRNA和蛋白表达水平分别为1．45±0．29和494．25±30．25,SG术后分别为0．65±0．25和345．25±28．13;假手术组术后GCK mRNA和蛋白表达水平分别为1．05±0．19和409．13±26．86,与DJB组和SG组比较,差异有统计学意义（均P〈0．01）;对照组GCK mRNA和蛋白表达水平分别为1．04±0．17和404．75±30．90,与DJB组和SG组比较,差异具有统计学意义（均P〈0．01）。SD大鼠术后GCK表达也呈现了相似的变化。结论DJB和SG均能明显改善糖尿病大鼠的术后血糖水平．但两种术式对肝脏GCK的表达却具有完全不同的影响。     

The role of Th1/Th2 cell chemokine expression in hypertrophic scar

The aim of this study was to study the role of Th1/Th2 cell‐associated chemokines in the formation of hypertrophic scars in rabbit ears. Twenty‐six New Zealand white rabbits were used to establish the hypertrophic scar model of rabbit ear and the normal scar model of rabbit's back. Two rabbits were sacrificed on days 0 and 21, 28, 35, 42, 49, 56, and 63 after operation. The specimens were stained with haematoxylin‐eosin (HE). Scar elevation index (SEI) was used to detect the expression of 10 chemokines related to Th1/Th2 cells in both scar formation expressions. Real‐time polymerase chain reaction (PCR) results showed that two chemokines (CXCL10, CXCL12) were highly expressed during the formation of normal scar, and there was almost no expression during the formation of hypertrophic scar (*P < 0.05). The chemokines (CCL2, CCL3, CCL4, CCL5, CCL7, CCL13, CX3CL1) were almost non‐expressed in the formation of normal scars but were expressed for a long time in the formation of hypertrophic scars. The four chemokines, CCL2, CCL4, CCL5, and CX3CL1, maintained a long‐term high expression level during the formation of hypertrophic scars (P < 0.01). There were also three chemokines (CCL14, CCL19, CCL21) that were almost undetectable in normal scarring, but there was transiently low‐level expression (P < 0.05) only during the peak proliferative phase in proliferative scarring. Th1/Th2 cell‐associated chemokines are different in the type, quantity and expression, and maintenance time of rabbit ear hypertrophic scars.     

维生素K2调节骨代谢的生物学研究回顾

维生素K(vitamin K,VK)是一类2-甲基-1,4-萘醌衍生物,维生素K2是一种人体必需的天然营养素,主要分布在肾、骨、生殖器及血管壁等组织,维生素K2除参与体内凝血功能外,还与人体的许多生理功能有关,在骨代谢的多个环节中具有重要作用,其调节骨代谢的机制错综复杂。本文回顾性综述了维生素K2对骨代谢调节的生理作用、动物实验及临床试验研究,以及维生素K2在防治骨质疏松中的重要作用。     

蛋白酪氨酸磷酸酶调节破骨细胞活性的作用

蛋白酪氨酸磷酸酶对破骨细胞活性具有重要的调节作用。与破骨细胞相关的,能够使c-SrcPY527去磷酸化起到关键作用的PTPs有:SHP-1、胞质PTP-ε、PTP-PEST以及PTP-oc。其中SHP-1对破骨细胞进行负调节,其它三种为正向调节。     

H2O2诱导肝细胞凋亡时p53、bcl—2 mRNA的表达及其意义

目的 探讨H2O2诱导人类正常肝细胞系（L02细胞）凋亡时p53、bcl-2 mRNA表达的意义及丹参有效成分Rxa对其表达的影响。方法 以正常L02细胞为对照（L组）,以10μmol/L H2O2诱导L02细胞凋亡为细胞模型（LH组）,观察Rxa处理（LaH组,Rxa 2mmol/L）对p53、bcl-2 mRNA表达（原位杂交结合图像分析处理）的影响。结果 LH组2－6h p53 mRNA表达随凋亡细胞指数增高而增强,6－8h表达下降;bcl-2 mRNA表达弱。相比较LaH组各时限检测点p53 mRNA表达均低于LH组,bcl-2 mRNA表达较强（P＜0．01）。结论 Rxa可通过p53的细胞周期调控、DNA损伤修复作用和bcl-2的抗凋亡作用减轻L02细胞过氧化损伤。     

茄碱通过调控Stat3信号通路影响胰腺癌细胞Panc-1中MMP-2和MMP-9表达

目的 探讨茄碱对胰腺癌细胞Panc-1中基质金属蛋白酶2（MMP-2）和基质金属蛋白酶9（MMP-9）表达的影响及Stat3信号通路在此过程中的作用。方法 实验分为对照组和药物组,药物组分别采用浓度为3.5、7.0和10.5 μmol/L的茄碱对Panc-1细胞进行干预,明胶酶谱法观察茄碱对细胞中MMP-2和MMP-9活性的影响;实时荧光定量PCR检测MMP-2和MMP-9基因的表达变化;Western blotting法检测总蛋白中信号转导及转录激活因子3（Stat3）蛋白的磷酸化表达变化。结果 与对照组相比,药物组（7.0、10.5 μmol/L）MMP-2活性明显下降（t值分别为2.4、6.4,P<0.05）;MMP-9活性亦下降（t值分别为3.2、4.8,P<0.05）。实时荧光定量PCR结果显示,药物组（7.0 μmol/L、10.5 μmol/L）MMP-2基因表达下调（t值分别为8.7、13,P<0.05）;MMP-9基因表达下调（t值分别为3.2、16.3,P<0.05）。Western blotting结果显示,随着茄碱浓度的增加,Panc-1细胞中Stat3蛋白磷酸化受到明显抑制（t值分别为10.2、6.4、6.2,P<0.05）,延长药物处理的时间（12 h、18 h、24 h）亦可抑制Stat3蛋白磷酸化（t值分别为11.4、26.1、20.4,P<0.05）。结论 茄碱可抑制胰腺癌细胞Panc-1中MMP-2和MMP-9的表达,发挥其抗肿瘤转移能力。其具体机制可能与调控Sata3信号通路有关。