激活型Akt1转染大鼠胰岛对移植物凋亡和再血管化的影响

目的 探讨腺病毒介导激活型蛋白激酶B(Akt1)基因(Adv-CA-Akt1)转染大鼠胰岛对同种糖尿病大鼠胰岛移植物凋亡和再血管化的影响.方法 分离纯化Wistar大鼠胰岛,转染Adv-CA-Akt1.36只糖尿病Wistar大鼠完全随机均分为3组: Adv-CA-Akt1组(Adv-CA-Akt1转染的胰岛移植); Adv-LacZ组(Adv-LacZ转染的胰岛移植); 未转染组(单纯胰岛移植).术后每日测血糖,隔日测血清胰岛素浓度,术后10 d行静脉糖耐量试验(IVGTT)观察胰岛功能; HE染色和胰岛素免疫组化检测胰岛功能,细胞凋亡原位检测胰岛凋亡,CD31免疫组化计数微血管密度(MVD).结果 Adv-CA-Akt1组大鼠血糖术后2 d恢复正常,Adv-LacZ组和未转染组血糖虽下降但仍高于正常; Adv-CA-Akt1组术后各时相血清胰岛素水平明显高于其他2组(P＜0.05).IVGTT示Adv-CA-Akt1组血糖下降较快,60 min恢复至空腹水平; 另2组下降慢,60 min仍未恢复至空腹水平.术后3 d,Adv-CA-Akt1组肾被膜下存活胰岛细胞数量多,胰岛素免疫组化证明为有功能的胰岛.Adv-CA-Akt1组胰岛凋亡率比Adv-LacZ组和未转染组降低约25%; 术后12 d,Adv-CA-Akt1组MVD比Adv-LacZ组及未转染组明显增高(P＜0.05).结论 激活型Akt1转染大鼠胰岛能够抑制胰岛细胞凋亡,提高胰岛β细胞功能,促进移植物早期再血管化.     

Myofibroblast Transdifferentiation in Obliterative Bronchiolitis: TGF-β Signaling Through Smad3-Dependent and -Independent Pathways

We have shown that Smad3, an intracellular signal transducer for transforming growth factor-beta1 (TGF-beta1), is required to elicit the full histological manifestations of obliterative airway disease in a tracheal transplant model. This suggests that chronic allograft rejection results in TGF-beta1-induced Smad3 activation that leads to airway obliteration through fibroproliferation and increased matrix deposition. In other systems, these latter events are causally related to the transdifferentiation of fibroblasts into myofibroblasts, but their role in obliterative bronchiolitis (OB) after lung transplantation is unknown. We confirmed the presence of myofibroblasts inside affected airways associated with experimental OB using immunohistochemistry. Studying airway fibroblasts in vitro, we observed increased myofibroblast transdifferentiation in response to TGF-beta1, evidenced by increased alpha-smooth muscle actin mRNA and protein expression. In Smad3-null fibroblasts, TGF-beta1 induction of myofibroblast transdifferentiation was greatly diminished but not abolished, suggesting the presence of Smad3-independent pathways. Further studies revealed that small molecule inhibitors of p38 (SB203580) and MEK/ERK (U1026) further reduced the remaining effect of TGF-beta1 in Smad3-deficient fibroblasts. Together, these studies suggest that in chronic allograft rejection, TGF-beta1 stimulates myofibroblast transdifferentiation through Smad3-dependent and -independent signals, contributing to the excessive matrix deposition that characterizes obliterative bronchiolitis.     

Engraftment of Adult Porcine Islet Xenografts in Diabetic Nonhuman Primates Through Targeting of Costimulation Pathways

Recent advances in human allogeneic islet transplantation have established beta-cell replacement therapy as a potentially viable treatment option for individuals afflicted with Type 1 diabetes. Two recent successes, one involving neonatal porcine islet xenografts transplanted into diabetic rhesus macaques treated with a costimulation blockade-based regimen and the other involving diabetic cynomolgus monkeys transplanted with adult porcine islet xenografts treated with an alternative multidrug immunosuppressive regimen have demonstrated the feasibility of porcine islet xenotransplantation in nonhuman primate models. In the current study, we assessed whether transplantation of adult porcine islet xenografts into pancreatectomized macaques, under the cover of a costimulation blockade-based immunosuppressive regimen (CD28 and CD154 blockade), could correct hyperglycemia. Our findings suggest that the adult porcine islets transplanted into rhesus macaques receiving a costimulation blockade-based regimen are not uniformly subject to hyperacute rejection, can engraft (2/5 recipients), and have the potential to provide sustained normoglycemia. These results provide further evidence to suggest that porcine islet xenotransplantation may be an attainable strategy to alleviate the islet supply crisis that is one of the principal obstacles to large-scale application of islet replacement therapy in the treatment of Type 1 diabetes.     

单侧睾丸扭转后生精细胞凋亡的分子途径

目的:研究大鼠单侧睾丸扭转复位后生精细胞凋亡的分子机制。方法:雄性SD大鼠16只,随机分为对照组和扭转组,每组8只。建立睾丸扭转动物模型(720°2h),术后24h留取手术侧睾丸。应用流式细胞术检测生精细胞凋亡和各级生精细胞计数,应用RT-PCR技术对Fas/FasL mRNA和Bax mRNA进行半定量分析,Western印迹技术检测细胞色素C含量。结果:两组间生精细胞凋亡及各类生精细胞计数均有显著性差异(P<0.01)。扭转组FCM直方图呈现高大凋亡峰,单倍体和四倍体细胞群计数下降,Fas/FasL mRNA和Bax mRNA表达上调,同时细胞质中细胞色素C含量亦明显升高,与对照组相比其差异均有显著性(P均<0.01)。结论:睾丸扭转复位后生精细胞凋亡存在着外源性和内源性两条基本途径。凋亡相关分子Fas/FasL表达上调和Bax介导的细胞色素C释放可能是睾丸扭转后生精细胞凋亡的重要环节。     

七氟烷通过ERK1／2／Nrf2信号通路诱导HO-1基因表达抑制神经元凋亡

目的：研究七氟烷（Sevoflurane）诱导神经元血红素氧合酶－1（HO－1）基因表达的信号转导通路，探讨七氟烷脑保护机制。 方法：将培养7d的新生Wistar大鼠海马神元随机分为5组：正常培养组（C组）、氧糖剥夺组（D组）、2％七氟烷＋氧糖剥压组（S1组）、4％七氟烷＋氧糖剥压组（S2组）、4％七氟烷＋U-012＋4％七氟烷＋氧糖剥压组（U组）。C组和S2组神经元分别给予2％或4％七氟烷预处理60min后同D组处理。U组在神经元给予4％七氟烷处理同时在培养液中加入U－0126使其终浓度为10μmol/L后同S2组处理。收集神经元进行HO－1－mRNA和ERK1/2、Nrf2、AP-1和HO－1蛋白表达的检测，检测神经元的存活率和凋亡率。 结果：与C组比较，D组神经无HO－1蛋白表达增加（P〈0.05）,ERK1/2,Nrf2和AP-1蛋白表达增加（P〈0.05），神经元存活率降低、凋亡率增加（P〈0.01）.与D组比较，S1组神经元HO－1－mRNA和HO-1蛋白表达增加（P〈0.01）,ERK1/2和Nrf2蛋白表达增加（P〈0.01）,AP－1蛋白表达变化不明显（P〉0.05），经元存活率长高、凋亡率降低（P〈0.01）.与S2组比较，U组神经元HO-1－mRNA和HO－1蛋白表达降低（P〈0.01）,ERK1/2和Nrf2蛋白表达降低（P〈0.01），AP-1蛋白表达表达变化不明显（P〉0.05）,神经元存活率降低、凋亡率增加（P〈0.01）. 结论：Sevoflurane通过ERK1/2/Nrf2信号通路诱导神经元HO-1-mRNA表达，抑制氧糖剥夺神经元的凋亡。     

Wnt信号通路成员APC抑癌基因在肾上腺皮质腺瘤中杂合性缺失的研究

目的：探讨APC基因杂合性缺失在。肾上腺皮质腺瘤中的意义。方法：采用聚合酶链反应-限制性酶切片段长度多态性（PCR—RFLP）方法研究Wnt信号通路成员APC基因11外显子RsaI酶切位点和15外显子MspI酶切位点多态性,分析42例肾上腺皮质腺瘤中APC基因杂合性缺失。结果：在42例肾上腺皮质腺瘤组中杂合子有30例（71．4％）,未发现杂合性缺失。结论 肾上腺皮质腺瘤未发现APC基因杂合性缺失现象,据此认为APC基因杂合性缺失在肾上腺皮质腺瘤发生发展过程中可能无明显关联。     

caveolin-1通过阻断PI3K/Akt信号的激活抑制胰腺癌细胞增殖

目的研究caveolin-1对胰腺癌Panc1细胞在体外生长和增殖的影响,并初步探讨其机理。方法选用人类胰腺癌Panc1细胞,通过基因转染技术培育过表达caveolin-1细胞株Panc1/cav-1作为实验组,空载体细胞株Panc1/vec和亲本细胞株Panc1作为对照组。Western blot方法检测各组细胞内caveolin-1、Akt和p-Akt的表达,绘制细胞生长曲线并计算细胞倍增时间,流式细胞仪分析细胞周期,软琼脂集落形成实验检测细胞增殖克隆的能力。结果①caveolin-1在实验组细胞中稳定表达,其表达量明显高于对照组细胞(P<0.01),而对照组的Panc1/vec细胞和Panc1细胞之间差异无统计学意义(P>0.05)。②实验组细胞的生长速度明显慢于对照组细胞(P<0.05),其倍增时间明显长于对照组细胞(P<0.01)。③细胞周期显示,实验组细胞被抑制于G0/G1期(P<0.05),进入S期的细胞比率明显减少(P<0.01),实验组细胞的增殖指数较对照组明显降低(P<0.01)。④实验组细胞在软琼脂中形成的集落数目较对照组明显减少(P<0.01),体积较小。⑤实验组细胞中Akt表达量与对照组之间差异无统计学意义(P>0.05),而实验组细胞中p-Akt表达量明显低于对照组(P<0.05)。结论 caveo-lin-1通过抑制PI3K/Akt信号激活抑制Panc1细胞生长和增殖。     

转染人血红素加氧酶-1基因抑制移植静脉血管内膜增生

目的　应用含人血红素加氧酶 - 1基因 (human Heme Oxygenase- 1,h HO- 1)的重组腺病毒 (Adeno- XTMh HO- 1,Ad- h HO- 1)转染静脉移植血管 ,观察 h HO- 1基因预防静脉移植血管内膜增生的作用。　方法　将 2 1只日本大耳白兔分为 3组 ,对照组 ,Ad- null组和 Ad- h HO- 1组 ,每组各 7只。在兔颈外静脉移植于颈总动脉的血管移植术前分别应用肝素生理盐水、Ad- null和 Ad- h HO- 1病毒液常温浸泡静脉移植血管 30 min。术后 2 8d病理切片观察移植血管内膜增生的情况 ,计算机图象分析仪计算新生内膜厚度、中膜厚度及二者比值 ;采用免疫组织化学染色方法 (S- P法 )观察术后 14 d、2 8d移植血管壁 h HO- 1蛋白表达情况。　结果　Ad- h HO- 1组内膜厚度、内膜厚度与中膜厚度比均显著低于Ad- null组和对照组 (P<0 .0 1) ,中膜厚度差别无统计学意义 (P>0 .0 5 )。 Ad- h HO- 1组静脉血管壁细胞 h HO- 1免疫组化染色阳性。　结论　 Ad- h HO- 1转染兔静脉旁路移植血管能够抑制内膜增生。     

Ginsenoside Rg3 Enhances Islet Cell Function and Attenuates Apoptosis in Mouse Islets

Background

The transplantation of isolated islets is thought to be an attractive approach for curative treatment of diabetes mellitus. Panax ginseng has been used in oriental countries for its pharmacologic effects, such as antidiabetic and antiinflammatory activities. 20(S)-ginsenoside Rg3 (Rg3), an active ingredient of ginseng saponins, has been reported to enhance insulin secretion–stimulating and antiapoptotic activities in pancreatic beta cells. We performed this study to examine the hypothesis that preoperative Rg3 administration can enhance islet cell function and antiapoptosis before islet transplantation.

Methods

Balb/c mice were randomly divided into 2 groups according to the administration of Rg3 after islet isolation. Mouse islets were cultured in medium supplemented with or without Rg3. In vitro, islet viability and function were assessed. After treatment of islets with a cytokine cocktail (tumor necrosis factor α, interferon-γ, and interleukin-1β), cell viability, function, and apoptosis were assessed.

Results

Cell viability was similar between the 2 groups. Islets cultured in medium supplemented with Rg3 showed 2.3-fold higher glucose-induced insulin secretion than islets cultured in medium without Rg3. After treatment with a cytokine cocktail, glucose-induced insulin release, total insulin content of islets, and apoptosis were significantly improved in Rg3-treated islets compared with cytokine-treated islets. Cytokine-treated islets produced significantly higher levels of nitric oxide (NO) than islets treated with Rg3.

Conclusions

These results suggest that preoperative Rg3 administration enhanced islet function before islet transplantation and attenuated both cytokine-induced damage associated with NO production and apoptosis. Rg3 administration might be a prospective management to enhanced islet function and ameliorate early inflammation after transplantation.     

Salidroside Inhibits Ischemia/Reperfusion-Induced Myocardial Apoptosis by Targeting Mir-378a-3p Via the Igf1r/Pi3k/Akt Signaling Pathway

BackgroundThe present study aimed to investigate the protective effects and mechanism of salidroside (SAL) on hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis and myocardial ischemia/reperfusion (I/R) injury.MethodsWe set up an H/R H9c2 cell model in vitro and an I/R rat model in vivo. Cell viability, apoptosis and histopathologic evaluation were conducted.ResultsThe cell viability of H/R-induced cardiomyocytes was increased by pretreatment of SAL, whereas the release of lactate dehydrogenase, reactive oxygen species production, and apoptosis were decreased accompanied with reduced Cleaved-caspase-3 and Bax, and increased Bcl-2 expressions. The SAL restored mitochondrial membrane potential both in vitro and in vivo, and improved electrocardiographic abnormality, and attenuated myocardial apoptosis and injury in I/R-induced rats. The transfection of miR-378a-3p inhibitor counteracted the effects of SAL-induced increase of cell viability and decrease of cell apoptosis and mitochondrial membrane potential. SAL reduced the expression of insulin-like growth factor 1 receptor (IGF1R), and increased the expressions of PI3K and Akt, however, these alterations were blocked by miR-378a-3p inhibitor.ConclusionsmiR-378a-3p might participate in the protective effect of SAL in I/R-induced myocardial apoptosis via the IGF1R/PI3K/AKT signaling pathway.     

Placental Insufficiency Decreases Pancreatic Vascularity and Disrupts Hepatocyte Growth Factor Signaling in the Pancreatic Islet Endothelial Cell in Fetal Sheep

Hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGFA) are paracrine hormones that mediate communication between pancreatic islet endothelial cells (ECs) and β-cells. Our objective was to determine the impact of intrauterine growth restriction (IUGR) on pancreatic vascularity and paracrine signaling between the EC and β-cell. Vessel density was less in IUGR pancreata than in controls. HGF concentrations were also lower in islet EC-conditioned media (ECCM) from IUGR, and islets incubated with control islet ECCM responded by increasing insulin content, which was absent with IUGR ECCM. The effect of ECCM on islet insulin content was blocked with an inhibitory anti-HGF antibody. The HGF receptor was not different between control and IUGR islets, but VEGFA was lower and the high-affinity VEGF receptor was higher in IUGR islets and ECs, respectively. These findings show that paracrine actions from ECs increase islet insulin content, and in IUGR ECs, secretion of HGF was diminished. Given the potential feed-forward regulation of β-cell VEGFA and islet EC HGF, these two growth factors are highly integrated in normal pancreatic islet development, and this regulation is decreased in IUGR fetuses, resulting in lower pancreatic islet insulin concentrations and insulin secretion.     

Survival and Metabolic Function of Syngeneic Rat Islet Grafts Transplanted in the Omental Pouch

Many sites have been tested in an effort to identify the most ideal site to support islet function and viability. The aim of this study was to evaluate an omental pouch site for islet transplantation and compare it with the renal subcapsular space. All streptozotocine-induced diabetic rats receiving 2000 syngeneic islets in the omental pouch (n = 13) or under the kidney capsule (n = 10) returned to normoglycemia. At 7 days post-transplant and throughout the follow-up period, the mean blood glucose value in both groups was < 9.0 mM. At 4 and 8 weeks post-transplant, both groups displayed normal and similar glucose tolerance curves. Gain in the recipient's body weight after transplantation was similar between the two groups. At the end of follow up prompt hyperglycemia was observed in all rats after removal of the islet graft. No significant differences were found in the insulin contents of the harvested grafts, irrespective of the transplantation site. Histological examination of the grafts showed numerous well-granulated insulin-containing cells in both sites. The results indicate that the omental pouch is a viable site which offers a safe, convenient and efficacious alternative to the renal subcapsular space to transplant islets in rodents.     

Angiotensin II Induces Vascular Endothelial Growth Factor in Pancreatic Cancer Cells Through an Angiotensin II Type 1 Receptor and ERK1/2 Signaling

Vascular endothelial growth factor (VEGF) is a crucial pro-angiogenic component in pancreatic ductal adenocarcinoma (PDA), and its high expression levels have been correlated with poor prognosis and early postoperative recurrence. We have recently shown that high levels of angiotensin II (AngII) type 1 receptor (AT1R) correlate and colocalize with VEGF in invasive PDA and that AngII induces VEGF expression in PDA cell lines. In this study, we explored the signaling mechanisms involved in the AngII-mediated VEGF induction and correlated AT1R and VEGF expression in noninvasive precursor lesions. An AT1R antagonist significantly (p < 0.05) inhibited the AngII-mediated induction of VEGF messenger RNA and protein in all PDA cell lines. AngII-VEGF induction was inhibited by the tyrosine kinase inhibitor genistein, suggesting a mitogen-activated protein kinase signaling mechanism. AngII activated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not p38 or c-Jun NH2-terminal MAP kinases. Inhibition of ERK1/2 activation reduced the AngII-induced VEGF synthesis. Immunohistochemical analysis of precursor lesions showed increased expression of AT1R in most ductal cells undergoing metaplasia. Pancreatic intraepithelial neoplasms showed more intense AT1R staining when compared to intraductal papillary mucinous neoplasms, which showed heterogeneous immunoreactivity. VEGF followed the same distribution pattern of AT1R in both lesions. AT1R expression in the premalignant pancreatic lesions suggests its involvement in tumor progression and angiogenesis. Our mechanistic findings provide the first insight into an AngII-initiated signaling pathway that regulates PDA angiogenesis. An AT1R-mediated VEGF induction suggests the possibility of AT1R blockade as a novel therapeutic strategy to control angiogenesis in PDA. Presented as a poster presentation at the 48th Annual Meeting of the Society for Surgery of the Alimentary Tract, May 19–23, 2007 and an oral presentation at the Pancreas Club May 21st, 2007; Washington, DC.