Diabetic Basement Membrane Thickening Does Not Occur in Myocardial Capillaries of Transgenic Mice When Metallothionein is Overexpressed in Cardiac Myocytes

Diabetic cardiomyopathy is a clinically distinct disease characterized by impaired cardiac function as a result of reduced contractility and hypertension‐induced athero‐ or arteriosclerosis. This may be due either to generalized vascular disease, tissue‐based injury such as focal cardiomyocyte dysmorphia, or microvascular damage manifested by myocardial capillary basement membrane (CBM) thickening. Hyperglycemia‐driven increases in reactive oxygen species (ROS) have been proposed to contribute to such damage. To address this hypothesis, we utilized light (LM) and transmission electron microscopy (TEM) to demonstrate cardiomyocyte morphology and myocardial CBM thickness in the left ventricles of four mouse genotypes: FVB (background Friend virus B controls), OVE (transgenic diabetics), Mt [transgenics with targeted overexpression of the antioxidant protein metallothionein (MT) in cardiomyocytes], and OVEMt (bi‐transgenic cross of OVE and Mt) animals. Mice were prepared for morphometric analysis by vascular perfusion. Focal myocardial disorganization was identified in OVE mice but not in the remaining genotypes. Not unexpectedly, myocardial CBM thickness was increased significantly in OVE relative to FVB (P < 0.05) and Mt (P < 0.05) animals (+28% and +39.5%, respectively). Remarkably, however, OVEMt myocardial CBMs showed no increase in width; rather they were ～3% thinner than FVB controls. Although the molecular mechanisms regulating CBM width remain elusive, it seems possible that despite a significant hyperglycemic environment, MT antioxidant activity may mitigate local oxidative stress and reduce downstream excess microvascular extracellular matrix (ECM) formation. In addition, the reduction of intra‐ and perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury. Anat Rec, 296:480–487, 2013. © 2013 Wiley Periodicals, Inc.     

One Injection of DsRed Followed by Bites from Transgenic Mosquitoes Producing DsRed in the Saliva Elicits a High Titer of Antibody in Mice

It has been proposed that transgenic mosquitoes can be used as a “flying syringe” for infectious disease control. We succeeded in generating a transgenic (TG) mosquito, Anopheles stephensi, excreting and discharging DsRed in saliva. DsRed was deposited on the membrane where the TG mosquito probed with its proboscis. Repeated feeding by the TG mosquitoes induced anti-DeRed as well as anti-SG antibodies in mice. This indicates that the TG mosquitoes can immunize the animal. Moreover, in this report, we employed a pre-immunization method before exposing mice to the TG mosquitoes. We injected DsRed to mice to prepare memory B cells and exposed the mice to bites by the TG mosquitoes excreting DsRed. The mice produced a higher titer of antibody to DsRed, suggesting that the bites from TG mosquitoes act as a booster and that primary immunization with a vaccine protein and exposure to TG mosquitoes excreting the vaccine protein in the saliva produces a synergistic effect.     

髓源抑制细胞对HBV转基因小鼠肝脏炎症损伤抑制作用的研究

目的：建立HBV抗原特异性细胞毒性T细胞（CTLs）介导的小鼠肝炎模型,探讨肿瘤诱导的髓源抑制性细胞（MDSCs）在免疫介导的HBV转基因小鼠肝损伤中的有效性。方法制备新鲜的HBV转基因小鼠肝脏匀浆,予普通小鼠腹腔注射,1次/周,连续4周,以诱导致敏小鼠（Sensitized-mice）产生HBV抗原特异性CTLs（HBV-specific CTLs,HBV-CTLs）。分离致敏小鼠脾脏来源HBV-CTLs,静脉回输给高复制型HBV转基因小鼠,分别在注射前,注射后1 d、3 d、6 d和9 d经眶后取血测血清ALT/AST水平。分离荷瘤小鼠骨髓来源的MDSCs,静脉注射给HBV-CTLs诱导的肝炎小鼠,并在注射后24 h,经眶后取血测血清ALT/AST水平,肝脏组织经固定、石蜡包埋、HE染色进行组织形态学检测。结果致敏小鼠脾脏来源的HBV-CTLs可诱导HBV转基因小鼠肝组织损伤,血清ALT、AST水平呈升高趋势;且与CTLs注射组小鼠相比,CTLs联合MDSCs注射组小鼠肝脏组织损伤程度减轻,小鼠血清转氨酶水平显著降低[ALT：（254.5±25.50）vs （80.67±11.57）,P ＜0.05;AST：（301.5±40.50）vs（249.0±79.00）,P ＞0.05）]。结论静脉回输肿瘤诱导的MDSCs可有效减轻HBV-CTLs诱导的肝炎小鼠中肝组织损伤。     

Expression of DeltaNp73 in hippocampus of APP/PS1 transgenic mice following GFP-BMSCs transplantation

Abstract

Objective: To study the effect of hippocampal bone marrow stromal cells (GFP-BMSCs) transplantation on spatial memory and DeltaNp73 expression in APP/PS1 transgenic mice.

Methods: Twelve APP/PS1 transgenic mice randomly received either 10 μl GFP-BMSCs suspension in medium (GFP-BMSCs transplantation group) or 10 μl complete medium (sham-operated group). Learning and memory function of mice in both groups were observed and tested in Morris water maze experiment at 2 weeks after surgery. Senile plaques and DeltaNp73 protein in hippocampuses were determined by immunohistochemistry and western blot at 3 weeks after surgery, respectively.

Results: APP/PS1 mice treated with BMSCs performed significantly better on the water maze test than those in sham-operated group (P<0·05). Immunohistochemistry showed that GFP-BMSCs distributed uniformly and the number of Alzheimer’s senile plaques reduced after transplantation. Western blot showed that quantified DeltaNp73 protein expression was significantly higher in BMSCs transplantation group when compared with sham-operated group (P<0·01).

Conclusions: Our results suggest that BMSCs transplatation could retard Alzheimer’s disease (AD) like pathology and upregulate DeltaNp73 expression in hippocampuses of APP/PS1 transgenic mice. GFP-BMSCs transplantation will be a potential treatment for AD.     

心脏过表达人源PRKAG2(G100S)转基因小鼠模型的建立

目的 建立心脏过表达人源PRKAG2 (G100S)的转基因小鼠模型,为进一步研究该人源基因点突变对小鼠心脏发育、形态和功能维持的作用奠定基础.方法 克隆人源基因PRKAG2并构建点突变质粒,将人源PRKAG2 (G100S)插入α肌球蛋白重链(α-MHC)启动子下游,构建转基因表达载体.选用C57BL/6J小鼠为遗传背景,通过显微注射法建立人源PRKAG2(G100S)转基因小鼠模型,利用特异引物PCR法鉴定转基因小鼠的基因型.采用实时荧光定量PCR(qPCR)和蛋白质印迹法检测人源PRKAG2 (G100S)的表达.结果 经过回交繁育后建立了2个品系的心脏特异表达人源PRKAG2(G100S)的转基因小鼠品系F2代,并通过qPCR、蛋白质印迹法检测,确认了转基因小鼠心脏组织中人源PRKAG2(G100S)在rmRNA和蛋白水平存在过表达,且该突变能在转基因小鼠中稳定传代.结论 本研究成功建立了心脏特异表达人源PRKAG2(G100S)转基因小鼠模型,人源PRKAG2 (Gl00S)基因在心脏组织的过度表达在小鼠心脏发育和功能维持中的作用需要进一步深入研究与探讨.     

Pyruvate slows disease progression in a G93A SOD1 mutant transgenic mouse model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by selective motor neuron death, and currently no effective treatment is available for ALS. In this study, we investigated the neuroprotective effects of pyruvate, which acts as an anti-oxidant and as an energy source. We treated G93A SOD1 transgenic mice with pyruvate (from 70 days of age, i.p., at 1000 mg/kg/week), and found that it prolonged average lifespan by 12.3 days (10.5%), slowed disease progression, and improved motor performance, but did not delay disease onset. Pyruvate treatment was also associated with reduced nitrotyrosine immunoreactivity, gliosis, and increased Bcl-2 expression in the spinal cords of G93A SOD1 transgenic mice. These results suggest that pyruvate treatment may be a potential therapeutic strategy in ALS.     

Development of leiomyosarcoma of the uterus in MMTV-CR-1 transgenic mice

Overexpression of Cripto-1 (CR-1) in FVB/N mice using the MMTV-LTR promoter results in increased mammary tumourigenesis in these female transgenic mice (MMTV-CR-1). Here, we characterize uterine tumours that developed in 15/76 (19.7%) of MMTV-CR-1 female nulliparous or multiparous mice during 24 months of observation compared with 0/33 (0%) of FVB/N normal control mice observed during the same time period (p < 0.01). The uterine tumours collected from the MMTV-CR-1 mice were classified as leiomyosarcomas and found to express the CR-1 transgene by polymerase chain reaction analysis and immunohistochemistry. Detection by western blot analysis showed higher levels of phosphorylated (P) forms of c-src, Akt, GSK-3beta, and dephosphorylated (DP)-beta-catenin in lysates from MMTV-CR-1 uterine leiomyosarcomas in comparison with lysates from normal control FVB/N uteri. Immunostaining showed increased nuclear localization of beta-catenin in the MMTV-CR-1 uterine leiomyosarcomas. Increased immunostaining for CR-1 was detected in 9/13 (69.2%) cases of human leiomyosarcoma compared with staining in 3/15 (20%) human leiomyoma sections. Stronger immunostaining for P-src, P-Akt, P-GSK-3beta and increased nuclear localization of beta-catenin was also seen in human leiomyosarcomas in comparison with leiomyomas. Normal human uterine smooth muscle (UtSM) cells treated with exogenous soluble rhCR-1 showed increased levels of P-src, P-Akt, P-GSK-3beta and dephosphorylated (DP)-beta-catenin and increased proliferation (p < 0.05) and migration (p < 0.01) in comparison with untreated control UtSM cells. Inhibitors against c-src, Akt or beta-catenin, individually or in combination, significantly reduced CR-1-induced migration. These results suggest a role for CR-1 during uterine tumourigenesis either directly by activating c-src and Akt and/or via cross-talk with the canonical Wnt signalling pathway, as suggested by the increased expression of P-GSK-3beta, DP-beta-catenin, and increased nuclear localization of beta-catenin in human and MMTV-CR-1 mice leiomyosarcomas.