db/db小鼠胰岛的分离与特征分析

目的 分离db/db小鼠胰岛,并对其特征进行检测和分析。方法 采用胶原酶V对10只db/db小鼠胰岛分离进行逆行胰管灌注和消化,用Ficoll-1077和Ficoll-1119进行胰岛的不连续密度梯度纯化,对分离的胰岛进一步进行手工挑选,并用DTZ进行胰岛和纯度鉴定,用透射电镜观察胰岛内部分泌颗粒等情况。结果 经本方法分离可得到db/db小鼠胰岛数量为（122.4±6.6）个/只,当量为（483.6±82.3）IEQ/只,与ICR小鼠胰岛分离结果差异有统计学意义（P<0.05）;db/db小鼠胰岛DTZ染色后显淡红色;胰岛大小指数为（3.96±0.64）,显著大于ICR小鼠胰岛（P<0.05）;透射电镜下显示β细胞中的胰岛素分泌颗粒减少,分泌颗粒颜色较浅。结论 采用本方法可分离得到db/db小鼠胰岛,为后续开展基于胰岛功能和特征变化的2型糖尿病患者治疗研究提供一种参考。     

升清降浊胶囊对db/db小鼠肾组织PDGF-B表达的影响

目的:观察升清降浊胶囊改善自发性2型糖尿病db/db小鼠肾损伤的作用及对肾组织血小板衍化生长因子-B(PDGF-B)表达的影响.方法:将16只8周龄雄性db/db小鼠随机分为db/db模型组(n=8)和升清降浊组(n=8),选取db/m小鼠为空白对照组(n=8),升清降浊组给予升清降浊胶囊1800 mg·kg-1·d-...     

Cytokines in the BALB／c mouse testis in various conditions

Aim: To investigate whether testosterone, estrogens, vasectomy, experimental cryptorchidism, varicocele or aging would induce changes in the cytokine environment of the mouse testis, Methods: In adult male BALB/c mice, testosterone implants, estradiol benzoate, vasectomy, unilateral cryptorchidism, unilateral varicocele were administered/performed. The mice were followed up for different periods of time and were then sacrificed with testes incised for examination. The control mice received the vehicle or sham-operation. Results: IL-10 was present in Leydig cells of nearly every testis and IL-10 macrophages in 39% of testes. IL-6 was found in the testes of intact adult mice, mice treated with testosterone for 70 days, cryptorchid testes and sham-operated testes. Conclusion: Results suggest that IL-10 might be involved in the generation of the immunologically privileged microenvironment in the testis.     

阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用

目的 观察肾素抑制剂阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用。 方法 8周龄db/db和db/m小鼠行单侧肾切除,16周进入实验,分为4组：db/m小鼠对照组（db/m组）、db/db糖尿病小鼠对照组（db/db组）、db/db糖尿病小鼠阿利吉仑3 mg&#8226;kg-1&#8226;d-1治疗组（db/db+A3组）和db/db糖尿病小鼠阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗组(db/db+A25组)。阿利吉仑溶于磷酸盐缓冲液（PBS,350 mg/L）皮下泵入（0.25 μl/h）给药,疗程４周。治疗前后检测体质量、血糖、糖化血红蛋白、尿蛋白量、血压水平;PAS染色观察肾脏组织学变化;ELISA法检测肾皮质转化生长因子β1（TGF-β1）和纤溶酶原激活抑制因子1（PAI-1）含量;间接免疫荧光检测肾小球Ⅳ型胶原（ColⅣ）和纤连蛋白（FN）表达;实时定量PCR检测TGF-β1、PAI-1、ColⅣ、FN和肾素mRNA表达;放射免疫法检测肾皮质肾素活性和血管紧张素Ⅱ（AngⅡ）水平。 结果 与db/m组小鼠比较,db/db组小鼠有大量蛋白尿,肾小球细胞外基质沉积增加,TGF-β1、PAI-1、ColⅣ和FN蛋白及mRNA表达增加,同时肾皮质肾素mRNA、肾素活性和AngⅡ水平增高（均P < 0.05）。阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗在没有影响血压情况下,显著减少db/db小鼠24 h尿蛋白量,减少肾小球细胞外基质沉积,减少TGF-β1、PAI-1、ColⅣ、FN蛋白和mRNA表达,同时降低肾皮质肾素活性和AngⅡ水平（均P < 0.05）。 结论 阿利吉仑对2型糖尿病db/db小鼠肾脏损伤有保护作用。     

Involvement of the renin–angiotensin system in the development of nephrogenic systemic fibrosis-like lesions in the RenTag mouse model

Background

Renin–angiotensin system (RAS) activation increases angiotensin II production stimulating profibrotic factors, especially in the setting of chronic kidney disease. Nephrogenic systemic fibrosis (NSF) has been associated with gadolinium (Gd) exposure and renal failure. RAS involvement in NSF is unclear compared to transforming growth factor beta and Smad. RenTag mice were chosen to investigate the role of RAS in NSF-like dermal fibrosis because they demonstrated dermal fibrosis at birth, perturbations of RAS in subcutaneous tissue, and renal failure within 4 weeks of age.

Methods

Wild-type and RenTag mice were injected weekly with a supratherapeutic dose of intravenous gadodiamide (3.0 mmol/kg body weight) and killed at 12 weeks of age for skin and kidney histology.

Results

RenTag mice had elevated BUN levels, pitted kidneys, and glomerular damage. RenTag mice skin revealed an increased density of fibroblasts, no mucopolysaccharide deposits, and increased collagen fibril density regardless of Gd exposure. Skin and kidney histopathology of wild-type mice were normal regardless of Gd exposure. CD34 positivity was higher in RenTag compared to wild-type.

Conclusions

Since RenTag dermal lesions remained unchanged after gadolinium exposure in the setting of renal failure, this animal model suggests perturbations of subcutaneous RAS may be involved in Gd-naïve dermal fibrosis.

     

Is high-dose estrogen-induced osteogenesis in the mouse mediated by an estrogen receptor?

Although estrogen is known to induce new bone formation in the long bones of female mice, this response is only thought to occur following administration of high doses, suggesting that it may not be mediated by a conventional estrogen receptor. To address this question further, we first examined the stereospecificity of this response by comparing the potency of 17beta-estradiol (E(2)) in stimulating cancellous bone formation at the proximal tibial metaphysis of intact female mice with that of the relatively inactive stereoisomer, 17alpha-estradiol (alphaE(2)). We found that E(2) was significantly more potent than alphaE(2), as assessed by histomorphometry. To provide further evidence for an estrogen-receptor-mediated process, we examined whether E(2)-induced osteogenesis in intact female mice could be inhibited by the estrogen receptor antagonist, ICI 182,780 (ICI). Although ICI itself had no effect on histomorphometric indices of the proximal tibial metaphysis when given alone, it significantly inhibited the osteogenic response to E(2). Finally, we examined the dose dependency of E(2)-induced osteogenesis at the proximal tibial metaphysis in intact mice. We found that E(2) stimulated cancellous bone formation in a dose-dependent manner over a wide dose range (i. e., 1-4000 microg/kg per day), with significant increases observed at doses of 4 microg/kg per day and beyond. Our results raise the possibility that estrogen-induced osteogenesis in the mouse represents an estrogen-receptor-mediated response that is not confined solely to supraphysiological estrogen levels.     

Islet β-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model

Type 1 diabetes is preceded by islet β-cell dysfunction, but the mechanisms leading to β-cell dysfunction have not been rigorously studied. Because immune cell infiltration occurs prior to overt diabetes, we hypothesized that activation of inflammatory cascades and appearance of endoplasmic reticulum (ER) stress in β-cells contributes to insulin secretory defects. Prediabetic nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID and CD1 strains were studied for metabolic control and islet function and gene regulation. Prediabetic NOD mice were relatively glucose intolerant and had defective insulin secretion with elevated proinsulin:insulin ratios compared with control strains. Isolated islets from NOD mice displayed age-dependent increases in parameters of ER stress, morphologic alterations in ER structure by electron microscopy, and activation of nuclear factor-κB (NF-κB) target genes. Upon exposure to a mixture of proinflammatory cytokines that mimics the microenvironment of type 1 diabetes, MIN6 β-cells displayed evidence for polyribosomal runoff, a finding consistent with the translational initiation blockade characteristic of ER stress. We conclude that β-cells of prediabetic NOD mice display dysfunction and overt ER stress that may be driven by NF-κB signaling, and strategies that attenuate pathways leading to ER stress may preserve β-cell function in type 1 diabetes.     

Aminated β-1,3-D-glucan has a dose-dependent effect on wound healing in diabetic db/db mice

Inflammatory responses are common in diabetes and are operative in angiopathy, neuropathy, and wound healing. There are indications of incomplete macrophage activation in diabetes and reduced expression of growth factors. We have previously found that up to 15 topical applications of the macrophage‐stimulant, aminated β‐1,3‐D‐glucan (AG), improved wound healing in db/db mice. The present open‐label study was undertaken to examine dose‐dependent effects of AG over 40 days in db/db mice. AG was given as a single dose (group 1), one dose every 10th day (group 2), five initial doses on consecutive days (group 3), and ≥15 doses (group 4). Controls were db/db mice receiving platelet‐derived growth factor + insulin‐like growth factor‐1 (group 5), topical placebo (NaCl 9 mg/mL) and insulin (group 6), placebo (group 7), and a nondiabetic group receiving placebo (group 8). Seven to 14 animals were allocated to each group. Percentage wound closure 17 days after surgery in groups 1 and 2 were (mean ± standard error of the mean) 25.5 ± 5.3 and 32.2 ± 6.3, respectively. Corresponding closure in groups 3, 4, and 5 was 55.7 ± 5.0, 57.3 ± 5.0, and 55.6 ± 4.8, respectively (p < 0.05 vs. groups 1 and 2). Groups 6, 7, and 8 closed 32.0 ± 4.5, 38.2 ± 5.3, and 98.5 ± 0.4%, respectively. Significant association between the number of AG‐dosages and wound closure indicates dose‐related effects in db/db mice.     

In Vivo Expression of HGF/NK1 and GLP-1 From dsAAV Vectors Enhances Pancreatic ��-Cell Proliferation and Improves Pathology in the db/db Mouse Model of Diabetes

OBJECTIVE

The purpose of the current study was to determine whether double-stranded adeno-associated virus (dsAAV)-mediated in vivo expression of β-cell growth factors, glucagon-like peptide-1 (GLP-1) and the NK1 fragment of hepatocyte growth factor (HGF/NK1) in β-cells, improves pathology in the db/db mouse model of type 2 diabetes.

RESEARCH DESIGN AND METHODS

The glucoregulatory actions of GLP-1 and full-length HGF are well characterized. Here, we test the ability of HGF/NK1 to induce proliferation of exogenous islets and MIN6 β-cells. In addition, we target both GLP-1 and HGF/NK1 to endogenous β-cells using dsAAV vectors containing the mouse insulin-II promoter. We compare the abilities of these gene products to induce islet proliferation in vitro and in vivo and characterize their abilities to regulate diabetes after AAV-mediated delivery to endogenous islets of db/db mice.

RESULTS

Recombinant HGF/NK1 induces proliferation of isolated islets, and dsAAV-mediated expression of both GLP-1 and HGF/NK1 induces significant β-cell proliferation in vivo. Furthermore, both GLP-1 and HGF/NK1 expressed from dsAAV vectors enhance β-cell mass and insulin secretion in vivo and significantly delay the onset of hyperglycemia in db/db mice.

CONCLUSIONS

A single treatment with dsAAV vectors expressing GLP-1 or HGF/NK1 enhances islet growth and significantly improves pathology in a mouse model of type 2 diabetes. This represents the first example of a successful use of HGF/NK1 for diabetes therapy, providing support for direct AAV-mediated in vivo delivery of β-cell growth factors as a novel therapeutic strategy for the treatment of type 2 diabetes.Apromising therapeutic for type 2 diabetes is the incretin family of proteins, particularly glucagon-like peptide-1 (GLP-1) (1). GLP-1 has a variety of glucoregulatory actions, which include enhancing insulin synthesis and secretion, improving insulin sensitivity, inhibiting glucagon secretion, and increasing β-cell mass (2). However, GLP-1 is rapidly degraded by the ubiquitous enzyme, dipeptidyl peptidase-IV, and thus has a short in vivo half-life that limits its therapeutic efficacy (3). We have recently shown that adeno-associated virus (AAV) administration of GLP-1 provides long-term, high-level GLP-1 expression, offering an alternative approach to peptide therapy (4).Another protein that has potential as a therapeutic for diabetes is hepatocyte growth factor (HGF). HGF is involved in the regeneration of multiple organs, including the liver, kidney, and lung (5), and a number of studies illustrate the efficacy of HGF in animal models of diabetes (6–10). Transgenic mice specifically overexpressing HGF in β-cells exhibit increased β-cell proliferation, function, and survival (5), and HGF improves islet transplant outcome in rodent models (6,7). Moreover, HGF has previously been delivered to isolated islets via adenovirus gene transfer, reducing β-cell death, reducing the minimal islet mass required for successful transplant, and improving overall transplant outcome (8,9).While full-length HGF has beneficial effects in animal models of diabetes, recent research has focused specifically on the N and K1 domains of HGF (HGF/NK1). HGF/NK1 comprises the NH₂-terminal 175 amino acids of HGF and is sufficient for binding and partial activation of the HGF receptor, as well as initiation of some mitogenic activity (11–13). HGF/NK1 has not previously been studied in relation to diabetes but may provide several advantages over full-length HGF. HGF/NK1 may improve the safety profile compared with full-length HGF by limiting stimulation of the HGF receptor, Met. Another advantage of using HGF/NK1 instead of full-length HGF is the ability to use double-stranded AAV (dsAAV) vectors for gene delivery. dsAAV vectors provide rapid, efficient, and stable gene expression (14), without the immunogenicity associated with adenovirus vectors. However, dsAAV vectors have limited packaging capacity, thus preventing their use for delivery of full-length HGF.The majority of studies using gene transfer strategies to enhance islet function or survival have used ex vivo transduction protocols. However, in vivo transduction of β-cells has been achieved by direct targeting of genes to β-cells of pancreatic islets using dsAAV vectors (4,15). There are many benefits of direct in vivo gene transfer compared with transduction of islets for transplantation, as well as compared with existing therapeutics. First of all, the applicability of islet transplantation is reduced by the limited availability of islets. Secondly, while existing antidiabetic agents and incretin drugs have therapeutic efficacy, they are often associated with adverse effects and multiple daily administrations. Direct in vivo gene transfer could be cost-effective and beneficial to patients'' quality of life because it may be therapeutic in as little as a single treatment.Here, we examine whether in vivo delivery of the growth factors GLP-1 and HGF/NK1 via dsAAV-mediated gene transfer can improve pathology in a mouse model of type 2 diabetes. We demonstrate that dsAAV-expressed GLP-1 and HGF/NK1 enhance islet proliferation and delay onset of diabetes in db/db mice, providing the first evidence that HGF/NK1 is a potential therapeutic for diabetes.     

Comparison of gene expression of the oncogenic Wnt/β-catenin signaling pathway components in the mouse and human epididymis

β-catenin is an integral part of the Wnt signaling pathway and has been linked to tumorigenesis and multiple developmental processes. The high β-catenin expression with low tumor incidence in the human epididymis is thus intriguing. In the present study, the β-catenin gene and protein was found to be highly expressed in the murine caput epididymidis, and the protein mainly localized along the lateral plasma membranes of adjacent epithelial cells throughout both human and mouse epididymides. Furthermore, the adult mouse epididymis was found to express almost all the Wnt/β-catenin signaling pathway genes that were determined previously by our group in the human organ. Despite the differences in epididymal structure, the similar location of β-catenin and the high concordance of this pathway''s components’ gene expression in both the adult human and mouse epididymides make the mouse a suitable animal model for studying the anti-tumor mechanism of the epididymis. In addition, both the mRNA and protein expression of β-catenin shared a similar spatial expression as the mRNA of Ros1, a proto-oncogene and a key developmental regulator of the initial segment of the mouse epididymis. The observations on the parallel temporal expression of β-catenin and Ros1 during postnatal development raise the possibility that the canonical Wnt signaling pathway has an additional role in the postnatal development of mouse epididymis.     

Progressive erosion of β-cell function precedes the onset of hyperglycemia in the NOD mouse model of type 1 diabetes

OBJECTIVE

A progressive decline in insulin responses to glucose was noted in individuals before the onset of type 1 diabetes. We determined whether such abnormalities occurred in prediabetic NOD mice—the prototypic model for human type 1 diabetes.

RESEARCH DESIGN AND METHODS

Morning blood glucose was measured every other day in a cohort of NOD females. Glucose tolerance and insulin secretion were measured longitudinally by intraperitoneal glucose tolerance tests in NOD/ShiLtJ and BALB/cJ mice 6 to 14 weeks of age. Arginine-stimulated insulin secretion and insulin sensitivity were assessed during intraperitoneal arginine or intraperitoneal insulin tolerance tests.

RESULTS

During prediabetes, NOD females displayed a progressive increase in glucose levels followed by an acute onset of hyperglycemia. First-phase insulin responses (FPIRs) during the intraperitoneal glucose tolerance test (IPGTT) declined before loss of glucose tolerance in NOD. The failure of FPIR could be detected, with a decline in peak insulin secretion during IPGTT. Arginine-stimulated insulin secretion remained unchanged during the study period. The decline in insulin secretion in NOD mice could not be explained by changes in insulin sensitivity.

CONCLUSIONS

There was an impressive decline in FPIR before changes in glucose tolerance, suggesting that impairment of FPIR is an early in vivo marker of progressive β-cell failure in NOD mice and human type 1 diabetes. We portend that these phenotypes in NOD mice follow a similar pattern to those seen in humans with type 1 diabetes and validate, in a novel way, the importance of this animal model for studies of this disease.Type 1 diabetes is an autoimmune disease resulting from the destruction of pancreatic insulin-producing β-cells. The NOD mouse is the most commonly used rodent model of the disease. Studies in this mouse strain have led to interventions that have been translated to clinical investigations with human type 1 diabetic patients (1–5). However, intervening at the right therapeutic window is critical to the efficacy of certain therapies. For example, anti-mouse thymocyte globulin can delay or reverse diabetes in NOD only when used in the late prediabetes stage or at onset of the disease (6).Islet autoimmunity can be identified in the prediabetic stage. Specifically, in NOD mice, insulin autoantibodies can be detected as early as at 3 weeks of age (7). However, as in humans, autoantibody positivity can be transient as well as observed in nonprogressors (8,9). Therefore, markers of β-cell mass and function are needed to identify progression from the onset of islet autoimmunity to and through the stages of prediabetes.Previous cross-sectional studies have demonstrated decreased first-phase insulin secretion in NOD females at different ages. Using pancreatic perfusion, Kano et al. (10) documented that NOD mice maintain normal fasting glucose even though a significant decrease was measured in first-phase insulin response (FPIR) to glucose. In these studies, increased fasting glucose and loss of FPIR was associated with the degree of insulitis. In cross-sectional studies using intravenous glucose tolerance test, Reddy et al. (11) showed an age-related progressive decline in FPIR to glucose. In another cross-sectional study using in situ pancreas perfusion, Sreenan et al. (12) reported progressive decreases in glucose- and arginine-stimulated insulin secretion in NOD females at 8, 13, and 18 weeks of age. Although single-point analyses were performed, there have not been reports involving sequential analysis of single mice over time to confirm this progression. In this study, we sought to determine whether similar metabolic signatures exist in both humans and in prediabetic NOD mice.     

A dose‐dependent dual effect of oestrogen on voiding in the male mouse?

OBJECTIVES: To explore the effect of different degrees of oestrogenization on male voiding, by treating adult castrated and 5alpha-dihydrotestosterone (DHT)-maintained male mice with different doses of oestrogens, as exposure of male mice to excessive amounts of oestrogens can cause bladder outlet obstruction (BOO); in addition, male mice lacking oestrogen receptor (ER)alpha (ERKO) or ERbeta (BERKO) were studied to assess the importance of ER subtypes. MATERIALS AND METHODS: Castrated, DHT-maintained adult mice were treated with 17beta-oestradiol (E(2); 50 and 250 microg/kg) or oestrone (E(1); 5, 50 and 500 microg/kg) daily for 10 days. Control mice were treated only with the vehicle. BERKO and ERKO mice, and their wild-type littermates used as their controls, remained untreated. Under anaesthesia, the bladder and distal urethra were exposed to record simultaneously the bladder pressure and urinary flow rate from the distal urethra. RESULTS: E(2)-treated mice showed obstructive voiding, seen as increased bladder pressure, decreased average flow rate and prolonged micturition time. This was also evident when a high dose (500 microg/kg) of E(1) was used. After treatment with a dose of 50 microg/kg, the urodynamic variables were similar to those in the control mice. Surprisingly, after treatment with a low dose (5 microg/kg) all urodynamic variables improved. There was a minor increase in the bladder pressure in BERKO mice; ERKO mice had a significantly lower urinary flow rate. CONCLUSIONS: High doses of oestrogens caused BOO in castrated, DHT-maintained male mice. A small dose of E(1) had a positive effect on voiding, suggesting that oestrogens are needed for normal male voiding. Reduced urinary flow rates in ERKO mice suggest that oestrogen effects on voiding are mediated at least partly via ERalpha.     

Age-dependent expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis

Aim： To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis during postnatal development. Methods： The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. Results： （1） In both the testis and epididymis, Cres mRNA was fast detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. （2） In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. （3） The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. Conclusion： The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.     

The apoptosis of the rat and mouse testicular cells could be induced by synthetic analogous of the prostaglandin F2 alpha?

Numerous actions of the synthetic analogous of the prostaglandins were used in human and veterinary therapy. The action of these analogous (even, initially the prostaglandins were put in evidence in the seminal plasma) on the male reproductive system are little known. This is the reason for our study, which tried to emphasize the effects of analogous of the prostaglandin F2 alpha on the apoptosis of the rat and mouse testicular cells. Optic active Cloprostenol and Cloprostenol Isopropyl ester, in a dose of 100 micrograms/kg/day induces significant histopathologic modifications in the testis of rats and mice. These changes are evident after 14 days of treatment, but especially after 28 days of treatment.