RECK基因在兔颈动脉壁损伤后再狭窄过程中的表达

目的:通过建立动物损伤模型,分析RECK基因在兔子颈动脉球囊损伤术后的表达与动脉内膜变化和官腔狭窄的相关性。方法:兔子手术损伤侧的动脉血管设置为实验组,未进行手术操作的一侧动脉作为对照组。建立动脉模型的四个时间点7、14、21、28 d,在麻醉状态下处死模型动物。取得所需长度的损伤动脉血管及对照组动脉血管。将取得的标本进行HE病理染色,通过计算机图像计算软件观察标本动脉内膜随时间的变化;同时通过western-blot方法测定RECK蛋白表达水平、real-time PCR测量RECK基因表达量。结果:血管手术损伤后,血管内膜面积在随着术后日期的增长呈逐渐增厚变化,血管内膜与中层比值逐渐增大,同对照实验组比较,有统计学意义(P0.05)。实验组及对照组的中膜无显著增生。结论:RECK基因在组织中表达变化影响MMPs基因表达。实验论证了在动脉损伤后RECK基因参与了血管再狭窄,为血管再狭窄研究寻得新的研究方向。     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Phosphodiesterase inhibitor 3-isobutyl-methyl-xanthine stimulates reproduction in rabbit females

The aim of our study was to examine the influence of 3-isobutyl-1-methyl-xanthine (IBMX), an inhibitor of cyclic adenosine monophosphate and cyclic guanosine monophosphate phosphodiesterases, on the reproductive efficiency of gonadotropin-stimulated rabbits (Oryctolagus cuniculus, Leporidae, Lagomorpha). The ovarian cycle and ovulation of control rabbits was induced by pregnant mare serum gonadotropin (PMSG) followed by administration of human chorionic gonadotropin (hCG; first series of experiments) or gonadotropin-releasing hormone (GnRH; second series of experiments). Experimental animals received PMSG and hCG together with IBMX (at 5 or 25 μg/animal) or GnRH together with IBMX (at 50 or 500 μg/animal). After ovulation and mating, in the first series of experiments, animals were killed; the pronuclear-stage eggs were flushed from the oviducts and cultured up to blastocyst cell stage. Numbers of ovarian corpora lutea, ovulated oocytes, and oocyte-derived embryos reaching blastocyst stage were determined. In the second series of experiments, all the animals were kept until parturition, when the pregnancy and birth rate, litter size, and number, viability, and body weight of pups were recorded. IBMX injections at doses of 5 or 25 μg/animal significantly increased the number of ovulations/corpora lutea, harvested zygotes, and embryos derived from these zygotes. Administration of IBMX at doses of 500 μg/animal or 50 μg/animal to nulliparous young animals (4.5 mo of age) significantly increased their pregnancy rate and birth rate, litter size, and litter weight. In multiparous old animals (2 yr of age), IBMX at a dose of 50 μg/animal, but not 500 μg/animal, significantly increased their pregnancy rate and litter size, but not the birth rate, number of pups per female, or litter weight. These data demonstrate that (1) IBMX can enhance the stimulatory effect of GnRH/gonadotropins on rabbit ovulation, oocyte maturation, embryo yield and development, pregnancy and birth rate, and number, viability, and body weight of pups; (2) nulliparous young females (4.5 mo of age) are more sensitive to IBMX treatments than the multiparous old animals (2 yr of age); and (3) cyclic nucleotides-dependent intracellular mechanisms are involved in control of rabbit reproductive functions and IBMX, an activator of these mechanisms, can be a stimulator of reproduction and fertility.     

Suppressed CD31 Expression in Sarcoma-180 Tumors after Injection with Toxoplasma gondii Lysate Antigen in BALB/c Mice

The anti-tumorigenic effects of Toxoplasma gondii (RH) antigens were studied in a murine sarcoma-180 tumor model. To determine the anti-tumor effects, the reduction in tumor size and expression of CD31 (an angiogenesis marker in the tumor tissue) were examined after injection of BALB/c mice with T. gondii lysate antigen (TLA) or formalin-fixed, proliferation-inhibited, T. gondii tachyzoites. Tumors were successfully produced by an intradermal injection of sarcoma-180 cells with plain Matrigel in the mid-backs of mice. After injection with TLA or formalin-fixed T. gondii tachyzoites, the increase in tumor size and weight nearly stopped while tumor growth continued in control mice that were injected with PBS. CD31 expression in TLA-treated or formalin-fixed T. gondii-injected mice was lower than the control mice. Accordingly, the present study shows that the treatment of mice with formalin-fixed T. gondii or TLA in the murine sarcoma-180 tumor model results in a decrease of both tumor size and CD31 expression.     

Effect of Garlic Supplementation on Erythrocytes Antioxidant Parameters, Lipid Peroxidation, and Atherosclerotic Plaque Formation Process in Oxidized Oil-Fed Rabbits

Effect of garlic supplementation on blood antioxidant status, lipid peroxidation, and coronary plaque formation process was investigated in oxidized oil-fed rabbits. Eighteen adult male mixed European rabbits were given a balanced diet (21 g% protein, 34 g% fat, 45 g% carbohydrate), which contained isocaloristic addition of nonoxidized or oxidized rapeseed oil in the presence and absence of garlic. The experiment lasted 24 weeks. At the beginning and every 6 weeks, rabbits were weighed, and blood was taken. To evaluate the antioxidant status of the rabbits, erythrocytes malondialdehyde (MDA) concentration, total superoxide dismutase (t-SOD), and glutathione peroxidase (GPX) activations were determined. After the experiment was completed, aortas were dissected for histological examinations. Changes in the contents of the above parameters and histological examinations showed that oxidized rapeseed, oil administered to rabbits, caused the development of atherosclerotic changes and disturbed antioxidant status. The addition of garlic in such diets inhibited atherosclerotic changes in the aorta wall, and it is related to the homeostatic activity of antioxidative enzymes and lipid peroxidation.     

Contrasting effects of rabbit exclusion on nutrient availability and primary production in grasslands at different time scales

Herbivores influence nutrient cycling and primary production in terrestrial plant communities. However, both empirical and theoretical studies have indicated that the mechanisms by which herbivores influence nutrient availability, and thus their net effects on primary production, might differ between time scales. For a grassland in southeast England, we show that the effects of rabbits on primary production change over time in a set of grazed plots paired with exclosures ranging from 0 to 14 years in age. Herbivore exclusion decreased net aboveground primary production (APP) in the short term, but increased APP in the long term. APP was closely correlated with N mineralization rates in both grazed and ungrazed treatments, and accumulation of litter within the grazing exclosures led to higher N mineralization rates in exclosures in the long run. Rabbit grazing did not influence litter quality. The low contrast in palatability between species and the presence of grazing-tolerant plants might prevent rabbits from favoring unpalatable plant species that decompose slowly, in contrast to results from other ecosystems. Our results indicate that it is essential to understand the effects on N cycling in order to predict the effect of rabbit grazing on APP. Rabbits might decrease N mineralization and APP in the long term by increasing losses of N from grasslands.     

Localisation of caveolin in mammary tissue depends on cell type

Caveolins, components of caveolae, are expressed in mammary tissue. In order to determine whether caveolins are present in different mammary cell types and whether their localisation depends on the physiological stage or species, cav-1 and cav-2 were characterised by immunoblotting in mammary tissues from the mouse, ewe and rabbit and localised, by immunofluorescence and electron microscopy, in mammary tissues from the mouse and ewe. At all the physiological stages studied, cav-1 and cav-2 were present in endothelial and myoepithelial cells in which flask-shaped caveolae were abundant. However, labelling of cav-1 and cav-2 associated with small vesiculo-tubular structures (including those close to lipid droplets) was low in epithelial cells. To study the possible association of cav-1 with lipid droplets, lactating ewe mammary fragments were treated in vitro with brefeldin A. This treatment did not modify the association of cav-1-labelled structures with lipid droplets. Finally, HC11 and MCF-10A mammary cell lines were treated with oleic acid. The total quantity of cav-1 was little affected by the treatment, although the lipid droplet labelling of cav-1 was amplified in MCF-10A cells. Thus, the synthesis and localisation of caveolins are mostly dependent upon the cell types of mammary tissue and upon their state of differentiation.     

Survival and integration of tissue-engineered corneal stroma in a model of corneal ulcer

Tissue-engineered replacement of diseased or damaged tissue has become a reality for some types of tissue, such as skin and cartilage. Tissue-engineered corneal stroma represents a promising concept to overcome the limitations of cornea replacement with allograft. In this study, porcine cornea was decellularized by a series of extraction methods, and the in vivo biocompatibility of the scaffold was measured subcutaneously in rabbits (n = 8). These were not acutely rejected and no abscesses were observed by hematoxylin and eosin staining at the 8th week, indicating that the scaffolds had good biocompatibility. To investigate the potential value of clinical applications, rabbit stromal keratocytes were implanted onto decellularized scaffolds to fabricate tissue-engineered corneal stroma. Allograft, tissue-engineered corneal stroma, or scaffolds were implanted into a model of corneal ulcer. The survival and reconstruction of corneal transplantation were morphologically evaluated by light and electron microscopy until the 32nd week after implantation. Experiments involving transplantation indicated that the epithelial and stromal defect healed quickly, with improvement in corneal clarity. The integration of the graft was accompanied by neurite ingrowth from the host tissue. By 16 weeks after transplantation, the cornea had gradually regained an intact state similar to that of normal cornea. Our results demonstrate that the tissue-engineered corneal stroma with allogenetic cells is a promising therapeutic method for corneal injury. This study was supported by the Nature Science Foundation of China (project no. 30572046) and the Development of High and New Science and Technology (863 Project) of China (2002AA205041, 2005AA205241).     

急性兔脑栓塞模型的建立

目的建立稳定的兔脑栓塞模型,以期为进一步开展脑栓塞的病理生理变化及影像学研究提供可靠实用的工具。方法30只健康新西兰兔,随机分成3组,其中A组3只,为空白对照组;B组5只,为假手术对照组;C组22只,为栓塞组。分离右侧颈部血管,经颈外动脉向颈内动脉注入直径约0.5~1.0 mm的SiO2颗粒10枚左右,栓塞后30 min行CT灌注检查,利用多层螺旋CT灌注成像对各组动物的脑缺血情况进行观察。24 h处死动物取脑组织进行病理研究。结果A、B组CT灌注及病理均未见异常。C组栓塞过程中3只兔死亡。16只兔CT灌注异常,表现为右侧局部CBF降低、MTT延长、CBV无明显变化或轻度上升、下降。3只兔灌注未见异常。HE染色可见8只兔脑梗塞,7只兔脑缺血,4只兔未见明显异常。结论采用该方法和技术能够建立稳定的兔脑栓塞模型,结果可靠,具有可操作性和重复性。     

Evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.