Objective To determine the effects of lensspecific overexpression of OSM on the eye development. Methods A truncated mouse OSM c DNA (661 bp) was linked to the αA-crystallin promoter. Transgenic mice were characterized by routine histological and immunohistochemical techiniques. TUNEL assays were used to de tect cell death. The mRNA expression of caspase-3 was detected by in situhybridization, Rabbit anti-cleavage caspase-3 antibody was used to detectactive capase-3. Results At embryonic day (E) 14.5 and 17.5, expression of the OSM transgenic protein was detected specifically in lens fiber cells. The onset of retinal degeneration in the mid portion of the transgenic retinae was observed started from E17.5. By the time of birth 50% or more of the retinal cells were missing. The OSM transgenic retinal cells underwent apoptosis indicated by TUNEL assays. Most strikingly, activation of caspase-3 protein were observed throughout the transgenic retinas. Conclusions Lens-specific overexpression of OSM activate caspase-3, leading to abnormal eye development,apoptosis and widespread retinal degeneration. (Chin J Ocul Fundus Dis，2003，19：201-268)
ObjectiveTo explore the dynamic changes of microvessels in the hippocampal CA3 area in mice model of temporal lobe epilepsy (TLE) induced by pilocarpine. MethodsEighteen health SPF male C57BL/6 mice were randomly divided into control group and status epilepticus (SE) group. The SE group was subdivided into three groups:SE-7 days, SE-28 days and SE-56 days. SE was induced by intraperitoneal injection of pilocarpine. And immunohistochemical staining was used to detected the localization of platelet endothelial cell adhesion molecule-1 (PECAM-1). ResultsIn the control group, PECAM-1 labeled microvessels arranged in a layered structure, and the microvessel of the orient layer was most prominent. After SE, the microvessels started to form an unorganized vascular plexus and appeared fibrous and fragmented, which was prominent at SE-28 days. Furthermore, the microvessels density increased the top at SE-28 days compared to the control (P < 0.001). ConclusionThe angiogenesis exists during the hippocampus formation in the mice model of TLE induced by pilocarpine, which could direct a new explanation for TLE formation and development.
Objective To induce embryonic stem cell (ESC) to differentiate into endothel ioid cells using a simple adhesive culture method, and to provide a new cells seed source for vascular tissue engineering or cell therapy. Methods SV129-derived ESC were seeded at 2 × 104/cm2 and maintained undifferentiated on ESC culture medium in the presence of 1 000 U/mL leukaemia inhibitory factor (LIF). Embryoid body (EB) formatted when ESC cultured in suspension in the lack of LIF. At 4 days, EB was transferred to 0.1% gelatin coated dish and cultured with medium supplementary of VEGFto be induced differentiation. The characteristics of differentiated cells were determined by immunohistochemistry staining, flow cytometry (FCM), 1, 1-dioctadecyl-3, 3, 3, 3-tetramethyl indocarbocyanine-labeled acetylated low density l ipoprotein (DiIAc- LDL) takeup test, and TEM detection. Results Differentiated cells were morphologically characterized as endothel ial cells. They could takeup DiI-Ac-LDL, be stained positive by Flk-1 and CD31. The CD31 positive cells reached above 90% when measured by FCM. Furthermore, Weibel-Palade bodies were detected and tight junctions were found when differentiated cells were examined by TEM. Conclusion Using a simple adhesive culture method and by suppl ied with VEGF alone, ESCs can be induced to differentiate into endothel ioid cells. The differentiation method is simple and economic, and can provide seed cells for vascular tissue engineering or cell-therapy.
OBJECTIVE:To examine the possibility of allogenic neural retinal transplantation. METHODS:Donor neural retinal tissue obtained from neonatal mice was implanted into the subretlnal space of 36 adult mice. Twelve recipients were sacrificed at day 6,day 12,and day 18 post-transplantation respectively, and the eyes were enucleated for histologic examination. RESULTS: The graft was failed to be found but the host retina remained normal in 16.7%(6/36),the graft was found survival and showed further differentiation into rosette formation in the host subretinal space and inner retina in 66.7%(24/36),in the vitreous cavity in 8.3% (3/36). Disastrous granulomatous inflammation occurred in 8.3%(3/36). CONCLUSION: Allogenic retina from neonatal mice implanted into the subretinal space might experience immune privilege .in a great extent ,and showed further differentiation during the experimental periods of time. （Chin J Ocul Fundus Dis,1996,12: 236-238 ）
Objective To study the progressive development of the retinas through an observation on the histological changes of the retinas from neonatal mice of different day-ages. Methods The retinas from the mice of 1 to 20 days of age were examined by light microscopy,and from 1 to 3 days,by autoradiography. Results The retinas of the mice below 3 days of age only had the RPE cells layer,the neuroblast layer and the ganglion cell layer.With the increase in dayage,the retinas developed gradually and would be mature in the 20th day. Conclusions The retinas of mice is a kind of immature tissue before the 20th days,so it can be considered as transplantation donors. (Chin J Ocul Fundus Dis, 1999, 15: 174-176)
ObjectiveTo investigate the effect of burn on brown adipose tissue (BAT) in BALB/c mice. MethodsForty 3-4 months old male BALB/c mice with initial body weight of (20±3) g were randomly divided into control group and burn group (n=20).BALB/c mice in burn group were subjected to a 30% total body surface area (TBSA) full-thickness thermal injury.BALB/c mice in control group were not treated.The body weight and temperature were observed before and after burn.At 7 days after burn,morphological changes of white adipose tissue (WAT) and BAT were observed,the gene and protein expressions of uncoupling protein 1 (UCP-1) were detected. ResultsThere was no significant difference in the body weight and body temperature before burn (P>0.05).At 1,2,3,and 4 weeks after burn,the body weight was significantly lower in burn group than in control group (P<0.05).At 1,2,3,and 7 days after burn,the body temperature was significantly higher in burn group than in control group (P<0.05).At 7 days after burn,the weight of WAT was significantly reduced,and the weight of BAT was significantly increased in burn group (P<0.05); WAT and BAT cells became smaller,cell number increased,the cytoplasm and mitochondria appeared as compact.The UCP-1 gene and protein expressions of burn group were significantly higher than those of control group (P<0.05). ConclusionBAT plays an important role in burn-induced hypermetabolism.
Objective The biological effects of fibroblast growth factor (FGF) may be different under different intensities and durations. To investigate the impact of sustained increasing FGF signal upon the development of epiphyseal plate. Methods Epiphyseal plates cultured in vitro were obtained from embryonic C57BL/6J mice, and were divided into control group (0.1% DMSO), basic FGF (bFGF) group (100 μg/L bFGF and 0.1% DMSO), and PD98059 group (100 μg/L bFGF and 50 μmol/L PD98059 with 0.1% DMSO). The total length (TL) and ossified tissue length (OSL) of the cultured bones weremeasured with Calcein staining 6 days after culture. The expressions of Indian hedgehog (Ihh), collagen type II (Col II), and Col X genes were detected by real-time fluorescent quantative PCR 7 days after culture. Results The embryonic bones cultured in vitro continued growth. At 6 days after culture, there was no significant difference in increased percentage of TL between bFGF group and control group (P gt; 0.05), the increased percentage of OSL in bFGF group was significantly less than that in control group (P lt; 0.05). There was no significant difference in the increased percentage of TL and OSL between PD98059 group and control group (P gt; 0.05), but they were significantly higher than those of bFGF group (P lt; 0.05). At 7 days after culture, the gene expressions of Ihh, Col II, and Col X in bFGF group significantly decreased when compared with those in control group (P lt; 0.05). There was no significant difference in the gene expressions of Col II and Col X between PD98059 group and control group (P gt; 0.05), but the gene expressions were significantly higher than those of bFGF group (P lt; 0.05); the expression of Ihh in PD98059 group was significantly higher than that in control group and bFGF group (P lt; 0.05). Conclusion Sustained increasing FGF signal may affect the Col II and Col X expressions by down-regulating Ihh, which may lead to the development retardation of epiphyseal plate cultured in vitro. The external signal regulated kinase pathway may play an important role in the process.
ObjectiveTo establisht a gut microbiota mice model for chronic obstructive pulmonary disease (COPD) with fecal microbiota transplantation (FMT) and its evaluation.MethodsThe mice received FMT from healthy individuals, COPD Ⅰ-Ⅱ subjects, or COPD Ⅲ–Ⅳ subjects. After microbiota depletion, the FMT was performed by a single oral administration of 100 μL per mouse every other day, for a total of 14 times in 28 days. On the 29th day, the peripheral blood mononuclear cells were analyzed, the gut microbiota of mice before and after FMT was analyzed by 16S rRNA sequencing, and the mice model were evaluated.ResultsThe operational taxonomic units, Chao 1 and Shannon indexes of mice all decreased significantly after antibiotic treatment (P<0.001), but increased significantly after FMT from healthy individuals, COPD Ⅰ-Ⅱ subjects, or COPD Ⅲ–Ⅳ subjects (P<0.05 or P<0.01). The abundance of Firmicutes, Proteobacteria and Actinobacteria in the guts of the mice in the healthy human FMT group, COPD Ⅰ-Ⅱ FMT group and COPD Ⅲ-Ⅳ FMT group were significantly different from those of the control group who only received phosphate buffer saline instead of FMT (P<0.05 or P<0.01). The auxiliary T lymphocytes and cytotoxic T lymphocytes were higher, but B lymphocytes decreased in the peripheral blood of the mice in the COPD Ⅰ-Ⅱ FMT group and COPD Ⅲ-Ⅳ FMT group (P<0.05 or P<0.01).ConclusionFMT can successfully establish a COPD gut microbiota research model.
Objective To observe the effect of Minocycline on RP process of retinal pigmentary degeneration rd mice［C3H/HeN (Pde6brd-/rd-)］. Methods 40 rd mice were divided into ten groups randomly: 5 experimental groups and 5 control groups, 4 rd mice in each group. The experimental group received intraperitoneal injection of minocycline 22.5mg/kg while the control group received saline 10ml/kg every day from the postnatal day 1 (P1) . Mice were sacrificed at P1, P7, P14, P21 and P28 respectively. Eyeballs were enucleated to carry out histology observation and apoptosis cell detection. Meanwhile, to statistically analyze the number of retinal photoreceptor cells,the thickness of outer nuclear layer (ONL)and the number of apoptosis cells. Results （1）Photoreceptor cell began to apoptosis on P7, peaked on P14, and totally disappeared on P28. （2）No statistically significant differences were found of the number of photoreceptor cells and the thickness of ONL on P7 between the experimental group and the control group. (3) The number of photoreceptor cells and the thickness of ONL in the experimental were more than that in the control group at P14, P21, P28 respectively, the differences are statistically significant（Plt;0.05）. (4) The apoptotic cells on ONL were less in the experimental group than that in the control group on P7 and P14 respectively, the difference are statistically significant（Plt;0.05）. Conclusions Minocycline appears to protect photoreceptor cell from apoptosis in the early stage of the retinal degeneration mice, but it may not completely prevent RP from occurrence.
Objective Lots of metal ions accumulation and over-expression of receptor activator of NF-κB l igand (RANKL) around the prosthesis could be found in revision of total hip arthroplasty. To investigate the relationship between metal ions and aseptic loosening by observing the effects of Co2+ and Cr3+ ions on the expression of RANKL and osteoprotegerin(OPG) from osteoblast. Methods Osteoblasts were cultured in vitro at the density of 1 × 105 cells/mL, and were divided into 2 groups according to different culture solutions. In control group, osteoblasts were cultured with normal medium without CoCl2 and CrCl3. In experimental group, osteoblasts were cultured with the medium including CoCl2 (10 mg/ L) and CrCl3 (150 mg/L) solutions. The RT-PCR and ELISA methods were appl ied to detect the mRNA expression of RANKL and OPG and protein level at 24 and 48 hours after co-cultured, respectively. Results RT-PCR revealed that the mRNA expression of RANKL and OPG could be found in two groups at 24 and 48 hours after co-cultured, the expression was higher in the experimental group than in control group, especially the expression of RANKL, showing significant difference (P lt; 0.05). At 24 and 48 hours after co- cultured, the ratios of RANKL mRNA to OPG mRNA in the experimental group were 0.860 and 1.232, respectively, which were significantly higher than those in the control group (0.695 and 0.688,P lt; 0.05). ELISA revealed that the protein level of RANKL and OPG in experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion Co2+ and Cr3+ can stimulate the mRNA expressions of RANKL, OPG and secretion of those protein from osteoblasts, especially increase of the RANKL, which promotes the formation and activation of osteoblasts and the generation of aseptic loosening.