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 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)
Objective To construct specifically expressed vascular endothelial growth factor (VEGF)165 gene in retina. Methods Rho promoter, specifically expressed in retina, was amplified by polymerase chain reaction (PCR) from the genomic DNA of a BLAB/C rat, then it was cut with restriction enzymes and cloned into the plasmid pcDNA3.1+-VEGF165 to form recombinant plasmid pcDNA3.1+-rho-VEGF165. The correct recombinant plasmid pcDNA3.1+-rho-VEGF165 was identified by restriction enzymes and PCR, and was transferred by jetPEI into cultured human navel vein endothelial cells and human retinal pigment epithelial (RPE) cells. The expression of VEGF protein in human navel vein endothelial and RPE cells was detected by immunocytochemical staining and protraction of the growth curve of the cells. Results In human RPE cells, the expression of VEGF protein was more in recombinant plasmidpcDNA3.1+-rho-VEGF165 than that in plasmidpcDNA3.1+-rho-VEGF165 ; in human navel vein endothelial cells, no obvious difference of the expression of VEGF protein between recombinant plasmid pcDNA3.1+-rho-VEGF165 and plasmid pcDNA3.1+-rho-VEGF165 was found. Conclusions The construction of pcDNA3.1+-rho-VEGF165 carrier may provide the basic material for the study of the nosogenesis of VEGF in retinal neovascularization, and establish the foundation to set up the model of transgenic mice with VEGF specific expressing in retina. (Chin J Ocul Fundus Dis, 2005,21:106-108)
Objective Isoflurane has an acute preconditioning effectiveness against ischemia in kidney, but this beneficial effectiveness can only last for 2-3 hours. To investigate whether isoflurane produces delayed preconditioningagainst renal ischemia/reperfusion (I/R) injury, and whether this process is mediated by hypoxia inducible factor 1α(HIF- 1α). Methods A total of 52 male C57BL/6 mice were randomly assigned to 4 groups (n=13 in each group): the controlgroup (group A), PBS/isoflurane treated group (group B), scrambled small interference RNA (siRNA)/isoflurane treated group (group C), and HIF-1α siRNA/isoflurane treated group (group D). In groups C and D, 1 mL RNase-free PBS containing 50 μg scrambled siRNA or HIF-1α siRNA was administered via tail vein 24 hours before gas exposure, respectively. Equivalent RNasefree PBS was given in groups A and B. Then the mice in groups B, C, and D were exposed to 1.5% isoflurne and 25%O2 for 2 hours; while the mice in group A received 25%O2 for 2 hours. After 24 hours, 5 mice in each group were sacrificed to assesse the expressions of HIF-1α and erythropoietin (EPO) in renal cortex by Western blot. Renal I/R injury was induced with bilateral renal pedicle occlusion for 25 minutes followed by 24 hours reperfusion on the other 8 mice. At the end of reperfusion, the serum creatinine (SCr), the blood urea nitrogen (BUN), and the histological grading were measured. Results The expressions of HIF-1α and EPO in groups B and C were significantly higher than those in group A (P lt; 0.01). The concentrations of SCr and BUN in groups B and C were significantly lower than those in group A, as well as the scores of tubules (P lt; 0.01), and the injury of kidney was amel iorated noticeably in groups B and C. The expressions of HIF-1α and the concentrations of SCr and BUN in group D were significantly lower than those in group A (P lt; 0.01). Compared with groups B and C, the expression of HIF- 1α and EPO in group D decreased markedly (P lt; 0.01), the concentrations of SCr and BUN were increased obviously, as well asthe scores of tubules (P lt; 0.01), and the renal injury was aggratived significantly. Conclusion Isoflurane produces delayed preconditioning against renal I/R injury, and this beneficial effectiveness may be mediated by HIF-1α.
Objective To observe the immune responses of T helper cells 17 ( Th17) to respiratory syncytial virus ( RSV) infection induced lung inflammation in mice, and explore its roles on the host immune responses to RSV.Methods Female BALB/ c mice aged 3 to 5 weeks were randomly divided into a RSV group ( n=18) and a control group ( n = 12) . The mice were intranasally administrated by a 107.5 50% tissue culture infective dose ( TCID50) of RSV in 0.1 mL of culture medium. Sterile medium ( 0.1 mL/ mouse) was used as control. After infected on 1st , 4th, 8th day, the mice were sacrificed, and specimens from the lungs and lymph nodes were collected. The lung sections were stained by hematoxylin-eosin to observe the changes of lung inflammation after RSV infection. IL-17A, IL-17F and IL-23p19 mRNA expressions in the lung tissue were determined by real-time PCR. The frequencies of Th17 subsets in hilar lymph node were analyzed by flow cytometry. Results On 4th day after RSV infection, a typical lung interstitial inflammation was observed. However, this inflammation was alleviated on 8th day after RSV infection. The viral load in the lung tissue on 4th day after RSV infection were 9.208 ±0.548, which was the highest among all RSV subgroups ( P lt;0.001) . IL-23p19 and IL-17A cytokine expressions in the lung tissue were significantly increased on 4th day and 8th day after RSV infection compared with control groups ( P lt;0.01) , and the peak was on 4th day. However, IL-17F mRNA expression in the lung tissue on different day after RSV infection had no significant difference compared with the control group ( P gt;0.05) . The frequencies of Th17 subsets in hilar lymph node on 4th day and 8th day after RSV infection were ( 0.37 ±0.043) % and ( 0.853 ±0.048) % respectively, which were higher than those in control groups ( P lt;0.05) . The frequencies of Th17 on 8th day after RSV infection were significantly higher than that on 4th day after RSV infection ( P lt; 0.01) . Conclusions The expression of IL-17A in the lung tissue is increased and the level of Th17 cells in hilar lymph nodes is also elevated in the lung infected by RSV, which indicates that Th17 cells might be involved in host antiviral immune.
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 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.
ObjectiveTo determine the signal pathway of specifically expressed oncostatin M(OSM) in lens inducing retinal degeneration in transgenic mice.MethodsA sequence-truncated OSM cDNA (661 bp) of mice was linked to αA-crytallin promoter, and was micro-injected into unicellular embryo to set up the model of transgenic mice. Reversal transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of gp130/OSMRβ receptor in the retinae of OSM transgenic and non-transgenic mice. Rabbit anti-phosphorylated STAT-3 antibody was used to detect the protein expression of phosphorylated STAT-3，and mouse anti-cytochrome C antibody was used to detect the distributing of cytochrome C in retinae. ResultsExpression of gp130/OSMRβmRNA was found in retina of non-transgenic mice. At the 17.5th day in the embryonic stage, significant accumulation of the phosphorylated STAT-3 was detected in the retinal nucleolus in OSM transgenic retina. At the first day after birth, intensive staining of cytochrome C in OSM transgenic retina was found. Conclusionsspecifically expressed OSM in lens may act on gp130/OSMRβ receptor in retinae, activate STAT-3, and cause the release of cytochrome C from mitochondria, which eventually induces widespread retinal degeneration.(Chin J Ocul Fundus Dis, 2005,21:167-169)
ObjectiveTo investigate the effect of electroacupuncture on the apoptosis of hippocampal neurons in C57BL/6J mice with status epilepticus by observing the changes of hippocampal subtle neuron pathology and apoptosis.MethodsMale C57BL/6J mice were used to prepare epileptic status models of lithium-pilocarpine mice, and then 7-day electroacupuncture stimulation (Baihui, Fengfu) were given to the mice model. Open field experiment and new object recognition experiment were performed to observe the changes of cognitive abilities. The pathological changes of hippocampal neurons were detected by HE staining. Hippocampal apoptosis protein (Caspase-3) and microtubule-associated protein (MAP-2) were detected by immunohistochemistry. Effect of electroacupuncture on apoptosis of hippocampal neurons in C57BL/6J mice with status epilepticus were recorded.Results① Compared with the control group, the vertical movement, modification times, and number of crossings of the model group all decreased significantly (P<0.000 1,P<0.000 1,P<0.000 1), and their cognitive ability decreased significantly (P<0.01). Compared with the model group, vertical movements, modification times, and number of crossings were increased in the electroacupuncture (EA) group (P<0.01,P<0.05,P<0.05), and the cognitive ability of new objects was increased (P<0.01). ② HE staining showed that the model group had significant damage to the hippocampal neurons of mice, and the cells swelled, nuclear collapsed and vacuoles appeared. In the EA group, the injury of hippocampal neurons was alleviated, and cell edema and vacuolization were alleviated. ③ Immunohistochemistry showed that compared with the control group, the IOD of the Caspase-3 positive cells in the hippocampus of the model group increased significantly (P<0.000 1), and the IOD of the MAP-2 positive cells decreased significantly (P<0.01); Compared with the electroacupuncture, the IOD of the Caspase-3 positive cells in the hippocampus of the mice decreased (P<0.05), and the IOD of the MAP-2 positive cells increased (P<0.05).ConclusionsElectroacupuncture can improve the pathological changes of hippocampal neurons in C57BL/6J mice with status epilepticus, promote cytoskeletal repair, reduce neuronal apoptosis in hippocampus, and antagonize the damage of hippocampal neurons induced by status epilepticus.
ObjectiveTo establish the co-culture model of mice's early embryo and tumor cells in Vitro to observe the embryonic development and biologic behavior of tumor cells in the same microenvironment and discuss their interaction. MethodsWe acquired 2-cell embryos from mice, and then co-cultured them with tumor cell lines of mice in Vitro. We observed the development of embryos in Vitro and the rates of 4-cell embryos, morula and blastocyst formation. The transwell chamber was used for culture. Methylthiazolyldiphenyl-tetrazolium method was used to test the proliferative activity of tumor cells, while the flow cytometry was used to test its apoptosis. The interaction of co-cultured embryos and tumor cells was analyzed by propidium iodide staining and immunohistochemical technique. ResultsThe co-cultured 2-cell embryos could continue surviving and developing. The rates of 4-cell embryos, morula and blastocyst formation increased significantly in the co-cultured group (P<0.05). There was no significant difference in the proliferative activity and apoptosis of tumor cells between the co-cultured group and the control group (P>0.05). Tumor-free ring formed between the trophoblast and tumor cells. We could observe tumor cells stacked around the tumor-free ring. However, no difference in expression of proliferating cell nuclear antigen and B-cell lymphoma leukemia-2 was observed in tumor cells stacking around the tumor-free ring compared with those elsewhere. ConclusionThe development of 2-cell embryos is enhanced in the co-culture model. The proliferative activity and apoptosis of tumor cells are not affected in this model. A tumor-free ring can form between trophoblast and tumor cells. However, the proliferative activity of tumor cells is not affected by this ring.