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 evaluate the effects and the molecular mechanism of Liuwei dihuang pills in preventing steroid-induced osteonecrosis of the femoral head (ONFH) so as to provide an expremental basis for preventing ONFH cl inically. Methods Thirty-six adult Kunming mice (weighing 40-50 g, 46 g on average) were randomly divided into three groups (n=12): group A (control group), group B (model group) and group C (prevention group). In groups B and C, ONFHmice models were produced by intraperitoneal injection of horse serum at first (10 mL/kg) and a second injection of horse serum intraperitoneally (5 mL/kg) and prednisolone intramuscularly [45 mL/(kg•day), for 5 days] 2 weeks later. At the same time, the mice in group C were given Liuwei dihuang pills intragastrically [2 g/(kg•day)] and were given normal sal ine [10 mL/(kg•day)] in group B. In group A, mice were given normal sal ine intramuscularly and intragastrically as controls. The animals were sacrificed 2, 4, and 8 weeks after first treatment with prednisone, and femoral heads and l ivers were harvested to do histopathology analysis and apoptosis assay. Results Other mice survived throughout the experiment period except two death at 7 and 11 days after second injection of horse serum intraperitoneally in group B and one death at 24 hours after second injection of horse serum intraperitoneally in group C. The appearance and shape of the femoral head and the surface of cartilages were all normal. The histological observation showed: normal structures of l iver and femoral head were seen in group A at each time point; swell ing l iver cells with small fat vacuole, unclear structure of hepatic cords and narrower sinus hepaticus were seen, the bone trabeculae of femoral head was thin, sparse and collapsed in some regions and the changes became more obvious with time in group B; group C had similar results to group A. The percentage of empty osteocyte lacunae was significantly higher in group B than in groups A and C (P lt; 0.01). The osteoprotegrin expression significantly decreased and the osteoprotegrin l igand expressionsignificantly increased in group B when compared with groups A and C (P lt; 0.01). Apoptosis analysis showed that the apoptosis index in group B was significantly higher than that in groups A and C (P lt; 0.01). Conclusion Liuwei dihuang pills can prevent steroid-induced ONFH by improving l ipid metabol ism, releiving bone lesion, and protecting against cell death.
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.
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.
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 investigate the role and mechanism of heat shock protein 60 (HSP60) in induction of murine skin allograft tolerance. Methods At the age of 8-12 weeks, inbred female BALB/C (H-2d) mice (n=45) and CBA/N (H-2k)mice (n=15) were used as transplantation donors and C57BL/6 (H-2b) mice (n=60) as recipients. Recipients C57BL/6 (H-2b) mice were randomized into 4 groups (n=15). In group A, 1 cm × 1 cm Wolfe-Krause skin graft was excised from the back of BALB/C (H-2d) mice and hypoderma was scraped off aseptically, and then transplanted to the back of C57BL/6 (H-2b)mice. The method of skin transplantation in the other 3 groups was the same as to group A. In group B, C57BL/6 (H-2b) mice were treated with imcompleted Freund’s adjuvant (IFA) administration into the back 2 weeks before transplantation of BALB/C (H-2d) mice skin. In group C, C57BL/6 (H-2b) mice were administered HSP60 emulsified in IFA into the back 2 weeks before transplantation of BALB/C (H-2d) mice skin. In group D, C57BL/6 (H-2b) mice were treated with HSP60 emulsified in IFA into the back and followed by skin transplantation of CBA/N (H-2k) mice 2 weeks later. The delayed type hypersensitivity was determined 7 days after transplantation. One-way mixed lymphocyte reaction, the concentration of cytokines in the mixed lymphocyte reaction culture supernatant was determined 7 days and 25 days after transplantation. The survival time of skin allograft was observed. Results The survival time of skin allograft in groups A, B, C and D was 12.4 ± 0.5, 11.6 ± 0.8, 29.3 ± 2.6 and 27.6 ± 2.1 days, respectively. There was significant difference between groups A, B and groups C, D (P﹤0.05), while there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). The counts of per minute impulse (cpm) of mixed lymphocyte reaction 7 days after transplantation in groups A, B, C and D was 12 836 ± 1 357, 11 876 ±1 265, 6 581 ± 573 and 6 843 ± 612, respectively. There was significant difference between groups A, B and group C and group D (P lt; 0.05), while there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). The cpm of mixed lymphocyte reaction at 25 days after transplantation in group A, B, C and D was 13 286 ±1 498, 12 960 ± 1 376, 11 936 ± 1 265 and 12 374 ± 1269, respectively. There was no significant difference among 4 groups (P gt;0.05).The concentration of IL-10 in the mixed lymphocyte reaction culture supernatant in groups C, D were higher than that in groups A, B, and IL-2 and IFN-γ were lower than that in groups A, B 7 days after transplantation (P lt; 0.05), while there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). There was no significant difference in cytokines among the 4 groups 25 days after transplantation (P gt; 0.05). The delayed type hypersensitivity in groups A, B, C and D 7 days after transplantation was 0.84 ± 0.09, 0.81 ± 0.07, 0.43 ± 0.05 and 0.46 ± 0.03 mm, respectively. There was significant differences between groups A, B and groups C, D (P lt; 0.05). While there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). Conclusion HSP60 may play a role in induction and maintenance of murine skin allograft tolerance.
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 observe the preventing effect of intraocular injection of Bevacizumab (Avastin) to retinal microvascular proliferation in non-obese diabetes mice. Methods In the study, thirty non-obese diabetes mice (NOD mice) were selected. The left eyes of mice were selected as treatment group with 1mu;l A vastin (25mg/1ml) injected, and right eyes were selected as control group with 1 mu;l saline injected. One week, one month, two months after injection, ten mice were selected randomly, and then enucleated two eyes, in which the retinal microvascular endothelial cells ultrastructure and immunohistochemistry of retinal CD34 and VEGF, were observed and measured. The differences of dense of positive sta ining between two groups were compared by digital image analysis. Results The positive expression of VEGF and CD34 were brown staining, and the positive staining of CD34 located in vascular endothelial cells. There was statistically significant difference in VEGF expression between two groups in 1 week and 1 month after injection（t=21.6, t=13.5; P＜0.01), and no statistically significant difference in 2 months after injection （t=0.9, P＞0.05）. There was statistically significant difference in CD34 expression between two groups in 1 month and 2 months af ter injection（t=3.2, P＜0.01; t=2.7, P＜0.05） and no statistically significant difference in 1 week after injection（t=1.3, P＞0.05）. In every time point after injection, there was no obvious change in the microstructure of retinal vascular endothelial cells. Conclusion Intraocular injection of Avastin could prevent the abnormal proliferation of retinal microvascular in NOD mice. （Chin J Ocul Fundus Dis，2008，24：180-183）
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)
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)