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)
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 investigate the expression and significance of Krüppel-like factor 4 ( KLF4) in the lung tissues of mice with bleomycin-induced pulmonary fibrosis.Methods C57BL/6 mice were randomly divided into a control group and a BLM group. The mice in the BLM group were given a single intratracheal injection of bleomycin ( 2.5 mg/kg) , while those in the control group were injected with isodose physiological saline. The mice were sacrificed at the 12h and on the day 1, 2, 3, 7, 14 and 28, then HE stain and Masson’s trichrome stain were used to detect the architecture of alveolar and the deposition of cellularity and collagen. Real time-polymerase chain reaction ( RT-PCR ) and immunohistochemical technology were performed to investigate the expression of KLF4. Results In the bleomycin-induced pulmonary fibrosis, acute inflammation was observed on the day 1, 2 and 3, the inflammation was exacerbated and the collagen deposition began to be observed on the day 7, the architecture of alveolar was destroyed and the collagen deposition was more obvious on the day 14, while the alveolar structure was nearly recovered to normal, and the inflammation and collagen deposition were attenuated on the day 28. The expression of KLF4 mRNA increased from the day 1, then decreased, arrived at the minimumon the day 3, and then gradually increased until the day 28. The trend of KLF4 protein expression showed roughly the same as the KLF4 mRNA level, which started to increase on the day 1, then decreased, arrived at the minimum on the day 3,then gradually increased until the day 14 and then decreased again. Conclusion The expressions of KLF4 mRNA and protein are dynamically changed in the process of experimental pulmonary fibrosis, suggesting KLF4 may contribute to the pathogenesis of pulmonary fibrosis.
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 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 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 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.
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）
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.
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.