Objective To observe the regulation effect of transforming growth factor alpha (TGFalpha;) on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muuml;ller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7～10 day, and then cultured Muuml;ller cells according to literature. The 3～4 generation cultured cells of the same primary cell were divided into two groups at random: ① TGFalpha; group: maintained in different concentrations of TGFalpha; as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;② Control group: cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFalpha; on GLAST activity in Muuml;ller cells were observed by L-3H-glutamate uptake detection; the expression of GLAST mRNA in Muuml;ller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFalpha; concentration, both L3H glutamate uptake and GLAST mRNA expression were increased. The L-3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFalpha; on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant (Plt;0.05). Conclusion TGF-alpha; can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.
Paclitaxel (PTX)-loaded self-assembling nano-micelles (PTX/NMs) were prepared based on amphiphilic cholesterol-bearing γ-polyglutamic acid (γ-PGA-graft-CH). The properties of PTX/NMs in vitro and in vivo were investigated. The results indicated that PTX could be entrapped in γ-PGA-graft-CH NMs. PTX/NMs was characterized with a size of (343.5 ± 7.3) nm, drug loading content of 26.9% ± 0.8% and entrapment efficiency of 88.6% ± 1.7% at the optimized drug/carrier ratio of 1/10, and showed a pH-sensitive sustainable drug-release and less cytotoxicity in vitro. In vivo release and the pharmacokinetics study in mice showed that the elimination half-life (t1/2β) and area under curve (AUC) of PTX/NMs were significantly higher than those of PTX/polyoxyethylene castor oil (PTX/PCO), and less clearance (CL) of PTX/NMs was also observed. PTX/NMs were distributed higher in liver and tumor than PTX/PCO, and showed a good tumor-inhibiting activity in tumor-bearing mice. This study would lay a foundation on the potential application of γ-PGA-graft-CH NMs were the antitumor drug-delivery.
Objective To evaluate the inhibiting effect of adenosine on rat retinal ganglion cells (RGC) death induced by P2X7 and N-methyl-D-aspartate (NMDA) receptor. Methods (1) Long-Evan neonatal rats were back labeled with aminostilbamidine to identify RGC. The viability of RGC affected by P2X7 excitomotor BzATP (50 mu;mol/L), glutamate receptor excitomotor NMDA (100 mu;mol/L) and adenosine (300 mu;mol/L) was detected. (2) RGC from the retinae of unlabeled neonatal rats were cultured in vitro. After labeled with Fura-2 methyl acetate, an intracellular calcium indicator, the effect of BzATP, NMDA and adenosine on intracellular Ca2+ level was detected byCa2+ imaging system. Results Both BzATP (50 mu;mol/L) and NMDA(100 mu;mol/L) could kill about 30% of the RGC. Cell death was prevented by adenosine (300 mu;mol/L) with the cell viability increased from (68.9plusmn;2.3)% and (69.9plusmn;3.2)% to (91.2plusmn;3.5)% (P＜0.001) and (102.1plusmn;3.9)% (P＜0.001), respectively. BzATP (50 mu;mol/L) led to a large, sustained increase of intracellular Ca2+ concentration to (1183plusmn;109) nmol/L. After the adenosine intervened, Ca2+ concentration increased slightly to (314plusmn;64) nmol/L (P＜0.001). Conclusion Adenosine may prevent RGC death and increase of intracellular Ca2+ concentration from P2X7and NMDA receptor stimulation. (Chin J Ocul Fundus Dis, 2007, 23: 133-136)
Objective To investigate the effect of pigment epitheliumderived factor (PEDF）on the expression of glutamine synthetase in retinal Muuml;ller cells of diabetic rats.Methods Diabetic rats were induced with streptozotocin injection.Before and after injection of 10 mu;l (0.1 mu;g/mu;l) PEDF (experimental group) or 10 mu;l PBS (control group) into the vitreous cavities of diabetic rats respectively for 48 hours,the expressions of GS and IL-1beta; in retina were analyzed by immunohistochemistry and real time RTPCR techniques. After being treated with 100 ng/ml PEDF for 24 hours in high glucose conditions,the expressions of GS and IL-1beta; in cultured Muuml;ller cells were studied by western blot and real time RT-PCR techniques. Apoptosis was analyzed by flow cytometry after Annexin V fluorescein isothiocyanate/Propidium idoium (Annexin V-FITC/PI) staining.Results By immunohistochemistry (the protein level) and real time RT-PCR (the mRNA level),it was found that the expression of GS decreased and the expression of IL-1beta; increased obviously (real time RT-PCR：GS:t=4.23,P＜0.01；IL-1beta;:t=16.73,P＜0.01；immunohistochemistry：GS: t=5.13,P＜0.01；IL-1beta;:t=9.32,P＜0.01) in diabetic rats. After injection of 10 mu;l (0.1 mu;g/mu;l) PEDF into the vitreous cavities of diabetic rats for 48 hours,it was found that the expression of GS increased and the expression of IL-1beta; decreased significantly(RT-PCR GS:t=3.87,P＜0.01IL-1beta;:t=3.61,P＜0.05;immunohistochemistry:GS:t=3.32, P＜0.05；IL-1beta;: t=2.63,P＜0.05). Under high glucose conditions, 100 ng/ml PEDF induced decreasing expression of IL-1beta; and increasing expression of GS significantly (RT-PCR：GS: t=2.89, P＜0.05；IL-1beta;: t=3.37,P＜0.05；Western blot：GS:t=2.66,P＜0.05;IL-1beta;:t=3.23,P＜0.05).Apoptosis of Muuml;ller cells under high glucose conditions was inhibited significantly by the treatment with 100 nmol/ml PEDF (t=3.21,P＜0.05). Conclusions In diabetic rats,PEDF may decrease expression of IL-1beta; in rat retinal Muuml;ller cells, which may result in increasing expression of GS.To some degree,it inhibits possibly the death of retinal ganglion cells.
ObjectiveExplore the mechanism of action of Kruppel-like factor 4 (KLF4) in the oxidative damage model of hippocampal neurons in mice induced by glutamate. MethodsTo clarify the role of KLF4 and glutamate in the oxidative toxicity of epilepsy, the mouse hippocampal neuron cell line (HT22) was adopted, and a neuronal death excitotoxicity cell model was formed by induction with glutamate as the in vitro epilepsy experimental model. The expression level of KLF4 was detected by Real-Time PCR. HT22 cells were transfected with KLF4-specific siRNA, and the experiments were grouped as follows: Ctrl group, Glu group, Glu + siKLF4-1 group, and Glu + siKLF4-2 group. The cell viability of each group was detected by the CCK8 method. ResultsKLF4 was significantly increased in the epilepsy model of HT22 cells induced by glutamate, while downregulation of KLF4 improved the proliferation and viability of neurons in the epilepsy model of HT22 cells induced by glutamate. ConclusionIn the hippocampal neuron cells of epileptic mice, KLF4 is highly expressed. The downregulation of KLF4 improves the proliferation function and vitality of glutamate-induced HT22 cells, indicating that KLF4 may contribute to the occurrence and development of epilepsy by participating in the regulation of oxidative stress responses.
Objective To investigate the expression of induced heat shock protein (HSP) 70 in ratprime;s retinal neurons (RNs) and Muuml;ller cells, and evaluate the protective effect of HSP 70 on RNs injured with glucose deprivation and glutamate. Methods Ratprime;s RNs and Muuml;ller cells cultured in vitro were treated with heat shock (42℃ for 1 hour), and duration of the expression of HSP70 was detected by immunocytochemical techniques. Viability of the cells was measured by methyl thiazolyl tetrazolium (MTT) chromatometry after incitant toxic injury with glucose deprivation (0.56 mmol/L glucose for 6 hours) and glutamate (100 mu;mol/L for 6 hours). Simultaneously, the expression was interdicted by HSP70. Results Hypereffective expression of HSP70 was found in cultured RNs and Muuml;ller cells after heat shock. The viability of RNs pretreated by heat shock after injured with glucose deprivation and glutamate significantly increased which could be interdicted by HSP70 antibody. Conclusion Hypereffective expression of HSP 70 may be induced by heat shock, which enhances the ability of tolerance of RNs to the incitant toxic injury by glucose deprivation and exitotoxicity. (Chin J Ocul Fundus Dis, 2005,21:110-113)
ObjectiveTo observe the expression of glutamate (Glu) andγ-aminobutyric acid (GABA) in the retina of diabetic rats which were intervened later by insulin intensive therapy, and to investigate the mechanism of metabolic memory of hyperglycemia which induced the retina neuropathy in diabetic rats. Methods60 Brown Norway rats were randomly divided into normal control (NC) group, diabetes mellitus (DM) group (6 weeks at DM1, 12 weeks at DM2) and metabolic memory (MM) group, 15 rats in each group. Diabetes was induced by intraperitoneal injection of streptozocin. After 6 weeks, MM group was treated with insulin intensive therapy for 6 weeks. DM1 group was sacrificed at the end of 6 weeks and other groups were sacrificed at the end of 12 weeks. High performance liquid chromatography was used to detect the amount of Glu and GABA in the rat retina. Real-time polymerase chain reaction was applied to quantify the mRNA expressions of Glutamate decarboxylase (GAD). TdT mediated dUTP nick ending labelling was used to detect cell apoptosis. ResultsThe concentration of Glu (t=6.963), GABA (t=4.385) and the ratio of Glu/GABA (t=4.163) in MM group were significantly higher than DM1 group, but the concentration of Glu (t=3.411) and GABA (t=3.709) were significantly lower than DM2 group (P < 0.05). And there was no significant difference in the ratio of Glu/GABA between MM and DM2 groups (t=1.199, P > 0.05). The level of expressions of GAD mRNA in MM group was significantly lower than DM1 group (t=3.496, P < 0.05), but higher than DM2 group (t=8.613, P < 0.05). The number of nerve cells apoptosis in MM group was significantly higher than DM1 group (t=2.584, P < 0.05), but lower than DM2 group (t=3.531, P < 0.05). ConclusionsIntensive therapy later by insulin can partially reduce the content of Glu and GABA and the rate of nerve cells apoptosis, which cannot return to normal levels, and has no effect on the rise in the ratio of Glu/GABA caused by the hyperglycemia. The disorders of Glu and GABA may participate in the metabolic memory of hyperglycemia.
ObjectiveTo analyze the protective mechanism of spinal cord ischemia-reperfusion injury mediated by N-methyl-D-aspartate (NMDA) receptor.MethodsA total of 42 SD rats were randomly assigned to 4 groups: a non-blocking group (n=6), a saline group (n=12), a NMDA receptor blocker K-1024 (25 mg/kg) group (n=12) and a voltage-gated Ca2+ channel blocker nimodipine (0.5 mg/kg) group (n=12). The medications were injected intraperitoneally 30 min before ischemia. The neural function was evaluated. The neuronal histologic change of spinal cord lumbar region, the release of neurotransmitter amino acids and expression of spinal cord neuronal nitric oxide synthase (nNOS) were compared.ResultsAt 8 h after reperfusion, the behavioral score of the K-1024 group was 2.00±0.00 points, which was statistically different from those of the saline group (5.83±0.41 points) and the nimodipine group (5.00±1.00 points, P<0.05). Compared with the saline group and nimodipine group, K-1024 group had more normal motor neurons (P<0.05). There was no significant difference in glutamic acid concentration in each group at 10 min after ischemia (P=0.731). The nNOS protein expression in the K-1024 group was significantly down-regulated compared with the saline group (P<0.01). After 8 h of reperfusion, the expression of nNOS protein in the K-1024 group was significantly up-regulated compared with the saline group (P<0.05).ConclusionK-1024 plays a protective role in spinal cord ischemia by inhibiting NMDA receptor and down-regulating nNOS protein expression; during the reperfusion, K-1024 has a satisfactory protective effect on spinal cord function, structure and biological activity of nerve cells.