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Objective@#To investigate the mechanisms of Pyrroloquinoline quinone (PQQ) against oxidative stress induced apoptosis in Schwann cells (SCs).@*Methods@#SCs were cultured in vitro, identified by S-100 immunofluorence staining. SCs were divided into control group, H2O2 induced group, H2O2 + PQQ treated group. CCK-8 assay was used to detect cell proliferation. Apoptosis was detected by flow cytometry with Annecin V-FITC/PI staining, mitochondrial transmembrane potential was detected by flow cytometry with JC-1 labeled staining, cytochrome C (CytC), Bax and Caspase-9 protein levels was detected by Western blot analysis.@*Results@#In this study, the S-100 positive cells were more than 95%, cell proliferation was decreased in H2O2 induced SCs, apoptotic rate was increased, mitochondrial transmembrane potential was decreased, CytC, Bax and Caspase-9 protein levels were increased. After PQQ added, cell proliferation was increased, apoptotic rate decreased, mitochondrial transmembrane potential increased, CytC, Bax and Caspase-9 protein levels decreased.@*Conclusions@#PQQ protects SCs from oxidative induced apoptosis by inhibiting mitochondrial signaling pathway.
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Objective To investigate the impact of sphingosine kinase 1 (SPK1) modified adipose tissue-derived stromal cells(ADSC)on tissue engineered bone osteogenesis.Methods ADSC cells isolated from SD rat fat cells were divided into CON group and SPK1 group,and then the cells were respectively infected with 10 MOI CON and SPK1 lentivirus for 48 h.The infection efficiency was confirmed by using flow cytometry.Alizarin red and oil red O was used to stain the cells 14 days after ADSC infection,and osteogenic and adipogenic ability was evaluated by detecting A595nm and A490 nm.In the meantime,the activity change of alkaline phosphatase(ALP)was detected.The SD rat femoral defect model was created,and then after combining ADSC with β-TCP,the tissue engineered bone was pressed to the defect site.The repairment of bone defect was detected by X-ray in 4,6 weeks.After infection of CON and SPK1 virus,bone morphogenetic protein(BMP7)expression in ADSC of these two groups was detected.Results The infection efficiency of CON and SPK1 lentivirus was 94.4％ vs.94.9％ respectively by flow cytometry.The SPK1 protein expression level in CON group and SPK1 group was (0.73±0.10) vs.(1.29±0.17)(P＜0.05).The A value of CON and SPK1 group at 595 nm was (0.20±0.02) vs.(0.41±0.01) (P＜0.05),respectively.The A value of CON and SPK1 group at 490 nm was (0.72±0.01) vs.(0.51±0.02)(P＜0.05),respectively.The expression level of ALP in CON and SPK1 group was (1.42±-0.09) vs.(2.68±0.09) (P＜0.01),respectively.In the repairment of bone defect,high density tissue at rat bone defect was significantly larger in SPK1 group than in CON group in 4 weeks,and in 6 weeks,bone defect of SPK1 group was healed,but CON group still had defect.The expression level of BMP7 in CON and SPK1 group was (1.13±0.16) vs.(4.46±0.23)(P＜0.05),respectively,48 h after infection.Conclusion SPK1 modified ADSC has an enhanced osteogenesis ability in vitro and in vivo,which was related to the activation of BMP7.
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<p><b>OBJECTIVE</b>To investigate the effects of Pyrroloquinoline quinine (PQQ) on hydrogen peroxide-induced apoptosis of Schwann cells (SCs) and its mechanism.</p><p><b>METHODS</b>SCs were isolated and cultured in vitro, and identified by S-100 immunofluorescence staining. The cultured SCs were divided into control group, hydrogen peroxide-treated group, hydrogen peroxide and PQQ treated groups. The intracellular superoxide dismutase (SOD) and malondialdehyde (MDA) content was detected; the apoptotic rate of SCs induced by hydrogen peroxide was determined by flow cytometry assay. The Hoechst33342 staining was used to detect the nuclear fragmentation and apoptotic nuclear condensation of SCs; the Rhodamine123 staining was used to detect the changes of mitochondrial membrane potential in SCs, the Western blot analysis was used to detect the expression of Bcl-2 in hydrogen peroxide induced SCs.</p><p><b>RESULTS</b>The SOD activity was significantly decreased and MDA level was increased in H2O2 induced SCs (P < 0.05), after addition of PQQ, the SOD content increased and MDA content decreased (P < 0.05). Flow cytometry results showed that the early apoptotic rate was 58.8% in H2O2 induced SCs, which has significant difference compared with the control group (P < 0.05), after addition of 10, 50, 100 nmol/L PQQ, the apoptotic rates were reduced to 33.7%, 18.7%, 3.9% respectively, showing significantly different with injured group (P < 0.05). Hoechst 33342 staining showed that H2O2 induced SCs had typical morphological characteristics, such as uptake of nuclear chromatin, nuclear shrinkage, nuclear fragmentation phenomenon. The proportion of apoptotic cells after PQQ treatment reduced. Rhodamine staining results showed that the H2O2 induced mitochondrial membrane potential reduction in SCs, which was reversed by addition of PQQ. Western blot analysis showed that the expression of Bcl-2 was decreased in H2O2 induced SCs, while it increased significantly after addition of PQQ (P < 0.05).</p><p><b>CONCLUSION</b>PQQ has a protective effect on oxidative stress-induced apoptosis of SCs.</p>
Subject(s)
Humans , Apoptosis , Benzimidazoles , Cell Nucleus , DNA Fragmentation , Fluorescent Dyes , Hydrogen Peroxide , Pharmacology , Malondialdehyde , Metabolism , Oxidants , Pharmacology , Oxidative Stress , Pyrroles , Pharmacology , Quinine , Pharmacology , Quinolines , Pharmacology , Schwann Cells , Cell Biology , Superoxide Dismutase , MetabolismABSTRACT
Objective To study the effect of interleukin (IL)-1β on mitochondria of chondrocytes and reactive oxygen species.Methods Rat chondrocytes were isolated and cultured in vitro,10 ng/ml IL-1β was added for establishing model of osteoarthritis.Then,ratio of apoptosis was surveyed by Annexin V-FITC and PI flow-cytometry.After Hoechst 33342 dyeing,morphology of nucleus was observed by fluorescence microscope.After rhodamine-123 luorescent staining applied,change of mitochondria membrane potential was observed by confocal microscopy.We evaluated the ability of ATP synthesizing in mitochondria by luciferase reaction.Content of active oxygen was observed by confocal microscopy.T test was used for statistical analysis.Results IL-1β could obviously reduce the mitochondrial membrane potential of chondrocytes and its ability to synthesize ATP,and could promote the expression of reactive oxygen species in cells.The values were changed to (24±4) U,(4.1±0.8) pmol/106 cells and (89±7) U from (86±10) U,(13.3±3.0) pmol/106 cells and (17±3) U.The difference had statistical significance.Conclusion IL-1β can induce chondrocyte differentiation by destroying the mitochondrial membrane integrity of chondrocytes and promoting the expression of reactive oxygen species.It can also promote the articular cartilage degeneration.
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[Objective] To investigate the effects of pyrroloquinoline quinine (PQQ) on proliferation and expression of NF - kB of Schwann cells. [Methods] Schwann cells were cultured from sciatic nerves of SD rats in vitro. The Schwann cells were identified and purified by immunofluorescence of S-100 and Ara-C. Experimental group with was 100 nmol/L of PQQ and control group were set up. The expression of NF-kB in Schwann cells was determined by RT-PCR and Western blotting. [ Results] The expression of NF - kB were up - ragulated by PQQ in cultured Schwann cells (P<0.05).[Conclusion ] PQQ could promote the proliferation of Schwann cells and up-regulation of NF-rd3 play an important role in this process.
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, which will provide enough seed cells for peripheral nerve tissus engineering research.
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37 ℃, cell growth became poor, and clone formation decreased. At 39 ℃, cells could not adhere. Under incubator CO2 concentration between 3% and 8%, there was no significant difference in cell clone formation. CONCLUSION: The optimized culture condition of Schwann cells were 7.2 pH, 10% fetal bovine serum, 37 ℃ of temperature and 5% of CO2(v/v) of incubator.
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[Objective]To investigate the effects of Wnt/?-catenin signal pathway on Schwann cells proliferation promoted by pyrroloquinoline quinine (PQQ) and its molecular mechanisms.[Method]Schwann cells were cultured and purified in vitro. The purity was identified by S-100. PQQ of 10 nmol/L and 100 nmol/L were added into culture medium for 24 hours,respectively. Then the morphological changes promoted by PQQ were observed by inverted microscope. The expression of ?-catenin was detected by RT-PCR and Western blot in Schwann cells promoted by PQQ of different concentration for 72 hours.[Result]Morphological change was observed in Schwann cells treated by PQQ of 10 nmol/L and 100 nmol/L. The most obvious morphological changes took place in the Schwann cells treated by 100 nmol/L of PQQ,the RT-PCR and Western blot results showed that PQQ of 1-1000 nmol/L could up-regulate the expression of ?-catenin,especially when Schwann cells was treated by PQQ of 100 nmol/L(P
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[Objective] To investigate the effects of pyrroloquinoline quinine(PQQ)on proliferation and expression of NF-?B of Schwann cells.[Methods]Schwann cells were cultured from sciatic nerves of SD rats in vitro.The Schwann cells were identified and purified by immunofluorescence of S-100 and Ara-C.Experimental group with was 100 nmol/L of PQQ and control group were set up.The expression of NF-?B in Schwann cells was determined by RT-PCR and Western blotting.[Results]The expression of NF-?B were up-ragulated by PQQ in cultured Schwann cells(P
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Aim To investigate the feasibility of using arnnion allograftcombined with nerve growth factor to bridge peripheral nerve deficit.Methods 48 SD rats as models of sciatic nerve deficit were randomlyassigned into one of the following groups: autograft (Group A), allograftwith Cyclosporine A (CSA) 5 mg/(kg @ d) for 5 weeks (Group B), deficitbridged by amnion allograft combined with nerve growth factor (Group C) ,and allograft with no immune treatment (Group D) . 6 Wistar rats weresacrificed as the donors of the sciatic nerve allograft. Observation wascarried under light microscope and electron microscope examination at 12weeks post-operatively for morphological studying; The latency, ampli-tude, conduct velocity and negative area under the curve were recordedas electrophysiologic index; Maximum contractility of gastrocnemiusmuscle and thickness of myelin of the regenenative nerve were measured;Axons counting were also performed to evaluate the quality of regenera-tion. Results At 12 weeks, group A, B and C recovered better (Indexessuch as latency, amplitude, conduct velocity , negative area under thecurve, maximum contractility of gastrocnemius muscle, thickness of myelinand axons counting were compared by ANOVA test, F= 12.87, P ＜0.05; F=19.54, P ＜0.05; F=35.21, P ＜0.01; F=56.33, P ＜0.01. F=75.26, P ＜0.05; F=14.83, P ＜0.05; F=96.11, P ＜0.01), and no significant difference was found among these groups ( P ＞0.05). Conclusion For long distance peripheral nerve deficit, usingamnion allograft combined with nerve growth factor to bridge the deficit canfacilitate the regeneration of nerve and the recovery of function.
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Objective To investigate therapeutic effects of muscle sliding operation treating Volkmann ischemic contracture of the forearm. Methods 32 cases Volkmann ischemic contracture were classified two types: the single type and the complex type. Three approaches were employed to treat different types which were single muscle sliding operation, muscle sliding plus skeletal operation, muscle sliding operation plus neurolysis.Results 29 cases were followed up, which excellent and good was 93.7 percent.Conclusions Muscle sliding operation was effective management for Volkmann ischemic contracture, and the skeletal operation or neurolysis could managed for the complex type in the meantime.