Conventional methods for harvesting, culturing and purifying olfactory ensheathing cells are complicated, time-consuming, and poorly reproducible. Olfactory bulbs were detached from adult Sprague Dawley rats and olfac...Conventional methods for harvesting, culturing and purifying olfactory ensheathing cells are complicated, time-consuming, and poorly reproducible. Olfactory bulbs were detached from adult Sprague Dawley rats and olfactory ensheathing cells were isolated using shearing, dispersion processes. After the primary cultures reached confluence, the cells were purified using a three-step process. The olfactory ensheathing cells attached and grew rapidly. The purity of the olfactory ensheathing cells increased following the three purification steps, eventually exceeding 95%. These cells could be maintained for an extended period time in culture. This simple, inexpensive, reproducible method of harvesting, culturing and purifying olfactory ensheathing cells shortens the culture cycle and provides sufficient olfactory ensheathing cells of controllable purity.展开更多
Cells have intrinsic mechanisms for cleaning harmful oxidants represented mainly by reactive oxygen species (ROS). Despite the antioxidant defense, ROS can cause serious damage to the retina that with age leads to var...Cells have intrinsic mechanisms for cleaning harmful oxidants represented mainly by reactive oxygen species (ROS). Despite the antioxidant defense, ROS can cause serious damage to the retina that with age leads to various eye diseases and even blindness. Among numerous cell sites of ROS generation, mitochondrial electron transport is of crucial importance. Recently, for the purpose of cleaning ROS in the mitochondrial matrix, powerful mitochondria- targeted antioxidant “SkQ1” has been invented. We studied SkQ1 effects upon tissues of rat posterior eye cup that consisted: retinal pigment epithelium (RPE) ? choroidal coat ? scleral coat. The eye cups were isolated from the eyes of adult albino rats and cultivated in rotary tissue culture system in the presence of 20 nM SkQ1 or without this compound. After 7 days - 1 month in vitro eye cup samples were studied by immunohistochemistry, routine histology, morphometry, and digital image analysis. We have found that under chosen, “in vitro like in vivo” conditions 20 nM SkQ1 effectively reduced cell death in RPE and choroid, protected RPE from disintegration caused by cell phenotypic transformation and withdrawal from the layer, suppressed transmigration of choroidal coat cells. In the ex vivo model we used degenerative processes were more pronounced in the eye cup center where SkQ1 effect was most vivid. All this give us hopes for effectiveness of SkQ1 treatment of retinal central part that is very susceptible to light-induced over-oxidation injury and mostly suffering in many age-related diseases, AMD, in particular.展开更多
BACKGROUND： Pseudomonas aeruginosa mannose-sensitive hemagglutinin （PA-MSHA） parenteral injection is used as a broad-spectrum immunomodulator. It remains unclear whether PA-MSHA exhibits inhibitory effects on tumor...BACKGROUND： Pseudomonas aeruginosa mannose-sensitive hemagglutinin （PA-MSHA） parenteral injection is used as a broad-spectrum immunomodulator. It remains unclear whether PA-MSHA exhibits inhibitory effects on tumor cell growth. OBJECTIVE： To investigate inhibitory mechanisms of PA-MSHA-induced proliferation in rat C6 glioma cells in vitro. DESIGN, TIME AND SETTING： Comparative observation and in vitro experiments were performed at the Key Laboratory of Natural Medicine, Kunming Medical College, China from July 2008 to April 2009. MATERIALS： Rat C6 glioma cell line （Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China） and PA-MSHA parenteral injection （Beijing Wanteer Bio-Pharmaceutical, China） were used in the present study. METHODS： Rat C6 glioma cells in logarithmic growth phase were harvested in vitro. Adherent monolayer cells were respectively treated with PA-MSHA at final colony-forming units （cfu） of 1 ×10^8 cfu/mL, 2 × 10^8 cfu/mL, 4 × 10^8 cfu/mL, 6 × 10^8 cfu/mL, and 8 ×10^8 cfu/mL following 24 hours of conventional culture. MAIN OUTCOME MEASURES： MTT colorimetric assay was utilized to determine the inhibitory rate of C6 glioma cells following treatment with various concentrations of PA-MSHA at different times. Cell apoptosis was detected by fluorescent microscopy following Hoechst 33258 staining. Flow cytometry was used to measure PA-MSHA effects on C6 cell cycle. RESULTS： Inhibitory rate of C6 glioma cells increased with prolonged time and increased dose. Hoechst 33258 staining revealed obvious morphological changes in apoptotic C6 glioma cells. Flow cytometry revealed hypodiploid peaks, Le., apoptotic peak, and the apoptotic rate in cells during S-phase significantly increased with increased concentrations in the experimental groups. CONCLUSION： With in vitro experiments, PA-MSHA preparations inhibited C6 glioma cell proliferation in a time- and dose-dependent manner. These mechanisms are likely associated with cell apoptosis induction and inhibition of the S phase.展开更多
During life human eye is constantly exposed to sunlight and artificial light, the sources of reactive oxygen species (ROS)—the main cause of age-related eye pathology. A novel mitochondria-targeted antioxidant SkQ1 h...During life human eye is constantly exposed to sunlight and artificial light, the sources of reactive oxygen species (ROS)—the main cause of age-related eye pathology. A novel mitochondria-targeted antioxidant SkQ1 has recently been invented to reduce mitochondrial ROS by cleaning the mitochondria matrix, “the dirtiest place in the cell” in respect of ROS production and accumulation. Earlier we studied SkQ1 effects upon retinal pigment epithelium and choroid in the rat eye posterior cups exposed to long-term 3D organotypic culturing. It was found that under in vitro conditions 20 nM SkQ1 effectively reduced cell death in retinal pigment epithelium and choroid and protected the tissues from disintegration and cell withdrawal. In the present study we used same ex vivo conditions to examine the effect of SkQ1 upon the rat neural retina kept in the content of the posterior eye cup. Eye cups were isolated and cultured in vitro during 7, 14, and 30 days under rotation in the presence and absence of 20 nM SkQ1 in the culture medium. Serial sections of cultivated eye cups were subjected to histology, computer morphometry and immunohistochemistry. Obtained results show that SkQ1 operates as a strong protective agent, preventing neuronal cell death and other degenerative processes in the neural retina. Cell rescue by SkQ1 was more vivid in the central part of the retina than at the periphery. That, in turn, suggests SkQ1 effectiveness in treatment of some age-related eye diseases when central part of the retina, including macula, is most susceptible to degeneration.展开更多
Methoxyl methyl ether isoamylene quercetin （MIAQ） is one of the newly synthesized quercetin derivatives. The present study investigated the effect of MIAQ on rat aorta endothelial cells （RAECs） injured by hydrogen...Methoxyl methyl ether isoamylene quercetin （MIAQ） is one of the newly synthesized quercetin derivatives. The present study investigated the effect of MIAQ on rat aorta endothelial cells （RAECs） injured by hydrogen peroxide （H2O2）, as well as the potential mechanisms. We observed that MIAQ at 2.5-10μmol/L significantly enhanced the viability of injured RAECs, and the effect was more potent than quercetin and ct-tocopherol. However, M1AQ at the same concentration failed to show anti-oxidant activity in a cell-free system. In H2O2-injured endothelial cells treated with MIAQ （5-10μmol/L）, the level of nitric oxide （NO） and malondialdehyde was decreased, and the activities of superoxide dismutase and glutathione peroxidase was enhanced. In addition, RAECs treated with MIAQ （2.5-10 μmol/L） exhibited significant inhibiting apoptosis. In conclusion, MIAQ had protective effect on RAECs, possibly through increasing NO production and antioxidases activities, as well as inhibiting apoptosis. These findings suggest that MIAQ is possibly beneficial in the prevention of atherosclerosis and other diseases related to endothelial injury.展开更多
目的探讨沉默信息调节因子2相关酶1(silent mating type information regulation 2 homolog-1,SIRT1)抑制剂EX-527与激动剂SRT1720对老化内皮细胞成血管能力的影响。方法采用体外培养大鼠原代主动脉内皮细胞衰老技术,通过培养细胞的自...目的探讨沉默信息调节因子2相关酶1(silent mating type information regulation 2 homolog-1,SIRT1)抑制剂EX-527与激动剂SRT1720对老化内皮细胞成血管能力的影响。方法采用体外培养大鼠原代主动脉内皮细胞衰老技术,通过培养细胞的自然倍增建立大鼠主动脉内皮细胞(rat aorta endothelial cell,RAEC)衰老的细胞模型。内皮细胞的衰老用SA-β-Gal染色进行鉴定。衰老细胞模型建立后,对衰老的RAEC分别给予SIRT1抑制剂EX527和SIRT1激动剂SRT1720干预。运用Ki-67免疫荧光染色检测内皮细胞增殖,采用基质胶内皮管形成实验检测内皮细胞内皮管形成,应用蛋白印迹技术检测内皮细胞eNOS表达。结果SIRT1激动剂抑制老化RAEC增殖能力、内皮管形成能力和eNOS表达的降低,而SIRT1抑制剂使老化RAEC的增殖能力、内皮管形成能力和eNOS表达降低。结论SIRT1促进RAEC的增殖和内皮管形成,并增强RAEC中eNOS的表达;SIRT1改善衰老内皮细胞受损的成血管能力可能与其抑制衰老RAEC eNOS表达的下调有关。展开更多
基金Research Innovation Program for College Graduates of Jiangsu Province, No ZY320717General Scientific Research of Health Department of Jiangsu Province, No H200920
文摘Conventional methods for harvesting, culturing and purifying olfactory ensheathing cells are complicated, time-consuming, and poorly reproducible. Olfactory bulbs were detached from adult Sprague Dawley rats and olfactory ensheathing cells were isolated using shearing, dispersion processes. After the primary cultures reached confluence, the cells were purified using a three-step process. The olfactory ensheathing cells attached and grew rapidly. The purity of the olfactory ensheathing cells increased following the three purification steps, eventually exceeding 95%. These cells could be maintained for an extended period time in culture. This simple, inexpensive, reproducible method of harvesting, culturing and purifying olfactory ensheathing cells shortens the culture cycle and provides sufficient olfactory ensheathing cells of controllable purity.
文摘Cells have intrinsic mechanisms for cleaning harmful oxidants represented mainly by reactive oxygen species (ROS). Despite the antioxidant defense, ROS can cause serious damage to the retina that with age leads to various eye diseases and even blindness. Among numerous cell sites of ROS generation, mitochondrial electron transport is of crucial importance. Recently, for the purpose of cleaning ROS in the mitochondrial matrix, powerful mitochondria- targeted antioxidant “SkQ1” has been invented. We studied SkQ1 effects upon tissues of rat posterior eye cup that consisted: retinal pigment epithelium (RPE) ? choroidal coat ? scleral coat. The eye cups were isolated from the eyes of adult albino rats and cultivated in rotary tissue culture system in the presence of 20 nM SkQ1 or without this compound. After 7 days - 1 month in vitro eye cup samples were studied by immunohistochemistry, routine histology, morphometry, and digital image analysis. We have found that under chosen, “in vitro like in vivo” conditions 20 nM SkQ1 effectively reduced cell death in RPE and choroid, protected RPE from disintegration caused by cell phenotypic transformation and withdrawal from the layer, suppressed transmigration of choroidal coat cells. In the ex vivo model we used degenerative processes were more pronounced in the eye cup center where SkQ1 effect was most vivid. All this give us hopes for effectiveness of SkQ1 treatment of retinal central part that is very susceptible to light-induced over-oxidation injury and mostly suffering in many age-related diseases, AMD, in particular.
文摘BACKGROUND： Pseudomonas aeruginosa mannose-sensitive hemagglutinin （PA-MSHA） parenteral injection is used as a broad-spectrum immunomodulator. It remains unclear whether PA-MSHA exhibits inhibitory effects on tumor cell growth. OBJECTIVE： To investigate inhibitory mechanisms of PA-MSHA-induced proliferation in rat C6 glioma cells in vitro. DESIGN, TIME AND SETTING： Comparative observation and in vitro experiments were performed at the Key Laboratory of Natural Medicine, Kunming Medical College, China from July 2008 to April 2009. MATERIALS： Rat C6 glioma cell line （Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China） and PA-MSHA parenteral injection （Beijing Wanteer Bio-Pharmaceutical, China） were used in the present study. METHODS： Rat C6 glioma cells in logarithmic growth phase were harvested in vitro. Adherent monolayer cells were respectively treated with PA-MSHA at final colony-forming units （cfu） of 1 ×10^8 cfu/mL, 2 × 10^8 cfu/mL, 4 × 10^8 cfu/mL, 6 × 10^8 cfu/mL, and 8 ×10^8 cfu/mL following 24 hours of conventional culture. MAIN OUTCOME MEASURES： MTT colorimetric assay was utilized to determine the inhibitory rate of C6 glioma cells following treatment with various concentrations of PA-MSHA at different times. Cell apoptosis was detected by fluorescent microscopy following Hoechst 33258 staining. Flow cytometry was used to measure PA-MSHA effects on C6 cell cycle. RESULTS： Inhibitory rate of C6 glioma cells increased with prolonged time and increased dose. Hoechst 33258 staining revealed obvious morphological changes in apoptotic C6 glioma cells. Flow cytometry revealed hypodiploid peaks, Le., apoptotic peak, and the apoptotic rate in cells during S-phase significantly increased with increased concentrations in the experimental groups. CONCLUSION： With in vitro experiments, PA-MSHA preparations inhibited C6 glioma cell proliferation in a time- and dose-dependent manner. These mechanisms are likely associated with cell apoptosis induction and inhibition of the S phase.
文摘During life human eye is constantly exposed to sunlight and artificial light, the sources of reactive oxygen species (ROS)—the main cause of age-related eye pathology. A novel mitochondria-targeted antioxidant SkQ1 has recently been invented to reduce mitochondrial ROS by cleaning the mitochondria matrix, “the dirtiest place in the cell” in respect of ROS production and accumulation. Earlier we studied SkQ1 effects upon retinal pigment epithelium and choroid in the rat eye posterior cups exposed to long-term 3D organotypic culturing. It was found that under in vitro conditions 20 nM SkQ1 effectively reduced cell death in retinal pigment epithelium and choroid and protected the tissues from disintegration and cell withdrawal. In the present study we used same ex vivo conditions to examine the effect of SkQ1 upon the rat neural retina kept in the content of the posterior eye cup. Eye cups were isolated and cultured in vitro during 7, 14, and 30 days under rotation in the presence and absence of 20 nM SkQ1 in the culture medium. Serial sections of cultivated eye cups were subjected to histology, computer morphometry and immunohistochemistry. Obtained results show that SkQ1 operates as a strong protective agent, preventing neuronal cell death and other degenerative processes in the neural retina. Cell rescue by SkQ1 was more vivid in the central part of the retina than at the periphery. That, in turn, suggests SkQ1 effectiveness in treatment of some age-related eye diseases when central part of the retina, including macula, is most susceptible to degeneration.
基金National Natural Science Foundation of China (Grant No. 61071002)
文摘Methoxyl methyl ether isoamylene quercetin （MIAQ） is one of the newly synthesized quercetin derivatives. The present study investigated the effect of MIAQ on rat aorta endothelial cells （RAECs） injured by hydrogen peroxide （H2O2）, as well as the potential mechanisms. We observed that MIAQ at 2.5-10μmol/L significantly enhanced the viability of injured RAECs, and the effect was more potent than quercetin and ct-tocopherol. However, M1AQ at the same concentration failed to show anti-oxidant activity in a cell-free system. In H2O2-injured endothelial cells treated with MIAQ （5-10μmol/L）, the level of nitric oxide （NO） and malondialdehyde was decreased, and the activities of superoxide dismutase and glutathione peroxidase was enhanced. In addition, RAECs treated with MIAQ （2.5-10 μmol/L） exhibited significant inhibiting apoptosis. In conclusion, MIAQ had protective effect on RAECs, possibly through increasing NO production and antioxidases activities, as well as inhibiting apoptosis. These findings suggest that MIAQ is possibly beneficial in the prevention of atherosclerosis and other diseases related to endothelial injury.
