全反式视黄酸对诱导为神经元样细胞的脐带间充质干细胞分化和凋亡的影响

Objective To evaluate the role of different concentration of all-trans retinoic acid (ATRA)on the morphology,proliferation and apoptosis in inducing umbilical cord mesenchymal stem cells (MSC) into neuron-like cells in vitro,and screen the optimal concentration of ATRA.Methods It was an experimental study.The third passage of MSC was placed in 24-well cell culture plates at a density of 1×104/well.After the adherent of cells,the medium was changed to DMEM/F-12 containing different concentration of ATRA(0.25 μmol/L,0.5 μmol/L,1.0 μmol/L,2.0 μmol/L,4.0 μmol/L)for 24 h respectively.The cells cultured without ATRA were taken as the coutrol group.After another 24 h,the morphologic changes of induced cells were observed by inverted microscope and cell proliferation,apoptosis of ATRA was analyzed using the MTT colorimetric assay.We take another control group and ATRA groups to detect the apoptotic and positive stained percentage of induced cells by Annexin V-FITC/PI combining flow cytometry.The optimal concentration of ATRA was determined by all the above-mentioned index.According to the nature of the material,analysis of variance (ANOVA) was employed for absorption value and apoptosis rate in different concentration of ATRA for 24 h,t test for further comparision between two groups.T-test were also used between the positive expression of induced neuron-like cells and the control group.Results Compared to the control group,ATRA at the concentration of 0.25 μmoL/L did not inhibit the proliferation of umbilical cord MSC obviously(t=0.72.1.32,P>0.05).Part of MSC wele floating instantly at the moment of adding ATRA of 4.0 μmoL/L and no adherent cells were observed after 24 h'culture.Exposed to ATRA at the concentration of≥1.0 μmol/L for 24 h,the proliferation of MSC were significantly inhibited,showing a dose-dependent manner(t=8.8,18.9,22.1;P<0.01).0.5 μmol/L of ATRA did not affect the proliferation of cells and its moephology remained normal;1.0 μmol/L of ATRA affected very few cells;but 2.0 μmoL/L of ATRA cultured for 24 h inhibited the proliferation of cells obviously than 1 h.and the cells increased in size and became flattened.Flow cytometry showed that the rate of apoptosis between the control group and≥1.0 μmol/L groups were significantly different(t=9.88,19.95,31.61;P<0.01).Conclusion In the process of inducing umbilical cord MSC into neuron-like cells,0.5 μmol/L ATRA was the optical concentration.≥1.0 μmol/L ATRA can inhibit the cell proliferation,increase the apoptosis of cells significantly and caused obvious damages.     

In vitro induction and differentiation of umbilical cord mesenchymal stem cells into neuron-like cells by all-trans retinoic acid

AIM: To determine the optimal concentration for inducing the differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) into neuron-like cells, although it is understood that all-trans retinoic acid (ATRA) regulates cell proliferation in the nervous system by modulating the balance between mitosis and apoptosis. METHODS: The abilities of ATRA to promote apoptosis as well as neural differentiation were assessed in cultured hUC-MSCs by morphological observation, MTT assay, annexin V-FITC/PI flow cytometry and immunocytochemistry. RESULTS: The data showed that low concentrations of ATRA (0.5 μmol, 0.25 μmol) had no effect on the number of cells. However, treatment with 1.0 μmol or 2.0 μmol ATRA induced a 24.16% and 52.67% reduction in cell number, respectively, compared with vehicle-treated cultures. Further, 4.0 μmol ATRA had a potent effect on cell number, with almost no adherent cells recovered after 24h. We further showed that 0.5 μmol ATRA caused these cells to express characteristic markers of neuronal progenitor cells. CONCLUSION: Taken together, we conclude that ATRA has a dose-dependent influence on the neural differentiation and apoptosis of hUC-MSCs. These findings have implications on the use of ATRA-differentiated hUC-MSCs for the study of neural degeneration diseases.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

Cells, cells, and more cells

A 64-year-old woman presented with bilateral optic nerve swelling, vitreous cells, and cerebrospinal fluid monocytic pleocytosis. A chest radiograph and computed tomography demonstrated a lesion in the left lung, which histologically was confirmed to be a small-cell lung carcinoma. The serum was positive for the anti-CV2 (anti-CRMP-5) antibody. Following treatment with chemoradiation the optic nerve swelling and vitritis resolved. The differential diagnosis of uveal-meningeal diseases is discussed and the pathophysiology and clinical manifestations of paraneoplastic syndromes reviewed.     

乳鼠视网膜细胞诱导骨髓间充质干细胞分化为视网膜神经节样细胞

目的 探讨乳鼠视网膜细胞对大鼠骨髓间充质干细胞(BMSCs)的诱导分化作用.方法 从出生8 d的大鼠骨髓中分离培养BMSCs,同时培养乳鼠视网膜神经细胞.在Transwell双层培养体系下,利用乳鼠视网膜细胞诱导BMSCs,诱导后细胞经RT-PCR及免疫荧光鉴定.结果 加入乳鼠视网膜细胞共培养第7 d有神经元样细胞形成,经nestin、NF、β-III Tubulin、Thy1.1行免疫组织化学、RT-PCR、免疫荧光鉴定,细胞呈阳性反应.结论 BMSCs在体外培养条件下,经过新生乳鼠视网膜细胞诱导,可以分化为视网膜神经节样细胞.     

Mesenchymal stem cells derived from human limbal niche cells

Purpose. We investigated whether human limbal niche cells generate mesenchymal stem cells. Methods. Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells. Results. Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α. Conclusions. In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.     

Amacrine cells,bipolar cells and ganglion cells of the cat retina: A Golgi study

Neuronal types contributing to the inner plexiform layer of the cat retina are described based primarily on light microscopy of Golgi-impregnated retinal whole-mounts. Cells have been characterized on morphological criteria that include dendritic branching patterns, dendritic tree sizes, cell body sizes and stratification of processes in the inner plexiform layer. Nine different types of bipolar cell, 22 different types of amacrine cell and 23 different types of ganglion cell can be distinguished using one or more of these morphological criteria. The significance of the different morphological types of cells is discussed, particularly in relationship to the functional bisublamination of the cat inner plexiform layer.     

兔羊膜成体干细胞培养及体外定向诱导神经细胞分化

目的 观察体外分离、培养的兔羊膜成体干细胞(ADSC)定向诱导神经细胞的分化结果.方法 新西兰雌兔羊膜分离、培养ADSC.实时定量聚合酶链反应(PCR)检测羊膜ADSC的纤连蛋白(Fibronectin)、巢蛋白(Nestin)、波形蛋白(Vimentin) mRNA的表达水平.单克隆形成实验证实羊膜ADSC是否具有自我复制能力.培养成功的ADSC中加入神经分化诱导培养基诱导神经细胞分化.诱导分化5d后,免疫荧光染色检测β-微管蛋白(tubulin)和胶质纤维酸性蛋白(GFAP)的表达.结果 培养的ADSC在24 h后逐渐从羊膜组织中移行出;72 h可见明显的多角形细胞富集在羊膜组织周边;120 h后多角形细胞致密地分布于羊膜四周.分裂后的子代细胞形态同父代细胞.单克隆形成实验结果证实羊膜ADSC具有自我复制能力.实时定量PCR检测结果显示,羊膜ADSC的Nestin、Vimentin、Fibronectin mRNA分别是脾脏细胞同种分子表达量的15.79、1.91、7.65倍,两者差异有统计学意义(Z=-5.243、-3.972、-2.524,P＜0.05).免疫荧光染色结果显示,β-tubulin在大部分细胞的胞质中表达;GFAP在部分细胞的细胞质中表达.结论 羊膜ADSC具有自我复制能力,在合适条件下能够分化成神经元和神经胶质细胞.     

RDS／peripherin表达载体的构建和在COS—1细胞的表达

目的 构建大鼠ＲＤＳ／ｐｅｒｉｐｈｅｒｉｎ真核表达载体ｐＡＬＴＥＲ－ＲＤＳ,并观察在ＣＯＳ－１细胞中的表达,方法 将全长度的ＲＤＳ／ｐｅｒｉｐｈｅｒｉｎｃＤＮＡ（１．５ｋｂ）插入到真核表达载体ｐＡＬＴＥＲ－ＭＡＸ中,构建了ＲＤＳ真核表达载体ｐＡＬＴＥＲ－ＲＤＳ,采用电穿孔技术将其转染ＣＯＳ－１细胞,转染４８ｈ后,采用ＲＴ－ＰＣＲ检测ＲＤＳ／ｐｅｒｉｈｐｅｒｉｎｃＤＮＡ的表达,结果 采用ＲＴ－ＰＣＲ