In thiis study,we show thai carbachol stimulates the accumulation of inositol phosphates(InsPs)in human rellnal pigment epithelium (RPE)cells and atropine blocks the carbachol-induced effect ,suggesting the existence of musearinie acelyleholine receptors in human RPE cells. In contrast,noradrenaline,serotonin, cpidermal growth factor (EGF),isoproterenol,and
NECA (5'-[N-ethyl]-carboxamido-adenosine)do not influence the basal levels of InsPs.Moreover,isoprmerenol and NECA do not affect the carhaehol elevated levels of InsPs.EGF,howcvcr,does potentiate the carhaehol stimulated elevation of InsPs in a dose-dependent manner ,suggesting an interaction between EGF and musearinie receptors in cultured human RPE cells.
（Chin J Ocul Fundus Dis,1994,10:220-222）
ObjectiveTo review the research progress of different cell seeding densities and cell ratios in cartilage tissue engineering. MethodsThe literature about tissue engineered cartilage constructed with three-dimensional scaffold was extensively reviewed, and the seeding densities and ratios of most commonly used seed cells were summarized. ResultsArticular chondrocytes (ACHs) and bone marrow mesenchymal stem cells (BMSCs) are the most commonly used seed cells, and they can induce hyaline cartilage formation in vitro and in vivo. Cell seeding density and cell ratio both play important roles in cartilage formation. Tissue engineered cartilage with good quality can be produced when the cell seeding density of ACHs or BMSCs reaches or exceeds that in normal articular cartilage. Under the same culture conditions, the ability of pure BMSCs to build hyaline cartilage is weeker than that of pure ACHs or co-culture of both. ConclusionDue to the effect of scaffold materials, growth factors, and cell passages, optimal cell seeding density and cell ratio need further study.
ObjectiveTo observe the effect of subretinal injection of retinal pigment epithelium (RPE) cells for RPE in mice.
MethodsA total of 30 postnatal day 7 C57BL/6J mice were randomly divided into normal mice group, OIR model group and OIR model cell transplanted group, 10 mice in each group. The OIR model was induced in mice of OIR model group and OIR model cell transplanted group. The RPE cells were subretinal injected into the RPE of mice in OIR model cell transplanted group. At 20 days after the injection, the RPE thickness was evaluated by fluorescence microscope. The expression of RPE65, Bestrophin and zonula occludens-1 (ZO-1) were estimated by Western blot and real-time quantitative PCR (RT-PCR).
ResultsThe thickness of RPE in OIR model mice was thinner than that in normal mice; the thickness of RPE in OIR model cell transplantation mice was significantly thicker than that in the OIR model mice. The results of Western blot and RT-PCR indicated that the differences of protein (F=8.597, 18.864, 25.691) and mRNA expression (F=39.458, 11.461, 34.796) of RPE65, Bestrophin, ZO-1 were statistically significant between OIR model group and OIR model cell transplanted group (P < 0.05).
ConclusionsSubretinal injection of RPE cells can promote RPE thickening. RPE65 and Bestrophin protein relative expression levels increased, ZO-1 protein relative expression levels reduced; mRNA expression levels of RPE65, Bestrophin and ZO-1 genes increased.
Objective
To detect the apoptosis of vascular endothelial cells and retinal pigment epithelial (RPE) cells in vitro induced by verteporfin-photodynamic therapy.
Methods
Cultured vascular endothelial cells and human RPE cells were incubated with verteporfin at a concentration of 1.0 mu;g/ml which was equivalent to the initial plasma level of verteporfin in clinical therapy. Each kind of cells were divided into 6 groups according to different time of incubation: 0, 5, 15, 30, 60, and 120 minutes group. After incubated, the cells were illuminated by the laser light with the maximum wavelength of absorption of verteporfin (wavelength: 689 nm, power density: 600 mW/cm2) with the power of 2.4 J/cm 2for 83 seconds. The percentage of cellular apoptosis was measured by flow cytometry 3 hours after PDT, and the measurement was repeated thrice.
Results
The proportion of cellular apoptosis 3 hours after PDT were 0.01plusmn;0.01, 0.25plusmn;0.02, 0.32plusmn;0.02, 0.41plusmn;0.04, 0.49plusmn;0.03 and 0.61plusmn;0.02, respectively in 0-120 minutes group of vascular endothelial cells; and 0.02plusmn;0.01, 0.22plusmn;0.01, 0.31plusmn;0.02, 0.38plusmn;0.03, 0.47plusmn;0.05 and 0.58plusmn;0.03 respectively in 0-120 minutes group of RPE cells. The proportion of cellular apoptosis of both kinds of the cells increased as the incubation time was prolonged. There was no significant difference of the percentage of cellular apoptosis between the accordant time groups in the two kinds of cells (P＞0.05).
Conclusions
Cellular apoptosis can be quickly induced by verteporfin-PDT both in human vascular endothelial cells and RPE cells; under the same condition in vitro, PDT has no obvious selection for the apoptosis of the two kinds of cells.
(Chin J Ocul Fundus Dis, 2006, 22: 253-255)
ObjectiveTo investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment.
MethodsThe 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen typeⅡ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide.
ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P < 0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P < 0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P < 0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen typeⅡand ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P < 0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen typeⅡprotein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P < 0.05); RCCS 1 group expressed high levels of collagen typeⅡand ANCN proteins at all stages (P < 0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups.
ConclusionUnder the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.
ObjectiveTo construct a lentiviral vector carrying rat sirt1 gene and observe the expression of sirt1 in retinal ganglion cell (RGC) of rat.
MethodsRat sirt1 cDNA was inserted into pLV5 vector. After identification by sequencing analysis and PCR, the recombinant sirt1expressinglentivirus vector was packaged by cotransfecting 293T cells with packaged plasmid.Then pLV5-sirt1 was used to infect the cultured Sprague-Dawley rat RGC cell in vitro.The expressions of sirt1 protein and mRNA in infected rat RGC were detected by quantitative real-time PCR and Western blot.
ResultsThe sirt1 expression vector pLV5 was successful constructed and sequence was proved to be correct. The expression of sirt1 protein and mRNA in RGC was significantly increased than that in cells infected with control lentiviruses(P < 0.05).
ConclusionWe have successful constructed a sirt1 expression lentivirus vector pLV5-sirt1 and it can increase the expression of sirt1 protein and mRNA in the rat retinal ganglion cells.
Objective To investigate the species distribution and antibiotic resistance of pathogens fromcatheter-related bloodstream infections ( CRBSI) in intensive care unit( ICU) , to provide evidence for the guidance of clinical rational administration.Methods A retrospective analysis was performed to review the microbiological and susceptibility test data of all CRBSI patients in ICU from January 2009 to December 2011. The patterns of antibiotic resistance among the top seven bacteria were compared. Results 67 cases of CRBSI were detected with 81 strains, including 40 Gram-positive ( G+ ) bacteria( 49.4% ) , 38 Gram-negative( G- ) bacteria ( 46.9% ) , and 3 fungi ( 3.7% ) . The main pathogens causing CRBSI were coagulase negative Staphylococci ( 27 strains, 33.3%) , Acinetobacter baumannii ( 12 strains, 14.8% ) , Klebsiella pneumoniae( 9 strains, 11. 1% ) , Staphylococcus aureus ( 8 strains, 9. 9% ) , Pseudomonas aeruginosa ( 7 strains, 8. 6% ) , Escherichia coli ( 6 strains, 7.4% ) , suggesting that Staphylococcus epidermidis was predominant pathogenic G+ bacteria, and Acinetobacter baumannii was predominant G- bacteria. The antibiotic resistance tests demonstrated that isolated G- bacillus was highly sensitive to carbopenem, while vancomycin-resistant G+ bacteria were not found. Conclusions Within the latest 3 years, the predominant pathogens of CRBSI in ICU are Staphylococcus epidermidis and Acinetobacter baumannii. Acinetobacter baumannii exhibited high drug resistance to all antibiotics.
Objective
To establish a simple and efficient method to isolate and culture the umbilical vein vascular endothelial cells in canine.
Methods
Twelve umbilical cords [(13.0 ± 1.5) cm in length] were taken from 12 newborn pups of Beagles. And then the vascular endothelial cells were isolated from these umbilical cords digested by 1% collagenase type I for 5, 7, and 10 minutes respectively (4 umbilical cords in each group). After cultured, the vascular endothelial cells were identified by morphology, immunofluorescence, and flow cytometry. And the growth curvature of umbilical vein vascular endothelial cells was detected by MTT assay.
Results
Few vascular endothelial cells were collected at 5 and 10 minutes after digestion; many vascular endothelial cells were seen at 7 minutes, and became cobblestone with culture time, with a large nucleus; after passage, cell morphology had no obvious change. Fluorescence microscope results showed that positive von Willebrand factor (vWF) and CD31 cells were observed in most of cells. The flow cytometry test displayed that the positive cell rates of vWF and CD31 were 99.0% ± 0.7% and 98.0% ± 1.2%, respectively. The above results indicated that cultured cells were vascular endothelial cells. MTT assay showed that vascular endothelial cells proliferation increased significantly with culture time.
Conclusion
Enzyme digestion is a convenient method to isolate vascular endothelial cells from canine umbilical vein, and a large number of cells and high purity of cells can be obtained by the method.
ObjectiveTo investigate the effect of silk fibroin-poly-L-lactic acid (SF-PLLA) microcarriers on the expansion and differentiation of adipose-derived stem cells (ADSCs).MethodsADSCs were extracted from adipose tissue donated voluntarily by patients undergoing liposuction by enzymatic digestion. The 3rd generation ADSCs were inoculated on CultiSpher G and SF-PLLA microcarriers (set up as groups A and B, respectively), and cultured in the rotary cell culture system. ADSCs cultured in normal two-dimensional plane were used as the control group (group C). Scanning electron microscope was used to observe the microcarriers structure and cell growth. Live/Dead staining and confocal fluorescence microscope was used to observe the distribution and survival condition of cells on two microcarriers. DNA quantification was used to assess cell proliferation on two microcarriers. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect chondrogenesis, osteogenesis, and adipogenesis related gene expression of ADSCs in 3 groups cultured for 18 days. Flow cytometry was used to identify the MSCs surface markers of ADSCs in 3 groups cultured for 18 days, and differential experiments were made to identify differentiation ability of the harvested cells.ResultsADSCs could be adhered to and efficiently amplified on the two microcarriers. After 18 days of cultivation, the total increment of ADSCs of the two microcarriers were similar (P>0.05). qRT-PCR results showed that chondrogenesis related genes (aggrecan, cartilage oligomeric matrix protein, SOX9) were significantly up-regulated for ADSCs on SF-PLLA microcarriers and adipogenesis related genes (peroxisome proliferator-activated receptor γ, lipoprotein lipase, ADIPOQ) were significantly up-regulated for ADSCs on CultiSpher G microcarriers, all showing significant differences (P<0.05). Flow cytometry and differentiation identification proved that the harvested cells of the two groups were still ADSCs.ConclusionThe ADSCs can be amplified by SF-PLLA microcarriers, and the chondrogenic differential ability of harvested cells was up-regulated while the adipogenic differential was down-regulated.
Objective
To investigate the effect of arginase (Arg) inhibitor N-ω-Hydroxy-L nor-Arginine (nor-NOHA) on high glucose cultured rhesus macaque retinal vascular endothelial cell line (RF/6A) in vitro.
Methods
The RF/6A cells were divided into the following 4 groups: normal control group (5.0 mmol/L of glucose, group A), high glucose group (25.0 mmol/L, group B), high glucose with 125 mg/L nor-NOHA group (group C), and high glucose with 1% DMSO group (group D). The proliferation, migration ability and angiogenic ability of RF/6A cells were measured by Methyl thiazolyl tetrazolium (MTT), transwell chamber and tube assay respectively. The express of Arg I, eNOS, iNOS mRNA of RF/6A cells were measured by real-time polymerase chain reaction (RT-PCR), Enzyme-linked immuno sorbent assay (ELISA) was used to detect the expression of NO and interleukine (IL)-1b of RF/6A cells.
Results
The proliferation, migration, and tube formation ability of group A (t=2.367, 5.633, 7.045;P＜0.05) and group C (t=5.260, 6.952, 8.875;P＜0.05) were significantly higher than group B. RT-PCR results showed the Arg I and iNOS expression in group B was higher than that in group A (t=6.836, 3.342;P＜0.05) and group C (t=4.904, 7.192;P＜0.05). The eNOS expression in group B was lower than that in group A and group C (t=4.165, 6.594;P＜0.05). ELISA results showed NO expression in group B was lower than that in group A and group C (t=4.925, 5.368;P＜0.05). IL-1b expression in group B was higher than that in group A and group C (t=5.032, 7.792;P＜0.05).
Conclusions
Nor-NOHA has a protective effect on cultured RF/6A cells in vitro and can enhance its proliferation, migration and tube formation. The mechanism may be inhibiting the oxidative stress by balancing the expression of Arg/NOS.
