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Objective To detect the protein expression change in the proliferation of human retinal microvascular endothelial cells (hRMECs) stimulated with 4-Hydroxynonenal (4-HNE).Methods hRMECs were in a logarithmic growth phase, and then were separated into 4-HNE-stimulated group and negative control group. The concentration of 4-HNE included 5, 10, 20 and 50 μmol/L in 4-HNE-stimulated group, while the negative control group was added in the same volume of ethanol (the solvent of 4-HNE). Then the cells were stimulated with 4-HNE for 24 hours following by CCK-8 kits incubating for 4 hours to detect absorbance. It was found that 10 μmol/L 4-HNE had the most obvious effect on the proliferation of hRMECs. Therefore, the cellular proteins from 10 μmol/L 4-HNE-stimulated group and negative control group were acquired and prepared by FASP sample preparation method. Data independent acquisition was used for data acquisition, and the GO analysis and pathway enrichment were performed for analysis of differentially expressed proteins. Results CCK-8 kits detection results showed that theA value of the 10 and 20 μmol/L 4-HNE-stimulated groups were significantly higher than negative control group and 5 μmol/L 4-HNE-stimulated group (F=25.42, P<0.01), while there were no differences between 10 and 20 μmol/L 4-HNE-stimulated groups, and theA value of 50 μmol/L 4-HNE-stimulated groups was lower than negative control. A total of 2710 quantifiable proteins were identified by peoteomics,and 118 proteins were differentially expressed (fold change>1.5,P<0.05). Seventy-two proteins were up-regulated after 4-HNE stimulation, whereas 46 proteins were down-regulated. Particularly, the expressions of Heme oxygenase-1, Sulfoxdoxin-1, Heat shock protein A1B, Thioredoxin reductase-1, Glutathione reductase, ATPase and prothrombin were increased when cells were added in 4-HNE, whereas the expressions of apolipoprotein A1 and programmed cell death protein 4 were decreased. These differentially expressed proteins were mainly involved in the biological processes such as oxidative stress, cell detoxification, and ATPase-coupled membrane transport.Conclusion After stimulated with 4-HNE, the oxidative stress, cell detoxification, and ATPase-coupled membrane transport protein expression may change in hRMECs in order to regulate oxidative stress and growth situation.
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Objective To investigate the effects of migration and expression from chemokine receptor 4 (chemokine receptor-4,CXCR4) of rat bone marrow mesenchymal stem cells (BMSCs) which were pretreated by atorvastatin (ATV) in vitro.Methods Isolated,cultivated,identified the BMSCs,pretreated P4-P6 of BMSCs with different concentrations of ATV for 12 hours.The experimental group was divided into control group,0.1 nM/L (group 0.1 nM),1 nM/L (1 nM group),10 nM/L (10 nM group),100 nM/L (100 nM group),1 000 nM/L (1 000 nM group).The mRNA and protein of CXCR4 were determined by real time-polymerase chain reaction and Western blot.Immunofluoreseence assay were used to detect the expression levels of CXCR4.The migration ability of BMSCs were measured by transwell chamber.Results Immunofluoreseence assay showed the protein level of CXCR4 of group 1 nM and 10 nM were significantly higher than the other group.RT-PCR and Western blot showed the protein and mRNA level of CXCR4 in 10 nM was higher than that in group 1 rM.The migration ability of group 10 nM was higher than 1 nM and control group.Conclusions ATV can be dose-dependent promote expression levels of CXCR4 of BMSCs cultivated in vitro.
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Background Researches showed that the increase of intraocular pressure (IOP) in glaucomatous eye is associated with the increasing resistance to aqueous humor outflow effects of transforming growth factor-β (TGF-β) and CD44.Qingguangan is a traditional Chinese medicine and used to treat glaucoma.However,its mechanism of lowing-IOP effect is not elucidated.Objective This study was to investigate the lowing-IOP effect and mechanism of qingguangan granule in DBA/2J mouse,a spontaneous glaucoma model mice.Methods Ten 3 month-old female DBA/2J mice with normal IOP were chosen as control group,and 20 spontaneous ocular hypertension mice aged 9 months were randomized into high IOP group and qingguangan-treated group,with 10 mice for each group.The qingguangan (2.5 g/kg) was administered by gavaging twice per day for consecutive 15 days in the qingguangan-treated group,and normal saline solution was used in the same way in the control group and high IOP group.IOP was measured by anterior chamber injection/suction system at a perfusion rate of 2.5 and 5.0 μl/min,respectively,and the coefficient of aqueous outflow facility (C value) and outflow resistance (R value) were calculated.Another 60 3-month-old DBA/2J mice were randomized into blank control group gavaged with normal saline solution and high-,middle-and low-dose qingguangan groups gavaged with 25.00,12.50 and 6.25 g/kg drugs,respectively,and the mouse serum containing drugs was extracted 7 days after treatment.The scleral tissue with trabecular meshwork were obtained for the culture of trabecular meshwork cells and the cells were identified by immunohistochemistry of fibronectin (FN),laminin (LN) and neuronspecific enolase (NSE).TGF-β was added into the medium for 24 hours with the final concentration of 0,5,10,20,50 and 100 ng/ml,and MMT chromatometry was employed to detect the cell vitality.The cells pre-treated with 20 ng/ml TGF-β were treated with different concentration of drug serum for 24,48 and 72 hours,and the level of TGF-β2 receptor in cell supernatant and the expression of CD44 protein in the cells were detected by ELISA and Western blot assay,respectively.Results The IOPs with perfusion both 2.5 μl/min and 5.0μl/min in the qingguangan-treated group and the control group were significantly lower than those in the high IOP group (all at P＜0.01).Compared with the high IOP group,the C value was significantly reduced (2.35±1.34 vs.1.08±0.36) and the R value was evidently elevated (0.64±0.55 vs.1.05± 0.47) in the qingguangan-treated group (all at P＜0.01).Cultured cells were spindle-shaped with the positive response to FN,LN and NSE antibody.The cell vitality was lower in the 5,10 and 20 ng/ml TGF-β group than that in the 0 ng/ml TGF-β group (all at P＜0.05).Compared with the blank control group,the TGF-β2 receptor content in the supernatant and the related expression level of CD44 protein in the cells were elevated in the TGF-β-treated group (all at P＜0.01),and TGF-β2 receptor contents and CD44 expression levels in the TGF-β+high dose drug serum group was significantly lower than those in the TGF-β group and TGF-β +low dose drug serum group 24,48 and 72 hours after culture (all at P＜0.01).Conclusions Qingguangan can lower IOP of spontaneous glaucoma mice by affecting aqueous humor dynamics.Serum containing qingguangan down-regulates the expressions of TGF-β2 receptors and CD44 in trabecular meshwork cells in vitro.
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Background Human LECs can express telomerase activity,which participates in the formation of cataract.It is reported that estrogen can increase the expression of telomerase activity in human endometrial cancer and breast cancer cells and play an important role in promoting proliferation and anti-apoptosis,but whether estrogen exerts its role on human LECs is still unclear.Objective This study aimed to investigate whether β-estradiol (β-E2) can increase the telomerase activity of human LECs and the influence of β-E2 on proliferation and apoptosis of human LECs.Method Human LECs line was cultured and passaged in vitro,and 1×10-6 mol/L β-E2 was added in the medium for 0,6,12,24 and 48 hours,and reverse transcription PCR was used to determine the optimal time of the expression of human telomerase reverse transcriptase (hTERT) mRNA in the cells.Cultured cells were divided into five groups.The cells in the blank contol group were cultured in the routin medium.Ethanol of 0.1％ was added in the solvent control group,and 1 × 10-8,1 × 10-7 or 1 × 10-6 mol/L β-E2 was added in the medium in different contents of β-E2 groups,respectively.The relative expression level of hTERT mRNA in different groups was detected by reverse transcription PCR.Telomere repeat amplification protocol (TRAP)-ELISA was employed to determine the telomerase activity.The proliferative value of the cells was assayed by cell counting kit-8,and the apoptosis rate of the cells was examined by Hoechst33258 staining.Results The optimal time of β-E2 to rise the expression of hTERT mRNA (absorbance) was at 24 hours under the 1×10-6 mol/L.The relative expression levels of hTERT mRNA in the cells were 0.477±0.015,0.712±0.013 and 0.914±0.031 in the 1 ×10-8,1 ×10-7 and 1 ×10-6 mol/L β-E2 group,which were signifincatly higher than 0.428±0.010 in the blank control group and 0.426±0.010 in the solvent control group (all at P＜0.05).The telomerase activity values (absorbance) were 0.711 ±0.015,0.941±0.010 and 1.249±0.047 in the 1×10-8,1×10-7and 1×10-6 mol/L β-E2 group,which were higher than 0.535±0.013 in the blank control group and 0.543 ±0.013 in the solvent control group (all at P =0.000).The proliferantive values of the cells (absorbance) were significantly raised in the 1 × 10-8 mol/L β-E2 group compared with l × 10-7 and 1 × 10-6 mol/L β-E2 group (both at P =0.000),and no significnant difference was found in the proliferetive values between the blank control group and the solvent control group (P =0.718,0.856).The apoptosis rates of the cells in the 1 × 10-8,1 × 10-7 and 1 × 10-6 mol/L β-E2 group were lower than those in the the blank control group and the solvent control group (all at P=0.000),and there was no significant difference between the blank control group and the solvent control group (P =0.777).No obvious correlation was found between the HLECs preliferative values and hTERT mRNA expression levels or telomerase activity values (r=-0.299,P=0.278;r=-0.157,P=0.576).However,significantly negative correlations were seen between apoptosis rates and hTERT mRNA expression levels or telomerase activity values (r =-0.975,P=0.000;r=-0.981,P=0.000).Conclusions β-E2can increase the activity of telomerase in human LECs,and high dose of β-E2can inhibit apoptosis,but it dose not promote proliferation.
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All organisms regulate their life activities through the biological clock, which makes a variety of activities regular.For example, many physiological activities such as sleep-wake cycle, temperature, heart rate, blood pressure, endocrine and metabolic activity of the kidney and liver are subject to the regulation of circadian rhythms, that is to say they are all under the control of circadian pacemaker.Physiological activity of cell cultured in vitro also possess rhythms.This paper conducts a brief overview of biological clock of cell cultured in vitro and analyzes the molecular mechanism of the biological clock of the neurons and peripheral tissue cell as well as the existing problems, which provide reference for comprehensive interpretation of the molecular mechanism of biological clock.
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Background Krüppel-like factor 6 (KLF6) is related to the physiological or pathological process,such as growth,cell differentiation,proliferation,apoptosis,angiogenesis,tissue repair,and so on.But in ophthalmology,it is less reported about the expressing level of KLF6 protein in lens epithelial cclls (LECs) or the effect of KLF6 on the proliferation of human LECs.Objective This study was to investigate whether KLF6 can inhibit proliferation of human LECs.Methods KLF6 eukaryotic expression plasmid (pEGFP-C2-KLF6) was constructed using reverse transcription PCR(RT-PCR) and identified by double enzyme digestion method and PCR.Human LECs strain (HLE-B3) was cultured and passaged using low glucose DMEM containing 10％ fetal bovine serum and then divided into 4 groups.KLE-B3 transfection reagents were added in the culture medium of all groups.In addition,no agent was used in the blank control group;only insulin-like growth factor-1 (IGF-1) was appended to the medium in only IGF-1 group ;null vector was transfected and IGF-1 was appended in the null plasmid transfection+ IGF-1 group;while pEGFP-C2-KLF6 eukaryotic expression plasmid was transfected into the cells,and simultaneously IGF-1 was added in the pEGFP-C2-KLF6 plasmid transfection+IGF-1 group.After 24 hours of intervene,water soluble tetrazolium salt-1 (WST-1) test was used to detect the growth status of the cells,and Western blot assay was used to assay the relative expressing level of KLF6 protein in the cells.In the other hand,the cells were cultured at the density of 1 ×104/piece,and 0,0.10 and 0.25 μg pEGFP-KLF6 were transfected into each piece of cells respectively,and then IGF-1 was added with a final concentration of 50 μg/L for 24 hours after cell culture.Expressions of Ki-67 protein and mRNA in the cell pieces were detected by immunocytochemistry and fluorescent semiquantitative PCR,respectively.Results The PCR product bands were consistent with KLF-6 gene in length,and the product fragments were corresponding with expectant ones via PCR and double enzyme digestion method,showing a successful construction of pEGFP-C2-KLF6 eukaryotic expression plasmid.After 24 hours of IGF-1 stimulation,the absorbance values of the cells were 0.86±0.00,2.10±0.01,2.24±0.12 and 1.06±0.02 in the blank control group,only IGF-1 group,null plasmid transfection + IGF-1 group and the pEGFP-C2-KLF6 plasmid transfection + IGF-1 group,with a significant difference among the 4 groups (F =38.322,P ＜ 0.05),and that in the pEGFP-C2-KLF6 plasmid transfection +IGF-1 group was significantly lowed in comparison with the only IGF-1 group and the null plasmid transfection+IGF-1 group (q=6.42,7.31,both at P＜0.05).Western blot assay showed that the relative expressing levels of KLF6 protein were statistically different among the four groups (F =591.858,P＜0.05),and those in the pEGFP-C2-KLF6 plasmid transfection+IGF-1 group were 1.47,2.04,3.27 folds higher than those in the blank control group,only IGF-1 group,respectively and null plssmid transfection+IGF-1 group,respectively.Immunocytochemistry revealed that the expressing intensity of Ki-67 protein was gradually weakened with the decrease of pEGFP-C2-KLF6 plasmid dose in the 0,0.10 and 0.25 μg pEGFP-C2-KLF6 plasmid transfection+IGF-1 groups.Fluorescence semiquantitative PCR results showed that the relative expression values of Ki-67 mRNA in the cells were O.15±0.08 and 0.11±0.03 in the 0.10 μg and 0.25 μg pEGFP-C2-KLF6 plasmid transfection+ IGF-1 groups,which were significantly lower than O.77± 0.12 of the 0 μg pEGFP-C2-KLF6 plasmid transfection group,with a statistically significant difference among the three groups (F =54.825,P＜0.05).Conclusions KLF-6 can effectively inhibit IGF-1-induced proliferation of human LECs,and it can be regarded as one of the regulatory factors of the proliferation of HLE-B3 cells.
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Objective To evaluate if 18F-FLT PET imaging could be used as a new clinical method to predict tumor radiosensitivity.Methods MDA-MB-231 and LN229 cells were irradiated with doses of 0,8 and 16 Gy of 6 MV photon energy,then soft agar assay and cellular uptake of 18F-FLT were performed on the 2 cell lines.The t test and one-way analysis of variance were used for the two groups and data before and after irradiation.The MDA-MB-231 and LN229 tumor xenografts were prepared by injecting the tumor cells into the right limbs of female BALB/c nu/nu mice.Once tumors reached a diameter of 10 mm,the two types of mice were divided randomly into 3 groups (20 mice per group) according to the irradiation doses (0,8 and 16 Gy).After irradiation,18F-FLT PET imaging and immunohistochemical staining were conducted.Then correlations between 18F-FLT SUVtumor/SUVmuscle ratio (T/M ratio) and TK1 labeling index percentages (LITK1) were tested using linear correlation analysis.Results The survival fraction of MDA-MB-231 and LN229 cells after irradiated with 8 Gy were (59.73 ± 4.3) ％ and (93.41 ± 3.75) ％,respectively (t =-13.20,P ＜ 0.001).When the dose increased to 16 Gy,the survival fraction decreased to (43.57 ±4.06) ％ and (81.77 ± 4.42) ％,respectively(t =-14.24,P ＜ 0.001).In MDA-MB-231 cells,the cellular uptake of 18F-FLT after irradiation with 8 Gy declined rapidly to (18.32 ± 1.38) kBq/105 cells ((128.22 ± 8.24) kBq/105 cells with the dose of 0 Gy,F =266.41,P ＜ 0.01),and maintained this low level till 72 h.For the LN229 cells,the cellular uptake decreased to (9.87 ± 1.30) kBq/105 cells after 8 Gy irradiation ((134.88 ± 6.59) kBq/105 cells with the dose of 0 Gy,F =346.06,P ＜ 0.01),then increased gradually to (127.17 ± 9.08) kBq/105 cells at 72 h (F =346.06,P ＞ 0.05).The dynamic changes of 18F-FLT cellular uptake in the two cells had the same pattern after being treated with 16 Gy irradiation.In the 18F-FLT PET image of MDA-MB-231 tumor mice after 8 Gy radiotherapy,the T/M ratio decreased to 0.78 ± 0.39 at the first day,but it was 2.84 ± 0.29 before radiotherapy (F =39.78,P ＜0.01).Then the ratio increased slowly,and it was still lower than the baseline at 7 d after radiation (F =39.78,P ＜0.01).The same pattern could be seen in the group of 16 Gy irradiation.In LN229 tumor mice treatment with 8 Gy irradiation,the T/M ratio increased to 2.41 ±0.47 at the first day,and it was 1.58 ±0.29 before radiotherapy (F =34.01,P ＜ 0.05).The ratio decreased steadily to 0.66 ± 0.32 (F =34.01,P＜0.05) at 7 d after radiotherapy.However,in the treatment group with 16 Gy,the T/M ratio decreased gradually and reached 0.44 ± 0.22 at 7 d (F =41.85,P ＜ 0.01).A correlation was found between 18F-FLT T/M ratio and LITK1 (8 Gy:r=0.67,0.73; 16 Gy:r=0.73,0.69; all P＜0.01) in both tumor models.Conclusion 18F-FLT PET imaging may be used as a new assay to predict tumor radiosensitivity,but further investigation is needed before clinical application.
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Objective To observe the different expression of somatomedin-receptor in cell membrane of prostate epithelial cells at anoxic or normoxic condition.Methods Human prostate epithelial cells line RWPE-1 were cultured in vitro.At 4,8,12,24,48 h after cells had been seeded,the gene and protein expression of epidermal growth factor receptor (EGFR),fibroblast growth factor receptor (FGFR),transforming growth factor β1 receptor (TGF- β 1R),insulin-like growth factor-1 receptor (IGF-1 R) and vascular endothelial growth factor receptor (VEGFR) in prostate epithelial cells were tested by RT-PCR and immunohistochem-istry methods,respectively.Results The expression of mRNA and protein of EGFR,FGFR,IGF-1R,TGF- β1R,VEGFR were significantly increased in anoxic and normoxic prostate epithelial cells (P ＜ 0.01 ).At different time point,the expression of mRNA and protein of EGFR,FGFR,IGF-1R,TGF- β1R,VEGFR significantly higher in anoxic than those in normoxic prostate epithelial cells (P＜ 0.01 )besides 4 h EGFR mRNA,12 h EGFR protein,4 h IGF-1R mRNA,4 and 8 h IGF-1R protein,4 and 8 h TGF-β 1R mRNA,4 and 8 h TGF-β 1R protein,4 h VEGFR mRNA (P ＞ 0.05).Conclusion Anoxic prostate epithelial cell can up-regulate the expression of somatomedin-receptor.
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Objective To investigate the expression of aquaporin-4 (AQP4) in cultured astrocytes after in vitro hypoxia induced by CoCl2. Methods After primary culture and subculture, the astrocytes were placed in a controlled atmosphere culture chamber. Both control group and hypoxia groups were established.These groups were further divided into seven sub-groups according to the different time intervals: 15, 30minutes and 1,2, 4, 6, 12 hours, respectively (6 apertures for each group). The shape of the astrocytes in each group was observed with light microscopy and transmission electron microscopy ( TEM ). All groups were examined using in situ hybridization, real time fluorescence quantitative reverse transcriptase polymerase chain reaction, immunocytochemistry and Western blot. The data was analyzed statistically with SPSS 13.0 software. Results There was significant consistency between the AQP4 mRNA and protein ( r =0. 85, P ＜0. 01 ). There was slight positive expression of AQP4 in a few astrocytes of the control groups. In the hypoxia groups, the expression of AQP4 increased within 15 minutes; the increase was most prominent between 1 and 4 hours( mRNA in hypoxia groups: 0. 26 ± 0. 04, 0. 31 ± 0. 02, 0. 36 ± 0. 04; control groups:0. 06 ±0. 01,0. 09 ±0. 01,0. 08 ±0. 01 )after hypoxia and became less between 6 and 12 hours; There was significant difference in the AQP4 expression between the hypoxia groups and control groups among different time points (t = 16. 51, 18.20, 15.26,all P＜0. 01 ). The corresponding pathological changes were cellular edema, which was most prominent between 1 and 4 hours. Under TEM, increase in size of the nucleolus and swelling of endoplasmic reticulum and mitochondria; these changes became more marked with time.Disruption of a few astrocytes was detected in the hypoxia groups at 12 hours. Conclusions The pathological change of astrocytes is cellular edema following hypoxia. There is a positive relationship between the presence and degree of cellular edema as well as the duration of hypoxia and the up-regulating of AQP4.These results imply that AQP4 expression is an important molecular mechanism of celluar edema of astrocytes.
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Objective To evaluate whether 18F-fluorothymidine(FLT) can be used to monitor early response to irradiation in colorectal cancer (CRC).Methods SW480 cells were cultured and irradiated with 0, 10, and 20 Gy.Twenty-four hours later, morphological changes, apoptosis, necrosis, proliferation,and cell cycle phases were observed.Uptake of 18F-FLT was measured in these tumors in vitro from 24 h to 72 h after irradiation.The one-way analysis of variance was used to analyze the data.Results Apoptotic and necrotic cells were detected 24 h after radiotherapy.SW480 cells proliferation was significantly delayed after irradiation in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTI) assay.Cell cycle analysis showed that SW480 cells had a decreased fraction of cells in S phase( from 33.23% to 9.24%,then to 5.43% ) and an arrested fraction in G0-G1.After SW480 cells were cultured for60 min, the uptake of 18F-FLT was (5.21 ± 1.60) %; and 24 h after irradiation of 10 Gy, the uptake decreased significantly to (4.27±0.48)% (F=8.253, P=0.009).And 72 h after irradiation, the uptake further decreased significantly to (3.39 ± 0.59) % ( F = 36.715, P＜0.001 ).In tumor tissue, the uptake of 18F-FLT reduced significantly 72 h after radiotherapy (10 Gy:F = 12.388, P = 0.007; 20 Gy:F = 16.744, P = 0.004) and the attenuation degree increased with the radiation dose.Conclusion The uptake of 18F-FLT in SW480 cells or in CRC could reflect the changes of SW480 cells in proliferation, cell cycle re-distribution, cell apoptosis and necrosis.The results suggest that 18F-FLT may be used for monitoring early response to irradiation of CRC.
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Objective To observe the effect of supernatant liquid from brain tissue on the differentiation of adipose-derived stem cells (ADSCs) into neural cells in normal brain tissues, the homogenate of infarcted cerebral hemisphere and the opposite side in the rats. Methods The ADSCs were obtained from rat retroperitoneal adipose tissue. The normal brain tissues, the homogenate of the infarcted cerebral hemisphere and thc opposite side got from middle cerebral artery occlusion in rats were used to induce ADSCs. Immunocytochemistry or immunofluorescence were used to identify the cell types at the 3rd day. Positive expression rate was counted by fluorescence microscope. Results (1)The neuron-specific enolase (NSE) positive cells, microtubule-associated protein 2 (MAP-2) positive cells and glial fibrillary acidic protein (GFAP) positive cells were much more in the homogenate of the infracted cerebral hemisphere than in others (P＜0.05). (2)The NSE positive cells, MAP-2 positive cells and GFAP positive cells were much more in the homogenate of the normal brain tissues and the opposite side than normal level ( P ＜ 0. 05 ) . Conclusions The homogenate of the infracted cerebral hemisphere and the opposite side can induce adipose-derived stem cells into neural-like cells and express neural cells markers in rats.
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Objective To approach the effect of fluoride on the expression of thyroid peroxidase(TPO)activity and TPO mRNA in primary porcine thyrocytes.Methods Purified cultured porcine thyrocytes waft made into sodium fluoride model,and were divided according to the final concentration of NaF into 0(control group),40,80,160 mg/L.After exposed to NaF for 48 h,the morphology of the porcine thyrocytes was investigated with acridine orange staining method,TPO activity was measured with upgrade guaiacol method and RT-PCR method was used to detect the ratio of TPO/β-actin.Results The major changes included apoptotic bodies and cell fragments in the 80,160 mg/L groups under phase contrast microscope.With the increasing dose of fluoride.TPO activity,being(3.103±0.090),(1.944±0.025),(1.361±0.008),(0.668±0.026)U/L,respectively,had obviously lowered with a statistical significance compared between the groups(F=1563.864,P＜0.05).The TPO activity had a negative correlation with the dose of fluoride(r=-0.955,P＜0.05).With the dose of fluoride increasing,the expression of TPO mRNA had obviously lowered,being(0.947±0.013),(0.634±0.018),(0.448±0.028)and (0.210±0.009)with a statistical significance in group comparison(F=2713.855,P＜0.05).Conclusion Fluoride affects the thyroid via inhibiting TPO activity and expression of TPO mRNA.
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Objective To investigate the effects of different concentrations of sodium fluoride on the morphologic characteristics of primarily cultured thyroid cells of SD rats and in order to obtain important proof for approaehing the mechani8m of thyroid gland damage caused by fluoride.Methods Thyroid cells of SD rat were primarily culture for 96 hours,and cell density was adjusted to 5.0×108/L Cell suspension with 5 ml Wills seeded into 6 weII plates,after 12 hours,0(contr01),10.100,1000 μmol/L of sodium fluoride was added into the well, witll each well representing different level of treatment group.Finally the cultured thyroid cells were collected for morph010gic study.Results Under microscope,the transparency of the control thyroid cells Was good,and cells gathered in cluster and adhered to wall.But a lot of cells treated with fluoride suspended,and lost their transparency-under scaning delectron microscope,the control calls showed integrated membrane and tightness to each other,as well as clear boundary between cells normal proliferation.While the thyroid cells treated with 10,100 μmol/L sodium fluoride 0bviouslv shrinked and deformed,and the cells treated with 1000 μmol/L of sodium fluoride were broken-Conclusions nuoride can affect the growth and development of thyroid cell and damage the structure and morphology.Sodium fluoride affects the morphologie characteristics of thyroid cells in a dose-response manner.
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Schwann cells play an important role in diabetic peripheral neuropathy.Recently,to investigate the effect of high glucose on Schwann cell in vitro has become a new research hotpoint with the development of cell culture technology.This review concentrates on the biological effect of high glucose on Schwann cell.
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Objective To observe the effects of Chinese herbal medicine of Yiqi Huoxue (YQHX) on proliferation and expression of E-CD and invasion of human lung cancer cell lines - small cell lung cancer (NC-H446) in vitro. Methods By cell measured method, immunohistochemistry method, technique of image and Boyden Chamber method, the effects of YQHX or all trans-tretinoin acid (ARTA) on proliferation and expression of E-CD and invasion of NC-H446 cell lines with different time cultured by YQHX or ARTA in vitro were studied. Results The proliferation of NC-H446 were inhibited significantly by 5% YQHX or 10-5 mmol/L ARTA (P0.05). The shape and structure of cell NC-H446 was occurred variant. YQHX enhanced remarkably expression of E-CD of NC-H446 with 36 hour cultured (P 0.05). The quantity of NC-H446 cells crossed Matrigel and polyvinylpyrrolidone-free polycarbonate filter were more than the control group (P 0.05). Conclusion YQHX can inhibit the proliferation, enhance expression of E-CD on NC-H446, and decrease invasion and metastasis of NC-H446.
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Objective:To improve previous method of primary rat cortical neuron culture to get purer and more long-lasting cells for study.Methods:Timed-pregnant Wistar rats at a gestational age of 16 or 17 days(16-17 d) were used.Fetal brains were removed and the cerebral cortices were dissected out.Papain digestion and mechanical dissociation were combined to conduct mono-cell suspending media.Four to six hours(4-6 h) post-plating,all plating media were removed from cultures and replaced with Neurobasal medium supplemented with B27.On the third day,10 ?mol/L cytosine arabinoside(Ara-C) was added to the culture for 24 h to inhibit the outgrowth of glial cells.Half of the culture medium was changed every week.The morphological changes of neuron cells were observed by light microscope.Double immuno-staining of microtubule-associated protein 2(MAP2) and karyon were applied to assess the culture purity.Evaluation of synapse formation was processed by immunocytochemical analysis using antibodies against both pre-and postsynaptic protein markers.Results:The improved method could remarkably increase the cell number and reduce neuronal damnification.The primary culture was characte-rized by high uniformity,purity,normal synapse formation and longtime livability.Conclusion:This is a simple and reliable technique for the in vitro primary culture of rat cortical neurons.
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Objective:To study the expression and function of Mitofusin 2(Mfn2)in cultured neonatal rat cardiomyocytes during PE-induced hypertrophy.Methods:The hypertrophy neonatal rat cardiomyocytes model was induced by 0.01 mol/L PE.RT-PCR and Western blot were applied to assess Mfn2 mRNA expression and protein level respectively.Cultured neonatal rat cardiomyocytes were treated by PE after Ad GFP or Ad Mfn2 infection,the protein synthesis was determined by 3H-leucine incorporation assay.Results:PE led to ANF mRNA level(by~1 fold,P
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Objective To evaluate the feasibilities of the potential donors in liver cell transplantation using the human fetal hepatocytes and immortalized L-02 hepatocytes by comparing their biological features. Methods Human fetal hepatocytes were isolated from aborted fetal livers (gestational ages from 14 w to 24 w) by an improved two-stage perfusion method and cultured in a conditioned medium without any growth factors.?-fetal protein (AFP) and albumin (ALB) were detected by radioimmunoassay (RIA) and cytokeratin-19 (CK-19) was identified by cellular immunochemistry study.Immortalized L-02 hepatocytes were cultured in the same condition and the characteristic proteins were detected by the same methods.Results The viability of human fetal hepatocytes was approximately 95% using the perfusion method, and the maximum survival time of the cultured hepatocytes was 3 weeks.The expression of AFP, ALB, and CK19 was detected at the same time, especially during Day 3 to Day 7 in the culture.By comparison, the proliferation ability of L-02 hepatocyte was greater, although with a lower level of ALB secretion.The expression of AFP and CK19 was not detected.Furthermore, during the long culture, L-02 hepatocytes may undergo a morphologic change and fail to express ALB.Conclusion Human fetal hepatocyte may be a practical donor for hepatocyte transplantation with its high-level protein expression and potential bi-differentiation ability.In view of the absent expression of ALB and the morphologic change in culture, although with better proliferation, L-02 hepatocyte seems not useful for hepatocyte transplantation.
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Objective To assess the regulating effects of interleukin-1? (IL-1?) on gene expression of Bax mRNA in human hyaline chondrocyte.Methods Hyaline chondrocytes of human were harvested enzymatically and cultured in DMEM supplemented with 20% bovine serum.In the experiment,various concentrations of IL-1? were added to the medium.The effects of IL-1? on the Bax mRNA were assessed by reverse transcriptase polymerase chain reaction (RT-PCR) in the passaged monolayer cell cultures of hyaline chondrocyte.Results IL-1? increased the Bax mRNA level in passaged cultures of hyaline chondrocyte.The difference was significant (P
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Objective To investigate the inhibitiory effect of tacrolimus(FK506) and adriamycin(ADM) on hepatocellular carcinoma.Methods HepG-2 cells were cultured,and divided into 4 groups,namely(control)、FK506、ADM、FK506+ADM groups,the cells were trated by the drugs for 24 to 48 hours(respectively).The inhibitory rate of the cells was measured by MTT assay,and cell cycle and cell apoptotic rate were detected by flow cytometry(FCM).Results The ability of tumor cell growth were inhibited by FK506 after 48h;the apoptosis ratio was increased when FK506 was below 100?g/L,and when it(exceeded) that value,the apoptosis ratio was decreased.FK506 and ADM significantly prolonged cell G_2 phase;combined tacrolimus and adriamycin had synergistic role effect on arresting the cell in G_2 phase,FK506 combined with adriamycin demonstrated synergistic effect on apoptosis.Conclusions FK506 could inhibit the growth of hepatocellular carcinoma,arrest the cell in G_2 phase,and increase apoptosis.FK506 combined with adriamycin demonstrated synergistic inhibitory effect on hepatocellular carcinoma.