Expression of PCNA, C-fos and Bax proteins in human embryonic tongue tissues
Journal of ZheJiang University(Medical Science), 2013, 42(5): 517-522.
Objective： To investigate of proliferating cell nuclear antigen (PCNA), C-fos and Bax proteins in human embryonic tongue tissue of different developmental stages.
Methods： Immunohistochemistry was used to detect the expressions of PCNA, C-fos and Bax proteins in embryonic tongue tissues of fetuses with 2, 3 and 4 month gestational age (n=16). One-way ANOVA and LSD-t test were employed to compare the number of positive expression cells in tongue tissues of fetuses with different gestational age.
Results： In the fetuses at 2, 3 and 4 months of gestation, the numbers of PCNA-positive cells in tongue epithelial tissues were 20.20±7.13, 39.10±13.44 and 26.00±9.02, respectively; those in tongue muscle and fiber tissues were 17.20±8.99, 22.30±6.57 and 32.40±14.72, respectively. In fetuses at 2 month of gestation, no C-fos-positive cells were found in tongue tissues; while at 3 and 4 months of gestation, the numbers of C-fos-positive cells in the tongue epithelial layers were 25.10±7.91, 17.40±2.80; those in tongue muscle and fiber tissues were 24.50±4.67 and 28.00±7.75, respectively. Only weak positive expression of Bax protein was observed in the third month of gestation in embryonic tongue tissues. A significant difference was noted in PCNA expression in tongue epithelial layers, the muscle and fiber tissues (P<0.01 and P<0.05) among 3 embryonic periods. A significant difference was found in C-fos expression in tongue epithelial layers (P<0.01), but not in tongue muscle and fiber tissues (P>0.05) among 3 periods.
Conclusion： Dynamic changes were seen in PCNA and C-fos expressions in embryonic tongue tissues in different gestational ages of fetus, indicating these two proteins may participate in regulation of the development and differentiation of tongue tissues in human embryos and fetuses.
Effects of sub-micro emulsion composition on cellular disposition of incorporated lipophilic drug
Journal of ZheJiang University(Medical Science), 2013, 42(5): 523-529.
Objective： To investigate the effects of sub-micro emulsion composition on cellular uptake and disposition of incorporated lipophilic drug.
Methods： Sub-micro emulsions containing 10% oil, 1.2% lecithin and 2.25% glycerol were prepared, and the fluorescent agent coumarin 6 was used as a model drug. The effects of oil types, co-surfactants and cationic lipid on uptake and elimination kinetics of 6-coumarin in HeLa cells were studied. The uptake mechanism of sub-micro emulsions was further investigated.
Results： Oil type and Tweens had no influence on the cellular uptake. Modifications of surfactants with Span series increased the cellular influx, among which Span 20 with hydrophilic-lipophilic balance (HLB) value of 8.6 was the best enhancer. The intracellular drug level reached up to (46.09±1.98)ng/μg protein which had significant difference with control group \[(38.54±0.34)ng/μg protein\]. The positively charged emulsions significantly increased the uptake rate constant and elimination rate constant which were 4 times and 1.5 times of those in anionic groups, respectively. The uptake enhancement was also observed in cationic emulsions, cellular concentrations at plateau were (42.73±0.84)ng/μg protein, which was about 3 times of that in anionic emulsions \[(15.71±0.74)ng/μg protein\], when extracellular drug concentration kept at 100 ng/ml. Cationic emulsions delivered the payload mainly by direct drug transfer to contacted cells, while the negative ones depended on both drug passive diffusion and clathrin-mediated endocytosis of drug containing oil droplets which accounted for 20% of the intracellular drug.
Conclusion： Interfacial characteristic of sub-micro emulsions such as co-surfactants HLB as well as zeta potentials can influence lipophilic drug both in cellular uptake and elimination.
Effect of Helicobacter Pylori lipopolysaccharide on expression of Gli and Ptch-1 proteins in sonic hedgehog signaling pathway of gastric mucosa GES-1 cells
Journal of ZheJiang University(Medical Science), 2013, 42(5): 543-549.
Objective： To investigate the effect of Helicobacter Pylori lipopolysaccharide (Hp-LPS) on expression of Gli and Ptch-1 proteins in sonic hedgehog (Shh) signaling pathway of gastric mucosa GES-1 cells.
Methods： The LPS was extracted from Hp by hot phenol water method, and then the concentration of LPS was detected by the kinetic turbidimetric assay. GES-1 cells were stimulated by different concentrations of Hp-LPS (0, 1, 10, 20, 30 and 40 μg/ml). The inhibition rates of cell growth were measured by MTT assay after treated with Hp-LPS for 24 h. The expression of Gli and Ptch-1 proteins were determined by Western Blot.
Results： MTT assay showed that the inhibition rates of GES-1 cell growth after treatment by different concentrations of Hp-LPS (1, 10, 20, 30 and 40μg/ml) were 25.8%±2.7%, 34.2%±3.1%, 46.3%±3.4%, 60.8%±2.1% and 82.9%±2.8% respectively (r=0.985, P<0.001). Western blot showed that the expressions of Gli and Ptch-1 proteins were decreased after Hp-LPS treatment (0, 1, 10, 20, 30 and 40 μg/ml): the relative expression values of Gli were 1.286±0.180, 0.963±0.067, 0.850±0.085, 0.566±0.058, 0.549±0.056 and 0.377±0.047, respectively (r=-0.945, P<0.001); those of Ptch-1 were 1.688±0.088, 1.466±0.061, 1.170±0.065, 1.042±0.064, 0.648±0.057 and 0.482±0.074, respectively (r=-0.985, P<0.001).
Conclusion： Hp-LPS can decrease the related protein expression of Shh signaling pathway, which indicates that Hp may interfere with the function of Shh signaling pathway in gastric mucosa via the effect of its LPS.
Effect of renal sympathetic denervation on left ventricular hypertrophy and inflammatory factors in spontaneously hypertensive rats
Journal of ZheJiang University(Medical Science), 2013, 42(5): 550-555.
Objective： To investigate the effect of renal sympathetic denervation on left ventricular hypertrophy and inflammatory factors in spontaneously hypertensive rats.
Methods： Thirty six spontaneously hypertensive rats (SHR) were divided into 3 groups with 12 animals in each group: SHR control group，operation group and sham operation group.Bilateral renal sympathectomy or sham operation were performed in operation and sham groups，respectively; another 12 WKY rats served as normal controls.The blood pressure and body weight were examined weekly.The animals were sacrificed at w1 and w6，rat hearts were collected and left ventricular mass index (LVMI) was calculated.The expression of TLR4，TNF-α and IL-6 in heart tissue were detected by immunohistochemistry and Western blot.
Results： The systolic blood pressure \[(201.67±11.09)mmHg vs (140.0±10.86)mmHg，P<0.05\]，diastolic blood pressure \[(144.50±10.48)mmHg vs (78.50±7.32)mmHg，P<0.05\]，LVMI (2.44±0.05 vs 1.93±0.05，P<0.05)，the expression of TLR4 (0.298±0.004 vs 0.126±0.004，P<0.05)，NF-κB (0.249±0.006 vs 0.195±0.005, P<0.05)，TNF-α (0.323±0.004 vs 0.146±0.004，P <0.05)，IL-6 (0.283±0.005 vs 0.207±0.006，P<0.05) in SHR control group were significantly higher than those in WKY group.Compared to sham operation group，the systolic blood pressure (157.30±9.35 vs 197.30±11.5，P<0.05)， diastolic blood pressure (112.50±6.25 vs 146.80±7.6,P<0.05)，LVMI (2.32±0.04 vs 2.57±0.09, P<0.05，TLR4 (0.198±0.006 vs 0.317±0.008，P<0.05)，NF-κB (0.208±0.006 vs 0.332±0.007,P<0.05)，TNF-α (0.27±0.009 vs 0.375±0.004，P<0.05)，IL-6 (0.218±0.004 vs 0.376±0.009,P<0.05) in operation group were all decreased at w1 after sympathectomy.Six weeks after the operation，there were no significant differences in systolic blood pressure (197.50±12.13 vs 208.83±10.23，P>0.05) and diastolic blood pressure (150.33±7.74 vs 151.50±8.22,P>0.05) between denervated and sham-operated SHRs; however，the LVMI (2.46±0.07 vs 2.81±0.05，P<0.05) and the expression of TLR4(0.301±0.009 vs 0.567±0.006, P<0.05)，NF-κB (0.251±0.004 vs 0.476±0.009，P<0.05)，TNF-α (0.324±0.005 vs 0.535±0.006,P<0.05，IL-6 (0.285±0.009 vs 0.549±0.007,P<0.05) in operation group were still significantly lower than those in sham operation group.
Conclusion： Renal sympathetic denervation can significantly delay the progression of LVH in SHR，which may associated with lowering blood pressure and decreasing expression of TLR4，NF-κB，TNF-α，IL-6 in myocardial tissue.
Crosstalk between canonical TGF-β/Smad and Wnt/β-catenin signaling pathway
Journal of ZheJiang University(Medical Science), 2013, 42(5): 591-597.
TGF-β signaling pathway plays a central role in the signaling networks that control the growth, differentiation of the cell, and the initiation of fibrosis and cancer. Wnt signaling pathway is critical for the embryonic development and the invasion and migration of cancer cells. TGF-β signaling and Wnt signaling, both of which play an important role in regulating embryonic development, fibrotic disease and tumor progression, have a close relationship. Researches find several typical cross points between these two signaling systems, such as Smad, Axin, Dvl and β-catenin. In this review, we focus on the crosstalk between TGF-β signaling and Wnt signaling through these typical factors, intending to better understand the process of fibrosis and tumor progression.