Objective To investigate the effects of protein kinase C (PKC) on proliferation, extracelluar matrix synthesis and transcriptional factor SOX9 (SRY-related high mobility group-box gene 9) expression of rat growth plate chondrocytes in vitro. Methods Rat costochondral growth plate chondrocytes (RGC) were isolated and cultured. The 1st serum free cultured passage RGCs were treated with 1, 10 and 100 nmol/L phorbol 12,13-dibutyrate (PDBu), PKC agonist, cell morphology were observed with inverted microscopy, cell proliferation, COLLAGEN and GAG synthesis were detected by isotope incorporation COLLAGEN TYPEⅡ and AGGRECAN mRNA transcription and SOX9 expression were revealed by RT-PCR and Western blot.Results 100 nmol/L PDBu treatment made the cell morphology of serum free cultured RGC closed to serum group and inhibited proliferation but promoted COLLAGEN and AGGRECAN synthesis, 3H-TdR、3H-proline and 35S-sulfate incorporation of 100nmol/L PDBu group were as 83%, 52.6% and 146.5% as those of serum free control(P

0.05). [3H]-proline incorporation in testing groups was 20% higher than that in control. CONCLUSION: The present study suggests EGF is able to enhance RGCs proliferation and collagen synthesis. Dedifferentiation caused by serial passage decreases proliferative effect of EGF on RGCs, but has no effect on collagen synthesis enhancement.

AIM: To establish the methods for rat costochondral growth plate chondrocyte (RGC) separation and culture and investigate their biological features, thereby provide experimental bases for studying the regulation of chondrocyte proliferation and differentiation. METHODS: RGC were obtained by microdissection and digestion, and cultured in monolayer. Morphological changes of the serial passage of RGC and the cell growth kinectics were observed. The cellular GAG and collagen type Ⅱ expression were detected by histochemistry and ICC. RESULTS: There were more than 98% viable cells in the obtained RGC. The morphology of primary cultured RGC was round or polygon. In this experiment, the sixth passage RGC was still maintained and showed polygonal morphology. The index of duplicatings/day increased in the preceding fourth passage RGC and decreased afterwards. There were more than 95% cells expressed collagen type Ⅱ and alcine blue stained positively in the primary RGC, as the passage number increased, the ratio of collagen type Ⅱ expression and alcine blue positive stained RGC dropped abruptly. CONCLUSION: The separation and culture methods adopted in this study can obtain high pure and viable RGC. The preceding three passage RGC maintains their in vivo phenotype, they are idle experimental materials for studying the regulation of proliferation and differentiation of chondrocyte and tissue engineering.

Objective To investigate the effects of laser irradiation on intracellular ROS(reactive oxidant species),intracellular calcium concentration(_i,and cell membrane integrity in the process of live cell imaging with confocal laser scanning microscopy. Methods The effects of a given laser irradiation on ROS,intracellular calcium concentration(_i and cell viability were revealed respectively by stained ECV-304 with H_2DCFDA,Fluo-4AM and calcein-AM/PI,and visualized and analyzed using ultra view LCI(live cell image)confocal microscopy. Results The irradiation of 488nm laser induced fluorescent intensity of DCF to increase abruptly and attain the climax in about 80 seconds,afterwards the fluorescent intensity fell and returned to the baseline.In the 70 minutes of the irradiation,the fluorescent intensity of intracellular Fluo-4 kept a slightly ascending tendency.The fluorescent intensity of calcein decreased 15minutes after the irradiation,and serval cells were PI positively stained.Conclusion 488nm laser irradiation induces intracellular reactive oxidant species(ROS) and calcium concentration to increase,but there is no significant influence on cell membrane integrity.

Aim To investigate effects of genistein (5, 7, 4′-trihydroxyisoflavone) on rat costochondral growth plate chondrocyte (RGC) collagen and Sox9 expression. Methods Primary cultured RGC, effects of genistein on collagen synthesis, col2a1 mRNA transcription and Sox9 protein expression of 1st passage RGC were detected by isotope incorporation, RT-PCR and western blotting. Results Genistein inhibited collagen synthesis of 1st passage RGC (P

Objective To detect systematic oxidative stress in preeclampsia.Methods (1)Morphological features of placenta hypoxia were observed by histological method ; (2) Level of granulocyte intracellular reactive oxygen species was monitored by dyeing full blood with 2' ,7'-dichlorodihydrofluorescein diacetate (H2DCFDA) ; (3) Level of H_2O_2 in sera was detected by special kits.Results Compared to normal pregnancy,placentas from preeclampsia showed distinct features of hypoxic stress injury,such as more syncytial knots formation,fibrosis emerged,vein in-jury and loss its normal configuration; Fluorescence values of ROS probe in neutrophils from different women were 45.61±12.20(n =49),51.02 ± 13.60(n =56,P <0.01)and 85.10 ± 16.30(n =47,P <0.01); Concentra-tions of H_2O_2were (24.57±5.17)μmol/L(n =49),(26.61±3.25)μmol/L(n =56,P 0.01) and (39.84±9.67)μmol/L(n=47,P<0.01) respectively.Conclusion With the help of histological method,flow cytometry and special kits,systematic oxidative stress can be detected through checking placentic tissues,netrophils and sera of preeclampsia.

Objective To investigate the effects of metabolic syndrome (MS) and the cardiocerebrovascular risk factors such as essential hypertension (EH), diabetes mellitus (DM), and lipid metabolise disturbance on the subtypes of acute cerebrovascular disease (ACVD). Methods A total of 502 ACVD patients were studied, including 121 cases of intracerebral hemorrhage (ICH) and 381 cases of cerebral infarct (CI). The effects of MS, EH, DM, and blood lipids on the subtypes of ACVD were analyzed. Results The ratio of all subtypes of ACVD combined with EH was significantly higher than that of the control group ( P 0.05). Additionally, the concentration of total cholesterol in the ischemic group was significantly lower than that in the control group ( P

AIM: To study the effects of ultraviolet (UV) on mitochondrial functions and apoptosis in HaCaT cells.METHODS: After irradiation by UV at low dose(UVA 2 J/cm~2,UVB 10 mJ/cm~2) and high dose(UVA 6 J/cm~2,UVB(30 mJ/cm~2),) HaCaT cells were cultured for 15 hours.Flow cytometry was used to measure mitochondrial membrane potential,mitochondrial mass and apoptotic rate.Annexin V-FITC/PI staining of apoptotic cells was analyzed by laser confocal microscopy.RESULTS: After UV irradiation,cell proportion with low mitochondrial membrane potential increased with irradiation doses.The proportion of control group,low dose group and high dose group were 7.94%?1.02%,25.87%?4.55% and 39.27%?5.32%,respectively.Cells proportion with low mitochondrial mass increased with irradiation doses.The proportion of control group,low dose group and high dose group were 15.19%?1.58%,40.36%?4.41% and 68.79%?5.46%,respectively.The hypodiploid peaks of DNA content analysis represented the apoptotic rate of HaCaT cells.The apoptotic rate of control group,low dose group and high dose group were 1.82%?0.51%,30.16%?5.47% and 58.49%?5.98%,respectively.To analyze the cells apoptosis by staining with annexin V-FITC and PI,the results were consistent with those of DNA content analysis.Cells in control group showed almost no positive staining cells.Single annexin V-FITC positive cells in low dose group and double positive cells in high dose group were predominant,respectively.CONCLUSION: UV irradiation induces HaCaT cell mitochondrial depolarization,as well as mitochondrial mass loss.These changes are related to cell apoptosis.

AIM: To confirm that CD4~+CD25~+ regulatory T cells don't have an instinctive defection in IL-2 secretion, and to have an insight into the maturation state of CD4~+CD25~+ T cells in cord blood. METHODS: CD4~+CD25~+ and CD4~+CD25~- T cells were purified from cord blood of term infants (CB) and adult peripheral blood (PB) by autoMACS, and stimulated with PDB plus ionomycin. After 45 hours of culture, cells were detected for expression of CD69 and CD25 by flow cytometry, and the supernatants were measured for 7 kinds of cytokines by Luminex. RESULTS: CD4~+CD25~+ T cells from both CB and PB proliferated comparably with CD4~+CD25~- T cells when stimulated with PDB plus ionomycin. After 45 hours of culture, however, the CD4~+CD25~+ T cells underwent a tendency of cell death. Expression of CD25 was further upregulated when CD25~+ cells were activated. Under stimulation of PDB plus ionomycin, both CD4~+CD25~+ and CD4~+CD25~- T cells in PB secreted high levels of IFN-?, IL-2 and TNF-?, with CD25~+ cells secreted much higher level of IL-5, IL-4 and IL-10 than those in CD25~- cells; CD4~+CD25~+ and CD4~+CD25~- T cells in CB also secreted high level of IL-2 and TNF-? but much lower level of IFN-? than those in PB, and no secretion of IL-5, IL-4 and IL-10 was observed. CONCLUSION: CD4~+CD25~+ regulatory T cells don't have an instinctive defection in IL-2 secretion, otherwise there may be a different TCR signaling pattern in CD4~+CD25~+ T cells from traditional T cells. The CD4~+CD25~+ T cells in cord blood have not fully matured in function.

AIM: To study the effect of VEGF on extracellular H2O2 production in HUVECs and the role of H2O2 in the VEGF-induced proliferation. METHODS: HUVECs was stimulated with 500 ?g/L VEGF. Products of extracellular H2O2 was detected by H2DCFDA staining. MTT method was used to value the influences of 3?106 U/L catalase and 5-20 mmol/L H2O2 to VEGF function. RESULTS: After treatment for 15 min with VEGF, HUVECs appeared fluorescence, and continued to become stronger, peaked at 45 min then decreased. HUVECs, which was treated simultaneity with VEGF and 3?106 U/L catalase, only appeared very faint fluorescence. The proliferation of HUVECs by VEGF was restrained when treated with 3?106 U/L catalase. The extrinsic H2O2 at concentration of 5-10 mmol/L promoted the proliferation of HUVECs but inhibited the proliferation effect of VEGF on HUVECs (P