Objective To study the implication of phosphoinositide-specific phospholipase C?2(PLC-?2) in gastric cancer MGC80-3 cells treated by TPA.Methods The effect of 12-O-tetradecanoylphorbol-1,3-acetate(TPA) on gastric cancer MGC80-3 cells was detected and analyzed by DAPI staining under fluorescence microscope.The effect of TPA on the expression level of PLC-?2 protein was detected by Western blotting,when nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation.Localization and translocation of PLC-?2 were observed under laser scanning confocal microscope with immune-fluorescence technique.After MGC80-3 cells were pretreated by U73122(PLC-?2 inhibitor),the effect of TPA on PLC-?2 was detected by Western blotting and laser scanning confocal microscope with immunefluorescence technique,and the effect of U73122 on MGC80-3 cells followed by TPA was observed and analyzed by DAPI staining under fluorescence microscope. Results TPA induced the apoptosis of gastric cancer MGC80-3 cells.Meanwhile,TPA increased the expression level of PLC-?2 protein and induced its translocation from cytoplasm to nucleus.U73122 inhibited the effect of TPA on the expression level of PLC-?2 protein,but it did not affect on the cell apoptosis induced by TPA.However,the translocation of PLC-?2 protein induced by TPA was not inhibited by its inhibitor(U73122).Conclusion Although TPA could increase the expression level of PLC-?2 protein,it is not directly related to the cell apoptosis induced by TPA.However,its translocation might be related with the apoptosis of gastric cancer.

Objective To probe into the remodeling and maturity process along with extension of time in anterior cruciate ligament (ACL) reconstruction with hamstring autograft in order to confirm the date of maturity postoperatively. Methods Thirty-three patients after arthroscopic ACL reconstruction with hamstring autograft, were undergone second-look arthroscopical surgery. Meantime, biopsy specimens were obtained from the mid-zone of the hamstring graft. As a control group, specimens of normal ACL were obtained from total knee replacement of four cases, and specimens of semitendinous tendons obtained from ACL reconstruction of the four cases. The interval from initial reconstruction to second-look arthroscopy ranged from 2 to 36 months(mean 11.9 months). Patients were divided into different groups by postoperative time such as 1-, 4-, 7-, 10-, 13-, 18- and 25- month group. Thirty-three specimens were ordinarily sliced up into longitudinal sections. The sections were stained with hematoxylin and eosin so that cell and collagen fibrils were observed under light microscope, then compared the results with those of control group. Results Under microscopical observation, it was showed that tissue of hamstring graft was remodeled from more blood vessels and cellular amount to normal one, and from an irregular orientated crimp pattern of collagen to regular orientated crimp pattern of collagen in a time-dependent manner. Decreasing cellular amount, changing cell type, and a regular orientated crimp pattern of collagen of the hamstring graft were observed in 7- month group primarily, comparing with the original ACL. However, there existed a slower process in the other groups that are older than that of 7- month. Conclusion There are well histological features in the process of remodeling and maturation of the hamstring autograft after operation following extension of time, such as better survival of early period, and remodel of vessels and collagen. The process of remodeling and maturation in the hamstring autograft is similar to BPB autograft. The maturation period of the hamstring autograft after ACL reconstruction in human patients appears between 7 month and 9 month postoperatively.

Objective To study the expression of protein kinase B (Akt/PKB) isoforms in the gastrocnemius muscle of rats at different ages. Methods The expression levels of mRNA and protein of three Akt isoforms in the gastrocnemius muscle of 30-month-old and 6-month-old rats were detected respectively by RT-PCR and Western blotting. Data were analyzed statistically. Results (1) The levels of Akt1 and Akt2 mRNA in the 30-month-old rats was significantly higher than those in the 6-month-old rats (t=7.124, P=0.000; t=2.598, P=0.021), however, no significant difference was found in the level of Akt3 mRNA between the two groups (t=0.460, P=0.653) . (2) The level of Akt-Thr308 phosphorylation in the 30-month-old rats was significantly lower than that in the 6-month-old rats (t=-9.861, P=0.000), while the level of Akt2 protein in the 30-month group was significantly higher than that in the younger rats (t=7.522, P=0.000). No significant differences were detected in the levels of Akt1 and Akt3 proteins between the two groups (t=0.469, P=0.646; t=0.058, P=0.955). Conclusion The expression levels of three Akt isoforms in the gastrocnemius muscle of rats change with age, suggesting that the three isoforms of Akt have different functions in the gastrocnemius muscle metabolism of rats at different ages.

Objective To investigate the expression and role of Caspase-3 during the development of non-traumatic avascular/aseptic necrosis of femoral head(ANFH).Methods Tissue samples were obtained from 22 femoral heads of 21 patients with ANFH.The samples were divided into three groups:early stage(6 cases),middle stage(8 cases) and late stage(8 cases) groups according to the progress of the disease.Meanwhile,19 tissue samples of 19 femoral heads were obtained from 19 cases of acute femoral neck fracture and set as a control group.The level of apoptosis of each group was compared in terms of the percentages of TUNEL positive osteocytes and empty osteocyte lacuna,and then the activity of Caspase-3 was detected by colorimetric method.Results Both the pecentages of empty osteocyte lacunae(F=45.43,P=0.000) and the TUNEL positive osteocytes(F=120.86,P=0.000) increased significantly with the progress of ANFH.The two indexes in the early and middle stage groups were significantly higher than those in the control(q=18.899,P0.05).Caspase-3 activity was not significantly related to the capacity of osteogenesis(r=0.126,P=0.425).Conclusions Caspase-3 is not the major executor of apoptosis in ANFH.It is very likely that it does not play a nonapoptotic role in the progress of the diseases.

Actins ; metabolism ; Animals ; Creatinine ; blood ; Kidney ; pathology ; Kidney Diseases ; metabolism ; pathology ; Male ; Radiation Injuries, Experimental ; metabolism ; pathology ; Rats ; Rats, Wistar ; Vimentin ; metabolism

AIM: To study p42/p44 mitogen - activated protein kinases ( MAPK ) signal transduction pathway effect on vascular endothelial growth factor ( VEGF ) expression induced by elevated glucose concentration in cultured human retinal pigment epithelium ( hRPE) .
METHODS:hRPE cells were cultured and divided into four groups:normal glucose group (NG) (5. 6mmol/L), high glucose group ( HG1:15mmol/L D-glucose, HG2:20mmol/L D - glucose, HG3:30mmol/L D - glucose ), PD98059 group: hRPE cells were treated by an efficient and selective inhibitor PD98059 (20μmol/L) of p42/p44MAPK signal transduction pathway and solvent dimethyl sulfoxide group ( DMSO group) . The expression of VEGF and pigment epithelium derived factor ( PEDF ) mRNA was detected by RT-PCR. VEGF protein expression in cultured hRPE supernatants was detected by enzyme-linked immumosorbent assay ( ELISA) .
RUSULTS: VEGF mRNA and protein expression induced by elevated glucose concentration increased significantly. VEGF mRNA and protein expression were restrained in PD98059 group. Ratio of ( VEGF/β-actine)/( PEDF/β - actine ) in PD98059 group decreased significantly compare with that in high glucose group.
CONCLUSION: p42/p44MAPK signal transduction pathway might play a part in VEGF expression induced by elevated glucose concentration in cultured hRPE cells.

Objective To understand the relationship between clinical manifestations and Kashin-Beck disease(KBD) and their contribution to diagnosis of KBD and to construct the diagnosis model for KBD in adolescents.Methods A total of 2248 subjects under the age of 18 were collected from 6 KBD endemic and 1 non-KBD areas of the Shaanxi province in China.Analysis of 32 indicators,including gender,age,and KBD clinical indicators.Indicators of the distribution of measurement data between the two groups using t test and analysis of variance,x2 test with count data,multi-category ordered response variables Logistic regression analysis for model building.Results It showed the KBD prevalence rate in adolescent had an increasing tendency with age.Analysis of indicators between the two groups,in addition to the age factor(P ＜ 0.05),the difference of ankle pain,knee pain,wrist movement disorder and other 5 indicators(P ＜ 0.05) and the last bend,elbow movement disorder,syndactyly and other 9 indicators(P ＜ 0.01 ) were statistically significant.Sixteen clinical and radiographic features in the clinical manifestations were significantly related with the clinical severity grading with KBD(P ＜ 0.01 ).Four models on the diagnostic indictors were constructed by cumulative logit model for adolescent KBD (-21ogL,Score,Wald x2 test,P ＜ 0.01 ).Conclusions The establishment of the diagnostic model based on their contribution of the joint involvement in systemic performance-related indicators has an important role for clinical diagnosis of KBD.

BACKGROUND: Previous studies demonstrated that eucommia bark can promote bone marrow stern cells (BMSCs) differentiated into osteoblasts, but relative mechanism is poorly understood. OBJECTIVE: To investigate the effects of eucommia bark water/methanol extracts on expressions of osteopontin (OPN) and osteoprotegedn (OPG) in rat BMSCs. METHODS: Totally 2 g eucommia bark powder were added into water or methanol to 16 mL and oscillated for 1 hour at room temperature. After soaked overnight, both extracts were centrifuged at 15 000 r/min for 10 minutes. Water extract was obtained from supernatant in water soaked powder. In methanol soaked powder, methanol extracts was obtained by concentrated supernatant in vacuo and resolved using 16 mL water. Water and methanol extracts were then filtered by 0.22 μm membrane, and conserved at -20℃. Six SD rats, aged 2 months, were selected, and the 3~(rd)passage of BMSCs were induced by water or methanol extracts with dilution of 1 × 10~(-2), 1 × 10~(-3), 1 × 10~(-4) and 1 × 10~(-5), respectively. PBS was added in the negative control group. All cells were cultured for 6 days. Expressions of OPN and OPG was measured by immunocytochemistry at 6 days with induction. The expression of OPN and OPG induced by water and methanol at 1 ×10~(-3) and 1×10~(-4) dilution was detected by RT-PCR. RESULTS AND CONCLUSION: Immunocytochemistrical results indicated that both water and methanol extracts of eucommia bark simulated OPN and OPG expression, in particular with dilution of 1×10~(-4). The methanol extracts had a stronger effect than water extract, but the expression of OPG did not change obviously. RT-PCR demonstrated that at the 3rd day of inducement, the level of OPN expression induced by water extract was higher than that of methanol extract, and no OPG expression was detected. Osteogenic differentiation of rat MSCs induced by eucommia bark water/methanol extracts relates to stimulating expression of OPN, which has no correlation to OPG. OPN expression induced by water extract is early than that of methanol extract.

Child ; Critical Care ; Humans ; Intensive Care Units, Pediatric ; Medical Records ; Severity of Illness Index

Objective To study the antitumor effect of toremifene on MCF7 cell lines,and investigate the role of mitogen-activated protein kinase pathway. Methods Inhibitory effect of toremifene alone or combined with MEK inhibitor PD98059 on MCF7 cells was measured by SRB test,and that on phosphorylated ERK was detected by Western blotting.Results Toremifene exhibited a concentration-dependent inhibitory effect on the activity of MCF7 cells.Phosphorylated ERK was significantly inhibited by 5,10 and 20 mmol/L toremifene.Combined with PD98059,toremifene had a significantly enhanced cytotoxity effect,which exceeded that of application alone. Conclusion Mitogen-activated protein kinase pathway may play an important role in the antitumor effect of toremifene which is independent of estrogens.Combined with PD98059,the antitumor effect of toremifene can be reinforced,indicating a synergistic effect of these two drugs.