Objective To analyze the direct interaction between mmu-miR-30b and mouse FoxO3 (mFoxO3) mRNA.Methods Three target gene fragments, which were respectively 402, 123 and 299 bp in length, were amplified from mouse cDNA by PCR using specific primers and site-direct mutant primers.A complete mutant fragment was obtained by joining together the 123 and 299 bp fragments.Recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed through inserting wild and mutant fragments of mFoxO3 into pmirGLO vector, respectively.HEK 293T cells were respectively co-transfected with the constructed recombinant plasmids and mmu-miR-30b/mmu-miR-30b inhibitor.Dual-luciferase reporter assay system was used to determine the Firefly-Renilla luciferase activity in different groups.Western blot assay was performed to evaluate the regulatory effect of mmu-miR-30b on mFoxO3 expression.ResultsRestriction enzyme analysis and gene sequencing showed that the two recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed successfully.The activity of Firefly-Renilla luciferase in HEK 293T cells transfected with pmirGLO, pmirGLO+mmu-miR-30b, pmirGLO+mmu-miR-30b inhibitor, pmirGLO-mFoxO3+mmu-miR-30b, pmirGLO-mFoxO3+mmu-miR-30b+mmu-miR-30b inhibitor, or pmirGLO-mFoxO3 mt+mmu-miR-30b was 13.18±0.97, 14.35±0.99, 12.46±1.20, 9.55±1.11, 13.71±0.89 and 10.99±0.92, respectively.Compared with pmirGLO+mmu-miR-30b group, the luciferase activity was significantly decreased in pmirGLO-mFoxO3+mmu-miR-30b group (P<0.05), but showed no significant change in pmirGLO-mFoxO3 mt+mmu-miR-30b group (P>0.05).In addition, the suppressed Firefly-Renilla luciferase activity in pmirGLO-mFoxO3+mmu-miR-30b group was restored by mmu-miR-30b inhibitor treatment (P<0.05).Enhancing the expression of mmu-miR-30b could markedly inhibit the expression of mFoxO3 at protien level (P<0.05), and that could be significantly attenuated by mmu-miR-30b inhibitor treatmeat (P<0.05).Conclusion mFoxO3 mRNA is a novel target gene of mmu-miR-30b.There is a direct interaction between mmu-miR-30b and mFoxO3 mRNA.

Objective To investigate the effect of CO2 pneumoperitoneum on the cell adhesion capability of cultured endometrium cancer cells.Methods After treated in a pneumoperitoneum model for 4 h,the changes in cell growth and adhesion of the endometrium cancer were detected by MTT.The expression of ?1-integrin and E-cadherin in the cancer cells were compared with the control that were not treated with CO2,by the immunohistochemical method.Results After treated with CO2 for 4 h,the cell growth was enhanced and the cell adhesion capability increased as compared with the control group.At the same time,the expression of ?1-integrin and E-cadherin was lower in treated cells than that in untreated ones.Conclusion The adhesion capability of endometrium cancer cells in CO2 pneumoperitoneum could be enhanced by the reduction of the expression of ?1-integrin and E-cadherin,and thereby influences the metastasis of cancer.

Objective To determine whether pupil response to dilute tropicamide could be used as a diagnostic test for Alzheimer's disease (AD). Methods Pupil diameters of both eyes were measured simultaneously by an infra red automatic pupil diameter recorder and analyzer every 0 1 second for 30 minutes after instillation of 0 01% tropicamide to one eye and normal saline to the other. Three groups of patients were studied: 52 patients with AD, 33 with vascular dementia (VD), and 58 elderly controls. The percent change in pupil diameter of the treated eye was examined automatically by the analyzer. After finding the cut off point for differential diagnosis by receiver operator characteristic curve (ROC), its sensitivity, specificity and Kappa coefficient were calculated. Results Mean percent change in diameters of the treated eye showed a trend of fastest maximum dilation in AD group, and was significantly different from other groups at all the measurement time points after 10th minute instillation. The difference was most significant at the 18th minute after instillation, and 15% was used as a cut off point, the sensitivity was 0 81,specificity 0 79 0 82, and Kappa coefficient 0 62 0 67. Conclusions Pupil dilation test could be used as a screening method in the diagnosis of Alzheimer's disease, or as a tool for differential diagnosis between AD and VD.

Objective:To observe the clinical effect of treatment of burning mouth syndrome in women by hormone replacement therapy (HRT).Methods:21 cases of climacteric women with burning mouth syndrome (BMS) were treated by HRT with nilestriol and medroxyprogesterone acetate.Another 21 cases were treated with vitamin B,C and oryzanol as the controls.The treatment and follow-up were conducted for 3～6 months.Results:The ratio of effectiveness in HRT group and control group was 85.71% and 14.27% (P

Objective To discuss the effect of CO2 artificial pneumoperitoneum on the invasion of the endometrial cancer in nude mouse resulted from the transplantation of the cancer cells and its mechanism.Methods Thirty nude mice were divided into 3 groups based on the time in the CO2 pneumoperitoneum circumstance.Control group: the small intestine of the nude mouse was exposed in air for 5 min,and the cancer cells were injected into right lower quadrant after suture.The gas was depleted after 40 min.40 min group: CO2 gas was poured into abdominal cavity to form a 4 mmHg artificial pneumoperitoneum for 5 min before cancer cells were injected into right lower quadrant.The gas were depleted after 40 min.80 min group: CO2 gas was poured into abdominal cavity to form a 4 mmHg artificial pneumoperitoneum for 5 min before cancer cells were injected into right lower quadrant.The gas were depleted after 80 min.The time that each group took to form a solid tumor was recorded.Four weeks later,the transplantation tumors were taken out and sliced into frozen sections and paraffin-embedded sections.The expression of ?1-intergrin and E-cadherins was detected by IMF.Results The time taken to form the solid cancer was shorter in the 40 min group and 80 min group than in the control group,with more blood vessel found(P0.05).Conclusion The CO2 pneumoperitoneum could enhance the abilities of invasion and adhesion of endometrial cancer cells,which is associated with the expression changes of ?1-intergrin and E-cadherins in the cancer cells.

Objective To establish the magnetic activated cell sorting system(MACS)for isolation and incubation of human ovarian carcinoma-derived microvascular endothelial cells(ODMECs) and to observe their ability to form tubule-like structure(TLS) in vitro.Methods MACS was used to purify ODMECs with antiCD31 antibody and ODMECs were identified according to their morphological,biochemical and phenotypic characteristics.The ability of ODMECs to intake acetylated-low-density lipoprotein(DIL-acLDL) and Ulex europeaus agglutinin-1(UEA-Ⅰ) was assayed to observe the formation of TLS in vitro.Results Flow cytometry showed that the purity of ODMECs was up to 99.6%.The ODMECs were characterized by cobble stones,contact inhibition,expressed CD31,CD105 and FⅧ-Rag,and exhibited the ability to bind to UEA-1,intake DIL-acLDL and form TLS during incubation.Conclusion The MACS we established can be used to isolate and incubate human ODMECs and study the formation characteristics of TLS.

OBJECTIVETo determine the effect of type I transmembrane protein (IRE1alpha) deletions on the cell cycle of human periodontal ligament fibroblasts (hPDLFs) cells.

METHODSBased on the IRE1alpha deletions, a full-length model was successfully constructed. Moreover, overlapping polymerase chain reaction mutagenesis facilitated the establishment of two deletion mutants of IREla (pD-Kinase, pD-Rnase). The full-length model and two mutant eukaryotic expression vectors were transfected into hPDLFs cells. Western blot analysis was performed to identify the expression in the cells. The changes in the cell cycle of hPDLFS cells were detected by flow cytometry (FCM).

RESULTSThe two deletion mutants of IRE1alpha with eukaryotic expression vectors were successfully constructed and correctly expressed in hPDLFs cells based on Western blot analysis. Under stress conditions, the FCM assay showed that cell percentage of S phases increased, whereas that of G1 phases decreased in the IRE1alpha group (P < 0.05) compared with the control group of tunicamycin (TM) treatment. Moreover, the cell percentage of the S phases decreased, whereas that of the G1 phases increased in the D-Rnase group (P < 0.05) compared with the control. The deletion mutant D-Kinase had no influence on hPDLFS cell proliferation and cycle (P>0.05).

CONCLUSIONUnder stress conditions, IRE1alpha can improve the cell cycle of hPDLFs cells from the G1 to the S phase. The deletion mutant D-Rnase cause hPDLFs cell growth arrest at the G1 phase, whereas deletion mutant D-Kinase has no significant effect.

Objective To establish an animal model to study endometriosis. Methods Six female Bama miniature pigs that were sexually mature were chosen and their endometrial tissues were acquired surgically. The endometrial tissues were implanted to abdominal cavity and subcutaneous tissues. Results The ectopic endometria in the implant location formed mass or cyst that was verified pathologically to be similar to normal endometrial tissues. Conclusion The establishment of ectopic endometriosis in Bama miniature pigs is successful, which provides an ideal model for further therapeutic study of endometriosis.

Objective To investigate the effects of nordihydroguaiaretic acid(NDGA)on the endometriosis established in Bama miniature pigs.Methods Six Bama pigs that had been successfully established into animal model of endometriosis subcutaneously received 3% NDGA at dose of 20 mg/kg(n=4)or PBS solution(n=2)for 20 d.The grafts in abdominal cavity and subcutaneous tissues were observed and the serum concentrations of hormone,were measured by chemiluminoimmunoassay before and after the period of NDGA or PBS injection.The expressions of FⅧAg and VEGF in the endometriotic tissues were observed.Results The endometriotic masses were reduced to some degree after the time period of NDGA injection,even partially disappeared,while those treated with PBS showed no obvious changes.Histoimmunochemical analysis showed NDGA decreased the number of endometrial glands,the proliferation rate of the glandular epithelial cells,the endometrial stroma cells and the vessels,and inhibited the microvascular density and VEGF in the endometriotic tissues(P

Objective To investigate the effect of statin therapy on pneumonia and stroke outcome after acute ischemic stroke.Methods The patients with pneumonia after acute ischemic stroke were enrolled retrospectively.The demographic and clinical data of the patients were collected,including whether using statins,the clinical features of pneumonia,the days of using antibiotics,and neutrophil count.They were divided into either a statin group or a control group according to whether they used statins or not.Stroke outcome was evaluated with the modified Rankin scale.Results A total of 53 patients with pneumonia after acute ischemic stroke were enrolled in the study,20 of them were in the statin group and 33 were in the control group.There were no significant differences in age and the proportions of male and complicated with hypertension,diabetes and coronary heart disease between the statin group and the control group (all P ＞ 0.05).The cure rate,improvement rate,invalid rate and mortality in the statin group were 50.00％,35.00％,15.00％ and 0.00％,respectively; and in the control group were 15.15％,39.40％,33.30％ and 15.15％,respectively.There were significant differences (P =0.003).The days of temperature returning to normal (2.10 ± 3.29 d vs.4.61 ±3.54 d; P =0.002),pulmonary rales disappearing (3.60 ±2.46 d vs.7.67 ±4.09 d; t =-4.019,P =0.000)and using antibiotics (7.05 ± 3.13 d vs.9.73 ± 4.00 d; t =-2.562,P =0.013) in the statin group were significantly shorter than those in the control group,while there were no significant differences in the days of cough and sputum production retuming to normal between the 2 groups.The proportions of diabetes (20.83％ vs.55.17％ ;x2 =6.473,P =0.011),posterior circulation stroke (4.7％ vs.27.59％ ; P =0.031)and bilateral lung shadows (29.17％ vs.55.17％ ;x2 =5.705,P=0.017),as well as baseline National Stroke Association NIH Stroke Scale (NIHSS) score (4.00 ± 4.54 vs.10.66 ± 6.33; t =1.898,P =0.001) in a good outcome group were significantly lower than those in a poor outcome group.But there was no significant difference in the proportion of the patients treated with statins between the good outcome group and the poor outcome group (41.67％ vs.34.48％ ;x2 =0.288,P =0.591).Multivariate logistic regression analysis showed that diabetes (odds ratio [OR] 5.146,95％ confidence interval [CI] 1.166-22.709; P =0.031) and baseline NIHSS score (OR 1.251,95％ CI 1.080-1.449; P =0.003) were the independent risk factor for patient with poor outcomes.Conclusions Statins may promote the pneumonia recovery in patients with acute ischemic stroke; however,it has no effect on stroke outcome.