This study examined the effects of over-expression of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the cell cycle and survival of human glioma cell line U87 and U251 and explored the possible mechanisms. The LRIG3 gene was transduced into U87 and U251 cells respectively by using lentivirus and the transduced cells were selected by puromycin. The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blotting. The apoptosis rate was detected by Annexin V-FITC/PI double labeling and the cell cycle was flow cytometrically analyzed. Compared with control cells, LRIG3 mRNA expression in U251 and U87 cells transduced with pLVX-DsRed-LRIG3-Monomer-N1 were increased by 77.6% and 129.7%, and LRIG3 protein expression was raised by 141.3% and 322.7%, respectively. Cell cycle analysis showed that LRIG3 over-expression increased the percentage of cells at G(0)/G(1) phase (P<0.01). Over-expressed LRIG3 could significantly promote the apoptosis of U87 and U251 cells (P<0.05). These findings suggest that the over-expression of LRIG3 could arrest the cell cycle in G(0)/G(1) phase, and promote apoptosis of U87 and U251 cells.

OBJECTIVETo study the molecular mechanism of Yangjing Zhongyu Decoction (YZD) n-butanol extracts (ZDC) and ethyl acetate extracts (YSYZ) in reducing androgen in porcine granulose cells by mitogen-activated protein kinase (MAPK) pathway.

METHODSPorcine granulose cells were isolated and cultured. They were inoculated by MAPK inhibitor PD98059 at different concentrations, and then they were divided into the blank control group (0), 1, 3, 10, and 25 micromol/L groups. After 24-h culture the cytochrome P450c17a (CYP17) mRNA expression level was detected using Real-time fluorescent quantitative PCR. Contents of androgen (testosterone) in the supernate were detected using RIA and optimal PD98059 concentration screened. After intervened by 10 micromol/L PD98059 for 24 h, the culture solution was intervened by effective ingredients of with or without YZD or YSYZ at various concentrations (0, 1 , 5, 25, 50 mg/mL) at various time points (3, 6, 18, 24 h). Expression levels of p-ERK1/2, c-Fos and CYP17 were detected by Western blot. Testosterone content in the supernate was determined by radioimmunoassay (RIA).

RESULTSTen pLmol/L PD98059 could obviously decrease p-ERK1/2 protein expression and increase CYP17 mRMA expression, and elevate testosterone content in the supernate (P < 0.05). ZDC and YSYZ at 25 ng/mL could increase p-ERK1/2 protein expression and c-Fos levels, and reduce CYP17 protein expression, and lower testosterone content in the supernate after 6-h intervention (P < 0.01).

CONCLUSIONEffective ingredients of YZD could reduce androgen production in porcine granulose cells through increasing activities of MAPK.

Androgens ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Flavonoids ; Granulosa Cells ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; RNA, Messenger ; Swine

This study examined the effect of GHRP-6, a known GHSs receptor agonist, on the phosphorylation of cAMP-responsive element-binding protein (CREB) and the underly mechanism. GH3 cells were cultured and subjected to different treatments as follows: GHRP-6, GHRP-6 plus GHRH, phorbol ester (PMA), an activator of PKC, alone or in combination with GHRP-6, Gö6983, a general inhibitor of PKCs, in the presence or absence of GHRP-6, rottlerin, an inhibitor of PKCs, alone or plus GHRP-6. The cells were transiently transfected with PKCsigma-specific siRNA and then treated with GHRP-6. GH level was measured by enzyme-linked immunosorbent assay (ELISA). The expression of phosphor-CREB, PKCsigma, PKCtheta and phosphor-PKCsigma was determined by Western blotting. The results showed that GHRP-6 stimulated GH secretion in both time- and dose-dependent manners and enhanced the effect of GHRH on GH secretion. GHRP-6 was also found to induce CREB phosphorylation. Moreover, GH secretion was enhanced by the PKC activator PMA and reduced by the PKC inhibitors (Gö6983, rottlerin) and knockdown of PKCsigma. PKCsigma could be activated by GHRP-6. It is concluded that PKC, especially PKCsigma, mediates CREB phosphorylation and GHRP-6-induced GH secretion.

Objective To evaluate the feasibility and outcomes of different surgical approaches on the basis of sentinel lymph node biopsy (SLNB) in treating early-stage vulvar cancer, and discuss the proper strategy for individualized treatment. Methods The medical charts of patients with early-stage vulvar cancer treated in Sun Yat-sen University between January 2004 and December 2013 were retrospectively collected. A total of 74 patients who received sentinel lymph node(SLN)detection in primary surgery were enrolled (average age 55). The surgical approaches contained SLNB, inguinal lymphadenectomy (IL), and extensive vulvectomy. The SLN were examed on intraoperative frozen sections. The treatment protocols, lymphatic metastasis, postoperative recovery condition, recurrence and survival data were collected and analyzed. Results At least one SLN was successfully detected in 68 (92%,68/74) patients. SLN were positive in 21 patients, of whom 12 (group A) underwent bilateral IL, and 9 (group B) received radiotherapy without performed IL. SLN were negative in 47 patients, of whom 26 (group C) underwent bilateral IL and one of them had a non-SLN metastasis, and 21 (group D) were advised to follow-up. The coincidence of pathological results between frozen and paraffin sections was 100%. The sensitivity and specificity of SLNB for diagnosis of lymph node metastasis were 95% and 100%, respectively. A total of 44 complications happened in patients underwent SLNB and IL (group A and C), including 16 poor wound healing, 14 lymphedema, 8 lymphatic fistulas, 3 phlebothrombosis and 3 infections. There were no complications happened in patients underwent SLNB alone (group B and D), among whom the operation time, bleeding amount, and hospital stay were also significantly less than those in patients underwent SLNB and IL. The median follow-up time was 41 months and the 3-year overall survival rate was 85% in the whole series. Recurrences were observed in 11 patients and 9 of them died of the tumor with the median survival time of 15 months. In patients with positive SLN (group A and B), the 3-year overall survival rate was 58% with 8 patients died of the disease, including 4 in group A and 4 in group B. In patients with negative SLN (group C and D), the 3-years overall survival rate was 97% with one patient in group D died of the tumor, and significantly higher than that of patients with positive SLN (P=0.003). The 3-year overall survival rate was significantly difference. In univariate analysis by log-rank test showed that, neither in patients with nor without SLN metastasis the prognosis differed with respect to surgical approaches (group A vs B, P=0.709;group C vs D, P=0.253). Univariate analysis by log-rank test showed that, lymph node metastasis, pathological grade, depth of invasion, and tumor location could significantly affected survival (P<0.05), whereas age, tumor diameter, and surgical approach didn′t (P>0.05). Multivariate analysis showed that lymph node metastasis (RR=21.57, 95%CI:2.68-173.10, P=0.002) and tumor location (RR=7.85, 95%CI:1.79-34.50, P=0.024) were the independent factors for overall survival. Conclusions Lymph node metastasis is an independent prognosis factor for patients with early-stage vulvar cancer. SLNB could accurately diagnose the status of lymph nodes and help to decide subsequent treatment. The omissions of IL in patients with negative SLN avoid surgical morbidity and shorten postoperative recovery period without an increased risk of recurrence.

BACKGROUND: Strontium-doped calcium polyphosphate (SCPP) is a new type of bone repair materials with good biocompatibility and controlled degradation. The preliminary studies of our group indicate their role in promoting angiogenesis,but its mechanism is unclear.OBJECTIVE: By co-culturing osteoblasts ROS17/2.8 with SCPP in vitro to observe cell proliferation and the secretion of vascular endothelial growth factor (VEGF).DESIGN, TIME AND SETTING: A contrast study was performed at the Laboratory of Tissue Engineering of Sichuan University from October 2008 to June 2009.MATERIALS: A series of calcium polyphosphate (CPP) respectively containing 0%, 1 %, 2%, 5%, 8%, and 10% Sr~(2+) were prepared. ROS17/2.8 osteoblastic cell strain was provided by Laboratory of Transplantation Immunity and Transplantation Engineering, West China Hospital, Sichuan University.METHODS: ①Preparation of cell scaffold complexes: The materials were placed in 24-well plates, then 300 μL cell suspension with a concentration of 2×10~7 cells/Lwas inoculated into each hole. These complexes were cultured for 14 days and the liquid was changed every two days. ②These complexes were measured by MTT assay to observe the proliferation of osteoblasts on the 1~(st), 3~(rd), 5~(th), 7~(th), 10~(th) and 14~(th) days, respectively. ③ The centrifugal supernatant of the complex cultured for seven days was measured by ELISA assay to check the secretion of VEGF.MAIN OUTCOME MEASURES: The proliferation of osteoblastic cells on SCPP and CPP was observed. The amount of VEGF protein secreting from osteoblastic cells was detected.RESULTS: The results of MTT showed that, compared with the CPP group, SCPP groups could promote the proliferation of osteoblasts, and 8% SCPP group was the best; ELISA results showed that, compared with the CPP group, SCPP groups could increase the amount of VEGF protein secretion, of which the promoting role of 8% SCPP was the most obvious (P < 0.05).CONCLUSION: When cultured with osteoblasts, SCPP can promote cell proliferation, and can significantly increase the secretion of VEGF; moreover, 8% SCPP is the best, which reveals a certain mechanism of its promoting angiogenesis.

Objective To prepare the survival motor neuron(SMN)polyclonal antibody and explore the localization of SMN protein in transfected cells and its expression in skeletal muscles of patients with spinal muscular atrophy(SMA).Methods A prokaryotic expressional plasmid named pET-28? (+)/SMN was constructed and SMN-His fusion protein was induced.The fusion protein was used to immunize New Zealadd rabbits to prepare SMN polyclonal antibody.A eukaryotic expressional plasmid named pcDNA3.1/myc-HisB-SMN was constructed and used to transfect CHO cells.Skeletal muscles were collected from 3 patients with bone fracture who were regarded as normal controls, and 3 SMA patients of type Ⅰ, 3 of type Ⅱ and 3 of type Ⅲ who were ascertained by genetic analysis.Western-blotting and immunofluorescence stain were applied to study the expression of SMN in transfected CHO cells and skeletal muscles of normal individuals and SMA patients.Results Correct pET-28a(+)/SMN prokaryotic expressive plasmid was constructed and SMN-His fusion protein was obtained from E coli BL21 transformed with pET-28a(+)/SMN.Then, rabbit anti-human full-length SMN polyclonal antibody of high specificity and sensitivity was obtained from rabbits immunized by SMN-His fusion protein.SMN proteins were shown diffusedly locating in the cytoplasm and nucleus of CHO cells transfected with pcDNA3.1/myc-HisB-SMN plasmid and mainly accumulating around the nucleus.The results of Western-blotting were as follows:the average ratio of SMN band density to glyceraldehyde phosphate dehydrogenase(GAPDH)band density (SMN/GAPDH)is 0.619 in skeletal muscles from normal controls, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type Ⅲ and Ⅱ were 0.347 and 0.340 respectively, which were lower than that of normal controls.However, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type I was only 0.079, which was quite lower than that of normal controls.Conclusions The rabbit anti-human full-length SMN polyclonal antibody is of high specificity and sensitivity, which makes the basis for the research of SMN function and SMA pathogenesis.There may be a correlation between the SMN level in skeletal muscle and the severity of disease.

Sanjie Zhentong capsules were scanned by using a near infrared spectra probe with different drug mass fraction and the spectral information of capsule shells and contents in it were obtained. Then partial least squares (PLS) models were developed for the prediction of mass fraction of Fritillariae Thunbergii Bulbus and Resine draconis in Sanjie Zhentong capsules. The correlation coefficient (r9c)) and root mean standard error( RMSEC) of 0.949 5, 0.958 2 and 4.742 4, 4.135 7. The models obtained correlation coefficient (r(v)) of 0.919 2, 0.936 7 and root mean square error (RMSECV) of 6.158 9, 5.037 3 respectively in the training set. The paired T test analysis of statistics showed that there were no significant difference between predictive values and measure values. The established models reflected a strong prediction performance and can meet the needs of the production.
Capsules ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Least-Squares Analysis ; Spectroscopy, Near-Infrared ; methods

This study examined the effects of over-expression of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the cell cycle and survival of human glioma cell line U87 and U251 and explored the possible mechanisms.The LRIG3 gene was transduced into U87 and U251 cells respectively by using lentivirus and the transduced cells were selected by puromycin.The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blotting.The apoptosis rate was detected by Annexin V-FITC/PI double labeling and the cell cycle was flow cytometrically analyzed.Compared with control cells,LRIG3 mRNA expression in U251 and U87 cells transduced with pLVX-DsRed-LRIG3-Monomer-N1 were increased by 77.6％ and 129.7％,and LRIG3 protein expression was raised by 141.3％ and 322.7％,respectively.Cell cycle analysis showed that LRIG3 over-expression increased the percentage of cells at G0/G1 phase (P＜0.01).Over-expressed LRIG3 could significantly promote the apoptosis of U87 and U251 cells (P＜0.05).These findings suggest that the over-expression of LRIG3 could arrest the cell cycle in G0/G1 phase,and promote apoptosis of U87 and U251 cells.

AIM To observe the oxidant stress and opoptotic effects of anisodine hydromide (AH) on chronic cerebral hypoperfusion (CCH) rats.METHODS In vivo CCH models were established in adult male SpragueDawley rats by permanent ligation of bilateral common carotid arteries [two-vessel occlusion (2-VO)] surgery.Rats were randomly divided into six groups,sham group,model group,positive group of n-butylphthalide and sodium chloride injection,and AH groups (1.2 mg/kg high-dose group,0.6 mg/kg medium-dose group,and 0.3 mg/kg low-dose group).Antioxidant indices including the activity of SOD,CAT,LDH and iNOS and the content of GSH and NO were measured.In the in vitro trial,PC12 cells were divided into control group,model group,positive group of n-butylphthalide,and AH groups (100 μmol/L high-dose group,50 μmol/L mediumdose group,and 25 μmol/L low-dose group),and the hypoxic models were established by treating PC12 cells with CoCl2.The cells had their release of NO and LDH detected,their cellular apoptosis determined by Hochest 33342 fluorescence staining,and the expression of P53 protein identified by IF (immunofluorescence) and Western blotting method.RESULTS The in vivo trial revealed AH's enhancement in serum SOD activity and inhibition in serum iNOS activityof the CCH rats,and its power in the cerebral GSH and LDH release reduction.The in vitro trial showed the resultant lower LDH and NO release,decreased number of neuro-apoptosis,and inhibited P53 pro tein expression after AH intervention.CONCLUSION The antioxidant and antiapoptotic effects of AH on CCH rats may be associated with down regulation of P53 protein.

Objective To investigate the role of Wnt/β-catenin signaling pathway in the process of adipose-derived stem cells (ADSCs)differentiating into neurons.Methods The third generation of ADSCs were divided into three groups.Neural induction medium was used in induction group and DDK-1 was added into neural induction medium in inhibition group.Normal culture medium was used in control group.Ten days after culture,Real-time PCR and Western blot were used to detect the expressions of NSE,β-catenin and GSK-3βin each group.Results The expressions of NSE and β-catenin were high but the expression of GSK-3 was low in induction group.The expression ofβ-catenin was lower but GSK-3 was higher in inhibition group;the expression of NSE was much lower than that in induction group,but higher than control group.Conclusion The differentiation of ADSCs into neurons is related to activation of Wnt signaling pathway,but Wnt signal pathway is not the only pathway to regulate ADSCs differentiation which may be controlled by different signaling pathways.