Nerve growth factor is one of the most important factors playing an important role in regulating the growth, development and survival of the neuron. The purified NGF from human placenta has been reported, the tissue from which can be isolated the NGF is very limited. It is important for basic research and clinic application to expression hNGF by genetic engineering. By polymerase chain reaction,gene fragment encoding the mature part of ?-NGF was amplified using the DNA of human placenta as template. The fragment was sequenced and inserted into expression vector pET-15b, and the recombinant expression vector pET15b-NGF was transformed into E.coli BL21(DE3)pLysS. After inducing with IPTG the NGF was higher expressed up to 25% of the total cell proteins. The expression product was purified with metal chelate affinity chromatography on Ni-IDA agarose under denaturing condition. The purity of rNGF was higher than 90% and yield of rNGF was 4.56mg/L expressing bacteria. SDS-PAGE revealed the NGF expression product had a Mr 16kDa. Western-blot displayed the recombinant product had strong immunological activity with rabbit anti-human ?-NGF polyclonic antibodies. The expression products were dealed with solubilizing inclusion bodies and refolding protein. The test of nerve fiber growth of chicken embryo DRG neurons displayed rNGF had biological activity.

OBJECTIVETo study and compare the prophylatic efficacy of levetiracetam, valproate and phenobarbital on febrile convulsions in rats.

METHODSSixty Wistar rats were randomly administered with levetiracetam (200 mg/kg), valproate (250 mg/kg), phenobarbital (30 mg/kg) or normal saline (8 ml/kg) for 5 days. Five days later, febrile convulsions were induced by hyperthermal bath (45 Celcius degree). The latency, duration and the severity of seizures were observed.

RESULTSIn all the three drug-treated groups, the latency was significantly prolonged, and the duration and the severity of seizures were notably reduced compared with the saline group (P<0.05 or 0.01). The phenobarbital group had the shortest duration of seizures and the least severe seizures among the three drug-treated groups. There were no significant differences between the levetiracetam and valproate groups.

CONCLUSIONSContinuous administration of levetiracetam, valproate or phenobarbital is effective in preventing recurrent febrile convulsions in rats. Phenobarbital appears to be more effective than levetiracetam and valproate. There were no significant differences in the prophylactic efficacy between levetiracetam and valproate.

Animals ; Anticonvulsants ; therapeutic use ; Male ; Phenobarbital ; therapeutic use ; Piracetam ; analogs & derivatives ; therapeutic use ; Rats ; Recurrence ; Seizures, Febrile ; prevention & control ; Valproic Acid ; therapeutic use

Objective To investigate the clinical value of cardiac troponin Ⅰ(cTnⅠ) and creatinine kinase MB(CK-MB) in early diagnosis of myocardial injury(MCI) in neonatal asphyxia.Methods The serum cTnⅠ and CK-MB in neonates [34 with asphyxia and MCI,38 with asphyxia but no MCI(NMCI)],and 30 cases of normal control(NC) were measured with direct immunoassay chemiluminometric technology and immunoinhibition enzymes-activated assay.Results The cTnⅠ level in NC group had no changes within 10 days after birth,MCI group were significantly higher than those in NMCI and NC groups(all P0.05).The diagnostic sensitivity,specificity and accuracy of cTnⅠ for neonates with MCI were 91%,88% and 89%,respectively;and of CK-MB were 85%,68% and 74%,respectively.Conclusions cTnⅠ and CK-MB can be taken as early diagnostic markers of MCI in neonates with asphyxia,(cTnⅠ) is better than CK-MB.

Objective To evaluate the precision of GFR using Gates method and compared with the results from renal pathological changes. Methods Twenty-seven patients whose 99Tcm-DTPA renograms had no obvious uptake phase were enrolled in Group A, and 27 patients whose 99Tcm-DTPA renograms had obvious uptake phase were enrolled in Group B. The measurement of GFR by Gates method was compared to the creatinine clearance measured and predicted by Cockroft-Gault (C-G), modification of diet in renal disease (MDRD) and SCr level. Renal pathological changes in two groups were compared using Pearson correlation and t test analysis. Results In Group A, GFR determined by Gates method did not show correlation with that estimated by C-G or 1/SCr (r = 0. 357,0. 376, both P ＞0.05), but was significantly correlated with GFR estimated by MDRD(r = 0. 440, P ＜ 0.05). In Group B, GFR determined by Gates method showed significantly correlation among GFR estimated by MDRD, C-G, and 1/SCr (r =0. 471, 0. 527,0. 452, all P ＜ 0.05). Renal tubulointerstitial damage score in Group A was higher than that in Group B (7.15±2.32, 3.70±3.06, t=4.66, P ＜0.001). Conclusions GFR determined by Gates method is less precise when 99Tcm-DTPA renogram has no obvious uptake phase than that when 99Tcm-DTPA renogram has obvious uptake phase. Renal tubulointerstitial damage is a strong indicator of no obvious uptake phase in 99Tcm-DTPA renogram.

OBJECTIVETo study the effects of sodium butyrate (SB) on growth, apoptosis and telomerase activity in Hep-2 cells.

METHODSGrowth inhibition effect of SB on Hep-2 cells was assessed by methyl thiazolyl tetrazolium (MTT) assay. Morphological alterations were observed by electronic microscope. Cell apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) method, DNA fragmentation and flow cytometry (FCM). Cell cycle was analyzed by FCM. Telomerase activity was examined by telomeric repeat amplification protocol (TRAP)-silver staining. The expression status of telomerase subunits was analyzed by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSA time-and dose-dependent inhibition was detected in cells treated with SB. Typical morphological changes of apoptotic cells were observed under electronic microscopy. The characteristic DNA fragmentation of apoptotic cells was detected by agarose gel electrophoresis. Apoptosis and the changes of cell cycle were confirmed by TUNEL method and FCM. The apoptosis indexes of the cells before treatment and at 72 h after SB (2.5 mmol/L) treatment were 2.27 +/- 1.18 and 33.50 +/- 2.75 respectively, the apoptosis rates were 2. 86% and 31. 28% respectively, the proportion of the cells at G0/G1 stage were 50.38% and 70.88% respectively, the proportion of the cells at S stage were 27.40% and 8.20% respectively, and the proliferation indexes of the cells were 49.62% and 29.12% respectively. Telomerase activity and expression level of human telomerase reverse transcriptase (hTERT), the key subunit of telomerase, decreased after SB treatment. No significant changes were observed in the expression of human telomerase RNA (hTR) and human telomerase associated protein (hTP1), the other two subunit of telomerase.

CONCLUSIONSB could inhibit growth of Hep-2 cells and induce apoptosis in the cells, and inhibit telomerase activity by decrease expression level of hTERT.

Apoptosis ; drug effects ; Butyrates ; pharmacology ; Cell Cycle ; drug effects ; Hep G2 Cells ; Humans ; Sodium ; pharmacology ; Telomerase ; metabolism

Adolescent ; Child ; Collagen Type I ; metabolism ; Extracellular Matrix ; metabolism ; Female ; Fibrillin-1 ; Fibrillins ; Heart Defects, Congenital ; metabolism ; pathology ; Humans ; Male ; Microfilament Proteins ; metabolism

OBJECTIVETo investigate the effect of effective parts of Zingiber officinal (EPZ) on the adhesion of ECV-304 cells with monocytes cultivated in vitro and on the expression of monocyte chemotactic protein-1 (MCP-1) and adhesion molecules.

METHODThe model of ECV-304 cell oxidative stress injury was established by hydrogen peroxide (H2O2). Then EPZ-contained blood serum was taken as experimental drug. The adherence of monocytes to endothelial cell were measured by method of rose Bengal. The total RNA of cells was extracted. The intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and MCP-1 mRNA expression in cells were detected by RT-PCR. MCP-1 protein expression were detected by ELISA.

RESULTEPZ could decrease the adhesion of monocytes with ECV-304 cells obviously. Meanwhile it could diminish the expression of ICAM-1, VCAM-1 and MCP-1 in injured ECV-304 cells.

CONCLUSIONEPZ could inhibit H2O2-induced ICAM-1, VCAM-1 and MCP-1 expression in ECV-304 and could inhibit the adherence of monocytes to endothelial cell, which may result in the protect effect in endothelial cells.

Animals ; Cell Adhesion ; drug effects ; Cell Line ; Cells, Cultured ; Chemokine CCL2 ; biosynthesis ; genetics ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Ginger ; chemistry ; Humans ; Hydrogen Peroxide ; pharmacology ; Intercellular Adhesion Molecule-1 ; biosynthesis ; genetics ; Monocytes ; cytology ; drug effects ; metabolism ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Cell Adhesion Molecule-1 ; biosynthesis ; genetics

Objective To evaluate the acute and delayed killing effect of high intensity focused ultrasound (HIFU) on Echinococcus granulosus(E. granulosus)protoscolices in vitro.Methods E. granulosus protoscolices were treated with different dosage of effective power(0,25,50,100,200,250 W)and time(5,10,20,30,40,50,60 s)of HIFU in vitro to obtain the dosage-effect curves.Then the survival pmtoscolices were incubated,and the mortality of each group was counted daily.The protoscolicidal effects were investigated by trypan blue exclusion assay.Results Compared with the untreated group,the Vitality of E.granulosus protoscolices significantly decreased immediately after treated by HIFU of different dosage(F=5201.59 vs 1865.65,P＜0.05),there were the interaction both different dosage and time(F=214.50,P＜0.05).The protoscolices were broken into pieces by HIFU of 250 W×40 s,whereas the growth of the surviving protoscolices after exposed to HIFU was obvious suppressed.Both the acute killing effect and the delayed inhibitory effect showed a dosage-dependant manner.The inhibitory effect increased along with the increased dosage of HIFU(P＜0.05).The inhibitory effect in 50 W×10 s group was stronger than 25 W×20 s group(P＜0.05).The mortality was increased in parallel with the increase of HIFU dosage.Conclusions HIFU show an effective immediately killing effect,as well as a growth-inhibiting effect on the E.granulosus protoscolices in vitro.

OBJECTIVEThe aim of this study was to investigate the expression of transforming growth factor-beta1 (TGF-beta1) and its signaling pathway molecules in oral squamous cell carcinoma (OSCC) and analyze the association between these factors and genesis and metastasis of OSCC.

METHODSThe express of TGF-beta1, TbetaRI, TbetaRII and Smad4, a pivotal downstream molecule of its signaling, in 10 normal oral mucosa tissues and 108 OSCC was detected by SP immunohistochemistry, and thier correlation with genesis and metastasis of OSCC were assessed.

RESULTSThe expressions of TbetaRII and Smad4 were lower in the tumors (34.3%, 38.9%) than those in the normal oral epithelium (80.0%, 100.0%, P < 0.05). The positive expression rates of TGF-beta1 and TbetaRI in the normal oral epithelium and OSCC were not significantly different (P > 0.05). There was an inverse correlation between TGF-beta1, Smad4, TbetaRII, TbetaRI expression and clinical stages (P < 0.01). The expression of TGF-beta1 was related with histological differentiation and tumor localization (P < 0.05). There was a relationship beteween Smad4 expression and histological differentiation and lymph node metastasis (P < 0.05). The expression of TbetaRII in the samples with lymph node metastasis was less than that in the ones without lymph node metastasis (P < 0.01), although there was no association between expression of TbetaRII and lymph node metastasis status.

CONCLUSIONThere is an important relationship between the abnormal TGF-beta1/Smad4 signal pathway and genesis and development of OSCC, while the low expressed Smad4 and TbetaRII may promote the metastasis of OSCC.

Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Membrane ; metabolism ; Cytoplasm ; metabolism ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Mouth Neoplasms ; metabolism ; pathology ; Neoplasm Staging ; Protein-Serine-Threonine Kinases ; metabolism ; Receptors, Transforming Growth Factor beta ; metabolism ; Signal Transduction ; Smad4 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the inhibitory effect of transforming growth factor (TGF)-β₁ on oral squamous cell carcinoma (OSCC) Tb cell line.

METHODSCell counting method was used to examine the inhibitory effect of TGF-β₁ on Tb cell and flow cytometry (FCM) assay performed to measure the changes of cell cycle. Superarray was used to screen the changing expression of genes in TGF-β₁/Smads signaling pathway.RT-PCR method was used to detect the results of Superarray.

RESULTSTGF-β₁ showed significant inhibiting effect on OSCC Tb cell line. TGF-β₁ blocked the cell cycle at G₁ phase. The expression level of activin receptor-like kinase-1 (ACVRL-1), anti-mullerian hirmine (AMH), cyclim-dependent kinase inhibitor-2B (CDKN-2B) and transforming growth factor-beta-indnced factor (TGIF) was higer in the cells treated with TGF-β₁ than in control, while TDGF-1 expression was down-regulated. ACVRL-1 and CDKN-2B gene expression was consistent with the results of Superarray.

CONCLUSIONSTGF-β₁ can inhibit the growth of OSCC Tb cell line. The mechanism may be related to the regulation of cell cycle and the expression of ACVRL-1 and CDKN-2B in TGF-β₁-Smads signaling pathway.

Activin Receptors, Type II ; metabolism ; Anti-Mullerian Hormone ; metabolism ; Carcinoma, Squamous Cell ; pathology ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Humans ; Neoplasm Metastasis ; Signal Transduction ; Transforming Growth Factor beta1 ; pharmacology