Objective To study the relationship between extracellular signal-regulated kinases（ERK）phosphorylation and indirect activa-tion of the epidermal growth factor receptor（EGFR）in cultured cerebellar granule neurons induced by homocysteine.Methods We pri-marily cultured 7-day-old CD-1 mouse cerebellar granule neurons for 7 days and treated with different agents（saline,AG1478,Hcy and AG1478＋Hcy）.The phosphorylation of ERK was analyzed by Western blot,and apoptotic rate by flow cytometry.Results Compared with saline and AG1478 groups,the phosphorylation of ERK was increased in Hcy group（P 〈 0.05）.When we pretreated the cells with tyr-phostin AG1478,an inhibitor of EGFR,there was no difference in the activation of ERK compared with the control group.We found obvious neuronal apoptosis when 7-day-old CD-1 mouse cerebellar granule neurons were cultured for 7 days and treated with Hcy for 6 hours,and the apoptosis induced by Hcy was aggravated when the neurons were pretreated with AG1478（P 〈 0.05）.No apoptosis was found in the saline and AG1478 groups.Conclusion The role of Hcy on ERK activation suggests the involvement of an indirect activation process of EGFR,which has neural potentially protective effect.

Objective To explore ear reconstruction using autologous rib cartilage ear framework by multi-layer spliced sculpture in microtia patients.Methods From Feb 2010 to May 2011,29 microtia patients were subjected to ear reconstruction using autologous rib cartilage ear framwork by four-layer spliced sculpture.Results In one operation 29 patients achieved 2 cm transverse height of reconstructed ears which were basically coincidence with the normal side.Patents and their families were all satisfied with the results.Follow-up of 3-12 months showed that only 1 case reconstructed-ear height was significantly lower transverse process.Owing to sleeping position,the patient did not protect the reconstructed ear,leading to frequent reconstructed-ear pressure.Conclusions The method of multi-layer spliced sculpture autologous rib cartilage ear reconstruction has good clinical effect.It can make reconstructed ear reach nomal transverse height and avoid the third rib cartilage transplant operation to continue increasing the transvers height.

Objective To evaluate the effect of angiotensin Ⅱ (Ang Ⅱ ) on the change of nephrin phosphorylation both in Ang Ⅱ-infused rat model and cultured podocytes.Methods Thirty Wistar rats were subcutaneously embedded with osmotic minipumps and randomly divided into 3 groups according to receiving either Ang Ⅱ at a dose of 400 ng· kg-1· min-1 or Ang Ⅱ +telmisartan at a dose of 3 mg·kg-1 ·d-1,or normal saline as a control group.Blood pressure and 24-hour urinary albumin were measured at 0 d,7 d,14 d,21 d and 28 d of the experiment.Renal histomorphology was evaluated through electron microscopy.The concentrations of Ang Ⅱ both in blood plasma and kidney were detected by radioimmunoassay.In vitro,cultured murine podocytes were exposed to Ang Ⅱ (10-6 mol/L) pretreated with or without losartan (10-5 mol/L) for different time periods.Nephrin and its phosphorylation expression were analyzed by Western blotting.The distribution of F-actin was presented by FITC-phallodin labeling.The change in phenomenon of F-actin was evaluated by cortical F-actin score index (CFS).Results (1)Ang Ⅱ-infused rats exhibited increased Ang Ⅱ concentration,significant hypertension and marked albuminuria.(2)In Ang Ⅱ-infusion group,nephrin expression was decreased (P＜0.05).Ang Ⅱ-receiving rats displayed diminished phosphorylation of nephrin.(3)In vitro,the phosphorylation of nephrin was significantly reduced after Ang Ⅱ stimulation for 3-6 hours (P＜0.05).(4)Ang Ⅱ stimulatation resulted in irregularly arrangement of F-actin followed by the redistribution of F-actin to podocyte periphery and formation of F-actin ring,in which the CFS obviously increased compared to control (P＜0.05).Conclusions Phosphorylation of nephrin is important for the survival status of podocytes.Ang Ⅱ-induced nephrin dephosphorylation may be an important molecular mechanism for Ang Ⅱ-induced podocyte cytoskeleton rearrangement and foot process effacement.

Serum cortisol levels during oral glucose tolerance test (OGTY) were measured in subjects of type 2 diabetic pedigrees. The results showed that cortisol levels during OGTF were higher in type 2 diabetic patients than those in non-diabetic first-degree relatives and normal controls. Fasting cortisol level was positively correlated with fasting plasma glucose level in type 2 diabetic pedigree members. These results suggest that the dysregulation of hypothalamic-pituitary-adrenal axis may coexist in type 2 diabetic patients.

Objective To investigate the change of serum non-esterified fatty acid (NEFA) level in nondiabetic first-degree relatives of type 2 diabetics, and to explore the related factors in the change.MethodsSerum lipid profile, plasma glucose and insulin levels were measured in 186 type 2 diabetic patients, 565 nondiabetic first-degree relatives of type 2 diabetics and 149 normal controls. Results (1) The fasting NEFA level in first-degree relatives was significantly lower than that of type 2 diabetic patients [(0.53±0.28 vs 0.63±0.31) mmol/L,P＜0.01]and HOMA-IR was significantly higher than that of normal controls (0.98±0.51 vs 0.89±0.47,P＜0.01). (2) The fasting NEFA level in the first-degree relatives with higher body mass index (BMI), plasma glucose or area under curve of glucose concentration (AUCglu) was higher than that in those with lower BMI, plasma glucose , blood pressure or AUCglu (all P＜0.05). (3) NEFA showed significantly positive correlations with BMI, systolic blood pressure, diastolic blood pressure (DBP), AUCglu in the first-degree relatives by correlative analysis (r=0.12, r=0.148, r=0.21 and r=0.281 respectively, all P＜0.05). Stepwise linear regression analysis showed that DBP, AUCglu and age were the independent risk factors of NEFA (all P＜0.01). Conclusion Insulin resistance exists in nondiabetic first-degree relatives of type 2 diabetics, which seems to be related to elevated NEFA levels.

Objective To explore the variation and influential factors of high sensitive C-reactive protein (hs-CRP) level in type 2 diabetic family members. Methods A total of 427 type 2 diabetic patients, 377 non-diabetic first-degree relatives of type 2 diabetics and 135 normal control subjects without diabetic family history were recruited. Serum hs-CRP, clinical and biochemical parameters were measured. The relations among indicators were analyzed. Results Compared with normal control subjects, serum hs-CRP levels in type 2 diabetics and first-degree relatives were significantly increased (both P＜0.05), and the increment was even marked in type 2 diabetics than that in first-degree relatives (P＜0.01). The serum hs-CRP levels in type 2 diabetics and first-degree relatives were positively associated with body mass index, waist-hip ratio, abdominal circumference, postgrandial 2 h plasma glucose, fasting and postgrandial 2 h serum insulin, HOMA-IR, triglyceride, creatinine and negatively correlated with high density lipoprotein-cholesterol. In first-degree relatives, serum hs-CRP level was positively associated with systolic blood pressure and HOMA-β. Conclusion As in type 2 diabetic patients, there exists inflammatory reaction in the non-diabetic first-degree relatives of type 2 diabetics, which may play an important role in the pathogenesis of type 2 diabetes mellitus.

Adolescent ; Adult ; Aged ; Cadherins ; genetics ; metabolism ; physiology ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; Ki-67 Antigen ; genetics ; metabolism ; Male ; Microtubule-Associated Proteins ; genetics ; metabolism ; physiology ; Middle Aged ; Neoplasm Invasiveness ; physiopathology ; Pituitary Neoplasms ; pathology ; physiopathology ; Young Adult

This article reviews the resultsof the basic research about epidermal growth factor and its receptor,and the development of the novel drug, EGF eyedrop, that containing chemically synthesised EGF gene,the construction of EGF expression vector, the transformation of the host cells , the purification of the recombinant protein EGF, the preparation of three batches of the EGF product and identification, the preclinical and clinical trials. Relevant studies show that recombinant EGF consisting of 51 amino acids can be secreted into the medium under the control of the α factor leading sequence in the yeast cells.The EGF can accelerate the growth of corneal-limbal epithelial cells and the healing of an alkali burned corneal. The EGF can be used in curing oral cavity ulcer and skin burned wound. And it has the preventive effects on experimental duodenal ulcer of rat. The antiserum was made for test of the con- centration of blood EGF and urine EGF by RIA. Data from studies demonstrate the inhibition effect of EGF on the growth of tumor cells, such as A431 and BT325 cells in the presence of high EGF concentration (＞10 ng/ml). The expression of EGFR and DNA ploidy in renal carcinoma has clinical significance. Crystallization and preliminary x-ray diffraction studies of the EGF has been made. The MW of the EGF product is 6000, and the pI is about 4. 6 and it has correct N-terminal amino acids sequences , immunogenicity and biological activity. There is no vestige of the DNA of the yeast cells. Animal experiments reveal that there is no cumulation of the EGF in the body, and EGF can promote corneal epithelial healing. There is no toxicological effect during cornea wound healing of rabbit. A randomized, double-blind, placebo-controlled, multi-center clinical trial was conducted in four hospitals to assess safety,ocular tolerance and efficacy of an ophthalmic solution of EGF for 200 cases of cornea transplantation and 247 cases of nebulae. Unequivocal results were obtained as the eyedrop really accelerate the wounded cornea healing. So, the EGF eyedrop as a novel drug of class I is approved by the National Drug Administration, and this is the first gene engineering drug that come from yeast expression system in China.

Tumor immune checkpoint therapy is a clinical treatment strategy developed based on the new principle of the inhibition of negative immune regulation. In this article, the tumor immune checkpoint therapy and the drug delivery strategies were reviewed, mainly including immunity and tumor therapy, tumor immune checkpoint therapy and its mechanism of action, clinical application of tumor immune checkpoint therapy and therapeutic drugs, immune resistance of programmed cell death protein 1 (PD1)/programmed cell death ligand 1 (PDL1) treatment and countermeasures, drug delivery strategies for tumor immune checkpoint therapeutic agents, etc. As a revolutionary new immunotherapy strategy, tumor immune checkpoint therapy has shown obvious superior therapeutic efficacy in a variety types of tumor. However, tumor immune checkpoint therapy is also faced with a big challenge, namely, immunotherapy resistance. With the discovery of new mechanism, the continuous development of new therapeutic drugs and delivery strategies, tumor immune checkpoint therapy is expected to further improve the clinical efficacy of tumor.

OBJECTIVETo isolate and clone the differentially expressed genes induced by epithelial growth factor (EGF) with inhibiting dosage in cultured glioma BT325 cells and understand the molecular mechanism that inhibits glioma cells growth.

METHODSUsing differential display reversed transcription polymerase chain reaction (DDRT-PCR) method to analyze the differentially expressed cDNA in BT325 cells induced by EGF with inhibiting dosage. After sequencing and homology research, the differentially expressed cDNA fragments were further confirmed by Dot blot analysis and one of them by Northern blot.

RESULTSUp-regulated genes cDNA fragments were isolated in growth inhibited BT325 cells. It was found that five cDNA fragments were highly homologous to the known human genes, while one was a fragment of a novel genes. Among these genes, one has coding sequence homology with transaldolase (TAL), which has been proved to be associated with apoptosis in recently research.

CONCLUSIONSHigh-dose EGF could change the expression of many genes in BT325 cells. EGF can inhibit the growth of BT325 cell growth, which may be resulted from its potential role in promoting TAL gene expression and thus inducing cell apoptosis.

Base Sequence ; Brain Neoplasms ; genetics ; pathology ; Cell Division ; drug effects ; Cloning, Molecular ; Epidermal Growth Factor ; pharmacology ; Gene Expression Regulation, Neoplastic ; drug effects ; Genes, Tumor Suppressor ; Glioma ; genetics ; pathology ; Humans ; Molecular Sequence Data ; Sequence Analysis, DNA ; Tumor Cells, Cultured