To express and identify fibronectin C-terminal heparin-binding domain (FNCH BD) polypeptides in Pichia pastoris expression system and study its function, the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector. After sequenced, the fragment was inserted into pAo815SM vector, and then cloned into the expression vector pPIC9k. The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115. The positive yeast clone was screened by G418 resistant, and the target protein was induced to express in the medium containing 0.5% methanol. The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography. The purified product was analyzed with mass spectrogram, SDS-PAGE, Western blotting and heparin affinity chromatography. The results showed that the target protein was around 32 kDa and the purity of the product was above 95%. FNCHBD could be specifically recognized by fibronectin polyclonal antibody. These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris, which provides a good strategy to further studies.
Fibronectins ; biosynthesis ; chemistry ; genetics ; Genetic Vectors ; Heparin ; metabolism ; Peptides ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

Objective To explore the guidance significance of the functional MRI and DTI (fMRI ,DTI) ,intraoperative ultra-sound(IOUS) ,neuronavigation ,electrocorticography(EcoG) monitoring used in surgical treatment of low-grade gliomas with epi-lepsy as main symptom located near the eloquent brain regions .Methods 23 neurosurgical patients in the First Affiliated Hospital of Chongqing Medical University during 2009-2010 were performed the retrospective analysis .The preoperative fMRI ,DTI deter-mined the positional relation between the lesions with the conduction bundle and the eloquent brain regions ,the electrophysiological and imageological examinations positioned the epileptogenic focus and lesions ,the MRI-mediated neuronavigation system was adopt-ed to formulate the surgical plan and choose the best surgical approach ,IOUS was used to perform the realtime monitoring for pre-cisely positioning the lesion range and determining the extent of resection ,and the intraoperative EcoG was adopted to determine the epileptogenic focus localization ,the lesions and the epileptogenic focus was dealed by the operating microscope for avoiding the func-tional region ,and the patient′s prognosis was evaluated and recored in detail after operation .Results By the precisely positioning the lesions and epileptogenic focus by fMRI ,DTI ,neuronavigation and ultrasound ,the lesion resection degrees by the operative mi-croscope and intraopertaive pathological guidance were 17 cases of Ⅰ-Ⅱ grade ,4 cases of Ⅲ grade and 2 case of Ⅳ-Ⅴ grade .1 case of motor aphasia ,4 cases of hemiplegia and monoplegia and 1 case of disturbance of consciousness after operation were improved by the treatment of neurotrophy ,dehydration and hyperbaric oxygen and discharged from hospital with rehabilitation .No death case occurred .The evaluation of the life quality :20 cases ofⅠ-Ⅱ grade ,3 cases of Ⅲ grade and no vegetable survival case of Ⅳ grade . The evaluation of resection clinical effect :20 cases of cure ,3 cases of improvement ,no case of as before and exacerbation .After fol-lowed up for 6-24 months ,according to Engel classification of seizure efficacy assessment :Ⅰ-Ⅱ grade in 21 cases ,Ⅲ grade in 2 case ,no case of Ⅳgrade .Conclusion fMRI ,DTI ,neuronavigation ,IOUS and EcoG for guiding the operation of low grade gliomas located near the eloquent brain regions can resect the lesion to the largest extent and simultaneously deal with epileptogenic focus , effectively protect the neurological function of the functional region ,greatly reduce the side-injury of the normal brain tissues in the functional region ,at the same time increase the curative effect of symptomatic epilepsy .

Objective:To evaluate the cytotoxicity of colorized zirconia ceramics.Methods:Zirconia ceramic 3Y-TZP blocks were colorized by NH4 VO3 ,FeCl3 ,Co(NO3 )3 ,Ni(NO3 )3 and MoCl3 respectively.The cytotoxicity of colorized zirconia to L-929 cells was examined by immersion method and MTT assay.Results:During 3 days culture,L929 cells in all groups showed similar prolifera-tion and normal morphology.The toxicity gradation of all groups was 0 -I.Conclusion:Zirconia ceramics colorized with the 5 kinds of colorants has no cytotoxicity.

This study was purposed to investigate the effects of siRNA targeting c-myc on apoptosis induction, proliferation in inhibition as well as c-myc protein and mRNA expression in human myeloid leukemia cell line HL-60 cells. C-myc siRNA synthesized in vitro was transfected into HL-60 cells by liposome. Changes of cell morphology were observed. Growth inhibition was detected by MTT assay and colony formation assay, and cell apoptosis was determined by DNA ladder. The expressions of c-myc mRNA and protein were detected by RT-PCR and Western-blot respectively. The results indicated that c-myc siRNA remarkably inhibited the cell proliferation, with an IC(50) value of 150 nmol/L. Data of DNA ladder showed that HL-60 cells apoptosis could be efficiently induced by c-myc siRNA, the apoptosis rate positively correlated with the time duration of treatment with drugs. The c-myc mRNA and protein expressions on HL-60 cells decreased after treatment with c-myc siRNA, which negatively correlated with time duration of treatment. It is concluded that c-myc siRNA can efficiently induce growth inhibition, decrease the expressions of c-myc mRNA and protein, and induce apoptosis in HL-60 cells.
Apoptosis ; genetics ; Cell Proliferation ; Gene Targeting ; HL-60 Cells ; Humans ; Leukemia, Myeloid ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection

Objective:To observe the expression of adenosine monophosphate activated protein kinase （AMPK）/transcription factor EB （TFEB） autophagy signaling pathway protein in type 2 diabetes （T2DM） complicated with nonalcoholic fatty liver disease （NAFLD） rats after intervention with Shihu mixture （SHM）. Method:Among 40 male SD rats， 10 rats were randomly selected as normal group according to body weight. The remaining 30 rats were fed with high-fat and high-sugar diet for 6 weeks， and then intraperitoneally injected with streptozotocin （STZ） to establish a T2DM NAFLD model. They were divided into normal control group （10 mL·kg^-1·d^-1）， model control group （10 mL·kg^-1·d^-1）， metformin group （100 mg·kg^-1·d^-1）， SHM group （11.3 g·kg^-1·d^-1）. The rats in each group were gavaged for 4 weeks. After gavage， the rats were euthanized. Abdominal aortic blood and liver tissue were collected to detect fasting blood glucose （FBS）， glycated serum protein （GSP）， insulin （INS）， triglyceride （TG）， total cholesterol （TC）， high density lipoprotein cholesterol（HDL-C）， low-density lipoprotein cholesterol（LDL-C） content. Htoxylin eosin （HE） staining was performed to observe changes in liver tissue morphology. Western blot was used to detect AMPK/TFEB signaling pathway-related protein expression. Result:Compared with the model control group， FBS and GSP of the SHM group and the DMBG group decreased （P<0.05）， while INS increased （P<0.05）. Compared with the model group， HDL increased in the SHM group and the DMBG group （P<0.05）， whereas TC， TG and LDL contents decreased （P<0.05）. Liver HE staining results showed that both SHM and Metformin could improve the liver morphology of T2DM and NAFLD rats to some extent. Western blot results showed that p-AMPK/AMPK of SHM and metformin increased （P<0.05）， while the expressions of TFEB and LC3Ⅱ increased （P<0.05）. Conclusion:SHM can improve glucose and lipid metabolism by activating AMPK/TFEB autophagy signaling pathway， so as to improve liver pathological morphology.