BACKGROUND:Tissue engineering requires a lot of seed cells. Osteoblasts have become important seed cells in bone tissue engineering. However, it is difficult to culture the osteoblasts, and cellnumber, purity, proliferation and differentiation activity are different obtained by different culture methods. OBJECTIVE:To identify and compare three common primary osteoblat culture methods, and to explore a method for the primary culture of osteoblasts which is easy to operate, economical and effective, in order to provide basis for the further experimental research. METHODS:Calvarias were dissected from newborn Sprague Dawley rats in 72 hours, and osteoblasts were isolated with col agenase digestion method, sequential digestion method and bone tissue method respectively. The morphological observation and cytochemical staining were performed, the growth curve of the cells was drawn with cellCounting Kit-8 method, and the rate of living osteobalsts was counted with trypan blue staining. RESULTS AND CONCLUSION:The proliferation of the insolated and cultured osteoblasts was wel with typical characteristics of osteoblasts, cytochemical staining results were positive. Compared with the sequential col agenase digestion method, the col agenase digestion method presented higher production of osteoblasts and higher cellsurvival rate (P<0.05), and the col agenase digestion method was easier than the sequential col agenase digestion method and cost less than sequential col agenase digestion method. Bone tissue method was the easiest method with less damage to cells, but bone tissue method presented lower production of osteoblasts and cost much more time, which cannot be used in large-scale osteoblast culture. The col agenase digestion method is a simple, efficient and ideal method for isolation and culture of primary osteoblasts.

Objective To study the internalization mediated by heat shock protein 70 (HSP70) receptors expressed on the surface of dendritic cells (DCs). Methods DCs from C57BL/6 mouse bone marrow were divided into HSP70 receptor-blocking group, non-receptor-blocking group and control group. The HSP70-FITC positive cells were detected by flow cytometry (FCM). Results FCM indicated that in HSP70 receptor-blocking group, the HSP70 receptors could be almost totally blocked by 50?g HSP70. With the increase of HSP70-FITC dosage, the number of HSP70-FITC positive cells increased. In non-receptor-blocking group, the rates of the HSP70-FITC positive cells were up to 90% either when 50?g HSP70-FITC or 100?g HSP70-FITC was used with no remarkable difference between them. Conclusions The internalization mediated by HSP70 receptors plays a major role in the stage of ingestion of HSP70 with the characteristics of saturation and priority. Once the receptors are blocked, non-receptor pathway participates in internalization in a dose-dependent manner.

Objective To study the clinical therapeutic effects of early rehabilitation training combined with acupuncture on dysphagia after acute cerebral infarction.Methods One hundred patients with dysphagia caused by acute cerebral infarction were randomly divided into 4 groups:a comprehensive treatment group(25 cases,received rehabilitation training,acupuncture and routine medication),a routine drug group(25 cases,routine medication),a rehabilitation group(25 cases,received comprehensive rehabilitation training and routine mediciation)and an acu-puncture group(25 cases,received acupuncture therapy and routine medication).Dysphagia was evaluated with videofluoroscopic swallowing study(VFSS),and the incidence of pneumonia was observed before treatment and at the 14th day after daily treatment. Results After treatment,the VFSS scores of the 4 groups were significantly higher than before treatment,and VFSS scores in the comprehensive treatment group were significantly higher than those in the other groups.The effectiveness rate in the comprehensive treatment group was significantly higher than that in the other three groups.and the incidence of pneumonia was significantly lower.Conclusions Early rehabilitation com-bined with acupuncture can improve swallowing and reduce the incidence of aspiration pneumonia in acute stroke pa-tients.

Objective To understand the genetic characterization of HA1 gene of influenza A H3N2 viruses circulated in recent years in Hebei.Methods Viral RNAs of 25 H3N2 strains were extracted and amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR).The products of PCR were purified and sequenced,and the sequences were analyzed though biometic software.Results Several amino acid substitutions located in antigenic sites or receptor binding sites were found more in the isolates than the current vaccine virus or the isolates from previous year.Amino acid substitution was found in 3,140,142,144,145,158,159,189,192,193,198,204,225,226 and 227 positions in the isolates during 2003-2008,more amino acid substitutions took place in antigenic determinant A,B and receptor binding site (RBS).New phylogenetic branches appeared continuously during 2003-2008.The H3N2 strains of the same year almost clustered in the same group on the phylogenetic tree.Conclusions Amino acid substitutions continuously occurred in the HA1 genes in influenza A H3N2 viruses isolated in Hebei from 2003 to 2008,it is meaningful to pay close attention to the HA1 variation in order to prevent and control influenza.

AIM To evaluate the effects of diazoxide on the structures and functions of the mitochondria after hypoxia injury. METHODS A myocardial hypoxia injury model on rat is established by subcutaneous injection of isoproterenol (ISO) 5 mg?kg -1. The degree of oxidative phosphorylation, membrane lipid fluidity (LFU), phospholipidase A 2 activity, and phosphlipid content of mitochondria were observed after hypoxia injury. RESULTS Compared with the controls, the levels of RCR, LFU and phosphlipid content were decreased 20.8% (P

Objective To investigate the influences of 5-azacytidine (5-Aza) with different concentrations on mesenchymal stem cells (MSCs) proliferation and differentiation into myoblasts. Methods Bone marrow-derived MSCs of 4 weeks Wistar rats were separated and purified, and then treated with 5-Aza with different concentrations. The growth ability of cell was assayed with methyl thiazolyl tetrazolium (MTT) method. The expression of skeletal muscle actin in MSCs was determined with reverse transcription polymerase chain reaction (RT-PCR) and electrophoresis after MSCs were induced. Results The cell proliferation was not affected by 0 and 1 ?mol ? L-1 5-Aza . There were expressions of skeletal muscle actin treated with 3 -12 ?mol ? L-1 5-Aza. Some cells were obviously enlarged at the 9th day after induction and myotubu-like cells were found at the 12th day when treated with 9 and 12 ?mol ? L-1 5-Aza. 20 - 30 ?mol ? L-1 5-Aza induced the toxic effect on proliferation of MSCs. With the increase of concentration, the proliferation ability of MSCs was weakened. Conclusion 5-Aza affects the expression of regulatory gene to the stem cells and regulate MSCs to orientationally differentiate into myotube-like cells.

This paper reviewed recent advances in clinical and basic researches of scalp acupuncture based on evoked potentials (EPs).The role and significance of various EPs in scalp acupuncture were analyzed and compared.Eventrelated potentials (ERPs) which occupied leading position in EPs application nowadays and the latest advances in the related components of its action were combined to make a prospect of the scientific research methods of scalp acupuncture in the treatment of cerebral apoplexy and other diseases.

Objective To investigate the differences of the correct memorization of a sentence with auditory between the hearing-impaired children after rehabilitation training and normal children.Methods Twenty-four hearing-impaired children and 14 normal hearing children at age 3~4 were selected.The hearing-impaired chil-dren were divided into two groups according to the length of the hearing device.The closed-set sentence test of e-valuation of auditory response to speech (EARS)was used.Results 1 .In the first test,the hearing-impaired chil-dren with hearing device 2 to 3 years whose correct rate was 57.14%were close to the normal children whose correct rate was 58.93%.The hearing-impaired children with hearing devices 1 to 2 years whose correct rate was 32.5%were worse than the children of other two groups 2 .In the second test,the hearing-impaired children with hearing devices 2 to 3 years whose correct rate was 44.64% were better than the hearing-impaired children with hearing devices 1 to 2 years whose the correct rate was 22.5%.The normal children whose the correct rate was 55.36%were better than the children of other two groups.3.There was no significant difference to correctly memorize a sen-tence between three groups in the first test,but there was significant difference to correctly memorize a sentence between the hearing-impaired with hearing device 1 to 2 years and the hearing-impaired children with hearing de-vice 2 to 3 years (P<0.05),and significant differences also existed between the hearing-impaired with hearing de-vice 1 to 2 years and normal children (P<0.05),but there was no significant difference between the hearing-im-paired children with hearing device 2 to 3 years and normal children (P>0.05).Conclusion The length with hear-ing device strengthens the ability of hearing-impaired children in correctly memorizing a sentence after rehabilitation training.And the content of test affects the result ,it provides reference to the hearing-impaired children's auditory training .

ObjectiveTo detect the expressions of β-catenin protein in different pancreatic tissues ( NP,PanINs,IPMNs and PDAC) and evaluate its significance during the carcinogenesis of PDAC.Methods The expression of β-catenin protein in 12 samples of normal pancreatic tissues,52 samples of IPMN (IPMA 20 foci,IPMB 13 foci,IPMC 19 foci),PanINs 118 foci (PanIN-1 73 foci,PanIN-2 29 foci,PanIN-3 16 foci),50 cases of PDAC was determined by using immunohistochemistry. The correlation between β-catenin expression and clinicopathologic characteristics of PDAC was analyzed. Results β-catenin was mainly expressed in cell membrane of NP,the quantity was 4.38 ± 2.11 ; in PanINs,PDAC and IPMN,β-catenin membrane expression was significantly decreased or absent,the β-catenin membrane expressions of PanIN-1,PanIN-2,PanIN-3,PDAC,IPMA,IPMB,IPMC were 5.22 ±2.21,2.24 ±2.31,1.44 ±1.37,2.71 ±2.08,4.85 ±2.28,4.15 ±2.51,2.68 ±2.75.The cytoplasm expression of PanIN-1 was 12.3％ (9/73),while the nuclear expression was 0 ; and the corresponding values were 34.5％ (10/29) and 3.4％ ( 1/29 ) in PanIN-2; 43.8％ (7/16) and 12.5％ (2/16) in PanIN-3; 44.0％ (22/50) and 10.0％ (5/50) in PDAC.The IHCS of β-catenin membrane expression decreased with the severe tissue atypia along the progressive multistage.The β-catenin membrane expression was significantly associated with tumor size,neural infiltration and lymphatic metastasis ( P ＜ 0.05 ).Ectopic cytoplasm expression was significantly associated with tumor size (P＜0.05 ). Ectopicnuclear expression was significantly associated with tumor differentiation (P＜0.01).The membrane or ectopic cytoplasm expression of β-catenin was significantly associated with postoperative survival.ConclusionsAbnormal Wnt/β-catenin signal activation induces decreased β-catenin membrane expression and increased ectopic expression,which is an important mechanism of pathogenesis and development of PDAC,and promotes the growth and metastasis of PDAC.The expression of β-catenin was associated with postoperative survival.

Objective To prepare melanoma antigen n(MAGEn)protein vaccine and to investigate the immune responses and anti-tumor effects of MAGE-n protein vaccine accompanied by CpG-containing oligodeoxynucleotide(CpG-ODN)adjuvant.Methods The DH5? containing the MAGE-n prokaryotic expression plasmid pGEX-MAGE-n was induced and the protein was purified as protein vaccine.The CpG-ODN was synthesized as adjuvant and the C57BL/6 mice were inoculated.The cellular and humoral immune responses were detected by ELISPOT,cytotoxicity assay and enzyme linked immunosorbent assay(ELISA).The antitumor effects were detected through tumor volume and life span.Results The MAGE-n protein accompanied by CpG-ODN could induce strong MAGE-n-specific cellular and humoral immune responses.In the MAGE-n positive B16 tumor model of C57BL/6,the growth velocity of tumor was decreased and the life span was prolonged with the treatment of vaccine.Conclusion MAGE-n protein vaccine accompanied by CpG-ODN adjuvant can induce strong immune responses and anti-tumor effects against MAGE-n positive B16 tumor,which provides a new way for tumor therapy.