Objective To explore an alternative strategy of ganciclovir-mediated cytotoxicity and study the mechanism of enhancing bystander effect of HSV-TK/GCV system by combination gene transfer of Rb gene and HSV-TK gene.Methods Recombinant eukaryotic expression plasmid pcDNA3.1-Rb harbour ing1.65kb of Rb gene was constructed.The pcDNA3.1-Rb and the plasmids tgCMV/HyTK car-ry ing HSV-TK gene were transfected respectively or co-transfected into the osteosarcoma cell line OS732by lipofection using DOTAP reagent.The mRNA expression of Rb gene and HSV-TK gene were detected by RT-PCR and Northern blot.Cell counting,cell cycle analysis and soft agar colony formation test were adopted to observe cell growth features.The expression of gap junction Connexin43gene was performed by RT -PCR and Western blot.The direct confirmation of gap junction intercellular commu ni cation was demonstrated by Lucifer yellow dye transfer.Cell sensitivity to GCV and″bystander effect″of HSV-TK/GCV system were measured by MTT assay.Results The eukaryotic expression plasmid pcD NA3.1-Rb was constructed successfully.The transfected cell lines OS732TK,OS732Rb and OS732RbTK were har-vested.mRNA expression of HSV-TK gene was demonstrated in OS732TK and OS732RbTK cells.Both exogenous and endogenous Rb gene expression could be detected in OS732Rb and OS732RbTK cells si-multa neously.Compared with parental cell OS732,OS732Rb and OS732RbTK cells changed their mor -phology and decreased the growth rate;the ability of soft agar colony formation reduced and the cell cycles were arrested at G 0 G 1 checkpoint.The Connexin43expression and gap junction in tercellular communica-tion en hanced in OS732Rb cell.GCV was of toxicity to only TK -positive cells,OS732TK and OS732RbTK,in a concentration dependent manner,the mixed coculture experiments of OS732and OS732Rb with the TK-negative cell,both showed sensitive to GCV,but the survival rate of OS732Rb cell was significantly lower than OS732cell under the same condition.Conclusion Coordinate ex pression of Rb andHSV-TK gene conferred a more po tent bystander effect by enhancing gap junction intercellular communica-tion and in hibiting prolifera tion.

Objective To investigate the effect of simvastatin on the mobilization and migration of endothelial progenitor cells(EPCs).Methods EPCs were harvested from the bone marrows of two rabbits,cultured with M199,and identified by immunohistochemistry.The identified EPCs were then treated with simvastatin with different concentrations(0,0.01,0.1,1.0 ?mol/L),and their migration induced by simvastatin was determined with Transwell chamber assay.Six rabbits models of cranial bone defect were established and divided into control and experiment groups(3 in each).In order to elicit the effects of simvastatin on mobilization of EPCs,simvastatin was embedded in polylactic acid compound,and implanted into the cranial bone defect area in the experiment group.Meanwhile,polylactic acid was implanted in the control animals.After 10 days,the expression rate of CD34+/CD133+ EPCs in the rabbit peripheral blood was counted by flow cytometry to determine the motivating effect of simvastatin.Results In Transwell experiment,16 hours after adding simvastatin(0,0.01,0.1 or 1.0 ?mol/L),the cell migration ability was obviously increased showing a dose-dependent trend(OD value:0.077?0.014 in control group and 0.075?0.013 in 0.01 ?mol/L group vs 0.097?0.011 in 0.1 ?mol/L group and 0.099?0.019 in 1.0 ?mol/L group,P

Objective To investigate the effect of simvastatin-polylactic acid compound on critical calvarial defects in rats.Methods Twenty male SD rats(150 g?10 g),were used to establish critical cranial defect(10 mm in diameter)model.The animals were randomly divided into control and experiment groups(10 in each).In the control group,40 mg of polylactic acid were implanted into the defect area;whereas in the experiment group,simvastatin-polylactic acid compound were used(20 mg simvastatin and 40 mg polylactic acid).Four and eight weeks after the implantation,the defect area of the rats was observed by X-ray and toluidine blue staining.Results Eight weeks after the operation,X-ray examination showed high-density regions in the defect area in the experiment group,while low-density regions in the control group.The radiopacity of cranial defects were 27.33%?2.54% in the control group,and 74.63%?2.42% in the experimental group(n=5,t=-30.148,P=0.000).Toluidine blue staining showed a few new bone tissues at 4 weeks and fully filled bone defect at 8 weeks in the experiment group.Meanwhile,in the control group,only a small quantity of new bone tissue could be seen on the edge of the cranial defects.Conclusion Locally implanted simvastain-polylactic acid compound is a promising method for the treatment of bone defect owing to its high osteogenic ability.

Bone morphogenetic protein-1(BMP-1) and its related molecules are members of metalloendoproteinase astacin family, including BMP-1, mTLD, mTLL-1 and mTLL-2. Even though all of them lack of the ability to induce bone or cartilage formation directly, they play key roles in numerable activities in ECM from embryo to adult, then affect the procedure and the result of osteogenesis and bone remodeling directly or indirectly. They are critical in maturation and deposition of some major collagen types, and in regulating the signaling of some growth factors in TGF-? superfamily by degradation of TGF-? inhibitor such as Chordin. The investigations about tissue distribution of BMP-1 and its related proteinases and also gene knock-out studies strongly indicate that they play key roles in osteogenesis and bone development.

OBJECTIVE To explore the clinical efficacy of adenoidectomy in treating pediatric secretory otitis media,sinusitis and snoring. METHODS A retrospective study was conducted to analyze the clinical features,therapeutic methods and prognosis of 68 children in hospital who underwent adenoidectomy to treat secretory otitis media,sinusitis or snoring. RESULTS The conditions of the 68 children were marked improved after the removal of hypertrophic adenoids. The total clinical effectiveness rate was 100 % and the cure rate was 86.8 %. CONCLUSION Hypertrophic adenoids is a fundamental cause of pediatric secretory otitis media,sinusitis and snoring. The removal of hypertrophic adenoids is a safe and effective method for treating pediatric secretory otitis media,sinusitis and snoring.

Objective: To analyze the expression of CMTM1-v17 in normal prostate tissue and prostate carcinoma originated cell lines, and study its impact on the transactivation of androgen receptor and the possible mechanism. Methods: The expression of CMTM1-v17 in normal prostate tissue was analyzed with immunohistochemistry method. In immounocytochemistry was used to analyze the expression of CMTM1-v17 in prostate carcinoma originated cell lines. Luciferase assay was used to study the impact of CMTM1-v17 on the transactivation of AR and its mechanism. Results: The results of immunohistochemistry showed that CMTM1-v17 was highly expressed in prostate. In prostate cancer originated cell lines, CMTM1-v17 could also be detected in prostate cancer originated cell lines PC3, Du145 and LNCaP. And the results of luciferase implied that the relative luciferase activity of the PC3 cells transfected with 1 ?g and 2 ?g pCDI-CMTM1-v17 plasmids separately were 70.8 and 34.7, compared with the control set as 100. When trichostatin A, the inhibitor for histone deacetylase, was used, the repression of androgen receptor could be recovered with trichostatin A treatment,for the relative luciferase activity of the PC3 cells transfected with 1 ?g and 2 ?g pCDI-CMTM1-v17 plasmids and treated with 100 nmol/L trichostatin A rebound to 90.9 and 86.4. Conclusion: CMTM1-v17 is highly expressed in both normal prostate and prostate carcinoma originated cell lines. It may recruit histone deacetylas to inhibit the function of androgen receptor.

Objective To study osteoporosis associated with hyperlipidemia;To research high cholesterol se-rum and Jiangzhizhuanggu-containing serum intervention rat bone marrow stromal cells BMP-2 expression changes. Methods Adult rat femur by density gradient centrifugation separation of bone marrow stromal cells, cultured cells to the third generation, and was identified by flow cytometry. The third generation cells, were randomly divided into three groups. Normal serum control group: two percent blank senun (volume ratio); High cholesterol serum injury group: fi-nal concentration of 4 mmol/L, high cholesterol serum; medicine + pretreatment serum high cholesterol serum injury, group :2% senun Chinese medicine(volume ratio) after pretreatment 2 hours + final concentration of 4 mmol/L, high cholesterol serum. Total RNA was isolated from cells recovered. BMP-2 mRNA expression was detected. And analyzed statistically. Results (1) Blank serum compared with the control group,high cholesterol serum cultured bone mar-row stromal cells proliferation was inhibited. BMP-2 mRNA expression significantly decreased(P < 0.01) . (2) Jiang-zhizhuanggu solution containing serum protection two hours later to join a high cholesterol serum group, the cell growth conditions improved significantly, BMP-2 mRNA expression significantly increased than pure high cholesterol serum injured group(P <0.01) . Conclusion Solution's Jiangzhizhuanggu containing serum can enhance high cholesterol serum environment bone manow stromal cells BMP-2 mRNA expression, significantly improved bone marrow stromal cell growth state is conducive to the treatment of osteoporosis.

AIM: To manufacture recombinant protein of the highly conserved domain in human bone morphogenetic protein-1(BMP-1) using gene engineering methods as antigen for making wide spectrum antibody to BMP-1. METHODS: We analyzed the gene sequences and protein structures of BMP-1 and its related proteins, and chose a highly conserved fragment as target gene. Total RNA was prepared from human osteosarcoma cell line Saos-2, then the target gene was amplified with RT-PCR. The PCR product was cloned into prokaryotic expression vector pMAL c2 to get recombinant vector BMP-1(322-588aa)-pMAL c2. After transforming the recombinant plasmid into DH5-alpha and screening, several prositive clones were got for sequencing. Finally the transformed cells was induced with IPTG to get fusion protein. RESULTS: The BMP-1 gene fragment was successfully cloned into vector pMAL c2, and was able to express efficiently with IPTG inducement. The amount of expressed fusion protein is about 66%-72% in total volume of bacterial proteins. CONCLUSIONS: The recombinant protein contains several key domains(2 CUB domains and 1 EGF domain), which are shared by BMP-1 and its related proteins. Specific wide spectrum antibody to human BMP-1 and its related proteins may be generated with this recombinant protein antigen.

BACKGROUND: Active bone morphogenetic protein (BMP-2) reduction can cause severe symptoms like osteoporosis. In addition, there is estrogen receptor (ER) in osteoblast, osteoclast and bone marrow stromal cells, indicating a direct effect of estrogen on bone tissues.OBJECTIVE: To investigate the effect of Chinese medicine of Jiangzhi Zhuanggu on BMP-2 and ER expression in rats with prey.hyperlipidemia induced osteoporosis.METHODS: A total of 27 adult SD rats, of either gender, weighing 180-230 g, were randomly divided into three groups. In the normal control group, rats were intragastrically infused with 5 mL/kg normal saline every morning and afternoon. In the model group, the rats were infused with 5 mL/kg high-fat diet in the morning and 5 mL/kg normal saline in the afternoon. In the Chinese medicine group, 5 mL/kg high-fat diet was infused in the morning and 5 mL/kg Chinese medicine of Jiangzhi Zhuanggu water extract in the afternoon. After 8 weeks, expression levels of BMP-2 and ER in bone tissue was detected with immunohistological methods, and ER mRNA level of bone tissue in rats was detected by in situ hybridization.RESULTS AND CONCLUSION: Compared with the model group, the BMP-2 and ER expression in the bone tissue was significantly increased (P ＜ 0.01), and ER mRNA level increased following Chinese medicine treatment. Results show that Chinese medicine of Jiangzhi Zhuanggu could increase BMP-2 and ER expression in the osteoporosis bone tissue, and improve osteoporosis effectively.

Objective To establish an improved model of exercise-induced glycometabolism in type II diabetic rats,and to provide a theoretical reference for the establishment of exercise prescription for type II diabetes.Methods Forty-five 8-week old SPF male Wistar rats were used in this study.Of which 32 were fed with high-fat diet for 7 weeks,and intraperitoneal injection of 30 mg/kg STZ was given to establish the rat model of type II diabetes.The normal rats and successful model rats were divided into four groups:The normal control group (C group),normal exercise group (CE group),diabetic group (DM group) and diabetic exercise group (DME group).The exercise group was assigned by the Ploug training protocol,6 days/week,60 min/day,for a total of 8 weeks.After the high fat diet fed for 7 weeks,blood sample was taken from the tail vein,FBG and serum insulin were detected after baseline and 8 weeks exercise,and blood sample was collected from the tail vein to determine the FBG.Serum insulin (FINS) was detected by orbital blood sampling at the end of 8 weeks of exercise,and HOMA-IR was calculated.Results 1.After 7 weeks of high fat diet,compared with the groups C and CE,the levels of FBG,FINS and HOMA-IR were significantly higher in the DM and DME groups.2.After 8 weeks of exercise intervention,compared with the groups C and CE,FINS was significantly lower in the groups DM and DME,but the FBG and HOMA-IR were higher.Compared with the DM group,the level of FINS was significantly higher in the DME group,and the levels of FBG and HOMA-IR were significantly lower.The body weights of DM and DME groups were significantly lower than those of the groups Cand CE,the body weight had no significant difference between the DME and DM groups,and similar result was between the groups CE and C.Conclusions 1.The rat model of type II diabetes is successfully established with high fat diet for 7 weeks plus STZ injection(30 mg/mL).2.Aerobic exercise 60 min/day for a total of 8 weeks can improve the glycometabolism in type 2 diabetic rats,to be an ideal animal model for study of the mechanism of prevention and amelioration of type II diabetes.