BACKGROUND:The microcosmic and submicroscopic organizations of tissue engineering scaffold matedals’superficial structure have all important effect on the eell adhesion and growth.By means of nano.Technique and three-dimensional porous technique,the resultant nano-hydroxyapatite/collagen(n-HAC)call imitate the component and microstructure of natural bone.OBJECTIVE:To observe the biocompatibility of human bone m arrow mesenchymal stem cells(MSCs)cultured in vitro with nHAC.DESIGN,TIME AND SETTING :Single samples observation was performed in the Experimental Center of School ofMedical Technology,Jiangsu University from September 2005 to December 2006. MATERIALD:nHAc was provided by the Material Science and Engineering Department of Tsinghua University.Humanbone marrow mesenchymal stem cells were derived from healthy adult volunteers.All the subiects signed the informedconsents. METHODS:Whole bone marrow culture and successive adherence method was used to culture MSCs in vitro,and the cells were then induced to differentiate into the phenotype of osteoblasts by the revulsants(methylprednisolone,vitamin C,β-glycerophosphate and basic fibroblast growth factor).MSCs at passage 3 were co-cultured with nHACfor 14 days.MAIN OUTCOME MEASURES:The cytological characteristics of the osteoblast were identified throue,alkalinephosphatase immunohistochemistry method and Von Kossa stain.The growth condition with or without nHAC wasevaluated through invert microscope and scanning electron microscope,respectively.RESULTS:The cultured MSCs proliferated into uniform fibroblast-like cells rapidly.MSCs reached confluence and started to form multilayers averaging from 10 to 12 days,passaged stably as well.Then the MSCs passaged from 7 to 9 days.Cytochemistry evaluation showed that MSCs in induced culture were positive for alkaline phosphatase and Von Kossa stain,and deposited calcified matrix.It showed a typical ostcoblast feature in morphology and biology.In coculture model ofMSCs with nHAC,cells would attach to the inner surface of nHAC.At 8 days,the osteoblasts proliferated in the nHAC and the secretion of the matrix was observed.Lots ofcells adheredon the surfaceand pores of nHAC at 14 days.There wereextensive prominent connections among cells. CONCLUSION:THE nHAC is suitable for MSCs to adhere,grow and proliferate,with a good compatibility.

The voltage-gated potassium channels Kvl.5 is widely expressed in the plasma membranes of numerous tumor cells and it can contribute to a variety of cellular functions such as proliferation,ap optosis,and cell cycle.Several types of antineoplastic drugs can change the expression of Kv1.5 and then affect the biological processes.Kv1.5 is identified as a novel target for therapy in human cancer.The researches of Kv1.5 will contribute to gain a further understanding of the molecular mechanism of tumor and provide therapeutic opportunity for the prevention and treatment of cancer.

Objective: To observe the clinical efficacy of leuprorelin combined with mifepristone in the treatment of endometriosis. Methods: From September 2014 to September 2016, 168 cases of endometriosis were selected in the research.The patients were divided into the control group and the research group according to the random number table method, with 84 patients in each group.All patients underwent laparoscopic endometrial debridement with fertility preservation.The control group was treated with mifepristone, while the study group was treated with combination of leuprorelin and mifepristone.The incidence of adverse drug reactions during treatment was observed, the levels of serum E2, LH, FSH, EmAb and CA125 were detected before and after treatment, the curative effect after treatment was evaluated, and the recurrence and pregnancy of endometriosis within 12 months after treatment were followed. Results: During the treatment, the incidence rate of adverse drug reactions in the study group(17/84, 20.24%) had no statistically significant difference compared with the control group (13/84, 15.48%) (χ²=0.649, P=0.420). The levels of serum estradiol (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), anti-endometrial antibody (EmAb) and carbohydrate antigen 125 (CA125) in the two groups were significantly lower than those before treatment (t=17.694, 13.271, 7.943, 4.315, 10.029, 7.952, 14.231, 10.610, 27.953, 22.436, all P<0.001), and the indicators in the study group were significantly lower than those in the control group (t=5.217, 3.859, 3.526, 4.337, 6.048, all P<0.001). The total effective rate of the study group (96.43%) was significantly higher than that of the control group (83.33%) (χ²=7.919, P=0.005). Within 12 months after treatment, the recurrence rate of endometriosis in the study group (2.38%) was significantly lower than that of the control group (15.48%), while the rate of pregnancy (48.81%) was significantly higher than that of the control group(27.38%), there were statistically significant differences between the two groups (χ²=8.858, 8.178; P=0.003, 0.004). Conclusion Compared with leuprorelin treatment alone, leuprorelin combined with mifepristone can significantly reduce the serum levels of E2, LH, FSH, EmAb and CA125 in patients with endometriosis, and improve the overall treatment of endometriosis, reduce recurrence, promote pregnancy, and has high safety.

Objective To investigate the mutation of type Ⅱ human hair basic keratin (hHb) gene in a family with monilethrix.Methods Scanning electron microscopy was used to observe the structure of hair shafts.With informed consent,blood samples were drawn from affected and unaffected membets in this family,as well as from 50 healthy controls.Genomic DNA was isolated from these samples.The exon 1 and exon 7 of hHb1,hHb3 and hHb6 were amplified by polymerase chain reaction (PCR).All the PCR products were sequenced directly using ABI3730 automated sequencer.DNA sequence alignment was carried out with BLAST software.Results A typical beaded appearance was observed in affected hairs by using scanning electron microscopy.There were obvious longitudinal ridges and sulcuses in hair node.and hair cuticles were irregularly shaped.Most cortex and medullary substance were absent in affected hairs of a patient.After sequence alignment,a G1289A point mutation in exon 7 of hHb6 gene,which led to a substitution of arginine for glutamide at codon 430,was detected in affected members of this family,but not in unaffected family members or 50 unrelated human controls.No mutation was observed in exon 1 or exon 7 of hHb1 and hHb3 gene or exon 1 of hHb6 gene.Conclusion The missense mutation of R430Q is a novel mutation.which may be associated with the pathogenesis of monilethrix in this pedigree.

Objective To detect the influence of miRNA-34a (miR-34a) on the proliferation of osteosarcoma and the mechanisms responsible for miR-34a regulation.Methods The osteoblastic cell line MG-63 and Saos-2,human osteoblastic cell line hFOB 1.19,10 osteosarcoma tissues and 10 normal bone tissues were selected.The expression of miRNA-34a in osteosarcoma cells and tissues was detected by quantitative real-time polymerase chain reaction (qPCR).Next,a eukaryotic expression vector named pcDNA/miR-34a was constructed.Then,osteosarcoma cells were transfected with this eukaryotic expression vector and the effects of miR-34a overexpression on the proliferation and growth of osteosarcoma were measured using CCK-8,colony formation and xenograft model of nude mice.Finally,Western blot analysis was used to detect the expression of ether-à-go-go 1 (Eag1) gene in osteosarcoma cells after transfected with pcDNA/miR-34a or a miR-34a inhibitor miR-34a-2'-O-Methyl antisense oligoribonucleotide (miR-34a-2'-O-Me).Results Compared with normal bone tissues and osteoblastic cell line,miR-34a was down-regulated in osteosarcoma cell lines and tissues.Compared with the blank group and the control group,the cell survival rates of miR-34a group of the two cell lines were significantly lower [MG-63 72 h:blank group (40.05±4.82) ％,control group (36.88± 4.66) ％,miRNA-34a group (26.24±6.22) ％;MG-63 96 h:blank group (83.55±5.95) ％,control group (80.13± 4.48) ％,miRNA-34a group (30.21±7.26) ％;Saos-2 72 h:blank group (46.45±8.15) ％,control group (43.33± 6.89) ％,miRNA-34a group (26.81±3.17) ％;Saos-2 96 h:blank group (84.79±4.10) ％,control group (80.14± 3.11) ％,miRNA-34a group (31.77±5.17) ％].The similar results were obtained from colony formation assay (MG-63:blank group 83.40±3.29,control group 80.00±3.06,miR-34a group 24.40±2.71;Saos-2:blank group 85.00±3.32,control group 80.60±3.29,miR-34a group 30.40±4.94).The tumor volumes of osteosarcoma xenograft in the miR-34a group was significantly smaller than that in the blank group and control group after 21 days treatment (all P ＜ 0.001).Overexpression of miR-34a could decrease Eag1 expression in osteosarcoma cell lines while inhibition of miR-34a induced the of expression Eag1 (P ＜ 0.001).Conclusion MiR-34a plays a tumor suppressor role in osteosarcoma and could suppress the proliferation and growth of osteosarcoma through the regulation of Eag1.Moreover,it may be a novel target for osteosarcoma therapy.

Objective To investigate the clinical efficacy of our self-designed adjustable weight-bearing brace for AO type B tibial shaft fractures managed by interlocking intramedullary nail.Methods A total of 68 consecutive patients with AO type 42-B tibial shaft fracture who had been managed from April 2013 to March 2015 hy interlocking intramedul]ary nail were recruited into our study.They were randomized into 2 equal groups (n =34).Group A received conventional therapy after operation while group B received auxiliary mauagement with our self-designed adjustable weight-bearing brace after conventional postoperative therapy for one week.The 2 groups were compared at postoperative 1,3 and 6 months and at the final follow-up in terms of visual analogue scale (VAS),weight-bearing status of the affected limb,time for fracture union,Radiographic Union Score for Tibial Fractures (RUST) and Johner-Wruhs scale.Results Of this series,62 cases were followed up for 12 to 18 months (average,14.7 months),5 ones were lost to the follow-up and one withdrew.The mean VAS scores at 3-month and 6-month follow-ups for group B were 2.5 ± 0.8 and 0.9 ± 0.6 respectively,significantly lower than those for group A (3.0 ± 0.9 and 1.4 ± 0.8 respectively) (P ＜ 0.05).In group A at 1-month,3-month and 6-month follow-ups,the weight-bearing status was 44.1％ ± 17.5％,72.0％ ±17.4％ and 86.4％ ±12.5％ while the mean RUST scores were 5.4±1.4,8.7±1.1 and 10.3 ± 1.1,respectively.In group B at 1-month,3-month and 6-month follow-ups,the weight-bearing status was 53.8％ ± 11.0％,84.1％ ± 12.2％ and 94.4％ ± 10.6％ while the mean RUST scores were 6.5 ± 0.8,9.9 ± 0.9 and 11.3 ± 0.8,respectively.There were significant differences between the 2 groups in the above indexes (all P ＜ 0.05).Group B achieved clinical fracture union after an average of 3.3 ±0.7 months,significantly faster than group A (3.9 ± 1.0 months) (P ＜ 0.05).According to the Johner-Wruhs scoring,group A had 19 excellent cases and 12 good ones while group B had 27 excellent ones and 4 good ones,showing a significant difference between the 2 groups (P ＜ 0.05).Conclusions Early application of our self-designed adjustable weight-bearing brace for patients with AO type B tibial shaft fracture managed by interlocking intramedullary nail can reduce postoperative pain,accelerate callus growth,shorten bony healing time and achieve satisfactory functional recovery.

Objective To detect the expression of HERG (human ether-à-go-go-related gene ) potassium channel in human osteosarcoma,and explore the effects of silencing HERG by small interfering RNA (siRNA)on the proliferation and apoptosis of osteosarcoma cells and the mechanisms responsible for HERG regulation.Methods The expressions of HERG in osteosarcoma MG-63 cells and tissues were detected by reverse transcription polymerase chain reaction (RT-PCR),Western blotting and immunohistochemistry.Next, osteosarcoma cells were divided into three groups:HERG-siRNA group,control-siRNA group and blank group. CCK-8,colony formation,flow cytometry and Tunel assay were used to measure the proliferation and apoptosis of the osteosarcoma cells.Finally,Western blotting analysis was performed to detect the expression of nuclear factor-κB (NF-κB)pathway in osteosarcoma cells treated with HERG siRNA.Results Osteosarcoma cells and tissues were found to highly express HERG.Inhibition of HERG in the osteosarcoma cells significantly inhibited the cell proliferation and induced cell apoptosis.Compared to control-siRNA group or blank group,HERG-siRNA could inhibit the proliferation of MG-63 cells significantly [HERG-siRNA group:(75.34 ± 4.45)%;compared to control-siRNA group:(100.60 ±5.31)%;t =3.64,P =0.007;compared to blank group:(100.00 ±5.66)%;t =3.43,P =0.009].The similar results were obtained from colony formation assay (HERG-siRNA group:134.30 ±11.82;compared to control-siRNA group:225.30 ±11.56;t =5.51, P =0.002;compared to blank group:232.80 ±12.21;t =5.80,P =0.001).HERG-siRNA transfected MG-63 cells demonstrated a significant increase of apoptotic rate compared to control-siRNA transfected cells or untreated cells [HERG-siRNA group:(28.10 ±2.21 )%;compared to control-siRNA group:(9.36 ± 2.42)%;t =5.72,P =0.005;compared to blank group:(10.92 ±2.51)%;t =5.14,P =0.007].This resultwas further confirmed by Tunel assay.The cells transfected with HERG-siRNA (31.57 ±2.08)% dem-onstrated extensive apoptosis,compared with the control-siRNA group [(10.35 ±1.82)%;t =7.69,P =0.002)]or blank group [(7.96 ±0.88)%;t =10.48,P =0.001].Silencing HERG gene down-regulated the cIAP-1,XIAP,Bcl-2,Survivin,P-IκBαand NF-κB p65 expression,compared to the control groups. Conclusion HERG is highly expressed in osteosarcoma.HERG silencing can suppress osteosarcoma progres-sion through NF-κB pathway and suggest that HERG may be a novel molecular target for osteosarcoma therapy and diagnosis.

Objective To explore the expression of ether à go-go 1 ( Eag1) in human osteosarcoma and its molecular mechanisms of regulating the malignant phenotype of osteosarcoma .Methods The expression levels of Eag 1 in osteosarcoma cell lines and human osteosarcoma tissues were detected by reverse transcription polymerase chain reaction ( RT-PCR), western blot analysis and immunohistochemistry .The small interfering RNA ( siRNA) was used to inhibit the expression of Eag 1.The abilities of proliferation and invasion in osteosarcoma cells transfected with Eag 1 siRNA were determined by CCK-8, colony formation assay, transwell assay and wound healing assay .The osteosarcoma xenograft model of nude mouse was established and tumor growth curve was drawn .Western blot analysis was performed to detect the expression of vascular endothelial growth factor ( VEGF) and signal transducer and activator of transcription 3 ( STAT3) in osteosarcoma cells transfected with Eag 1 siRNAs.Results Eag1 was overexpressed in the osteosarcoma cells and tissues .Compared with the scrambled siRNA group , the cell survival rates of Eag 1 siRNA1 and Eag1 siRNA2 groups of the two cell lines were significantly lower .[ MG-63 cells: scrambled siRNA group (100.0±4.65)%, Eag1 siRNA1 group (63.57±3.89)%, and Eag1 siRNA2 group (54.13±3.70)%;Saos2-cells:scrambled siRNA group (100.00±5.46)%, Eag1 siRNA1 group (56.70±5.34)%, and Eag1 siRNA2 group (40.27±5.28)%(P<0.001 for all)].Similar results were obtained from colony formation assay .The colony formation rates of MG-63 cells: the scrambled siRNA group was (92.00±3.46)%, Eag1 siRNA1 group (60.00±3.06)%, and Eag1 siRNA2 group (53.67±2.40)%; the colony formation rates of Saos-2 cells: the scrambled siRNA group was (92.00±5.57)%, Eag1 siRNA1 group (52.33±5.13)%, and Eag1 siRNA2 group (41.67±27.3 )%.Compared with the scrambled siRNA group , P<0.001 for all.The tumor volumes of osteosarcoma xenograft in the Eag 1 siRNA1 and Eag1 siRNA2 groups were significantly smaller than that in the scrambled siRNA group after 10 days treatment (P<0.01 for all).The invasion assay data showed that MG-63 and Saos-2 cells transfected with Eag 1siRNAs exhibited the ability of cell invasion , when compared with the cells transfected with scrambled siRNA .( Invasive cell number of MG-63 cells:the scrambled siRNA group was 134.00 ±3.61, Eag1 siRNA1 group 105.20 ±2.52, and Eag1 siRNA2 group 91.00±3.01; Invasive cell number of Saos-2 cells: the scrambled siRNA group was 132.30±3.23, Eag1 siRNA1 group 114.30±3.48, and Eag1 siRNA2 group 82.67±6.33.Compared with the scrambled siRNA group, P<0.01 for all.The migration rates were (62.48±1.83)%, (35.98±1.23)%and (32.30±1.20)%in the three groups of MG-63 cells, and (70.15±1.42)%, (41.38±1.34)%and (32.40±1.92)%in the three groups of Saos-2 cells, respectively.Compared with the scrambled siRNA group , P<0.001 for all.Notably, the expression levels of VEGF decreased evidently after Eag 1 siRNAs transfection , paralleled with reduction in the expression levels of STAT 3.Conclusions Eag1 may promote osteosarcoma cell proliferation and invasion by targeting STAT 3-VEGF pathway and may be a potential therapeutic target for osteosarcoma .

Objective To explore the expression of ether à go-go 1 ( Eag1) in human osteosarcoma and its molecular mechanisms of regulating the malignant phenotype of osteosarcoma .Methods The expression levels of Eag 1 in osteosarcoma cell lines and human osteosarcoma tissues were detected by reverse transcription polymerase chain reaction ( RT-PCR), western blot analysis and immunohistochemistry .The small interfering RNA ( siRNA) was used to inhibit the expression of Eag 1.The abilities of proliferation and invasion in osteosarcoma cells transfected with Eag 1 siRNA were determined by CCK-8, colony formation assay, transwell assay and wound healing assay .The osteosarcoma xenograft model of nude mouse was established and tumor growth curve was drawn .Western blot analysis was performed to detect the expression of vascular endothelial growth factor ( VEGF) and signal transducer and activator of transcription 3 ( STAT3) in osteosarcoma cells transfected with Eag 1 siRNAs.Results Eag1 was overexpressed in the osteosarcoma cells and tissues .Compared with the scrambled siRNA group , the cell survival rates of Eag 1 siRNA1 and Eag1 siRNA2 groups of the two cell lines were significantly lower .[ MG-63 cells: scrambled siRNA group (100.0±4.65)%, Eag1 siRNA1 group (63.57±3.89)%, and Eag1 siRNA2 group (54.13±3.70)%;Saos2-cells:scrambled siRNA group (100.00±5.46)%, Eag1 siRNA1 group (56.70±5.34)%, and Eag1 siRNA2 group (40.27±5.28)%(P<0.001 for all)].Similar results were obtained from colony formation assay .The colony formation rates of MG-63 cells: the scrambled siRNA group was (92.00±3.46)%, Eag1 siRNA1 group (60.00±3.06)%, and Eag1 siRNA2 group (53.67±2.40)%; the colony formation rates of Saos-2 cells: the scrambled siRNA group was (92.00±5.57)%, Eag1 siRNA1 group (52.33±5.13)%, and Eag1 siRNA2 group (41.67±27.3 )%.Compared with the scrambled siRNA group , P<0.001 for all.The tumor volumes of osteosarcoma xenograft in the Eag 1 siRNA1 and Eag1 siRNA2 groups were significantly smaller than that in the scrambled siRNA group after 10 days treatment (P<0.01 for all).The invasion assay data showed that MG-63 and Saos-2 cells transfected with Eag 1siRNAs exhibited the ability of cell invasion , when compared with the cells transfected with scrambled siRNA .( Invasive cell number of MG-63 cells:the scrambled siRNA group was 134.00 ±3.61, Eag1 siRNA1 group 105.20 ±2.52, and Eag1 siRNA2 group 91.00±3.01; Invasive cell number of Saos-2 cells: the scrambled siRNA group was 132.30±3.23, Eag1 siRNA1 group 114.30±3.48, and Eag1 siRNA2 group 82.67±6.33.Compared with the scrambled siRNA group, P<0.01 for all.The migration rates were (62.48±1.83)%, (35.98±1.23)%and (32.30±1.20)%in the three groups of MG-63 cells, and (70.15±1.42)%, (41.38±1.34)%and (32.40±1.92)%in the three groups of Saos-2 cells, respectively.Compared with the scrambled siRNA group , P<0.001 for all.Notably, the expression levels of VEGF decreased evidently after Eag 1 siRNAs transfection , paralleled with reduction in the expression levels of STAT 3.Conclusions Eag1 may promote osteosarcoma cell proliferation and invasion by targeting STAT 3-VEGF pathway and may be a potential therapeutic target for osteosarcoma .

Objective:To investigate the antibacterial effect of iodophor on Staphylococcus aureus biofilm (BBF).Methods:Staphylococcus aureus were cultured in vitro and 480 pieces of titanium alloy plates were selected. On the surface of titanium plates, in vitro models of Staphylococcus aureus biofilms were established at days 7, 14, 21 and 28 respectively with 120 pieces of titanium plates at each time points. The biofilms at each time point were assigned to no iodophor immersion (PBS group), 5 g/L iodophor immersion for 5 minutes (5-min group) and 5 g/L iodophor immersion for 10 minutes (10-min group), according to the random number table method. FITC-ConA, propidium iodide (PI) and SYT09 were used to dye Staphylococcus aureus in PBS group. After dyeing, confocal laser scanning microscopy and scanning electron microscopy were used to observe the morphological structure of bacterial biofilms, and the Colony forming unit (CFU) was counted by the viable count method. In the other two groups, PI and SYT09 were applied to dye Staphylococcus aureus, and then confocal laser scanning microscopy and scanning electron microscopy were used to observe the changes of biofilms and bacterial viability after iodophor immersion. The antibacterial effect of iodophor was evaluated by the viable count method.Results:After dyeing Staphylococcus aureus with FITC-ConA and PI in PBS group, confocal laser scanning microscopy showed that the extracellular polymers of the bacteria increased gradually with the extension of culture time. The space structure of biofilm was gradually mature, changed significantly at day 21 and became mature at day 28. After staining Staphylococcus aureus with PI and SYT09 in PBS group, confocal laser scanning microscopy showed that the number of bacteria increased, and had a mountain-like shape. Scanning electron microscopy showed that the number of bacterial extracellular polymers increased gradually with the extension of culture time and a structured microenvironment was formed and gradually matured. In 5-min and 10-min groups, all bacteria were killed at days 7 and 14 [0(0, 0)CFU/ml], the antibacterial effect was weakened at 21 days, but the antibacterial effect of iodophor immersion in 10-min group [100 (100, 125)CFU/ml] was better than that in 5-min group [300 (275, 425)CFU/ml] ( P<0.05). There was no significant difference in iodophor immersion in 5-min group [500 (375, 700)CFU/ml] and 10-min group [250 (175, 400)CFU/ml] at 28 days ( P>0.05). Conclusions:The maturation of biofilm is the overall maturation of bacteria and bacterial extracellular polymers and the formation of a spatialized microenvironment. Bounded by the 21st day, biofilms are divided into young biofilms and mature biofilms. The main difference between them lies in the maturation of extracellular polymers and microenvironment. For the bacterial biofilm with culture time less than 21 days, the antibacterial effect of the iodophor immersion for 10 min is better than that of 5 min. However, for the bacterial biofilm with culture time greater than 21 days, there is no significant difference in the antibacterial effect of the bacterial biofilm of prolonged iodophor immersion time.