BACKGROUND:The in vitro construction, maturation and differentiation of cellscaffold complexes into tissue-engineered bone is the necessary process of bone tissue engineering construction, but there are no uniform
methods and standards.
OBJECTIVE:To summarize the basic method and technology to build bone tissue engineering at present and to discuss the related development.
METHODS:A compute-based online search was conducted on the PubMed database and CNKI database for the articles related to the in vitro construction and culture of bone tissue engineering bone from January 1997 to
January 2013 with the key words of“bone tissue engineering, cellbiological scaffold, cellinoculation, seeding density, culture in vitro, bioreactor”in English and Chinese. Final y, 44 articles were included according to the inclusion and exclusion criteria.
RESULTS AND CONCLUSION:As the carrier of bone tissue engineering seed cells, the primary prerequisite of biological scaffold is sterile, because the sterile biological scaffold can be able to survive. Sterilization of biological scaffolds includes ultraviolet sterilization, 60Coγ-ray sterilization, soaking in ethanol with the volume fraction of 75%, autoclave method, and ethylene oxide sterilization. 60Coγ-ray sterilization is the common method in the biological scaffold sterilization. The inoculation density of seed cells is the key factors that influence the adhesion growth and proliferation of seed cells on the scaffolds. The adhesion between cells and scaffold materials wil be affected by the affinity of scaffolds, celladhesion and gravity, and other factors. The method for the inoculation of bone tissue engineering seed cells includes static inoculation and dynamic inoculation. Each construction method has its advantages and disadvantages. Overcomeing these disadvantages, forming a uniform construction method and ful y clinical application are the direction of future development.

Objective To investigate the clinical roles of lysine methyltransferase 5A(KMT5A)in human hepatocellular carcinoma(HCC)and its functions in cell migration and invasion. Methods The expression levels of KMT5A of 60 cases were detected by immunohistochemistry(IHC). KMT5A siRNA was used to down-regulate the expression of KMT5A in SMMC-7721 cells. Cell migration and invasion were measured by wound healing assays and transwell assays,respectively. Immunoblotting was used to detect the expression of MMP-2 after siRNA trans-fection. miR-186 mimics were transfected into SMMC-7721 cells and the mRNA levels of KMT5A was detected by qRT-PCR after transfection. Results High expression of KMT5A was associated with large tumor diameter (>5 cm,P=0.047)and advanced TNM stage(Ⅲ+Ⅳ,P=0.035). The expression of KMT5A was knocked down by siRNA in SMMC-7721 cells. Down-regulation of KMT5A suppressed cell migration(P=0.031,P=0.006)and invasion(P=0.010),and impaired MMP-2 expression(P=0.040). Overexpression of miR-186 could significantly inhibit the expression of KMT5A(P = 0.007). Conclusions Over-expression of KMT5A in HCC tissues associ-ates with poor clinical features. KMT5A knockdown inhibits the migration and invasion on HCC cells.

Objective To discuss clinical effect of minimally invasive surgery, on herniated lumbar disc.Methods 50 patients of herniated lumbar disc were selected, and 25 patients were treated by micro endoscopy discectomy (MED).Results The treatment effect was better in treatment group, and the surgery time was shorter.1 case in treatment group had erector spinae hematoma, and 9 patients in control group had lumbago, while 2 cases had sciatica behavior.Conclusion The injury of MED was small,the surgery time was shorter,and the recovery was quicker,and it could be popularized in the clinic.

Objective:To study the rule of spontaneous behavior and to explore the effect on emotion of mice peripherally infected with influenza A WSN33 virus( H1N1).Methods: Mice were intranasal inoculated with H1N1 WSN33 or saline.Then mice bodyweight change,and total distance movement,average movement speed distance in the central area and feces in the open field test in 5 minutes were recorded in two weeks.Results: Mice following WSN33 infection bodyweight declined sharply until day 7 post-inoculation,and mice bodyweight recovered from influenza infection at day 8 post-inoculation.Total distance movement of mice following H1N1 WSN33 infection decreased in the open field test,and difference of the reduction was significant from day 5 to day 10 post-inocu-lation.The average movement speed had no statistical difference.The range of numbers of fecal grains was large, and they were no significant difference.Conclusion:The total distance movement decreased,but average movement speed did not change following mice infected with H1N1 WSN33.They told us that mice infected with H1N1 WSN33 had anxiety,depressed and nervous emotion which is more evident in acute stage and early recovery stage,whereas the change of the nervous emotion was small and not obvious.

BACKGROUND:Study confirms that bone morphogenetic protein can induce osteogenesis;however the ultrastructure of periosteal cells induced by bone morphogenetic protein-7 remains poorly reported.
OBJECTIVE:To study the bioactivity and ultrastructure of periosteal cells induced by bone morphogenetic protein-7 in vitro.
METHODS:The primary periosteal cells isolated from adult tibial bone were in vitro cultured, and then divided into experimental group and control group. In the experimental group, cells were cultured with bone morphogenetic protein-7 and culture adjuvant;while cells in the control group were only cultured with the adjuvant. Three samples in each group were tested at 5, 10, 15 days, respectively. The general structure of cultured cells was observed using von Kossa staining, and the ultrastructure was observed under transmission electron microscopy.
RESULTS AND CONCLUSION:The periosteal cells in the two groups grew wel in vitro, showing uniform morphology. Early cells were spindle-shaped, with strong three-dimensional sense and ful transparency;mitotic cells were short columnar or cubic shaped, there were a lot of rough endoplasmic reticulum and Golgi complex in osteoblasts under electron microscope. Later stage of cells developed from long fusiform into wide shuttle and irregular shape, there were a large number of matrix vesicles within the cells under the electron microscope. The membrane coating, alkaline phosphatase and calcium-binding protein in the cytoplasm, as wel as calcium crystals were found. The osteogenesis basement and lateral sides appeared projections, which were connected with adjacent bone cells. Induction of bone morphogenetic protein-7 in vitro promotes the osteoblasts proliferation, division and bone formation speed. The results suggest that bone morphogenetic protein-7 can significantly enhance the proliferation ability of osteoblasts in vitro.

Objective To explore biological characteristics of chondrogenic differentiation of human periosteum-derived cells and the role of BMP4 in chondrogenic differentiation of these cells.Methods From October 2009 to September 2012,periosteum was obtained from tibia of patients undergoing leg amputation surgery,and isolated periosteum-derived cells by tissue culture method.Cells were cultured in DMEM/F12 containing 10％ fetal bovine serum,and morphology of cells were observed under inverted microscope.Periosteum-derived cells growth and the effect of BMP4 on cells growth examined by cell count using trypan blue,and cells growth curve was made.Experiment was divided into control group,chondrogenic differentiation group and BMP4 group,cells were expanded and differentiated in the presence or absence of BMP4 and complete medium.Then toluidine and immunohistochemical staining analyzed proteoglycan and collagenⅡ expression of these cells after 14 and 21 days.The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of these cells using real-time PCR.Results (1) Periosteumderived cells adhered to growth in vitro,the shape of cell presented fibroblast-like morphology changing into polygonal after 1 week and round cell formation after 2 weeks chondrrogenic differemtiation.Growth curve showed that the passage 3 and 9 cells had similar reproductive activity.The passage 3 cells were positive for CD90 (21.07％) and CD105 (25.84％).(2)Toluidine bule staining and type Ⅱ collagen immunohistochemical staining showed BMP4 group (40.29 ± 4.29,56.74 ± 5.12) and chondrogenic differentiated group (19.27 ± 3.71,38.31 ± 4.25) ccould secrete proteoglycan and collagen Ⅱ,control group were negative (10.24 ± 1.21,15.28 ± 2.23),BMP4 group were significantly than chondrogenic differentiated group.(3) The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of BMP4 group(25.76 ±0.57,6.48 ±0.48,2.91 ±0.18)were significantly higher than that of control group(2.37 ±0.24,1.12 ± 0.31,1.07 ± 0.22)and chondrogenic differentiated group(11.12 ± 0.38,2.24 ± 0.41,1.54 ± 0.35)using real-time PCR.Conclusion Periosteum-derived cells have strong proliferative,and have good potentials of differentiating into chondroblasts like mesenchymal stem cells.BMP4 can promote chondrogenic differentiation of periosteum-derived cells in vitro cultures.

Objective:Studies on the immune-enhancing activity of Polysaccharides from the fruits of Borojoa sorbilis cuter ( polysaccharides BSCP) in vivo.Methods:54 male BALB/c mice were randomly divided into six groups.The immune suppression mice in the three experimental groups,which were induced by cyclophosphamide ( CY) at 80 mg/kg/d via intraperitoneal injection, were perfused with 100,200,400 mg/kg/d BSCP for 30 d.The effect of BSCP on cellular immune function,humoral immune function and mononuclear macrophage function were measured.Results:CY-treated mice showed a significant decrease in immune function( P<0.05),humoral immune function(P<0.01) and mononuclear macrophage function(P<0.01).The administration of BSCP was able to overcome the CY-induced immunosuppression,treatment with BSCP-L,BSCP-M and BSCP-H groups significantly enhanced the cellular immune function( each was P<0.05 ) and mononuclear macrophage function ( each was P<0.05 ) , and treatment with BSCP-M and BSCP-H significantly enhanced humoral immune function(each was P<0.01).Conclusion:BSCP could significantly increase immune responses and reduce the side effects of CY in immune system.Therefore,the BSCP could be an immunomodulatory agent.

OBJECTIVETo investigate the association between receptor-interacting kinase protein 4 (RIPK4) relative copy number (RCN) and prognosis of stage III( colorectal cancer (CRC) patients treated with oxaliplatin-based chemotherapy.

METHODSRIPK4 RCN was determined by real-time PCR and then dichotomized into high RIPK4 RCN group(n=35) and low RIPK4 RCN group (n=104) using the third quartile as the cut-off point. Overall survival (OS) and recurrence-free survival (RFS) were compared between high and low RIPK4 RCN groups. The subgroup prognostic analysis was also conducted based on tumor site.

RESULTSThe median follow-up period was 49 months (ranged 4 to 98 months). Patients with high RIPK4 RCN had poorer OS than those with low RIPK4 RCN, which reached marginal significance(median OS, 43.0 months vs. 53.5 months, P=0.074). Meanwhile there was no significant difference of RFS between two groups (P=0.352). In colon cancer subgroup, high RIPK4 RCN was significantly associated with poor OS (median OS, 31.5 months vs. 56.6 months, P=0.015) but not with RFS (P=0.135). In rectal cancer subgroup, RIPK4 RCN was not associated with both OS and RFS (P=0.981, P=0.738). Multivariate analysis revealed that high RIPK4 RCN was an independent prognostic factor of OS in stage III( CRC patients treated with oxaliplatin-based chemotherapy (HR=2.903, 95% CI: 1.275 to 6.610).

CONCLUSIONRIPK4 RCN is significantly associated with OS in stage III( colon cancer patients receiving oxaliplatin-based chemotherapy and may be a novel biomarker that can predict the efficacy of oxaliplatin in colon cancer patients.

OBJECTIVETo investigate the expression of microRNA-218 (miR-218) and its role in hepatocellular carcinoma (HCC).

METHODSForty-six pairs of fresh surgical specimens of HCC and adjacent tissues were examined for miR-218 expression using qRT-PCR. A miR-218 mimic was transfected into HepG2 cells, and the cell viability and apoptosis were analyzed by MTT assay and flow cytometry, and the potential targets of miR-218 were detected by qRT-PCR and Western blotting.

RESULTSThe expressions of miR-218 in HCC tissues were significantly down-regulated compared to those in the adjacent tissues (P<0.05). Down-regulation of miR-218 was found to correlate significantly with the tumor size (>5 cm) and an advanced TNM stage (III+IV) (P<0.05). Ectopic expression of miR-218 in HepG2 cells resulted in suppressed cell proliferation and enhanced cell apoptosis as well as the down-regulation of Bmi-1 and CDK6 mRNA and protein expressions (P<0.05).

CONCLUSIONThe low-expression of miR-218 is correlated with malignant clinicopathological characteristics of HCC, and miR-218 may inhibit cell proliferation and promote cell apoptosis by down-regulating Bmi-1 and CDK6 in HCC.

Adult ; Aged ; Apoptosis ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Proliferation ; Cyclin-Dependent Kinase 6 ; metabolism ; Female ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Polycomb Repressive Complex 1 ; metabolism

Circular RNA (circRNA) is a ubiquitous class of noncoding RNAs (ncRNAs) in all kingdoms of life with diverse structures.CircRNA is an endogenous noncoding RNA molecule with covalently close cyclic structure which does not have 5'end cap and 3'poly (A) tail.It is found to be evolutionary conservative and stable with tissue specificity.CircRNAs have many biological functions.In addition to acting as miRNA sponges,many other functions are being revealed,including regulator of gene transcription,and splicing and protein translation.It is involved in ovarian epithelial neoplasms,pre-eclampsia and other development.CircRNAs could regulate the expression of numerous gynecological cancer-related microRNA (miRNAs).The circRNA-miRNA-messenger RNA(mRNA) axis is a known regulatory pattern of several cancer-associated pathways,with both agonist and antagonist effects on carcinogenesis.Numerous studies have shown that circRNAs may become potential targets and clinical diagnostic markers for reproductive and gynecological diseases.This review focuses on the biogenesis and properties of circRNAs,databases of circRNA,their functions and potential significance in gestational,tumor and gestational diseases.