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 To study the role of bone morphogenetic protein-7 in the osteogenic differentiation of periosteal cellsin vitro. Methods Periosteal cells, obtained from adult tibial periosteum, were cultured by routine method in vitro, and divided into two groups. One group cultured with BMP7 and the supplements of 100 nmol dexametasone, 10 mmol b-glycerophosphate and 50 mg/mL L-ascorbic acid (BMP7 group), the other cultured with the supplements alone as the control (control group). Ultrastructure and morphological changes of periosteal cells were observed by contrast phase microscope and electron microscope. In order to test the expression of markers of osteoblastic differantiation in periosteal cells, involved mineralized node and alkaline phosphatase. Each group was tested at the time of 5 d, 10 d, 15 d, 20 d, respectively, using ALP kit stain and Von Kossa stain with 3 samples at each time. Results The periosteal cells cultured by routine method and induced into osteoblast differentiation with BMP7 were both growing well, in vitro. Microscope observations showed that the periosteal cells were spindle-shaped, well-stacked, transparent and three-dimensional in the early stage, and cube-shaped or puncheon shaped in the mitotic phase, gradually became wide shuttle and irregular shape with a lot secretion in telophase. The positive cells were visible by the ALP kit staining and Von Kossa staining of calcium nodules at 5 d, 10 d, 15 d and 20 d in both groups.A difference of positive rate at each time point was found between BMP7 group and control group at 5 d, 10 d, 15 d, 20 d, and the difference was statistically significant (P ＜ 0.01). Conclusion It displayed well regeneration and osteogenesis ability in the periosteal cell. BMP7 has definite osteo-inductive activity, which can obviously enhance the proliferation and ossifyng differentiation of periosteal cells.

Objective To observe different hypoglycemic treatments on glucagon-like peptide-1 (GLP-1) and its correlation with β cells function in newly diagnosed type 2 diabetes mellitus (T2DM).Methods A total of 76 patients with newly diagnosed T2DM were randomly divided into insulin aspart 30 group and oral drugs group.OGTT test were measured at 0,30,120 min,the serum levels of GLP-1 and corresponding C peptide,and insulin levels were measured,HOMA insulin secretion and insulin resistance index were calculated.Then 30 healthy cases were as the control group,GLP-1 levels were analyzed,and the changes of the index were compared.Results At each time point,blood glucose and glucose area under the curve (AUCg) was significantly decreased (P ＜0.05).Insulin and C-peptide and the AUC (AUCins,AUCc-p) were increased (P ＜ 0.05).HOMA-IR was statistically decreased compared with pre-therapy (P ＜ 0.05),HOMA-IS and △INS30/G30,△ INS120/G120 were significantly increased (P ＜ 0.05).Compared with pre-therapy,GLP-1 levels and AUCglp (area under the curve GLP-1) were significantly increased (P ＜0.05),and reached the peak at 120 min,closed to secretion curve of the control group.Conclusion GLP-1 levels were lower than normal in newly diagnosed T2DM patients,and different hypoglycemic drugs makes GLP-1 levels increase.Insulin,C-peptide and AUCins,AUCc-p,HOMA-IS,HOMA-IR,etc.,suggest that β-cell function is restored and insulin resistance is reduced.

Objective To observe different hypoglycemic treatments on glucagon-like peptide-1 (GLP-1) and its correlation with β cells function in newly diagnosed type 2 diabetes mellitus (T2DM).Methods A total of 76 patients with newly diagnosed T2DM were randomly divided into insulin aspart 30 group and oral drugs group.OGTT test were measured at 0,30,120 min,the serum levels of GLP-1 and corresponding C peptide,and insulin levels were measured,HOMA insulin secretion and insulin resistance index were calculated.Then 30 healthy cases were as the control group,GLP-1 levels were analyzed,and the changes of the index were compared.Results At each time point,blood glucose and glucose area under the curve (AUCg) was significantly decreased (P ＜0.05).Insulin and C-peptide and the AUC (AUCins,AUCc-p) were increased (P ＜ 0.05).HOMA-IR was statistically decreased compared with pre-therapy (P ＜ 0.05),HOMA-IS and △INS30/G30,△ INS120/G120 were significantly increased (P ＜ 0.05).Compared with pre-therapy,GLP-1 levels and AUCglp (area under the curve GLP-1) were significantly increased (P ＜0.05),and reached the peak at 120 min,closed to secretion curve of the control group.Conclusion GLP-1 levels were lower than normal in newly diagnosed T2DM patients,and different hypoglycemic drugs makes GLP-1 levels increase.Insulin,C-peptide and AUCins,AUCc-p,HOMA-IS,HOMA-IR,etc.,suggest that β-cell function is restored and insulin resistance is reduced.