Aim To investigate the effects of osthol on bone marrow stromal stem cells in vitro under the con-ditions of the ability to differentiate into osteoblasts and the case of proliferation. Methods The rat bone marrow sample was obtained,and the all bone marrow cell culture methods were used to separate and collect the stratum of mononuclear cells. The cells were cultured in DMEM containing 10% fetal bovine serum. Three days later the culture medium was changed for the first time. Nine days later,serial subcultivation proceeded. The final concentration of osthol was 1 ? 10 -4,1 ? 10 -5,1 ? 10 -6,1 ? 10 -7 mol?L -1 respectively. MTT method was adopted in proliferation analysis. Under the induced condition,the Alkaline phosphatase activity, calcium salt sediment yield and osteocalcin were meas-ured on 4th,8th,12th,16th day. On the fifteenth day,his-tochemistry dyeing proceeded for calcified tubercle. Total RNA was isolated and the gene expression of bFGF, IGF-1,Osterix and Runx-2 was investigated by RT-Real Time PCR. Results The BMSCs proliferation was refrained by osthol dose-dependently. But it evidently led to osteogenesis. The ALP activity,calcium salt sediment yield and osteocalcin were raised,and calcified tubercle amount was increased. Besides,it also could enhance the mRNA level of bFGF,IGF-1,Osterix and Runx-2. Conclusion The osthol with final concentra-tion of 1 ? 10 -5 mol?L -1 can markedly promote BM-SCs differentiation to osteogenesis. which proves osthol is an active constituent of the traditional Chinese medi-cine Common Cnidium Fruit.

Objective Pulmonary fibrosis ( PF) is a group of pulmonary interstitial pathological changes caused by various factors, and its pathogenesis is not yet fully elucidated .This article discussed the effect of lycium barbarum polysaccharide ( LBP) on ble-omycin-induced PF in mice and its action mechanisms . Methods The C57/BL6 mice were randomly divided into 6 groups:sham-opera-tion, PF model, dexamethasone ( DM), high-dose LBP, medium-dose LBP, and low-dose LBP.The mice in the sham-operation group were injected into the trachea with isotonic saline and those in the oth-er groups with 5 bleomycin at mg/kg for establishing the PF model . Two days after modeling , the mice in the DM and high-, medium-,and low-dose LBP groups were treated with DM at 5 mg/kg and LBP at 0.8, 0.4, and 0.2 g/kg, while those in the sham-operation and PF model groups with the same volume of isotonic saline , respectively , qd, for 4 consecutive weeks .Then, the pathological chan-ges of the lung tissue were observed and the hydroxyproline ( HYP) content in the lung tissue was detected for all the animals .RT-PCR was used to determine the relative expressions of the PF-related COL1A1 and α-SMA genes. Results Compared with the PF mod-els, the the high-, medium-, and low-dose LBP groups showed significant increased body weight after 4 weeks of medication ([14.29 ±0.38] vs [16.12 ±0.37], [15.58 ±0.25] and [15.07 ±0.21] g, P<0.01), the high-and medium-dose groups ex-hibited remarkably decreased lung indexes ([0.887 ±0.13] vs [0.847 ±0.22] and [0.859 ±0.18]%, P<0.05), and the high-dose group presented markedly reduced alveolitis score (3.40 ±0.23 vs 3.09 ±0.22, P<0.05), PF score (3.57 ±0.27 vs 3.07 ± 0.31, P<0.01) and HYP content ([0.831 ±0.05] vs [0.786 ±0.07] μg/mg wet weight, P<0.05).In comparison with the mod-el group, the DM, high-dose LBP and medium-dose LBP groups showed significantly decreased gene expressions of COL 1A1 (1.53 ± 0.13 vs 1.26 ±0.10 and 0.98 ±0.17, P<0.05) and α-SMA (5.67 ±0.47 vs 4.19 ±0.28 and 2.29 ±0.31, P<0.05). Conclusion LBP can suppress the progression of bleomycin-induced pulmonary fibrosis by inhibiting the gene expressions of COL 1A1 andα-SMA and decreasing the HYP content in the lung tissue .

This study is to investigate the effect of osthol on osteoclasts' activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1 x 10(-5) mol x L(-1) osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1 x 10(-5) mol x L(-1) osthol. There is significant difference between control group and experiment group treated by 1 x 10(-5) mol x L(-1) osthol. Therefore, the osthol through RANK+RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption.

This study is to investigate the effects on the expression of iNOS and production of NO in the osteogenic differentiation process of rat bone marrow stromal cells (rBMSCs) by icariside II. rBMSCs were cultured by adherence screening method. When the culture dishes were covered with 80% cells, the osteogenic induced cultures were adopted. Icariside II was supplemented into the culture at 1 x 10(-5) mol x L(-1). The activity of iNOS, content of NO and osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-FALP and mineralized bone nodules were compared among the icariside II-supplemented group, L-NMAE group, icariside II + L-NAME group and the control. Total RNA was isolated and the gene expression of iNOS, Osterix and Runx-2 was investigated by real-time PCR. Total protein was also isolated and the secretion of iNOS and collagen I was examined by Western blotting. Icariside II can significantly improved ALP activity, CFU-FALP amount and mineralized nodules. Besides, the mRNA level of factors related to the osteogenic differentiation includes Osterix and Runx-2 also enhanced. The secretion of collagen I also promoted significantly. But all of these effects can be inhibited by L-NAME which can specifically inhibit the activity of iNOS. Icariside II enhances the osteogenic differentiation of rBMSCs significantly, but if the activity of iNOS was blocked by L-NAME, the osteogenic differentiation markers decrease accompanied with iNOS and NO decrease, suggesting that icariside II stimulates the osteogenic differentiation via enhancing the activity of iNOS and promoting the generation of NO.

OBJECTIVETo investigate the effects of icariin on the osteogenic differentiation of rat bone marrow stromal cells (rBMSCs).

METHODrBMSCs were cultured by adherence screening method. Icariin was supplemented into the culture at 1 x 10(-5) mol x L(-1). The osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-F(ALP) and mineralized bone modulus were compared between the icariin-supplemented group and the control group. Total RNA was isolated and the gene expression of bFGF, IGF-1, Osterix(OSX) and Runx-2 was investigated by RT Real-time PCR.

RESULTIcariin significantly improved ALP activity, CFU-F(ALP) amounts and mineralized modulus. It also can enhance the mRNA level of bFGF, IGF-1, Osterix and Runx-2.

CONCLUSIONIcariin enhances the osteogenic differentiation of rBMSCs significantly, which suggested that icariin has the potentiality to be a new drug of anti-osteoporosis or fracture healing.

Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Male ; Osteogenesis ; drug effects ; Rats ; Rats, Wistar ; Stromal Cells ; cytology ; drug effects ; metabolism

The present research was to investigate the time effect of sinusoidal electromagnetic fields (SEMFs) at different exposure time on the proliferation and differentiation of osteoblasts (OB) in vitro. The newborn rat calvarial OB were isolated by enzyme digestion and divided randomly into 7 groups after one passage. The exposure times of the SEMFs were 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h and 3.0 h, respectively, and the frequency was 50 Hz. The cells were exposed in the SEMFs of 1.8 mT. Those without SEMFs exposure were used as the control group. They were observed under the contrast phase microscope each day. After 48 h, cell proliferation was assayed by MTT method. The alkaline phosphatase (Alkaline Phosphatase, ALP) activities were measured after the exposure of SEMFs for 3 d, 6 d, 9 d and 12 d, respectively. The calcified nodules were stained by Alizarin Bordeaux after 10 d. The cells exposed in the SEMFs were arranged in Spiral appearance after 8 d. The SEMFs exposure time at 2.0 h, 2.5 h and 3.0 h significantly inhibited cell proliferation (P < 0.01) and 0.5 h, 1.0 h, 1.5 h groups more significantly than control groups (P < 0.05). When the 3 d, 6 d and 12 d the ALP activities of the 0.5 h, 1.0 h, 1.5 h and 2.0 h, times group were significantly higher than those in the control group (P < 0.05), and after 9 d the 1.0 h, 1.5 h and 2.0 h activity of ALP higher significantly than control and other groups (P < 0.01). Other groups had no effect on the ALP activity. Alizarin Bordeaux staining result showed the amounts of calcified nodules 1.0 h, 1.5 h and 2.0 h higher than control groups. The SEMFs at 50 Hz, 1.8 mT different time exposure groups inhibits the proliferation of OB, but they enhances the maturation and mineralization of the OB and SEMFs at 1.8 mT of the 1.5 h has the strongest activity.
Animals ; Animals, Newborn ; Calcification, Physiologic ; radiation effects ; Cell Differentiation ; radiation effects ; Cell Proliferation ; radiation effects ; Cells, Cultured ; Electromagnetic Fields ; Osteoblasts ; cytology ; Osteogenesis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigated the effect of icariin and genistein on proliferation and mineralization of cultured rat osteoblasts (rat calvarial osteoblasts, ROB). And to contrast the pharmacological activity of icariin and genistein.

METHODBone cells were obtained by enzyme digestion from the segregated neonatal SD rat skull, and were cultured in MEM containing 10% FBS which was changed after three days later. Serial subcultivation was proceeded when cells covered with 90% culture dish. The final action concentration of icariin and genistein were both 1 x 10(-5) mol x L(-1). Proliferation was analyzed by MTT on 96-well plates, while differentiation was analyzed on 24-well plates. Under the induced condition, the alkaline phosphatase activity, calcium salt sediment yield and osteocalcin were measured at the 3, 6, 9, 12 d. At 12th day, ALP staining, alizarin red staining and calcified nodule count were preceded. Total RNA was isolated at 0, 6, 12, 24, 48, 72 h. The gene expression of bFGF, IGF-1, Osterix and Runx-2 was analyzed by Real-time RT-PCR.

RESULTWith the concentration of 1 x 10(-5) mol x L(-1), icariin and genistein have no significant effect on the ROB' s proliferation. The osteogenesis, ALP activity, calcium salt sediment yield and osteocalcin, calcified tubercle amount were significantly increased. And they enhanced the mRNA level of bFGF, IGF-1, Osterix and Runx-2. On the level of osteoblasts, the activity of icariin is stronger than that of genistein.

CONCLUSIONWhen the final concentration of icariin and genistein is 1 x 10(-5) mol x L(-1), they can significantly promoted ROB maturation. And on the level of osteoblasts, the activity of icariin is stronger than that of genistein.

Alkaline Phosphatase ; metabolism ; Animals ; Calcification, Physiologic ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; genetics ; Fibroblast Growth Factor 2 ; genetics ; Flavonoids ; pharmacology ; Genistein ; pharmacology ; Osteoblasts ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; genetics

OBJECTIVETo investigated the effects of isopsoralen on bone marrow stromal stem cells (BMSCs) differentiate and proliferate in vitro.

METHODThe stratum of mononuclear cells were separated and collected from the rat bone marrow sample by the all bone marrow cell culture methods. The cells were cultured in DMEM contained 10% fetal bovine serum. The culture medium was changed after three days. Nine days later, cells were treatment by isopsoralen with the concentration 1 x 10(-5), 1 x 10(-4), 1 x10(-6), 1 x 10(-7) mol x L(-1). MTT method was used for the proliferated analyzing. Under the induced condition, the alkaline phosphatase (ALP) activity, calcium salt sediment yield and osteocalcin were measured at the 4, 8, 12, 16 d. At the fifteenth day, histochemistry dyeing for calcified tubercle and ALP was proceeded. Total RNA was isolated and the gene expression of bFGF, IGF-1, Osterix and Runx-2 were investigated by Real Time PCR.

RESULTThe BMSCs proliferation refrained by isopsoralen with dose dependent. But it significantly enhanced osteogenesis, which was represented by the promotion of the ALP activity, calcium salt sediment yield, osteocalcin, and calcified tubercle amount. Besides, it also enhanced the mRNA level of bFGF, IGF-1, Osterix and Runx-2.

CONCLUSIONThe isopsoralen with the concentration 1 x 10(-5) mol x L(-1) can promote BMSCs differentiation to osteoblasts. It demonstrated the isopsoralen can prevent antiosteoporotic, which is an active part of the traditional Chinese medicine psoralea corylifolia.

Alkaline Phosphatase ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Core Binding Factor Alpha 1 Subunit ; genetics ; Fibroblast Growth Factors ; genetics ; Furocoumarins ; pharmacology ; Gene Expression Regulation ; drug effects ; Insulin-Like Growth Factor I ; genetics ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Osteocalcin ; metabolism ; Osteogenesis ; drug effects ; RNA, Messenger ; genetics ; Rats ; Stromal Cells ; cytology ; drug effects ; metabolism ; Transcription Factors ; genetics

Objective To investigate the myogenic differentiation of laryngeal mucosal mesenchymal stem cells (LM-MSCs) and the possibility of LM-MSCs as new alternative seed cells for laryngeal tissue engineering.Methods LM-MSCs were separated from normal epiglottis mucosa and the cell surface markers including CD44,CD105,CD90,CD29,CD34 and CD45 were analyzed through flow cytometry.The osteogenesis and adipogenesis differentiation of LM-MSCs were investigated by oil red staining and alizarin red S staining.Immunofluorescence staining and RT-PCR were used to detect the expressions of myogenic differentiation markers including Myod1,Myogenin and myosin heavy chain (MyHc).Results The separated LM-MSCs were in a fibrocyte-like form with long fusiform shape and grew adherently.The expression rates of cell surface markers LM-MSCs were CD44 (100.0％),CD105 (90.4％),CD90 (99.9％),CD29 (93.0％),CD34 (0.4％) and CD45 (1.3％) respectively.A number of beaded lipid drops and mineral deposition were observed after 14 days of adipogenesis differentiation and 21 days of osteogenesis differentiation.Myod1,Myogenin and MyHc genes appeared after 1 week and 3 weeks of myogenesis differentiation respectively.Conclusions The LM-MSCs have the properties of mesenchymal stem cells and could be differentiated into myoblasts,providing with the possibility to repair the damaged vocal cords with LM-MSCs through tissue engineering techniques.