OBJECTIVE: Animal experiments prove that alcohol may cause adipo-accumulation in femoral head and then induce osteonecrosis,but its mechanism of action is not known. NIH 3T3 fibroblast is a kind of pluripotent stem cell.There are not relative reports on the effect of alcohol on NIH 3T3fibroblast at present.OBJECTIVE: To observe the effect of alcohol on the differentiation of NIH 3T3 fibroblast into adipocyte and the influence of that on the expression of adipogenic transcription factor-peroxisome proliferator activated receptor gamma (PPAR-γ).DESIGN: Single sample observation.SETTING: Department of Biochemistry, Basic Medical College,Zhengzhou University and Department of Orthopaedics, First Affiliated Hospital, Zhengzhou University.MATERIALS: The experiment was completed in the Laboratory of Department of Biochemistry, Basic Medical College, Zhengzhou University from April 2002 to June 2003.NIH 3T3 fibroblasts were provided by the Laboratory of Department of Orthopaedics,University of Virginia Hospital.METHODS: The NIH 3T3 fibroblasts were divided into 6 groups randomly.The alcohols (volume fraction was 0.998) with the dosage 0(control group),0.03,0.06,0.09,0.15,0.21 mol/L were added respectively to the NIH 3T3 fibroblasts daily.The mixtures were cultured for 14 days and then stained with Sudan Ⅳ .The percentage of adipocyte was determined with image analysis software of computer,Image Pro Plus 4.1.The expression of PPAR-γ mRNA was determined with the technology of reverse transcription-polymerase chain reaction.alcohol and control groups.RESULTS: The cells were processed with the alcohols of progressive inpercentages of adipocytes in all the alcohol groups were 11.9% ,23.8%,28.2% ,31.1% ,42.6% respectively and 1.2,2.5,2.9,3.2,4.4 times that in control group(9.6%) respectively. The differences between 0.03 mol/L alcohol group and control group were not significant (P ＞ 0.05),but the differences between the other 4 groups and control group,0.03 mol/L alcohol ues of PPAR-γ mRNA to 18S gene in the cells of all the groups were 0.218,0.411,0.486,0.473,0.453 respectively and were 1.1,2.1,2.5,2.4,2.3times that in control group (0.197) respectively. The differences between 0.03 mol/L alcohol group and control group were not significant(P ＞ 0.05),but the differences between the other 4 groups and control group and 003 mol/L alcohol group were significant extremely(P ＜ 0.001).CONCLUSION: Alcohol can induce differentiations of a large quantity of NIH 3T3 fibroblasts into adipocytes directly, which might be one of the factors of lipotrophy in bone marrow wheri alcohol-induced osteonecrosis happens.

Objective The mechanism of osteonecrosis induced by alcohol was not clear, lipoidosis in bone cell could be one of reasons to cause osteonecrosis. The study was to observe the effect of alcohol on the adipocyte-specific gene, 422 (aP2), and osteoblastic gene, type-I collagen of marrow stromal cells in vitro experimentally. Methods The primary marrow stromal cells of the femur of 6 to 8 weeks mouse were procured and cultured in DMEM culture fluids, and the samples were isolated after adherent growth culture in vitro. The cells were divided into two groups, one of which was experimental group treated with 0.09 mol/L alcohol added with the culture fluids once for two days; another one was control group. The mixed culture discontinued 10 days later, the cells were collected after the combined handling of 0.22% EDTA and 0.25% trypsinase, and the concentration of the cell suspension was adjusted to 1?109/L. A volume of 10 ?L of the cells suspension was dropped on the nitric cellulose membrane. The expression level of 422(aP2) and type-I collagen mRNA in the cells were investigated by means of intact cell RNA dot blot hybridization. Results The expression scanning value of the dot blot hybridization of 422(aP2)mRNA was 7 207.8?331.3 in the experimental group, that were as 11 times as that in the control group( 652.2?62.6), the difference was statistical significant between the two groups (P

Objective To study the effect of simvastatin and dexamethasone on the expression of endothelial nitric oxide synthase(eNOS)mRNA and the proliferation in osteoblast.Methods The osteoblast was collected from newborn SD rats,skull and cultured,and then randomly divided into 3 groups(8 samples for each group):in group A,the cells were treated with simvastatin of 1?10-7 mol/L,dexamethasone of 1?10-7 mol/L and DMSO of 1‰;in group B,the cells were treated with dexamethasone of 1?10-7 mol/L and DMSO of 1‰;in group C,the cells were treated with only DMSO of 1‰.The cells were collected and total RNA were obtained 96 h after culture.The expression of eNOS mRNA in the cells was analysed with Reverse Transcription-Polymerase Chain Reaction(RT-PCR).The amount of nitric oxide(NO)in culture medium was detected by the method of nitric acid reduction enzyme.The ratio of proliferation of osteoblast was measured using MTT reduction assay.Results The expression of eNOS mRNA in group A,B,C was 0.491?0.014,0.421?0.018,0.489?0.014.The level of NO in the culture medium in group A,B,C was(14.37?1.24)?mol/L,(12.94?0.69)?mol/L,(14.14?1.23)?mol/L.The ratio of osteoblast proliferation in group A,B,C was 0.3055?0.0178,0.2659?0.0105,0.3044?0.0213.There were significant differences of the expression of eNOS mRNA,level of NO and ratio of osteoblast proliferation between group A and group B,group B and group C(P 0.05).Conclusion It demonstrates that the lower expression of eNOS in the osteoblasts induced by dexamethasone may be an important pathogenesis of glucocorticoid-induced osteoporosis.Simvastatin may inhibit the effect of dexamethasone and regulate the expression of eNOS mRNA in the cells and protect the proliferation of osteoblast.

Objective To explore the expression of microRNA‐34a(miR‐34a) in sodium iodoacetate‐induced rat model of os‐teoarthritis rat model and to initially clarify its function .Methods Thirty male Wistar rats were selected .The right knees were in‐jected with sodium iodoacetate (model group) and the left knees served as the control group without injecting by sodium iodoace‐tate .The expression levels of miR‐34a in the synovial fluid and articular chondrocytes were compared between two groups after con‐structing the model .Articular chondrocytes in the two groups were separated for conducting the primary culture ,after miR‐34a knockdown ,the flow cytometry was used to determine cell apoptosis .Results The Makin score in the model group was(5 .09 ± 1 .35)points ,which was (1 .27 ± 0 .64)points in the control group ,the difference had statistical significance (P<0 .05) .Compared with the control group ,the expression levels of miR‐34a in the synovial fluid and articular chondrocytes in the model group were in‐creased ,the difference was statistically significant(P<0 .05) .After the miR‐34a knockdown by using its complementary antisense fragment ,articular chondrocytes apoptosis in the model group was significantly inhibited (P<0 .05) .Conclusion The expression of miR‐34a in the synovial fluid of sodium iodoacetate‐induced knee osteoarthritis is increased ,moreover inhibiting the microRNA‐34a expression could reduce the apoptotic rate of articular chondrocytes in osteoarthritis rat .

BACKGROUND:Pueraria is a member of isoflavone characterizing by decreasing the toxic effects of alcohol,relieving the effect of alcohol withdrawal,anti-oxidative effect,removing oxygen free radicals,and preventing cell injury induced by lipid peroxidation.OBJECTIVE:To investigate the inhibitory effect of puerarin on adipogenic differentiation of alcohol-induced human bone marrow mesenchymal stem cells (hBMSCs).DESIGN,TIME AND SETTING:An in vitro observation based on cytology was performed at Laboratory of Biochemistry and Molecular Biology,Basic Medical College of Zhengzhou University in October 2008.MATERIALS:The bone marrow was taken from the adult healthy volunteers from the Department of Orthopedics of the First Affiliated Hospital of Zhengzhou University.Puerarin standard was provided by the National Institute for the Control of Pharmaceutical and Biological Products.METHODS:The primary hBMSCs were purified from the adult marrow by gradient centrifugation.The second passaged cells were divided into 3 groups.Blank control group:Cells were cultured with DMEM culture media containing 10% fetal bovine serum;Model group:Cells were cultured with 0.09 mol/L alcohol;Experimental group:Cells were cultured with 0.09 mol/L alcohol and 10-6 mol/L puerarin;while,the culture liquid was changed every two or three days.Cells were cultured for 7 days in each group.MAIN OUTCOME MEASURES:PPARγ 2 gene expression with RT-PCR;amount of adipocyte with oil red "O" staining;alkaline phosphatase activity.RESULTS:PPAR-γ2 mRNA expression in the model group was significantly greater than experimental group and blank control group (P ＜0.05),while the expression in the experimental group was slightly greater than blank control group,but there was no significant difference (P ＞0.05).The number of adipocytes was the most in the model group,and the lipid droplet was large and abundant.The number of adipocytes in the experimental group was significantly less than model group (P ＜ 0.05),and there were few adipocytes in the blank control group.Compared with blank control group,alkaline phosphatase activity was decreased in the model group (P＜0.05);but the activity in the experimental group was increased compared to model group (P ＜ 0.05).CONCLUSION:Puerarin inhibits differentiation of hBMSCs into adipocytes induced by alcohol.

BACKGROUND: Animal tests verified that alcohol could cause lipid accumulation in femoral head, leading to osteonecrosis. Molecular biology tests verified that alcohol could induce differentiation of marrow stromal cells (MSCs) into adipocytes. If a drug can resist the differentiation of MSCs into adipocytes induced by alcohol, it can prevent and treat alcoholinduced osteonecrosis.OBJECTIVE: To observe the effects of inhibition of puerarin on differentiation of MSCs into adipocytes induced by alcohol from the aspect of cell biology.DESIGN: Randomized controlled experiment.SETTING: Department of Orthopaedics, First Affiliated Hospital,Zhengzhou University and Department of Biochemistry and Molecular Biology, Basic Medical College, Zhengzhou University.MATERIALS: The experiment was conducted at the Department of Biochemistry and Molecular Biology, Basic Medical College, Zhengzhou University from April 2000 to December 2002. A total of 50 clean healthy Kunming mice aged 6-8 weeks, of either sex, were selected. Bone marrow cells of the bilateral femur were obtained, with the inoculated density of 1.5×106/cm2, implanted into 6 wells and 24 wells cultured plate, randomized into groups, a well as a specimen and 24 specimens in each group.METHODS: MSCs were separated, cultured and assigned into 3 groups at random: model group (cells were treated with 0.09 mol/L alcohol), experimental group (0.09 mol/L alcohol and 0.01 mg/mL final concentration of puerarin) and controlled group (no alcohol and puerarin). Sudan Ⅲ staining was performed and adipose cells was counted under light microscope.Content of triacylglycerol, activity of alkaline phosphatase and content of osteocalcin in cell culture fluid were measured.MAIN OUTCOME MEASURES: ①Result of differentiation of MSCs into adipocytes in micé of the three groups, ②content of triacylglycerol in MSCs of mice of the three groups, ③activity of alkaline phosphatase in MSCs of mice of the three groups, ④content of osteocalcin in cell culture fluid in the three groups.RESULTS: ①After being disposed and cultured for 21 days, number of adipocytes in the experimental group and control group decreased markedly compared with the model group. The number of adipocytes in the model group was 8.9 times of that in the experimental group, and 15.6 times of that in the control group [(319.17±19.92), (35.92±23.77)(20.42±12.15)per cm2,P＜0.001]. ②After being disposed and cultured for 21 days, coutent of triacylglycerol in the experinental group and control group was remarkably lower than that in the model group [(4.15±1.92) and (3.42±1.60),(11.55±4.42) μgin a well, P ＜ 0.001]. ③After being disposed and cultured for 12 days, activity of alkaline phosphatase in the experimental group and control group was distinctly higher than that in the model group [(9.51±2.96),(11.18±3.11), (4.84±2.23) μkat/g,P ＜ 0.001]. ④After being disposed and cultured for 14 days, content of osteocalcin in the experimental group and control group was 2.2 times and 2.7 times of that in the model group, respectively, and the difference was very significant com pared with themodel group [(11.11±4.57),(13.43±5.29), (4.95±2.31) μg/g,P ＜ 0.001].CONCLUSION: Puerarin can inhibit the differentiation of MSCs into adipocytes induced by alcohol, and maintain the osteogenic differentiation,which may prevent the development of osteonecrosis.

BACKGROUNDSteroids inhibit osteogenic differentiation and decrease bone formation while concomitantly inducing adipose deposition in osteocytes. This leads to the fatty degeneration and necrosis of bone cells commonly seen in osteonecrosis of the femoral head. The peroxisome proliferator-activated receptor-γ (PPARγ) is an adipogenic transcription factor linked to the development of this disease and responsible for inducing adipogenesis over osteogenesis in bone marrow mesenchymal stem cells (BMSCs). The aim of this study was to assess whether adipogenic differentiation could be suppressed, and thus osteogenic potential retained, by inhibiting PPARγ expression in BMSCs.

METHODSCells from the bone marrow of New Zealand rabbits were treated with 10(-7) mol/L dexamethasone and infected with one of three small interference RNA (siRNA) adenovirus vectors (S1, S2, and S3) or non-targeting control siRNA (Con) and compared with dexamethasone-treated (model) and untreated (normal) cells. Cells were grown for 21 days and stained with Sudan III for adipocyte formation. At various time points, cells were also assessed for changes in PPARγ, osteocalcin (OC), Runx2, alkaline phosphatase (ALP) activity, and triglyceride (TG) content.

RESULTSDexamethasone-treated model and control groups showed a significant increase in fatty acid-positive staining, which was inhibited in cells treated with PPARγ siRNA-treated, similar to normal untreated cells. All three siRNA groups significantly inhibited PPARγ mRNA and protein, adipocyte number, and TG content compared with the dexamethasone-treated model and control groups, matching that seen in normal cells. OC and Runx2 mRNA and protein, as well as ALP activity, were significantly higher in cells treated with siRNA against PPARγ, similar to that seen in the normal cells. These osteogenic markers were significantly lower in the dexamethasone-treated cell cultures.

CONCLUSIONSThe siRNA adenovirus vector targeting PPARγ can efficiently inhibit steroid-induced adipogenic differentiation in rabbit BMSCs and retain their osteogenic differentiation potential.

Adenoviridae ; genetics ; Adipogenesis ; drug effects ; genetics ; Animals ; Cell Differentiation ; drug effects ; genetics ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; PPAR gamma ; genetics ; metabolism ; pharmacology ; RNA, Small Interfering ; Rabbits ; Steroids