OBJECTIVETo establish a reliable model for drug screening and therapy by culturing rat femoral head and inducing cartilage degeneration quickly in vitro.

METHODSThe femoral heads from the same SD rats of two-month old were divided into control group and experimental group respectively. They were cultured with DMEM medium plus 10% fetal bovine serum or DMEM medium plus 10% fetal bovine serum plus 50 ng/ml IL-1β for three days. Femoral heads were fixed in 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin and cut into slices. Specimens were stained with Toluidine blue and Safranine O-Fast Green FCF. The protein expression levels of type II collagen, MMP13, Sox9 and ADAMTS5 were analyzed by immunofluorescence.

RESULTSBoth the Toluidine blue and Safranine O staining were pale in the margin of femoral heads which were stimulated with IL-1β for three days compared to that in control group. The Fast Green FCF staining was positive at the edge of the femoral head in experimental group, which indicated that cartilage became degenerated. The expression levels of both type H collagen and Sox9 were decreased significantly while the expression levels of MMP13 and ADAMTS5 were increased in experimental group.

CONCLUSIONThe model of cartilage degeneration is established by culturing and inducing the degeneration of the femoral heads quickly in vitro.

Animals ; Cartilage Diseases ; genetics ; metabolism ; Collagen Type II ; genetics ; metabolism ; Disease Models, Animal ; Femur Head ; metabolism ; Humans ; In Vitro Techniques ; Interleukin-1beta ; genetics ; metabolism ; Male ; Matrix Metalloproteinase 13 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; SOX9 Transcription Factor ; genetics ; metabolism

OBJECTIVETo investigate the effects of serum containing Gumibao (Chinese character: see text) on the proliferation and differentiation of osteoblast induced by dexamethasone.

METHODSOsteoblasts were extracted from skulls in newly born (within 24 hours) SD rats, and digested with collagenase. The first passage of cells were used for experiments. Cells were cultured in the medium containing different concentrations of dexamethasone (0, 10(-8), 10(-7), 10(-6), 10(-5) ,10(-4) mol/L). Alkaline phosphatase staining were carried out after 1 week and numbers of mineralized nodes with alizarin red staining were observed after 3 weeks. Accordingly, following the treatment of 10(-5) mol/L dexamethasone for 1 week, cells were cultured in the medium with serum containing Gumibao (Chinese character: see text). One week after Cumibao (Chinese character: see text) treatment, cells were stained with Alkaline phosphatase and collagen I and PCNA were examined by Western-blot. However, the observation of numbers of mineralized nodes with alizarin red stain required one more week.

RESULTSHigh concentration of dexamethasone could inhibit the expression of PCNA, collagen I, alkaline phosphatase and reduce the number of mineralized nodes of osteoblast, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.

CONCLUSIONHigh concentration of dexamethasone could inhibit the proliferation and differentiation of osteoblastic cells, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.

Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; analysis ; Dexamethasone ; pharmacology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Female ; Osteoblasts ; cytology ; drug effects ; physiology ; Proliferating Cell Nuclear Antigen ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate regulation function of anodonta glucan HBP-A on chondrocytes through Wnt pathway in vitro.

METHODSRat chondrocytes were cultured and differentiated induced with IL-1beta (10 ng/ml) in vitro. Chondrocytes were divided into five groups:IL-13 group,IL-1beta + IWP-2 (5 microM,Wnt pathway inhibitor) group, IL-1beta + HBP-A (0.3 mg/ml) group and IL-1beta + IWP-2 + HBP-A group. Wnt-3a, beta-catenin (24 h,48 h,72 h) and MMP-13(72 h) genes expression were detected by Rt-PCR, while beta-catenin, MMP-13, Sox-9 and coll-II (48 h) protein expression were measured by Western-blot.

RESULTSAfter induction of IL-1beta, gene expression of Wnt-3a, beta-catenin and MMP-13 were increased,so were the protein expression of beta-catenin and MMP-13. In contrast,protein expression of Sox-9 and Coll-II were declined. Following addition of HBP-A, Wnt-3a, beta-catenin and MMP-13 were shown as induction of IL-1beta, but protein expression of Sox-9 and Coll-II were upgraded. Combining HBP-A with IWP-2 led to the lowest level in Wnt-3a, beta-catenin gene and beta-catenin protein expression and highest expression of Sox-9 protein.

CONCLUSIONHBP-A could not only delay the differentiation of chondrocytes through downgrading the signal expression of Wnt/beta-catenin,but also adjust the expression of Wnt-3a, beta-catenin and Sox-9 when combinated with the Wnt inhibitor.

Animals ; Anodonta ; chemistry ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; metabolism ; Glucans ; pharmacology ; Interleukin-1beta ; metabolism ; Rats ; Wnt Signaling Pathway ; drug effects ; Wnt3A Protein ; genetics ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo compared the activity and yield rate of osteoblast obtained by different collagenase digestion methods, to find a better way to extract osteoblast for the experimental researches of osteoporosis.

METHODSTen 24-hour-old SD rats were were euthanized. The cranium of rats were removed and cuted into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, all the cranium were divided into two equal parts, and randomly divided into two groups which would be digested by type I collagenase and type II collagenase separately for two times. The rat cells of the two groups were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. The primary culture osteoblasts were counted by using a haemacytometer after digestion and 72 hours later. The second generation osteoblasts cultured 48 h were dyed by NBT/BCIP staining solution, and were detected by quantitative measurement with PNPP.

RESULTSThe cells had irregular shapes. The results of cell counting showed that the cell number of type I group was larger than type 11 group. Alkaline phosphatase dyeing were positive. Detecting of alkaline phosphatase using the method of PNPP showed that the absorbance value in type I group were higher than type II group (P<0.05).

CONCLUSIONTwo types of collagenase are both suitable for the in vitro culture of rat osteoblasts. The activity and yield rate of osteoblasts in type I group are higher which could provide more stable seed cells for the treatment of osteoporosis.

Animals ; Cell Count ; Cell Culture Techniques ; methods ; Collagenases ; metabolism ; Female ; Male ; Osteoblasts ; cytology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the clinical value of glycosylated G-CSF combined with middle-high dose cyclophosphamide (Cy) or conventional chemotherapy with increased dose of Cy for mobilizing peripheral blood progenitor cells in patients with tumor.

METHODSThirty patients from four hospitals in Beijing region were enrolled in this clinical study. Diagnoses of the patients were non-Hodgkin' lymphoma (n = 21), Hodgkin disease (n = 1), breast cancer (n = 7) and ovary cancer (n = 1). Autologous peripheral blood progenitor cells (APBPC) were mobilized by middle-high dose Cy or conventional chemotherapy with increased dose of Cy combined with G-CSF. G-CSF was given subcutaneously from the nadir of the white blood cell (WBC) count to the end of PBPC collection. The dosage of G-CSF was 250 microg/d in 29 patients and 500 microg/d in 1 patient. When WBC count was > 5 x 10(9)/L, APBPC were harvested with CS 3000 plus/COBE Spectra.

RESULTSThe average dosage of Cy was 3.95 g (2.3 g/m(2)). The doses of G-CSF were 3.1 approximately 6.4 microg x kg(-1) x d(-1). Thirteen patients (43%) were collected twice, 14 patients (47%) three times and 3 patients (10%) four times. All of the patients could tolerate the treatment regimens. Seven patients had bone pain after G-CSF injection and one was severe, one patient had headache and one had nausea and vomiting.

CONCLUSION250 microg glycosylated G-CSF combined with middle-high Cy or conventional chemotherapy with increased dose of Cy combined G-CSF is an optimal method for APBPC mobilization in tumor patients.

Adolescent ; Adult ; Antigens, CD34 ; analysis ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Colony-Forming Units Assay ; Cyclophosphamide ; administration & dosage ; Dose-Response Relationship, Drug ; Female ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; Hematopoietic Stem Cell Mobilization ; Humans ; Leukocyte Count ; Leukocytes, Mononuclear ; cytology ; drug effects ; immunology ; Male ; Middle Aged ; Neoplasms ; blood ; drug therapy ; pathology ; Platelet Count ; Treatment Outcome

OBJECTIVETo investigate the effects of osthole on proliferation of neonatal rat osteoblast and the mechanism.

METHODSTen 24 hours old SD rats were executed by dislocating. The cranium of rats were removed and cut into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, the cranium were digested by type I collagenase for one hour two times. The mixed cells were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. To identify the cells, ALP staining and alizarin red staining were performed after cultured 48 h and 28 d. The osteoblasts were randomly divided into five groups. Cells were treated with osthole at concentrations of 100, 50, 25, 12.5, 0 micromol/L. CCK-8 method was used to evaluate the proliferation after 24 h,48 h and 72 h. The expression of PCNA and beta-catenin protein were detected through the method of Western Blot after one week.

RESULTSThe cells had irregular shapes and showed typical features of osteoblast. The results of ALP staining and alizarin red staining were both positive. CCK-8 detection showed that the osthole with final concentration of 100 micromol/L inhibited the proliferation of osteoblast after 24 h, while the osthole with final concentrations of 50 micromol/L and 25 micromol/L displayed the inhibition effect after 48 h. The osthole of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast. The result of Western Blot showed that osthole reduced the expression of PCNA and beta-catenin protein in a dose-dependent manner.

CONCLUSIONThe osthole with final concentrations of 100, 50, 25 micromol/L inhibited the proliferation of osteoblast (P < 0.05). The osthole with final concentrations of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast (P > 0.05). These findings demonstrate that osthole may inhibit the proliferation of osteoblast by regulating the Wnt/beta-catenin signaling in osteoblast.

Animals ; Animals, Newborn ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coumarins ; pharmacology ; Female ; Male ; Osteoblasts ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Sincalide ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo investigate the effects of resveratrol (RV) in reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) and the related mechanism.

METHODSPrimary MEFs were isolated from E13.5 embryos and used within three passages. Retroviruses expressing Sox2 and Oct4 were produced by transfecting GP2-293t cells with recombinant plasmids (MSCV)-Sox2 and MSCV-Oct4. Supernatants containing retroviruses were obtained after 48-hour transfection and MEFs were then infected. Different concentrations (0, 5, 10 and 20 μmol/L) of RV were added to embryonic stem cell (ESC) medium to culture MEFs 48 h post-infection. iPSC clones emerged and were further cultured. Expression of pluripotent markers of iPSCs was identified by cell immunofluorescence and reverse transcription-polymerase chain reaction. Both cytotoxicity and cell proliferation were assayed by Western blot analysis after RV was added into ESC medium. The ultrastructure change of mitochondria was observed by electron microscopy.

RESULTSMore than 2.9-fold and 1.3-fold increases in colony number were observed by treatment with RV at 5 and 10 μmol/L, respectively. The reprogramming efficiency was significantly decreased by treatment with 20 μmol/L RV. The proliferation effect on MEFs or MEFs infected by two factors Sox2/Oct4 (2 factors-MEFs, 2F-MEFs) was investigated after RV treatment. At 20 μmol/L RV, induced cell apoptosis and proliferation inhibition were more obvious than those of 5 and 10 μmol/L treatments. Clones were selected from the 10 μmol/L RV-treated group and cultured. Green fluorescent protein expression from one typical clone was silenced one month later which expressed ESC-associated marker genes Gdf3, Nanog, Ecat1, Fgf4 and Foxd3. Electron transmission microscope showed obvious cavitations in mitochondria. The expression of hypoxia-inducible factor-1α was up-regulated when 2F-MEFs were treated with RV compared to the control group.

CONCLUSIONRV improved the efficiency of reprogramming 2F-MEFs into iPSCs at low and moderate concentrations (5 and 10 μmol/L). The effect of 10 μmol/L RV on reprogramming was much greater than that of 5 μmol/L RV. However, high concentration of RV (20 μmol/L) led to more severe cavitations in mitochondria and caused cytotoxic effects. Taken together, these findings suggest that RV mimics hypoxia in cells and promotes reprogramming at a low concentration.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; Induced Pluripotent Stem Cells ; drug effects ; Mice ; Octamer Transcription Factor-3 ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; SOXB1 Transcription Factors ; physiology ; Stilbenes ; pharmacology

OBJECTIVETo observe the changes in activities of superoxide dismutase (SOD) and glutathione peroxide (GSH-Px) in the induced sputum of silicosis patients, and to investigate the roles of SOD and GSH-Px in the development and progression of silicosis and the significance of measuring activities of SOD and GSH-Px in induced sputum among silicosis patients.

METHODSFifty hotel attendants were chosen as control group, 50 workers with more than one year of silica dust exposure as dust exposure group, 32 silica dust-exposed workers as observation subject group, and 52 silicosis patients as silicosis group. The activities of SOD and GSH-Px in their induced sputum were measured by enzyme-linked immunosorbent assay.

RESULTSCompared with the control group, the observation subject group and silicosis group had significantly decreased SOD activity (68.16 ± 30.17 and 66.38 ± 47.32 U/ml vs 75.81 ± 11.92 U/ml, P < 0.05); compared with the dust exposure group, the silicosis group had significantly decreased SOD activity (66.38 ± 47.32 U/ml vs 70.12 ± 14.31 U/ml, P < 0.05). Compared with the control group and dust exposure group, the observation subject group and silicosis group had significantly increased GSH-Px activity (268.21 ± 15.45 and 279.34 ± 29.26 U/ml vs 224.22 ± 12.64 and 236.41 ± 14.54 U/ml, P < 0.05 or P < 0.01).

CONCLUSIONThe SOD activity in dust exposure group and silicosis group decreased, but there were no significant differences between patients with different stages of silicosis. The GSH-Px activity in dust exposure group and silicosis group was significantly higher than that in control group, and there were significant differences between patients with different stages of silicosis. These suggest that the imbalance of oxidative/antioxidant systems is associated with the development and progression of silicosis.

Adult ; Case-Control Studies ; Glutathione Peroxidase ; metabolism ; Humans ; Middle Aged ; Silicosis ; enzymology ; Sputum ; enzymology ; Superoxide Dismutase ; metabolism

OBJECTIVE: To study the epidemiologic characteristics of human herpes virus (HHV) activated infection in the diseases of blood system and patients received allo-HSCT by statistically analyzing the screening results of 8 human herpes viruses (HHVs) of 4164 patients in Hebei Yanda LU Dao-Pei Hospital from 2012 to 2017. METHODS: PCR was used to screen 8 HHVs. RESULTS: Two thousand and fifty-two patients (49.28%) were HHV-positive among 4164 patients screened. Among these patients screened, the infection spectra of 8 human HHVs in hematological diseases as well as patients received allogeneic hematopoietic stem cell transplantation of totally 2994 patients were summarized as follows: the positive rate of EBV (29.49%) was the highest, that of HCMV (23.15%), HHV-6 was 18.77% and HHV-7 was 17.64%, while the remaining 4 HHVs all≤2.1%. The rate of co-infection of various HHVs was significantly higher than that of single infection of HHV among all these disease groups except familial hemophagocytic lymphohistiocytosis, for which single EBV infection was the most common. The differences of positive rates among these 8 human HHVs in hematological diseases as well as patients received allogeneic hematopoietic stem cell transplantation were statistically significant by Chi-square test of R*C tables (χ=54.99, P＜0.05). For each HHV, the differences of positive rates among the above-mentioned disease groups were also statistically significant except HHV-8 (P＜0.05). CONCLUSION The patients with various blood diseases have different activated infection spectra of HHVs. EBV, HCMV, HHV-6 and HHV-7 are most common in HHVs infection. Different HHVs infections correlate with different hematologion diseases.

OBJECTIVETo investigate the effect of HBP-A on meniscal injuries and the expressions of genes associated with pathological hypertrophy and calcification of the meniscusinduced by abnormal loading.

METHODSBovine meniscus explants were subjected to 25% strain at 0.3 Hz for 3 h and treated with 0.6 mg/mL of HBP-A. The cell viability in the meniscus explants after 72 hin culture was determined using live/dead staining and the expression levels of genes associated with pathological hypertrophy and calcification of the meniscus (ANKH, ENPP1, ALP, MMP13, and IL-1) were measured using real-time PCR and Western blotting. The conditioned medium was collected for testing sulfated glycosaminoglycan (GAG) release.

RESULTSThe number of dead cells, loss of proteoglycan content, and the expressions of ANKH, ENPP1, ALP and MMP13, and IL-1 at both the mRNA and protein levels were all significantly lower in the meniscus explants treated with 0.6 mg/mL HBP-A than in the explants with only 25% abnormal pressure stimulation (n=3, P<0.05).

CONCLUSIONHBP-A can effectively alleviate meniscal injuries induced by abnormal loading and suppress the expressions of genes related with pathological hypertrophy and calcification of the meniscus, and can serve as a potential drug for treatment of knee osteoarthritis.

Animals ; Calcinosis ; drug therapy ; Cattle ; Glucans ; pharmacology ; Hypertrophy ; Menisci, Tibial ; drug effects ; Osteoarthritis, Knee ; drug therapy ; Real-Time Polymerase Chain Reaction ; Tibial Meniscus Injuries ; drug therapy