By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 micromol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.
Cartilage/*cytology ; Cell Differentiation/*drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Oligodeoxyribonucleotides/genetics ; Oligodeoxyribonucleotides/*pharmacology ; Rats, Sprague-Dawley ; Stathmin/*genetics ; Stathmin/pharmacology ; Stem Cells/*cytology

Objective To investigate the effects of magnetic stimulation (MS) on the proliferation and differentiation of endogenous neural stem cells (NSCs)/progenitor cells after spinal cord injury (SCI) in rats. Methods Forty-six Wistar rats were used, of which 40 were used to make an animal model of spinal cord injury (SCI) by administering a 10 g x 12.5 cm impact at the T8 level. The other 6 served as the normal controls. The SCI model rats were evenly divided into a magnetic stimulation (MS) group ( n = 20) and a control group ( n = 20). The rats in the MS group received 0.5 Hz and 1.44 T magnetic stimulation 24 h post injury, then 30 pulses per day for 7 days. The rats in the other groups were not exposed to MS. The scale of Basso, Beatti and Bresnahan (BBB) was used to assess hindlimb neurological function. Rats were sacrificed at the 24th hour, and at the 1st, 4th and 8th weeks after SCI. The ratio of nestin to microtubule associated protein 2 (MAP2)/nestin in the cells of the spinal cord was determined by immunofluorescence. Results The BBB scores in the MS group were signifi-cantly higher than those of the control group at 1, 4 and 8 weeks post SCI. Nestin and the MAP2/nestin ratios were mild in the normal spinal cords, but increased after SCI. They were higher in the MS group than that in the control groups at all time points. Conclusions MS can promote nestin expression in the spinal cord after SCI and facili-tate neural differentiation.

Objective To investigate the biological effects of low intensity pulsed ultrasound (LIPUS) on the proliferation and osteogenic differentiation of adipose tissue-derived stem cells (ADSCs) in vitro.Methods Primary ADSCs were harvested from the inguinal fat pads of 4-week-old female Sprague-Dawley rats,cultured in vitro and purified by magnetic-activated cell sorting.Surface ADSC markers were identified by flow cytometry.LIPUS at 100 mW/cm2 was used to stimulate the cultured cells.Flow cytometry was performed for cell cycle analysis.Cellular proliferation was evaluated via CCK8 chromatometry,and a proliferation index was calculated.ADSCs were assigned to 4 groups:a negative control group,a LIPUS group,an osteoinduction group and a LIPUS plus osteoinduction group,and treated accordingly.Alkaline phosphatase (ALP) activity was determined at the 7th and 14th day in each group,and calcium nodes were marked by Von Kossa staining.The levels of osteogenic differentiation in the different groups were evaluated.Results The ADSCs of passage 3 expressed CD 34low,and CD29high CD44high,which was consistent with the characteristics of ADSC surface markers.Proliferation was upregulated significantly in the LIPUS group compared with the negative control group.ALP activity was also elevated significantly and it resulted in mine-ralization.The highest mineralization rate was observed in the LIPUS plus osteoinduction group.Conclusions LIPUS not only can stimulate the proliferation of rat ADSCs,it also promotes their osteogenic differentiation.

This study is to investigate the effect of FK506 on expression of hepatocyte growth factor (HGF) in rats' spinal cord following peripheral nerve injury and to elucidate the mechanisms for neuroprotective property of FK506. Fifty male rats were randomly divided into normal group, injury group and treatment group. Models of peripheral nerve injury were established by bilateral transection of sciatic nerve 0.5 cm distal to piriform muscle. Then the treatment group received subcutaneous injection of FK506 (1 mg/kg) at the back of neck, while the injury group was given 0.9% saline. The L(4-6) spinal cords were harvested at various time points after the surgery. Western blotting and immunofluorescent staining were used to detect the level and position of HGF in spinal cord. Immunofluorescent staining showed that HGF-positive neurons were located in anterior horn, intermediate zone and posterior horn of gray matter in normal spinal cord. Western blotting revealed that there was no significant difference in the expressions of HGF between the injury group and the normal group, while the expression of HGF was significantly higher in the treatment group than in the injury group 7 and 14 days after surgery. It is suggested that peripheral nerve injury does not result in up-regulation of the expression of HGF in spinal cord, while FK506 may induce high expression of endogenous HGF after injury thereby protecting neurons and promoting axonal outgrowth.
Cells, Cultured ; Gene Expression Regulation ; Hepatocyte Growth Factor/metabolism ; Immunosuppressive Agents/metabolism ; Immunosuppressive Agents/*pharmacology ; Microscopy, Fluorescence/methods ; Neurons/metabolism ; Peripheral Nervous System/*metabolism ; Sciatic Nerve/metabolism ; Spinal Cord/*cytology ; Spinal Cord/metabolism ; Spinal Cord Injuries/*drug therapy ; Tacrolimus/metabolism ; Tacrolimus/*pharmacology

The influence of short hairpin RNA (shRNA)-mediated osteopontin (OPN) gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated. Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids, which were transferred into the cultured ACHN cells by Lipofectamine 2000. The cells transfected by shRNA expression vectors (ACHN/OPN) were visualized under an inverted microscope and screened by G418. Untreated cells (ACHN) and cells transfected by mock vectors (ACHN/Vect) were used as control groups. The expression levels of OPN mRNA and protein were detected by real-time PCR and Western blot respectively. The cell cycle and ratios of apoptotic cells were assessed by flow cytometry. MTT method was used for drawing the growth curve and observing cell proliferation in vitro. The abilities of migration and invasion in three groups were measured by Transwell chamber test. The expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in three groups were examined by Western blot. Our results showed that the recombinant plasmid could be successfully transferred into ACHN cells by LipofectamineTM 2000. Compared with untreated cells, the expression levels of OPN mRNA and protein in ACHN/OPN cells were decreased by 59.68% and 76.42%, respectively (P<0.05), ACHN/OPN cells were blocked in S phase and apoptotic ratio increased significantly (P<0.05), however, no significant differences were found between ACHN/Vect and ACHN. Recombinant plasmid significantly attenuated expression levels of MMP-2 and MMP-9 proteins and suppressed the proliferation, migration, and invasion of ACHN cells. This study suggested that OPN may play an important role in the growth and invasion of human renal cancer ACHN cells, and these processes are correlated with the activations of MMP-2 and MMP-9. Our data provided preliminary experimental evidence for the feasibility of RNA interference technology in gene therapy of human renal cancer.

This study examined the effect of small interfering RNA-mediated β-catenin knockdown on the survival, invasion and chemosensitivity of human osteosarcoma cells (U2-OS cells). The siRNA against β-catenin was constructed and transfected into U2-OS cells. The expression of β-catenin was detected by qRT-PCR and Western blotting. Cell growth and apoptosis was detected in the presence or absence of doxorubicin by MTT and flow cytometry, respectively. Cell invasion ability was measured by transwell assay. The results showed that the transfection of β-catenin siRNA resulted in decreased expression of β-catenin, suppression of invasion and motility of U2-OS cells, reduced chemosensitivity to doxorubicin in vitro, and little change in cell growth and apoptosis. Additionally, down-regulated MT1-MMP expression was found after transfection. It was concluded that knockdown of β-catenin gene may decrease the invasive ability of human osteosarcoma cells through down-regulated MT1-MMP expression, and the chemosensitivity of osteosarcoma cells against doxorubicin.

Matrix metalloproteinase-2 (MMP-2) level and the ERK1/2 signal pathway are dependent factors for the growth and metastasis of cancer. However, the impact of MMP-2 in combination with ERK1/2 in tumor patients with drug resistance is unknown. To determine the relationship between MMP-2 and the ERK1/2 signal pathway, we established an adriamycin (ADM)-induced MG-63 (ADM-MG-63) cell line. With the increase of the ERK1/2 pathway blocker PD98059, we detected the expression levels of MMP-2 and p-ERK1/2 by Western blot in ADM-MG-63 cells. In ADM-MG-63 cells transfected with MMP-2-siRNA, the expression of ERK1/2 was detected for understanding the function of the ERK1/2 signal pathway. Three siRNAs for MMP-2 (MMP-2-siRNA) were designed, and the optimal one was selected and tested at different time points of 24, 48 and 72 h. Under an ADM-induced condition, ADM-MG-63 cells were finally stable living in the medium of ADM (200 ng/mL). PD98059 could effectively suppress the expression levels of p-ERK1/2 and MMP-2. When the MMP-2 was silenced by using MMP-2-siRNA, the expression of p-ERK1/2 was enhanced. It is concluded that MMP-2 may be involved in ADM resistance dependent on ERK1/2 signal pathway, suggesting interference in ERK1/2 may be a new method of targeted therapy for tumor resistance.

Objective To establish a doxycycline-controlled immortalized pre-cartilaginons stem cells (IPCSCs) strains, clone parathroid hormone-related peptide[PTHrP(1-36)] gene and construct re- sponsive plasmid, pTRE-PTHrP (1-36). Methods Plasmid pTet-on was transfected into IPCSCs by using LipoinfectaminTM 2000 and then the stable clones were obtained by G418 screening. The doxycyc- line was added into the medium of monoclonal cells that were transiently transfected with plasmid pTRE- 2Hyg-Lue. The total RNA was extracted from PCSCs and the PTHrP(1-36) gene obtained by RT-PCR method. Then, the PTHrP (1-36) gene was subcloned to plasmids of Tet-responsive element with the se- lection marker of hygromycin pTRE-2Hyg to construct recombinant eukaryotic expression plasmid pTRE- PTHrP(1-36). After transferred into E. coli-DH5α, the clone was amplified, the recombinant plasm0ids were purified and identified by double-enzyme digestion. Results The doxycycline induced IPCSCs line was obtained, with 50 times higher than the non-induced cell line. Double enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant plasmid. Conclusions The induced IPCSCs line can be used to highly express alien genes. The responsive plasmid containing PTHrP (1-36) gene may be premising for rigorous control of PTHrp (1-36) gene expression.

Objective To investigate the effect of direct intercell contact on the bone mesenchymal stem cells(MSCs)differentiate into nucleus pulposus cells(NPs)when cocultured with NPs in constant magnetic field.Methods The primary NPs labeled by DAP1 were cocultured with the 3 rd generation of MSCs through direct and indirect intercell contact in the presence or absence of constant magnetic field(0.05,0.10,0.50 and 1.00 mT,respectively). Observation of morphological changes was performed every 24 hours.The method of MTT was employed to evaluate the level of proliferation.The gene expression of collagen Ⅱ,Sox-9 and Aggrecan was measured by using RT-PCR. Results MSCs cocultured with direct intercell contact with the NPs rounded up and presented a round ring-like structure appearance.The expression of marker genes including Collagen type Ⅱ,Aggrecan and Sox-9 were significantly increased when cells cocultured in constant magnetic field of 0.05 mT compared with those without constant magnetic field(P＜0.05).There were no significantly changes with regard to the expression of the above genes in 0.10 mT field(P＞0.05).The growth of NP-like cells was suppressed when the intensity of magnetic field was higher than 0.10 mT(P＜0.05).Conclusion It is suggested that 0.05 mT constant magnetic field and direct intercell contact facilitate differentiation of MSCs into NPs.

Objective To detect the expression levels of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-3 (MMP-3),MMP-9,MMP-13 and MMP-14 in the patients with osteoarthritis(OA)before and after arthroscopic debridement,and to explore the influence of arthroscopic debridement in the expressions of uPA, MMP-3,MMP-9,MMP-13, and MMP-14.Methods 420 cases of synovial fluid from knee OA patients undergoing arthroscopic debridement were obtained before operation. After six months follow-up, 350 cases of synovial fluid samples were obtained and according to inclusion and exclusion criteria, 228 synovial fluid were selected to analyze.The expression levels of uPA,MMP-3,MMP-9,MMP-13,and MMP-14 were measured by ELISA assay.Pain intensity of these patients before operation and six months after operation were recorded using the Visual Analogue Scale/Score(VAS).The differences of the expression levels of uPA,MMP-3,MMP-9, MMP-13,and MMP-14 between before operation and after operation were compared.The relationship between the expression levels of uPA, and MMP-3, MMP-9, MMP-13, MMP-14 and VAS was analyzed with Spearman analysis.Results All the patients were followed up for 36.5 months. Compared with before operation, the expression levels of uPA and MMP-3 in the synovial fluid of the patients after arthroscopic debridement were significantly decreased(P<0.01),the expression levels of MMP-9 and MMP-13 were also decreased (P<0.05), but the MMP-14 expression level showed no significant change.The expression levels of uPA,MMP-3,MMP-9, MMP-13,MMP-14 were positively associated with VAS before arthroscopic debridement (r=0.361,r=0.417, r=0.136,r=0.514,r=0.156,P<0.05 );uPA and MMP-3 were positively correlated with VAS after arthroscopic debridement(r=0.981,r=0.831,P<0.01),as well as the expression level of MMP-13 and VAS, but there were no significant differences between the expression levels of MMP-9, MMP-14 and VAS. Conclusion The decreased levels of uPA,MMP-3 and MMP-13 in synovial fluid may contribute to the pain-relief effects of arthroscopic debridement.