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.

Some studies indicate that adipose derived stem cells (ADSCs) can differentiate into adipogenic, chondrogenic, myogenic, and osteogenic cells in vitro. However, whether ADSCs can be induced to differentiate into neural cells in vitro has not been clearly demonstrated. In this study, the ADSCs isolated from the murine adipose tissue were cultured and transfected with the EGFP gene, and then the cells were induced for neural differentiation. The morphology of those ADSCs began to change within two days which developed into characteristics of round cell bodies with several branching extensions, concomitantly expressing EGFP fluorescence. Approximately 60% of the total cell populations were bipolar or multipolar in shape. Some of them appeared to make contact with their neighboring cells. RT-PCR, Western blot and Immunocytochemistry revealed that the expression levels of the markers of neurons and oligodendrocytes such as MAP2, NF-70, Neu N and RIP upon neural induction were increased, but the expression of the special marker of astrocytes, GFAP, was undetectable until 96 h after induction when a small signal was observed. It was concluded that the ADSCs transfected with EGFP possessed the ability to undergo morphologic and phenotypic changes consistent with neural differentiation in vitro. It suggests that these cells might provide an ideal source for further stem cell research with possible therapeutic application for spinal cord injury.

Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2 expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal cells differentiated into the precartilaginous stem cells.
Base Sequence ; Bone Marrow Cells/*cytology ; Cartilage/*cytology ; Cell Differentiation/genetics ; Cells, Cultured ; Cloning, Molecular ; Genetic Vectors/genetics ; Molecular Sequence Data ; Receptor, Fibroblast Growth Factor, Type 3/metabolism ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; SOX9 Transcription Factor/biosynthesis ; SOX9 Transcription Factor/*genetics ; Stem Cells/*cytology ; Stromal Cells/*cytology ; Transfection

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.