ABSTRACT
The osteogenic in vitro effect of low intensity pulsed ultrasound (LIPUS) on SD rat adipose-derived stem cells (ADSCs) was investigated.Rat ADSCs underwent LIPUS (intensity=100 mW/cm2) or sham exposure for 8 min per treatment once everyday in vitro,and then the alkaline phosphatase (ALP) activity and mineralized nodule formation were assessed to evaluate the osteogenic effect of LIPUS on ADSCs.To further explore the underlying mechanism,the osteogenic-related gene mRNA expression was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR) at 1st,3rd,5th,7th day after exposure repectively.Westen blot was used to evaluate the protein expression levels of two osteogenic differentiation associated genes at 7th and 14th day repectively.It was found that ALP activity was increased after LIPUS exposure and LIPUS resulted in mineralized nodule formation of ADSCs in vitro.LIPUS-treated ADSCs displayed higher mRNA expression levels of runt-related transcription factor 2 (Runx2),osteocalcin (OCN),ALP and bone sialoprotein (BSP) genes than controls,and the protein levels of Runx2 and BSP were also increased.The results suggested that LIPUS may induce the osteogenic differentiation of ADSCs in vitro.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3 (hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells (PSCs) into chondroblasts.hTGF-β3 gene was amplified by using polymerase chain reaction (PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3.Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system.pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines.The expression of TGF-β3 and cartilage-specific extracellular matrix (ECM)components was detected after transfection by real-time quantitative PCR,ELISA,immunochemistry and Western blotting,respectively.The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing.Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs.Real-time quantitative PCR,immunochemistry and Western blotting showed that the cartilage-specific ECM markers,i.e.,cartilage oligomeric matrix protein (COMP),Aggrecan,collagen type Ⅹ and Ⅱ were intensely expressed in the pcDNA3.1(+)-hTGF-β3-transfected cells.It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.
ABSTRACT
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 LipofectamineTM 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 by59.68% 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.
ABSTRACT
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 Immanocytochemistry 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.
ABSTRACT
-tiated into the precartilaginons stem cells.
ABSTRACT
The roles of Indian hedgehog (Ihh) signaling pathway in the proliferation and apoptosis of precartilaginous stem cells (PSCs) were investigated.PSCs,labeled with fibroblast growth factor receptor 3 (FGFR-3),were isolated from neonatal rats by immanomagnetic separation.After identifi-cation with FGFR-3 and Col Ⅱ,the cells were incubated with different concentrations of cyclopamine (cyclo),the specific inhibitor of lhh signaling pathway.The morphologic changes of the cells were observed under the inverted phase contrast microscope.The mRNA expression levels of Ibh,para-thyroid hormonerelated peptide (PTHrP),protein Patched (Ptch),Bcl-2 and p21 were detected by RT-PCR.The protein expression levels of Ihh and Ptch were measured by Western blot.MTT assay was used to examine the effects of cyclo on proliferation of PSCs.Apoptosis rate of PSCs was exam-ined by Annexin V/PI assay of flow cytometric analyses.After PSCs were incubated with cyclo,ob-vious morphologic changes were observed as compared with the control group.The mRNA expres-sion levels of PTHrP,Ptch and Bcl-2 were decreased to varying degrees in a cyclo dose-dependent manner.However,the expression levels of lhh and p21 mRNA were increased.The protein expres-sion of Ptch and Ihh had the same change as the mRNA expression.Meanwhile,cyclo could obvi-ously inhibit the proliferation and promote the apoptosis of PSCs.The results indicated that Ihh sig-naling pathway plays an important role in regulating the proliferation and apoptosis of PSCs,which is probably mediated by Bcl-2 and p21.
ABSTRACT
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 FKS06. 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 subcutaneons injection of FK506 (1 mg/kg) at the back of neck, while the injury group was given 0.9% saline. The L4-6 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. Lm munofluorescent staining showed that HGF-positive neurons were located in anterior horn, interme- diate 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.
ABSTRACT
To investigate the preventive effect of epimedium-defivod phytoestrogen (PE) on osteoporosis induced by ovariectomy (OVX) in rats, 11-month-old female Wistar rats were randomly di- vided into Sham, OVX and PE groups. One week after OVX, daily oral administration of PE (0.4 g·kg-1·day·-1) started in PE group, and rats in Sham and OVX groups were given vehicle accordingly. The administrations lasted for 12 weeks. The biological markers including serum osteocalcin (OC) and urinary deoxypyridinoline (DPD) for bone turnover were evaluated at the end of the 12th week. On the 13th week, all the rats were sacrificed. The right proximal tibiae were removed, subjected to micro CT for determination of trabeonlar bone structure and then bone histomorphometry was per- formed to assess bone remodeling. The OVX rats were in a high bone turnover status as evidenced by increased bone formation markers and bone resorption markers. Treatment with PE could suppress the high bone turnover rate in OVX rats. Micro CT data revealed that PE treatment could ameliorate the deterioration of the micro-architecture of proximal tibiae induced by OVX, as demonstrated by greater bone volume, increased trabecular thickness and less trahecular separation in PE group in comparison with OVX group. The static and dynamic parameters of bone histomorphometry indi- cated that there were significant increases in bone formation variables and significant decreases in bone resorption variables between PE and OVX groups. The findings suggest that PE has a beneficial effect on trabecular bone in OVX rat model and this effect is possibly associated with stimulation of bone formation as well as inhibition of bone resorption.
ABSTRACT
<p><b>OBJECTIVE</b>To obtain seed cells for cartilage repair through constructing recombinant human transforming growth factor beta3 vector (hTGF-beta3) and transfecting it into rat's precartilaginous stem cells (PSCs).</p><p><b>METHODS</b>Gene engineering technique was introduced to construct eukaryotic expression plasmid pcDNA3.1 (+)-hTGF-beta3. PSCs of rats were isolated and purified with method of immunomagnetic microbeads. Then PSCs were cotransfected with plasmid hTGF-beta3 and pcDNA3.1 (+)-enhanced green fluorescence protein (EGFP) by liner polyethyleneimine (PEI). And 48 hours later the transient expression of EGFP was observed under a fluorescence microscope, and the expression of hTGF-beta3 was detected with reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The sequences of the recombinants were consistent with that from Genebank. Cotransfection of EGFP provided fast visual confirmation of successful transduction. The hTGF-beta3 mRNA and protein expression could be detected by RT-PCR and ELISA.</p><p><b>CONCLUSIONS</b>The recombinant plasmid is correctly constructed and successfully transfected into rat's PSCs, which is an important step to treat epiphyseal injury or other osteo-cartilage diseases with transgenic therapy.</p>