The primary neural stem cells were isolated from SD rat and formed the neuropheres, the neuropheres were passaged and planted on the dish coated with 0.1% gelatin, the colony was picked up under the microscope, then dispersed and cultured, to obtain the clone proliferated from one cell, passaging and picking up the cells 5～6 times at least. The NSC and its differentiated cells were identified with the marker genes respectively. The results showed that the neural stem cells were isolated from the SD rat embryos and the real clone were obtained by picking up the cells again and again, and then cultured in the form of monolayer. The marker genes of the neural stem cells and its differentiated cells could be detected at last. It will provide the rat model the resource of the cells for the treatment and the basic research for the morphology standard.

Objective To study the clinical efficacy of acupuncture combined with medication plus joint mobilization in the treatment of lumbar intervertebral disc protrusion accompanied with secondary lumbar spinal stenosis.MethodsTotally 66 cases of lumbar intervertebral disc protrusion accompanied with secondary lumbar spinal stenosis were collected randomly and divided into treatment group (34 cases) and control group (32 cases). The control group was given treatment of simple acupuncture and TCM medication, while the treatment group was given joint mobilization treatment besides acupuncture and TCM medication. Functions of lumbar vertebra were evaluated by ODI scale and the degrees of pain were evaluated by VAS. The clinical efficacy in the two groups was compared. Results ODI in both groups were significantly improved after treatment compared with that before treatment. However, the changing range of the ODI of the treatment group was more significant than that in control group (P<0.01). After treatment, VAS scores were relieved (P<0.05) in both groups, and treatment group was more significant than that in control group (P<0.05). The total clinical efficacy was 97.06% (33/34) in the treatment group, and 84.38% (27/32) in the control group, with statistical significance (P<0.05).Conclusion Acupuncture combined with medication plus joint mobilization in the treatment of lumbar intervertebral disc protrusion accompanied with secondary lumbar spinal stenosis has good efficacy.

The Wnt signaling pathway plays an important role in bone metabolism. Inducing the Wnt signaling pathway promotes bone formation while restraining it results in osteopenic states. Although the regulation of this signaling pathway may bring enormous therapeutic potential, it still requires cautious approach because of the risks of tumorigenesis. The role of the Wnt signaling pathway in bone metabolism and the molecular targets of therapeutic potential for osteoporosis are discussed in this review.

10.3969/j.issn.2095-4344.2013.23.008

To investigate the effects of osthole on chondrocyte proliferation in vitro.

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

Transplantation of embryonic stem cells could promote nerve repair by providing nutrients to the injured spinal cord, or differentiating into remyelinating oligodendrocytes or neurons integrating with the host neurons, which has a bright future in the treatment of spinal cord injury.

OBJECTIVETo investigate the role of bone morphogenetic protein 4 (BMP4) in culturing induced pluripotent stem cells (iPSCs) and the related signal pathways.

METHODSWe amplified the mature peptide of BMP4 from the placenta through RT-PCR, and IgK secretion peptide was ligated to the N-terminal of BMP4 mature peptide. The recombinant plasmid pPYCAG-IgK-BMP4 was transfected into 293T cells and screened with puromycin, and the positive clones for expressing BMP4 were verified by cell immunofluorescence and Western blotting. To test the bioactivity of BMP4, iPSCs were cultured in the medium supplemented with leukemia inhibitory factor (LIF) plus the supernatant containing BMP4, and the cell phenotype, cell differentiation capacity into lineages of the 3 germ layers and expression levels of pluripotency-associated genes were investigated.

RESULTSSmad1 was phosphorylated by BMP4 from the culture medium. iPSCs cultured in the medium supplemented with LIF plus the supernatant containing BMP4 for 3 passages maintained the phenotype of stem cells with the expression levels of pluripotency-associated genes not affected. These iPSCs also maintained the capacity to differentiate into cell lineages of the 3 germ layers.

CONCLUSIONBMP4 can be efficiently expressed in mammalian cells to maintain the multipotent differentiation capacity of the iPSCs in in vitro culture.

Animals ; Bone Morphogenetic Protein 4 ; genetics ; Cell Differentiation ; genetics ; Cells, Cultured ; Female ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin kappa-Chains ; genetics ; Induced Pluripotent Stem Cells ; cytology ; Mice

OBJECTIVETo investigate the role of human Wnt7b gene in rat chondrocyte degeneration.

METHODSWnt7b gene obtained by PCR was cloned to PCDH-GFP. 293ft cell line was transfected with PCDH-GFP and PCDH-Wnt7b, and the supernatant and transfected cells were collected. The expression level of Wnt7b in 293ft cells was detected by Western blotting. The first passage of chondrocytes were isolated from articular cartilages of newborn born (within 24 h) SD rats were cultured in the supernatants from the transfected cells (at 10- and 50-fold dilutions). The cell morphology of the rat chondrocytes was observed under inverted microscope, and the protein expressions of MMP13, MMP3 and type II collagen and mRNA expressions of A-can, ADAMTS5, Col X and Sox9 were examined by Western blotting or real-time PCR.

RESULTSHuman Wnt7b gene cloned to PCDH-GFP was expressed efficiently in 293ft cell line. Rat chndrocytes cultured for 24 h in the supernatants from PCDH-Wnt7b-transfected 293ft cells underwent changes from a polygonal to a spindle-shaped morphology. The protein expression levels of MMP13 and MMP3 increased while type II collagen decreased significantly, and the mRNA levels of A-can and Sox9 were down-regulated while Col X and ADMATS5 up-regulated in ratchondrocytes after incubation in supernatants from PCDH-Wnt7b-transfected 293ft cells.

CONCLUSIONHuman Wnt7b gene can be expressed efficiently in 293ft cell line and can induce rat chondrocyte degeneration in vitro.

Animals ; Cartilage, Articular ; cytology ; Cell Line ; Chondrocytes ; pathology ; Humans ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Transfection ; Wnt Proteins ; genetics

OBJECTIVETo investigate the effect of inhibition and activation of MAPK-ERK1/2 pathway on the expression of osteogenic genes and proliferation of rat osteoblasts in vitro.

METHODSPrimarily cultured rat osteoblasts, identified by cell morphology studies and ALP staining, were exposed to 1% or 5% rat serum for 24 h or to the specific MAPK-ERK1/2 inhibitor PD0325901. The downstream molecules of MAPK-ERK1/2 pathway including p-ERK1/2 and ERK1/2, osteogenic genes such as Runx2 and Type I collagen, and proliferating cell nuclear antigen (PCNA) were detected by Western Blotting, and alkaline phosphatase activities were analyzed quantitatively.

RESULTSCompared with 1% rat serum-treated cells, exposure of the cells to a higher concentration (5%) of rat serum caused a significantly increased phosphorylation level of p-ERK1/2 (P<0.05) and obviously enhanced expressions of the osteogenic genes (Runx2, type I collagen and ALP) and PCNA (P<0.05). Inhibition of the MAPK-ERK1/2 pathway with PD0325901 resulted in suppressed expressions of the osteogenic genes and PCNA.

CONCLUSIONThe activation of MAPK-ERK1/2 pathway promotes the expression of osteogenic genes such as Runx2, type I collagen and ALP and enhances the proliferative activity of the osteoblasts, while inhibition of this pathway suppresses the expressions of these genes and the cell proliferation, suggesting that this pathway may potentially serve as a therapeutic target for osteoporosis.

Alkaline Phosphatase ; metabolism ; Animals ; Antineoplastic Agents ; pharmacology ; Benzamides ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Diphenylamine ; analogs & derivatives ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; MAP Kinase Signaling System ; drug effects ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Osteoblasts ; cytology ; metabolism ; Phosphorylation ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Sprague-Dawley