Effects of Er-Zhi-Wan on Microarchitecture and Regulation of Wnt/β-catenin Signaling Pathway in Alveolar Bone of Ovariectomized Rats简

Recent studies have shown that Er-Zhi-Wan（EZW）, a traditional Chinese medicine consisting of Herba Ecliptae（HE） and Fructus Ligustri Lucidi（FLL）, had a definite antiosteoporotic effect on osteoporotic femur, but its effect on osteoporosis of alveolar bone remains unknown. In the present study, we investigated the effects of Er-Zhi-Wan（EZW） on the microarchitecture and the regulation of Wnt/β-catenin signaling pathway in the alveolar bone of ovariectomized rats. Thirty Sprague-Dawley rats were randomly divided into three groups： sham operation group（sham, n=10）, ovariectomy（OVX） group（n=10）, and OVX with EZW treatment group（EZW group, n=10）. From one week after ovariectomy, EZW（100 mg/mL） or vehicle（distilled water） was fed（1 mL/100 g） once per day for 12 weeks until the sacrifice of the rats. The body weights were measured weekly. After sacrifice, the sera and mandible were collected and routinely prepared for the measurement of alveolar trabecular microarchitecture, serum levels of E2, bone-specific alkaline phosphatase（BALP） and tartrate-resistant acid phosphatase 5b（TRAP5b）, as well as mandibular mRNA expression of Wnt/β-catenin signaling pathway molecules wnt3a, low-density lipoprotein receptor-related protein 5（LRP5）, β-catenin and dickkopf homolog 1（DKK1）. The results showed that EZW treatment significantly prevented the body weight gain, degradation of alveolar trabecular microarchitecture and alveolar bone loss in the OVX rats. Furthermore, we observed that EZW could increase the serum levels of E2 and BALP, and decrease levels of serum TRAP5b in EZW group compared with vehicle group. In addition, RT-PCR results revealed that EZW upregulated the expression levels of wnt3a, LRP5 and β-catenin, and reduced the expression of DKK1 in OVX rats. Taken together, our results suggested that EZW may have potential anti-osteoporotic effects on osteoporotic alveolar bone by stimulating Wnt/LRP5/β-catenin signaling pathway.     

Differentiation character of adult mesenchymal stem cells and transfection of MSCs with lentiviral vectors

This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lenti- viral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and trans- fection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Ag- gracan was positive in chondrogenic lineages and the expression of aggracan and type Ⅱ collagenwas significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.     

Effect of rhBMP-2 and Osteogenic Revulsants on Proliferation and Differentiation of Bone Marrow Stromal Cells in Rats

The effects of recombinant human bone morphogenetic protein-2 （rhBMP-2） and osteogenic revulsants alone or in combination at different time points and in different dosages on proliferation and osteogenesis of bone marrow stromal cells （BMSCs） in SD rats were investigated. Rat BMSCs were cultured in vitro and induced by rhBMP-2 in different dosages （10, 50, 100 and 200μg/L） alone or in combination with osteogenic revulsants. MTT colorimetric assay was used to evaluate The proliferation, activity of alkaline phosphoric （ALP） and osteocalcin were measured at 3rd, 6th, 9th, 12th day respectively. The results showed that rhBMP-2 and osteogenic revulsants could promote the differentiation of BMSCs towards osteoblast phenotype. The proliferation of BMSCs could be enhanced by rhBMP-2 in a dose-dependent manner. The expression of osteoblast phenotype was significantly higher by using both of them than by using them alone, which was verified by the activity of ALP and osteocalcin. It was suggested that the combined use of rhBMP-2 and osteogenic revulsants could promote the proliferation and simultaneously induce and maintain the expression of osteoblast phenotype of BMSCs in rats.     

Influence of Exogenous TGFβ_1 on the Expression of Smad2 and Smad3 in Rat Bone Marrow-derived Mesenchymal Stem Cells

Bone marrow derived mesenchymal stem cells(MSCs) have multipotential to differentiate intoosteogenic, myogenic, and adipogenic cell linea ges, depending on signals derived from the cellularenvironment. At present, MSCs have been regar ded to be one kind of preferred seed cells in the re generation of bone defects. The TGFβ1 protein ismultifunctional peptide and has a broad range ofcellular activities including the control of cell pro liferation and differentiation …     

Effects of Zuogui Pill (左归丸) on Wnt Singal Transduction in Rats with Glucocorticoid-induced Osteoporosis

Objective: To reveal the mechanism of Zuogui Pill (左归丸) in treatment of glucocorticoid-induced osteoporosis from the angle of the Wnt signal transduction pathway and to provide further experimental evidence for expounding the scientific connotation of "the kidney dominating the bones" in TCM. Methods: Forty-two male Wistar rats were selected and randomly divided into three groups, control group (n=12), model group (n=15) and Zuogui Pill group (n=15). Form the beginning, The rats were injected dexamethasone for eight weeks to make the model of osteoporosis, and the Zuogui Pill were administered intragastrically to the rats of Zuogui Pill group for eight weeks. The relative morphological parameters were measured in the undecalcified tibial slices. And the protein expression levels of Wnt1, LRP-5 and β-catenin in rat tibial osteoblasts (OB) and bone marrow stromal cells (BMC) were detected by immunohistochemistry. Results: Compared with the control group, TBV% and TFS% decreased significantly, while TRS% increased significantly, and the protein expression of Wnt1, LRP-5 and β-catenin in OB and BMC decreased significantly in the model group. And compared with the model group, TBV% and TFS% increased significantly, and expression levels of Wnt1, LRP-5 and β-catenin proteins increased significantly in the Zuogui pill group. Conclusion: Zuogui Pill can prevent and treat glucocorticoid-induced osteoporosis in rats by up-regulating the expression of the key signal molecules Wnt1, LRP-5 and β-catenin in Wnt signal transduction pathway.     

Craniocerebral trauma

208077 The experimental study on promoting vascularization by bone marrow stromal cells transplantation after local brain injury in rats/Zhang Pengfei(张鹏飞,Beijing Neurosurg Inst,Affil Tiantan Hosp,Cap Univ Med Sci,Beijing 100050)…∥Chin J Neurosurg.-2007,23(8).-633～635Objective To observe the effects of bone marrow stromal cells transplantation on vascularization after local brain injury in rats,and to investigate the mechanism of     

In vitro Culture of Bone Marrow Mesenchymal Stem Cells in Rats and Differentiation into Retinal Neural-like Cells

In order to study the in vitro culture and expansion of bone marrow mesenchymal stem cells in rats(rMSCs) and the possibility of rMSCs differentiation into retinal neural cells,the bone marrow-derived cells in SD rats were isolated and cultured in vitro.The retinal neural cells in SD rats were cultured and the supernatants were collected to prepare conditioned medium.The cultured rMSCs were induced to differentiate by two steps.Immunofluorescence method and anti-nestin,anti-NeuN,anti-GFAP and anti-Thy1.1 antibodies were used to identify the cells derived from the rMSCs.The results showed that the in vitro cultured rMSCs grew well and expanded quickly.After induction with two conditioned media,rMSCs was induced to differentiate into neural progenitor cells,then into retinal neural-like cells which were positive for nestin,NeuN,GFAP and Thy1.1 detected by fluorescence method.The findings suggested that rMSCs could be culture and expanded in vitro,and induced to differentiate into retinal neural-like cells.     

Human adipose-derived mesenchymal stem cells: a better cell source for nervous system regeneration

Background In order to suggest an ideal source of adult stem cells for the treatment of nervous system diseases,MSCs from human adipose tissue and bone marrow were isolated and studied to explore the differences with regard to cell morphology,surface markers,neuronal differentiation capacity,especially the synapse structure formation and the secretion of neurotrophic factors.Methods The neuronal differentiation capacity of human mesenchymal stem cells from adipose tissue （hADSCs） and bone marrow （hBMSCs） was determined based on nissl body and synapse structure formation,and neural factor secretion function.hADSCs and hBMSCs were isolated and differentiated into neuron-like cells with rat brain-conditioned medium,a potentially rich source of neuronal differentiation promoting signals.Specific neuronal proteins and neural factors were detected by immunohistochemistry and enzyme-linked immunosorbent assay analysis,respectively.Results Flow cytometric analysis showed that both cell types had similar phenotypes.Cell growth curves showed that hADSCs proliferated more quickly than hBMSCs.Both kinds of cells were capable of osteogenic and adipogenic differentiation.The morphology of hADSCs and hBMSCs changed during neuronal differentiation and displayed neuronlike cell appearance after 14 days＇ differentiation.Both hADSCs and hBMSCs were able to differentiate into neuron-like cells based on their production of neuron specific proteins including β-tubulin-Ⅲ,neuron-specific enolase （NSE）,nissl bodies,and their ability to secrete brain derived neurotrophic factor （BDNF） and nerve growth factor （NGF）.Assessment of synaptop hysin and growth-associated protein-43 （GAP-43） suggested synapse structure formation in differentiated hADSCs and hBMSCs.Conclusions Our results demonstrate that hADSCs have neuronal differentiation potential similar to hBMSC,but with a higher proliferation capacity than hBMSC.Adipose tissue is abundant,easily available and would be a potential ideal source of adult stem cells for neural-related clinical research and application.     

In Vitro Chondrogenic Phenotype Differentiation of Bone Marrow-derived Mesenchymal Stem Cells

In order to study the chondrogenic phenotype differentiation of adult sheep bone marrowderived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering, MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCS were induced with H-DMEM containing TGF-β3 .IGF-I, Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindlelike appearance to a polynal shape, a large amount of endoplasmic reticulum, Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when induced in vitro and can be used as optimal seed cells for cartilage tissue engineering.     

Human Adipose-derived Mesenchymal Stem Cells: A Better Cell Source for Nervous System Diseases Application

In order to suggest an ideal source of adult stem cells for treatment of nervous system diseases, neuronal differentiation capacity of human mesenchymal stem cells from adipose tissue (hADSCs) and bone marrow (hBMSCs) were studied in terms of nissl body and synapse structure formation, neural factors secretion function for the first time. The hADSCs and hBMSCs were isolated and differentiated to the neuron-like cells with rat brain-conditioned medium which is potentially a rich source of signals promoting neuronal differentiation. The specific proteins of neurons and neural factors secreted by the cells were detected with immunohistochemistry and ELISA analysis, respectively. Flow cytometric analysis showed that these cells had similar phenotypes and cell growth curves showed that hADSCs proliferated more quickly than hBMSCs. Both kinds of cells were capable of osteogenic and adipogenic differentiation. The morphology of hADSCs and hBMSCs changed during neuronal differentiation and presented neuron-like cell appearance after 14 days differentiation. hADSCs and hBMSCs could differentiated into neuronal-like cells, which were identified by neuron specific proteins: β-Tubulin-Ш, neuron-specific enolase (NSE), and nissl body and their ability to secrete brain derived neurophic factor (BDNF) and nerve growth factor (NGF). Assessment of synaptophysin and growth-associated protein-43 (GAP-43) suggested synapse structure formation in differentiated hADSCs and hBMSCs. The present results demonstrated that hADSCs have a similar neuronal differentiation potential with hBMSCs but with higher proliferation capacity. Adipose tissue is an abundant and easily available, which would be a potential ideal source of adult stem cells for neural-related clinical research and application.     

Osteogenic Differentition of Adipose-derived Stem Cells Isolated from Rabbit Subcutaneous Adipose Tissue

Objective To fully evaluate the osteogenic potential of adipose-derived stem cells （ADSCs） isolated from rabbit subcutaneous adipose tissue. Methods Rabbit ADSCs （rADSCs） were prepared by collagenase I digestion of subcutaneous fat from the suprascapular site of Japanese white rabbit after being excised and finely minced. Meanwhile, the bone marrow stem cells （BMSCs） were isolated from the ventral ilium by bone marrow aspiration from the same rabbit as control. The in vitro cell proliferation of rADSCs and rabbit BMSCs （rBMSCs） were firstly examined. After which, the osteogenic differentiation of rADSCs and rBMSCs were identified by von Kossa staining after cultured under osteogenic medium for 28 days. During the osteogenic differentiation process, the alkaline phosphatase （ALPase） activity and extracellular matrix mineralization of rADSCs was analyzed quantitatively compared with the rBMSCs. Results The in-vitro cultured rADSCs assumed similar fibroblast-like morphology similar to rBMSCs, but have a higher proliferation rate. The von Kossa staining indicated that calcium nodules were formed in both rADSCs and rBMSCs after having been induced under osteogenic medium for 28 days. Furthermore, quantitative analyses of ALPase and extracellular matrix mineralization implied that the rADSCs have a favorable osteogenic potency similar to that of rBMSCs. Conclusion The rADSCs have excellent osteogenic differentiation capacity and can be used as seed cells in bone tissue engineering so as to provide valuable data for the potential application of human ADSCs in clinical area.     

Implication of Receptor Activator of NF-κB Ligand in Wnt/β-Catenin Pathway Promoting Osteoblast-like Cell Differentiation

Recent studies showed that activation of Wnt/β-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/β-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/β-catenin pathway was activated by LiCl, the expression of RANKL was significantly in-creased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/β-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.     

Bone Marrow Urokinase Plasminogen Activator Receptor Levels are Associated with the Progress of Multiple Myeloma△

Objective To determine the mRNA and protein levels of urokinase plasminogen activator receptors (uPAR) in bone marrow fluid and bone marrow tissue from multiple myeloma (MM) patients and assess association of uPAR level with prognosis of MM. Methods uPAR levels in bone marrow fluid of 22 MM patients at the stable and progressive stages and 18iron deficiency anemia patients with normal bone marrow (control) were examined by ELISA. Furthermore, uPAR expression in bone marrow tissue was investigated by RT-PCR and Western blot, respectively. The distribution of uPAR in MM cells was examined using immunofluorescence staining. The pathological changes in different stages of MM patients were studied by HE staining. Results uPAR level in bone marrow fluid of MM patients (1.52±0.32μg/ml) was found to be higher than that in the control group (0.98±0.15μg/ml). Interestingly, uPAR protein (0.686±0.075vs. 0.372±0.043, P<0.05) and mRNA (2.51±0.46vs. 4.46±1.15,P<0.05) expression levels of MM patients at the progressive stage were significantly higher than those at the stable stage. The expression of uPAR in MM bone marrow was confirmed by immunofluorescence staining.Moreover, HE staining revealed a great increased number of nucleated cells and severe impairment of hematopoietic function in the bone marrow of patients with progressive-stage myeloma. Conclusion Our study reveals that uPAR expression is positively correlated with the development and progress of MM.     

Effect of gamma irradiation on nuclear factor—kappa B in cultured bone marrow stromal cells

Objective: To explore the effect of gamma irradiation on nuclear factor-kappa B in cultured bone marrow stromal cells. Methods: Immunocytochemistry, Western blot and electrophoretic mobility shift assay (EMSA) were used. Results: The expression of NF-kB in cultured mouse bone marrow stromal cells (BM-SCs) on the level of protein was elevated after exposure to 60Co in the dosage of 8. 0 Gy with the use of im-munocytochemistry and Western blot. The activity of nuclear factor-kappa B in cultured BMSCs was significantly increased after exposure to gamma irradiation by using EMSA. The activity peak was at the 4th h after irradiation. Conclusion: Our results suggest that the activation of nuclear factor-kappa B in the BMSCs after irradiation may be involved in the protection of BMSCs against apoptosis and in the recovery of hematopoiesis after radiation.