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Objective To study the mechanical effects of cyclic strain on neural differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). Methods The rBMSCs were subjected to cyclic strain for 24 hours andthen cultured for 5 days. The expression of neural markers and the phosphorylation of relative signaling pathway proteins were evaluated. The stress distribution on cell surface was analyzed by finite element method. The differentially expressed genes induced by strain were identified by RNA sequencing analysis. Results The 0. 5 Hz strain with 5% magnitude could significantly induce higher expression of neural markers and elevated phosphorylation level of extracellular-signal-regulated kinase (ERK), protein kinase B (AKT) and mammalian target of rapamycin ( mTOR). KEGG pathway analysis showed that the focal adhesion and ECM-receptor interaction were significantly enriched under cyclic strain. Conclusions Cyclic strain could change the interaction of cells with the extracellular matrix ( ECM) and enhance the AKT/ mTOR and ERK pathway, finally promote rBMSC neural differentiation. Knowledge about the impact of mechanical stimulation on BMSC neural differentiation is expected to improve the efficiency of stem cell differentiation, shed light on device design for tissue engineering, and promote clinical application of mesenchymal stem cells in neural issue repair and regeneration.
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Stroke is one of the major diseases threatening human health， with ischemic stroke accounting for about 70%. Ischemic stroke is characterized by complex pathological mechanism and high incidence， mobility and mortality. At present， the effective clinical treatment measures for ischemic stroke are limited， and it is urgent to develop new and effective treatment measures to improve the prognosis of patients. In recent years， bone marrow mesenchymal stem cells （BMSCs） transplantation has shown great therapeutic potential for a variety of diseases， including ischemic stroke， and has become a new research hotspot. However， due to the low homing and survival rate of BMSCs in human body after transplantation， their clinical effect on ischemic stroke needs to be further improved. With the characteristics of multi-components， multi-channels and multi-targets， Chinese medicine displays desirable curative effect on ischemic stroke， which has been widely concerned. Both Chinese medicine and BMSCs transplantation have good overall brain protection， and their combined effect on ischemic stroke is significantly better than that of single application. The mechanisms include improving the transplantation efficiency of BMSCs， promoting angiogenesis， enhancing neuroplasticity， ameliorating neuroinflammation， enhancing neuroprotection， and regulating the blood-brain barrier and exosomes. The combination of Chinese medicine and modern cutting-edge cell therapy reflects the advantages of integrative medicine， providing a new model and idea for preventing and treating ischemic stroke and improving the efficacy of BMSCs transplantation.
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Objective:To investigate the effects of different concentrations of Astragali Radix containing serum on the expression of 24-hydroxylase（CYP24A1），1α-OHase（CYP27B1） mRNA and protein in rat bone marrow mesenchymal stem cells （BMSCs）， and to explore the mechanism of primary osteoporosis （OP）. Method:The experiment was divided into 6 groups，like normal group， model group， low ，middle and high dose group of Astragali Radix containing serum（20%，40%，60%），Vitamin D group. Cell proliferation toxicity assay（CCK-8） was used to detect the effect of different concentrations of Astragali Radix containing serum on survival rate of aging BMSCs.Real-time quantitative PCR（Real-time PCR） and Western blot was used to detect the expression of CYP24A1 CYP27B1 mRNA and protein in senile BMSCs osteogenic differentiation cells by different concentrations of Astragali Radix containing serum. Result:Compared with normal group， the proliferation and survival rate of BMSCs osteoblasts induced by D-galactose in model group was significantly lower than that in normal group （P<0.01）. Compared with model group， medium and high dose groups and Vitamin D group could improve the proliferation and differentiation of aging BMSCs into osteoblasts in different degrees（P<0.01）. The relative expression of CYP27B1 mRNA and protein in model group was significantly lower than that in normal group， while the relative expression of CYP24A1 mRNA and protein in model group was significantly higher than that in normal group. Compared with model group， high dose Astragali Radix containing serum group could increase the relative expression of CYP27B1 mRNA and protein， and decrease the relative expression of CYP24A1 mRNA and protein in a dose-dependent manner（P<0.01）. Conclusion:The mechanism of different concentrations of Astragali Radix containing serum in the treatment of osteoporosis may be related to the regulation of CYP24A1， CYP27B1 mRNA and protein in the osteogenic differentiation of aging BMSCs.
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Supercritical large area or large segmental bone defects are still a clinical problem. Researchers are committed to the development of artificial bone materials, but in order to solve the problem of poor bone formation of artificial bone materials, people are paying more and more attention to application of bone marrow mesenchymal stem cells in bone tissue engineering. In this review, bone marrow mesenchymal stem cells, osteogenic differentiation, osteoblasts cells, clinical application were used as keywords to search CNKI database, Wanfang database, Weipu database and PUBMED database by computer. The isolation and culture of bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cell identification method, osteogenic induction method, osteogenic differentiation identification and clinical applicationt were comprehensively summarized in order to provide a theoretical basis for its use as a seed cell in the treatment of bone tissue diseases. Scholars have preliminarily studied the treatment of bone and cartilage defects, osteoarthritis, femoral head necrosis and other diseases with bone marrow mesenchymal stem cells combined with transplantation, and obtained good clinical efficacy.However, bone marrow mesenchymal stem cells have certain advantages and disadvantages, and further clinical studies and long-term efficacy verification are needed.
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Bone Marrow Cells , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , OsteogenesisABSTRACT
SUMMARY: Matrigel is a basement membrane matrix extracted from the EHS mouse tumor containing extracellular matrix protein, its main components are laminin, type IV collagen, nestin, heparin sulfate, growth factor and matrix metalloproteinase.At room temperature, Matrigel polymerized to form a three dimensional matrix with biological activity. It can simulate the structure, composition, physical properties and functions of the cell basement membrane in vivo, which is beneficial to the culture and differentiation of the cells in vitro, and can be used for the study of cell morphology, biochemical function, migration, infection and gene expression. In this study, Matrigel three-dimensional culture model of bone marrow mesenchymal stem cells(BMSCs) was established, and its morphology, proliferation and survival were observed. BMSCs were isolated and cultured with whole bone marrow adherence method. The Second generation BMSCs with good growth condition were selected and mixed with Matrigel to form cell gel complexes. The morphology and proliferation of mesenchymal stem cells were observed by phase contrast microscope and HE staining,Live/Dead staining was used to evaluate the cell activity.Phase contrast microscopy showed that BMSCs were reticulated in Matrigel and proliferated well, After 7 days, the matrix gel gradually became soft and collapsed, a few cell reticular crosslinking growth was seen at 14 days; HE staining showed that the cytoplasm of the cells was larger on the fourth day and the cells were elongated and cross-linked on the seventh day; Live/dead staining showed that most cells showed green fluorescence with the prolongation of culture time, on the first, 4 and 7 days, the activity of bone marrow mesenchymal stem cells in Matrigel gradually increased, and the percentages were 92.57 %, 95.54 % and 97.37 %, respectively. Matrigel three-dimensional culture system can maintain the morphology, function and proliferation ability of bone marrow mesenchymal stem cells.
RESUMEN: Matrigel es una matriz de membrana basal extraída del tumor de ratón EHS que contiene proteína de matriz extracelular. Los componentes principales son laminina, el colágeno tipo IV, nestina, sulfato de heparina, factor de crecimiento y metaloproteinasa de matriz. A temperatura ambiente, Matrigel se polimerizó para formar una matriz tridimensional. Es posible simular la estructura, la composición, las propiedades físicas y las funciones de la membrana basal celular in vivo, lo que es beneficioso para el cultivo y la diferenciación de las células in vitro, y se puede utilizar para el estudio de la morfología celular, la función bioquímica, la migración, infección y expresión génica. En este estudio, se estableció el modelo de cultivo tridimensional Matrigel de células madre mesenquimales de médula ósea (BMSC), y se observó su morfología, proliferación y supervivencia. Las BMSC fueron aisladas y cultivadas con el método de adherencia de la médula ósea completa. Se seleccionaron las BMSC de segunda generación con buenas condiciones de crecimiento y se mezclaron con Matrigel para formar complejos de gel de células. La morfología y la proliferación de las células madre mesenquimales se observaron con microscopio de contraste de fase y se tiñó con Hematoxilina-Eosina (HE); para evaluar la actividad celular se usó la tinción Live/Dead. La microscopía de contraste mostró que las BMSC se reticularon en Matrigel y proliferaron bien. Después de 7 días, se observó que el gel de matriz gradualmente se volvió blando y colapsó, y se visualizó un cruce transversal de algunas células reticulares a los 14 días. La tinción mostró que la mayoría de las células mostraron una fluorescencia verde con la prolongación del tiempo de cultivo; en los primeros 4 y 7 días, la actividad de las células madre mesenquimales de la médula ósea en Matrigel aumentó gradualmente y los porcentajes fueron de 92,57 %, 95,54 % y 97,37 %, respectivamente. El sistema de cultivo tridimensional de Matrigel puede mantener la morfología, la función y la capacidad de proliferación de las células madre mesenquimales de la médula ósea.
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Animals , Dogs , Proteoglycans/chemistry , Collagen/chemistry , Laminin/chemistry , Cell Culture Techniques/methods , Mesenchymal Stem Cells/cytology , Tissue Engineering , Drug CombinationsABSTRACT
Objective: To construct a novel tissue engineering complex, BMSCs sheet-RADA16 scaffold, by combining cell sheet and self-assembled peptides.. Methods: The self-assembled peptide RADA16 scaffold was wrapped with the BMSCs cell sheet. The morphology of the cells and the complex were observed by SEM and confocal laser microscopy, and the proliferation of cells was assessed by CCK-8. The osteogenic differentiation of BMSCs was examined by detection of related gene expression with RT-PCR. Results: Compared with the BMSCs cell sheet, the numbers of cells on RADA16 scaffold growth rapidly at 3 rd-8 th day, and BMSCs were more on the scaffold than those on the cell sheet (P＜0. 05) . RT-PCR results showed that the expression level of osteogenesis-related genes was higher in the complex (P＜0. 05) . Conclusion: BMSCs Sheet-RADA16 Scaffold may promote proliferation and osteogenic differentiation of BMSCs.
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Objective To study the effects of osteopontin (OPN) on the nuclear mechanics of bone marrow-derived mesenchymal stem cells (BMSCs) as well as its involved mechanisms. Methods The BMSC migration was evaluated using the Transwell assay. An atomic force microscope (AFM) was used to determine the elastic modulus of the BMSC nucleus and analyze the changes in the nuclear mechanics of the BMSCs after treatment with OPN. The activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase1/2 (ERK1/2) was measured by Western blot. The role of the FAK-ERK1/2 signaling pathway in mediating the OPN-affected BMSC nuclear mechanics was investigated by employing a specific inhibitor. RT-PCR and Western blot were used to detect the expression of Lamin A/C at mRNA and protein levels in the BMSCs, respectively. Results The elastic modulus of the BMSC nucleus exhibited a significant decrease after OPN treatment compared with that of the control group. OPN could upregulate the phosphorylation level of FAK and ERK1/2, but the inhibitor of FAK or ERK1/2 restored the OPN-decreased elastic modulus of the BMSC nucleus and inhibited the BMSC migration significantly. After treatment with OPN, the expression of Lamin A/C in the BMSCs reduced significantly, and such a reduced expression could be suppressed by the inhibitor of FAK or ERK1/2. Conclusions OPN could probably downregulate the expression of Lamin A/C of the BMSCs via the FAK-ERK1/2 signaling pathway, decrease the stiffness of the BMSC nucleus, and promote the migration of the BMSCs. The research outcomes provide the experimental evidence for further understanding the mechanism of the OPN-regulated BMSC migration and its potential clinical application.
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Objective: To study the bone regeneration capacity of the BMSCs cell sheets combined with PRP used with dental implant. Methods: BMSCs were isolated from young SD rats and induced to form cell sheets(BMSCs); PRP were prepared from the fresh blood of rats; PRP gel with BMSCs fragments(BMSCs + PRP) were injected into the space of dental implant of Ti and β-TCP. BMSCs, PRP and BMSCs + PRP with the implant were respectively transplanted into nude mice(n = 6). 8 weeks after transplantation bone regeneration were examined by Micro CT and hard tissue slicing. Results: The group of BMSCs + PRP showed more new bone formation around implants with blood vessel than the group of BMSCs and PRP. Conclusion: BMSCs sheets with PRP can improve the bone regeneration around dental implants.
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Objective: To investigate the effects of 25-hydroxyvitamin D3[25(OH) D3,D3] on NLRP3 inflammasome activation and inflammatory response of bone marrow mesenchymal stem cells (BMSCs) from diabetic rats. Methods: BMSCs were isolated from diabetic rats and identified by immunocytochemical staining. The cells were divided into diabetic control group (without D3 treatment),low concentration group(treated with 1 × 10-5 mmol /L of D3),intermediate concentration group(treated with 1 × 10-4 mmol /L of D3),and high concentration group(treated with 1 × 10-3 mmol /L of D3) (n = 10),BMSCs from normal rats were used as the normal control group(without D3 treatment). Inflammation-related proteins including NLRP3 in BMSCs were examined by western blot analysis. Results: The cultured cells expressed biomarkers of BMSCs. VDR expression in normal control,diabetic control and low concentration groups was less than that in intermediate concentration and high concentration groups(P ＜ 0. 05). Compared with all diabetc groups,normal control group expressed less NF-κB,NLRP3,ASC,Caspase-1,IL-1β,IL-18 and IL-6(P ＜ 0. 05). Additionally,diabetic control and low concentration groups showed stronger expression of NF-κB,NLRP3,ASC,Caspase-1,IL-1β,IL-18 and IL-6 than intermediate concentration and high concentration groups(P ＜ 0. 05). Conclusion: 25-(OH)2-D3 can inhibit the activation of NLRP3 in BMSCs and suppress the inflammatory response of BMSCs from the diabetic rats.
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Objective To investigate the functional recovery and the effect of CNTF antibody block to the bone marrow mesenchymal stem cells (BMSCs) transplantated by Abdominal aortic on the downstream signaling pathways STAT3/Caspase-9 in spinal cord ischemic reperfusion injury in rats.Methods Adult female SD ratswere assigned randomly to 4 groups. The neurological functional status of the animals was assessed with BBB scores, the Motor evoked potentials (MEP) and Cortical somatosensory evoked potentials (CSEP).The IHC were used to detect the the expressional changes of CNTF, then Western blot and RT-PCR were used to detect the expressional changes of STAT3、p-STAT3、CNTF and Caspase-9 in the ischemic segments of spinal cord.Results Compared with the sham group, in the SCIRI rats, the BBB scores were markedly decreased at all time points (P＜0.01), the latency and the amplitude of MEP and CSEP was longer and lower at 14 d post operation (P＜0.01), and this change was the most significant in the control group the second in the CNTF block group, and the last in the transplantation group, Resutts between each two groups were statistically significant (P＜0.05). At 7 d post operation, compared with the sham group, the immunoreactive products of CNTF were decreased in the CNTF block group (P＜0.05), but were increased (P＜0.05) in the control group and the transplantation group (P＜0.05), and results in the transplantation group were higher than in the control group (P＜0.05). At 7 d post operation, compared with the sham group, the m RNA and protein level of CNTF、STAT3、 p-STAT3 were decreased obviously in CNTF block group (P＜0.05), the levels were increased in the control group and the transplantation group (P＜0.05), and the levels in the transplantation group were higher than that in the control group (P＜0.05); but the m RNA and protein level of Caspase-9 were only decreased in the transplantation group (P＜0.05), the level was increased in the CNTF block group and the control groups (P＜0.05), and the level in the CNTF block group was more significantly increased than that in the control group (P＜0.05). At 14 d post operation, in CNTF block group, the m RNA and protein level of CNTF、STAT3、p-STAT3 were significantly higher than that in the sham group and the control group (P＜0.05), and the m RNA and protein level of caspase-9 was higher than that in the sham group (P＜0.05), but lower than that in the control group (P＜0.05), there were not statistically different in the level of each factor compared with transplantation group (P＞0.05). Conclusions BMSCs, transplanted by the abdominal aorta, can promote the expression of CNTF in the injuried spinal cord and significantly improve the hind limb function recovery by CNTF-mediated signaling pathway downstream of STAT3/Caspase-9 SCIRI in rats, but the role of BMSCs can be weakening by CNTF block that inhibited STAT3/Caspase-9 signaling pathway.
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Objective:To study the bone regeneration capacity of the complex of bone marrow mesenchymal stem cells(BMSCs) and platelet rich plasma (PRP) with decellularized cartilage matrix (DCM).Methods:BMSCs were isolated from young rabbit and cultured;PRP were prepared from the fresh blood of rabbit and the DCM were come from the fresh ears of rabbits and processed into a mixture of BMSCs-PRP-DCM.The mixture was injected subcutaneously into nude mice,8 weeks after injection bone regeneration was examine by HE staining and Massons staining decellularization.Results:The BMSCs show good differentiation capacity in vitro and the cartilage fragments were well decellularized.Excellent endochondral ossification ability of the complex was observed by in vivo experiment.Conclusion:The BMSCs-PRP-DCM complex has good capacity of endochondral ossification.
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Objective:To observe the effects of low intensity pulsed ultrasound(LIPUS) on the osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs) on titanium surface.Methods:BMSCs from Wistar rat bone marrow were respectively cultured on the flat titanium surface and the large grain blast acid etched(SLA) titanium surface,and induced by mineralization medium.Then,the cells were interfered by LIPUS and a control condition.Alkaline phosphatase(ALP) were quantitative determinated after 3 and 7 d mineralization induction respectively,ALP staining were observed after 14 d induction.Alizarin red staining were observed after 21 d mineralization induction.Osteogenic related protein and gene expressions were detected after mineralization induction.Results:ALP in culture medium of LIPUS group was higher than that of the control group after 3 d and 7 d mineralization induction(P＜0.05).LIPUS group showed stronger ALP staining and alizarin staining,and more mineralized nodules than control group.The expression of osteogenic related proteins,including Runx2,BMP2,OPN in LIPUS group increased.Osteogenic related genes expression,including ALP,Runx2,BMP2,OPN,OCN and Col-1 of the LIPUS group increased.Conclusion:The osteogenic differentiation of BMSCs on the fiat titanium surface or SLA titanium surface can be promoted by LIPUS.
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Objective:To observe the effect of human bone marrow mesenchymal stem cel s (BMSCs) on the growth, metastasis and apoptosis of hepatocarcinoma cells with low metastatic potential. Methods: Mouse model with low metastatic liver cancer cells were used. Mice were randomly divided into the model experimental group and the control group. In the experimental group, BMSCs (5 × 105) were injected into the tail vein of each mouse on the 7th day after inoculation with hepatocarcinoma cells. Mice in the control group were injected with the culture solution (0.2 mL per mouse) of BMSCs through the tail vein at the same time. The subcutaneous tumor volume was measured every week after the start of the experiment. Animals were sacrificed at day 14 (2 weeks), day 21 (3 weeks), day 28 (4 weeks), day 35 (5 weeks), and day 42 (6 weeks) after inoculation of tumor cells. Complete dissection of the tumor blocks was done and the tumor inhibition rate was calculated by weight and volume. Real-time PCR was used to detect the expression of the osteopontin, bone salivary protein, and integrinαⅤgenes and the expression of Bcl-2, Bax, and caspase3 genes. Results:The inhibitory effect of BMSCs on tumor weight was statistically significant at 3 weeks. The tumor weight of the samples from the experimental group was significantly lower than that of the samples from control group. The inhibitory effect of BMSCs on tumor volume was statistically significant at 2, 3, 5, and 6 weeks. The tumor volume of the samples from the experimental group was significantly lower than that of the samples from the control group. The expression of pro-apoptotic factors, Bax and caspase3, in BMSCs was increased, and the expression of anti-apoptotic factor, Bcl-2, decreased gradual y. Conclusion:BMSCs inhibited the growth of low metastatic hepatocel ular carcinoma cel s. The inhibition rate on tumor weight and volume was highest at the third week, and the tumor inhibition rate decreased gradually with time.
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AIM To observe the effects of Zuogui Pills and Yougui Pills on the proliferation of BMSCs and the expression of apoptosis after osteogenic and adipogenic differentiation in ovariectomized rats.METHODS Of sixty female SD rats,forty rats followed by bilateral ovariectomy were randomly divided into ovariectomy group (1.0 g/kg distilled water),Zuogui Pills group (9.45 g/kg Zuogui Pills) and Yougui Pills group (10.26 g/kg Yougui Pills) and Bujiale group (0.09 mg/kg estradiol valerate),another ten rats as control group (1.0 g/kg distilled water),ten rats bilateral excision of a small amount of fat around the ovary was treated as sham operation group (1.0 g/kg distilled water).After 12 weeks of administration,the rats were killed,BMSCs were cultured in vitro.Cell proliferation was detected by MTT assay.Western blot was used to detect the protein expressions of Caspase-3 and Bcl-2.RE-SULTS MTT assay showed that the proliferation of BMSCs were promoted in Zuogui and Yougui Pills groups,and the proliferation effect in Zuogui Pills group was better than that in Yougui Pills group.After osteogenic differentiation,as compared with the control group,the Caspase-3 expression of ovariectomy group was up-regulated (P ＜0.05) and the expression of Bcl-2 was down-regulated (P ＜0.01).As compared with ovariectomy group,the expression of Caspase-3 was decreased and the expression of Bcl-2 was up-regulated in Zuogui Pills group and Yougui Pills group.After adipogenic differentiation,as compared with the control group,the expressions of Caspase-3 were significantly up-regulated and Bcl-2 were down-regulated in the ovariectomy group,Zuogui Pills group and Yougui Pills group (P ＜ 0.05).After osteogenic and adipogenic differentiation Zuogui Pills group and Yougui Pills group were significant different (P ＜ 0.05).CONCLUSION Zuogui Pills and Yougui Pills both can inhibit the apoptosis of BMSCs after osteogenic and adipogenic differentiation in ovariectomized rats.Zuogui Pills can promote BMSCs osteogenesis differentiation while Yougui Pills can promote adipogenic differentiation.
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AIM To observe the effects of Zuogui Pills and Yougui Pills on the proliferation of BMSCs and the expression of apoptosis after osteogenic and adipogenic differentiation in ovariectomized rats.METHODS Of sixty female SD rats,forty rats followed by bilateral ovariectomy were randomly divided into ovariectomy group (1.0 g/kg distilled water),Zuogui Pills group (9.45 g/kg Zuogui Pills) and Yougui Pills group (10.26 g/kg Yougui Pills) and Bujiale group (0.09 mg/kg estradiol valerate),another ten rats as control group (1.0 g/kg distilled water),ten rats bilateral excision of a small amount of fat around the ovary was treated as sham operation group (1.0 g/kg distilled water).After 12 weeks of administration,the rats were killed,BMSCs were cultured in vitro.Cell proliferation was detected by MTT assay.Western blot was used to detect the protein expressions of Caspase-3 and Bcl-2.RE-SULTS MTT assay showed that the proliferation of BMSCs were promoted in Zuogui and Yougui Pills groups,and the proliferation effect in Zuogui Pills group was better than that in Yougui Pills group.After osteogenic differentiation,as compared with the control group,the Caspase-3 expression of ovariectomy group was up-regulated (P ＜0.05) and the expression of Bcl-2 was down-regulated (P ＜0.01).As compared with ovariectomy group,the expression of Caspase-3 was decreased and the expression of Bcl-2 was up-regulated in Zuogui Pills group and Yougui Pills group.After adipogenic differentiation,as compared with the control group,the expressions of Caspase-3 were significantly up-regulated and Bcl-2 were down-regulated in the ovariectomy group,Zuogui Pills group and Yougui Pills group (P ＜ 0.05).After osteogenic and adipogenic differentiation Zuogui Pills group and Yougui Pills group were significant different (P ＜ 0.05).CONCLUSION Zuogui Pills and Yougui Pills both can inhibit the apoptosis of BMSCs after osteogenic and adipogenic differentiation in ovariectomized rats.Zuogui Pills can promote BMSCs osteogenesis differentiation while Yougui Pills can promote adipogenic differentiation.
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Objective:To investigate the regulation of proliferation and differentiation of bone marrow mesenchymal stem cells(BM-SCs)by CKIP-1 in vitro.Methods:BMSCs from CKIP-1 nock out(KO)and wild type(WT)C57 mice were isolated and cultured u-sing adherence method in vitro.BMSCs of the 3rd passage were induced to osteogenic and adipgenic differentiation.Cell proliferation was examined by MTT assay.Cell surface markers were tested by FCM.The osteogenic and adipogenic differentiation was studied by alkaline phosphatase (ALP)staining,alizarin red staining and oil red O staining.Results:The proliferation and cell marke expression of the 2 groups were similar.ALP staining of KO group was strong than that of WT group after osteogenic induction.Alizarin red stai-ning showed that there were more mineralized nodules in BMSCs of KO group than in those of WT group.Oil red O staining of KO mice BMSCs was stronger than that of WT.Conclusion:CKIP-1 deficiency can enhance the osteogenic and adipogenic differentiation without influence on the proliferation of BMSCs.
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Objective:To study the effects of endothelial progenitor cells(EPCs)on the osteogenesis of bone marrow mesenchymal stem cell (BMSCs)sheet-implant complex.Methods:EPCs were added to the BMSC sheets,and the expression of osteogenesis-relat-ed genes was examined by real time PCR.Cell sheets were wrapped around implants to construct cell sheet-implant complexes and the complexes were subcutaneously transplanted into SCID mice.The complexes were harvested 8 weeks after operation and observed by micro-CT and histological examination.Results:The BMSC sheet with EPCs showed higher expression of Runx2,ALP,BMP2 and VEGF in the in vitro test;higher bone volume ratio,greater amount of new bone tissue and higher expression of Runx2 and BMP2 in the in vivo test.Conclusion:EPCs can improve the osteogenesis of BMSC sheet-implant complex.
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Objective To establish immortalized bone marrow mesenchymal stem cells (BMSCs) from SD rats in vitro for further research on the characteristics and clinical application of BMSCs.Methods By using L ipo-fectamine TM 2000 -mediated gene transfection, plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigene gene (SV40Tag) was transfected into BMSCs.BMSCs were screened by puromycin and then cultured on an extended scale.Their cell morphology and growth conditions were observed.Growth curve of cells was graphed.The expression of SV40Tag in transfected cells was identified by enzyme digestion method for the detection of tumor forma-tion.Results The growth rate of the experimental group of BMSCs after the 7th generation was significantly higher than that of the control group.No tumorigenicity was found in BMSCs after the 7th generation.Conclusion In vitro, the immortalized BMSCs by pCMVSV40T/PUR can provide basis on a large scale for its application in clinic and sci-entific research.
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Objective To evaluate the gene therapeutic effects of guinea pigs model with immune -mediated inner ear disease(IMIED)after locally injection of bone -marrow mesenchymal stem cells(BMSCs) decorated by in-terleukin-4 gene .Methods Guinea pigs were immunized with keyhole limpet hemocyanin (KLH) and caused 55 animal models ,divided into five groups ,each group with 11 animals :groupA(BMSCs carrier) ,group B(BMSCs emp-ty -carrier control group) ,group C(recombinent lentivirus IL -4 gene) ,group D(lentivirus empty -carrier control group) ,group E(simulation operation control group) .Groups were all injected with the corresponding suspension (20 μl)[includs BMSCs ceas of (1 .5~2 .0) × 106 ,the concentration of (entivirus) is 0 .5 × 108 pfu] by the scala tympani window into the inner ear .The fluorography immunohistochemistry test and enzyme immunohistochemistry test for the situation of IL -4 gene express and productive protein distribution in inner ear .Auditory functions and the KLH level of guinea pigs blood were monitored respectively by auditory brainstem responses (ABRs) and ELISA test .Results The threshold of ABR wave Ⅲ decreased in group A ,group B and group C .The result were more significant in group A and group B than that in group C ,but results in group A was more prominent (P<0 .05) . The results of immunohistochemistry test showed that fluorescence positive BMSCs mainly scattered in scala tymani and scala vestibule .The microscope results showed that for the group A ,B and C ,there were only few foccule and red and white blood cells in scala tympani floc ,but for group D and group E ,with different levels of labyrinthine hy-drops and some mononuclear cells around the spiral ganglion and small blood vessels .Conclusion Restructuring lentiviral vector with IL -4 gene can be successfully transfected into BMSCs in vitro ,compared to inplangting into inner ear in scala tympani approach ,the cells can migrate and generate gene product of IL -4 ,to significantly im-prove the auditory functions and inflammatory reaction of inner ear disease ,and BMSCs can be used as a carrier to migrate to the damaged part with therapeutic gene .
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Objective Observe the influence on the Gilial cell-line derived neurotrophic factor (GDNF) after treatment stroke rats with Bone marrow mesenchymal stem cells (BMSCs) or Bone marrow mononuclear cells (BMNCs)combined with traditional Chinese medicine (TCM).Methods Separating and cultivating BMSCs and BMNCs were.140 Wistar rats were divided into 7 groups randomly,normal group,pretended surgery group,model group,BMNC group,BMNC+TCM group,BMSC group,BMSC+TCM group.The other five groups were performed for 2 hours middle cerebral arterial occlusion (MCAO) except normal group and pretended surgery group.Intervention methods in each group after 24 hours of MCAO:model group:Subarachnoid injection of 100 μl 0.01M PBS,BMNC group and BMNC + TCM group,Subarachnoid injection of 2 × 107 BMNCS,BMSC group and BMSC+TCM group,Subarachnoid injection of 2 × 107 BMSCS,BMNC +TCM group and BMSC+TCM group were treated united with TCM on the transplantation day (po.Qd.).GDNF level in all groups' rat brain were analyzed by Enzyme-linked immuno sorbent assay (ELISA) method at the 4th day and the 28th day after transplantation.Results The GDNF level of model group [(62.60±4.05) pg/ml] is higher than normal group's [(53.46 ± 3.91)pg/ml] at the 28th day (P＜ 0.05).The GDNF levels of BMNC group [(194.21 ±39.56)pg/ml,(67.70±4.73)pg/ml] and BMSC group [(169.83±28.84)pg/ml,(82.66±32.23)pg/ml] are higher than model group's at the 4th and 28th day(P＜0.05).The GDNF level of BMSC group is higher than BMNC group's at the 28th day(P＜0.05).The GDNF levels of group BMNC+TCM group[(560.61 ± 194.84) pg/ml,(265.83 ±93.58) pg/ml and BMSC+TCM group[(370.93 ±46.19) pg/ml,(247.34±98.02)pg/ml] are higher significantly than BMNC group's or BMSC group's at the 4th and 28th day(P＜0.05).At the 4th day the GDNF level of the BMNC+TCM group is higher than BMSC+TCM group's(P＜0.05).Conclusion Subarachnoid transplantation of BMNCs or BMSCs will increase the GDNF level in brain of MCAO rats.The transplantantion combined with TCM can inprove the capability of the enhance.That reflect the advantage of transplantantion bone marrow origin stem cell united with TCM.