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@#：【Objective】Toinvestigatetheeffectsofinterleukin-17（IL-17）ontheproliferationandmigrationof bronchialsmoothmusclecells（BSMC）andtheroleofJAK/STAT3signalingpathwayinthisprocess.【Methods】BSMC weretreatedwithdifferentconcentrationsofIL-17fordifferenttimestodeterminethebestoftheexperimentalcondition. ThenMTTassaywasusedtodetectcellviability.CellproliferationstatesweredetectedbyBrdUstaining，andthecell cyclewasassessedbyPIstainingusingaflowcytometer.Transwellcellmigrationassaywasfurtherusedtodetectcell migrationability.TheexpressionofJAK，p-JAK，STAT3andp-STAT3inBSMCafterbeingtreatedwithIL-17was detectedbyWesternblotting.JAK/STAT3signalingpathwayspecificblockerAG490wasusedtoinvestigatetheroleof JAK/STAT3signalingpathwayinIL-17-inducedBSMCproliferationandmigration.TheeffectsofIL-17oncellproliferation， migration and JAK/STAT3 signaling pathway related protein expression were evaluated after blocking the JAK/STAT3 signaling.【Results】IL-17enhancedtheproliferation（P<0.05），promotedthecellcycletransitions（P<0.05）andsig⁃nificantlyincreasesthemigrationability（P<0.05）inBSMC.ThisprocesswasaccompaniedbytheenhancementofJAK/ STAT3signalingpathwayinBSMC（P<0.05）.InhibitionofJAK/STAT3signalingpathwayalleviatedBSMCproliferation andmigrationinducedbyIL-17（P<0.05） .【Conclusions】JAK/STAT3signalingpathwayparticipatesinthestimulation processofIL-17ontheproliferationandmigrationofBSMC.AG490inhibitstheenhancementofJAK/STAT3signaling pathwayinBSMCinducedbyinterleukin-17.
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Although, antineoplastic therapies have now been developed reduction of tumor progression,itis necessarytofind new therapeutic alternatives to suppress angiogenesis.Thus celecoxib (Cx) has been used for its antiangiogenic action in combination with certain polymeric compounds such as poly (lactic co-glycolic acid) (PLGA) acid, which help to improve the bioavailability and avoid effects of long drug administrations. For this purpose we used a murine tumor modelinduced by mammary adenocarcinoma cells resistant to chemotherapy (TA3-MTXR). CX/PLGA inhibits the microvascular density, VEGF expression and cell proliferationinaddition to increased apoptosis (P <0.0001). Cx reduces tumor progression in a concentration of 1000 ppm associated with PLGA, reducing cell proliferation, the presence of VEGF and promoting apoptosis of multiresistant TA3 tumor cells.
Si bien actualmente se han desarrollado terapias antineoplásicas que permiten reducir de cierta manera el avance tumoral, es necesario buscar nuevas alternativas terapéuticas que permitan suprimir la angiogénesis. Es así como el Celecoxib (Cx) ha sido utilizado por su acción antiangiogénica en combinación con algunos compuestos poliméricos, tal como el ácido poli (láctico co-glicólico) (PLGA), el cual ayudaría a mejorar la biodisponibilidad y evitaría efectos derivados de largas administraciones del fármaco. Para tal efecto se ha utilizado un modelo tumoral murino, inducido por células tumorales de adenocarcinoma mamario resistente a la quimioterapia (TA3-MTXR). Los resultados indican que CX/PLGA inhibe la microvascularización, expresión de VEGF y la proliferación celular además del aumento de la apoptosis (P<0,0001). El efecto antitumoral del Cx está bien reportado en la literatura; este sumado a la microencapsulación con PLGA, aportarían un sistema de administración útil, ya que nos otorga una administración sostenida en el tiempo, los cual podría ayudar a mantener los niveles de droga durante un período más prolongado, lo cual sería beneficioso en la terapia tumoral.
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Animals , Female , Mice , Antineoplastic Agents/administration & dosage , Celecoxib/administration & dosage , Neovascularization, Pathologic/drug therapy , Apoptosis/drug effects , Cell Line, Tumor/drug effects , Cell Proliferation/drug effects , Drug Delivery Systems , Immunohistochemistry , Lactic Acid/administration & dosage , Neoplasm Invasiveness/prevention & control , Polyglycolic Acid/administration & dosage , Polymers/administration & dosage , Vascular Endothelial Growth Factor A/drug effectsABSTRACT
Objective:To study the effects of panax notoginsenosides on the proliferation and oxidation indices of cisplatin-induced nephroxicity in HK-2 cells. Methods:HK-2 cells were cultured in vitro till the number was up to 1 × 106/ml. The cells were inoculated in 96-well culture plate and randomly divided into six groups:normal saline ( NS) group,the model group, the positive control group and the high dose group , medium dose group and low dose group of panax notoginsenosides ( PNS) . The nephroxicity model was dupli-cated with the addition of cisplatin (the final concentration was 6. 25μg·L-1). The model group, positive control group and the three panax notoginsenosides groups was treated with saline solutions, amifostine, panax notoginsenosides at the dose of 100,50 and 25 mg· L-1 , respectively. The cell viability was detected with an MTT method, the content of MDA and the activity of SOD, GSH-PX and LDH were measured and the cell structure was observed. DCFH-DA was used as the fluorescence probe to detect the level of ROS by a fluorescence microplate reader. Results:Compared with those in the model group, the cell viability and the activity of SOD and GSH-PX in the three PNS groups and the positive control group significantly increased (P<0. 05);the content of MDA, the level of ROS and the activity of LDH significantly decreased (P<0. 05); the cell structure was significantly improved. Conclusion: PNS can pro-mote the proliferation of HK-2 cells in vitro, and improve the biochemical parameters and enzyme levels. The results suggest that PNS has a protective effect on HK-2 cell,and the protective mechanisms may be related with its antioxidant effect.
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AIM:ToinvestigatetheeffectofIkarosisoformsontheproliferationofhumanovariancancerSK-OV3 cells.METHODS:Three isoforms of Ikaros, IK1, IK2 and IK6, were transfected into ovarian cancer SKOV3 cells. CCK-8 assay and cell counting were used to detect the effects of Ikaros isoforms on the proliferation of SKOV 3 cells.The cell cycle was analyzed by flow cytometry .The cell cycle-related proteins were detected by Western blot .RESULTS:IK1 and IK2 expression inhibited SKOV 3 cells proliferation .Flow cytometry analysis indicated that IK 1 and IK2 induced SK-OV3 cell cycle arrest at the G 1 phase.IK6 isoform exerted no obvious effect on the proliferation or cell cycle of SKOV 3 cells.Compared with control EV group , IK1 group and IK2 group showed a dramatic elevation in the expression of the cell cycle inhibitor p21, along with a substantial decrease in the expression of the cell cycle inducers cyclin D 1 and cyclin D2, which did not change in IK 6 group.CONCLUSION:IK1 and IK2 significantly inhibit the proliferation of ovarian cancer SKOV3 cells and induce cell cycle arrest at G 1 phase by regulation of cell cycle-related proteins cyclin D1, cyclin D2 and p21, while IK6 isoform exerts no obvious effect on the proliferation and cell cycle of SKOV 3 cells.
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Objective To study the expression of T cell immunoglobulin mucin-3(Tim-3)in hepatocellular carcinoma(HCC)cell line and its influence on the oncobiological behavior of HCC cells.Methods The expression of Tim-3 mRNA and protein in human normal liver cell line L02 and HCC cell line HepG2 and SMMC7721 was assessed by FQ-PCR and Western blot.The siRNA Tim-3 fragments were designed to silence the Tim-3 gene expression in HepG2 cel1.HepG2 cells were transfected with siRNA by using LipofectamineTM 2000.The expression of Tim-3 protein was detected after transfection by Western blot to screen the effective siR-NA fragment.The proliferation and migration potential of hepG2 cells was evaluated after Tim-3 gene silence by the cell growth curve MTT assay and the wound healing assay.Results Both expressions of Tim-3 mRNA and protein in human HCC cell line HepG2 and SMMC7721 were significantly higher than those in normal liver cell line L02(P<0.05).After siRNA transfection,the protein expression of Tim-3 in experimental group was significantly lower than that of the control group.Compared with control group,the proliferative activity and the migration ability were decreased significantly.Conclusion Expressions of Tim-3 mRNA and protein are increased in HCC cell line.Tim-3 expression promotes HCC cell proliferation and migration.
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BACKGROUND:As mesenchymal stem cells are commonly used as seed cells in studies of regenerative medicine and tissue engineering, the regulatory mechanism of their biological characteristics is a current research focus. OBJECTIVE:To summarize the regulations of Wnt signaling pathway on proliferation, senescence and differentiation of mesenchymal stem cells. METHODS:PubMed database and CNKI database were retrieved by computer using the key words of“mesenchymal stem cells, Wnt signaling pathway, proliferation, senescence, differentiation”in Chinese and English, respectively, between 2002 and 2014. Final y, 44 articles were included in result analysis. RESULTS AND CONCLUSION:Wnt signaling pathway is widely involved in the regulations of the biological characteristics of mesenchymal stem cells. Canonical Wnt signaling pathway reveals a bi-directional regulation effect on cellproliferation and osteogenic differentiation, and enhances senescence and neural differentiation, but inhibits adipogenic differentiation;non-canonical Wnt signaling pathway enhances senescence and osteogenic differentiation, and inhibits proliferation and adipogenic differentiation of mesenchymal stem cells, but it takes no part in neural differentiation of mesenchymal stem cells. So the regulations of Wnt signaling pathway on the biological characteristics of mesenchymal stem cells can be used as the new therapeutic targets of bone tissue engineering, nerve injury repair, and so on.
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BACKGROUND:Modern research shows that Drynaria can delay celldegeneration and reduce the incidence of osteoarthritis. OBJECTIVE:To observe the proliferation and differentiation of rabbit bone marrow mesenchymal stem cells induced by different dosages of Drynaria freeze-dried powder, and to explore the optimum induction concentration. METHODS:Rabbit bone marrow mesenchymal stem cells were isolated and cultured in vitro by density gradient centrifugation and adherence screening methods, and then divided into blank group, positive control group (transforming growth factorβ1), high-, middle-, low-dosage Drynaria groups (0.4 mg, 0.1 mg, 5μg). Passage 3 cells were selected and cultured in different media. After 1 week, cellviability was detected by MTT method, and expression of type II col agen by immunohistochemical method. RESULTS AND CONCLUSION:Both transforming growth factorβ1 and Drynaria could improve the proliferation of bone marrow mesenchymal stem cells, and the increase in cellproliferation was ranked as fol ows:positive control group>low-dosage group>middle-dosage group>high-dosage group>blank group. Bone marrow mesenchymal stem cells were differentiated into chondrocytes under induction of transforming growth factorβ1 and Drynaria, and induced cells significantly expressed type II col agen. The expression of type II col agen was ranked as fol ows:positive control group>low-dosage group>middle-dosage group>high-dosage group>blank group. These findings suggest that Drynaria can promote the proliferation and differentiation of rabbit bone marrow mesenchymal stem cells, and the optimal dosage is 5μg.
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BACKGROUND:Neural stem cells have self-renewal and multidirectional differentiation potential, but under normal circumstances, the number of neural stem cells is less, and most cells are in the resting state. Thus, to promote the proliferation of neural stem cells is the key to the treatment of neurodegenerative diseases. OBJECTIVE:To investigate the effects of osthole on the proliferation of neural stem cells cultured in vitro, and to analyze its mechanism underlying promoting the proliferation. METHODS:Neural stem cells were cultured in vitro, and passage 3 cells were cultured with different concentrations of osthole(10, 50 and 100μmol/L). After 24 hours, cellvitality was determined by cellcounting kit-8. After 3, 5, 7 days of further culture, the radius of neurospheres was measured, and Ki67-positive cells were counted by immunofluorescence staining. Meanwhile, after 3 days of further culture, the gene expression of Notch 1, Hes 1 and Mash 1 in neural stem cells was detected by RT-PCR. RESULTS AND CONCLUSION:Compared with the control group, 50, 100μmol/L osthole could obviously promote the proliferation ability of neural stem cells. 100μmol/L osthole had the most significant effect and increased the expression of Notch 1 gene, Hes 1 gene, but it had no effect on Mash 1 gene. These results suggest that osthole can promote proliferation of neural stem cells cultured in vitro and its mechanism may be associated with activation of Notch 1 gene and Hes 1 gene in Notch signaling pathway.
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BACKGROUND:Endogenous stem cells have no repair effects on the process of disc degeneration. Authors assumed that this maybe associate with abnormal effects of related etiological factor, resulting in an inhibitory effect on the function of nucleus pulposus-derived mesenchymal stem cells. OBJECTIVE:To investigate the effects of inflammatory cytokine interleukin-1βon biological characteristics of nucleus pulposus-derived mesenchymal stem cells of rats. METHODS:Lumbar spinal nucleus pulposus was obtained from 3-month-old male Sprague-Dawley rats. Nucleus pulposus-derived mesenchymal stem cells were isolated and cultured with col agenase and sequential trypsin digestion. The expression of CD24, CD34, CD45, CD90 and CD105 was detected using flow cytometry. Stem cellgene SOX2 and Nanog expression was measured using RT-PCR. Adipogenic, osteogenic and chondrogenic abilities of nucleus pulposus-derived mesenchymal stem cells were observed. The apoptotic rate of interleukin-1β-treated nucleus pulposus-derived mesenchymal stem cells was detected using flow cytometry. Fluorescent quantitative PCR was used to measure the expression of SOX9, proteoglycan, type II col agenase and caspase-3 gene after nucleus pulposus-derived mesenchymal stem cells were treated with interleukin-1β.
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BACKGROUND:The biological function of human periodontal ligament stem cells is a hot area of research in the treatment of periodontal disease. Human periodontal ligament cells are one of the end cells derived from human periodontal ligament stem cells;meanwhile, it can also provide supports to the development of human periodontal ligament stem cells. However, few studies are reported about the difference of biological characteristics between human periodontal ligament stem cells and human periodontal ligament cells. OBJECTIVE:To compare the differences of biological characteristics between human periodontal ligament stem cells and human periodontal ligament cells. METHODS:The human periodontal ligament stem cells and human periodontal ligament cells were isolated and purified using tissue explant method and cellclone method, respectively, and then were observed under light microscope to compare the differences of morphology. cellproliferation curves of human periodontal ligament stem cells and human periodontal ligament cells were drawn respectively with cellcounting kit 8 assay. Flow cytometry analysis was used to detect their cellcircles and their surface markers expressions. The alkaline phosphatase gene, proliferating cellnuclear antigen gene and Scleraxis gene of human periodontal ligament stem cells and human periodontal ligament cells were detected by Real-time PCR assay.RESULTS AND CONCLUSION:The human periodontal ligament stem cells and human periodontal ligament cells showed a notable difference in morphology under the light microscope observation. During the first 5 days, the cellproliferation curve of human periodontal ligament stem cells was lower than that of human periodontal ligament cells, but 5 days later, the curve of human periodontal ligament stem cells was significantly higher than that of human periodontal ligament cells. The cellcircles of human periodontal ligament stem cells and human periodontal ligament cells were 41.1%and 23.9%, respectively. The surface markers of human periodontal ligament stem cells and human periodontal ligament cells were similar, but their expression rates had significant difference. The expressions of alkaline phosphatase gene, proliferating cellnuclear antigen gene and Scleraxis gene of human periodontal ligament stem cells were significantly higher than those of human periodontal ligament cells. The above results suggest that human periodontal ligament stem cells have much stronger potential ability than human periodontal ligament cells in osteogensis and cellproliferation.
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BACKGROUND:Bone marrow mesenchymal stem cells are considered as commonly used seed cells to construct tissue-engineered for repair of bone and cartilage defects. It is of great significance for cytology and tissue engineering experiments to study the common problems existing in the basic operation and how to avoid these problems in a timely manner. OBJECTIVE:To summarize the common problems existing in the process of operation in order to provide reliable methods about separation, culture and identification of bone marrow mesenchymal stem cells for beginners and researchers. These can reduce or avoid some errors and problems during operation. METHODS:Sixteen New Zealand white rabbits were selected as experiment objects, and bone marrow mesenchymal stem cells were separated from rabbits by iliac puncture, purified and augmented by using density gradient centrifugation combined with adherent culture method. Then cellmorphology was observed by inverted phase contrast microscope, growth curve detected by MTT method and cellphenotype identified by flow cytometry. RESULTS AND CONCLUSION:We encountered some problems in the process of separation and culture, when we operated the first five rabbits. After careful y summarizing and analysis of the reasons, the operation was successful y completed on the rest 11 rabbits. Bacteria pol ution and cellaging were not found in the process of cellculture. What is more, the cells at passage 3 appeared with high-expression of CD29, and CD44, but low expression of CD14 and CD34. The cellgrowth curve showed that the proliferation activity of cells at passages 3 and 5 was higher than that at passage 10. Although the technology of separation, culture and identification of bone marrow mesenchymal stem cells is mature, the failure wil be happen if we do not pay attention to the details of operation. By strictly carrying out normal operations, we can get high purity of bone marrow mesenchymal stem cells, which lays a good foundation for celland animal experiments in the future.
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BACKGROUND:Phytohemagglutinin (PHA) can stimulate the peripheral blood mononuclear cells (PBMCs) into cellcycle, and cause their immune activation, which is a common immune proliferation model. However, the role of non-PBMC ingredient of peripheral blood is unclear, as wel as the expression of endothelial cells related cytokines. OBJECTIVE:To study the effect of whole blood culture and PBMCs alone culture with PHA on the PBMC proliferation and apoptosis, expression of inflammatory cytokine and endothelial cellsecreted cytokine markers. METHODS:Morphological changes of PBMCs separated from normal karyotype human peripheral blood individual y cultured with or without PHA were observed. The PBMCs were col ected by whole blood culture or PBMC separated culture. mRNA was extracted for the fluorescence quantitative RT-PCR, which was applied to detect the cellproliferation, apoptosis, and expression of inflammatory cytokine and endothelial cellsecreted cytokines. The statistic analysis was used for the significance explication. RESULTS AND CONCLUSION:PBMCs alone cultured ere different from those undergoing whole blood culture. The PHA could up-regulate the gene expression of Ki67, proliferating cellnuclear antigen, Caspase 3, interferon-γ, tumor necrosis factor-βand interleukin-6, but down-regulate Protein C. This indicted that PHA could promote the proliferation and apoptosis of PBMCs and up-regulate the expression of inflammatory cytokines, but down-regulate the expression of endothelial cells secreted coagulation cytokines.
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BACKGROUND:Currently, the research about effect of non-dextran coated superparamagnetic iron oxide nanoparticles on cellproliferation and cytotoxicity is relatively much less. OBJECTIVE:To evaluate the effects of 0, 25, 50, 75, 100 mg/L non-dextran coated superparamagnetic iron oxide nanoparticles on the proliferation and cytotoxicity of rat bone marrow mesenchymal stem cels. METHODS:Culture media containing 0, 25, 50, 75, 100 mg/L non-dextran coated superparamagnetic iron oxide nanoparticles were prepared for culture of bone marrow mesenchymal stem cels. After 24 hours of culture, the cels were confirmed using Prussian blue staining, and cellcounting was detected using cellcounting kit-8. Meanwhile, lactate dehydrogenase activity in the supernatant and intracelular superoxide dismutase activity were detected. RESULTS AND CONCLUSION:Loading of non-dextran coated superparamagnetic iron oxide nanoparticles in BMSCs was confirmed by Prussian blue staining. The percentage of cels labeled with non-dextran coated superparamagnetic iron oxide nanoparticles was up to 100% when the cels were incubated with a non-dextran coated superparamagnetic iron oxide nanoparticle solution of 50 mg/L and above, but 25 mg/L was insufficient to label al of the cels. Furthermore, as the concentration of non-dextran coated superparamagnetic iron oxide nanoparticles decreased, the cellproliferation rate decreased gradualy. The 25 mg/L group had a minimum cellproliferation rate, but the 25 and 50 mg/L groups showed no statisticaly significant difference (P > 0.05). Therefore, 50 mg/L is considered as the appropriate concentration of non-dextran coated superparamagnetic iron oxide nanoparticles, under which, the labeling efficiency is higher and the cytotoxicity is lower.
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BACKGROUND:The effects and molecular mechanism of simvastatin on the proliferation and differentiation of osteoblasts remain unclear. Especial y, we do not know much about the effects of connexin 43. OBJECTIVE:To evaluate the effects of simvastatin on the proliferation and differentiation of osteoblasts and the regulatory effect of simvastatin on the expression of osteogenic genes and connexin 43. METHODS:Newborn Sprague-Dawley rats were chosen and the cranium digestion method was used to culture osteoblasts. The different concentrations of simvastatin (0.062 5, 0.125, 0.25, 0.5 and 1.0μmol/L) were used to deal with osteoblasts. The proliferative effect of simvastatin on osteoblasts was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The effect of simvastatin on osteoblast differentiation was measured with alkaline phosphatase activities. The mRNA and protein expression of osteogenic genes and connexin 43 were measured by real time quantitative RT-PCR and western blot assay. RESULTS AND CONCLUSION:There were no significant differences in absorbance values of simvastatin groups at 3 days (P>0.05). However, at 4 and 5 days, absorbance values were lower in the simvastatin groups than those in the control group (P<0.05). Compared with the control group, alkaline phosphatase activities of osteoblasts were greater in the simvastatin groups (P<0.05). Moreover, the effects of 0.25μmol/L simvastatin on alkaline phosphatase activities of osteoblasts were most significant. Osteocalcin, alkaline phosphatase activities, type I col agen and connexin 43 mRNA and protein expressions were increased after treatment with 0.25μmol/L simvastatin (P<0.05). These results indicated that simvastatin may inhibit the proliferation and improve the differentiation of osteoblasts by upregulating the mRNA and protein expression of osteogenic genes and connexin 43. These data may provide the new intervention target for osteoporosis treated with statins.
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BACKGROUND:A variety of embryonic stem cells induction and differentiation systems have been established so far, while the research that promotes embryonic stem cells to differentiate into hematopoietic stem cells is stil at an initial stage, and the induction efficiency needs to be improved. OBJECTIVE:To active the Wnt/β-catenin signal pathway in mouse embryonic stem cells with exogenous win3a as an inducer, and then to observe whether the activation of this pathway wil promote the directional differentiation of embryonic stem cells into hematopoietic progenitor cells. METHODS:The ES-E14TG2a mouse stem cells were cultured with the exogenous wnt3a (100 μg/L) for 21 days, the content ofβ-catenin was tested by cellimmunofluorescence and western blot, and expression of Wnt downstream target gene was detected by quantitative reverse transcription PCR to determine the activation of Wnt/β-catenin signal pathway. Single-layer adherent culture method was used to induce the directional differentiate of above-mentioned cells into hematopoietic stem cells, and detection of hematopoietic development associated surface marker CD34+/Sca-1+was achieved by flow cytometry;meanwhile, the expression of hematopoietic associated gene was measured by quantitative reverse transcription PCR. RESULTS AND CONCLUSION:We found thatβ-catenin accumulated in ES-E14TG2a mouse stem cells after cultured with wnt3a (100 μg/L) for 21 days;the expressions of Wnt downstream target genes such as Pitx2, Frizzled, Sox17 and Oct4 showed the different degrees in increase, meaning the activation of Wnt/β-catenin signal pathway. Furthermore, during the time that we used single-layer adherent culture method to induce hematopoietic stem cells, the CD34+/Sca-1+cells accounted for 20.2%of total cells at day 14, and control cells only accounted for 11.9%. Again, expression quantity of hematopoietic associated gene BMP4, FLK2 and CD34 increased while Smad5 was suppressed significantly. Our data suggest that sustaining action by wnt3a wil active Wnt/β-catenin signal pathway, and also promote the directional differentiation of ES-E14TG2a mouse stem cells into hematopoietic progenitor cells.
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BACKGROUND:Mesenchymal stem celltransplantation promoted skin repair in trauma via various regulatory mechanisms and inhibited scar formation. At present, many scholars believed that bioactive factors secreted by mesenchymal stem cells played an important role. OBJECTIVE:To investigate the effects of bone marrow mesenchymal stem cellconditioned medium on the proliferation and col agen synthesis of hypertrophic scar fibroblasts. METHODS:Human bone marrow mesenchymal stem cells and hypertrophic scar fibroblasts were isolated and cultured, and bone marrow mesenchymal stem cellconditioned medium was prepared. Hypertrophic scar fibroblasts were cultured in vitro with 12, 24, and 48 hour-col ected conditioned medium for 24 hours, which was compared with blank control group. The proliferation of cells was determined by CCK-8. Type I and type III col agen expression in hypertrophic scar fibroblasts was detected using real-time PCR. RESULTS AND CONCLUSION:Compared with the blank control group, 24 and 48 hour-col ected conditioned medium significantly inhibited the proliferation of hypertrophic scar fibroblasts (P<0.01), and also suppressed col agen synthesis of hypertrophic scar fibroblasts (P<0.01). Results suggested that bone marrow mesenchymal stem cellconditioned medium inhibited the proliferation and col agen synthesis of hypertrophic scar fibroblasts by secreting anti-fibrotic bioactive factors, which may provide new theoretical supports for celltherapy to reduce cutaneous scarring.
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OBJECTIVE:To investigate the paracrine effects of bone marrow mesenchymal stem cells on osteoblast biological function. METHODS:Bone marrow mesenchymal stem cells were isolated using standard Ficol-Paquedensity gradient centrifugation. Mesenchymal stem cellconditioned medium was prepared to cultivate osteoblasts, MG63. Proliferation of MG63 cells was analyzed by cellcounting kit-8. Migration of MG63 cells was analyzed by cellscratch method. Alkaline phosphatase activity of MG63 cells was analyzed by microplate test kit. Real-time PCR was performed to evaluate osteoblast differentiation markers, alkaline phosphatase, col agen type I and osteocalcin. Alizarin red staining was performed to evaluate osteoblast mineralization. RESULTS AND CONCLUSION:The cells were strongly positive for CD44, CD73 and CD90, but negative for CD34. MG63 cells cultured in the conditioned medium showed better proliferation and migration than those cultured in the Dulbecco’s modified Eagle’s medium. The activity and mRNA expression of alkaline phosphatase were much higher after induction of 4, 7 days (P<0.01). There was no significant difference in expression of col agen type I and osteocalcin after induction of 4 days, but they were significantly higher than those in the control group after induction of 7 days (P<0.05). Alizarin red staining showed that the number of calcium nodules was increased and the mineral apposition was enhanced after induction of 21 days with the conditioned medium. These findings suggest that the paracrine substance of bone marrow mesenchymal stem cells can significantly promote osteoblast proliferation, migration, differentiation and mineralization.
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BACKGROUND:Stem cells expansion technology in vitro is affected by the microenvironment of the culture system. So, to find an effective method is particularly important to promote celladherent and growth. OBJECTIVE:To compare the effects of different culture media on cellexpansion. METHODS:Human umbilical cord mesenchymal stem cells at a density of 5.0×104 cells/cm2 were inoculated into Corning? polystyrene culture dishes coated with or without poly-L-ornithine and Corning? cellBIND culture dishes. celladhesion and proliferation were observed, and expressions of celladhesion proteins and cellmarkers were detected. RESULTS AND CONCLUSION:celladhesion was promoted when cells were cultured in Corning? cellBIND? Surface medium coated with poly-L-ornithine for 24 hours, and the cultured cells grew at the logarithmic phase. The cellproliferation was also enhanced, and the cells expressed celladhesion protein but not the cellmarkers of CD73, CD90, CD105. Corning? cellBIND? Surface medium was superior to Corning? polystyrene culture medium in the improvement of celladhesion and proliferation. Additional y, both of these two media showed no influence on cellphenotype. These findings indicate that Corning? cellBIND? Surface medium can promote celladhesion and proliferation, but shows no effects on cyclin and cellphenotype. This medium coated with poly-L-ornithine can further accelerate celladhesion and proliferation, and stably express cellphenotype of human umbilical cord mesenchymal stem cells.
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BACKGROUND:Carboxymethylated chitosan is shown to promote some kinds of cells proliferation, but its effects on proliferation of Schwann cells need further studies. OBJECTIVE:To investigate the effects of carboxymethylated chitosan on proliferation of Schwann cells and expression of nuclear factor-κB in cultured Schwann cells. METHODS:Schwann cells from Sprague-Dawley rats at logarithmic growth phase were seeded in 96-wel plates, and cultured respectively with PBS, 0, 10, 50, 100, 200, 500, 1 000 mg/L carboxymethyl chitosan for 24 hours. cellproliferation was detected using the cellcounting kit-8 assay. After trypsin digestion, Schwann cells from Sprague-Dawley rats at logarithmic growth phase were used to prepare cellsuspensions, which were seeded in 6-wel cellculture plates and cultured respectively with 50, 100 and 200 mg/L carboxymethyl chitosan and PBS for 24 hours. Then, 5-bromo-2-deoxyuridine, real-time PCR and western blot assay were performed. RESULTS AND CONCLUSION:cellcounting kit-8 and 5-bromo-2-deoxyuridine detection results showed that carboxymethyl chitosan at 50-1000 mg/L, especial y at 200-500 mg/L, could promote Schwann cellproliferation. Real-time PCR and western blot results showed 50-200 mg/L carboxymethyl chitosan could promote nuclear factorκB mRNA and protein expression in Schwann cells in a dose-dependent manner, suggesting carboxymethyl chitosan can promote Schwann cellproliferation and expression of nuclear factor-κB in Schwann cells cultured in vitro.
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BACKGROUND:In traditional view, the germ cels in mammal ovaries were considered to lose the capacity of self-renew after birth. However, recent studies have showed the existence of female germline stem cels (FGSCs) which take the responsibility for regenerating oocytes in the ovaries of postnatal mammals. OBJECTIVE:To establish the FGSC line and to explore the biological characterization of the FGSC line from neonatal CD-1 mouse ovaries. METHODS:Using two-step enzymatic digestion and immunomagnetic purification, Fragilis-positive cels were isolated from neonatal CD-1 mice ovaries and subjected to long-term culture. Then their biological characteristics were studies with RT-PCR, fluorescent immunocytochemistry and cytogenetic analysis. RESULTS AND CONCLUSION:FGSCs from neonatal CD-1 mice aged 3-5 days were isolated and long-term cultured for 70 passages to establish a cellline. This cellline expressed Mvh, Dazl, Oct-4, Stela, Blimp1, Fragilis, and was proven to be mitoticaly active germ cels by BrdU/Fragilis dual immunofluorescence analysis. Moreover, cytogenetic analysis by Gimsa staining confirmed their normal karyotype. In al, these results indicate the existence of FGSCs with mitotic activity in neonatal CD-1 mouse ovaries.