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Objective@#This study aims to explore the influence of the serine and arginine rich splicing factor 2 (SRSF2) on the biological characteristics of keloid fibroblast, by comparing the expression levels of SRSF2 in normal skin and keloid, with the purpose to provide a new method to study the pathogenesis of keloid.@*Methods@#Samples of normal skin were derived from excess tissue of skin grafts, collected from 8 patients, aged 8-53 years old, while the specimens of keloid were from 12 keloid patients, aged 18-24 years old. All the patients were admitted in the Plastic Surgery Hospital, Chinese Academy of Medical Sciences. The expression of SRSF2 was assessed by immunohistochemistry and immunofluorescent staining in both normal and keloid tissue. Keloid fibroblasts were cultured in vitro to detect the relationship between TGF-β1 stimulation and SRSF2 expression. After constructing the lentiviral sh-RNA-expression vector, targeting SRSF2 and infecting keloid fibroblast, the apoptosis and proliferation of cells were analyzed by the MuseTM Cell Analyzer. The expression of CyclinE1 was analyzed by real-time PCR and western blot, and the secretion and expression of extracellular matrix and TGF-β1 were detected by real-time PCR and ELISA.The software SPSS 21.0 was used to do the statistical analysis.@*Results@#The expression of SRSF2 was significantly higher in the keloid samples than in the normal skin samples. The percentage of SRSF2 positive cells in keloid was (77.04±4.37)% , while the percentage of SRSF2 positive cells in normal skin was (25.10±1.24)%. TGF-β1 promotes the expression of SRSF2 to (159.73±17.03)% times in keloid fibroblasts. After the SRSF2 knock-out, the apoptosis rate of the keloid fibroblasts increased, its cell cycle arrest was observed at the G0/G1 phase, and the expression of Cyclin E1 decreased. Apoptotic cells in control group was about (18.83±1.24)% , while (25.81±7.09)% and (26.71±6.14)% were in the two knock-out groups respectively. Cells in G0/G1 phase was about (58.97±1.73)% in control group, while (63.95±2.07)% and (64.65±3.23)% were in the two knock-out groups respectively. Compared to the control group, the expression of Cyclin E1 mRNA in the two knock-out groups were (31.60±6.81)% and (33.01±11.39)% respectively. Additionally, the expression of COL3A1 FN1 mRNA decreased, and the expression and secretion of TGF-β1 was declined. Compared to the control group, the expression of COL3A1 mRNA in the two knock-out groups were (64.90±23.71)% and (67.97±13.50)%, the expression of FN1 mRNA in the two knock-out groups were (59.10±8.11)% and (70.70±18.26)%, the expression of TGFB1 mRNA in the two knock-out groups were (53.37±15.51)% and (67.53±19.33)% respectively. The concentration of TGF-β1 in the medium of control group was (115.60±18.17)pg/ml, while (75.35±12.25) pg/ml and (72.06±14.66) pg/ml were in the two knock-out groups respectively.@*Conclusions@#The expression of SRSF2 increased in keloid. The inhibition of SRSF2 in keloid fibroblast resulted in the growth restriction and apoptosis of cells, and decreased the secretion of extracellular matrix and TGF-β1, which providing a new insight into keloid pathogenesis, and suggesting that SRSF2 could be a new therapeutic target for keloid conditions.
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Objective@#To investigate the tissue structure, chondrocyte characteristics, and the differential expression of related genes and cell surface markers of auricular cartilage of patients in different ages, in order to provide a basis for the age selection of tissue engineered cartilage repair defects.@*Methods@#The auricular cartilage tissue was obtained from 22 patients with microtia in the Plastic Surgery Hospital, Chinese Academy of Medical Science, ranged from 6 to 28 years old, and divided into the child group (6-12 years old), the adolescent group (13-18 years old) and the adult group (21-28 years old). The proliferation and differentiation features of chondrocytes which from different-aged patients were detected. Furthermore, quantitative real-time PCR was used to detect the differences in the expression of genes related to cell proliferation and chondrocyte extracellular matrix. Flow cytometry, immunofluorescence and immunohistochemistry were used to detect the differences in the expression of mesenchymal stem cell markers CD90, CD44, CD73 and CD105 in chondrocytes. SPSS Statistics 21.0 software was used to process statistics.@*Results@#The proliferation capability of auricular chondrocytes of children was stronger than adolescents and adults, the child group vs the adult group P<0.05, the child group vs the adolescent group P<0.01. The expression of cartilage extracellular matrix related gene COL2A1 increased with age, the child group vs the adult group P<0.01, the adolescent group vs the adult group P<0.01. While the capability of cell osteogenic differentiation decreased with age(P<0.05). However, there was no significant difference in the capability of adipogenic differentiation when considering the ages of patients. The results of both flow cytometry and real-time PCR showed that the expression of mesenchymal stem cell markers decreased with age, with the most significant decrease in CD90(P<0.01).@*Conclusions@#The biological characteristics and stem cell content of cells derived from auricular cartilage tissue was influenced by the patients′age.
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This paper summarizes the research progress of epidermal stem cells, mesenchymal stem cells, embryonic stem cells and induced pluripotent stem cell in the regeneration and repair of skin, bone and cartilage, and discusses the direction for future research on stem cells in plastic surgery.
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Objective@#To clarify the status of proliferation and differentiation of RCJ3.1C5.18 cells in chondrogenesis and explore insulin-like growth factor binding proteins (Igfbps) gene expression profile during the process. The results provided a research foundation for understanding the role of Igfbps in chondrogenesis.@*Methods@#Fetal rat derived mesenchymal chondrogenic cell line RCJ3.1C5.18 was used, which progresses spontaneously to differentiated growth plate chondrocytes. The proliferation and differentiation status of cells were determined by cell staining, real-time PCR and Muse™ Cell Analyzer at days 1, 4, 7 and 10 of culture respectively. Igfbps gene expression was assessed by real-time PCR.@*Results@#In the early period (1-4 days), cells proliferated rapidly and the expression of chondrocytes marker genes, such as typeⅡ collagen, SRY-box 9 and aggrecan increased gradually. Igfbp4 and Igfbp6 gene expression paralleled the expression of chondrocytes marker genes. On days 7-10 of culture, cells viability decreased and gradually developed hypertrophic-like and osteogenic-like characteristics, which was confirmed by specific staining and gene expression of alkaline phosphatase, matrix metallopeptidase 13.Igfbp1, Igfbp3 and Igfbp5 mRNA levels were up regulated gradually at this stage.@*Conclusions@#The status of proliferation and differentiation of cells are different in the process of chondrogenesis. Changes in Igfbp gene expression are associated with chondrocyte differentiation, suggesting that they have different role during chondrocyte proliferation and differentiation.
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Objective@#To treat the depressed scars by injecting nanofat and investigate its therapeutic effect.@*Methods@#Autologous fat was harvested from abdomen or thigh using low-pressure suction. The lipoaspirate was mechanically emulsified after rinsing. Emulsification of the fat was achieved by shifting the fat between two 5 ml syringes connected to each other by a three direct connector. After this emulsification process, the fatty liquid was again filtered over the sterile nylon cloth. Nanofat was injected into the dermis of depressed scars using a 26-gauge needle and the injection volume was 1-2 ml/cm2. After three months, another injection would be performed if the depressed scar remained obvious.@*Results@#From January 2016 to October 2017, eighteen patients and thirty-three depressed scars were treated. There was a temporary erythema of the injected area that lasted two to three weeks. The clinical result gradually improved over time and were maximal from three months postoperatively for most cases. Three months after nanofat injecting, the cavity of scars was significantly decreased; The color of scars were significantly improved and more close to the adjacent skin; The stiffness of scars was also obvious decreased. The follow-up ranged 4 months to 18 months and the average was 11.0±4.6 months. Seventeen patients were satisfied with the result, one patients was not satisfied and the satisfaction rate was 94%. No infections, fat cysts, granulomas, or other unwanted side effects were observed.@*Conclusions@#Nanofat injecting is a definite and effective treatment for depressed scars with fewer complications.
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Kinases are a class of enZymes that catalyZe the transfer of a phosphate group from a high-energy molecule to their substrates ( i.e., phosphorylation ).Kinase-induced intracellular phosphorylation plays important roles in a variety of cellular processes including metabolism, cell signaling, protein regulation, DNA replication and repair.Consequently, kinases have become potential biomarkers for disease diagnosis and drug discovery, and the development of simple and sensitive methods for kinase assay is highly desirable.In this review, we summariZe the recent advances in kinase assays with protein kinase A (PKA), casein kinase-2 ( CKⅡ) and T4 polynucleotide kinase ( T4 PNK) as the models.We focus on the newly emerging methods for kinase assays including fluorescent, single-molecule detection, colorimetric, chemiluminescent, bioluminescent, and electrochemical and photoelectrochemical methods.Furthermore, we give a new insight into the future direction of kinase assays.
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Objective To compare the expression of CD 49f splicing isoforms in different human stem cells and colon cancer cell lines , and construct lentiviral vectors overexpressing specific isoform .Methods The expression of CD49f isoforms in different cells was detected by RT-PCR and real-time PCR.The lentiviral vectors overexpressing CD49f isoforms were constructed by molecular cloning method .The overexpression of CD49f in colon cancer cell line HT29 was confirmed by FCM, Western blot and real-time PCR, the invasive ability was examined by transwell assay.Results CD49f splicing isoform B was highly expressed in H 9 human embryonic stem cell line , while iso-form A expressed in epithelial cells .Both isoform A and B were expressed in mesenchymal cells .CD49f isoforms A and B were also expressed in colorectal cancer cell lines , while HT29 and HCT116 showed higher expression of iso-form A than isoform B , HCT8 and LoVo showed higher expression of isoform B than isoform A .Overexpression of CD49f isoform B greatly increased the invasive ability of HT 29 cells, while isoform A showed no effect .Conclusions The expressions of CD49f splicing isoforms A and B in different types of cells are significantly different , which sug-gests that CD49f isoforms play different biological functions in cells .
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Cell biology is a highly experimental discipline, and is a discipline with theoretical knowledge closely combined with practical operation.Most of the medical graduate students should master the cell biology theory in the graduate stage, and get on a series of cell biology researches.Therefore, it is important to master the most basic cell culture techniques.As a technical instructor in the cell culture laboratory, I have been summarizing and optimizing the teaching content and teaching mode during the past few years.The aim of the teaching work is to train the students' scientific attitude of seeking truth from facts so as to develop good experimental habits and to master the basic skills of cell culture.
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Objective@#To compare the expressions of ALX gene family members (ALX1, ALX3, ALX4) in human embryonic stem cells (hESCs) and their differentiated neural crest cells (NCCs), as well as human mesenchymal stem cells (MSCs) and peripheral neurons derived from NCCs. The result provided a research foundation for understanding the expressions of ALX gene family in the process of NCCs differentiation.@*Methods@#The characteristics of hESCs, NCCs, MSCs and peripheral neurons were identified by RT-PCR, immunofluorescence staining and flow cytometry analysis. The expressions of ALX1, ALX3 and ALX4 were quantified by Real-time PCR.@*Results@#The expressions of ALX1 were significantly lower in MSCs than in NCCs (P < 0.05). However, the expression of ALX3 was significantly higher in MSCs than in hESCs, NCCs, peripheral neurons and osteoblasts (P < 0.05). The expressions of ALX4 were higher in MSCs, peripheral neurons and osteoblasts.@*Conclusions@#The expression profiles of different ALX genes in stem cells at different development stages are different, suggesting the regulation of ALX genes expressions is different in diverse cells.
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SUMMARY Thehumanembryonicstemcells(hESCs)serveasaself-renewable,genetically-healthy, pluripotent and single source of all body cells,tissues and organs.Therefore,it is considered as the good standard for all human stem cells by US,Europe and international authorities.In this study,the standard and healthy human mesenchymal progenitors,ligament tissues,cardiomyocytes,keratinocytes,primary neurons,fibroblasts,and salivary serous cells were differentiated from hESCs.The human cellular health-safety of NaF,retinoic acid,5-fluorouracil,dexamethasone,penicillin G,adriamycin,lead ace-tate PbAc,bisphenol A-biglycidyl methacrylate (Bis-GMA)were evaluated selectively on the standar-dized platforms of hESCs,hESCs-derived cardiomyocytes,keratinocytes,primary neurons,and fibro-blasts.The evaluations were compared with those on the currently most adopted cellular platforms.Parti-cularly,the sensitivity difference of PM2.5 toxicity on standardized and healthy hESCs derived fibroblasts, currently adopted immortalized human bronchial epithelial cells Beas-2B and human umbilical vein endo-thelial cells (HUVECs)were evaluated.The results showed that the standardized hESCs cellular plat-forms provided more sensitivity and accuracy for human cellular health-safety evaluation.
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<p><b>OBJECTIVE</b>To construct and characterize the TGF-β1, induced epithelial-mesenchymal transition (EMT) model of keloid epithelial cells in vitro, and to investigate the expression of epithelial stem cells related surface markers in keloid epithelial cells during EMT induction.</p><p><b>METHODS</b>The epithelial cells from 3 keloid samples of ears were cultured in vitro and induced by transforming growth factor betal (TGF-β1, 1 ng/ml) for 5 days, which was the experimental group, the same cells untreated were considered as the negative control group. The expressions of EMT-associated markers and regulative genes were detected using immunofluorescence staining, real-time PCR and western blot analysis. Then the surface markers of epithelial stem cells were detected using real-time PCR. Statistical significance was determined using Independent-Samples t Test, a p value less than 0. 05 was considered statistically significant.</p><p><b>RESULTS</b>The mRNA expression of transcription factor snail2 and mesenchymal-specific marker vimentin increased significantly in TGF-β1, induced keloid epithelial cells (P < 0. 05), in which snail2 increasing from 0. 91 ± 0. 23 to 1. 69 ± 0. 10, and vimentin from 5. 86 ± 2. 07 to 24. 29 ± 5. 39. Whereas the mRNA expression of epithelial-specific marker E-cadherin decreased from 1. 06 ± 0. 19 to 0. 65 ± 0. 09. The mRNA expression of CD29 and Lgr6, two surface markers of epithelial stem cells, significantly increased after induction of the TGF-β1, (P < 0. 05), from 0. 55 ± 0. 14 and 1. 61 ± 0. 31 to 1. 19 ± 0. 12 and 3. 84 t 0. 62 respectively. In induced cells, the immunofluorescence results showed staining of E- cadherin became faint, but the number of positive staining cells of vimentin increased. Western blot confirmed the protein expression of E-cadherin weakened, and the vimentin and p-Smad3 enhanced (P < 0. 05).</p><p><b>CONCLUSIONS</b>TGF-β1, initiated EMT in keloid epithelial cells by inducing the up-regulation of snail2, and TGF-β1,/Smad3 signaling pathway was involved in EMT. EMT could change the phenotype of epithelial stem cells in keloid.</p>
Subject(s)
Humans , Biomarkers , Metabolism , Cadherins , Genetics , Metabolism , Epithelial Cells , Physiology , Epithelial-Mesenchymal Transition , Physiology , In Vitro Techniques , Keloid , Pathology , RNA, Messenger , Metabolism , Signal Transduction , Smad3 Protein , Genetics , Metabolism , Snail Family Transcription Factors , Transcription Factors , Genetics , Metabolism , Transforming Growth Factor beta1 , Metabolism , Pharmacology , Up-Regulation , Vimentin , Genetics , MetabolismABSTRACT
Objective To observe the effectiveness of introducing Diazepam into combined aesthesia of Sumianxin and ketamine hydrochloride .Method A total of 80 rabbits of both genders for operation were randomly divided into A , B and C groups .The A group was injected with Sumianxin intramuscularly ( 0.3 mL/kg by weight ) .The B group was injected with Sumianxin and ketamine hydrochloride intramuscularly ( 0.3 mL/kg by weight ) .The C group was injected with Diazepam intravenously ( 1.5 mL/kg by weight ) combined with Sumianxin and ketamine hydrochloride injected intramuscularly (0.3 mL/kg by weight).The aesthetic effects, induction time, anesthesia maintaining time, total anaesthetic dose and operation time were observed , recorded and compared .Result The induction time of the C group was significantly shorter than A and B groups (P<0.01).The initial anesthesia maintaining time of the C group was the longest among the three (P<0.01) with least total anaesthetic dose (P<0.01).The operation time of the C group was the least with best aesthetic effects (P<0.01).Conclusion Introducing Diazepam into combined aesthesia of Sumianxin and ketamine hydrochloride can improve the aesthetic effects .Therefore , this is an optional aesthetic method for time-consuming animal operation or sensitive surgical sites of rabbits .
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<p><b>OBJECTIVE</b>To compare the tissue engineered cartilage constructed with chondrocytes derived from auricular and articular cartilage.</p><p><b>METHODS</b>Chondrocytes were isolated from porcine auricular and articular cartilage, and BMSCs were obtained from bone marrow aspirate and cultured. Each kind of chondrocytes were resuspended alone or mixed with BMSCs at a ratio of 1:1, and seeded onto PGA/PLA scaffolds to construct tissue engineered cartilage (n = 4). The constructs were cultured for 8 weeks in vitro and then subcutaneously implanted into nude mice for 6 weeks. The differences between chondrocytes monoculture from articular and auricular cartilage or between each of them co-cultured with BMSCs were evaluated by gross view, measurement of thickness and wet weight, histological examinations including H&E, Safranin O, type II collagen, and Ponceau's & Victoria blue staining, and gene expression analysis of cartilage related genes.</p><p><b>RESULTS</b>No obvious differences were found histologically among the complexes constructed in vitro 8 weeks except for few elastic fibers secreted in the auricular chondrocytes + BMSCs co-culture group. Neo-cartilage is thicker in the groups of articular chondrocytes (38. 1% than the group of auricular chondrocytes, P < 0.05) and articular chondrocytes + BMSCs co-culture (19.3% than the group of auricular chondrocytes + BMSCs, P < 0.05). However, after 6 weeks in vivo the elastic fibers were found positive in the complexes constructed by auricular chondrocytes, and its staining was even stronger and more homogenous in the group of auricular chondrocytes + BMSCs co-culture. The tissues generated by articular chondrocytes alone and co-cultured with BMSCs both formed the characteristic features of three-layer structure of hyaline cartilage and ossified in vivo with significant up-regulation of COL10A1 and MMP-13. To summarize, auricular chondrocytes formed the elastic cartilage while articular chondrocytes formed the hyaline cartilage during the development of tissue engineered cartilage either by monoculture or the co-culture with BMSCs.</p><p><b>CONCLUSIONS</b>The chondrogenic response of chondrocytes from different cartilage origins demonstrates that an initial chondrocyte and cartilage type recapitulates the same in later tissue-engineered development.</p>
Subject(s)
Animals , Bone Marrow Cells , Cell Biology , Cartilage, Articular , Cell Biology , Cells, Cultured , Chondrocytes , Coculture Techniques , Ear Auricle , Cell Biology , Mesenchymal Stem Cells , Cell Biology , Mice, Nude , Swine , Tissue Engineering , Methods , Tissue ScaffoldsABSTRACT
<p><b>OBJECTIVE</b>To analyze the influence of retinoic acid (RA) on the undifferentiated state and EB formation abilities of human embryonic stem cells.</p><p><b>METHODS</b>The biological characteristics of H9 ESCs after RA treatment were characterized by real-time PCR, MTS proliferation assay and immunofluorescence staining. The expression of three germ layers markers, osteogenic differentiation markers and adipogenic differentiation markers in H9-differentiated embryoid bodies (EBs) with RA treatment were quantified by real time PCR.</p><p><b>RESULTS</b>The proliferation of H9 ESCs in the early logarithmic growth phase was accelerated by RA treatment. In addition, RA induced differentiation of H9 ESC coupled with morphology changes, decreased expression of undifferentiated markers Oct4, Nanog, Sox2 and OCT4 mRNA binding protein Lin28 at mRNA level, and reduced expression of Oct4 at protein level. RA induced formation of cavities in EBs. Real time PCR results showed that the expressions of ectodermal markers: NeuroD1, Noggin; mesodermal markers: Brachyury, Twist and endodermal markers: AFP, GATA-4 were significantly increased (P < 0.05), especially for AFP (P < 0.01), by RA treatment in a dose-dependent manner. In addition, the expression of adipogenic differentiation marker C/EBPalpha was increased while the osteogenic differentiation marker OPN was decreased in EBs after RA treatment for 5 days.</p><p><b>CONCLUSIONS</b>High concentrations of RA induced the loss of stemness in H9 ESCs and excessive differentiation in EBs, and damaged the balance between osteogenic and adipogenic differentiation during early EB differentiation, which may be relevant to the congenital malformations.</p>
Subject(s)
Humans , Cell Differentiation , Cell Proliferation , Cells, Cultured , Embryonic Stem Cells , Cell Biology , Tretinoin , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of HE4 gene knockdown on the proliferation, adhesion and invasion of the ovarian cancer cells SKOV3.</p><p><b>METHODS</b>The knockdown of HE4 gene was performed by RNAi technology. The recombinant plasmids (pSUPER-HE4 shDNAs) were constructed and transfected into human ovarian cancer cells SKOV3. HE4 expression was then identified by real-time PCR and Western blot analysis. The invasion and adhesion ability of transduced cells were determined. In addition, cell proliferation and growth were analyzed by colonies formation assay.</p><p><b>RESULTS</b>Knockdown of HE4 was achieved, and further confirmed by real-time PCR and Western blot. The proliferation of HE4-down-regulated cells was not affected, but the invasion ability of the transfected cells was reduced (P < 0.05) and the adhesion ability was also reduced to 27.3%.</p><p><b>CONCLUSION</b>HE4 expression is down-regulated effectively by the constructed HE4 shDNA, and thus knockdown of HE4 inhibits the adhesion and invasion of SKOV3 cells.</p>
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Female , Humans , Cell Adhesion , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cystadenocarcinoma, Serous , Metabolism , Down-Regulation , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Ovarian Neoplasms , Genetics , Metabolism , Pathology , Plasmids , Proteins , Genetics , Metabolism , RNA Interference , RNA, Messenger , Metabolism , RNA, Small Interfering , Genetics , Recombinant Proteins , Genetics , Metabolism , TransfectionABSTRACT
Objective To investigate the effect of tourniquet compression on axonal transport in sciatic nerve of rats.Methods Twenty-four 12-week old male SD rats weighing 250-300 g were randomly divided into 4groups according to the duration of tourniquet compression(n=6 each):1,2,4 and 12 h.The tourniquet was applied to the middle 1/3 of thigh.In each animal whether the left or right thigh was compressed was determined by a flip of coin.The tourniquet was released for 10 min after every hour of compression.A 3-cm segment of sciatic nerve was removed at the end of tourniquet compression(1.5 cm proximal and 1.5 cm distal to the site of compression).Immuno-histochemistry was used to measure the expression of insulin-like growth factor-1(IGF-1)in the sciatic nerve.The ratio of average optic density of the compressed sciatic nerve to that of control was used to estimate the degree of IGF-1 accumulation.The regression equation of the interaction between the duration of compression and accumulation of IGF-1 was analyzed.Results There was significant accumulation of IGF-1 in the sciatic nerve proximal to the compressed site.The accumulation increased with the duration of compression.There was no significant accumulation of IGF-1 in the sciatic nerve distal to the compressed site.The regression equation of the interaction between the duration of compression(X)and accumulation of IGF-1(Y)was Y=0.422X+0.887.Conclusion Tourniquet compression of sciatic nerve can inhibit axonal transport.The accumulation increases with the duration of compression.
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BACKGROUND: Classic isolation method of bone marrow mesenchymal stem cells (BMSCs) is Percoll density gradient centrifugation. Blood cell component was removed. However, this method is complicated. Preparation density was needed when isolating dog bone marrow. Moreover, centrifugation was frequent, which had a great damage to cells. OBJECTIVE: To establish methods of the isolation, proliferation, culture and osteoinduction of canine BMSCs, and observe the in vitro proliferation and ability to osteoinduction differentiation. METHODS: 10 mL bone marrow was extracted from dog posterior superior iliac spine, heparin anticoagulation, diluted using Hanks juice, treated with 1.077 g/mL Ficoll solution 3 mL, and centrifuged at 2 000 r/min for 20 minutes. Karyocytes were absorbed to form white cloudlike layering interface, and then centrifuged twice using DMEM supplemented with fetal bovine serum, incubated at 12×10~4/cm~2 at 37 ℃ in a 5% CO_2 incubator. Following subculture, cells were incubated in DMEM containing dexamethasone, β-sodium phosphoglycerol and ascorbic acid 2-phosphate. Immunocytochemical staining and immunofluorescence staining were utilized to detect osteocalcin, osteopontin and type Ⅰ collagen expression in osteoblasts. Alkaline phosphatase staining and alizarin red staining were performed. RESULTS AND CONCLUSION: 1.077 g/mL Ficoll density gradient centrifugation was used to isolate karyocytes that were significant compared with Percoll solution. Obtained BMSCs had high purity, good growth and the mean doubling time was 24 hours. Following in vitro osteogenic incubation of dog BMSCs, osteocalcin, osteopontin and type Ⅰ collagen showed positive expression. Alkaline phosphatase staining demonstrated bluish-green cytoplasm. Alizarin red staining showed red nodes in extracellular matrix, and could differentiate into osteoblasts in vitro.