Objective To study the effect of core-binding factor α1(Cbfa1)on the mesenchymal stem cells(MSCs) osteoblastic differentiation.Methods The MSCs were isolated from Japan white rabbits and cultured in vitro. The 3rd generation MSCs were infected with Cbfa1 recombinant adenovirus. The expression of Cbfa1 was detected by immunofluorescence after being infected for 3 days and the proliferation was estimated by MTT method from the 1st day to the 7th day. Then the MSCs were divided into four groups: the commonly cultured group, the simply induced group, the control adenovirus treatment group, and the Cbfa1 adenovirus treatment group. The expressions of mRNA for a various of osteoblast gene markers such as alkaline phosphatase, osteocalcin, osteopontin and type I collagen were analyzed based on reverse transcriptase polymerase chain reaction (RT-PCR). The change of adipose and myoblastic differentiation gene marker PPARγ2 and MyoD expression were detected by RT-PCR respectively.Results Positive staining of Cbfa1 was found in the MSCs infected with Cbfa1 adenovirus, and there was no significant difference in cell proliferation among the experimental groups（Pgt;0.05）. The RT-PCR indicated that all the osteoblast gene markers except type I collagen were up-regulated in the Cbfa1 adenovirus treatment group. In contrast, the expressions of PPARγ2 and MyoD were restrained. Conclusion Cbfa1 can directly promote the differentiation of MSCs into osteoblasts.
Objective To explore the method that can inducethe mesenchymal stem cells (MSCs) to differentiate into the neuronlike cells in vitro．Methods The neuron-like cells were isolated froman SD rat (age, 3 months; weight, 200 g). They underwent a primary culture; theinduced liquid supernatant was collected, and was identified by the cell immunohistochemistry. The C3H1OT1/2 cells were cultured, as an MSCs model, and they were induced into differentiation by β-mercaptoethanol (Group A) and by the liquid supernatant of the neuron-like primary cells (Group B), respectively. The cells were cultured without any induction were used as a control (Group C). Immunohistochemistrywas used to identify the type of the cells． Results The result of the immunochemistry showed that the cells undergoing the primary culture expressed the neurofilament protein (NF) and the neuronspecific enolase (NSE), and they were neuron-like cells. β-mercaptoethanol could induce the C3H1OT1/2 cells toexpress NF and NSE at 2 h, and the expression intensity increased at 5 h. The liquid supernatant of the primarily-cultured neuron-like cells could induce theC3H1OT1/2 cells to express NF and NSE at 1 d, but the expression intensity induced by the liquid supernatant was weaker than that induced by β-mercaptoethanol. The positivity rate and the intensity expression of NSE were higher than those of NF. Conclusion MSCs can differentiate into the neuron-like cells by β-mercaptoethanol and the microenvironment humoral factor, which can pave the way for a further study of the differentiation of MSCs and the effectof the differentiation on the brain trauma repair.
Objective To explore the effects of bone marrow mesenchymal stem cells ( BMMSCs) on pulmonary fibroblasts of patients with nonspecific interstitial pneumonitis ( NSIP) , and investigate the therapeutic mechanism of BMMSCs for interstitial pulmonary fibrosis. Methods Human BMMSCs, human pulmonry fibroblasts ( HPFs) from NSIP patients, and normal HPFs were primary cultured in vitro. Then HPFs fromNSIP patients were co-cultured with BMMSCs or normal HPFs using Transwell co-culture system. After 24 hours, levels of transforming growth factor β1 (TGF-β1) and interferon inducible protein 10 ( IP-10) in culture supernatants were detected by ELISA method. Meanwhile, interleukin-6 ( IL-6) , IL-8, and monocyte chemotactic protein-1 ( MCP-1) in co-culture supernatants were detected by liquid chip. After co-cultured for 48 hours, total protein of HPFs was extracted and the expression level of alpha smooth muscle actin ( α-SMA) secreted by HPFs were detected by Western blot.Results HPFs from NSIP patients secreted higher level of IL-6, IL-8, and MCP-1 than normal HPFs, and secreted high level of α-SMA. In the Transwell co-culture system, human BMMSCs significantly reduced the levels of IL-6, IL-8, and MCP-1 secreted from HPFs of NSIP patients, and reduced the high expression of α-SMA in HPFs of NSIP patients. Conclusion Human BMMSCs can significantly reduce the secretion of IL-6, IL-8, MCP-1, and the expression of α-SMA in HPFs from NSIP patients.
Objective To establish a method of isolating and culturing adult human bloodderived mesenchymal stem cells(MSCs) and to investigate their osteogenic potential in vitro. Methods Thirty peripheral blood sampleswere collected from 30adult volunteers(15 ml per person).Adult human MSCs derived from peripheral blood were isolated from the lymphocyte separation fluid fraction of mononuclear cells, cultured in α-Modified Eagle’s Medium with low glucose containing 20% fetal bovine serum, and proliferated through a process of subculturing. The phenotype of MSCs was analyzed with flow cytometry. For in vitro osteogenic differentiation, MSCs from the second passage grew in the presence of osteogenic supplements (100 nmol/L dexamethasone,10 mmol/L β-glycerophosphate,50 μmol/L vitamin C, and 10 nmol/L 1,25-2-hydroxide vitamin D3). In the fifth passage cells, the activity of alkaline phosphatase, the expression level of collagen typeI, osteocalcin and osteonectin were determined. And the calcium tubercle formation would be examined after the continual one-month culture of the fifth passage. Results MSCs exsited in the pheripheral blood of adult human. And the clone forming efficiency of blood-derived MSCs was 0.27±0.22/106 mononuclear cells. The MSCs expressed CD44,CD54,CD105,and CD166，but did not CD14, CD34, CD45,and CD31.Under the function of osteogenic supplements, the MSCs were found to be higher activity of alkaline phosphatase and higher expression levels of collagen type Ⅰ, osteocalcin and osteonectin. And the calcium tubercle formation was examined throughtetracycline fluorescence labeling method. Conclusion The isolation and cultureconditions established for adult human MSCs may select a distinct population of peripheral blood-derived adherent cells. Adult human blood-derived MSCs possess osteogenic potential in vitro, and may be used as seed cells for bone tissue engineering.
Objective Bone marrow mesenchymal stem cells （MSCs） have been suggested to play an important role in the treatment of a variety of pulmonary diseases. The present study was aimed at evaluating the therapeutical effect of MSCs transplantation on emphysematous rats，and explore its influence in local and systemic inflammation. Methods Emphysema rat model was established by cigarette smoking. MSCs were transfected with lentivirus vector carrying green fluorecent protein （GFP） and the transfected MSCs in lung of smoke rats were detected by imaging system for small animals. Thirty-six SD rats were randomly divided into a control group，an emphysema group，and a MSCs transplantation group. The total and differential cell counts in bronchoalveolar lavage fluid （BALF） were measured. TNF-α and IL-1β levels in BALF and serum were measured by ELISA. Malonaldehyde （MDA） level in lung tissue was detected by chromatometry. Emphysema changes were evaluated by mean linear intercept （MLI） of lung under light microscope by HE staining. Results The transfected MSC in different lung lobes were found to be alive at four weeks after intrapulmonary delivery. Compared with the emphysema group，the total cell count in BALF，TNF-α and IL-1β levels in BALF and serum，MDA level in lung tissue and MLI were significantly reduced intheMSCs transplantation group（Plt;0.01）. Conclusions Transplantation of MSCs can mediate down-regulation of TNF-α and IL-1β in BALF and serum，attenuate inflammation，oxidative stress and emphysema change of lung，suggesting that MSCs have significant therapeutic effects on emphysema.
Objective To explore the in vitrodifferentiation of the rat mesenchymal stem cells (MSCs ) into the skeletal muscle cells induced by the myoblast differentiation factor (MyoD) and 5-azacytidine. Methods The MSCs were taken from the rat bone marrow and the suspension of MSCs was made and cultured in the homeothermia incubator which contained 5% CO2at 37℃. The cells were observed under the inverted phase contrast microscope daily. The cells spreading all the bottom of the culture bottle were defined as onepassage. The differentiation of the 3rd passage of MSCs was induced by the combination of 5-azacytidine, MyoD, transforming growth factor β1, and the insulin like growth factor 1. Nine days after the induction, the induced MSCs were collected, which were analyzed with the MTT chromatometry, theflow cytometry, and the immunohistochemistry. Results The primarily cultured MSCs grew as a colony on the walls of the culture bottle; after the culture for 5-7 days, the cells were shaped like the fibroblasts, the big flat polygonal cells, the medium sized polygonal cells, and the small triangle cells; after the culture for 12 days, the cells were found to be fused, spreadingall over the bottle bottom, but MSCs were unchanged too much in shape. After the induction by 5-azacytidine, some of the cells died, and the cells grew slowly. However, after the culture for 7 days, the cells grew remarkably, the cell volume increased gradually in a form of ellipse, fusiform or irregularity. After theculture for 14 days, the proliferated fusiform cells began to increase in a great amount. After the culture for 18-22 days, the myotubes increased in number and volume, with the nucleus increased in number, and the newly formed myotubes and the fusiform myoblst grew parallelly and separately. The immunohistochemistry for MSCs revealed that CD44 was positive in reaction, with the cytoplasm ina form of brown granules. And the nucleus had an obvious border，and CD34 was negative. The induced MSCs were found to be positive for desmin and specific myoglobulin of the skeletal muscle. The flow cytometry showed that most of the MSCs and the induced MSCs were in the stages of G0/G1，accounting for 79.4％ and 62.9％，respectively; however, the cells in the stages of G2/S accounted for 20.6％ and 36.1％. The growth curve was drawn based on MTT，which showed that MSCs weregreater in the growth speed than the induced MSCs. The two kinds of cells did not reach the platform stage，having a tendency to continuously proliferate.ConclusionIn vitro,the rat MSCs can be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine, with a positive reaction for the desmin and the myoglobulin of the skeletal muscle. After the induction, the proliferation stage of MSCs can be increased, with a higher degree of the differentiation into the skeletal muscle.
Objective Toreview theresearch progress of nucleus pulposus cells phenot ypic markers. Methods The domestic and international l iterature about nucleus pulposus cells phenotypic markers was reviewed extensively and summarized. Results Due to different biomechanical properties，nucleus pulposus cells and articular chondrocytes have differences in morphology and extracellular components such as the ratio of aggrecan to collagen type II α1. Nucleus pulposus cells can be identified by surface marker (CD24), gene markers (hypoxia inducible factor 1α, glucosetransporter protein 1, matrix metalloproteinase 2, vascular endothel ial growth factor A, etc), and various markers (keratin 19 and glypican 3，paired box 1, forkhead box F1 and integrin-binding sialoprotein, etc). Conclusion Nucleus pulposus cells and articular chondrocytes have different phenotypic markers, but nucleus pulposus cells are still lack of specific markers.
ObjectiveTo observe the effects of exosomes derived from rat mesenchymal stem cells (MSC-exosomes) on the rat experimental autoimmune uveitis (EAU) model.MethodsTwelve Lewis rats were randomly divided into experimental group and control group by random number table, with 6 rats in each group. Rats in the experimental group were established with EAU model, 100 μl of MSC-exosomes (50 μg) were periocular injected on the 9th day after modeling while the control rats were injected with the same volume of phosphate buffer. At different time points after modeling, the retinal structure was observed by hematoxylin and eosin (HE) staining, and the clinical and pathological manifestations were evaluated. T cells from the two groups were analyzed by flow cytometry. Immunohistochemical staining was used to observe the expression of macrophage surface marker CD68. The effect of MSC-exosomes on T cells was measured by lymphocyte proliferation assays. And flow cytometry was used to detect Th1, Th17 and regulatory T cells Variety. Electroretinogram (ERG) was used to evaluate the retinal function. Data were compared between the two groups using the t test.ResultsHE staining showed that the retina structure of the experimental group was more complete than that of the control group on the 15th day after modeling. Immunohistochemical staining showed that the positive expression of CD68 in the experimental group was significantly less than that in the control group. On the 15th day after modeling, the retinal pathological score of the experimental group was lower than that of the control group. On the 9th to 13th day after modeling, compared to the control group, the average clinical scores of the retina in the experimental group were lower, and the difference was statistically significant (t=3.665, 3.21, 3.181, 4.121, 3.227; P＜0.01). The results of T cell proliferation assay showed that exosomes (1.0, 10.0 μg/ml) inhibited the proliferation of T cells under different concentrations of R16 (1, 10, 30 μg/ml), and the difference was statistically significant (F=11.630, 4.188, 6.011; P＜0.05). The results of flow cytometry showed that the number of Th1, Th17 and Treg cell subsets in the experimental group was decreased compared with the control group, and the difference was statistically significant (t=7.374, 4.525, 6.910; P＜0.01). There was no difference in the proportion of cells in the T cells and lymph nodes (t=1.126, 0.493, 0.178; P=0.286, 0.632, 0.862). The results of ERG showed that, compared with the control group, the amplitudes of 0.01, 3.0 cd/m2 a wave and b wave of the experiment group were all increased on the 15th day after modeling, and the differences were statistically significant (t=3.604, 4.178, 4.551, 2.566, P＜0.05).ConclusionsMSC-exosomes can reduce the clinical and pathological manifestations of EAU, protect retinal function, reduce ocular macrophage infiltration, down-regulate the proportion of inflammatory cells in the eye, and inhibit T cell proliferation.
OBJECTIVE: From the point of view of material science, the methods of tissue repair and defect reconstruct were discussed, including mesenchymal stem cells (MSCs), growth factors, gene therapy and tissue engineered tissue. METHODS: The advances in tissue engineering technologies were introduced based on the recent literature. RESULTS: Tissue engineering should solve the design and preparation of molecular scaffold, tissue vascularization and dynamic culture of cell on the scaffolds in vitro. CONCLUSION: Biomaterials play an important role in the tissue engineering. They can be used as the matrices of MSCs, the delivery carrier of growth factor, the culture scaffold of cell in bioreactors and delivery carrier of gene encoding growth factors.
ObjectiveTo comprehensively analyze the recent advancements in the field of mesenchymal stem cells (MSCs) aging,and summary its achievements and its difficulty at the present. MethodsThe literature about MSCs aging was reviewed and analyzed. ResultsInducible telomerase reactivation of MSCs is successful to extend the life span of senescent cells,but it also has potential safety hazard.The age range presented in the research of age-related cell senescence is inconsistent,resulting in different outcomes.Many ways to improve cell in vitro culture conditions will help delay aging.Recent research indicates that oxidative stress theory is seemed to not completely explain cell aging. ConclusionFurther research of MSCs aging mechanism will help the tissue engineering transform to clinical application.