Objective To investigate the myogenic differentiation of mesenchymal stem cells (MSCs) after being transplanted into the local muscle tissues. Methods The serious muscleinjured model was established by the way of radiation injury, incising, and freezing injury in 36 mouses. Purified MSCs derived from bone marrow of male mouse and MSCs induced by5-azacytidine(5-Aza-CR) were transplanted into the local of normal muscle tissues and injured muscle tissues of femal mouse. The quantity of MSCs and the myogenic differentiation of implanted MSCs were detected by the method of double labeling, which included fluorescence in situ DNA hybridization (FISH) and immuno-histochemistry on the 1st, 3rd, 6th, 9th, 12th, and 15th day after transplantation. Results The quantity of implanted MSCs decreased as timepassed. MSCs’ differentiation into myoblasts and positive expression of desmin were observed on the 15th day in purified MSCs group and on the 6th day in induced MSCs groups. Conclusion MSCs could differentiate into myoblasts after being implanted into the local of muscle tissues. The differentiationoccurs earlier in the induced MSCs group than that in purified MSCs group.
Retinal degeneration mainly include age-related macular degeneration, retinitispigmentosa and Stargardt’s disease. Although its expression is slightly different, its pathogenesis is photoreceptor cells and/or retinal pigment epithelial (RPE) cel1 damage or degeneration. Because of the 1ack of self-repairing and renewal of retinal photoreceptor cells and RPE cells, cell replacement therapy is one of the most effective methods for treating such diseases.The stem cells currently used for the treatment of retinal degeneration include embryonicstem cells (ESC) and various adult stem cells, such as retinal stem cells (RSC), induced pluripotent stem cells (iPSC). and mesenchyma1 stem cells (MSC). Understanding the currentbasic and clinical application progress of ESC, iPSC, RSC, MSC can provide a new idea for the treatment of retinal degeneration.
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 investigate the feasibility of differentiation of the marrow mesenchymal stem cells (MSCs) into the cells of the skin appendages andthe mechanism of their involvement in the wound healing. Methods The bone marrow was collected from Wistar rats by the flushing of the femurs, MSCs were isolated and purified by the density gradient centrifugation. Then, the MSCs were amplified and labelled with 5-bromo-2′-deoxyuridine (BrdU). The full-thickness skin wounds with an area of 1 cm×1 cm were made on the midback of the homogeneous male Wistar rats. At the same time, 1×106/ml BrdU-labelled MSCs were infused from thepenile vein. The specimens were harvested from the wound tissues on the 3rd dayand the 7th day after operation and were immunohistochemically stained by either BrdU or BrdU and pan-keratin. Results The BrdU positive cells appeared in thehypodermia, the sebaceous glands, and the hair follicles of the wounds, as wellas the medullary canal of the femurs. The double-staining showed the BrdU positive cells in the sebaceous glands and the hair follicles of the wounds expressedpan-keratin simultaneously. Conclusion During the course of the wound healing, MSCs are involved in the wound repair and can differentiate into the cells ofthe skin appendages under the microenvironment of the wound.
ObjectiveTo study immunodepression effect of bone marrow-derived mesenchymal stem cell (BMSC) on acute asthmatic airway inflammation by galectin-1 (gal-1) in vivo.MethodsEighty-five female BALB/c mice were equally randomized into normal control group, asthmatic group, BMSC treatment group, gal-1 treatment group and BMSC and gal-1 inhibitor group. Ovalbumin (OVA) was used to establish acute asthmatic model. Total cell number and differential cell analysis in each group in bronchoalveolar lavage fluid (BALF) were determined. Furthermore, hematoxylin-eosin and periodic-acid Schiff staining was used to compare airway inflammation among five groups. Measurement of cytokines, including interleukin (IL) -4, IL-5 and gal-1 in BALF and OVA specific IgE (OVA-IgE) in serum were evaluated by enzyme linked immunosorbent assay. Moreover, dendritic cell (DC) in lung tissue was sorted by immunomagnetic beads and its MAPK signal pathway was analyzed by western blotting among five groups.ResultsAccumulation of inflammation cells, particularly eosinophils around airway and in BALF was evident in asthmatic mouse model, meanwhile hyperplasia of Goblet cell was also obvious in asthmatic group. BMSC engraftment or gal-1 infusion significantly reduced airway inflammation and hyperplasia of Goblet cell and the number of inflammation cells in BALF, especially eosinophils attenuated dramatically. However, there was no effect on airway inflammation and hyperplasia of Goblet Cell by simultaneous infusion BMSC engraftment and gal-1 inhibitor. Compared to normal control group, the level of IL-4, IL-5 in BALF and OVA-IgE in serum was increased remarkably in asthmatic group, but the level of gal-1 reduced obviously. Moreover, infusion of BMSC or gal-1 could mitigate the level of IL-4, IL-5 in BALF and OVA-IgE in serum and increase the level of gal-1 in asthmatic mouse. However, infusion with both BMSC and gal-1 inhibitor exerted no effect on cytokine and OVA-IgE in asthmatic mouse. DC was sorted by immunomagnetic beads and western blotting was used to detect the expression of MAPK signal pathway among five groups. The expression of ERK phosphorylation in asthmatic group was much lower than that in normal control group. On the contrary, the expression of p38 phosphorylation was much higher than that in normal control group. BMSC engraftment or gal-1 infusion significantly activated the ERK pathway and inhibited the p38 MARP pathway on asthmatic mouse DC. Nevertheless, the expression of ERK phosphorylation and p38 phosphorylation for group with BMSC and gal-1 inhibitor infusion was between the level of asthmatic group and normal control group.ConclusionsBMSC infusion alleviates airway inflammation in asthmatic mouse, especially weakens eosinophils infiltration, and the underlying mechanism might be protective effect of gal-1 secreted by BMSC which plays a role in lung tissue DC and regulates the DC expression of MAPK signal pathway.
Objective To monitor the stem cell migration into the bone defect following an injection of the labeled mesenchymal stem cells (MSCs) by the enha nced green fluorescent protein (EGFP)technology and to provide insights into an application of MSCs for the fracture healing. Methods Isolated MSCs from the rabbit femur marrow were culture-expanded and were labeled by the transfection with the recombinant retrovirus containing the EGFP gene. Then, some labeled MSCs were cultured under the osteogenic differentiation condition and the phenotype was examined. After the fracture of their bilateral ulna, 18 rabbits were divide d into two groups. The labeled MSCs were injected into the aural vein at 1×107 cells／kg in the experimental group and the unmarked MSCs were injected in the control group 24 hours before surgery, and 1 and 24 hours after surgery, res pectively. Necropsies were performed 2 days after surgery in the two groups. The sections from the left defects were observed under the fluorescence microscope and the others were analyzed by the bright-field microscopy after the HE staining. Results The EGFP did not affect the MSCs viability. After the labeled cells were incubated in the osteogenic medium alkaline phosphatase, the calcium nodule s were observed. All the rabbits survived. The tissue of haematoma was observed in the bone defects and the fluorescent cells were found in the experimental gr oup, but no fluorescent cells existed in the control group. Conclusion The EG FP labeled MSCs can undergo osteogenic differentiation in vitro and can mig rate into bone defects after their being injected into the peripheral vein.
Objective To explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.
ObjectiveTo investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene. MethodsThe adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs.The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A),pcDNA3.1 empty vector transfection (group B),and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C),respectively.At 48 hours after transfection,the cells in groups B and C were selected with G418.The cell morphology changes were observed under the inverted microscope.Pax6 protein and level of corneal epithelial cells specific molecular-cytokeratin 12 (CK-12) were measured by Western blot.Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12. ResultsNo morphology change was observed in groups A and B.Two different cell clones were found in group C.No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells;No.2 selected clone showed a net-like appearance,with 3-7 cell processes.The Western blot results showed the Pax6 protein expression in 2 clones of group C,but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C,and no expression in the others.The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64±0.73,which was significantly higher than that of No.2 selected clone of group C (0.55±0.42),group B (1.36±0.40),and group A (1.00±0.00) (P<0.05),and there was no significant difference among groups A,B and No.2 selected clone of group C (P>0.05). ConclusionPax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels.This result provides a promising strategy of generating corneal epithelilcm-like cells for construction of tissue engineered cornea.
Objective To analyze the advances of cancer stem cell (CSC) in recent years, and to propose a prospect for CSC research and cancer therapy. Methods Articles about important advances of CSC theory and cancer therapy were reviewed, and then selected and summarized. Results In 2001, CSC was first put forward as a concept, till now, which has been confirmed in many tissues. In recent years, efforts were dedicated to such topics including: identification of CSC in sol id tumors, the origin of CSC, its niche and growth mechanism, cancer therapy, etc. According to the CSC theory, traditional therapeutic methods have deficiencies, and new treatment targeting CSC may thoroughly el iminate tumors. Conclusion At present, CSC theory is still controversial, while it proposed revolutionary methods and directions for the therapy of cancer.
Objective To review the advances in repair of spinal cord injury by transplantation of marrow mesenchymal stem cells(MSCs). Methods The related articles in recent years were extensively reviewed,the biological characteristic of MSCs,the experimental and clinical studies on repair of spinal cord injury by transplantation of MSCs,the machanisms of immigration and therapy and the problems were discussed and analysed. Results The experimental and clinical studies demonstrated that the great advances was made in repair of spinal cord injury by transplantation of MSCs. After transplantation, MSCs could immigrate to the position of spinal cord injury, and differentiate into nervelike cells and secrete neurotrophic factors.So it could promote repair of injuryed spinal cord and recovery of neurologicalfunction. Conclusion Transplantation of MSCs was one of effective ways in repair of spinal cord injury, but many problems remain to be resolved.