- [Show abstract] [Hide abstract] ABSTRACT: The objective of this study was to investigate the expression of exogenous hPDGF-A and hBD(2) in gene-modified bone marrow mesenchymal stem cells (BM-MSCs) in vitro and in vivo. Recombinant adenovirus vector expressing hPDGF-A/hBD(2) genes was constructed and packaged into virion. Primary isolated and cultured BM-MSCs were transfected by using hPDGF-A hBD(2), then the expressions of exogenous hPDGF-A/hBD(2) were detected by immunocytochemical staining in vitro. The conditioned medium (serum-free cultured supernatant of BM-MSCs transfected with recombinant adenovirus) collected from gene-modified BM-MSCs was applied to scratch wound on monolayer cells of multipotential cell line 10T1/2 in order to confirm the stimulative effect of hPDGF-A on cell migration. Gene-modified BM-MSCs were topically transplanted on wound of rats with radiation and skin excision combined injury. The distribution of BM-MSCs and expression of hPDGF-A/hBD(2) on the wound was observed by fluorescent microscopy and immunohistochemical staining respectively. The results indicated that the rat BM-MSCs transfected with recombinant adenovirus could express the EGFP in vitro. The immunofluorescent cytochemistry assay showed that the gene-modified BM-MSCs expressed the hPDGF-A and hBD(2). The scratch test confirmed that the percentage of healing area of wound in cultured supernatant group of gene-modified BM-MSCs was significant higher than that in control group on 8, 12, 24 and 48 hours (p < 0.05). The fluorescence microscopy of exogenous gene-modified BM-MSCs transplanted on wound revealed that the gene-modified BM-MSCs could higher express exogenous genes of EGFP at least within 2 weeks. The immunohistochemistry staining of wound indicated that the expression of exogenous genes began from day 3, reached to peak on day 7, and still visible on day 21 even though the expression became weak because of the possible dilution of the exogenous genes during cell division. It is concluded that efficient expression of exogenous hPDGF-A/hBD(2) in gene-modified BM-MSCs are demonstrated both in vitro and in vivo, which suggests that the molecular mechanism underlying chronic wound-healing accelerated by the strategy combining cell therapy with gene therapy.
- [Show abstract] [Hide abstract] ABSTRACT: To explore the effect of calpain inhibitor I(CI-I) on ikappaBalpha expression and cytokine secretion in RAW264.7 cells after LPS attack. RAW264.7 cells were pretreated with CI-I for 1 h and attacked with LPS. The ikappaBalpha expression and TNF-alpha and IL-6 secretion were detected by Western blot and ELISA, respectively. CI-I inhibited the decrease of ikappaBalpha expression in RAW264.7 cells treated with LPS. The secretion of both TNF-alpha and IL-6 secretion was notably increased after RAW264.7 cells were treated with LPS for 4 h or 8 h. CI-I and dexamethasone(DEX) inhibited these effects, and the combination of DEX and CI-I had synergistic effect. CI-I and DEX can inhibit the decrease of ikappaBalpha expression and prevent TNF-alpha and IL-6 secretion in RAW264.7 cells attacked with LPS, which contributes to the alleviation of cellular injury.
- [Show abstract] [Hide abstract] ABSTRACT: To observe the effects of burn serum on the erythropoiesis and granulopoiesis in bone marrow in mice, and to explore the possible underlying mechanism. Murine bone marrow cell (BMC) strain was prepared routinely and was employed in the establishment of the culture system of colony forming units of erythrocytes, or granulocytes and monocytes. To both sets of culture system normal murine serum (N group) and burn serum, which was collected from the mice with 15% full thickness burn at 12 postburn hours (PBH) and 1, 3, 5, 7 and 10 postburn days (PBD), (burn serum group) was added. In addition, positive control and blank control groups were set accordingly. The stimulating activity of all kinds of sera on the BMCs in the two sets of culture system was determined. The changes in the burn serum concentrations of EPO and GM-CSF were detected by radioimmunoassay, and the data were analyzed by logarithmic linear fitting correlation with the former influence of burn sera on the erythrocytes and granulocytes. (1) Burn sera exhibited obvious stimulation promoting activity on the erythropoiesis and granulopoiesis in BMC, and the activity peaked (384 +/- 60 and 127 +/- 16 CFU) on 1 PBD and decreased thereafter to approach the values found in normal sera group (125 +/- 14 and 34 +/- 20 CFU) on 7 PBD. (2) The EPO content in burn serum was evidently higher than the normal value (P < 0.01) during 12 PBH to 7PBD period. The GM-CSF concentration was obviously higher than the normal value (P < 0.05) at 12 PBH and on 1 PBD. (3) The EPO concentration in burn serum was significantly and logarithmically correlated with the stimulation promoting activity of burn serum on erythropoiesis (r = 0.8570, P = 0.0137). But the GM-CSF concentration in culture with burn serum was not correlated with the stimulation promoting activity of burn serum on granulopoiesis (r = 0.7049, P > 0.05). The sera harvested from burned mice during early postburn stage exhibited strong stimulation promoting activity on the erythropoiesis and granulopoiesis in bone marrow. The increased EPO level in burn serum might be the important factor contributing strong stimulation action on erythropoiesis, while increased GM-CSF level was not.
- [Show abstract] [Hide abstract] ABSTRACT: Inducing mesenchymal stem cells (MSCs) differentiate to myoblasts with 5-azacytidine(5-Aza-CR), investigating the expression of Myf5 and the role of the signal transduction case of p38 in all the course of differentiation. Seperating and purifying bone marrow-derived MSCs, inducing MSCs differentiation to myoblasts with 10 micromol/L 5-Aza-CR, assaying the gene expression time of Myf5 with RT-PCR method, the antigen expression of myosion with immunohistochemistray method and observing the changes of the activity of phosphorylation p38 before and after inhibited by SB203580 with Western-blot method. MSCs begin to express Myf5 delaied to the 9th day after inhibited by SB203580. Some of MSCs express myosion at the 7th day after induced; The phosphorylation p38 activity of MSCs enhanced after induced by 5-Aza-CR but obviously decreased after inhibited by SB203580. MSCs can express myogenic regulator factors and orientation differentiate to myoblasts after induced by 5-Aza-CR, p38 really have a positive signal transduction effection in this course.
- [Show abstract] [Hide abstract] ABSTRACT: To explore the effects of bone marrow cells expanded under different conditions on hematopoietic reconstitution, in the liquid expanded cultural system with several cytokines and/or bone marrow stromal cell layers, the BMMNC of mice were expanded for 5 days. Then the expanded cells were transplanted into the lethal-dose irradiated mice via the caudal vein. The hematopoietic reconstitution of transplanted mice were evaluated by detecting the number of bone marrow nuclear cells and various colony forming cells. The results showed that ex vivo expansion of bone marrow mononuclear cells mediated with cytokines under cultural conditions could not improve the hematopoietic engraftment in post-irradiated mice, but the expansion supported by bone marrow stromal cells could benefit the reconstruction significantly regardless of addition with cytokines. In conclusion, the ex vivo hematopoietic cell expansion supported by bone marrow stromal cells can maintain the properties of the hematopoietic stem/progenitor cells for hematopoietic reconstitution.
- [Show abstract] [Hide abstract] ABSTRACT: AIM: To probe into the possibility of dendritic cells (DC) in preventing solid carcinomas such as gastric cancer (GC). METHODS: Immunotherapy was proformed by DC sensitized by mRNA of gastric cancer cells. RESULTS: The indices such as the production rate of ascites of mice (75%), the metastasis rate of tumor (25%), the survival rate of animals (75%) and the average weight of tumor (2.04±0.33 g) showed that the condition of the mice treated by DC was better than that in the control group (P <0.01). And the level of gene expression of IL-12, IFNγ and IL-18 in local tumor tissue was significantly raised (P <0.01) and the amount of local CD4+ (0.71±0.25/μm2) and CD8+ (0.67±0.22/μm2) cells was increased (P <0.05) and the cytotoxicity of TIL was remarkably enhanced. CONCLUSION: DC transfected with mRNA isolated from mouse tumor cells may effectively control the growth of tumor and ameliorate the symptoms, suggesting that a certain therapeutic efficiency for mice carrying tumor can be realized.
Southern Medical UniversityShengcheng, Guangdong, China