Objective To study the influence of autologous bone mesenchymal stem cells (BMSCs) on myocardial structure and cardiac function after being implantated into acute infarcted myocardial site. Methods Bone marrow was aspirated from the posterosuperior iliac spine of Guizhou Xiang swine. After being isolated, cultured and co cultured with 5 azacytidine, either autologous BMSCs (total cells 2×10 6, experimental group, n =12), or a comparable volume of culture medium (control group, n =12), was injected into the left anterior descending(LAD) branch of coronary artery just distal to the ligation site of the LAD. The same volume of BMSCs or culture medium was injected into several spots in the infarcted myocardium. Echocardiographic measurements were performed three or six weeks after implantation to assess the myocardial structure and cardiac function. Results Left ventricular function, including eject fraction(EF), fractional shortening and wall thickening, were higher in experimental group when compared with control group. The thickness of the ventricular wall and septum was also found increased while the left ventricular chamber size was smaller in experimental group. Conclusion Implantation of BMSCs into the infarcted myocardium is believed to attenuate the remodeling process, inhibit the extent of wall thinning and dilatation of the ventricular chamber. BMSCs implantation may also improve the contractile ability of the myocardium and cardiac function.
Objective To solve the shortage of hepatocytes for l iver tissue engineering, to explore the possibil ity of prol iferation of rat bone marrow mesenchymal stem cells (BMSCs) and the feasibil ity of differentiation of BMSCs into hepatocyteswith a culture system containing cholestatic rat serum and hepatocyte growth factor (HGF) in vitro. Methods Myeloid cellsof femur and tibia were collected from the female healthy Wistar rats at the age of 6 weeks, the BMSCs were isolated, purified and identified. Normal and cholestatic rat serum were prepared from 40 healthy Wistar rats at the age of 12-14 weeks. The 3rd passage of BMSCs were harvested and added different cultures according to the following grouping: group A, DMEM plus 10%FBS; group B, hepatocyte growth medium (HGM) plus 5%FBS; group C, HGM plus 5% normal rat serum; group D, HGM plus 5% cholestatic rat serum; group E, HGM plus 5% cholestatic rat serum plus 25 μg/L HGF. The changes of cell morphology were observed, MTT assay was used to measure cell growth; the expression of alpha-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunocytochemistry; the glycogen deposit was examined by periodic acid-schiff (PAS) staining; and the urea content in culture supernatant was determined by glutamate dehydrogenase. Results Polygonal cells and binuclear cells were observed in groups D and E, while the shapes of cells in groups A, B, and C did not obviously change. The cell growth curve demonstrated that the speed of cells proliferation in group C was the fastest, the one in group B was the slowest; showing significant differences when compared with groups A, D, and E (P lt; 0.05). On the 7th day in groups D and E, the positive expressions of AFP and CK18 emerged, on the 14th day the positive expression of glycogen emerged. At the same period, the expression ratio was higherin group E than in group D (P lt; 0.05). The urea concentration increased gradually with induction time in groups D and E, the concentration was higher in group E than in group D (P lt; 0.05). No expressions of AFP, CK18, glycogen, and change of the urea concentration were observed in groups A, B, and C. Conclusion Normal rat serum can obviously promote the growth of BMSCs; cholestatic rat serum which promote the growth of BMSCs can induce to differentiate into hepatocyte; and a combination of cholestatic serum and HGF can increase the differentiation ratio.
Objective To study the time effect of the gene expression of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes so as to lay a theoretical foundation for gene therapy of osteonecrosis. Methods The best multipl icity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 × 106 cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sl iced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo. Results The best MOI of BMSCs transfected with rAAV was 5 × 104 v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the b level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and b expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 ± 75.58) pg/mL and (4.80 ± 0.61) ng/mL respectively in experimental group 1, and were (32.28 ± 8.42) pg/mL and (0.64 ± 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.
Objective To investigate the effect of bone mesenchymal stem cells (MSCs) transfected with pEGFP-C1/Akt after intramuscular injection on angiogenesis in ischemic hindlimb of mice. Methods A total of 30 Wistar mice used in the present experiments were divided into gene therapy group (injected MSCs transfected with pEGFP-C1/Akt), non-gene therapy group （injected MSCs） and control group (injected PBS). Mice skin temperature in left hindlimbs was measured by a infrared ray temperature meter before and right after ischemia and 1-7 days after transfection, respectively. Digital subtraction angiography was carried out to observe angiogenesis in hindlimb of mice after 28 days. The capillary density was determined with immunohistochemical analysis in the resected specimen. The expressions of Akt mRNA and protein, VEGF mRNA and protein were measured via RT-PCR and Western blot. Results Skin temperature increased significantly on 3 d after transfection in gene therapy group. On 28 d after transfection， angiography showed that the number of collateral vessels markedly increased in gene therapy group. Green fluorescence cells were observed in ischemic tissue from the adductor and semimembranous muscles of gene therapy group under fluorescent microscope. The result of immunohistochemical analysis of capillary density showed that the capillary density was significantly higher in gene therapy group 〔(7.1±0.3)/high power〕 than that in non-gene therapy group 〔(4.2±0.4)/high power〕 and control group 〔(1.3±0.2)/high power〕, with significantly statistical differences among three groups (P＜0.01). Akt mRNA (2.44±0.14) and protein (1.1±0.13), VEGF mRNA (1.1±0.11) and protein (0.97±0.13) of gene therapy group markedly increased compared with Akt mRNA (1.58±0.13) and protein (0.78±0.12), VEGF mRNA (0.78±0.14) and protein (0.67±0.11) of non-gene therapy group as well as Akt mRNA (0.64±0.11) and protein (0.36±0.12), VEGF mRNA (0.56±0.11) and protein (0.33±0.13) of control group (P＜0.01). Conclusion The effect of MSCs transfected with pEGFP-C1/Akt on angiogenesis is significantly better than only MSCs therapy, which may offer a new way for the ischemic diseases.
ObjectiveTo review the development of cell sheet engineering technology in engineering vascularized tissue. MethodsThe literature about cell sheet engineering technology and engineering vascularized tissue was reviewed, analyzed, and summarized. ResultsAlthough there are many methods to engineer vascularized tissue, cell sheet engineering technology provides a promising potential to develop a vascularized tissue. Recently, cell sheet engineering technology has become a hot topic in engineering vascularized tissue. Co-culturing endothelial cells on a cell sheet, endothelial cells are able to form three-dimensional prevascularized networks and microvascular cavities in the cell sheet, which facilitate the formation of functional vascular networks in the transplanted tissue. ConclusionCell sheet engineering technology is a promising strategy to engineer vascularized tissue, which is still being studied to explore more potential.