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 study the effect of bone marrow mesenchymal stem cells(MSCs) implantation into infarcted myocardium on cytokine secretion and angiogenesis. Methods 24 Guizhou xiang porcine were equally divided into experimental group and control group randomly. Three ml bone marrow was extracted from the posterior superior iliac spine. MSCs were cultured according to the methods of Wakitani’s. After being co-cultured with 5-azacytidine for 24 hours, these cells were labeled with bromodeoxyuridine(BrdU). Autologus MSCs were implanted into the acute myocardial infarct site both via the distal segment of the ligated left anterior descending artery (LAD) and topical injection. 3 amp; 6 weeks after transplantation, the samples from experimental group and control group were collected to detect the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and Ⅷ factor by immunohistology and video image digital analysis system. Results The expression of VEGF, bFGF and the microvessel counts in the experimental group were much higher than those of control group (Plt;0.01) at 3 and 6 weeks after transplantation. Conclusion MSCs, after being implanted into infarcted myocardium, shows the ability of secreting VEGF, bFGF, with subsequent angiogenesis.
Objective To investigate the possibility of establishing the human bone marrow mesenchymal stem cells (hMSCs) bank as to provide an alternative source for the seed cells of tissue engineering. Methods The cell surface antigensof the purified, expanded hMSCs and the ones following cryopreservation were detected by flow cytometry, cultured in a special medium to induce the ostoegenic and chondrocytic- differentiation. Morphology was studied by light and electronic microscopes. The detection of alkaline phosphatase, collagen type Ⅰ, osteocalcin, and collagen type Ⅱ were also performed by immunochemistry and molecular biology.Results The phenotype and expansion possibility of hMSCs after cryopreservation were remained. It could expand for 10 generations. The doubling time was 40 h.Conclusion The bank of hMSCs is inipiently established and can provide eligible seed cells for tissue engineering.
Objective To optimize the culture conditions of the human bone marrow mesenchymal stem cells(hMSCs). Methods The influence of the primary culture method, planting density, the time of the first medium changing , culture medium and serum concentration on growth of the hMSCs were analyzed. Results When the other conditions were the same, the density gradient isolation was better than whole-marrow isolation;2.5×105 cells/cm2 was the best planting density;the best first medium changing was the fifth day at primary culture, DMEM medium was better than α-MEM, serum C was the best of four serums compared, 10% was the suitable serum concentration. The hMSCs under the optimal conditions could expand over 15 passages, remaining their normal modality and differentiation potentials. Conclusion The optimal culture condition of the hMSCs is established and it is a new investigation on application of hMSCs to tissue engineering.
Objective To evaluate the osteogenic potential of human bone marrow mesenchymal stem cells (MSCs) transferred with human bone morphogenetic protein 2(BMP 2) gene by adenovirus. Methods The MSCs were isolated from human bone marrow and cultured in vitro. They were divided into 3 groups: Adv hBMP 2 transduced group; Adv βgal transduced group; untransduced group. Western immunoblot analysis, alkaline phosphatase(ALP) staining, Von Kossa staining, and a quantitative ALP activity assay were performed. Nine unde mice received injection into a thigh muscle to test the osteoinductivity of the three types of cells. Results In the Adv-hBMP-2 transprotein; most MSCs were stained positively for ALP activity 9 day after transduction; the MSCs reached the peak of ALP activity 12 day after transduction; the calcified nodes formed 21 days after transduction. The ectopic bones formed in the thigh muscles of the nude mice. Little bone formation was observed in the other groups 4 weeks after cell injection. Conclusion Adenovirus mediated hBMP-2 gene transfection can induce osteogenesis of human bone marrow mesenchymal stem cells.
Objective To investigate the possibility of sheep joint cartilage defect repair with tissue engineered cartilage constructed by using porous bioceramics as scaffold and TGF-β induced autologous bone marrow derived mesenchymal stem cells(MSCs) as seed cell. Methods In the experimental group(n=12), autologous MSCs were isolated and expanded in vitro and then implanted into the pre molded porous β-TCP; the cell β-TCP complex was implanted into sheep right humeral cartilage defect. The defects in β-TCP (n= 12) group were repaired by B-TCP only, while defects in the control group (n= 4) were left un-repaired. Samples were extracted 12 and 24 weeks after operation for histological, histochemical and immunohistochemical analysis. Results In the experimental group, cartilage-like tissue formation could be seen on the surface of the implants. Microscopic analysis demonstrated obvious degradation of B-TCP and extensive new cartilage formation 12 weeks after operation, containing rich extracellularmatrix. The cells were stained positively with type II collagen. The bioceramics had almo st completely been degraded and abundant cartilage formation could be seen in the whole defects 24 weeks later. In the B-TCP group, marginal cartilage ingrowth could be seen 12 weeks after operation and the number of chondrocytes increasedmarkedly after 24wee s. However, no cartilage can be found in the middle of the material. In the control group, only a small quantity of new cartilage formation could be seenalong the margin of defects. Conclusion It is feasible to generate tissue engineered cartilage with porous B-TCP and auto logousM SCs for cartilage defect repair.
OBJECTIVE: To determine whether culture expanded bone marrow derived mesenchymal stem cells (MSCs) in combination with beta-tricalcium phosphate(beta-TCP) can repair critical cranial defects in New Zealand rabbits. METHODS: In group A(n = 20), MSCs from homogeneous rabbits were isolated and expanded in vitro and then implanted onto the pre-molded porous beta-TCP. The MSCs-beta-TCP complexes were implanted into rabbit critical cranial defects. In group B (n = 10), The defects were repaired with beta-TCP only. In group C(n = 4), the defects were left un-repaired. Samples were extracted 6 and 12 weeks after operation for histological, histochemical and immunohistochemical analysis. RESULTS: In group A, bone-like tissue formation could be seen on the surface of the implants. Microscopic analysis demonstrated certain degradation of beta-TCP and extensive new bone filling in rich extracellular matrix after 6 weeks. The cells were stained positively for type I collagen. After 12 weeks, the bioceramics had almost completely degraded and abundant bone formation could be seen in the whole defects. In group B, marginal bone ingrowth was observed after 6 weeks and the number of osteoblasts increased significantly after 12 weeks. However, no new bone formation could be detected in the middle of the material. In group C, only a small quantity of new bone formation was found along the margin of defects. CONCLUSION: Transplantation of MSCs with beta-TCP can serve as an example of a cell-based treatment for bone regeneration in skeletal defects.
目的 构建小鼠甲状腺转录因子-2（TTF-2）转基因动物表达载体（pBROAD3-TTF-2），观察其在小鼠骨髓间充质干细胞（BMSC）中的表达。 方法 从C57BL/6J小鼠肝脏组织中提取基因组DNA，利用聚合酶链式反应方法扩增出TTF-2基因1 113 bp开放阅读框，通过DNA重组技术将TTF-2基因片段插入克隆载体pMD18-T中，经测序正确后，再重组于pBROAD3-mcs中，构建转基因动物表达载体pBROAD3-TTF-2，用酶切电泳分析对其进行鉴定。运用脂质体转染试剂将其转染BMSC后，蛋白质印迹法检测TTF-2基因的表达。 结果 ① DNA测序证实目的基因序列正确无突变，酶切电泳分析得到相应的目的片段，大小与理论计算值一致，成功构建转基因动物表达载体pBROAD3-TTF-2。② 蛋白质印迹法显示转染的BMSC高表达TTF-2蛋白。 结论 成功构建了pBROAD3- TTF-2转基因动物表达载体，显示其转染BMSC后TTF-2基因的表达，为下一步建立TTF-2转基因小鼠模型奠定了基础。
目的：建立并探讨一种体外分离、培养和扩增骨髓间质干细胞的优化方法。方法：采用密度梯度离心法和全骨髓贴壁法分离培养大鼠骨髓干细胞，应用倒置显微镜和免疫组化法观察和鉴定骨髓间质干细胞。结果：大鼠骨髓干细胞呈均一的梭形成纤维细胞样，形成集落样生长，显微结构表现出干细胞特征；免疫组化显示CD14、CD45阴性，CD44、CD90阳性。与密度梯度离心法比较，贴壁法获得的骨髓干细胞活性高，增殖力强，克隆形成早，传代时间短。结论：全骨髓贴壁法和密度梯度离心法均可获得高纯度贴壁生长的骨髓干细胞，其中全骨髓贴壁法方法简单易行，骨髓干细胞增殖快，活性好，传代力持久，是一种有实用价值的骨髓干细胞培养方法。