Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small ...Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )展开更多
OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression o...OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. RESULTS: The exogenous gene could be expressed efficiently in transduced BMSCs. CONCLUSION: The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.展开更多
Increasing the osteogenic differentiation ability and decreasing the adipogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs)is a potential strategy for the treatment of osteoporosis(OP).Natural...Increasing the osteogenic differentiation ability and decreasing the adipogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs)is a potential strategy for the treatment of osteoporosis(OP).Naturally derived oligosaccharides have shown significant anti-osteoporotic effects.Nystose(NST),an oligosaccharide,was isolated from the roots of Morinda officinalis How.(MO).The aim of the present study was to investigate the effects of NST on bone loss in ovariectomized mice,and explore the underlying mechanism of NST in promoting differentiation of BMSCs to osteoblasts.Administration of NST(40,80 and 160 mg/kg)and the positive control of estradiol valerate(0.2 mg/kg)for 8 weeks significantly prevented bone loss induced by ovariectomy(OVX),increased the bone mass density(BMD),improved the bone microarchitecture and reduced urine calcium and deoxypyridinoline(DPD)in ovariectomized mice,while inhibited the increase of body weight without significantly affecting the uterus weight.Furthermore,we found that NST increased osteogenic differentiation,inhibited adipogenic differentiation of BMSCs in vitro,and upregulated the expression of the key proteins of BMP and Wnt/β-catenin pathways.In addition,Noggin and Dickkopf-related protein-1(DKK-1)reversed the effect of NST on osteogenic differentiation and expression of the key proteins in BMP and Wnt/β-catenin pathway.The luciferase activities and the molecular docking analysis further supported the mechanism of NST.In conclusion,these results indicating that NST can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.展开更多
目的观察骨髓间充质干细胞(BMSCs)来源的外泌体(exosome)对小鼠乳腺癌细胞4T1增殖、侵袭的影响,并探讨其可能机制。方法将小鼠乳腺癌细胞4T1随机分为3组,4T1+vehicle组仅加入400μL无血清培养基,4T1+exosome组加入400μL由无血清培养基...目的观察骨髓间充质干细胞(BMSCs)来源的外泌体(exosome)对小鼠乳腺癌细胞4T1增殖、侵袭的影响,并探讨其可能机制。方法将小鼠乳腺癌细胞4T1随机分为3组,4T1+vehicle组仅加入400μL无血清培养基,4T1+exosome组加入400μL由无血清培养基配置的exosome,4T1+exosome+磷脂酰肌醇3激酶(PI3K)/Akt信号通路阻断剂(Y294002)组加入400μL终浓度为5μmol/m L Y294002及400μg/m L exosome的培养基。分别采用MTT法、细胞划痕实验、Western blotting法检测各组细胞增殖、迁移和侵袭能力以及PI3K/Akt信号通路相关蛋白。结果 4T1+exosome组、4T1+vehicle组、4T1+exosome+Y294002组细胞增殖抑制率分别为0.713%±0.050%、0.401%±0.030%、0.459%±0.800%,4T1+exosome组分别与4T1+vehicle组、4T1+exosome+Y294002组比较,P均<0.05。4T1+exosome组、4T1+vehicle组、4T1+exosome+Y294002组细胞迁移距离分别为(388.0±36.1)、(295.0±34.2)、(275.0±63.5)μm,4T1+exosome组分别与4T1+vehicle组、4T1+exosome+Y294002组比较,P均<0.05。4T1+exosome组p-AKT、β-catenin OD值分别为0.30±0.11、0.30±0.08,4T1+vehicle组分别为1.10±0.41、0.70±0.08,4T1+exosome+Y294002组分别为0.40±0.13、0.30±0.07,4T1+exosome组分别与4T1+vehicle组、4T1+exosome+Y294002组比较,P均<0.05。结论 BMSCs来源的exosome能够增加小鼠乳腺癌细胞4T1的增殖、迁移及侵袭能力,其机制可能与上调PI3K/Akt信号通路有关。展开更多
目的:观察血管生成素-1/重组质粒(pEGFP/Ang-1)转染的大鼠骨髓间充质干细胞(BMSCs)对高浓度葡萄糖损伤猴脉络膜-视网膜内皮细胞(RF/6A)的保护作用。方法:以pEGFP/Ang-1转染BMSCs,倒置荧光显微镜下观察增强型绿色荧光蛋白的表达,再利用Tr...目的:观察血管生成素-1/重组质粒(pEGFP/Ang-1)转染的大鼠骨髓间充质干细胞(BMSCs)对高浓度葡萄糖损伤猴脉络膜-视网膜内皮细胞(RF/6A)的保护作用。方法:以pEGFP/Ang-1转染BMSCs,倒置荧光显微镜下观察增强型绿色荧光蛋白的表达,再利用Transwell模型,将转染的BMSCs与RF/6A共培养于高浓度葡萄糖培养基中。3d后MTT法检测RF/6A活力,Western blot检测磷酸化蛋白激酶B(phosphorylated protein kinase B,P-PKB)的表达,从而探讨转染pEGFP/Ang-1的BMSCs对高浓度葡萄糖培养中的RF/6A的保护作用。结果:成功转染pEGFP/Ang-1的BMSCs可见增强型绿色荧光蛋白表达,与转染pEGFP/Ang-1的BMSCs共培养的RF/6A细胞活力及P-PKB的表达均高于未转染组(均P<0.01),与对照组无明显差别(均P>0.05)。结论:质粒pEGFP/Ang-1转染的BMSCs对高糖环境中的RF/6A具有保护作用,其机制可能与P-PKB表达上调有关。展开更多
基金supported by research grants from State 863 high technology R&D Project of China(2002AA205051and 2003AA205160)the National Key Rroject for Basic Research of China(2001CB509906)
文摘Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )
文摘OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. RESULTS: The exogenous gene could be expressed efficiently in transduced BMSCs. CONCLUSION: The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.
基金support from the Public Platform of Medical Research Center,Academy of Chinese Medical Science,Zhejiang Chinese Medical Universitysponsored by the National Natural Science Foundation of China(81973534,U1505226)。
文摘Increasing the osteogenic differentiation ability and decreasing the adipogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs)is a potential strategy for the treatment of osteoporosis(OP).Naturally derived oligosaccharides have shown significant anti-osteoporotic effects.Nystose(NST),an oligosaccharide,was isolated from the roots of Morinda officinalis How.(MO).The aim of the present study was to investigate the effects of NST on bone loss in ovariectomized mice,and explore the underlying mechanism of NST in promoting differentiation of BMSCs to osteoblasts.Administration of NST(40,80 and 160 mg/kg)and the positive control of estradiol valerate(0.2 mg/kg)for 8 weeks significantly prevented bone loss induced by ovariectomy(OVX),increased the bone mass density(BMD),improved the bone microarchitecture and reduced urine calcium and deoxypyridinoline(DPD)in ovariectomized mice,while inhibited the increase of body weight without significantly affecting the uterus weight.Furthermore,we found that NST increased osteogenic differentiation,inhibited adipogenic differentiation of BMSCs in vitro,and upregulated the expression of the key proteins of BMP and Wnt/β-catenin pathways.In addition,Noggin and Dickkopf-related protein-1(DKK-1)reversed the effect of NST on osteogenic differentiation and expression of the key proteins in BMP and Wnt/β-catenin pathway.The luciferase activities and the molecular docking analysis further supported the mechanism of NST.In conclusion,these results indicating that NST can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.
文摘目的观察骨髓间充质干细胞(BMSCs)来源的外泌体(exosome)对小鼠乳腺癌细胞4T1增殖、侵袭的影响,并探讨其可能机制。方法将小鼠乳腺癌细胞4T1随机分为3组,4T1+vehicle组仅加入400μL无血清培养基,4T1+exosome组加入400μL由无血清培养基配置的exosome,4T1+exosome+磷脂酰肌醇3激酶(PI3K)/Akt信号通路阻断剂(Y294002)组加入400μL终浓度为5μmol/m L Y294002及400μg/m L exosome的培养基。分别采用MTT法、细胞划痕实验、Western blotting法检测各组细胞增殖、迁移和侵袭能力以及PI3K/Akt信号通路相关蛋白。结果 4T1+exosome组、4T1+vehicle组、4T1+exosome+Y294002组细胞增殖抑制率分别为0.713%±0.050%、0.401%±0.030%、0.459%±0.800%,4T1+exosome组分别与4T1+vehicle组、4T1+exosome+Y294002组比较,P均<0.05。4T1+exosome组、4T1+vehicle组、4T1+exosome+Y294002组细胞迁移距离分别为(388.0±36.1)、(295.0±34.2)、(275.0±63.5)μm,4T1+exosome组分别与4T1+vehicle组、4T1+exosome+Y294002组比较,P均<0.05。4T1+exosome组p-AKT、β-catenin OD值分别为0.30±0.11、0.30±0.08,4T1+vehicle组分别为1.10±0.41、0.70±0.08,4T1+exosome+Y294002组分别为0.40±0.13、0.30±0.07,4T1+exosome组分别与4T1+vehicle组、4T1+exosome+Y294002组比较,P均<0.05。结论 BMSCs来源的exosome能够增加小鼠乳腺癌细胞4T1的增殖、迁移及侵袭能力,其机制可能与上调PI3K/Akt信号通路有关。
文摘目的:观察血管生成素-1/重组质粒(pEGFP/Ang-1)转染的大鼠骨髓间充质干细胞(BMSCs)对高浓度葡萄糖损伤猴脉络膜-视网膜内皮细胞(RF/6A)的保护作用。方法:以pEGFP/Ang-1转染BMSCs,倒置荧光显微镜下观察增强型绿色荧光蛋白的表达,再利用Transwell模型,将转染的BMSCs与RF/6A共培养于高浓度葡萄糖培养基中。3d后MTT法检测RF/6A活力,Western blot检测磷酸化蛋白激酶B(phosphorylated protein kinase B,P-PKB)的表达,从而探讨转染pEGFP/Ang-1的BMSCs对高浓度葡萄糖培养中的RF/6A的保护作用。结果:成功转染pEGFP/Ang-1的BMSCs可见增强型绿色荧光蛋白表达,与转染pEGFP/Ang-1的BMSCs共培养的RF/6A细胞活力及P-PKB的表达均高于未转染组(均P<0.01),与对照组无明显差别(均P>0.05)。结论:质粒pEGFP/Ang-1转染的BMSCs对高糖环境中的RF/6A具有保护作用,其机制可能与P-PKB表达上调有关。
