AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate(TCP) on osteogenesis.METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential mediu...AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate(TCP) on osteogenesis.METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium(MEM) containing ascorbic acid phosphate(AscP) and dexamethasone(Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase(ALP) activity and osteocalcin(OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs wereimplanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk.RESULTS In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group(P < 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group(P > 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. CONCLUSION This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.展开更多
Background Most of the basic and clinical studies of osteonecrosis of the femoral head （ONFH） are restricted to bone tissues only, whereas various systems are involved in the onset and development of ONFH, including...Background Most of the basic and clinical studies of osteonecrosis of the femoral head （ONFH） are restricted to bone tissues only, whereas various systems are involved in the onset and development of ONFH, including nervous system. Peptidergic nerve participates in the neuronal regulation of bone metabolism and anabolism, and plays key roles in the growth, repair and reconstruction of bone. Calcitonin gene-related peptide （CGRP）, which is secreted by peptidergic nerve, is the main mediator of bone metabolism. It dramatically promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells （BMSCs）. Additionally, it enhances the osteoblast mass and the rate of osteoblast formation, and reduces the bone resorption by acting on osteoblasts and osteoclasts. Hence, we aimed to construct recombinant retrovirus vector pLNCX2-hCGRPα and to investigate the proliferation and osteogenic potential of hCGRPα-producing BMSCs （BMSCs/pLNCX2-hCGRPα） after virus infection. Methods The constructed recombinant retrovirus vector pLNCX2-hCGRPα was transfected into PT67 packaging cells by lipofectamine 2000. Virus was collected for BMSCs infection. The mRNA and protein expression of hCGRPα was examined by reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, respectively. The cell proliferation was determined by methyl thiazoleterazolium （MTT） assay. The osteogenic potential of BMSCs was evaluated by alkaline phosphatase （ALP） activity. Results Both mRNA and protein expression of hCGRPα was detected in BMSCs/pLNCX2-hCGRPα cells. These cells exhibited significantly elevated proliferation and ALP value as compared with control BMSCs （P 〈0.05）. Conclusion BMSCs/pLNCX2-hCGRPα cells could stably express hCGRPα and showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.展开更多
Background Alzheimer’s disease is a neurodegenerative disorder.Therapeutically,a transplantation of bone marrow mesenchymal stem cells(BMMSCs)can play a beneficial role in animal models of Alzheimer’s disease.Howeve...Background Alzheimer’s disease is a neurodegenerative disorder.Therapeutically,a transplantation of bone marrow mesenchymal stem cells(BMMSCs)can play a beneficial role in animal models of Alzheimer’s disease.However,the relevant mechanism remains to be fully elucidated.Main body Subsequent to the transplantation of BMMSCs,memory loss and cognitive impairment were significantly improved in animal models with Alzheimer’s disease(AD).Potential mechanisms involved neurogenesis,apoptosis,angiogenesis,inflammation,immunomodulation,etc.The above mechanisms might play different roles at certain stages.It was revealed that the transplantation of BMMSCs could alter some gene levels.Moreover,the differential expression of representative genes was responsible for neuropathological phenotypes in Alzheimer’s disease,which could be used to construct gene-specific patterns.Conclusions Multiple signal pathways involve therapeutic mechanisms by which the transplantation of BMMSCs improves cognitive and behavioral deficits in AD models.Gene expression profile can be utilized to establish statistical regression model for the evaluation of therapeutic effect.The transplantation of autologous BMMSCs maybe a prospective therapy for patients with Alzheimer’s disease.展开更多
目的:体外构建人C型利钠利尿肽(C N P,前体基因名N PPC)和增强型绿色荧光蛋白(EG FP)重组腺病毒,并检测其在兔骨髓间充干细胞(B M SC s)中的表达。方法:将N PPC基因克隆至含有EG FP的穿梭质粒,获得重组腺病毒A d-N PPC-EG FP颗粒,测定...目的:体外构建人C型利钠利尿肽(C N P,前体基因名N PPC)和增强型绿色荧光蛋白(EG FP)重组腺病毒,并检测其在兔骨髓间充干细胞(B M SC s)中的表达。方法:将N PPC基因克隆至含有EG FP的穿梭质粒,获得重组腺病毒A d-N PPC-EG FP颗粒,测定病毒滴度。转染兔骨髓间充质干细胞,测定感染复数(M O I)值,荧光显微镜、流式细胞仪检测转染效率及传代稳定性。结果:经PC R、酶切鉴定、测序分析,重组腺病毒构建成功,效价为1.2×1011PFU/m l,转染兔B M SC s最适M O I值为1000。感染细胞后效率较高,且稳定遗传。结论:成功构建C N P基因N PPC重组腺病毒,可在兔B M SC s中稳定表达,为进一步研究C N P的作用奠定基础。展开更多
基金Supported by Grant-in-Aid for scientific research from the Ministry of Health,Labour and Welfare,Japan
文摘AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate(TCP) on osteogenesis.METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium(MEM) containing ascorbic acid phosphate(AscP) and dexamethasone(Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase(ALP) activity and osteocalcin(OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs wereimplanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk.RESULTS In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group(P < 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group(P > 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. CONCLUSION This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.
基金the National Natural Science Foundation of China
文摘Background Most of the basic and clinical studies of osteonecrosis of the femoral head （ONFH） are restricted to bone tissues only, whereas various systems are involved in the onset and development of ONFH, including nervous system. Peptidergic nerve participates in the neuronal regulation of bone metabolism and anabolism, and plays key roles in the growth, repair and reconstruction of bone. Calcitonin gene-related peptide （CGRP）, which is secreted by peptidergic nerve, is the main mediator of bone metabolism. It dramatically promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells （BMSCs）. Additionally, it enhances the osteoblast mass and the rate of osteoblast formation, and reduces the bone resorption by acting on osteoblasts and osteoclasts. Hence, we aimed to construct recombinant retrovirus vector pLNCX2-hCGRPα and to investigate the proliferation and osteogenic potential of hCGRPα-producing BMSCs （BMSCs/pLNCX2-hCGRPα） after virus infection. Methods The constructed recombinant retrovirus vector pLNCX2-hCGRPα was transfected into PT67 packaging cells by lipofectamine 2000. Virus was collected for BMSCs infection. The mRNA and protein expression of hCGRPα was examined by reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, respectively. The cell proliferation was determined by methyl thiazoleterazolium （MTT） assay. The osteogenic potential of BMSCs was evaluated by alkaline phosphatase （ALP） activity. Results Both mRNA and protein expression of hCGRPα was detected in BMSCs/pLNCX2-hCGRPα cells. These cells exhibited significantly elevated proliferation and ALP value as compared with control BMSCs （P 〈0.05）. Conclusion BMSCs/pLNCX2-hCGRPα cells could stably express hCGRPα and showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.
基金This work was supported by grants Beijing Natural Science Foundation(No.517100)National Key Research and Development Project(No.2017YFA0105200)CAMS Innovation Fund for Medical Sciences(CIFMS)(2016-I2M-2-006).
文摘Background Alzheimer’s disease is a neurodegenerative disorder.Therapeutically,a transplantation of bone marrow mesenchymal stem cells(BMMSCs)can play a beneficial role in animal models of Alzheimer’s disease.However,the relevant mechanism remains to be fully elucidated.Main body Subsequent to the transplantation of BMMSCs,memory loss and cognitive impairment were significantly improved in animal models with Alzheimer’s disease(AD).Potential mechanisms involved neurogenesis,apoptosis,angiogenesis,inflammation,immunomodulation,etc.The above mechanisms might play different roles at certain stages.It was revealed that the transplantation of BMMSCs could alter some gene levels.Moreover,the differential expression of representative genes was responsible for neuropathological phenotypes in Alzheimer’s disease,which could be used to construct gene-specific patterns.Conclusions Multiple signal pathways involve therapeutic mechanisms by which the transplantation of BMMSCs improves cognitive and behavioral deficits in AD models.Gene expression profile can be utilized to establish statistical regression model for the evaluation of therapeutic effect.The transplantation of autologous BMMSCs maybe a prospective therapy for patients with Alzheimer’s disease.
文摘目的:体外构建人C型利钠利尿肽(C N P,前体基因名N PPC)和增强型绿色荧光蛋白(EG FP)重组腺病毒,并检测其在兔骨髓间充干细胞(B M SC s)中的表达。方法:将N PPC基因克隆至含有EG FP的穿梭质粒,获得重组腺病毒A d-N PPC-EG FP颗粒,测定病毒滴度。转染兔骨髓间充质干细胞,测定感染复数(M O I)值,荧光显微镜、流式细胞仪检测转染效率及传代稳定性。结果:经PC R、酶切鉴定、测序分析,重组腺病毒构建成功,效价为1.2×1011PFU/m l,转染兔B M SC s最适M O I值为1000。感染细胞后效率较高,且稳定遗传。结论:成功构建C N P基因N PPC重组腺病毒,可在兔B M SC s中稳定表达,为进一步研究C N P的作用奠定基础。
