骨髓间充质干细胞的磁标记成像及移植治疗骨缺血坏死的研究
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摘要
股骨头缺血性坏死是临床常见病、多发病,治疗上除手术外都是基于改善股骨头周围血液循环。骨髓间充质干细胞取材方便,能在体外大量扩增,具有多种分化潜能,来源于自体骨髓的细胞移植时不存在免疫排斥,同种异体的间充质干细胞免疫原性很低,为组织损伤修复提供了新的种子细胞。
本研究采用密度梯度离心分离法对rMSCs进行分离扩增,通过形态学和成骨、成脂方向诱导分化,证实分离得到的细胞为骨髓间充质干细胞。采用菲立磁-多聚赖氨酸对得到的rMSCs进行磁标记,标记率为100%;用1.5TMRI对标记细胞进行了体外成像研究,表明GRE T2*WI序列可以作为磁标记细胞研究的首选序列。通过注射马血清及甲泼尼龙的方法建立了兔激素性股骨头缺血坏死模型,该模型骨坏死处于0～I期,是研究早期骨坏死的理想模型。采用介入导管技术将骨髓间充质干细胞移植到兔股骨头缺血坏死模型体内,显示移植后病变部位可见明显骨修复,移植细胞可以在病变局部存活。在此基础上采用全骨髓培养法对胎儿fMSCs进行分离和扩增,并进行fMSCs磁标记成像,获得了与兔MSCs相似的结果。本研究从兔骨髓间充质干细胞研究获得经验并逐步应用到人骨髓间充质干细胞研究上,期望为进一步的骨髓间充质干细胞移植治疗股骨头缺血坏死的临床应用做些有益的探索。
Bone marrow mesenchymal stem cells(BMSCs) divide from bone marrow is a kind of progenitor cell and keep capacity for both self-renewal and multilineage differentiation. These cells can be ex vivo expanded and induced, either in vitro or in vivo,to terminally differentiate into osteoblasts, chondrocytes, adipocytes, cardicytes, myotubes, neural cells, tenocytes and hematopoietic-supporting stroma, and so on. It was considered to play an important role in cell therapy, gene therapy and regenerative medicine field. The therapeutic cells used in clinical medicine will require techniques that can monitor their migration and biodistribution in tissue after transplantation. Thus, the tracking of MSCs in vivo after transplantation has great value and attracts the attentions of the academic circle. MSCs labeling by combining superparamagnetic iron oxide (SPIO) and poly-l-lysine (PLL) can be detected by MR imaging. Avascular necrosis of femoral head (ANFH) is a usually and multiply disease in clinical, and the interventional therapy may improve the vessel supply and benefit to absorption of osteonecrosis and bone renewal. We always consider whether the interventional therapy combining with the MSCs infuse can increase the process of bone regeneration and repair in local.
1 Study on the basic biological characteristics of rMSCs.
Bone marrow were obtained from two Japanese big-ears albino rabbits aging two weeks, then the rMSCs were isolated from BM sample by using Ficoll(1.077g/L) density gradient separation method. Separated cells were add into L-DMEM supplemented with 20% fetal bovine serum, 10000U/L penicillin and 10000U/L streptomycin, plated into 50 ml plastic flasks at a density of 2×105cells/cm2 and incubated at 37℃in 5%CO2. At 80% confluence, cells were harvested with 0.25% trypsin and replanted in new flasks. The rMSCs were indentified by growth conditation, cell growth curves and cell cycle analysis, induced and differentiated into osteocytes and adipocytes. Most adhere cells have spindle shape and obviously polar characters. The culture cells remained dormant for 1 to 2 days and began to multiply rapidly at 3 days and reach flat phase at 7 days. Cell cycle analysis showed that 96% of exponential phase of growth cells were in G0 and S phase. The alkaline phosphates activity increased after induced by osteogenic medium and Von Kossa staining detected the formation of calcium nodus. Adipogenic differentiation was induced in the expanded rMSCs. The accumulation of lipid-rich vacuoles within cells was apparent at 10 days, and positive express by Oil-red-O staining.
2 Study on the labeling rMSCs combining with ferumoxides and poly-l-lysine.
The P3 exponential phase of growth rMSCs was labeled with ferumoxides (25μg/ml) and PLL(0.75μg/ml) for 12h at 37℃in a 95% air per 5% atmosphere. The cell growth curve of labeled cells was similar to the unlabeled cells, and there was no difference in the rate of apoptosis. Labeling efficiency was reproducible in approximately 100% of rMSCs by Prussian blue staining and no stainable iron was detected in the unlabeled cells. Analysis of transmission electron microscope showed that a great lot of iron particles located in labeled cells. MR imaging of cell suspensions was performed at 1.5T by using surface coil, and a dramatic signal decrease was observed in the T2 weighted MR imagines of labeled cells compared with unlabeled control cells, specially in the T2*weighted MR imagines. The results showed that the concentration of ferumoxides used in labeling stem cells was safe. 1.5T MRI scanner was able to show magnetic stem cells, which greatly promoted research and application of the magnetic stem cell. Moreover T2*WI imaging of GRE sequences can be used as the preferred method of tracking stem cells.
3 Interventional treatment of avascular necrosis of femoral head in rabbit by transplantation of rMSCs.
The rabbit model of avascular necrosis of femoral head was established by injecting the horse serum and methylprednisolone. The femoral heads were taken out and evaluated by pathology at 1, 4, 7 weeks after imageological examination (CR, CT, MRI and vascular angiography). CR and CT had no difference of bone density difference at 7 weeks. MR showed bone marrow edema in femoral head and wire-like, spot-like middle signals in T1-weighted imaging and asymmetrical high signals in T2-weighted imaging. Vascular angiography showed a decrease in vascular supply and fewer obstructed blood vessels in the femoral head. HE staining of pathological sections showed necrosis and rupture of bone trabeculae, an increased proportion of empty osteocyte lacunae, hematopoietic decreased and fat cell hypertrophy. On the study of stem cells transplantation by interventional method, the 2.7F micro-catheter was superselected into artery of femoral head, infusion the dilatation and thrombus dissolution drugs after angiography, then inject the rMSCs about 5×106 /5ml into the target vascular. At 1, 4, 7 weeks after cell transplantation, the femoral heads were taken out and pathological examination was made after imagological examination. Compared with the control group, MR showed hat abnormal signals significantly decreased area of in femoral head in the therapy group, and hydrarthrosis disappeared. Vascular angiography showed the increase of vascular supply and richness of vessel branch. Pathological examination showed the decrease of bone trabeculae necrosis and number of empty osteocyte lacunae, active bone formation, the thickness and number of chondrocyte increased in the therapy group, no significantly difference between the labeled and unlabeled groups. Blue staining particles ware detected in cartilage and subchondral bone tissues in labeled groups by Prussian Blue staining, which indicated that rMSCs might live in the necrosis area of femoral head after transplantation by interventional method. The results suggested that simply perfusion of dilatation and thrombus dissolution drugs obviously improved local blood supply and promoted bone repair and reconstruction. Furthermore, after a certain amount of perfusion through catheter,MSCS could survive topically, which provided a theoretical and practical basis for the clinical application.
4 Isolation and differentiation of human fetal mesenchymal stem cells in vitro
Bone marrow were obtained from aborted fetal (4-6 months) four limbs, and washed bone marrow by DMEM, then the separated cells were added into L-DMEM /F12 supplemented with 20% fetal bovine serum, 10000U/L penicillin and 10000U/L streptomycin, plated into 100mm plastic dish at a density of 2-5×105cells/cm2 and incubated at 37℃in 5%CO2. At 80% confluence, cells were harvested with 0.25% trypsin and replanted in new dish. The fMSCs were indentified by growth conditation, cell growth curves, cell cycle analysis, immune-phenotypical characterization and differentiation into osteocytes and adipocytes. Most adhered cells have spindle shaped with obviously polar characters. The culture cells remained dormant for 1 to 2 days and then began to multiply rapidly at 3 days and reach flat phase at 7 days. Cell cycle analysis showed that 85% of exponential phase of P3 growth cells were in G0 and S phase. They were positive for CD29 and CD105, negative for CD45. The Von Kossa staining detected the formation of calcium nodus at 21 days after induced by osteogenic medium, and the osceocalcin (OC) mRNA was expressed in the inductor group but no expressed in the control group assayed with the reverse transcript polymerase chain reaction. Adipogenic differentiation was induced in the expanded fMSCs, the accumulation of lipid-rich vacuoles within cells was apparent at 10 days, and positive express by Oil-red-O staining. Our study indicated that the cells isolated from fetal bone marrow have the characteristic of MSCs.
5 Study on the labeling fMSCs combining with ferumoxides and poly-l-lysine
The P3 exponential phase of growth fMSCs labeled with ferumoxides(25μg/ml) and PLL(0.75μg/ml) for 12h at 37℃in a 5% CO2 atmosphere. The cell growth curve of labeled cells was similar to the unlabeled cells, and there was no difference in the rate of apoptosis. Labeling efficiency was reproducible in approximately 98% of fMSCs by Prussian blue staining and no stainable iron was detected in the unlabeled cells. Analysis of transmission electron microscope showed that a great lot of iron particles locate in endosoma of labeled cells. MR imaging of cell suspensions was performed at 1.5T by using surface coil, and a dramatic signal decrease was observed in the T2 weighted MR imagines of labeled cells compared with unlabeled control cells, specially in the T2*weighted MR imagines. The fMSCs were successfully labeled with Ferumoxides and PLL, which provides experimental basis for tracking magnetic labeled MSCs in clinical application.
We isolated and expanded rMSCs and fMSCs by density gradient separation method and whole bone marrow culture methods, and confirmed the characteristic of MSCs by growth conditation, immunephenoty and multipotentiality. The model of ANFH was established in rabbit and investigated rMSCs transplantation by interventional therapy. Our results may provide references for clinical research on magnetic labeling MCSs and necrosis therapy of stem cells transplantation.
本研究采用密度梯度离心分离法对rMSCs进行分离扩增,通过形态学和成骨、成脂方向诱导分化,证实分离得到的细胞为骨髓间充质干细胞。采用菲立磁-多聚赖氨酸对得到的rMSCs进行磁标记,标记率为100%;用1.5TMRI对标记细胞进行了体外成像研究,表明GRE T2*WI序列可以作为磁标记细胞研究的首选序列。通过注射马血清及甲泼尼龙的方法建立了兔激素性股骨头缺血坏死模型,该模型骨坏死处于0～I期,是研究早期骨坏死的理想模型。采用介入导管技术将骨髓间充质干细胞移植到兔股骨头缺血坏死模型体内,显示移植后病变部位可见明显骨修复,移植细胞可以在病变局部存活。在此基础上采用全骨髓培养法对胎儿fMSCs进行分离和扩增,并进行fMSCs磁标记成像,获得了与兔MSCs相似的结果。本研究从兔骨髓间充质干细胞研究获得经验并逐步应用到人骨髓间充质干细胞研究上,期望为进一步的骨髓间充质干细胞移植治疗股骨头缺血坏死的临床应用做些有益的探索。
Bone marrow mesenchymal stem cells(BMSCs) divide from bone marrow is a kind of progenitor cell and keep capacity for both self-renewal and multilineage differentiation. These cells can be ex vivo expanded and induced, either in vitro or in vivo,to terminally differentiate into osteoblasts, chondrocytes, adipocytes, cardicytes, myotubes, neural cells, tenocytes and hematopoietic-supporting stroma, and so on. It was considered to play an important role in cell therapy, gene therapy and regenerative medicine field. The therapeutic cells used in clinical medicine will require techniques that can monitor their migration and biodistribution in tissue after transplantation. Thus, the tracking of MSCs in vivo after transplantation has great value and attracts the attentions of the academic circle. MSCs labeling by combining superparamagnetic iron oxide (SPIO) and poly-l-lysine (PLL) can be detected by MR imaging. Avascular necrosis of femoral head (ANFH) is a usually and multiply disease in clinical, and the interventional therapy may improve the vessel supply and benefit to absorption of osteonecrosis and bone renewal. We always consider whether the interventional therapy combining with the MSCs infuse can increase the process of bone regeneration and repair in local.
1 Study on the basic biological characteristics of rMSCs.
Bone marrow were obtained from two Japanese big-ears albino rabbits aging two weeks, then the rMSCs were isolated from BM sample by using Ficoll(1.077g/L) density gradient separation method. Separated cells were add into L-DMEM supplemented with 20% fetal bovine serum, 10000U/L penicillin and 10000U/L streptomycin, plated into 50 ml plastic flasks at a density of 2×105cells/cm2 and incubated at 37℃in 5%CO2. At 80% confluence, cells were harvested with 0.25% trypsin and replanted in new flasks. The rMSCs were indentified by growth conditation, cell growth curves and cell cycle analysis, induced and differentiated into osteocytes and adipocytes. Most adhere cells have spindle shape and obviously polar characters. The culture cells remained dormant for 1 to 2 days and began to multiply rapidly at 3 days and reach flat phase at 7 days. Cell cycle analysis showed that 96% of exponential phase of growth cells were in G0 and S phase. The alkaline phosphates activity increased after induced by osteogenic medium and Von Kossa staining detected the formation of calcium nodus. Adipogenic differentiation was induced in the expanded rMSCs. The accumulation of lipid-rich vacuoles within cells was apparent at 10 days, and positive express by Oil-red-O staining.
2 Study on the labeling rMSCs combining with ferumoxides and poly-l-lysine.
The P3 exponential phase of growth rMSCs was labeled with ferumoxides (25μg/ml) and PLL(0.75μg/ml) for 12h at 37℃in a 95% air per 5% atmosphere. The cell growth curve of labeled cells was similar to the unlabeled cells, and there was no difference in the rate of apoptosis. Labeling efficiency was reproducible in approximately 100% of rMSCs by Prussian blue staining and no stainable iron was detected in the unlabeled cells. Analysis of transmission electron microscope showed that a great lot of iron particles located in labeled cells. MR imaging of cell suspensions was performed at 1.5T by using surface coil, and a dramatic signal decrease was observed in the T2 weighted MR imagines of labeled cells compared with unlabeled control cells, specially in the T2*weighted MR imagines. The results showed that the concentration of ferumoxides used in labeling stem cells was safe. 1.5T MRI scanner was able to show magnetic stem cells, which greatly promoted research and application of the magnetic stem cell. Moreover T2*WI imaging of GRE sequences can be used as the preferred method of tracking stem cells.
3 Interventional treatment of avascular necrosis of femoral head in rabbit by transplantation of rMSCs.
The rabbit model of avascular necrosis of femoral head was established by injecting the horse serum and methylprednisolone. The femoral heads were taken out and evaluated by pathology at 1, 4, 7 weeks after imageological examination (CR, CT, MRI and vascular angiography). CR and CT had no difference of bone density difference at 7 weeks. MR showed bone marrow edema in femoral head and wire-like, spot-like middle signals in T1-weighted imaging and asymmetrical high signals in T2-weighted imaging. Vascular angiography showed a decrease in vascular supply and fewer obstructed blood vessels in the femoral head. HE staining of pathological sections showed necrosis and rupture of bone trabeculae, an increased proportion of empty osteocyte lacunae, hematopoietic decreased and fat cell hypertrophy. On the study of stem cells transplantation by interventional method, the 2.7F micro-catheter was superselected into artery of femoral head, infusion the dilatation and thrombus dissolution drugs after angiography, then inject the rMSCs about 5×106 /5ml into the target vascular. At 1, 4, 7 weeks after cell transplantation, the femoral heads were taken out and pathological examination was made after imagological examination. Compared with the control group, MR showed hat abnormal signals significantly decreased area of in femoral head in the therapy group, and hydrarthrosis disappeared. Vascular angiography showed the increase of vascular supply and richness of vessel branch. Pathological examination showed the decrease of bone trabeculae necrosis and number of empty osteocyte lacunae, active bone formation, the thickness and number of chondrocyte increased in the therapy group, no significantly difference between the labeled and unlabeled groups. Blue staining particles ware detected in cartilage and subchondral bone tissues in labeled groups by Prussian Blue staining, which indicated that rMSCs might live in the necrosis area of femoral head after transplantation by interventional method. The results suggested that simply perfusion of dilatation and thrombus dissolution drugs obviously improved local blood supply and promoted bone repair and reconstruction. Furthermore, after a certain amount of perfusion through catheter,MSCS could survive topically, which provided a theoretical and practical basis for the clinical application.
4 Isolation and differentiation of human fetal mesenchymal stem cells in vitro
Bone marrow were obtained from aborted fetal (4-6 months) four limbs, and washed bone marrow by DMEM, then the separated cells were added into L-DMEM /F12 supplemented with 20% fetal bovine serum, 10000U/L penicillin and 10000U/L streptomycin, plated into 100mm plastic dish at a density of 2-5×105cells/cm2 and incubated at 37℃in 5%CO2. At 80% confluence, cells were harvested with 0.25% trypsin and replanted in new dish. The fMSCs were indentified by growth conditation, cell growth curves, cell cycle analysis, immune-phenotypical characterization and differentiation into osteocytes and adipocytes. Most adhered cells have spindle shaped with obviously polar characters. The culture cells remained dormant for 1 to 2 days and then began to multiply rapidly at 3 days and reach flat phase at 7 days. Cell cycle analysis showed that 85% of exponential phase of P3 growth cells were in G0 and S phase. They were positive for CD29 and CD105, negative for CD45. The Von Kossa staining detected the formation of calcium nodus at 21 days after induced by osteogenic medium, and the osceocalcin (OC) mRNA was expressed in the inductor group but no expressed in the control group assayed with the reverse transcript polymerase chain reaction. Adipogenic differentiation was induced in the expanded fMSCs, the accumulation of lipid-rich vacuoles within cells was apparent at 10 days, and positive express by Oil-red-O staining. Our study indicated that the cells isolated from fetal bone marrow have the characteristic of MSCs.
5 Study on the labeling fMSCs combining with ferumoxides and poly-l-lysine
The P3 exponential phase of growth fMSCs labeled with ferumoxides(25μg/ml) and PLL(0.75μg/ml) for 12h at 37℃in a 5% CO2 atmosphere. The cell growth curve of labeled cells was similar to the unlabeled cells, and there was no difference in the rate of apoptosis. Labeling efficiency was reproducible in approximately 98% of fMSCs by Prussian blue staining and no stainable iron was detected in the unlabeled cells. Analysis of transmission electron microscope showed that a great lot of iron particles locate in endosoma of labeled cells. MR imaging of cell suspensions was performed at 1.5T by using surface coil, and a dramatic signal decrease was observed in the T2 weighted MR imagines of labeled cells compared with unlabeled control cells, specially in the T2*weighted MR imagines. The fMSCs were successfully labeled with Ferumoxides and PLL, which provides experimental basis for tracking magnetic labeled MSCs in clinical application.
We isolated and expanded rMSCs and fMSCs by density gradient separation method and whole bone marrow culture methods, and confirmed the characteristic of MSCs by growth conditation, immunephenoty and multipotentiality. The model of ANFH was established in rabbit and investigated rMSCs transplantation by interventional therapy. Our results may provide references for clinical research on magnetic labeling MCSs and necrosis therapy of stem cells transplantation.
引文
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