SPIO对两种干细胞生物学特性影响及利用磁靶向技术进行细胞迁移的初探
发布时间：2018-09-10 13:18
【摘要】：背景和目的:龋病和牙周病是临床常见疾病、多发病,往往造成牙髓坏死和牙槽骨吸收以致牙齿缺失。现有的诊疗技术往往无法真正意义上实现牙髓、牙周组织的生理性和功能性再生。因此,在当代口腔医学领域,牙髓和牙周组织再生治疗仍然是学者们研究的热点。随着组织工程技术的不断发展,外源性干细胞植入和自体干细胞归巢疗法在许多组织器官再生的实验研究中已经被广泛开展。然而,由于牙齿解剖结构的特殊性,学者们往往难以将干细胞有效的迁移并富集在牙髓或者牙周组织,同时在进行干细胞治疗过程中也无法对植入的干细胞进行示踪观察,这些都对牙髓和牙周组织原位再生带来了极大的挑战。近年来出现的磁靶向技术可以极大的促进植入的干细胞在靶器官的迁移和定植,同时与之相辅相成的临床MRI检测手段还可以对植入细胞进行无创的示踪观察,这些都为学者进行牙髓和牙周组织再生指明了新的研究方向。在本试验中,我们首先选择了一种新的自带荧光且无需转染剂的SPIO(Molday ION Rhodamine-B,MIRB?)对人牙髓干细胞进行了标记,探讨了对其活力、增殖、分化等一系列生物学影响,进一步通过MRI技术对裸鼠皮下植入的细胞进行无创示踪观察。接下来我们用SPIO标记了大鼠骨髓间充质干细胞,在体外磁场环境下诱导细胞迁移,同时构建大鼠牙槽骨缺损模型,从静脉注射以及局部注射两种细胞输入途径进行研究,观察体内环境下磁靶向技术在促进干细胞在牙槽骨缺损处的迁移和定植。本实验实现了MRI活体示踪观察植入的干细胞,还丰富了磁靶向技术在牙周组织再生中的运用,为未来牙周再生干细胞疗法提供了新思路。结果如下:第一部分MIRB标记对h DPSCs的生物学影响以及体内体外MRI成像1.人牙髓干细胞(h DPSCs)培养和鉴定本研究采用酶消化法/组织块法从成人第三磨牙牙髓组织中分离培养人DPSCs,利用单细胞克隆法纯化细胞,通过体外成骨、成脂诱导分化以及流式细胞仪鉴定确定了其干细胞属性。2.MIRB标记h DPSCs分别用终浓度为12.5,25,50,100μg Fe/m L的SPIO(MIRB?)标记h DPSCs,利用激光共聚焦、普鲁士蓝染色光镜观察以及透射电镜等手段观察了SPIO纳米颗粒在h DPSCs细胞内的分布情况,分光光度计测量了各组细胞内铁含量。结果发现,MIRB可以不需要转染剂即可高效的标记h DPSCs,各种浓度标记后对细胞的形态没有影响,且随着标记浓度的升高细胞内铁含量也逐渐上升。3.MIRB对h DPSCs体外生物学特性的影响采用台盼蓝排斥实验、CCK8实验、流式细胞仪、体内外成骨诱导等手段分别检测了MIRB标记对h DPSCs的活力、增殖、干细胞表型、细胞周期、凋亡以及成骨分化的影响。结果发现,12.5-50μg/m L的MIRB标记不影响h DPSCs的活力且可以促进细胞增殖,而100μg/m L则对细胞有毒性,最佳标记浓度为12.5μg/m L;12.5μg/m L的MIRB标记不影响h DPSCs的干细胞表型;12.5μg/m L-50μg/m L的MIRB标记可以加快细胞周期而对细胞凋亡无影响;12.5μg/m L-50μg/m L的MIRB对h DPSCs成骨分化无影响。4.MIRB标记牙髓干细胞的体外、体内MRI成像观察通过MRI检测手段,体外观察了MIRB标记的h DPSCs细胞团块,体内观察了MIRB标记的细胞膜片在裸鼠皮下的生长情况。体外结果发现,12.5g/m L的MIRB标记1×10~5个细胞无法在MRI清晰显影,而用12.5g/m L-100g/m L的MIRB标记1×106个细胞均可清晰显影。体内结果发现,MIRB标记的h DPSCs细胞膜片在MRI下可以清晰呈负性对比影像,且随着植入时间延长,无信号影像所占比率也逐渐下降。组织学检查发现,膜片内普鲁士蓝阳性染色细胞数量也随着时间的延长而逐渐下降。第二部分SPIO对大鼠骨髓间充质干细胞的生物学影响1.大鼠骨髓间充质干细胞(r BMMSCs)培养和鉴定成功采用全骨髓贴壁法培养了大鼠骨髓间充质干细胞(r BMMSCs)。通过克隆形成实验、干细胞表型测定以及多向分化能力检测确定其干细胞属性。2.Resovist标记r BMMSCs分别用终浓度为25、50、100g/m L的SPIO(Resovist?)标记r BMMSCs,利用普鲁士蓝染色光镜观察以及透射电镜等手段观察了Resovist纳米颗粒在r BMMSCs细胞内的分布情况。结果发现,Resovist同样可以不需要转染剂即可高效的标记r BMMSCs。3.Resovist对r BMMSCs生物学特性的影响采用台盼蓝排斥实验、CCK8实验、流式细胞仪、体外成骨诱导等手段分别检测了Resovist标记对r BMMSCs的活力、增殖、细胞周期、凋亡以及成骨分化的影响。结果发现,25-50g/m L的Resovist对r BMMSCs活力没有影响,而100g/m L对细胞有毒性;25g/m L不影响细胞增殖、细胞周期以及凋亡,而50-100g/m L则抑制细胞增殖、减缓细胞周期并促进细胞凋亡,说明25g/m L的Resovist是标记r BMMSCs的合适浓度;25g/m L的Resovist对r BMMSCs的成骨分化有促进作用。第三部分SPIO标记的大鼠骨髓间充质干细胞在体外体内磁场环境下迁移和定植的实验研究1.体外磁场环境下SPIO标记的r BMMSCs迁移实验利用Transwell小室,在体外磁场环境下观察SPIO标记的r BMMSCs的穿膜能力。结果发现,磁场可以明显促进SPIO标记的细胞穿膜。另外,我们在培养皿底部正中心粘贴磁铁,将SPIO标记的r BMMSCs接种后观察细胞在培养皿内的分布情况,发现细胞围绕磁铁周围呈圆环状分布。2.体内磁场环境下SPIO标记的r BMMSCs向牙槽骨缺损处的磁靶向迁移或定植构建大鼠牙槽骨缺损模型,分别从静脉途径和局部注射途径输入SPIO标记的干细胞。结果发现,从静脉输入的干细胞大部分被脾脏内巨噬细胞所吞噬,在牙槽骨缺损处未见到远距离迁移的干细胞,而局部注射标记后的干细胞由于有磁场作用可以大量聚集在牙槽骨缺损处。结论:1.第一部分研究结果表明,目前市售的最新的纳米铁颗粒MIRB可以无需添加额外的转染剂即可高效标记人牙髓干细胞;12.5g/m L-50g/m L的MIRB标记人牙髓干细胞是安全可靠的;MRI可以对植入体内的h DPSCs细胞膜片进行无创实时观察,是干细胞移植治疗中进行细胞示踪安全有效的手段,为未来利用牙髓干细胞移植进行牙髓原位再生提供了实验依据。2.第二部分研究结果发现,德国Schering的纳米铁颗粒Resovist同样可以无需转染剂即可高效标记大鼠骨髓间充质干细胞;25g/m L的Resovist不影响大鼠骨髓间充质干细胞的增殖和活力,50-100g/m L的Resovist则抑制r BMMSCs增殖、减缓细胞周期并促进细胞凋亡,说明25g/m L的Resovist是标记r BMMSCs的合适浓度;25g/m L的Resovist对r BMMSCs的成骨分化有促进作用,其具体机制还需进一步深入探究。3.第三部分研究结果表明,体外磁场环境下可以完美模拟出标记了SPIO的干细胞在磁场作用下发生靶向迁移;体内环境下,构建了牙槽骨缺损的磁靶向诱导细胞归巢实验,结果发现远距离磁靶向诱导SPIO标记的干细胞归巢至牙槽骨缺损处没有得到理想的预期效果,而磁场促进了局部注射的SPIO标记的干细胞在牙槽骨缺损处的定植。定植的干细胞是否可以在骨缺损处继续增殖、分化为新的骨细胞并继而参与牙槽骨的重建尚需要进一步更深入的研究去证实。
[Abstract]:BACKGROUND AND OBJECTIVE: Dental caries and periodontal diseases are common clinical diseases, frequently occurring, often resulting in pulp necrosis and alveolar bone resorption, resulting in tooth loss. Existing diagnostic and therapeutic techniques often fail to truly achieve the physiological and functional regeneration of pulp and periodontal tissue. With the development of tissue engineering technology, exogenous stem cell transplantation and autologous stem cell homing therapy have been widely used in many experimental studies of tissue and organ regeneration. In-situ regeneration of dental pulp and periodontal tissues is challenged by the fact that implanted stem cells can not be traced during stem cell therapy, and magnetic targeting technology can greatly promote the migration and colonization of implanted stem cells in target organs. In this study, we first selected a new self-fluorescent SPIO (Molday ION Rhodamine-B, MIRB?) for human dental pulp stem. Then we used SPIO to label rat bone marrow mesenchymal stem cells to induce cell migration in vitro and construct rat alveolar bone defect. In this study, we observed the effect of magnetic targeting technique on the migration and colonization of stem cells in the alveolar bone defect in vivo. MRI in vivo was used to observe the implanted stem cells and enrich the application of magnetic targeting technique in periodontal tissue regeneration. The results are as follows: Part 1: Biological effects of MIRB labeling on H DPSCs and MRI imaging in vivo and in vitro 1. Human dental pulp stem cells (h DPSCs) culture and identification This study used enzymatic digestion/tissue block method to isolate and culture human DPSCs from adult third molar pulp tissue, using single cell gram. MiRB labeled h DPSCs were labeled with SPIO (MIRB?) of 12.5,25,50,100 UG Fe/ml at the final concentration, respectively. Confocal laser, Prussian blue staining light microscopy and transmission electron microscopy were used to observe SPIONA. Distribution of rice granules in H DPSCs cells was measured by spectrophotometer. The results showed that MIRB could efficiently label h DPSCs without transfection. The morphology of cells was not affected by different concentrations of MIRB, and the content of iron in cells increased gradually with the increase of the concentration of MIRB. The effects of MIRB on the viability, proliferation, stem cell phenotype, cell cycle, apoptosis and osteogenic differentiation of H DPSCs were investigated by trypan blue exclusion test, CCK8 test, flow cytometry and osteogenic induction in vitro and in vivo. The optimal labeling concentration was 12.5 ug/ml; the 12.5 ug/ml MIRB labeling did not affect the stem cell phenotype of H DPSCs; the 12.5 ug/ml-50 ug/ml MIRB labeling could accelerate the cell cycle but had no effect on the cell apoptosis; the 12.5 ug/ml-50 ug/ml MIRB had no effect on the osteogenic differentiation of H DPSCs. RB labeled dental pulp stem cells in vitro, in vivo MRI imaging observation through MRI detection, in vitro observation of MIRB labeled h DPSCs cell mass, in vivo observation of MIRB labeled cell membrane in nude mice subcutaneous growth. In vitro, 12.5g/ml of MIRB labeled 1 *105 cells can not be clearly developed in MRI, but with 12.5g/ml-100g/m. In vivo, MIRB-labeled h DPSCs showed a clear negative contrast image, and the proportion of non-signal images decreased with the implantation time. Histological examination showed that the number of Prussian blue positive cells in the membrane also decreased with the implantation time. The second part is the biological effects of SPIO on rat bone marrow mesenchymal stem cells (BMMSCs). The culture and identification of rat bone marrow mesenchymal stem cells (r BMMSCs) were successfully carried out by whole bone marrow adherence method. The phenotype of stem cells and the ability of multi-directional differentiation were determined by clone formation test. Resovist-labeled R BMMSCs were labeled with SPIO (Resovist?) of 25,50,100 g/ml, respectively. The distribution of Resovist nanoparticles in R BMMSCs was observed by Prussian blue staining and transmission electron microscopy. The effects of Resovist on the biological characteristics of R BMMSCs were studied by trypan blue exclusion test, CCK8 test, flow cytometry and osteogenesis induction in vitro. The results showed that the effect of Resovist on the activity, proliferation, cell cycle, apoptosis and osteogenic differentiation of R BMMSCs was 25-50g/ml. BMMSCs activity was not affected, but 100g/ml was toxic to cells; 25g/ml did not affect cell proliferation, cell cycle and apoptosis, while 50-100g/ml inhibited cell proliferation, slowed cell cycle and promoted cell apoptosis, suggesting that 25g/ml of Roesovist was the appropriate concentration for labeling R BMMSCs; 25g/ml of Roesovist could promote osteogenic differentiation of R BMMSCs. Part 3. Experimental study on the migration and colonization of SPIO-labeled rat bone marrow mesenchymal stem cells in vitro and in vivo magnetic field 1. The migration of SPIO-labeled R BMMSCs in vitro magnetic field was studied by using Transwell chamber. The permeability of SPIO-labeled R BMMSCs in vitro magnetic field was observed. In addition, the distribution of SPIO-labeled R BMMSCs in the culture dish was observed after inoculation. The cells were circularly distributed around the magnets. 2. The magnetic targeted migration or colonization of SPIO-labeled R BMMSCs to the alveolar bone defect was observed in vivo. SPIO-labeled stem cells were injected into alveolar bone defect model of rats through intravenous route and local injection route respectively. Conclusion: 1. The results of the first part of the study show that the latest nano-iron particles MIRB on the market can effectively label human dental pulp stem cells without additional transfectants; 12.5g/ml-50g/ml MIRB labeling human dental pulp stem cells is safe and reliable; MRI can be used to label human dental pulp stem cells in vivo; 2. Non-invasive real-time observation of PSCs cell membrane is a safe and effective method for cell tracing in stem cell transplantation. It provides an experimental basis for in-situ regeneration of dental pulp using dental pulp stem cell transplantation in the future. 2. The results of the second part show that the Resovist of Schering nanoparticles can also be high without transfection agent. Rat bone marrow mesenchymal stem cells were effectively labeled; 25g/m L Roesovist did not affect the proliferation and viability of rat bone marrow mesenchymal stem cells, while 50-100g/m L Roesovist inhibited the proliferation of R BMMSCs, slowed cell cycle and promoted cell apoptosis, indicating that 25g/m L Roesovist was the appropriate concentration for labeling R BMMSCs; 25g/m L Roesovist did not affect the osteogenesis of R BMMSCs. The third part of the study shows that the magnetic field can simulate the target migration of SPIO-labeled stem cells under the magnetic field in vitro. In vivo, a magnetic targeted induction cell homing experiment of alveolar bone defect was constructed, and the results showed that the long-distance migration of the cells labeled with SPIO could be simulated perfectly. Magnetic field can promote the implantation of SPIO-labeled stem cells in alveolar bone defects. Whether the implanted stem cells can continue to proliferate, differentiate into new bone cells and then participate in the weight of alveolar bone Jian Shang needs further in-depth study to prove it.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R781
，
本文编号：2234554
[Abstract]:BACKGROUND AND OBJECTIVE: Dental caries and periodontal diseases are common clinical diseases, frequently occurring, often resulting in pulp necrosis and alveolar bone resorption, resulting in tooth loss. Existing diagnostic and therapeutic techniques often fail to truly achieve the physiological and functional regeneration of pulp and periodontal tissue. With the development of tissue engineering technology, exogenous stem cell transplantation and autologous stem cell homing therapy have been widely used in many experimental studies of tissue and organ regeneration. In-situ regeneration of dental pulp and periodontal tissues is challenged by the fact that implanted stem cells can not be traced during stem cell therapy, and magnetic targeting technology can greatly promote the migration and colonization of implanted stem cells in target organs. In this study, we first selected a new self-fluorescent SPIO (Molday ION Rhodamine-B, MIRB?) for human dental pulp stem. Then we used SPIO to label rat bone marrow mesenchymal stem cells to induce cell migration in vitro and construct rat alveolar bone defect. In this study, we observed the effect of magnetic targeting technique on the migration and colonization of stem cells in the alveolar bone defect in vivo. MRI in vivo was used to observe the implanted stem cells and enrich the application of magnetic targeting technique in periodontal tissue regeneration. The results are as follows: Part 1: Biological effects of MIRB labeling on H DPSCs and MRI imaging in vivo and in vitro 1. Human dental pulp stem cells (h DPSCs) culture and identification This study used enzymatic digestion/tissue block method to isolate and culture human DPSCs from adult third molar pulp tissue, using single cell gram. MiRB labeled h DPSCs were labeled with SPIO (MIRB?) of 12.5,25,50,100 UG Fe/ml at the final concentration, respectively. Confocal laser, Prussian blue staining light microscopy and transmission electron microscopy were used to observe SPIONA. Distribution of rice granules in H DPSCs cells was measured by spectrophotometer. The results showed that MIRB could efficiently label h DPSCs without transfection. The morphology of cells was not affected by different concentrations of MIRB, and the content of iron in cells increased gradually with the increase of the concentration of MIRB. The effects of MIRB on the viability, proliferation, stem cell phenotype, cell cycle, apoptosis and osteogenic differentiation of H DPSCs were investigated by trypan blue exclusion test, CCK8 test, flow cytometry and osteogenic induction in vitro and in vivo. The optimal labeling concentration was 12.5 ug/ml; the 12.5 ug/ml MIRB labeling did not affect the stem cell phenotype of H DPSCs; the 12.5 ug/ml-50 ug/ml MIRB labeling could accelerate the cell cycle but had no effect on the cell apoptosis; the 12.5 ug/ml-50 ug/ml MIRB had no effect on the osteogenic differentiation of H DPSCs. RB labeled dental pulp stem cells in vitro, in vivo MRI imaging observation through MRI detection, in vitro observation of MIRB labeled h DPSCs cell mass, in vivo observation of MIRB labeled cell membrane in nude mice subcutaneous growth. In vitro, 12.5g/ml of MIRB labeled 1 *105 cells can not be clearly developed in MRI, but with 12.5g/ml-100g/m. In vivo, MIRB-labeled h DPSCs showed a clear negative contrast image, and the proportion of non-signal images decreased with the implantation time. Histological examination showed that the number of Prussian blue positive cells in the membrane also decreased with the implantation time. The second part is the biological effects of SPIO on rat bone marrow mesenchymal stem cells (BMMSCs). The culture and identification of rat bone marrow mesenchymal stem cells (r BMMSCs) were successfully carried out by whole bone marrow adherence method. The phenotype of stem cells and the ability of multi-directional differentiation were determined by clone formation test. Resovist-labeled R BMMSCs were labeled with SPIO (Resovist?) of 25,50,100 g/ml, respectively. The distribution of Resovist nanoparticles in R BMMSCs was observed by Prussian blue staining and transmission electron microscopy. The effects of Resovist on the biological characteristics of R BMMSCs were studied by trypan blue exclusion test, CCK8 test, flow cytometry and osteogenesis induction in vitro. The results showed that the effect of Resovist on the activity, proliferation, cell cycle, apoptosis and osteogenic differentiation of R BMMSCs was 25-50g/ml. BMMSCs activity was not affected, but 100g/ml was toxic to cells; 25g/ml did not affect cell proliferation, cell cycle and apoptosis, while 50-100g/ml inhibited cell proliferation, slowed cell cycle and promoted cell apoptosis, suggesting that 25g/ml of Roesovist was the appropriate concentration for labeling R BMMSCs; 25g/ml of Roesovist could promote osteogenic differentiation of R BMMSCs. Part 3. Experimental study on the migration and colonization of SPIO-labeled rat bone marrow mesenchymal stem cells in vitro and in vivo magnetic field 1. The migration of SPIO-labeled R BMMSCs in vitro magnetic field was studied by using Transwell chamber. The permeability of SPIO-labeled R BMMSCs in vitro magnetic field was observed. In addition, the distribution of SPIO-labeled R BMMSCs in the culture dish was observed after inoculation. The cells were circularly distributed around the magnets. 2. The magnetic targeted migration or colonization of SPIO-labeled R BMMSCs to the alveolar bone defect was observed in vivo. SPIO-labeled stem cells were injected into alveolar bone defect model of rats through intravenous route and local injection route respectively. Conclusion: 1. The results of the first part of the study show that the latest nano-iron particles MIRB on the market can effectively label human dental pulp stem cells without additional transfectants; 12.5g/ml-50g/ml MIRB labeling human dental pulp stem cells is safe and reliable; MRI can be used to label human dental pulp stem cells in vivo; 2. Non-invasive real-time observation of PSCs cell membrane is a safe and effective method for cell tracing in stem cell transplantation. It provides an experimental basis for in-situ regeneration of dental pulp using dental pulp stem cell transplantation in the future. 2. The results of the second part show that the Resovist of Schering nanoparticles can also be high without transfection agent. Rat bone marrow mesenchymal stem cells were effectively labeled; 25g/m L Roesovist did not affect the proliferation and viability of rat bone marrow mesenchymal stem cells, while 50-100g/m L Roesovist inhibited the proliferation of R BMMSCs, slowed cell cycle and promoted cell apoptosis, indicating that 25g/m L Roesovist was the appropriate concentration for labeling R BMMSCs; 25g/m L Roesovist did not affect the osteogenesis of R BMMSCs. The third part of the study shows that the magnetic field can simulate the target migration of SPIO-labeled stem cells under the magnetic field in vitro. In vivo, a magnetic targeted induction cell homing experiment of alveolar bone defect was constructed, and the results showed that the long-distance migration of the cells labeled with SPIO could be simulated perfectly. Magnetic field can promote the implantation of SPIO-labeled stem cells in alveolar bone defects. Whether the implanted stem cells can continue to proliferate, differentiate into new bone cells and then participate in the weight of alveolar bone Jian Shang needs further in-depth study to prove it.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R781
，
本文编号：2234554
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