同步辐射技术检测干细胞移植后大鼠后肢新生血管生成的实验研究
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摘要
第一部分同步辐射成像技术检测大鼠后肢微血管的实验研究
目的：目前临床应用的血管成像技术无法检测直径在200微米以下的微小血管。同步辐射(Synchrotron Radiation, SR)成像技术的发展为检测更细的微血管提供了新的手段。本实验旨在利用同步辐射微血管成像技术对大鼠后肢的微血管进行成像观察,并且和传统的X线成像对比,为进一步的实验提供必要的成像参数和基础。
材料和方法：利用同步辐射单色光对F344近交系大鼠的左后肢微血管进行活体和离体成像观察。活体成像为吸收成像,从对侧髂动脉穿刺置管至腹主动脉分叉,注射含碘造影剂同时造影成像,单色光能量设为33.4Kev。离体成像的标本制备采用硫酸钡和生理盐水作为对比剂注入大鼠后肢微循环,采用相位衬度成像,能量和物像距离分别为15Kev和800mm,同时进行微CT血管三维重建。活体成像和离体成像分别采用数字减影血管造影(Digital subtraction angiography,DSA)造影和普通x线摄片作为对照,比较血管的直径和造影评分的差异。同步辐射成像分别用分辨率为9μm/pixel和2.25μm/pixel的探测器进行检测。血管直径的计算和图像编辑用NIH Image-pro plus软件进行处理。
结果：同步辐射微血管活体成像可清晰显示大鼠髂动脉的4级以上分支,最细血管直径约40μm；而普通DSA造影则无法显示如此清晰的微血管,只能显示髂动脉的3级分支。离体标本中,灌注生理盐水的标本可辨认血管,但衬度和“边缘增强”效应不明显。灌注硫酸钡的样本血管清晰可见,高分辨率CCD(2.25μm)可测量的最小血管直径为9μm。而标本经普通X光成像,血管显影差。活体和离体同步辐射成像的血管造影评分明显高于普通X线成像。对灌注硫酸钡的整体肢体进行同步辐射微血管三维成像发现,肢体血管树的三维结构清晰显示,可以测量和定位大鼠后肢中微米级别的微血管。
结论：应用同步辐射成像技术以及合适的对比剂可清晰显示大鼠后肢的微循环,和普通X线成像相比,同步辐射血管成像可显示更为细微的内容,最细可检测到直径10μm左右的大鼠后肢血管。同步辐射成像技术可为进一步检测干细胞移植后生成的新生血管提供有效手段。
第二部分大鼠骨髓间充质干细胞的培养和定向分化能力的研究
目的：骨髓来源的间充质干细胞(Mesenchymal stem cells, MSCs)移植为治疗下肢缺血性疾病开辟了一个新的思路,但是关于它的分离培养方法以及定向分化能力目前仍然缺乏统一的观点。本实验旨在从大鼠骨髓中获取间充质干细胞并进行原代培养和鉴定,研究培养方法和接种密度对细胞形态和定向分化能力的影响,为进一步的干细胞移植实验提供基础。
材料和方法：取6-8周龄F344大鼠的骨髓以全骨髓贴壁法进行原代培养,并以不同的比例和接种密度(3×10,3×102,3×103 cells/cm2)进行传代,观察接种密度对细胞形态和生长特点的影响。按适宜的接种密度对细胞进行传代,取第3代对数生长期的rMSCs,对其表面标志物用流式细胞仪和免疫荧光进行鉴定。同时在传代的rMSCs培养液中分别添加不同的化学试剂和细胞因子,观察细胞的形态结构变化,诱导其向成骨细胞、脂肪细胞和内皮细胞分化,并对分化的细胞用碱性磷酸酶、油红O和DiI-Ac-LDL吞噬实验进行鉴定。
结果：原代培养的rMSCs呈较强的贴壁生长特性,形态上呈纺锤形或扁平多角形。当细胞按接种密度3×10 cells/cm2进行传代时,细胞的倍增速度最快,并且形态较均一,纺锤形的细胞数量多,呈菌落样生长。对低密度接种培养的rMSCs进行流式细胞仪和免疫荧光鉴定,提示rMSCs的表面抗原为CD34(-)、CD45 (-)、CD90(+)、CD29 (+)、CD105 (+)。定向分化实验表明,在不同的诱导条件下,低密度接种培养的rMSCs可以向成骨细胞、脂肪细胞和内皮细胞定向分化,分化后的细胞用碱性磷酸酶、油红O和DiI-Ac-LDL吞噬实验鉴定为阳性反应。
结论：全骨髓贴壁培养法和较低接种密度培养可获得数量较多的相对较纯的大鼠骨髓间充质干细胞,其表面标志物符合间充质干细胞标准,并且具有多能定向分化能力。
第三部分同步辐射技术检测干细胞移植后大鼠后肢新生血管生成的实验研究
目的：目前干细胞移植后新生血管的生成缺乏切实有效的评价方法。本研究应用同步辐射技术检测大鼠骨髓间充质细胞(Rat bone marrow-derived mesenchymal stem cells, rBMSC)移植到大鼠缺血肢体后新生血管(Neovascularization)的生成情况,观察分析干细胞移植后新生血管空间密度分布和形态结构的变化,为干细胞移植治疗缺血性疾病的效果评价提供新的思路和有效检测手段。
材料和方法：取F344近交系大鼠24只,制作左后肢缺血模型后随机分为rBMSC移植组(A组)、PBS对照组(B组)和假手术组(C组)接受治疗。在细胞移植后第3天、14天和21天采用同步辐射微血管吸收成像技术对各组动物的左后肢新生血管进行活体成像观察,于细胞移植后第4周进行同步辐射离体成像分析和检测,评估各阶段新生血管形态和密度的变化。同时对各组动物用激光多普勒血流灌注成像(Laser-Doppler perfusion imaging, LDPI)检测缺血后肢血流情况和对缺血后肢损伤功能进行评分,并用免疫组化法检测毛细血管密度,比较分析各组血流灌注指数(Perfusion ratio, PR)和新生血管密度的差异。
结果：同步辐射微血管活体成像提示,术后第14天和第21天,A组和B组的血管密度和造影评分均较第3天明显增高,且A组的血管造影评分明显高于B组(14天,0.54±0.036 vs 0.34±0.032;21天,0.64±0.037 vs 0.49±0.027,P<0.05)。比较第21天A、B两组的血管形态发现,A组的新生血管形态和排列较B组血管规则。C组的血管形态密度在术后的随访中基本保持一致。LDPI检测提示细胞移植第7天以后,A组动物缺血肢体的血流灌注指数明显高于B组。缺血后肢损伤评分的结果显示A组损伤评分明显低于B组。同步辐射离体成像分析提示A组的新生血管密度较B组高,和免疫组化检测的毛细血管密度结果一致。
结论：同步辐射微血管成像技术是干细胞移植后肢体新生血管检测的有效手段,其检测结果提示干细胞移植后能促进新生血管密度和形态结构的发生发展。
Part One Detection of Microvasculature in Rat Hind Limb Using Synchrotron Radiation
Objective:Conventional radiography cannot provide sufficiently clear images of vessels with diameters of 200μm or less. New X-ray microangiography and third-generation synchrotron radiation-based microcomputed tomography have opened new perspectives for microvascular imaging of extremity. Here we aimed to visualize deep-level microvascular structure in rat hind limb by microangiographic technique, and compare images with those by conventional method.
Materials and Methods:Inbred strain rats (F344) were used for in vivo and ex vivo study. Microangiography in vivo and ex vivo was performed and images were compared with those by conventional method. In microangiography in vivo study, the catheter was connected to an automated angiographic injector, through which the nonionic contrast media was injected into blood vessels in rat hind limbs. A monochromatic 33.4 keV X-ray source just above the K-absorption edge of iodine was used for imaging. In vitro study, normal saline and barium sulfate solution was infused through catheter to the left iliac artery of rats, and the phase contrast imaging were performed with the energy of 15 Kev and the object-to-image distance of 800mm. Synchrotron radiation-based micro-computed tomography (SRmCT) was also performed to reveal three-dimensional (3D) morphology of the blood vessel in rat hind limb.
Results:Using microangiographic technique in vivo and in vitro (with barium sulfate), blood vessels in the rat limb muscle could be visualized with high resolution and more detail compared with those by conventional technique, and the fifth to sixth branches of iliac artery in rat hind limb could be detected with the minimum visualized blood vessels about 40μm and 9μm in diameter, respectively. Accordingly, higher angiographic scores were achieved than those by conventional X-ray. In addition, the vascular network could be defined and analyzed at the micrometer scale from the 3D renderings of limb vessel as shown by SRmCT.
Conclusions:Synchrotron radiation-based microangiography and SRmCT thus provided a practical and effective means to observe the microvasculature of rat hindlimb, which might be useful in assessment of angiogenesis in lower limbs.
Part Two
The study of culturing and multilineage potential of rat marrow-derived mesenchymal stem cells
Objective:Bone marrow mesenchymal stem cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in limb ischemic disease. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of plating density and culturing method on proliferation, cell morphology, and differentiation potential of MSC, and to provide an effective approach of cell obtaining for stem cell transplantiation.
Materials and Methods:Rat MSCs were isolated from rat bone marrow relying on their ability to adhere to plastic, and replated at appropriate seeding density (3x10, 3x102 and 3x103 cells/cm2) at each passage. Cell morphology and growth characteristic were evaluated and compared among different seeding density. Passage 3 MSCs from cultures seeded at optimal plated density were used for identification of their surface marker by flow cytometry and immunofluorescence. For differentiation assays, cells from passage 3 were cultured in induction medium supplemented with different chemical agents and cytokines. Differentiation of MSCs into adipogenic, osteogenic, and endothelial lineages were identified by Oil Red-O stain for lipid deposits, stained for kaline phosphatase by an indoxyltetrazolium method, and incubated in the endothelial differentiation medium containing Dil-Ac-LDL and stained with BS-1-lectin, respectively.
Results:Primary cultur of rMSCs showed a spindle-like or flattened polygonal appearance with the characteristic of adherence to plastic. The low density culture consisted of a relatively homogenous monolayer of spindle-like cells while high density cultures appeared heterogeneous in which nonfibroblastic cells were present. Cell colonies were observed in low density culture with high population doubling speed. Flow cytometric analysis of the low density culture at passage 3 demonstrated to be positive for CD90, CD29, CD105, but negative for CD34, CD45. Under certain conditions, rMSCs of low density culture were capable of differentiating into multilineage cell types, including adipogenic cells which were positive for oil red-0 stain, osteogenic lineages with positive stain for kaline phosphatase by an indoxyltetrazolium method, and endothelial cells with positive result of both LDL-uptaking assay and BS-1-lectin stain.
Conclusion:Based on the results, we concluded that adherent culturing method combined with the low density culture system is a more appropriate system to isolate and expand murine MSCs from marrow samples. With low density culture system, more purified mMSCs which express putative surface maker and have multiple differential potential could be produced. density (AMVD) was increased at 14 and 21 days in both group A and group B, and significant higher in group A compared with group B (0.54±0.036 vs 0.34±0.032 and 0.64±0.037 vs 0.49±0.027, P＜0.05, for 14 days and 21 days, respectively). In addition, the microvascular pattern and arrangement was more regular in group A, when compare with group B at 21 days after transplantation. There were no significant changes of microvasculature in the sham operation group (group C). A significant improvement of blood flow perfusion and lower injury score was found in group A, when compared with group B since 7 days after cell transplantation. The results of microvascular density by microangiography ex vivo indicated there was a higher microvascular density in group A than in group B, which were consistent with the results of immunohistochemistry stain.
Conclusion:SR based microangiography is an effective method for the detection of angiogenesis after stem cell transplantation in a rat model of hindlimb ischemia, which suggested a higher microvascular density and more regular branching pattern of neovascularization in rMSC transplantation group compared with PBS group.
目的：目前临床应用的血管成像技术无法检测直径在200微米以下的微小血管。同步辐射(Synchrotron Radiation, SR)成像技术的发展为检测更细的微血管提供了新的手段。本实验旨在利用同步辐射微血管成像技术对大鼠后肢的微血管进行成像观察,并且和传统的X线成像对比,为进一步的实验提供必要的成像参数和基础。
材料和方法：利用同步辐射单色光对F344近交系大鼠的左后肢微血管进行活体和离体成像观察。活体成像为吸收成像,从对侧髂动脉穿刺置管至腹主动脉分叉,注射含碘造影剂同时造影成像,单色光能量设为33.4Kev。离体成像的标本制备采用硫酸钡和生理盐水作为对比剂注入大鼠后肢微循环,采用相位衬度成像,能量和物像距离分别为15Kev和800mm,同时进行微CT血管三维重建。活体成像和离体成像分别采用数字减影血管造影(Digital subtraction angiography,DSA)造影和普通x线摄片作为对照,比较血管的直径和造影评分的差异。同步辐射成像分别用分辨率为9μm/pixel和2.25μm/pixel的探测器进行检测。血管直径的计算和图像编辑用NIH Image-pro plus软件进行处理。
结果：同步辐射微血管活体成像可清晰显示大鼠髂动脉的4级以上分支,最细血管直径约40μm；而普通DSA造影则无法显示如此清晰的微血管,只能显示髂动脉的3级分支。离体标本中,灌注生理盐水的标本可辨认血管,但衬度和“边缘增强”效应不明显。灌注硫酸钡的样本血管清晰可见,高分辨率CCD(2.25μm)可测量的最小血管直径为9μm。而标本经普通X光成像,血管显影差。活体和离体同步辐射成像的血管造影评分明显高于普通X线成像。对灌注硫酸钡的整体肢体进行同步辐射微血管三维成像发现,肢体血管树的三维结构清晰显示,可以测量和定位大鼠后肢中微米级别的微血管。
结论：应用同步辐射成像技术以及合适的对比剂可清晰显示大鼠后肢的微循环,和普通X线成像相比,同步辐射血管成像可显示更为细微的内容,最细可检测到直径10μm左右的大鼠后肢血管。同步辐射成像技术可为进一步检测干细胞移植后生成的新生血管提供有效手段。
第二部分大鼠骨髓间充质干细胞的培养和定向分化能力的研究
目的：骨髓来源的间充质干细胞(Mesenchymal stem cells, MSCs)移植为治疗下肢缺血性疾病开辟了一个新的思路,但是关于它的分离培养方法以及定向分化能力目前仍然缺乏统一的观点。本实验旨在从大鼠骨髓中获取间充质干细胞并进行原代培养和鉴定,研究培养方法和接种密度对细胞形态和定向分化能力的影响,为进一步的干细胞移植实验提供基础。
材料和方法：取6-8周龄F344大鼠的骨髓以全骨髓贴壁法进行原代培养,并以不同的比例和接种密度(3×10,3×102,3×103 cells/cm2)进行传代,观察接种密度对细胞形态和生长特点的影响。按适宜的接种密度对细胞进行传代,取第3代对数生长期的rMSCs,对其表面标志物用流式细胞仪和免疫荧光进行鉴定。同时在传代的rMSCs培养液中分别添加不同的化学试剂和细胞因子,观察细胞的形态结构变化,诱导其向成骨细胞、脂肪细胞和内皮细胞分化,并对分化的细胞用碱性磷酸酶、油红O和DiI-Ac-LDL吞噬实验进行鉴定。
结果：原代培养的rMSCs呈较强的贴壁生长特性,形态上呈纺锤形或扁平多角形。当细胞按接种密度3×10 cells/cm2进行传代时,细胞的倍增速度最快,并且形态较均一,纺锤形的细胞数量多,呈菌落样生长。对低密度接种培养的rMSCs进行流式细胞仪和免疫荧光鉴定,提示rMSCs的表面抗原为CD34(-)、CD45 (-)、CD90(+)、CD29 (+)、CD105 (+)。定向分化实验表明,在不同的诱导条件下,低密度接种培养的rMSCs可以向成骨细胞、脂肪细胞和内皮细胞定向分化,分化后的细胞用碱性磷酸酶、油红O和DiI-Ac-LDL吞噬实验鉴定为阳性反应。
结论：全骨髓贴壁培养法和较低接种密度培养可获得数量较多的相对较纯的大鼠骨髓间充质干细胞,其表面标志物符合间充质干细胞标准,并且具有多能定向分化能力。
第三部分同步辐射技术检测干细胞移植后大鼠后肢新生血管生成的实验研究
目的：目前干细胞移植后新生血管的生成缺乏切实有效的评价方法。本研究应用同步辐射技术检测大鼠骨髓间充质细胞(Rat bone marrow-derived mesenchymal stem cells, rBMSC)移植到大鼠缺血肢体后新生血管(Neovascularization)的生成情况,观察分析干细胞移植后新生血管空间密度分布和形态结构的变化,为干细胞移植治疗缺血性疾病的效果评价提供新的思路和有效检测手段。
材料和方法：取F344近交系大鼠24只,制作左后肢缺血模型后随机分为rBMSC移植组(A组)、PBS对照组(B组)和假手术组(C组)接受治疗。在细胞移植后第3天、14天和21天采用同步辐射微血管吸收成像技术对各组动物的左后肢新生血管进行活体成像观察,于细胞移植后第4周进行同步辐射离体成像分析和检测,评估各阶段新生血管形态和密度的变化。同时对各组动物用激光多普勒血流灌注成像(Laser-Doppler perfusion imaging, LDPI)检测缺血后肢血流情况和对缺血后肢损伤功能进行评分,并用免疫组化法检测毛细血管密度,比较分析各组血流灌注指数(Perfusion ratio, PR)和新生血管密度的差异。
结果：同步辐射微血管活体成像提示,术后第14天和第21天,A组和B组的血管密度和造影评分均较第3天明显增高,且A组的血管造影评分明显高于B组(14天,0.54±0.036 vs 0.34±0.032;21天,0.64±0.037 vs 0.49±0.027,P<0.05)。比较第21天A、B两组的血管形态发现,A组的新生血管形态和排列较B组血管规则。C组的血管形态密度在术后的随访中基本保持一致。LDPI检测提示细胞移植第7天以后,A组动物缺血肢体的血流灌注指数明显高于B组。缺血后肢损伤评分的结果显示A组损伤评分明显低于B组。同步辐射离体成像分析提示A组的新生血管密度较B组高,和免疫组化检测的毛细血管密度结果一致。
结论：同步辐射微血管成像技术是干细胞移植后肢体新生血管检测的有效手段,其检测结果提示干细胞移植后能促进新生血管密度和形态结构的发生发展。
Part One Detection of Microvasculature in Rat Hind Limb Using Synchrotron Radiation
Objective:Conventional radiography cannot provide sufficiently clear images of vessels with diameters of 200μm or less. New X-ray microangiography and third-generation synchrotron radiation-based microcomputed tomography have opened new perspectives for microvascular imaging of extremity. Here we aimed to visualize deep-level microvascular structure in rat hind limb by microangiographic technique, and compare images with those by conventional method.
Materials and Methods:Inbred strain rats (F344) were used for in vivo and ex vivo study. Microangiography in vivo and ex vivo was performed and images were compared with those by conventional method. In microangiography in vivo study, the catheter was connected to an automated angiographic injector, through which the nonionic contrast media was injected into blood vessels in rat hind limbs. A monochromatic 33.4 keV X-ray source just above the K-absorption edge of iodine was used for imaging. In vitro study, normal saline and barium sulfate solution was infused through catheter to the left iliac artery of rats, and the phase contrast imaging were performed with the energy of 15 Kev and the object-to-image distance of 800mm. Synchrotron radiation-based micro-computed tomography (SRmCT) was also performed to reveal three-dimensional (3D) morphology of the blood vessel in rat hind limb.
Results:Using microangiographic technique in vivo and in vitro (with barium sulfate), blood vessels in the rat limb muscle could be visualized with high resolution and more detail compared with those by conventional technique, and the fifth to sixth branches of iliac artery in rat hind limb could be detected with the minimum visualized blood vessels about 40μm and 9μm in diameter, respectively. Accordingly, higher angiographic scores were achieved than those by conventional X-ray. In addition, the vascular network could be defined and analyzed at the micrometer scale from the 3D renderings of limb vessel as shown by SRmCT.
Conclusions:Synchrotron radiation-based microangiography and SRmCT thus provided a practical and effective means to observe the microvasculature of rat hindlimb, which might be useful in assessment of angiogenesis in lower limbs.
Part Two
The study of culturing and multilineage potential of rat marrow-derived mesenchymal stem cells
Objective:Bone marrow mesenchymal stem cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in limb ischemic disease. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of plating density and culturing method on proliferation, cell morphology, and differentiation potential of MSC, and to provide an effective approach of cell obtaining for stem cell transplantiation.
Materials and Methods:Rat MSCs were isolated from rat bone marrow relying on their ability to adhere to plastic, and replated at appropriate seeding density (3x10, 3x102 and 3x103 cells/cm2) at each passage. Cell morphology and growth characteristic were evaluated and compared among different seeding density. Passage 3 MSCs from cultures seeded at optimal plated density were used for identification of their surface marker by flow cytometry and immunofluorescence. For differentiation assays, cells from passage 3 were cultured in induction medium supplemented with different chemical agents and cytokines. Differentiation of MSCs into adipogenic, osteogenic, and endothelial lineages were identified by Oil Red-O stain for lipid deposits, stained for kaline phosphatase by an indoxyltetrazolium method, and incubated in the endothelial differentiation medium containing Dil-Ac-LDL and stained with BS-1-lectin, respectively.
Results:Primary cultur of rMSCs showed a spindle-like or flattened polygonal appearance with the characteristic of adherence to plastic. The low density culture consisted of a relatively homogenous monolayer of spindle-like cells while high density cultures appeared heterogeneous in which nonfibroblastic cells were present. Cell colonies were observed in low density culture with high population doubling speed. Flow cytometric analysis of the low density culture at passage 3 demonstrated to be positive for CD90, CD29, CD105, but negative for CD34, CD45. Under certain conditions, rMSCs of low density culture were capable of differentiating into multilineage cell types, including adipogenic cells which were positive for oil red-0 stain, osteogenic lineages with positive stain for kaline phosphatase by an indoxyltetrazolium method, and endothelial cells with positive result of both LDL-uptaking assay and BS-1-lectin stain.
Conclusion:Based on the results, we concluded that adherent culturing method combined with the low density culture system is a more appropriate system to isolate and expand murine MSCs from marrow samples. With low density culture system, more purified mMSCs which express putative surface maker and have multiple differential potential could be produced. density (AMVD) was increased at 14 and 21 days in both group A and group B, and significant higher in group A compared with group B (0.54±0.036 vs 0.34±0.032 and 0.64±0.037 vs 0.49±0.027, P＜0.05, for 14 days and 21 days, respectively). In addition, the microvascular pattern and arrangement was more regular in group A, when compare with group B at 21 days after transplantation. There were no significant changes of microvasculature in the sham operation group (group C). A significant improvement of blood flow perfusion and lower injury score was found in group A, when compared with group B since 7 days after cell transplantation. The results of microvascular density by microangiography ex vivo indicated there was a higher microvascular density in group A than in group B, which were consistent with the results of immunohistochemistry stain.
Conclusion:SR based microangiography is an effective method for the detection of angiogenesis after stem cell transplantation in a rat model of hindlimb ischemia, which suggested a higher microvascular density and more regular branching pattern of neovascularization in rMSC transplantation group compared with PBS group.
引文
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