骨髓间充质干细胞复合可注射型纤维蛋白凝胶TGF-β1移植治疗兔椎间盘退变的实验研究
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摘要
第一部分骨髓间充质干细胞的体外培养及鉴定
目的:建立兔骨髓间充质干细胞(MSCs)体外培养、扩增和鉴定的方法,了解其作为椎间盘组织工程种子细胞的可行性。方法:穿刺兔股骨大转子抽取骨髓4ml,培养、扩增、传代,倒置显微镜下观察原代和传代细胞的生长特性,流式细胞仪分析细胞表面标记物CD44、CD45。细胞经过诱导分化后进行Ⅱ型胶原免疫组化染色、阿利新蓝染色、碱性磷酸酶染色及钙结节染色。结果:体外成功建立兔骨髓间充质干细胞培养扩增的方法,并成功诱导出成骨、软骨细胞。骨髓MSCs表达CD44,不表达CD45。Ⅱ型胶原免疫组化染色,阿利新蓝染色,ALP染色及钙结节染色阳性。结论:全骨髓法能有效分离纯化和扩增骨髓MSCs,培养的细胞具有骨髓MSCs的基本表型和特征,该细胞经诱导后能表现类软骨细胞的特性,是一种有效的椎间盘组织工程种子细胞。
第二部分针刺抽吸法诱导椎间盘退变动物模型的建立
目的:探讨针刺抽吸法建立椎间盘退变动物模型的可行性。方法:新西兰大白兔18只,用21G皮肤穿刺针从椎间隙正前方刺入L3/4、L4/5、L5/6椎间盘的纤维环,深度控制在5mm,拔出针芯,抽吸出部分髓核组织。术前及术后4、8、12周分别对造模后椎间盘及对照椎间盘(L2/3)行MRI及CR检查,并行免疫组化及组织学观察。结果:术后第4周到第12周,造模后的椎间盘MRI的T2W1信号呈现持续减弱趋势;椎间盘高度指数持续下降;免疫组化及组织学检查显示髓核细胞的数量及Ⅱ型胶原含量较对照间盘进行性减少,细胞凋亡率明显增加(P<0.01)。结论:针刺抽吸法可诱导兔椎间盘的缓慢退变,为研究椎间盘退行性变的机理及治疗提供有效的载体。
第三部分兔骨髓间充质干细胞在退变椎间盘环境中存活及迁移能力的实验研究
目的:探讨兔骨髓间充质干细胞在退变椎间盘环境中存活及迁移能力,为进一步研究提供实验基础。方法:新西兰大白兔9只,穿刺法诱导椎间盘退变模型建立2周后每个退变椎间盘移植入BrdU标记的骨髓间充质干细胞纤维蛋白凝胶TGF-β1复合物0.04ml,术后2、4、8周将兔处死,取L3/4,L4/5,L5/6的9个椎间盘髓核组织进行BrdU免疫组织化学染色,随机取9个L2/3髓核作为对照组,计数10个高倍视野中BrdU阳性细胞数。结果:BrdU免疫组化染色可见在2、4、8周均有阳性染色细胞,细胞数为157.11±17.45、68.56±16.89、79.78±22.11,4周及8周组间无统计学意义,P>0.05,其余各组间有统计意义,F=74.59,P<0.01。纤维蛋白移植区阳性细胞数量随时间推移逐渐减少后保持一定数量,部分细胞向髓核组织迁移,正常对照组未见阳性细胞。结论:兔骨髓间充质干细胞复合纤维蛋白凝胶TGF-β1移植后能在退变椎间盘环境中存活及迁移,为椎间盘退变的组织工程学研究奠定了实验基础。
第四部分骨髓间充质干细胞复合可注射型纤维蛋白凝胶TGF-β1移植治疗兔椎间盘退变的实验研究
目的:探讨兔骨髓间充质干细胞移植抑制椎间盘退变的可行性,为后续的临床研究提供实验基础。方法:新西兰大白兔54只,随机分为3组:(1)退变模型组,(2)单纯纤维蛋白凝胶TGF-β1移植组(,3)骨髓间充质干细胞纤维蛋白凝胶TGF-β1移植组,每组18只。退变模型组单纯用针刺法诱导建立退变模型,单纯纤维蛋白凝胶TGF-β1移植组及骨髓间充质干细胞纤维蛋白凝胶TGF-β1移植组退变模型诱导两周后分别移植入纤维蛋白凝胶TGF-β1复合物及骨髓间充质干细胞纤维蛋白凝胶TGF-β1复合物,分别于术后4、8、12周(移植术后2、6、10周)行CR、MRI及病理检查。结果:椎间盘高度指数(DHI)在退变模型组及单纯支架移植组中下降明显,与时间呈正相关;正常对照组及干细胞移植组下降较缓慢。MRI结果显示退变模型组及支架移植组随时间的推移,退变逐渐加重,干细胞移植组退变不明显。免疫组化及组织学检查显示退变模型组髓核细胞的数量及Ⅱ型胶原含量较对照间盘进行性减少,细胞凋亡率明显增加,单纯支架移植组与退变模型组相似,干细胞移植组髓核细胞的数量及Ⅱ型胶原含量较退变组及单纯支架移植组明显增多,细胞凋亡率下降。结论:干细胞移植能明显抑制椎间盘退变,为进一步的研究奠定了实验基础。
Part I In vitro Culture and Identification of Marrow Mesenchymal Stem Cells
Objective: We aim to develop a method for in vitro culture, proliferation and identification of the mesenchymal stem cells (MSCs) for the purpose of further application of the MSCs. Methods: The rabbit thighbone was punctured and 4 ml marrow was extracted from it. The marrow MSCs were processed by culture, proliferation and passage. The primary and passage cells were then observed under an inversion microscope for their growth characteristics. In addition, a flow cytometry was applied to assay the cell surface markers, i.e., CD44 and CD45. Finally, after induction and differentiation, the cells were processed by Collagen II immunohistochemistry, Alcian Blue stain, ALP stain and mineralization node stain. Results: We developed a method for in vitro culture and proliferation of the rabbit marrow MSCs and successfully induced osteoblast and cartilage. The marrow MSCs expressed CD44, however, did not express CD45. In addition, the results of Collagen II immunohistochemistry and staining using Alcian Blue, ALP and mineralization node stain were positive. Conclusions: The method of marrow culture can be used to effectively separate, purify and proliferate the marrow MSCs. The cultured cells have the essential phenotypes and characteristics of the marrow MSCs.
PartⅡConstruction of rabbit model of intervertebral disc degeneration induced by puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs
Objective: to establish a slowly progressive reproducible rabbit model of intervertebral disc degeneration induced by puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs(IVDs). Methods: The L3/4、L4/5andL5/6 lumbar intervertebral discs of 18 New Zealand white rabbits were stabbed by 21-gauge hypodemic needle into a depth of 5mm in the antero annulus fibrosus, pulled out the needle valve, and aspirated out some nucleus pulposus tissue. Magnetic resonance imaging scans (MRI) Computed radiography (CR) immunohistochemical and histologic analyses of the stabbed discs and intact L2/3 disc were performed preoperatively and at 4, 8, 12weeks respectively after surgery. Results: In the magnetic resonance imaging the stabbed discs exhibited a progressive decrease of sigal intensity in T2-weighted images which start at 4 week after stabing and last for 12 weeks. Computed radiography analyses indicates progressive decrease of the disc height index Immunohistochemical and histologic analyses revealed progressive decrease of chondrocyte-like cells and typeⅡcollagen.Conclusion and increase of cells apoptosis(P<0.01). Conculation: The stabbing and asprirating approach results in a slowly progressive intertebral disc degeneration in rabbit model.This model is available for studying the status intervertebral disc degeneration and treatment.
PartⅢA Research on Existence and Migration Capability of Rabbit Mesenchymal Stem Cells in Intervertebral Disc Degeneration
Objective: to explore the existence and migration capability of rabbit mesenchymal stem cells in intervertebral disc degeneration and lay the experimental foundation for further researches. Method: with nine white New Zealand rabbits, by means of puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs, induce intervertebral disc degeneration model. After two weeks, transplant each degenerated intervertebral disc to mesenchymal stem cells fibrinousgelatin TGF-β1 complex 0.04ml with symbol of BrdU. After operation, in 2nd, 4th and 8th weeks, kill the rabbits and take nine intervertebral disc nucleus pulposus tissues of L3/4,L4/5,L5/6, carry out BrdU immunohistochemical stain and randomly take nine L2/3 nucleus pulposus as the control group. Result: From BrdU immunohistochemical stain, masculine staining cells can be seen in the 2nd, 4th and 8th weeks and cell counts are respectively 157.11±13.26, 68.56±13.16, 79.78±22.11, statistics of 4th and 8th weeks group have indifferent meanings P>0.05,and each of rest group have different meanings F=74.59, P<0.01. Along with the time, masculine staining cell count in fibrous protein implanted region decreases, until keeping a certain quantity, part of cells immigrate to nucleus pulposus tissue and there is no masculine staining cell found in normal control group. Conclusion: Rabbit mesenchymal stem cells can survive and immigrate in intervertebral disc degeneration. This lays the experimental foundation for tissue engineering researches of intervertebral disc degeneration.
PartⅣA Research on Mesenchymal Stem Cells Compound Injectable Fibrinousgelatin TGF-β1 Transplantation Treatment of Rabbit Intervertebral Disc Degeneration
Objective: To explore the feasibility of rabbit mesenchymal stem cell transplantation’s inhibiting intervertebral disc degeneration. Method: 54 white New Zealand rabbits are randomly divided into three groups: (1) degeneration model group; (2) pure fibrinousgelatin TGF-β1 transplanted group; (3) mesenchymal stem cells TGF-β1 transplanted group. Every group has 18 rabbits. In the degeneration model group, induce and establish the degeneration model merely by means of puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs; in the pure fibrinousgelatin TGF-β1 transplanted group and the mesenchymal stem cells TGF-β1 transplanted group, 2 weeks after inducing degeneration model, transplant fibrinousgelatin TGF-β1 complex and mesenchymal stem cells fibrinousgelatin TGF-β1 complex respectively. Carry out CR, MRI and histologic examinations respectively in the 4th, 8th and 12th weeks (2nd, 6th and 10th weeks after transplant operation). Result: the disc height index (DHI) obviously decreases in the degeneration model group and the pure fibrinousgelatin TGF-β1 transplanted group, which is positively correlated with time; the normal control group and the stem cells transplanted group decrease slowly. MRI results show an increasingly aggravated degeneration in the degeneration model group and the stents transplanted group along with time, while the degeneration in the stem cells transplanted group is not obvious. An immunohistochemistry study and a histological examination indicate a decreasing quantity of the nucleus pulposus cells and the content of typeⅡcollagen in the degeneration model group, compared with that of the disc as well as an obviously increasing rate of cells apoptosis. The pure fibrinousgelatin TGF-β1 transplanted group is similar to the degeneration model group with an obviously increasing quantity of stem cells apoptosis in the stem cells transplanted group and typeⅡcollagen content compared with the degeneration group and the pure stents transplanted group while the cells apoptosis rate decreases. Conclusion: Stem cells can prohibit intervertebral disc degeneration. This lays the experimental foundation for further researches.
目的:建立兔骨髓间充质干细胞(MSCs)体外培养、扩增和鉴定的方法,了解其作为椎间盘组织工程种子细胞的可行性。方法:穿刺兔股骨大转子抽取骨髓4ml,培养、扩增、传代,倒置显微镜下观察原代和传代细胞的生长特性,流式细胞仪分析细胞表面标记物CD44、CD45。细胞经过诱导分化后进行Ⅱ型胶原免疫组化染色、阿利新蓝染色、碱性磷酸酶染色及钙结节染色。结果:体外成功建立兔骨髓间充质干细胞培养扩增的方法,并成功诱导出成骨、软骨细胞。骨髓MSCs表达CD44,不表达CD45。Ⅱ型胶原免疫组化染色,阿利新蓝染色,ALP染色及钙结节染色阳性。结论:全骨髓法能有效分离纯化和扩增骨髓MSCs,培养的细胞具有骨髓MSCs的基本表型和特征,该细胞经诱导后能表现类软骨细胞的特性,是一种有效的椎间盘组织工程种子细胞。
第二部分针刺抽吸法诱导椎间盘退变动物模型的建立
目的:探讨针刺抽吸法建立椎间盘退变动物模型的可行性。方法:新西兰大白兔18只,用21G皮肤穿刺针从椎间隙正前方刺入L3/4、L4/5、L5/6椎间盘的纤维环,深度控制在5mm,拔出针芯,抽吸出部分髓核组织。术前及术后4、8、12周分别对造模后椎间盘及对照椎间盘(L2/3)行MRI及CR检查,并行免疫组化及组织学观察。结果:术后第4周到第12周,造模后的椎间盘MRI的T2W1信号呈现持续减弱趋势;椎间盘高度指数持续下降;免疫组化及组织学检查显示髓核细胞的数量及Ⅱ型胶原含量较对照间盘进行性减少,细胞凋亡率明显增加(P<0.01)。结论:针刺抽吸法可诱导兔椎间盘的缓慢退变,为研究椎间盘退行性变的机理及治疗提供有效的载体。
第三部分兔骨髓间充质干细胞在退变椎间盘环境中存活及迁移能力的实验研究
目的:探讨兔骨髓间充质干细胞在退变椎间盘环境中存活及迁移能力,为进一步研究提供实验基础。方法:新西兰大白兔9只,穿刺法诱导椎间盘退变模型建立2周后每个退变椎间盘移植入BrdU标记的骨髓间充质干细胞纤维蛋白凝胶TGF-β1复合物0.04ml,术后2、4、8周将兔处死,取L3/4,L4/5,L5/6的9个椎间盘髓核组织进行BrdU免疫组织化学染色,随机取9个L2/3髓核作为对照组,计数10个高倍视野中BrdU阳性细胞数。结果:BrdU免疫组化染色可见在2、4、8周均有阳性染色细胞,细胞数为157.11±17.45、68.56±16.89、79.78±22.11,4周及8周组间无统计学意义,P>0.05,其余各组间有统计意义,F=74.59,P<0.01。纤维蛋白移植区阳性细胞数量随时间推移逐渐减少后保持一定数量,部分细胞向髓核组织迁移,正常对照组未见阳性细胞。结论:兔骨髓间充质干细胞复合纤维蛋白凝胶TGF-β1移植后能在退变椎间盘环境中存活及迁移,为椎间盘退变的组织工程学研究奠定了实验基础。
第四部分骨髓间充质干细胞复合可注射型纤维蛋白凝胶TGF-β1移植治疗兔椎间盘退变的实验研究
目的:探讨兔骨髓间充质干细胞移植抑制椎间盘退变的可行性,为后续的临床研究提供实验基础。方法:新西兰大白兔54只,随机分为3组:(1)退变模型组,(2)单纯纤维蛋白凝胶TGF-β1移植组(,3)骨髓间充质干细胞纤维蛋白凝胶TGF-β1移植组,每组18只。退变模型组单纯用针刺法诱导建立退变模型,单纯纤维蛋白凝胶TGF-β1移植组及骨髓间充质干细胞纤维蛋白凝胶TGF-β1移植组退变模型诱导两周后分别移植入纤维蛋白凝胶TGF-β1复合物及骨髓间充质干细胞纤维蛋白凝胶TGF-β1复合物,分别于术后4、8、12周(移植术后2、6、10周)行CR、MRI及病理检查。结果:椎间盘高度指数(DHI)在退变模型组及单纯支架移植组中下降明显,与时间呈正相关;正常对照组及干细胞移植组下降较缓慢。MRI结果显示退变模型组及支架移植组随时间的推移,退变逐渐加重,干细胞移植组退变不明显。免疫组化及组织学检查显示退变模型组髓核细胞的数量及Ⅱ型胶原含量较对照间盘进行性减少,细胞凋亡率明显增加,单纯支架移植组与退变模型组相似,干细胞移植组髓核细胞的数量及Ⅱ型胶原含量较退变组及单纯支架移植组明显增多,细胞凋亡率下降。结论:干细胞移植能明显抑制椎间盘退变,为进一步的研究奠定了实验基础。
Part I In vitro Culture and Identification of Marrow Mesenchymal Stem Cells
Objective: We aim to develop a method for in vitro culture, proliferation and identification of the mesenchymal stem cells (MSCs) for the purpose of further application of the MSCs. Methods: The rabbit thighbone was punctured and 4 ml marrow was extracted from it. The marrow MSCs were processed by culture, proliferation and passage. The primary and passage cells were then observed under an inversion microscope for their growth characteristics. In addition, a flow cytometry was applied to assay the cell surface markers, i.e., CD44 and CD45. Finally, after induction and differentiation, the cells were processed by Collagen II immunohistochemistry, Alcian Blue stain, ALP stain and mineralization node stain. Results: We developed a method for in vitro culture and proliferation of the rabbit marrow MSCs and successfully induced osteoblast and cartilage. The marrow MSCs expressed CD44, however, did not express CD45. In addition, the results of Collagen II immunohistochemistry and staining using Alcian Blue, ALP and mineralization node stain were positive. Conclusions: The method of marrow culture can be used to effectively separate, purify and proliferate the marrow MSCs. The cultured cells have the essential phenotypes and characteristics of the marrow MSCs.
PartⅡConstruction of rabbit model of intervertebral disc degeneration induced by puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs
Objective: to establish a slowly progressive reproducible rabbit model of intervertebral disc degeneration induced by puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs(IVDs). Methods: The L3/4、L4/5andL5/6 lumbar intervertebral discs of 18 New Zealand white rabbits were stabbed by 21-gauge hypodemic needle into a depth of 5mm in the antero annulus fibrosus, pulled out the needle valve, and aspirated out some nucleus pulposus tissue. Magnetic resonance imaging scans (MRI) Computed radiography (CR) immunohistochemical and histologic analyses of the stabbed discs and intact L2/3 disc were performed preoperatively and at 4, 8, 12weeks respectively after surgery. Results: In the magnetic resonance imaging the stabbed discs exhibited a progressive decrease of sigal intensity in T2-weighted images which start at 4 week after stabing and last for 12 weeks. Computed radiography analyses indicates progressive decrease of the disc height index Immunohistochemical and histologic analyses revealed progressive decrease of chondrocyte-like cells and typeⅡcollagen.Conclusion and increase of cells apoptosis(P<0.01). Conculation: The stabbing and asprirating approach results in a slowly progressive intertebral disc degeneration in rabbit model.This model is available for studying the status intervertebral disc degeneration and treatment.
PartⅢA Research on Existence and Migration Capability of Rabbit Mesenchymal Stem Cells in Intervertebral Disc Degeneration
Objective: to explore the existence and migration capability of rabbit mesenchymal stem cells in intervertebral disc degeneration and lay the experimental foundation for further researches. Method: with nine white New Zealand rabbits, by means of puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs, induce intervertebral disc degeneration model. After two weeks, transplant each degenerated intervertebral disc to mesenchymal stem cells fibrinousgelatin TGF-β1 complex 0.04ml with symbol of BrdU. After operation, in 2nd, 4th and 8th weeks, kill the rabbits and take nine intervertebral disc nucleus pulposus tissues of L3/4,L4/5,L5/6, carry out BrdU immunohistochemical stain and randomly take nine L2/3 nucleus pulposus as the control group. Result: From BrdU immunohistochemical stain, masculine staining cells can be seen in the 2nd, 4th and 8th weeks and cell counts are respectively 157.11±13.26, 68.56±13.16, 79.78±22.11, statistics of 4th and 8th weeks group have indifferent meanings P>0.05,and each of rest group have different meanings F=74.59, P<0.01. Along with the time, masculine staining cell count in fibrous protein implanted region decreases, until keeping a certain quantity, part of cells immigrate to nucleus pulposus tissue and there is no masculine staining cell found in normal control group. Conclusion: Rabbit mesenchymal stem cells can survive and immigrate in intervertebral disc degeneration. This lays the experimental foundation for tissue engineering researches of intervertebral disc degeneration.
PartⅣA Research on Mesenchymal Stem Cells Compound Injectable Fibrinousgelatin TGF-β1 Transplantation Treatment of Rabbit Intervertebral Disc Degeneration
Objective: To explore the feasibility of rabbit mesenchymal stem cell transplantation’s inhibiting intervertebral disc degeneration. Method: 54 white New Zealand rabbits are randomly divided into three groups: (1) degeneration model group; (2) pure fibrinousgelatin TGF-β1 transplanted group; (3) mesenchymal stem cells TGF-β1 transplanted group. Every group has 18 rabbits. In the degeneration model group, induce and establish the degeneration model merely by means of puncturing the annulus fibrosus with needles of defined gauges and aspirating some nucleus pulposus tissue from the intervertebral discs; in the pure fibrinousgelatin TGF-β1 transplanted group and the mesenchymal stem cells TGF-β1 transplanted group, 2 weeks after inducing degeneration model, transplant fibrinousgelatin TGF-β1 complex and mesenchymal stem cells fibrinousgelatin TGF-β1 complex respectively. Carry out CR, MRI and histologic examinations respectively in the 4th, 8th and 12th weeks (2nd, 6th and 10th weeks after transplant operation). Result: the disc height index (DHI) obviously decreases in the degeneration model group and the pure fibrinousgelatin TGF-β1 transplanted group, which is positively correlated with time; the normal control group and the stem cells transplanted group decrease slowly. MRI results show an increasingly aggravated degeneration in the degeneration model group and the stents transplanted group along with time, while the degeneration in the stem cells transplanted group is not obvious. An immunohistochemistry study and a histological examination indicate a decreasing quantity of the nucleus pulposus cells and the content of typeⅡcollagen in the degeneration model group, compared with that of the disc as well as an obviously increasing rate of cells apoptosis. The pure fibrinousgelatin TGF-β1 transplanted group is similar to the degeneration model group with an obviously increasing quantity of stem cells apoptosis in the stem cells transplanted group and typeⅡcollagen content compared with the degeneration group and the pure stents transplanted group while the cells apoptosis rate decreases. Conclusion: Stem cells can prohibit intervertebral disc degeneration. This lays the experimental foundation for further researches.
引文
1. Saad L, Spector M.Effects of collagen type on the behavior of adult canine annulus fibrosus cells in collagen-glycosamino-glycan scaffolds[J].J Biomend Mater Res,2004, 71(2):233-241.
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