【作者】 张彦男；

【导师】 邵增务；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 第一部分脊索细胞和骨髓间充质干细胞的分离培养和鉴定目的分离兔髓核脊索细胞及骨髓间充质干细胞,通过形态学及流式细胞术对两种细胞进行鉴定,为下一步细胞试验奠定基础。方法4-6周龄新西兰兔4只,取胸腰段脊柱的髓核,用密度梯度离心法提取脊索细胞,同时取其股骨骨髓用FICOLL液分离得到骨髓间充质干细胞,光镜观察脊索细胞和骨髓间充质干细胞的生长情况,取原代脊索细胞脊进行电镜观察；对第3代的骨髓间充质干细胞进行表面抗原CD34、CD44、CD29、CD45的检测。结果光镜下观察原代脊索细胞呈圆形或椭圆形,胞体大,细胞增殖不明显。骨髓间充质干细胞呈梭形贴壁生长,旋涡状排列,细胞贴壁后折光性较差。电镜扫描发现脊索细胞大小约为20-30um,细胞形态呈圆形或者椭圆形,细胞膜表面可见大量糖原分布,细胞内可见大量的大小不一的囊泡存在。对第3代的骨髓间充质干细胞进行表面抗原的检测发现：细胞表达CD34、CD44阴性,CD29、CD45阳性。其中CD34的平均表达率为3.9%,CD44的平均表达率为4.4%,CD45的平均表达率为96.2%,CD29的平均表达率为88.1%。结论使用密度梯度离心法可以分离出脊索细胞和骨髓间充质干细胞,经过细胞鉴定两种细胞均表现出特征性特点,为进一步试验奠定了基础。第二部分接触性共培养脊索细胞促进骨髓间充质干细胞增值及定向诱导分化的实验研究目的分离兔髓核脊索细胞及骨髓间充质干细胞,通过共培养探讨脊索细胞对骨髓间充质干细胞增值能力及细胞表型的影响。方法4-6周龄新西兰兔4只,取胸腰段脊柱的髓核,用密度梯度离心法提取脊索细胞,同时取其股骨骨髓用FICOLL液分离得到骨髓间充质干细胞,光镜观察脊索细胞和骨髓间充质干细胞不同比例(1：2、1：1、2：1)共培养条件下细胞的生长情况、CCK-8法检测细胞增殖。脊索细胞和骨髓间充质干细胞共培养(1:1)后行甲苯胺蓝染色及Ⅱ型胶原染色检测骨髓间充质干细胞的细胞表型的改变情况。共培养后的骨髓间充质干细胞进行相关基因表达的检测。结果光镜下观察原代脊索细胞呈圆形或椭圆形,胞体大,细胞增殖不明显。骨髓间充质干细胞呈梭形贴壁生长,旋涡状排列,细胞贴壁后折光性较差。CCK-8检测发现脊索细胞/骨髓间充质干细胞1:1组细胞增殖明显高于其余各组。甲苯胺蓝染色骨髓间充质干细胞单独培养组呈阴性,共培养组呈阳性。Ⅱ型胶原染色骨髓间充质干细胞单独培养组呈阴性,共培养组呈阳性。RT-PCR检测发现共培养组蛋白聚糖及Ⅱ型胶原的表达分别为脊索细胞的2倍、1.35倍,而单独培养的MSC则表达阴性。结论在共培养条件下脊索细胞可以促进骨髓间充质干细胞增值,且细胞比例为1:1时更为显著；同时可以诱导其产生Ⅱ型胶原及聚集蛋白聚糖,表现出类软骨细胞表型。第三部分非接触共培养脊索细胞诱导骨髓间充质干细胞向类软骨细胞分化的实验研究目的分离兔髓核脊索细胞(NC)及骨髓间充质干细胞(MSC),通过非接触共培养探讨脊索细胞对MSC细胞表型的影响。方法取4-6周龄新西兰兔8只,取胸腰段脊柱的髓核,用密度梯度离心提取脊索细胞,同时取其股骨骨髓用FICOLL液分离,光镜观察脊索细胞和MSC等比例(1:1)非接触共培养条件下的生长情况。对非接触共培养(1:1)和单独培养的MSC行免疫组化及RT-PCR、Western-blot检测MSC的细胞表型的改变情况。结果原代脊索细胞呈圆形或椭圆形,细胞体积大,细胞增殖不明显。MSC呈梭形贴壁生长,呈三角形或梭形,旋涡状排列,两端呈尖性突起,细胞折光性较差。非接触共培养后,行甲苯胺蓝染色MSC单独培养组呈阴性,与NC非接触共培养的MSC组呈阳性。Ⅱ型胶原免疫组化MSC单独培养组呈阴性,与NC非接触共培养的MSC组呈阳性呈阳性。通过RT-PCR、Western-blot在基因及蛋白水平检测后发现：非接触共培养的MSC组其Ⅱ型胶原及蛋白聚糖的表达明显增多,与对照组有显著性差异(p<0.05)。结论在非接触共培养条件下脊索细胞可以诱导MSC向类软骨细胞方向分化,为组织工程化髓核的种子细胞筛选提供新选择。第四部分联合移植脊索细胞和MSC阻止兔椎间盘退行性变的实验研究目的使用纤维环穿刺抽吸法造模兔椎间盘退行性变,联合移植脊索细胞(NC)及骨髓间充质干细胞(MSC)到退变的椎间盘内,观察退变椎间盘的变化。方法取1-1.5kg新西兰兔12只,手术对兔脊柱L3-4、L4-5、L5-6、L6-7四个椎间盘行穿刺抽吸髓核组织造模,造模2周后取等比例共培养3天脊索细胞和MSC移植到L4-5；单独的脊索细胞移植到L5-6；单独的MSC移植到L6-7。动物饲养2周后处死行髓核组织的免疫组化及Western-blot检测椎间盘退变的改变情况。结果穿刺抽吸髓核组织后2周行MRI检测兔椎间盘退行性变模型%ST2WI值较2周前相比明显下降,经过细胞移植后各组%ST2WI值均较对照组增高,尤其以联合细胞组为著。HE染色及甲苯胺蓝染色显示联合细胞移植后,椎间盘髓核内软骨样细胞明显增多,细胞外基质较各组丰富。Ⅱ胶原免疫组化：联合细胞移植组在髓核内较其它3组可见大片棕色区域,其间存在深染的棕黄色颗粒,基质内可见黄染阳性的软骨样细胞,靠近细胞处染色较深。通过Western-blot在蛋白水平检测后发现：对照组Ⅰ型胶原呈现高表达,分别是联合细胞移植组、单独移植脊索细胞组、单独移植MSC组的24.78倍、13.63倍和14.03倍(p<0.05)。联合细胞移植组Ⅱ型胶原的表达较C组和D组增高(p<0.05)。结论脊索细胞和MSC共培养后联合移植可以阻逆兔椎间盘的退行性变,为组织工程治疗椎间盘退行性变提供新的思路。更多还原

【Abstract】 PartⅠIsolation and characterization of notochordal cells(NCs) and mesenchymal stem cells(MSCs)Objective To isolate and co-culture notochordal cells(NC) and mesenchymal stem cells(MSCs), to assess the cell characteristic of notochordal cells and mesenchymal stem cells.Methods Notochordal cell were obtained from immature NP of 4 New Zealand rabbits(4-6 week-old) and purified by discontinuous gradient density centrifugation, MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation. Cells culture expanded to passage 3, Investigate expression of CD29, CD34, CD44, CD45 in MSCs by flow cytometry (FCM). Results NCs were round or oval, the diameter between 20-30um, filled with vesicles, the cells aggregated commonly. Observed by electron microscope, there are many different size vesicles, but a few organelles in NCs, glycogen on the surface of membrane. MSCs were spindle shape or triangular, the original cells covered the flask for 7-10 days, grown rapidly by passage. The surface molecules of MSC were detected by FCM, the expression of CD29 and CD45 was positive compare to the negative expression of CD34 and CD44 that provided the evidence for identification of MSC.Conclusions Notochordal cell were obtained from immature NP and purified by discontinuous gradient density centrifugation, MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation.PartⅡNotochordala cell stimulates proliferation and differention of mesenchymal stem cell toward nucleus pulposus phenotype Objective:To isolate and co-culture notochordal cells(NC) and mesenchymal stem cells(MSCs) from New Zealand rabbit immature nucleus pulposus(NP), to assess the contribution of notochordal cells to proliferation and differention of mesenchymal stem cells.Methods:Notochordal cell were got from immature NP of 4 New Zealand rabbits(4-6 week-old) and purified by discontinuous gradient density centrifugation, MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation. MSCs were cultured alone and co-cultured with NC(1:1/1:2/2:1), evaluated cell proliferation by CCK8-kit and expressions of collagenⅡand proteoglycan by toluidine blue and immunocytochemistry staining. Assessed the expression of collagenⅡand proteoglycan in gene and protein level.Results:NCs and MSCs were isolated and purified. NC were with diameter of 20-30un, had abundant intracytoplasmic vesicles and poor proliferation. MSCs were adherent growth with fusiform, arrayed with whirlpool. By co-cultured, the group of cell ration at 1:1(NC:MSC) increased significantly in proliferation compared with other groups. After co-culture, MSCs from the group co-culture were observed expression of collagenⅡand proteoglycan compared with negative expression in the group of MSCs cultured alone. Conclusion:By coculture, NC can stimulate MSCs’proliferation that will be significant at cell ration 1:1, and differention toward nucleus pulposus cell.Part IIIInducing mesenchymal stem cells to differentiate toward nucleus pulposus phenotype with indirect co-cultureObjective To isolate and co-culture notochordal cells(NC) and mesenchymal stem cells(MSCs) from New Zealand rabbit immature nucleus pulposus(NP), to assess the contribution of notochordal cells to differentiation of mesenchymal stem cells.Methods Notochordal cell were got from immature NP of 8 New Zealand rabbits (4-6 week-old) and purified by discontinuous gradient density centrifugation, MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation. MSCs were cultured alone and non-contact co-cultured with NC(1:1), evaluated expressions of collagenⅡand proteoglycan by toluidine blue and immunocytochemistry staining. Assessed the expression of collagenⅡand proteoglycan in gene and protein level. Results NCs and MSCs were isolated and purified. NC were with diameter of 20-30un, had abundant intracytoplasmic vesicles and poor proliferation. MSCs were adherent growth with fusiform, arrayed with whirlpool. After indirect co-cultured, MSCs from the group co-culture were observed expression of collagenⅡand proteoglycan compared with negative expression in the group of MSCs cultured alone.Conclusion By indirect coculture, NC can stimulate MSCs’proliferation and differentiation toward nucleus pulposus cell, These findings maybe supply a new choice to cell therapy strategy of intervertebral disc regeneration.Part IVRestraining Degeneration of Punctured Intervertebral Discs by Transplantation of Rabbit notochordal cells and Marrow Stroma CellsObjective:To assess the ability of transplanted notochordal cells (NCs) and marrowstroma cells (MSCs) in restraining the degeneration of punctured intervertebral discs in rabbits.Methods The first passage NCs and third passage MSCs were harvested for transplantation. Twelve New Zealand white rabbits were invited in this experimentation. The L3/4, L4/5, L5/6, and L6/7 discs of the rabbits were stabbed and punctured using a 18G needle. After two weeks,The L3/4 discs were then untreated, L4/5 discs were treated by transplantation NCs and MSCs, L5/6 discs treated by transplantation NCs, L6/7 discs were treated by transplantation MSCs. Magnetic Resonance Images of the lumbar vertebral T2 weighed signals Were collected after 2 weeks by the operations. The DHI and standardized T2WI(ST2wI) were Measured using Image-proplus6.0 and Mergee Film Workstation. Nucleus pulposus were evaluated expressions of collagenⅡand proteoglycan by toluidine blue and immunocytochemistry staining. Assessed the expression of collagen II and collagen I in protein level.Results:Animals survived above 2 weeks after been transplanted. The transplantation effectively restrained the degeneration of the punctured discs. The L4/5 discs had higher DHI in 2 weeks by operation than other discs (P<0.05). The L4/5 discs demonstrated significantly positive compared with control by stained with toluidine blue and in Immunocytochemistry. The L4/5 discs were observed expression of collagen II compared with less expression in the L5/6, L6/7 disc.Conclusion:Transplantation of Rabbit NCs and MSCs restrain the degeneration of punctured discs. This cell therapy have shown stronger potential of extracellular matrixsynthesis, and height and water content Recovery of discs.更多还原