【作者】 陈文坚；

【导师】 李锋；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分平面及三维培养髓核细胞的生物学特性目的运用藻酸钠微球(alginate beads)培养法和平面培养法对兔椎间盘髓核细胞进行体外培养，研究其体外生物学特性。方法4月龄健康新西兰大耳白兔6只，取髓核细胞(nucleus pulposus cells，NPCs)原代培养，传代后分为藻酸钠微球培养组(实验组)和平面培养组(对照组)，通过倒置相差显微镜对髓核细胞进行形态学观察，MTT法测定两组髓核细胞增殖情况，运用RT—PCR技术检测两组髓核细胞中Ⅱ型胶原和聚集蛋白聚糖的表达。结果经过两周的培养，两组髓核细胞增殖率无明显差异，藻酸钠微球培养组在Ⅱ型胶原和聚集蛋白聚糖的表达上均高于平面培养组(P＜0．05)。结论藻酸钠微球培养法能促进髓核细胞中Ⅱ型胶原和聚集蛋白聚糖的表达，在维持其表型稳定方面优于平面培养法。第二部分体外诱导标记绿色荧光蛋白兔骨髓间充质干细胞分化成骨及成脂的实验研究目的绿色荧光蛋白(green fluorescent protein，GFP)标记兔骨髓间充质干细胞(mesenchymal stem cells，MSCs)，观察其诱导成骨及成脂多向分化的潜能。方法取四月龄新西兰大白兔6只，雌雄不限，实验过程中对动物处置符合动物伦理学标准。应用密度梯度离心法与贴壁培养法从兔股骨中分离、纯化骨髓间充质干细胞并在体外进行培养，以形态学及细胞表面标志的方法鉴定间充质干细胞，在倒置显微镜下观察细胞的形态特征。用脂质体介导法将绿色荧光蛋白质粒转染入骨髓间充质干细胞，经G418筛选得到稳定转染细胞株。利用成骨诱导剂和成脂诱导剂诱导稳定转染的骨髓间充质干细胞向成骨及成脂方向分化，采用茜素红染色及油红O染色检测MSCs分化的情况。结果经原代及传代培养的骨髓间充质干细胞多呈纺锤形或梭形，成纤维细胞样，流式细胞仪分析MSCsCD44表达阳性，CD34表达阴性。标记绿色荧光蛋白的骨髓间充质干细胞经成骨诱导21 d后有较多的钙盐沉积，茜素红染色呈红色。成脂诱导3天后，细胞内有小脂滴出现，2周后脂滴数量增加并相互融合，细胞由长梭形变为圆形或多边形，油红O染色显示细胞含有丰富的脂肪颗粒。结论通过密度梯度离心法与贴壁培养法可大量扩增、纯化MSCs，用脂质体介导法将绿色荧光蛋白质粒标记的骨髓间充质干细胞经诱导培养后具有多向分化潜能。第三部分三维共培养兔髓核细胞诱导骨髓间充质干细胞定向分化的实验研究目的研究髓核细胞(nucleus pulposus cells，NPCs)与骨髓间充质干细胞(mesenchymalstem cells，MSCs)在藻酸盐微球构建的三维环境下进行共培养时，髓核细胞与骨髓间充质干细胞的近距离接触后骨髓间充质干细胞表型的变化，为构建组织工程髓核提供种子细胞建立基础。方法自健康新西兰大耳白兔取髓核细胞与骨髓间充质干细胞原代培养；将传代分离纯化的骨髓间充质干细胞通过转染绿色荧光蛋白(GFP)进行标记并随机分为实验组与对照组；将转染后的骨髓间充质干细胞单独培养(对照组)或以1：1的比例和髓核细胞于海藻酸盐凝胶微球中进行共培养(实验组)；共培养14天后，溶解海藻酸盐凝胶珠，通过流式分选收集骨髓间充质干细胞并分别利用RT—PCR技术和免疫组织化学技术检测骨髓间充质干细胞中Ⅱ型胶原(ⅡCollagen)和聚集蛋白聚糖(Aggrecan)的表达。结果实验组骨髓间充质干细胞经过14天在海藻酸盐凝胶微球中与髓核细胞共培养后，RT—PCR结果显示有Ⅱ型胶原和聚集蛋白聚糖表达，而对照组则无Ⅱ型胶原和聚集蛋白聚糖表达；免疫组织化学染色显示实验组Ⅱ型胶原表达阳性而对照组为阴性。结论通过三维共同培养，髓核细胞能够在体外诱导骨髓间充质干细胞向髓核样细胞方向分化，进一步支持利用未分化骨髓间充质干细胞治疗椎间盘退行性疾病。更多还原

【Abstract】 PartⅠCulture of rabbit nucleus pulposus cells in alginate beads andstudy the biological characteristicsObjective To culture rabbit nucleus pulposus cells using alginate beads and plates, thendetected the biological character in vitro.Method We got the primary nucleus pulposus cells (NPCs) from 6 healthy New Zealandrabbits of 4-month-old, and then divided the cells into the experiment group (culture inalginate beads) and the control group (culture on plates) after passage. Cell morphology ofNPCs was observed by inverted phase contrast microscope. MTT was used to determine thecells proliferation, and RT-PCR was applied to detect the expression of collagenⅡandaggrecan in NPCs.Results After two weeks cultivation, there was no significant difference in the cellsproliferation rate of NPCs between the experiment group and the control group. But theexpression of collagenⅡand aggrecan in the experiment group was superior to thecontrol(P＜0.05).Conclusion Culturing NPCs in alginate beads can promote the expression of collagenⅡand aggrecan, it was better than that on plates to maintain phenotypic stability of NPCs.PartⅡExperimental study on differentiation of the rabbit mesenchymal stem cells labeled by green fluorescent protein into osteoblasts andlipoblasts in vitroObjective To label rabbit mesenchymal stem cells (MSCs) by green fluorescentprotein(GFP), and then observe the potency of multi-directional differentiation byosteoblast and lipoblast formation induction.Method We got six New Zealand rabbits aged 4 months after birth, of either sex. Thedisposition of the rabbits met the ethics standards. MSCs were isolated from the femurs ofrabbits, then they were purified and cultured in vitro by density gradient centrifugation andadherent culture. MSCs were evaluated by morphology and surface marker. MSCs culturewas observed with an inverted microscope. Green fluorescent protein plasmid wastransfected into mesenchymal stem cells using Lipofectamine. The stable transfection cellswere obtained using G418 selection after transfection. The stable transfection MSCs wereinduced to osteoblasts and lipoblasts by osteogenic inductor and adipogenic inductor. Oilred O staining and alizarin red staining were used to determine the differentiation.Results The MCSs were mostly fusiform in shape, to be similar to fibroblast cell aftercultivation of primary and subcultured. The flow cytometer analysis showed that themembrane mark CD44 was positive, CD34 was negative. MSCs labeled by greenfluorescent protein were apparent calcium mineralization after 21 days osteogenic induction.Alizarin red staining showed positive with red. Intra-cellular lipid droplet appeared after 3days adipogenic induction. The quantity of lipid droplet increased and were fused eachother 2 weeks later. The morphous of fusiform shape became round or polygon, and oil redO staining showed a great quantity lipid deposited.Conclusion MSCs can be isolated and cultured by the method of density gradientcentrifugation and adherent culture in vitro. MSCs labeled by green fluorescent proteinusing Lipofectamine have the potency of multi-directional differentiation after induction. PartⅢStudy on rabbit nucleus pulposus cells induces thedifferentiation of mesenchymal stem cells byco-cultured in a 3-dimensional environmentObjective To investigate the changes of mesenchymal stem cells phenotype afterco-cultured with nucleus pulposus cells in a 3-dimensional environment by alginate Beadsand find a new source of seed cells for constructing tissue engineered intervertebral disc.Method We got the primary nucleus pulposus cells (NPCs) and mesenchymal stem cells(MSCs) from healthy New Zealand rabbits. After passage and purification, the MSCs werelabeled by transduction with green fluorescent protein (GFP) and divided into theexperiment group and the control group. After transduction MSCs were cultured eitheralone (controls) or at a NPCs-to-MSCs ratio of 1:1 in alginate beads. The beads weredissolved after 14 days co-culture. Fluorescence-activated cell sorting was used to collectthe green-MSCs, and then RT-PCR and immunohistochemical staining were applied todetect the expression of collagenⅡand aggrecan in MSCs.Results The RT-PCR showed that the experiment group MSCs expressed collagen-Ⅱand aggrecan after 14 days co-culture with NPCs while the control group MSCs did not.Immunohistochemical staining of the experiment group MSCs for collagen-Ⅱwerepositive and the control group was negative.Conclusion MSC differentiation toward NP-like cells in vitro can be induced by co-culturewith NPCs in a 3-dimensional environment. These data support the use of undifferentiatedMSC for stem cell therapy for intervertebral disc degeneration treatment.更多还原