髓核细胞诱导骨髓间充质干细胞的分化与永生化研究

发布时间：2018-01-03 20:41

本文关键词：髓核细胞诱导骨髓间充质干细胞的分化与永生化研究　出处：《华中科技大学》2010年博士论文　论文类型：学位论文

【摘要】： 第一部分：利用抽吸法诱导兔椎间盘椎退变模型目的：探讨利用抽吸法构建兔椎间盘退变模型,并观察其病理及影像表现。方法：10月龄健康新西兰大耳白兔28只,对照组7只,实验造模组21只。利用21G皮肤穿刺针分别在造模组兔L4/5单节段刺入,抽吸髓核越0.008-0.012g。造模完毕,实验兔继续培养4-20周。造模前后利用,X-ray平片、MRI及Alcian blue组织染色观察退变椎间盘的变化。结果：造模前后退变椎间盘高度指数百分比(DHI%)：对照组、4周组、12周组、20周组分别为(100±0)%、(81±3.2)%、(75±2.5)%、(71±1.8)%(P0.05)。退变椎间盘T2加权像信号明显降低,Alcian blue组织染色显示退变椎间盘组织中聚集蛋白多糖含量明显降低。结论：抽吸法构建兔椎间盘退变模型简便可靠,其退变过程的病理及影像表现与人椎间盘退变过程的病理及影像表现十分相似,该退变模型可用于人椎间盘退变的机制与临床治疗研究。第二部分：髓核细胞对骨髓基质干细胞的诱导作用目的：探讨在藻酸盐微球的介导下,髓核细胞与骨髓间充质干细胞(bone marrow stromal cells, BMSCs)非接触性共培养时,髓核细胞对骨髓间充质干细胞的诱导作用。方法：①4月龄健康新西兰大耳白兔5只,取髓核细胞与骨髓间充质干细胞原代培养,利用传代法分离纯化；②将纯化的骨髓间充质干细胞经胰蛋白酶消化、收集,并与适量的藻酸钠溶液混合,形成106个/ml的单细胞悬液,将混合好的单细胞悬液经注射器滴加至3.5%的CaCl2溶液中,形成海藻酸钙凝胶珠；③将海藻酸钙凝胶微球与髓核细胞共培养。在7天和15天时,分组溶解海藻酸钙凝胶珠,收集骨髓间充质干细胞,分别利用免疫组化技术、rt-PCR技术和、Western blot技术检测骨髓间充质干细胞中Ⅱ型胶原和聚集蛋白聚糖的表达,用以判断髓核细胞对骨髓基质干细胞在非接触条件下的诱导作用。结果：在骨髓间充质干细胞的细胞爬片免疫组化染色中可见,细胞Ⅱ型胶原和聚集蛋白聚糖染色阳性,rt-PCR和、Western blot结果显示经诱导后骨髓间充质干细胞中已有Ⅱ型胶原和聚集蛋白聚糖基因的表达,且诱导15天组的目的条带明显亮于诱导7天组的目的条带。结论：在体外非接触共同培养时,髓核细胞能够实现对骨髓基质干细胞的诱导作用,将骨髓基质干细胞分化为髓核细胞,这必将为椎间盘退行性变的移植治疗提供可靠的种子细胞来源。第三部分：骨髓间充质干细胞向类髓核细胞的分化及永生化目的：尝试利用共培养法和SV40Tag;永生化基因导入法构建一种来源于骨髓基质干细胞(MSCs)的永生化型类髓核细胞。方法：取实验白兔原代MSCs和髓核细胞(NPCs),荧光标记NPCs;将标记的NPCs与MSCs直接共培养6、9、12、15、18天；共培养完毕,利用流式细胞仪分选出荧光标记阴性细胞,即MSCs;观测共培养后MSCs细胞形态变化,检测胞内Ⅱ型胶原和聚集蛋白多糖mRNA及蛋白的表达变化；共培养15天时,将含有SV40Tag永生化基因的pCMVSV40T/PUR质粒转染MSCs,利用MTT法观测转染后MSCs增殖活性。结果：共培养后,MSCs胞内表达大量的Ⅱ型胶原和聚集蛋白多糖,细胞形态也从长椭圆形变为多角形；当共培养15天时,胞内Ⅱ型胶原和聚集蛋白多糖mRNA表达浓度达到最大值,但低于原代NPCs胞内Ⅱ型胶原和聚集蛋白多糖mRNA含量(P0.05)。导入SV40Tag基因后,MSCs-SV增殖能力与原代NPCs相同,传代20次后其增殖能力无衰退(P0.05)。结论：利用共培养法和SV40Tag永生化基因导入法可构建出一种来源于MSCs的永生化型类髓核细胞；该类髓核细胞可能会对椎间盘退变的细胞移植治疗研究起到一定得帮助作用。
[Abstract]:Part 1: induced intervertebral disc degeneration model in rabbits by suction method
Objective: to construct a rabbit disc degeneration model by suction method, and to observe its pathological and imaging findings.
Methods: October age healthy New Zealand white rabbits 28, 7 rats in the control group, the model group 21. Respectively in the model of rabbit L4/5 single segment pierced the skin using 21G puncture needle aspiration of nucleus pulposus more 0.008-0.012g. modeling is completed, the experimental rabbits cultured for 4-20 weeks. Before and after modeling using X-ray flat to observe the changes of intervertebral disc degeneration, MRI and Alcian blue staining.
Results: rats before and after the degeneration of the intervertebral disc height index percentage (DHI%): control group, 4 week group, 12 week group, 20 week group respectively (100 + 0)% and (81 + 3.2)% and (75 + 2.5)% and (71 + 1.8)% (P0.05) of degenerative intervertebral. T2 weighted image signal was significantly reduced, Alcian blue staining showed significantly lower aggrecan content in degenerative intervertebral discs.
Conclusion: the rabbit model of intervertebral disc degeneration induced by aspiration is simple and reliable. The pathological and imaging findings of degeneration are very similar to the pathological and imaging findings of human intervertebral disc degeneration. The degenerative model can be applied to the study of the mechanism and clinical treatment of human intervertebral disc degeneration.
The second part: the induction of marrow stromal cells by nucleus pulposus cells
Objective: To investigate the induction of bone marrow mesenchymal stem cells (MSCs) induced by nucleus pulposus cells and bone marrow stromal cells (BMSCs) during co culture with alginate microspheres.
Methods: the April age healthy New Zealand white rabbits 5, nucleus pulposus cells and bone marrow mesenchymal stem cells were cultured, isolated and purified by passage method; the purification of bone marrow mesenchymal stem cells by trypsin digestion, collection, and mixed with appropriate amount of sodium alginate solution, suspension form solution 106 /ml single cell, the mixed single cell suspension by syringe dropping to 3.5% CaCl2 in solution, the formation of calcium alginate gel beads; the calcium alginate microspheres and nucleus pulposus cells were co cultured. In 7 days and 15 days. The group dissolved calcium alginate gel beads, bone marrow collection mesenchymal stem cells, respectively by immunohistochemistry technology, rt-PCR technology and Western blot technology, the detection of bone marrow mesenchymal stem cells in type II collagen and aggrecan expression, to determine the nucleus pulposus cells induced by stem cells in the non contact condition of bone marrow.
Results: the bone marrow mesenchymal stem cells cell smear immunohistochemical staining in visible cells, type II collagen and aggrecan positive staining, rt-PCR and Western, the results showed that blot expression after induction of bone marrow mesenchymal stem cells for type II collagen and aggrecan gene, and cultured for 15 days the purpose of the group of bands were brighter than 7 days induction group band.
Conclusion: when cultured in vitro, nucleus pulposus cells can induce bone marrow stromal cells and differentiate bone marrow stromal cells into nucleus pulposus cells, which will provide reliable source of seed cells for transplantation of intervertebral disc degeneration.
The third part: differentiation and immortalization of bone marrow mesenchymal stem cells to nucleus pulposus cells
Objective: to construct an immortalized nucleus pulposus cell derived from bone marrow stromal stem cells (MSCs) by means of co culture and SV40Tag, and immortalized gene introduction.
Methods: the experimental rabbits were primary MSCs and nucleus pulposus cells (NPCs), fluorescent NPCs; NPCs and MSCs labeled directly cultured for 6,9,12,15,18 days; after co culture, the use of selected fluorescence labeled negative cells, flow cytometry MSCs; morphology changes of MSCs cells after co culture observation, detection of changes in expression intracellular type II collagen and aggrecan mRNA and protein; co cultured for 15 days, the transfection of pCMVSV40T/PUR plasmid containing MSCs SV40Tag immortalized gene, the proliferation of MSCs was observed by MTT after transfection.
Results: after co culture, expression of type II collagen and aggrecan, a large number of MSCs cells in cell morphology from long oval shape to polygon; when co cultured for 15 days, intracellular type II collagen and aggrecan mRNA expression reached the maximum concentration, but lower than that of the original generation of intracellular NPCs type II collagen and aggrecan content of mRNA (P0.05). SV40Tag gene, the proliferation ability of MSCs-SV with the same generation of NPCs, after 20 generations the proliferation ability decline (P0.05).
Conclusion: co culture and SV40Tag immortalization can be used to construct immortalized nucleus pulposus cells derived from MSCs. These nucleus pulposus cells may play a role in the study of cell transplantation therapy for intervertebral disc degeneration.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【参考文献】