【作者】 杨茂光；

【导师】 杨小玉；

【作者基本信息】 吉林大学 ， 外科学， 2012， 博士

【摘要】 微管相关蛋白1B（Microtubule associated proteins;MAP1B）的生物学活性受其磷酸化修饰调节，蛋白磷酸酶2A(Protein Phosphotase2A;PP2A)对MAP1B去磷酸化具有调控作用，但磷酸化修饰后MAP1B对骨髓间充质干细胞(Bonemsenchymal stem cells;BMSCs)靶向损伤脊髓迁移的作用及BMSCs内调控MAP1B磷酸化修饰的机制尚未见相关报道。本论文旨在验证BMSCs内PP2A介导的I型MAP1B磷酸化形式（Type I of phosphorylated MAP1B; P1-MAP1B）去磷酸化在脊髓缺血性损伤(Spinal Cord Ischemia Injury;SCII)后BMSCs靶向脊髓迁徙中的作用，及BMSCs内调控MAP1B磷酸化修饰分子机制。研究内容包括：1、兔BMSCs体外培养、鉴定。2、应用CM-Dil对BMSCs进行体外荧光标记和体内示踪。3、PP2A抑制剂OA和激动剂C2-ceramide预处理BMSCs；通过同位素32P标记蛋白底物法和Western-blot分别检测PP2A活性与I型MAP1B磷酸化形式P1-MAP1B含量。3、应用节段性腰动脉阻断术建立兔SCII模型，随机分为四组：IG组（抑制剂组）、AG组（激动剂组）、UG组（未治疗组）和CG组（对照组）；经耳缘静脉移植BMSCs后观察各组BMSCs活体内靶向脊髓迁移情况。4、应用磷酸化抑制剂LY294002与U0126分别阻断BMSCs内的PI3K和ERK1/2通路，Western-blot定量检测BMSCs内P1-MAP1B。结果显示：1、所收获BMSCs形态典型，具有间充质干细胞生长特性；2、CM-Dil荧光标记BMSCs后标记率达到99%以上；OA与C2-ceramide处理细胞后PP2A活性分别降低61%和增长78%（P<0.05），P1-MAP1B表达分别增高和降低（P<0.01），而MAP1B表达无明显变化（P>0.05）。干预效果可维持48h。OA与C2-ceramide预处理细胞后测得生长曲线较低平，BMSCs增殖略减缓（P<0.05）；3、成功建立兔脊髓缺血性损伤模型并移植BMSCs后，在激光共聚焦显微镜下观察可见：移植2天后，IG组和AG组在脊髓损伤部位均未发现荧光标记BMSCs聚集，UG组相应部位可见少量散在BMSCs；移植4天后IG组和AG组的脊髓损伤部位可见BMSCs聚集；4、与对照组相比较，抑制PI3K活性后MAP1B磷酸化水平升高；而抑制ERK1/2后MAP1B磷酸化水平降低（P<0.05）。研究表明：证明BMSCs内PP2A介导的MAP1B去磷酸化在调节BMSCs靶向脊髓损伤迁移过程中发挥重要作用。其中作用机制可能是MAP1B与P1-MAP1B维持动态平衡参与调节BMSCs靶向迁移，为MAP1B蛋白功能研究提供新的理论依据。目前尚无关于MAP1B与BMSCs靶向脊髓损伤迁移之间关系的报道。更多还原

【Abstract】 【objective】Axon guidance and upregulated of MAP1B after SCI are utilized as the topicresearch background. Based on fluorescent tags, western blotting, andimmunocytochemistry, we focus on the relativity between phosphorylated level ofMAP1B and migration of BMSCs towards injured spinal cord, especially on detectingand analyzing the signaling pathways and molecular mechanisms for the migrationregulated by MAP1B, which was designed to verify the correlation betweenMAP1B and BMSCs migration and tentatively elaborate the relevant signalingmechanisms which could provide an important theoretical basis to improve BMSCstherapy effect for SCI.【Methods】Part I: To establish an optimal culture system for cultivating the bone marrowmesenchymal stem cells (BMSCS) of rabbit in vitro and via microscopicmorphological observation, the best cell was chosen to analyze BMSCs activity. Todraw a cellular growth curve by MTT colorimetry, and analyze the expression of cellsurface antigens and cell cycle by flow cytometry and antigen-antibody reaction.Part II: Fluorescent dye CM-DIL to label rabbits BMSCs in vitro was used, afterwhich determinating the labelling rate by flow cytometry, analyzing and comparingBMSCs activity by MTT colorimetry and growth curve. The highest fluorescentlylabeling rate and the best growth activity of rabbit BMSCs were selected to pre-dealwith OA and C2-ceramide, and cells were divided into the inhibitor group, agonistgroupand control group, respectively. PP2A inhibitor OA and PP2A agonistC2-ceramide could regulate activity of PP2A directly and change phosphorylationlevels of MAP1B indirectly, after that analyzing and comparing BMSCs activity byMTT colorimetry again were proceeded. And PP2A activity via isotope-labeledenzyme activity assay and MAP1B phosphorylated levels via western blot weredetected. Part III: Animal models about experimental SCI via blocking segmental lumbarartery for25minutes was established. Based on modified Tarlov score, the behavioraldifference of animal models from the surgery group and the sham group wereobserved and compared before live sampling at2, and7days after surgery. Animalethology and pathological morphological observation could validate the model whichwas established successfully. The surgical group models are divided into three groupsand transplantated with BMSCs via the marginal ear vessels of the rabbits.Respectively, each group will be lively sampled at2,4and7days aftertransplantation and observed by using laser confocal microscope to detect fluorescentmarker of BMSCs in spinal cord.Part IV: To use inhibitors to block the PI3K and ERK pathway in rabbit BMSCsand detect MAP1B phosphorylation level of BMSCs via immunoblotting, themodification function of regulating the phosphorylation of MAP1B was analyzed.【Results】Part I: The rabbit BMSCs cultured system was established successfully.Comparing the different passages of BMSCs’s morphology, there was no distinctdifference.The BMSCs harvested showed absence of CD34, CD45and the greatmajority of which were at the G0/G1point in the cycle. The detection of BMSCs’proliferation has shown that BMSCs accumulat greatly in the period of logarithmicphase for3-5days.Part II: The labeling yield of CM-Dil is more than99%on the condition that thecells’ proliferation wasn’t affected. Regulation of OA and C2-ceramide, however,made the activity of PP2A drop to39%and increases to178%, respectively, comparedwith that in the untreated group (p<0.05). The phosphorylation of BMSCs wasaffected accordingly by the C2-ceramide PP2A activity, according to immunoblotting.However, the changes above were back to normal at48hours after pretreatment(p>0.05). BMSCs’ proliferation was affected by C2-ceramide and OA, the growthcurve of BMSCs showed that the BMSCs of AG group still kept the most of growingability.Part III: Rabbit ischemic spinal cord injury model was successfully established,and the assessment of hindlimb locomotor function have shown that surgery groupwas2.7~3.2points (P<0.01). After ischemic injury, pathomorphological observationsshowed that the majority of nuclear membrane is integral and available, and the nuclei was slightly swelling. Part of the nuclear was translocation, deeply stained;condensation, fragmentation and the neurite reduce or disappear. Via transmissionelectron microscopy(TEM), rough endoplasmic reticulum and mitochondrial werefound swelling, and part of the mitochondrial membrane ruptured. Wide gapsappeared between layers of myelin shea. The cavitation and other pathologicalchanges were found in nuclear matrix. By laser scanning confocal microscope, theBMSCs in the injured spinal cord was observed and the difference between inhibitiongroup and agonists group, comparing to untreated group, was recorded. At the3rddayafter transplantation, there was no BMSCs in the injured spinal cord in the inhibitiongroup and agonists group, which was different until the fifth day.Part IV: Compared with the control group, inhibition of PI3K resulted in higherphosphorylated levels of MAP1B, while inhibition of ERK led to lowerphosphorylated levels of MAP1B.【Conclusion】BMSCs were successfully cultured and maintained proliferation in theexperiment, which successfully established the rabbit segmental ischemic spinal cordinjury model. BMSCs that were transplanted have successfully migrated into theinjured spinal cord which could be affected by phosphorylated level of MAP1B. Asincreasing or reducing of P1-MAP1B could break the dynamic equilibrium ofMAP1B/P1-MAP1B, the number of BMSCs within the injured spinal cord will befallen off. All of these factors have shown that dynamic equilibrium of MAP1B playsan important role in the regulation of BMSCs migration. PI3K, ERK and PP2A werein charge of the dynamic equilibrium and participated in the signal transductionpathways which regulate MAP1B phosphorylation.【Innovation】The paper innovatively relates the axon guidance of MAP1B and itsphosphorylated I type with BMSCs migration. And it proves that the form ofMAP1B and its phosphorylated I type can play an important role in the process ofBMSCs migration by experiment. Maintaining homeostasis between MAP1B andP1-MAP1B is essential for BMSCs athletic ability, which provides a new theoreticalbasis for the MAP1B protein function. There has not been reported about relativitybetween MAP1B and BMSCs migration ability.In the past, MAP1B was mainly as a neural differentiation marker proteins of BMSCs. For the first time, we aims at studying the signal transduction pathways ofthe regulation of MAP1B phosphorylation in BMSCs, analyzing the signalmechanism of MAP1B in the cell migration-oriented function in BMSCs, enrichingand perfecting the migration signal transduction pathway of BMSCs migration.更多还原