【作者】 林乐；

【导师】 范广宇；

【作者基本信息】 中国医科大学 ， 外科学， 2009， 博士

【摘要】 前言糖皮质激素具有良好的抗炎、免疫抑制作用,在骨科中对类风湿骨性关节炎,脊髓损伤有良好的治疗效果,临床上应用广泛。随着临床应用的增多,缺血性骨坏死发生率明显上升,是非创伤性缺血性骨坏死首要诱发因素,但其引起缺血性坏死的详细机理还不是十分清楚。目前糖皮质激素引起缺血性骨坏死的发病机理主要包括：骨髓中脂肪严重蓄积,造成骨内高压,血流灌注降低；血小板增多,粘滞性增大,使髓内静脉瘀滞,骨内压上升,骨内血流减少,骨细胞坏死；间充质细胞受损,成骨细胞的祖代细胞来源减少,向成骨、软骨细胞分化障碍,骨修复重建与骨吸收不能维持动态平衡。其中,对于糖皮质激素引起的髓内脂肪蓄积是由机体脂肪代谢异常在髓内形成的的脂肪沉淀,还是由于激素直接作用于骨髓间充质细胞,使细胞发生了脂肪分化以及具体生成的过程目前还没有定论。骨髓间充质细胞(Bone Marrow Mesenchymal Cell, BMSC)是存在于骨髓中的一种具有多向分化潜能的干细胞,能够分化为中胚层和神经外胚层来源的多种组织细胞,包括成骨细胞,软骨细胞,脂肪细胞和神经细胞等。研究报道,体外培养的BMSC可以在体外、体内向成骨细胞、脂肪细胞分化,且这种分化会随着体内外微环境的变化而发生改变,而糖皮质激素对细胞的成骨和脂肪分化有着显著影响。骨髓间充质细胞在向各种组织细胞分化中受多种因子的调控,目前已知的调控细胞成骨分化的关键因子Runχ2和脂肪分化早期的特异性因子(PPARγ-2)的出现与表达的变化,均能够客观反应骨髓间充质细胞分化进程,决定骨髓间充质细胞的分化方向。本课题通过地塞米松对骨髓间充质细胞脂肪分化和成骨分化的影响,探讨糖皮质激素引起骨坏死的发生机理,阐述骨髓间充质细胞的脂肪生成在非创伤缺血性骨坏死发生中的作用和影响。并通过进一步观察糖皮质激素对Runχ2基因转染骨髓间充质细胞的成脂分化作用,探讨成骨调控基因过表达时,糖皮质激素对骨髓间充质细胞分化作用的效果。立题一：糖皮质激素对骨髓间充质细胞分化的作用骨髓间充质细胞具有多向分化潜能,在不同的培养条件下,能够分化成多种组织和细胞,在体外正常培养中,大多数BMC能够分化为成骨细胞。本实验以剂量和时间方式观察地塞米松对骨髓间充质细胞分化的影响,同时观察地塞米松对细胞和细胞内细胞器形态学上的显微及超微变化的影响。立题二：地塞米松影响骨髓间充质细胞Runχ2和PPARγ-2基因的表达基因Runχ2是调控骨髓间充质细胞向成骨分化的关键性因子；PPARγ-2是脂肪细胞早期特异性标志因子。基因表达的不同代表了细胞分化方向的不同。本立题通过检测成骨基因(Runχ2)和成脂基因(PPARγ-2)mRNA和蛋白的表达,阐述地塞米松在基因和蛋白水平上对骨髓间充质细胞分化的影响。立题三：上调Runχ2对糖皮质激素诱导骨髓间充质细胞成脂分化的抑制作用在糖皮质激素促进骨髓基质细胞脂肪分化,抑制成骨分化过程中,成骨基因Runχ2表达减少,成脂基因PPARγ-2表达增高。如何拮抗糖皮质激素对骨髓间充质细胞的这种作用,从而为临床上应用糖皮质激素引起的缺血性骨坏死找到解决方法。本立题利用基因转染技术,检测Runχ2基因转染骨髓间充质细胞后,成骨标志性因子(ALP.Col I和OCN)基因mRNA的表达,探讨大剂量糖皮质激素对基因转染后的细胞成脂分化的作用。材料与方法立题一：糖皮质激素对骨髓间充质细胞分化的作用1、骨髓间充质细胞的分离、培养取3-4周龄SD大鼠双侧股骨和胫骨,10%FBS低糖DMEM培养液冲洗骨髓腔,过滤,离心,培养,0.25%胰酶消化传代培养。2、碱性磷酸酶染色和SudanⅢ复染含有不同浓度地塞米松培养液培养骨髓间充质细胞,碱性磷酸酶、SudanⅢ复染,观察SudanⅢ染色的脂肪细胞和碱性磷酸酶染色阳性成骨细胞数量变化的关系。3、细胞形态学的变化倒置相差光学显微镜和透射电镜观察地塞米松刺激后,细胞和细胞内细胞器形态上的显微及超微变化。4、细胞碱性磷酸酶活性检测改良的Kaplow氏法检测：分别以剂量方式和时间方式的地塞米松对细胞碱性磷酸酶活性的影响,比较不同组别的吸光度值差异。5、MTT方法检测地塞米松对细胞增殖的影响：检测各组细胞的吸光度值,比较不同浓度地塞米松对细胞增殖能力的抑制效果。立题二：地塞米松对骨髓间充质细胞Runx2和PPARγ-2基因表达的影响1、运用Real-Time PCR方法,检测成骨基因(Runx2)和脂肪基因(PPARγ-2) mRNA的表达：比较不同浓度地塞米松对骨髓间充质细胞分化的作用。2、Western blot印迹法检测成骨分化Runx2蛋白的表达：从蛋白表达变化上说明大剂量地塞米松抑制骨髓间充质细胞的成骨分化。立题三：上调Runx2对糖皮质激素诱导骨髓间充质细胞成脂分化的抑制作用1、构建pCMV/flag-Runx2质粒2、pCMV/flag-Runx2质粒转染骨髓间充质细胞,分别采用Western blot印迹和RT-PCR法以及免疫荧光化学方法确定重组质粒的转染是否成功。3、通过RT-PCR方法检测Runx2基因转染间充质细胞后,成骨分化标志性基因(ALP, ColⅠ、OCN)和脂肪分化基因(PPARγ-2、aP2)的mRNA表达；同时在高表达Runx2基因的情况下,大剂量地塞米松对上述基因表达的影响。4、Western blot印迹测定地塞米松对Runx2基因转染后,骨髓间充质细胞骨钙素表达的影响。实验结果1、地塞米松刺激后,细胞形态和排列发生变化,早期,细胞由梭型变为多角形或不规则性,出现细胞团聚,改变其致密的排列,团聚的细胞折光性增强。后期,细胞团聚现象消失,细胞形态呈圆型,细胞稀疏。2、随着地塞米松浓度增高,细胞内SundanⅢ着色的脂肪颗粒明显增加3、对照组的碱性磷酸酶活性与10-8 10-7 10-6M的地塞米松中,增高分别为1.57,4.49,5.0倍。4、电镜显示：细胞核缩小,核膜皱折增多,核内染色质发生固缩,电子密度增强,胞质明显减少。5、地塞米松减少Runχ2的表达88-37%(P<0.05),增加PPARγ-2的表达108-230%(P<0.05)；明显减少Runx2蛋白表达,(P<0.05)。6、.Runχ2基因转染后,细胞Runx2mRNA表达增加257%(P<0.05),下游成骨基因的表达增加明显(P<0.05)；PPARγ-2的表达减少76%(P<0.05)。结论1、地塞米松促进骨髓间充质细胞的脂肪分化,抑制成骨分化。2、地塞米松通过减少成骨调控因子Runχ2,增加脂肪分化因子PPARγ-2的基因和蛋白表达,促进细胞脂肪分化,抑制其成骨分化。3、Runχ2基因修饰的骨髓间充质细胞对地塞米松的促脂分化作用具有拮抗效果。更多还原

【Abstract】 IntroductionThe application of glucocrticoid occupied the primary position among many causes of induced avascular osteonecrosis. Glucocorticoids have fine characteristics of anti-inflammation and immunosuppression, and have good action in therapy of rhemuatoid arthritis and spinal cord injury. The extensive application of glucocorticiods has resulted in the increasing incidence of non-traumatic avascular osteonecrosis. However, the precise pathobiological mechanism underlyig the induction of avascular osteonecrosis by steriods has not been eluciated to our knowledge. Various pathomechanism of avascular osteonecrosis by glucocorticoids include progressive accumulation of marrow fat stores, intraosseous hypertension, decreased infusion of blood supply, the increased amount of platelet and viscosity resulted in marrow vessel obliteration. These changes would make stem cells injuried, and then they could not differentiate into osteoblasts or chondrocytes, leading to the reduced resource of osteoblasts’precuors, or the dynamic imbalance of bone reconstruction and resorption, and with osteocytes apoptosis by steroid, further generating avascular osteonecrosis. In these theories, the research about accumulatoion of fat and increasing size of adipocyte in medullary cavity due to fafty metabolism or to the direct influence on differentiation of mesenchymal cell by steroid is becoming a main issue.Bone marrow mesenchymal cells(BMC) located in medullary cavity are the multipotentional cells, and able to differentiate into various tissuese and cells that derived from mesoderm or neuroectoderm, including osteoblast, chondrocyte, adipocyte and neurone. Cultured BMCs invitro have been verified that may be differentiate into osteoblasts and adipocytes in vivo and vitro, furthermore, the trend of differentiation would be changed with influence of microenviroment. The significant effect of glucocorticoid on the adipogenic and osteoblastic differentiation of BMCs is a hotspot recently. Core binding factor al (Runx2/Cbfal) is a key regulatory factor that is required to commit mesenchymal progenitors to the osteoblast lineage. By contrast, the expression of PPARy-2, the early specific adipocytic marker, destines cells for adipocyte differentiation. The expression and occurrence of the two genes could really govern and respond to the progress of mesenchymal cells. The dissertation observe and detect the effect of differentiation of bone marrow mesenchymal cell by glucocorticoid,to study and research the pathomechanism of non-traumatic avascular osteonecrosis with glucocorticoid. Furthermore, interpretation of the accumulation and increasing volume of fat is adipogenesis of mesenchymal cells by glucocorticoid.Study 1:The effect of dexamethasone on differentiation of bone marrow mesenchymal cellsBMCs have the abilities of multipotentional differentiation. Under different conditions, it can be induced to differentiate into various tissuese and cells. Most BMCs could differentiate into osteoblasts under normal culture conditions without dexamathasone in vitro. Therefore the experiment by dose and time-manners detect the effect of different concentration dexamethasone on differentiation of mesenchymal cells, and observe the microvariation of cells and supermicrovariation of cellular organs with a inverted phase contrast microscope and transmission electron microscope in the differentiation course.Study 2:Dexamethasone effects on the expression of osteogenetic and adipogenic genes of bone marrow mesenchymal cellsRunx2 gene, a key factor, regulates osteoblastic differentiation. PPARy-2 gene is fat-cell early specific marker. If expression of these genes has disparity, it will suggest diffferent trend of differentiation of mesenchymal cells. The experiment tests the variant expression of Runx2 and PPARy-2 genes under different concentration of dexamethasone stimulation using real-time PCR and Western blot methods, and compare with control group without dexamethasone, indicating the effect of glucocorticoid on cell differentiation at the levels of gene and protein.Study 3:Up-regulation of Runx2 gene supresses glucocorticoid-induced the adipogenic differentiation of mesenchymal cellGlucocorticoids enhanced adipogenic differentiation at the expense of osteoblastic differentiation in BMC, with increasing the expression of PPARγ-2 and decreased the expression Runx2 genes. How can we reverse the direction of adipogenic differentiation and find a solution for clinical administration? Therefore, we investigate expression of osteoblast marker genes(ALP, Col I, OCN) under Runx2 gene transfected condition, and explore influence of glucocorticoid on differentiation of mesenchymal cell transfected gene.Materials and MethodsStudy 1:1. Isolation and culture of bone marrow mesnechymal cellsThe bone marrow cells were flushed from the shat of bi-femur and tibia of 3-4w SD(SPF) with DMEM containing 10% fetal bovine serum, filtered, centrifuged and cultured. The stromal cells were cultured 7 to 10 days until they reached 80 to 90% confluence, and then digested with 0.25% trypsin and subcultured.2. Alkaline phosphatase and Sudan III counterstained:Divide four concentratiations of Dex duration to 21d and then Alkaline phosphatase, Sudan III counterstained, and to observe the number of adipocyte and osteoblast, compared with control without dexamethasone.3. Morphologic changes:Observe the microvariation of cell and supermicrovaria-tion of cellular organs under dexamethasone stimulation with a invert phase contrast and TEM microscopes.4. The activity of phosphatase assay:Use the improved Kaplow’s method to test the activity of alkaline phosphatase of each group and then compare with control group without dexamethasone.5. MTT method determination of different concentrations of dexamethasone on the ability of cell proliferation.Study 2:1. We tested the expression of osteoblastic genes Runx2 and adipogenic gene PPARy-2 by using Real Time PCR, and then compare different concentration dexame-thasone groups with control.2. Western blot analysis was applied to investigate the differential expression of proteins (Runx2), and then at seven day after cultuerd with dexamethasone 10-7mol/L compare with control.Study 3:1. pCMV/flag-Runx2 plasmid reconstruction2. In order to confirm that Runx2 gene was transfected into BMC, we detect the expression of protein Flag, a plasmid tag, and protein Runx2 and osteocalcin by western blot and immunofluoresence techni, respectively.3. We test expression of osteoblast-related down-stream genes including ALP、ColⅠ、OCN and adipogenic genes of PPARy-2 and aP2 of cells transfected Runx2 gene by RT-PCR.4. By using western blot analysis, we test the expression of proteins OCN of BMC transfected Runx2 gene under stimulation of dexamethasone, to compare with control.Results1. BMCs exhibited fibroblast-like spindle phenotype without obvious deformation among subculture passages. However, when they were treated with dexamethasone, their shapes were became polygonal or irregular phenotype, and compacted alignment became agglomeration as well as increasing refractivity.2. With the increased concentration of Dex, the number of Sudan III stained lipochondria is significantly to rise.3. The activity of ALP without Dex is 1.57,4.49,5.0 times compared with 10-8, 10-7,10-6mol/l Dex groups.4. The expression of Runx2 gene decreased to 88-37% with 10-9M mol/1 to 10-6mol/l dexamethasone(P<0.05) and increased adipogenic genes 108-230% (P< 0.05) compared with control group without Dex.5. The result of TEM:appearance of nuclear pyknosis and membrance reductus, sparse cytoplasm, decreased the number of cellular organelles.6. Expression of Runx2 gene in BMCs with transfected technique has increased to 257%(P<0.05) compared with control, and adipogenic genes reached to 76% (P<0.05) and osteoblast-related down-stream genes distinctly increased (P<0.05).Conclusion 1.Dexamethasone enhance adipogenic differentiation of bone marrow mesenchymal cells.2. Demathasone affect the osteogenesis through mesnechymal cell gene experssion with decreasing the expression of Runx2 and increasing the expression of PPARy-2.3.Mesenchymal cell transfected Runx2 gene was resistant to adipogenic differentiation of BMC treated with high concentratrion Dex.更多还原