【作者】 刘长剑；

【导师】 刘建国；

【作者基本信息】 吉林大学 ， 外科学， 2008， 博士

【摘要】 现已知各种物理因素及化学刺激在成骨过程中起重要的调节作用,这些因素综合作用,共同完成对成骨的调节。目前医学界尚未完全探明这些机制,这些机制的阐明对于临床上骨病及骨创伤的治疗皆有重要意义。成骨细胞和骨细胞一直被认为是主要的应力感受细胞。低能体外冲击波(ESW)作为一种物理能量,能够穿透外层组织直接作用于成骨细胞,引起多种生物效应从而影响成骨。近来研究证实,ESW作用于成骨细胞后会激活蛋白激酶C(PKC)、丝裂酶原激活蛋白激酶(MAPK)等促进细胞增殖。但其作用的具体细胞内信号转导机制尚有待于进一步明确。目前研究表明甲状旁腺素PTH也对骨形成具有调节作用。正常人体内PTH的分泌遵循两种时相原则:①PTH分泌量在频繁活动中,这种浓度特点可以调节血钙平衡。②PTH分泌状态高度稳定,每天的分泌次数、分泌量都有一定规律,这一浓度变化特点有助于骨形成。研究也证实:连续给予较高剂量PTH可使实验动物体内骨形成减少;而周期性低剂量给予PTH可使实验动物体内成骨增加。但其机制并未完全明了。本研究采用可促进成骨的物理刺激—ESW和能促进成骨的化学刺激—低剂量间歇性人重组甲状旁腺素1-34(rhPTH1-34)刺激作用于体外培养的大鼠成骨细胞(ROB),来观察它们对ROB体外成骨的影响,并探讨其分子机制。另外,PTH作用于成骨细胞的体内环境中,由于液体流动、活体运动等因素,也是处于持续的应力刺激之下;因而本课题进一步在体外将应力刺激ESW和低剂量间歇式rhPTH1-34刺激因素结合起来,模拟ROB生长的体内环境观察两者对体外培养ROB成骨的影响,在体外证明ESW刺激可通过“ESW→PKC(某些亚型)→p38 MAPK→ROB成骨分化”信号转导通路起作用;而间歇性人重组甲状旁腺素1-34(rhPTH1-34)刺激可通过“间歇性rhPTH1-34→PKC(另外一些亚型)→ROB成骨分化”信号转导通路起作用;两种刺激共同作用的效果比各自单独作用效果显著增强。这也为临床应用ESW和rhPTH1-34促进骨折愈合提供理论依据。更多还原

【Abstract】 It has been widely known that many physical and chemical factors contribute to bone formation. Various effects of different stimulates are shown according to different periods and different organs they take effect. Detailing of these mechanisms are of vital importance to medical research.As a kind of sound wave, ESW can produce tremendous pressure variation in an extremely short period of time. When ESW traveling through medias of different densities, the relief of energy will result in kinds of biochemical effect to stress sensitive cells, such as osteoblast. In vivo, changes of local micro environment and fluid shearing stress(FSS) caused by ESW can cause series of bio-effect to these cells at molecular level. Many research have confirmed that osteoblast can be activated by ESW stimulate to contribute to bone formation. Therefore, the conception of stress construction in bone tissue engineering arises in recent years.The balance of bone formation and bone absorption is modulated by many kinds of chemicals, including PTH. In vivo, the secretion model of PTH are of 2 kinds. First, the quantity of secreted PTH is frequently altered, which contribute to the maintenance of constant calcium concentration in serum. Second, the amount and period of PTH secretion are constant, which contribute to bone formation. Thus, different patterns of PTH stimulates may cause different result to bone formation. Some researches show that continual high dose rhPTH1-34 inhibit bone formation, while low dose impulse rhPTH1-34 stimulate enhance it. But the mechanism is still unclear.ESW has been proved to be effective to enhance bone formation. But, as a kind of stress stimulate, its mechanism is still unclear. Many aspects of its clinical application, such as suitable energy density, the frequency, etc, are all left to be identified. The same as ESW, PTH’s mechanism of enhancing bone formation is not clear either. Exploring of these mechanisms, especially the intracellular signal transduction, are of key importance to Bone Tissue Engineering research and clinical application.1、Primary culture and identification of rat osteoblast.1.1 Aims: to establish reasonable and effective new method and process of in vitro osteoblast culture and identification.1.2 Methods: modified isolation method of osteoblast by type I collagenase was adopted to get rice’s calvarial bone cell. The cells were cultured invitro and identified. The growth line was drew to observe the proliferation of cells. Cell morphology was observed and recorded by microscope. Cells’Alkaline Phoshatase (ALP) and type I collagen expression was detected by method of Immunohistochemical staining.1.3 Results: by modified isolation method, purified bone cells were obtained. Growth line shows excellent proliferation rate of the cells. Immunohistochemical staining of ALP shows brown and yellow region exist in plasma of positive cells and the positive percentage is 77.3%. Type I collagen’s immunohistochemical staining shows the positive cells’plasma turn into brown and yellow and the positive rate is 77.3%. Calcic node was identified in the matrix of osteoblast had been cultured invitro for longer than 3 weeks.1.4 Conclusions: Modified isolation by type I collagenase is an excellent osteoblast primary culture method.2、Low Density Extracorporeal Shock Wave and Low Dose Pulsed rhPTH1-34’s Effect to proliferation and differentiation of Rat Osteoblast2.1 objective to investigate low density ESW and low dose pulse rhPTH1-34 stimulation’s effect on proliferation and differentiation of ROB. 2.2 method After stimulations of different times of 0.18mJ/mm2ESW, different pattern of rhPTH1-34 and ESW+pulse rhPTH1-34 (10-11M), ROB cells were collected to observe cell proliferation by cell counting, MTT and DNA cycle analysis and to observe cell differentiation by measuring ALP activity and type I collagen expression.2.3 result 0.18mJ/mm2ESW(60-150 times), pulse rhPTH1-34 (10-11and 10-10M) and ESW(60-150 times)+pulse rhPTH1-34 (10-11M) stimulation can all improve proliferation and differentiation of cultured ROB(p<0.05, compared with control). The effect of ESW+pulse rhPTH1-34 stimulation was stronger than ESW alone and pulse rhPTH1-34 (10-11M) alone(p<0.05).2.4 conclusion Suitable ESW stimulation and low dose pulse rhPTH1-34 stimulation can improve proliferation and differentiation of cultured ROB significantly. The effect of combining ESW with pulse rhPTH1-34 (10-11M) stimulation was the most strongest.3、The intracellular signal transduction of ROB stimulated by suitable ESW and Low Dose Pulsed rhPTH1-343.1 objective To have a study on the mechanism of ESW and low dose pulse rhPTH1-34’s contribution to proliferation and differentiation of ROB, and to investigate the role of PKC and p38MAPK in the intracellular signal transduction path.3.2 Method Added with specific blocker of PKC or p38MAPK, proliferation of ROB are measured by Typan Blue Dye, MTT and DNA circle analysis by Flow Cytometry to observe the role of PKC and p38MAPK during the process. Added with specific blocker of PKC or p38MAPK, bone formation of ROB are observed by ALP activity detection and expression percentage of collagen I to observe the role of PKC and p38MAPK during the process. Added with specific blocker of PKC or p38MAPK, activation of p38MAPK is measured by Western Blot to define the role of PKC and p38MAPK in the related intracellular signal transduction path.3.3 Result 3.3.1 Cell proliferation and bone formation of ROB measured by methods mentioned above are significantly high in cells treated by low dose pulse rhPTH1-34, ESW and cooperation of both stimulates(P<0.05). The effect of cooperation of low dose pulse rhPTH1-34 and ESW is the strongest, and is significantly higher than low dose pulse rhPTH1-34 and ESW(P<0.05).3.3.2 Added with H7, the specific PKC blocker, proliferation and bone formation of ROB treated with low dose pulse rhPTH1-34 become significantly less than treated with rhPTH1-34 alone(P<0.05), which indicates that PKC may play a key role. But SB203580 can not affect proliferation and bone formation of ROB treated with low dose pulse rhPTH1-34(P>0.05), which indicates that p38MAPK don’t take part in the process.3.3.3 Added with H7, the specific PKC blocker, proliferation and bone formation of ROB treated with ESW become significantly less than treated with ESW alone(P<0.05), which indicates that PKC may play a key role. SB203580, a specific p38MAPK blocker, can also affect proliferation and bone formation of ROB treated with ESW(P<0.05), which indicates that p38MAPK take part in the process too.3.3.4 Measured by Western Blot, P-p38MAPK expression of ROB treated with ESW alone and cooperation of ESW and rhPTH1-34 are higher than that of the control and treated with rhPTH1-34 alone(P<0.01), which confirms that p38MAPK can be activated by ESW, but not low dose pulse rhPTH1-34 stimulation. Activation of p38MAPK by ESW can be inhibited by H7(P<0.01), which shows that PKC take part in the activating. All these come together to indicate exist of the signal path: ESW→PKC→p38MAPK→ROB’s proliferation and differentiation. While H7 can significantly inhibit the proliferation and bone formation of ROB treated with low dose pulse rhPTH1-34, p38MAPK can not be activated by rhPTH1-34, which indicate exist of another signal path: rhPTH1-34→PKC→ROB’s proliferation and differentiation.3.4 Conclusion Suitable ESW stimulation and low dose pulse rhPTH1- 34 stimulation can improve proliferation and differentiation of cultured ROB significantly. The effect of cooperation of ESW and rhPTH1-34 stimulation was the most strongest. Proliferation and bone formation of ROB treated with ESW can be inhibited by H7 and SB203580, and activation of p38MAPK by ESW can be inhibited by H7, which indicate exist of the signal path: ESW→PKC(some isoforms)→p38MAPK→ROB’s proliferation and differentiation. While H7 can significantly inhibit the proliferation and bone formation of ROB treated with low dose pulse rhPTH1-34, p38MAPK can not be activated by rhPTH1-34, which indicate exist of another signal path: rhPTH1-34→PKC(other isoforms)→ROB’s proliferation and differentiation.更多还原