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rhG-CSF动员和骨骼肌电刺激促进大鼠骨折愈合及其可能机制探讨
【作者】 马德媛；
【导师】 田振军；
【作者基本信息】 陕西师范大学 ， 运动人体科学， 2009， 硕士
【摘要】 目的:在骨科领域,如何加快骨折愈合、治疗骨不连和骨折的延迟愈合是该领域的热点问题,本文研究目的旨在探讨经皮骨骼肌电刺激联合干细胞动员对大鼠骨折愈合过程中骨重建及细胞增殖的影响及其可能机制。方法:选取3月龄大鼠（由西安交通大学实验动物管理中心提供）80只,体重180g～220g。随机分为4组,每组20只:骨折模型组（A组）;模型+动员剂组（B组）;模型+电刺激组（C组）;模型+动员剂+电刺激组（D组）。各组均采用国家标准啮齿类动物干燥饲料喂养,自由饮食。用自制造模支架建立大鼠胫骨闭合性骨折模型,刺激模型参照吕晓宇和彭亮刺激模式相结合并略加改动建立大鼠的电刺激模型。骨折后分2周、4周、6周和8周取材,制成不脱钙硬组织切片和脱钙骨切片,运用四环素和茜素红双荧光标记、特殊染色、免疫组织化学SABC法检测骨组织在骨折愈合过程中结构的变化、钙的沉积、成骨情况及细胞因子和细胞增殖相关蛋白的表达。结果:1用大鼠闭合性骨折造模支架造成大鼠胫骨中段骨折,经X光片判断,成功建立了大鼠胫骨中段闭合性骨折模型。2 rhG-CSF和（或）骨骼肌电刺激可促进大鼠骨折愈合后的功能恢复。骨折后单纯rhG-CSF作用和单纯骨骼肌电刺激以及rhG-CSF联合骨骼肌电刺激作用均可促进骨折愈合后的功能恢复,而rhG-CSF联合骨骼肌电刺激促进大鼠骨折愈合后的功能恢复效果最显著。3 rhG-CSF和（或）骨骼肌电刺激可使大鼠骨折愈合后软骨骨痂数量增多,骨组织钙化发生较早,骨折愈合程度较好,尤其以电刺激组和电刺激+动员剂组效果更明显。提示,骨折后进行骨骼肌电刺激协同骨髓干细胞动员,可以增加骨折愈合早期的软骨细胞数量促进软骨内成骨,加速骨折愈合。4单纯rhG-CSF动员和单纯电刺激均可使患肢骨2次标记线间距随愈合时间延长而逐渐增加,和模型组相比较显著性升高,单位面积相对荧光强度结果显示,单纯干预组可影响骨折愈合进程中钙盐的沉积,促进骨基质钙化。5 rhG-CSF动员和骨骼肌电刺激联合作用可使患肢骨2次标记线间距逐渐增大,在造模后第6周达到峰值。提示在骨折中后期钙盐沉积量最大;与单纯干预组比较患肢骨2次标记线间距显著性增大,表明联合干预比单纯干预更有助于骨折愈合。6 rhG-CSF动员和骨骼肌电刺激对骨折大鼠骨组织细胞生长因子表达产生影响。骨折愈合初期软骨和骨细胞bFGF和c-kit显著性升高;单纯电刺激和电刺激联合动员干预,可促进软骨和骨细胞的c-kit表达在第4周达到峰值,而单纯动员剂组在第6周到峰值;各组TGF-β1和bFGF均在第6周阳性表达达到峰值;在骨折愈合后期各细胞因子表达均有所下降,但仍高于对照组。表明单纯刺激和联合刺激方式均可不同程度促进骨诱导因子的表达,促进骨折愈合。7 rhG-CSF动员和骨骼肌电刺激对骨折大鼠骨组织增殖相关蛋白表达产生影响。电刺激和动员剂联合作用后,PCNA、BrdU和ki-67在骨折愈合过程中表达逐渐升高,表明细胞增殖相关蛋白在骨折愈合各时期均有表达,但在不同时期表达细胞的种类不同,早期主要是分裂期的间充质干细胞及软骨细胞,后期是成骨细胞。在rhG-CSF作用的大鼠骨折愈合早期BrdU阳性表达并不显著,后期有所升高,PCNA和ki-67则持续高表达;单纯电刺激组细胞增殖相关蛋白的表达也随骨折愈合表达逐渐升高,尤其BrdU表达升高具有极显著性差异。表明,单纯电刺激与单纯干细胞动员相比更有利于骨髓干细胞的增殖及分化。二者联合作用后PCNA、BrdU和ki-67表达峰值出现在第6周,提示双重促进作用使细胞增殖数量增加,且处于有丝分裂期的细胞数量较多,有利于骨组织的重建。结论:骨骼肌电刺激改善骨折处周围的力学环境,在骨折部位形成微动效应,为成骨作用提供力学条件,可使大鼠骨折愈合后软骨骨痂数量增多,骨组织钙化发生较早,患肢骨2次标记线间距随愈合时间延长而逐渐增加,骨折愈合程度较好,促进骨折愈合后的功能恢复。在此过程中,TGF-β1、PCNA和BrdU表达显著性升高,电刺激促进局部细胞因子的分泌和释放,加速骨折愈合。因此,采用早期微动方式的骨折愈合效果优于传统的石膏夹板绝对固定方式。rhG-CSF动员自体骨髓干细胞,促进骨髓间充质干细胞和造血干细胞的释放并向骨折部位迁移,增加干细胞向成骨、成软骨方向的分化,促进软骨内成骨、骨基质钙化和骨折部位血管新生,加速骨折愈合,促进大鼠骨折愈合后的功能恢复。在此过程中,骨折愈合初期软骨和骨细胞TGF-β1、bFGF、PCNA、BrdU和ki-67在第6周达到峰值;在骨折愈合后期各细胞因子表达均有所下降,但仍高于对照组。因此,采用早期自体骨髓干细胞动员方式促进骨折愈合的效果优于传统的石膏夹板绝对固定方式。rhG-CSF动员和骨骼肌电刺激联合作用后,既可促进骨髓间充质干细胞和造血干细胞的释放并向骨折部位迁移,增加干细胞向成骨、成软骨方向的分化,又保证了骨生长所需要的力学环境,改善局部的血液循环,加快骨改建进程:二者均可加强骨诱导因子和细胞增殖相关蛋白的表达,进而缩短骨折愈合时间。因此,骨骼肌电刺激引起的微动效应和干细胞动员促进骨折愈合的效果优于单纯骨骼肌电刺激、rhG-CSF动员和传统的石膏夹板绝对固定等方式。
【Abstract】 Objective: In the orthopedic field, how to accelerate fracture healing, treatment of nonunion and delayed union fractures is a hot issue. This paper aims to investigate the effect of percutaneous electrical stimulation of skeletal muscle combine stem cell mobilization on bone remodeling and cell proliferation in rat fracture healing process and its possible mechanismMethod: we used 80 male SD rats, 3-month-age(provided by Xi’an JiaoTong University Medical School animal administrative center), and the weight was about 280～320g. then randomly divided them into 4 groups, each 20: Fracture model group (A group); model + mobilizing agent group (B group); model + electrical stimulation group (C group); model + mobilizing agent + electrical stimulation group (D group). All of them used the national standards murine dry feed, free drink and eat. Find support from the manufacturer with the establishment of rat model of closed fracture of tibia and stimulate lv xiao yu reference model and the Peng Liang stimulation mode and slightly changes the combination of a rat model of electrical stimulation. After fracture at 2,4,6,8 weeks to produce non-decalcified hard tissue and decalcified bone slices, the use of tetracycline and alizarin red double-fluorescent labeling, special staining, immunohistochemical SABC assay of bone tissue in the fracture healing process of structural change in calcium deposition and osteoblast cell proliferation and cytokine-related protein expression.Results:1 Rats closed fracture model caused by stent fracture of the middle tibia in rats by X-ray to determine: successful establish a rat closed fracture model of the middle tibia2 rhG-CSF and (or) electrical stimulation of skeletal muscle can promote functional recovery after fracture healing in rats. After fracture,the role of rhG-CSF alone and a simple electrical stimulation of skeletal muscle, as well as rhG-CSF combined of skeletal muscle stimulation all promote functional recovery after fracture healing, and the effect of rhG-CSF combined electrical stimulation of skeletal muscle is the most significant3 rhG-CSF and (or) electrical stimulation of skeletal muscle to cause the increase in the number of cartilage after fracture healing in rat. Bone tissue calcification occurs earlier, better fracture healing, especially to electrical stimulation and electrical stimulation + mobilizing agent group more obvious effects. Suggest that: after fracture electrical stimulation and bone marrow stem cell mobilization synergy, to increase the early fracture healing of the cartilage cells in the promotion of cartilage into bone, to accelerate fracture healing.4 pure rhG-CSF mobilization and simple electrical stimulation make the affected limb 2 labeling line spacing with the healing time and gradually increased, and compared to the model group significant increase,the unit area relative fluorescence intensity showed that: a simple intervention group can affect the fracture healing process of the deposition of calcium to promote bone matrix calcification. 5 rhG-CSF mobilization combined electrical stimulation of skeletal muscle can make 2 labeling line spacing is gradually increasing, and the peak to appeared in the six weeks after fracture. It suggests that calcium deposition in the latter half of the largest; compared with the pure intervention group 2 tag line spacing increased significantly, indicating that associate intervention is more than simply contribute to fracture healing.6 rhG-CSF mobilization and skeletal muscle electrical stimulation on fractured rats the expression of cell growth factor have an impact. Early fracture healing, in cartilage and bone cells, bFGF and c-kit significantly increased; simple electrical stimulation and electrical stimulation of joint mobilization intervention can promote cartilage and bone cells of c-kit expression in the fourth weeks to reach the peak, and the simple mobilization agent group reach peak in 6 weeks; each group TGF-β1 and bFGF to reach peak expression in the 6 weeks; fracture healing in the latter part of the expression of cytokines has declined, but still higher than control group. This showed that the simple way to stimulate and co-stimulation may be different levels to promote the expression of bone-inducing factor to promote fracture healing.7 rhG-CSF mobilization and skeletal muscle electrical stimulation have an impact on bone fracture in rats the expression of proliferation-related proteins.After electrical stimulation and combine with mobilization agents, PCNA, BrdU, and ki-67 in fracture healing process of a gradual increase in expression, indicating that cell proliferation-related proteins in fracture healing are the expression of the period, but at different times in different types of cells, the early period of the main division of mesenchymal stem cells and cartilage cells, the latter is the osteoblast. RhG-CSF in the role of early fracture healing in rats the expression of BrdU-positive is not significant, the latter has increased, PCNA and ki-67 expression continued high; simple electrical stimulation of group cell proliferation-associated protein expression with the progressive fracture healing increased, particularly, increased expression of BrdU with a very significant difference. Showed that electrical stimulation alone and compared to pure stem cell mobilization of bone marrow stem cells is more conducive to the proliferation and differentiation. After the joint role of both, PCNA, BrdU, and ki-67 expression peak appeared in the first six weeks, suggesting a dual role in promoting cell proliferation increase in the number of mitosis and in view of the cells are more conducive to the bone remodeling.Conclusions:Electrical stimulation of skeletal muscle to improve the fracture mechanics of the environment around the fracture site.The formation of micro-effect role for osteoblast-mechanical conditions of fracture healing in rats can callus after the increase in the number of cartilage, bone tissue calcification occurred in early, limb 2nd bone markers with the line spacing to extend the healing time and a gradual increase in the degree of fracture healing is better, the promotion of fracture healing after functional recovery. In this process, TGF-81, PCNA and BrdU expression increased significantly, and promote local cytokine secretion and release, to accelerate fracture healing. Therefore, the use of early forms of micro-fracture healing better than the traditional way of plaster splint is absolutely fixed.rhG-CSF mobilized autologous bone marrow stem cells to promote bone marrow-derived mesenchymal stem cells and the release of hematopoietic stem cells migrate to the fracture site, increasing stem cells into osteoblasts, the direction of differentiation into cartilage, promoting cartilage endochondral bone, bone matrix calcification and fracture of parts of new the number of blood vessels, accelerate fracture healing, fracture healing in rats after the promotion of functional recovery. During this process, the early fracture healing of cartilage and bone cells, TGF-β1, bFGF, PCNA, BrdU, and ki-67 in the first six weeks to reach the peak; fracture healing in the latter part of the expression of cytokines has declined, but still higher than the control group. Therefore, early use of autologous bone marrow stem cell mobilization to promote fracture healing is better than the traditional plaster splint means absolutely fixed.rhG-CSF mobilization and the joint role of skeletal muscle after electrical stimulation, can promote bone marrow-derived mesenchymal stem cells and the release of hematopoietic stem cells migrate to the fracture site, increasing stem cells into osteoblasts, the direction of differentiation into cartilage and bone growth by ensuring the needs for the mechanical environment, improve local blood circulation, speed up the process of bone remodeling; both of which will enhance bone-inducing factor and cell proliferation-related protein expression, thereby shortening the time to fracture healing. Therefore, the skeletal muscle caused by electrical stimulation of the micro-effects and the mobilization of stem cells to promote fracture healing better than the effect of electrical stimulation of skeletal muscle, rhG-CSF mobilization and traditional plaster splint, such as absolutely fixed.