局部应用P物质调控骨髓间充质干细胞迁移能力促进大鼠下颌骨牵张成骨的实验研究

发布时间：2018-09-01 18:18

【摘要】：骨骼中有丰富的感觉神经支配,这些神经除了传递痛觉之外,还能够分泌神经递质P物质、降钙素基因相关肽等调控骨形成,为了研究外源性P物质对牵张成骨中骨形成的影响及其机理,我们设计了以下实验:实验一:外源性P物质对大鼠下颌骨牵张成骨的影响目的:P物质是感觉神经纤维分泌的神经肽,其具有调控骨形成的功能,但其对牵张成骨的影响,尚未有报道。本实验通过局部注射10-7M的P物质来研究P物质对牵张大鼠下颌骨牵张成骨的影响。方法:20只大鼠行右侧下颌骨牵张成骨后经过5天的延迟期,在牵张期10天中,每12小时牵张0.2mm,实验组在牵张期内每日向牵张区注射0.2ml 10-7M的P物质溶液(对照组注射相同剂量的生理盐水);动物分别在固定期第0、14天处死取材行Micro-CT检测、HE染色。结果:在术后15日,Micro-CT检测骨密度结果显示实验组高于对照组,具有统计学意义p0.05;术后29日,实验组的骨密度和骨体积分数均高于对照组。HE染色发现:实验组的骨小梁结构较对照组成熟,骨小梁面积高于对照组。结论:局部注射10-7M的P物质能够促进大鼠下颌骨牵张成骨新骨形成及成熟程度。实验二外源性P物质在牵张成骨中对间充质干细胞动员的实验研究目的:实验一结果显示局部注射10-7M的P物质可以促进大鼠牵张成骨中新骨形成,但其机制并不清楚,本实验通过检测牵张局部的间充质干细胞迁移和外周血中的CD29+细胞数目来研究外源性P物质在大鼠牵张成骨中对间充质干细胞的动员影响。方法:20只大鼠行右侧下颌骨牵张成骨后经过5天的延迟期,在牵张期10天中,每12小时牵张0.2mm,实验组在牵张期10天内每日向牵张区注射10-7M的P物质溶液0.2ml(对照组注射相同剂量的生理盐水);动物分别在固定期第0、14天处死取材行Nestin免疫组织化学染色,并在手术后第术后5、6、11、15、22、29d经鼠尾静脉采血0.1ml经流式细胞仪检测外周血中的CD29+细胞数目。结果:Nestin免疫组织化学染色发现,实验组Nestin阳性细胞分布于牵张间隙中,而对照组主要位于微血管周围;流式细胞仪检测发现在术后第11、15、22天SP组外周血中的CD29+细胞明显高于对照组(p0.05),术后第29天实验组低于对照组(p0.05)。结论:局部注射10-7M的P物质可以更有效的动员间充质干细胞向牵张区域迁移。实验三局部应用P物质对大鼠下颌骨牵张成骨中SDF-1表达的影响目的:以上结果显示局部注射10-7M的P物质可以促进间充质干细胞动员影响大鼠牵张成骨中新骨形成,而SDF-1的浓度对干细胞迁移起到重要作用。方法:30只大鼠行右侧下颌骨牵张成骨后经过5天的延迟期,在牵张期10天中,每12小时牵张0.2mm,实验组每日向牵张区注射0.2ml 10-7M的P物质,对照组注射相同剂量的生理盐水,动物分别在术后第15天取材行SDF-1免疫组化染色,在术后第6、15、29天取材行实时定量PCR并在手术后第6、11、15、29d经鼠尾静脉采血0.5ml后ELISA试剂盒检测血浆中SDF-1浓度。结果:免疫组织化学染色标记SDF-1后观察到SP组SDF-1表达高于对照组;实时定量PCR和ELISA均显示在术后第15天检测到实验组牵张区和外周中的SDF-1及其m RNA表达均处于峰值并明显高于对照组(p0.05)。结论:局部注射10-7M的P物质可以提高牵张区和外周血中SDF-1的表达。实验四外源性P物质对大鼠骨髓间充质干细胞体外迁移的影响目的:上述实验说明,局部注射外源性的P物质可以增强局部和全身间充质你干细胞的动员,干细胞动员后迁移到创伤区域也是干细胞参与牵张区新骨形成的重要一步,本实验研究外源性P物质对大鼠间充质干细胞体外迁移的影响。方法:10只大鼠(80±20g,4周龄)处死后取双侧下颌骨剪碎后胶原酶消化法取原代细胞进行培养,扩充至第三代后流式细胞术测定表面抗型进行鉴定,实验组培养液中含有10-7 M P物质,在接种后第1、3、5、7天取培养基行ELISA SDF-1浓度测定,并获取细胞行实时定量PCR检测SDF-1m RNA表达,培养7日后鉴定表面CXCR4的表达,并行tranwell迁移实验比较细胞体外迁移能力。结果:从下颌骨中分离的原代培养细胞呈梭型贴壁生长,对第三代细胞表型经流式细胞仪鉴定后发现CD90、CD29、CD44阳性,CD34、CD45阴性,加入10-7M SP组,SDF-1及其m RNA表达均高于对照组(p0.05),培养7天后BMSCs表面CXCR-4表达倍增(p0.05),transwell迁移实验组,实验组穿膜细胞数量远远高于对照组(p0.05)。结论:外源性10-7M的P物质在体外实验中可以促进BMSCs分泌SDF-1,并增加BMSCs膜表面的CXCR4表达,同时可以增强BMSCs的体外迁移能力。实验五外源性P物质对大鼠骨髓间充质干细胞增殖活性和成骨能力的影响目的:骨髓间充质干细胞受到机体动员后迁移到牵张间隙黏附于基质网开始增殖和分化参与骨形成的过程,本实验观察加入外源性P物质后大鼠骨髓间充质干细胞增殖活性和成骨能力的变化。方法:实验四培养的第三代骨髓间充质干细胞,实验组培养液中含有10-7 M P物质,培养3日后Brd U染色,14天后观察集落形成,成骨分化中培养7天后加入含有P物质的成骨诱导液,分别在1、7、14天检测碱性磷酸酶活性、ALP、Runx2的m RNA表达,第14天茜素红染色。结果:实验组Brd U+细胞的数目高于对照组,集落形成能力高于对照组(p0.05),在成骨诱导过程中外源性的10-7 M P物质对ALP活性、ALP及Runx2的m RNA表达及茜素红染色未见明显差异(p0.05)。结论:体外实验中外源性10-7M的P物质作用于第三代骨髓间充质干细胞在可以促进其增殖活性但对成骨能力无明显作用。
[Abstract]:There are abundant sensory innervations in bone. These nerves can not only transmit pain, but also secrete substance P and calcitonin gene-related peptide to regulate bone formation. In order to study the effect of exogenous substance P on bone formation during distraction osteogenesis and its mechanism, we designed the following experiments: Experiment 1: Exogenous substance P under rats. OBJECTIVE: Substance P is a neuropeptide secreted by sensory nerve fibers, which can regulate bone formation, but its effect on distraction osteogenesis has not been reported. The effect of substance P on mandibular distraction osteogenesis in rats was studied by local injection of 10-7M substance P. METHODS: Twenty rats underwent right mandibular distraction osteogenesis. After 5 days of delayed distraction osteogenesis, 0.2 mm of substance P was injected into the distraction zone every 12 hours during the distraction period. The animals were sacrificed on the 0 th and 14 th day of the fixation period and tested by micro-CT and HE staining. The bone mineral density and bone volume fraction of the experimental group were higher than those of the control group on the 29th day after operation. HE staining showed that the bone trabecular structure of the experimental group was more mature than that of the control group, and the bone trabecular area of the experimental group was higher than that of the control group. Experimental study on mobilization of mesenchymal stem cells by exogenous substance P during distraction osteogenesis Objective: The results of Experiment 1 showed that local injection of 10-7M substance P could promote the formation of new bone during distraction osteogenesis in rats, but the mechanism was not clear. METHODS: Twenty rats underwent right mandibular distraction osteogenesis for 5 days. During the distraction period of 10 days, the distraction time was 0.2 mm every 12 hours, and the experimental group was daily for 10 days. Substance P solution of 10-7M was injected into the distraction area with 0.2ml (the control group was injected with the same dose of normal saline); Nestin immunohistochemical staining was performed on the 0 and 14 days of the fixed period, and the number of CD29 + cells in peripheral blood was detected by flow cytometry on the 5th, 6th, 11th, 15th, 22nd and 29th days after operation. Nestin immunohistochemical staining showed that Nestin positive cells were distributed in the distraction space in the experimental group, while those in the control group were mainly located around the microvessels. Flow cytometry showed that CD29 + cells in the peripheral blood of SP group were significantly higher than those in the control group on the 11th, 15th and 22th day after operation (p0.05), and lower than those in the control group on the 29th day after operation (p0.05). Experiment 3 Effect of topical application of substance P on the expression of SDF-1 in rat mandibular distraction osteogenesis Objective: The above results show that topical injection of substance P of 10-7M can promote the mobilization of mesenchymal stem cells and affect the formation of new bone in rat distraction osteogenesis, while SD can affect the formation of new bone in rat mandibular distraction osteogenesis. F-1 concentration plays an important role in the migration of stem cells. Methods: 30 rats underwent right mandibular distraction osteogenesis for 5 days. During the distraction period of 10 days, 0.2 mm was stretched every 12 hours. Substance P of 0.2 ml 10-7 M was injected into the distraction area daily in the experimental group and normal saline was injected into the control group. The animals were taken at the 15th day after operation. SDF-1 immunohistochemical staining, real-time quantitative PCR and ELISA kit were used to detect the concentration of SDF-1 in plasma on the 6th, 11th, 15th and 29th day after operation. The expression of SDF-1 and its m RNA in the distraction zone and peripheral blood of the experimental group were both at the peak value and significantly higher than that of the control group (p0.05). Conclusion: Local injection of 10-7M substance P can increase the expression of SDF-1 in the distraction zone and peripheral blood. Experiments show that topical injection of exogenous substance P can enhance the mobilization of local and systemic mesenchymal stem cells, and the migration of stem cells to traumatic areas is also an important step for stem cells to participate in the formation of new bone in distraction areas. The primary cells were cultured by collagenase digestion after mandibular crushing and then expanded to the third generation for identification of surface resistance. The culture medium of the experimental group contained 10-7 M P substance. The concentration of SDF-1 was determined by ELISA on the 1st, 3rd, 5th and 7th day after inoculation. The expression of SDF-1m RNA was detected by quantitative PCR, and the expression of CXCR4 on the surface of SDF-1m RNA was identified after 7 days of culture, and the migration ability of the cells in vitro was compared by tranwell migration test.Results: Primary cultured cells isolated from mandible grew in spindle-like adherence. The phenotype of the third generation cells was identified by flow cytometry, and CD90, CD29, CD44, CD34 and CD45 were positive. The expression of SDF-1 and its m RNA in-7MSP group was higher than that in control group (p0.05). The expression of CXCR-4 on the surface of BMSCs doubled (p0.05) after 7 days of culture. The number of penetrating cells in the experimental group was much higher than that in the control group (p0.05). Conclusion: Exogenous 10-7M substance P can promote the secretion of SDF-1 and increase the expression of CXCR 4 on the surface of BMSCs membrane in vitro. Experiments 5 Effects of exogenous substance P on proliferation and osteogenesis of rat bone marrow mesenchymal stem cells Objective: Bone marrow mesenchymal stem cells (BMSCs) after mobilization migrate to the distraction space, adhere to the matrix network and begin to proliferate and differentiate and participate in the process of bone formation. METHODS: The third generation bone marrow mesenchymal stem cells cultured in Experiment 4 contained 10-7 M P substance in the culture medium of experiment group. The colony formation was observed after 3 days of culture and Brd U staining after 14 days of culture. Alkaline phosphatase activity, expression of ALP and Runx2 m RNA and alizarin red staining were detected on day 1, 7 and 14 respectively in bone induction medium. Results: The number of Brd U + cells in the experimental group was higher than that in the control group, and the colony forming ability was higher than that in the control group (p0.05). Conclusion: Exogenous 10-7M substance P can promote the proliferation of the third generation bone marrow mesenchymal stem cells in vitro, but has no obvious effect on osteogenic capacity.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R782

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