【摘要】：骨骼中有豐富的感覺(jué)神經(jīng)支配,這些神經(jīng)除了傳遞痛覺(jué)之外,還能夠分泌神經(jīng)遞質(zhì)P物質(zhì)、降鈣素基因相關(guān)肽等調(diào)控骨形成,為了研究外源性P物質(zhì)對(duì)牽張成骨中骨形成的影響及其機(jī)理,我們?cè)O(shè)計(jì)了以下實(shí)驗(yàn):實(shí)驗(yàn)一:外源性P物質(zhì)對(duì)大鼠下頜骨牽張成骨的影響目的:P物質(zhì)是感覺(jué)神經(jīng)纖維分泌的神經(jīng)肽,其具有調(diào)控骨形成的功能,但其對(duì)牽張成骨的影響,尚未有報(bào)道。本實(shí)驗(yàn)通過(guò)局部注射10-7M的P物質(zhì)來(lái)研究P物質(zhì)對(duì)牽張大鼠下頜骨牽張成骨的影響。方法:20只大鼠行右側(cè)下頜骨牽張成骨后經(jīng)過(guò)5天的延遲期,在牽張期10天中,每12小時(shí)牽張0.2mm,實(shí)驗(yàn)組在牽張期內(nèi)每日向牽張區(qū)注射0.2ml 10-7M的P物質(zhì)溶液(對(duì)照組注射相同劑量的生理鹽水);動(dòng)物分別在固定期第0、14天處死取材行Micro-CT檢測(cè)、HE染色。結(jié)果:在術(shù)后15日,Micro-CT檢測(cè)骨密度結(jié)果顯示實(shí)驗(yàn)組高于對(duì)照組,具有統(tǒng)計(jì)學(xué)意義p0.05;術(shù)后29日,實(shí)驗(yàn)組的骨密度和骨體積分?jǐn)?shù)均高于對(duì)照組。HE染色發(fā)現(xiàn):實(shí)驗(yàn)組的骨小梁結(jié)構(gòu)較對(duì)照組成熟,骨小梁面積高于對(duì)照組。結(jié)論:局部注射10-7M的P物質(zhì)能夠促進(jìn)大鼠下頜骨牽張成骨新骨形成及成熟程度。實(shí)驗(yàn)二外源性P物質(zhì)在牽張成骨中對(duì)間充質(zhì)干細(xì)胞動(dòng)員的實(shí)驗(yàn)研究目的:實(shí)驗(yàn)一結(jié)果顯示局部注射10-7M的P物質(zhì)可以促進(jìn)大鼠牽張成骨中新骨形成,但其機(jī)制并不清楚,本實(shí)驗(yàn)通過(guò)檢測(cè)牽張局部的間充質(zhì)干細(xì)胞遷移和外周血中的CD29+細(xì)胞數(shù)目來(lái)研究外源性P物質(zhì)在大鼠牽張成骨中對(duì)間充質(zhì)干細(xì)胞的動(dòng)員影響。方法:20只大鼠行右側(cè)下頜骨牽張成骨后經(jīng)過(guò)5天的延遲期,在牽張期10天中,每12小時(shí)牽張0.2mm,實(shí)驗(yàn)組在牽張期10天內(nèi)每日向牽張區(qū)注射10-7M的P物質(zhì)溶液0.2ml(對(duì)照組注射相同劑量的生理鹽水);動(dòng)物分別在固定期第0、14天處死取材行Nestin免疫組織化學(xué)染色,并在手術(shù)后第術(shù)后5、6、11、15、22、29d經(jīng)鼠尾靜脈采血0.1ml經(jīng)流式細(xì)胞儀檢測(cè)外周血中的CD29+細(xì)胞數(shù)目。結(jié)果:Nestin免疫組織化學(xué)染色發(fā)現(xiàn),實(shí)驗(yàn)組Nestin陽(yáng)性細(xì)胞分布于牽張間隙中,而對(duì)照組主要位于微血管周圍;流式細(xì)胞儀檢測(cè)發(fā)現(xiàn)在術(shù)后第11、15、22天SP組外周血中的CD29+細(xì)胞明顯高于對(duì)照組(p0.05),術(shù)后第29天實(shí)驗(yàn)組低于對(duì)照組(p0.05)。結(jié)論:局部注射10-7M的P物質(zhì)可以更有效的動(dòng)員間充質(zhì)干細(xì)胞向牽張區(qū)域遷移。實(shí)驗(yàn)三局部應(yīng)用P物質(zhì)對(duì)大鼠下頜骨牽張成骨中SDF-1表達(dá)的影響目的:以上結(jié)果顯示局部注射10-7M的P物質(zhì)可以促進(jìn)間充質(zhì)干細(xì)胞動(dòng)員影響大鼠牽張成骨中新骨形成,而SDF-1的濃度對(duì)干細(xì)胞遷移起到重要作用。方法:30只大鼠行右側(cè)下頜骨牽張成骨后經(jīng)過(guò)5天的延遲期,在牽張期10天中,每12小時(shí)牽張0.2mm,實(shí)驗(yàn)組每日向牽張區(qū)注射0.2ml 10-7M的P物質(zhì),對(duì)照組注射相同劑量的生理鹽水,動(dòng)物分別在術(shù)后第15天取材行SDF-1免疫組化染色,在術(shù)后第6、15、29天取材行實(shí)時(shí)定量PCR并在手術(shù)后第6、11、15、29d經(jīng)鼠尾靜脈采血0.5ml后ELISA試劑盒檢測(cè)血漿中SDF-1濃度。結(jié)果:免疫組織化學(xué)染色標(biāo)記SDF-1后觀察到SP組SDF-1表達(dá)高于對(duì)照組;實(shí)時(shí)定量PCR和ELISA均顯示在術(shù)后第15天檢測(cè)到實(shí)驗(yàn)組牽張區(qū)和外周中的SDF-1及其m RNA表達(dá)均處于峰值并明顯高于對(duì)照組(p0.05)。結(jié)論:局部注射10-7M的P物質(zhì)可以提高牽張區(qū)和外周血中SDF-1的表達(dá)。實(shí)驗(yàn)四外源性P物質(zhì)對(duì)大鼠骨髓間充質(zhì)干細(xì)胞體外遷移的影響目的:上述實(shí)驗(yàn)說(shuō)明,局部注射外源性的P物質(zhì)可以增強(qiáng)局部和全身間充質(zhì)你干細(xì)胞的動(dòng)員,干細(xì)胞動(dòng)員后遷移到創(chuàng)傷區(qū)域也是干細(xì)胞參與牽張區(qū)新骨形成的重要一步,本實(shí)驗(yàn)研究外源性P物質(zhì)對(duì)大鼠間充質(zhì)干細(xì)胞體外遷移的影響。方法:10只大鼠(80±20g,4周齡)處死后取雙側(cè)下頜骨剪碎后膠原酶消化法取原代細(xì)胞進(jìn)行培養(yǎng),擴(kuò)充至第三代后流式細(xì)胞術(shù)測(cè)定表面抗型進(jìn)行鑒定,實(shí)驗(yàn)組培養(yǎng)液中含有10-7 M P物質(zhì),在接種后第1、3、5、7天取培養(yǎng)基行ELISA SDF-1濃度測(cè)定,并獲取細(xì)胞行實(shí)時(shí)定量PCR檢測(cè)SDF-1m RNA表達(dá),培養(yǎng)7日后鑒定表面CXCR4的表達(dá),并行tranwell遷移實(shí)驗(yàn)比較細(xì)胞體外遷移能力。結(jié)果:從下頜骨中分離的原代培養(yǎng)細(xì)胞呈梭型貼壁生長(zhǎng),對(duì)第三代細(xì)胞表型經(jīng)流式細(xì)胞儀鑒定后發(fā)現(xiàn)CD90、CD29、CD44陽(yáng)性,CD34、CD45陰性,加入10-7M SP組,SDF-1及其m RNA表達(dá)均高于對(duì)照組(p0.05),培養(yǎng)7天后BMSCs表面CXCR-4表達(dá)倍增(p0.05),transwell遷移實(shí)驗(yàn)組,實(shí)驗(yàn)組穿膜細(xì)胞數(shù)量遠(yuǎn)遠(yuǎn)高于對(duì)照組(p0.05)。結(jié)論:外源性10-7M的P物質(zhì)在體外實(shí)驗(yàn)中可以促進(jìn)BMSCs分泌SDF-1,并增加BMSCs膜表面的CXCR4表達(dá),同時(shí)可以增強(qiáng)BMSCs的體外遷移能力。實(shí)驗(yàn)五外源性P物質(zhì)對(duì)大鼠骨髓間充質(zhì)干細(xì)胞增殖活性和成骨能力的影響目的:骨髓間充質(zhì)干細(xì)胞受到機(jī)體動(dòng)員后遷移到牽張間隙黏附于基質(zhì)網(wǎng)開始增殖和分化參與骨形成的過(guò)程,本實(shí)驗(yàn)觀察加入外源性P物質(zhì)后大鼠骨髓間充質(zhì)干細(xì)胞增殖活性和成骨能力的變化。方法:實(shí)驗(yàn)四培養(yǎng)的第三代骨髓間充質(zhì)干細(xì)胞,實(shí)驗(yàn)組培養(yǎng)液中含有10-7 M P物質(zhì),培養(yǎng)3日后Brd U染色,14天后觀察集落形成,成骨分化中培養(yǎng)7天后加入含有P物質(zhì)的成骨誘導(dǎo)液,分別在1、7、14天檢測(cè)堿性磷酸酶活性、ALP、Runx2的m RNA表達(dá),第14天茜素紅染色。結(jié)果:實(shí)驗(yàn)組Brd U+細(xì)胞的數(shù)目高于對(duì)照組,集落形成能力高于對(duì)照組(p0.05),在成骨誘導(dǎo)過(guò)程中外源性的10-7 M P物質(zhì)對(duì)ALP活性、ALP及Runx2的m RNA表達(dá)及茜素紅染色未見明顯差異(p0.05)。結(jié)論:體外實(shí)驗(yàn)中外源性10-7M的P物質(zhì)作用于第三代骨髓間充質(zhì)干細(xì)胞在可以促進(jìn)其增殖活性但對(duì)成骨能力無(wú)明顯作用。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R782

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