【摘要】：背景：牽引成骨（distraction osteogenesis, DO）被認(rèn)為是體內(nèi)組織工程技術(shù)的最佳類型，已廣泛應(yīng)用于臨床，矯治了四肢及常規(guī)正頜外科手術(shù)難以矯治的諸多復(fù)雜顱頜面畸形、骨缺損等。盡管如此，DO也存在某些不足之處，固定治療周期長，牽引器長期留置體內(nèi)可能導(dǎo)致如局部感染、牽引器或螺釘松脫、骨折、局部延遲愈合及給病人和家屬帶來心理負(fù)擔(dān)和諸多煩惱。為此，許多學(xué)者進(jìn)行了促進(jìn)下頜骨牽引區(qū)新骨生成的相關(guān)研究。 目的：本研究在前期研究的基礎(chǔ)上，在不同時(shí)點(diǎn)通過電穿孔技術(shù)將構(gòu)建的重組質(zhì)粒pIRES-hBMP2-hVEGF165導(dǎo)入兔下頜骨牽引間隙內(nèi)，觀察基因轉(zhuǎn)染后成骨因子在下頜骨牽引區(qū)的局部表達(dá)，探索基因?qū)胱罴褧r(shí)機(jī)的理論依據(jù)。 方法：選用48只6月大小、健康新西蘭大白兔，隨機(jī)分為四組每組12只，在全麻下行雙側(cè)下頜骨截骨，植入兔專用下頜骨牽引器，外置牽引器旋轉(zhuǎn)桿，分層縫合切口。經(jīng)過3d潛伏期后開始牽引，牽引速度0.8mm/d，頻率1次/d，連續(xù)牽引10d后進(jìn)入固定期。各組在不同階段進(jìn)行基因?qū)耄?A、B、C組分別于術(shù)后即刻（潛伏期）、術(shù)后3d（牽引開始時(shí)）、術(shù)后14d（牽引結(jié)束固定開始）在雙側(cè)牽引區(qū)注射2μg，（0.1μg/μl）重組質(zhì)粒pIRES-hBMP2-hVEGF165； A、B、C三組均予電脈沖刺激（電脈沖參數(shù)：電壓是200V,電容是10μF，頻率是0.2Hz，平均脈沖寬度是2.7ms，單脈沖刺激，共為6個(gè)脈沖，3個(gè)脈沖更換正負(fù)極。），D組單純牽引不行基因轉(zhuǎn)染。各組分別在固定第7、14、28d處死4只實(shí)驗(yàn)動(dòng)物，獲取延長的下頜骨行組織學(xué)觀察牽引區(qū)新骨生成情況，免疫組化檢測BMP2、TGF-β1的表達(dá)情況，并用CMIAS系列多功能真彩色病理圖像分析系統(tǒng)對BMP2和TGF-β1的平均光密度和積分光密度分析。 結(jié)果：組織學(xué)檢查發(fā)現(xiàn)，固定期第7天，截骨部位有軟骨痂和鈣化組織形成的早期橋連，并伴有軟骨細(xì)胞和成纖維細(xì)胞；第14天，軟骨細(xì)胞和狹長的成纖維細(xì)胞在牽引間隙在形態(tài)學(xué)上被鑒別，并且在牽引方向上生長；第28天，牽引間隙的骨碎片產(chǎn)生橋接，伴隨著牽引間隙明顯的骨和鈣化組織沉積。B組與A、C、D組比較間隙內(nèi)有更多的新生血管、間充質(zhì)細(xì)胞和成骨細(xì)胞等成分。 免疫組化顯示：1、BMP-2表達(dá)于細(xì)胞胞漿中。固定期第7天，BMP-2主要表達(dá)于肉芽組織中的炎細(xì)胞（如間質(zhì)細(xì)胞、單核細(xì)胞）和成纖維細(xì)胞、軟骨細(xì)胞、成骨細(xì)胞、骨細(xì)胞（骨端骨細(xì)胞、骨痂骨細(xì)胞）以及周圍結(jié)締組織中。固定期第14天，BMP-2陽性染色主要在肥厚性軟骨細(xì)胞和成骨細(xì)胞的細(xì)胞的細(xì)胞質(zhì)中，與牽引方向排列一致新生的幼稚骨小梁表面成骨細(xì)胞、軟骨細(xì)胞、骨細(xì)胞和周圍結(jié)締組織中。在固定期第28天，BMP-2陽性染色逐漸減少，主要在成骨細(xì)胞和成纖維細(xì)胞中。半定量分析發(fā)現(xiàn)，在固定期第7天時(shí)，四組BMP-2表達(dá)達(dá)到高峰。其他3組比較,B組(0.31±0.04，0.68±0.06)是最強(qiáng)的，其中有顯著差異(P 0.01，P 0.05)。C組和A、D之間仍有顯著性差異(P 0.05)，A與D組相比,有顯著差異(P 0.05)。在固定期14天,陽性染色細(xì)胞的數(shù)量減少，B組與A、C、D之間具有顯著差異(P 0.05)。BMP-2在28天的表達(dá)很弱,各組間比較無顯著性差異(P0.05)。 2. TGF-β1表達(dá)各個(gè)時(shí)間點(diǎn)上與BMP-2密切相關(guān)。在固定期第7天，TGF-β1表達(dá)主要在成纖維細(xì)胞、骨細(xì)胞、成骨細(xì)胞和沿著截骨邊緣的軟骨細(xì)胞。還有大量不明確的間充質(zhì)細(xì)胞陽性染色。TGF-β1的表達(dá)在固定期第14天和28天BMP-2表達(dá)相似。在第14天, TGF-β1在肥厚性軟骨細(xì)胞,骨細(xì)胞和再生的成骨細(xì)胞以及間充質(zhì)細(xì)胞中表達(dá)。在第28天, TGF-β1在骨痂表面成骨細(xì)胞以及新生的編織骨痂骨細(xì)胞染色仍呈陽性，，而在肉芽組織中的陽性染色的細(xì)胞減少明顯。TGF-β1在四組在不同時(shí)間的表達(dá)強(qiáng)度在表2中列出。半定量分析發(fā)現(xiàn)，在固定期第7天時(shí)，四組TGF-β1表達(dá)達(dá)到高峰，B組(0.37±0.07，0.90±0.06)是最強(qiáng)的其次是C組(0.22±0.02，0.69±0.02)和A組(0.16±0.05，0.41±0.03)。B組與其余各組間有顯著差異(P 0.01，P 0.05)。在14-28天基因轉(zhuǎn)染后,統(tǒng)計(jì)分析TGF-β1在不同組織的表達(dá)情況類似BMP-2的情況，在固定期14天,陽性染色細(xì)胞的數(shù)量減少，B組與A、C、D之間具有顯著差異(P 0.05)，28天的表達(dá)很弱,各組間比較無顯著性差異(P0.05)。 結(jié)論：在下頜骨牽引區(qū)局部進(jìn)行基因轉(zhuǎn)染能上調(diào)成骨因子BMP2、TGF-β1的表達(dá)，不同時(shí)機(jī)轉(zhuǎn)染基因效果不同，在牽引期進(jìn)行基因轉(zhuǎn)染BMP2、TGF-β1各時(shí)間點(diǎn)的表達(dá)較潛伏期和固定期進(jìn)行基因轉(zhuǎn)染強(qiáng)烈，高表達(dá)的BMP2、TGF-β1可能通過一系列生物效應(yīng)促進(jìn)牽引間隙的新骨生成，這可能是牽引期為基因治療最佳時(shí)機(jī)的分子機(jī)制之一。
[Abstract]:BACKGROUND: Distraction osteogenesis (DO) is considered to be the best type of tissue engineering technology in vivo, and has been widely used in clinic to correct many complex craniomaxillofacial deformities and bone defects which are difficult to be corrected by limbs and conventional orthognathic surgery. Long-term retention of the distractor may lead to local infection, loosening of the distractor or screw, fracture, local delayed union, psychological burden and many troubles to patients and their families.
AIM: On the basis of previous studies, the recombinant plasmid pIRES-hBMP2-hVEGF165 was transfected into the mandibular traction space of rabbits by electroporation at different time points. The expression of osteogenic factors in the mandibular traction area after gene transfection was observed and the theoretical basis for the optimal time of gene transfection was explored.
Methods: Forty-eight healthy New Zealand white rabbits of 6 months old were randomly divided into four groups: 12 rabbits in each group. Under general anesthesia, bilateral mandibular osteotomy was performed, special mandibular distractor was implanted, external distractor rotating bar was inserted, and the incision was sutured in layers. Phase A, B and C were injected with 2 UG (0.1 ug/ml) recombinant plasmid pIRES-hBMP2-hVEGF 165 into bilateral traction area on the 14th day after operation. A, B and C were all given electric pulse stimulation (voltage 200 V, capacitance 1). The average pulse width was 2.7 ms, 6 pulses and 3 pulses were used to replace the positive and negative poles. Mean and integral optical densities of BMP2 and TGF-beta 1 were analyzed by CMIAS multifunctional true color pathological image analysis system.
Results: Histological examination showed that early bridging of cartilage callus and calcified tissue with chondrocytes and fibroblasts was found on the 7th day of fixation; on the 14th day, chondrocytes and narrow fibroblasts were identified morphologically in the traction space and grew in the traction direction; on the 28th day, the bone in the traction space was found. The debris bridged with bone and calcified tissue deposition in the traction gap. There were more neovascularization, mesenchymal cells and osteoblasts in group B than in group A, C and D.
BMP-2 was mainly expressed in inflammatory cells (such as stromal cells, monocytes) and fibroblasts, chondrocytes, osteoblasts, osteoblasts (bone-end osteocytes, callus osteocytes) and surrounding connective tissues in granulation tissues on the seventh day of fixation. BMP-2 positive staining was observed on the fourteenth day of fixation. In the cytoplasm of hypertrophic chondrocytes and osteoblasts, osteoblasts, chondrocytes, osteocytes and adjacent connective tissues on the surface of neonatal immature bone trabeculae aligned with the direction of traction. On the 28th day of fixation, BMP-2 positive staining gradually decreased, mainly in osteoblasts and fibroblasts. The expression of BMP-2 in group B was the strongest among the other three groups, and there was a significant difference between group C and group A, D (P 0.05). There was a significant difference between group A and group D (P 0.05). The number of positive staining cells decreased in group B and group A, C, D at 14 days after fixation. BMP-2 expression was weak at 28 days, and there was no significant difference among the groups (P 0.05).
2. The expression of TGF-beta 1 was closely related to BMP-2 at various time points. On the 7th day of fixation, the expression of TGF-beta 1 was mainly in fibroblasts, osteoblasts, osteoblasts and chondrocytes along the osteotomy margin. TGF-beta 1 was expressed in hypertrophic chondrocytes, osteoblasts, regenerated osteoblasts and mesenchymal cells. On the 28th day, TGF-beta 1 was still positive in osteoblasts on the callus surface and in newly-formed braided callus osteocytes, but decreased in granulation tissue. TGF-beta 1 was strongly expressed in the four groups at different times. Semi-quantitative analysis showed that the expression of TGF-beta 1 reached its peak at the 7th day of fixation, and the strongest in group B was at the 14th-28th day after gene transfection. There was a significant difference between group B and other groups (P 0.01, P 0.05). The expression of beta-1 in different tissues was similar to that of BMP-2. On the 14th day of fixation, the number of positive staining cells decreased. There was a significant difference between group B and A, C and D (P 0.05). The expression of beta-1 was weak on the 28th day, and there was no significant difference among the groups (P 0.05).
Conclusion: Local gene transfection can up-regulate the expression of osteogenic factors BMP2 and TGF-beta 1 in mandibular distraction area. The effect of gene transfection is different at different time. The expression of BMP2 and TGF-beta 1 at different time points during distraction period is stronger than that at latent and fixed period. The high expression of BMP2 and TGF-beta 1 may pass through a series of organisms. The effect promotes the formation of new bone in the traction gap, which may be one of the molecular mechanisms by which the traction period is the best time for gene therapy.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R782.23

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王曉霞,王興,李自力,武登誠;轉(zhuǎn)化生長因子β1在下頜骨牽引成骨過程中的作用[J];北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2003年01期

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