不同时机转染基因对兔下颌骨牵引区成骨因子表达的影响
发布时间：2018-08-11 16:31
【摘要】：背景：牵引成骨（distraction osteogenesis, DO）被认为是体内组织工程技术的最佳类型，已广泛应用于临床，矫治了四肢及常规正颌外科手术难以矫治的诸多复杂颅颌面畸形、骨缺损等。尽管如此，DO也存在某些不足之处，固定治疗周期长，牵引器长期留置体内可能导致如局部感染、牵引器或螺钉松脱、骨折、局部延迟愈合及给病人和家属带来心理负担和诸多烦恼。为此，许多学者进行了促进下颌骨牵引区新骨生成的相关研究。 目的：本研究在前期研究的基础上，在不同时点通过电穿孔技术将构建的重组质粒pIRES-hBMP2-hVEGF165导入兔下颌骨牵引间隙内，观察基因转染后成骨因子在下颌骨牵引区的局部表达，探索基因导入最佳时机的理论依据。 方法：选用48只6月大小、健康新西兰大白兔，随机分为四组每组12只，在全麻下行双侧下颌骨截骨，植入兔专用下颌骨牵引器，外置牵引器旋转杆，分层缝合切口。经过3d潜伏期后开始牵引，牵引速度0.8mm/d，频率1次/d，连续牵引10d后进入固定期。各组在不同阶段进行基因导入， A、B、C组分别于术后即刻（潜伏期）、术后3d（牵引开始时）、术后14d（牵引结束固定开始）在双侧牵引区注射2μg，（0.1μg/μl）重组质粒pIRES-hBMP2-hVEGF165； A、B、C三组均予电脉冲刺激（电脉冲参数：电压是200V,电容是10μF，频率是0.2Hz，平均脉冲宽度是2.7ms，单脉冲刺激，共为6个脉冲，3个脉冲更换正负极。），D组单纯牵引不行基因转染。各组分别在固定第7、14、28d处死4只实验动物，获取延长的下颌骨行组织学观察牵引区新骨生成情况，免疫组化检测BMP2、TGF-β1的表达情况，并用CMIAS系列多功能真彩色病理图像分析系统对BMP2和TGF-β1的平均光密度和积分光密度分析。 结果：组织学检查发现，固定期第7天，截骨部位有软骨痂和钙化组织形成的早期桥连，并伴有软骨细胞和成纤维细胞；第14天，软骨细胞和狭长的成纤维细胞在牵引间隙在形态学上被鉴别，并且在牵引方向上生长；第28天，牵引间隙的骨碎片产生桥接，伴随着牵引间隙明显的骨和钙化组织沉积。B组与A、C、D组比较间隙内有更多的新生血管、间充质细胞和成骨细胞等成分。 免疫组化显示：1、BMP-2表达于细胞胞浆中。固定期第7天，BMP-2主要表达于肉芽组织中的炎细胞（如间质细胞、单核细胞）和成纤维细胞、软骨细胞、成骨细胞、骨细胞（骨端骨细胞、骨痂骨细胞）以及周围结缔组织中。固定期第14天，BMP-2阳性染色主要在肥厚性软骨细胞和成骨细胞的细胞的细胞质中，与牵引方向排列一致新生的幼稚骨小梁表面成骨细胞、软骨细胞、骨细胞和周围结缔组织中。在固定期第28天，BMP-2阳性染色逐渐减少，主要在成骨细胞和成纤维细胞中。半定量分析发现，在固定期第7天时，四组BMP-2表达达到高峰。其他3组比较,B组(0.31±0.04，0.68±0.06)是最强的，其中有显著差异(P 0.01，P 0.05)。C组和A、D之间仍有显著性差异(P 0.05)，A与D组相比,有显著差异(P 0.05)。在固定期14天,阳性染色细胞的数量减少，B组与A、C、D之间具有显著差异(P 0.05)。BMP-2在28天的表达很弱,各组间比较无显著性差异(P0.05)。 2. TGF-β1表达各个时间点上与BMP-2密切相关。在固定期第7天，TGF-β1表达主要在成纤维细胞、骨细胞、成骨细胞和沿着截骨边缘的软骨细胞。还有大量不明确的间充质细胞阳性染色。TGF-β1的表达在固定期第14天和28天BMP-2表达相似。在第14天, TGF-β1在肥厚性软骨细胞,骨细胞和再生的成骨细胞以及间充质细胞中表达。在第28天, TGF-β1在骨痂表面成骨细胞以及新生的编织骨痂骨细胞染色仍呈阳性，，而在肉芽组织中的阳性染色的细胞减少明显。TGF-β1在四组在不同时间的表达强度在表2中列出。半定量分析发现，在固定期第7天时，四组TGF-β1表达达到高峰，B组(0.37±0.07，0.90±0.06)是最强的其次是C组(0.22±0.02，0.69±0.02)和A组(0.16±0.05，0.41±0.03)。B组与其余各组间有显著差异(P 0.01，P 0.05)。在14-28天基因转染后,统计分析TGF-β1在不同组织的表达情况类似BMP-2的情况，在固定期14天,阳性染色细胞的数量减少，B组与A、C、D之间具有显著差异(P 0.05)，28天的表达很弱,各组间比较无显著性差异(P0.05)。 结论：在下颌骨牵引区局部进行基因转染能上调成骨因子BMP2、TGF-β1的表达，不同时机转染基因效果不同，在牵引期进行基因转染BMP2、TGF-β1各时间点的表达较潜伏期和固定期进行基因转染强烈，高表达的BMP2、TGF-β1可能通过一系列生物效应促进牵引间隙的新骨生成，这可能是牵引期为基因治疗最佳时机的分子机制之一。
[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.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.23
本文编号：2177581
[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.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.23
【参考文献】
相关期刊论文 前1条
1 王晓霞,王兴,李自力,武登诚;转化生长因子β1在下颌骨牵引成骨过程中的作用[J];北京大学学报(医学版);2003年01期
本文编号：2177581
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