IGF-Ⅰ凝胶对自体移植带软骨膜肋软骨增殖及代谢的作用

发布时间：2018-04-23 00:23

本文选题：肋软骨 + 自体移植　；参考：《复旦大学》2010年硕士论文

【摘要】： 耳廓再造术中,肋软骨以其取材方便,便于雕刻成型而被广泛采用。自体软骨移植虽无排斥反应,但仍存在再造耳廓变形、挛缩,与正常耳廓外观不对称等不良手术后果。胰岛素样生长因子-Ⅰ(IGF-Ⅰ)是调节软骨细胞合成的重要生长因子之一,国内外学者离体实验证实它能刺激细胞的分裂增殖,增强细胞蛋白多糖及Ⅱ型胶原的合成。但针对IGF-Ⅰ对体内自体移植的肋软骨起何种作用,尚未见有文献报道。研究目的：评估IGF-Ⅰ凝胶对自体移植带软骨膜肋软骨增殖及代谢的作用。材料和方法实验动物及分组：健康新西兰白兔24只,幼年及成年各12只,雌雄不限,幼年兔为1月龄,体重lkg左右,成年兔为6月龄,体重2.5kg左右。按年龄分为幼年组及成年组,各组中再随机分为手术对照组、10ng/ml实验组及50ng/ml实验组,每组各4只。手术对照组直接将自体肋软骨取出,移植于背部皮下；10ng/ml实验组将自体肋软骨取出后,与0.5ml的10ng/ml IGF-Ⅰ凝胶混合为软骨-生长因子复合物后移植于背部皮下；50ng/ml实验组将自体肋软骨取出后,与0.5ml的50ng/mlIGF-Ⅰ凝胶混合为软骨-生长因子复合物后移植于背部皮下。在手术后8周,再取术侧对侧生理情况下生长的肋软骨作为空白对照组共同进行检测。凝胶制备及手术方法：纤维蛋白原(200mg/ml)、凝血酶(10u/ml)、IGF-Ⅰ(10、50ng/ml)混合制备成IGF-Ⅰ纤维蛋白凝胶,现用现配。实验兔麻醉、消毒后取出第8、9肋软骨,截为3段,按不同组别直接移植于背部皮下或混合不同浓度凝胶后移植于背部皮下。标本采集及结果评估：移植8周后,动物处死,取出移植的肋软骨,与空白对照组共同进行检测。检测项目包括：大体观察、HE染色、透射电镜检查及软骨羟脯氨酸分析。统计学方法：使用stata 7.0统计软件进行数据统计处理。对于各组所得到的软骨羟脯氨酸含量的结果,运用完全随机设计的单因素方差分析评价其4组间有无差异,各组间差异的评价通过t检验来实现。结果 1.大体观察：术后8周,幼年兔中,50ng/ml实验组移植软骨与周围组织粘连较牢固,不易分离；手术对照组与10ng/ml实验组移植软骨与周围组织无明显粘连,较易分离。手术对照组与50ng/ml实验组移植软骨呈乳白色,半透明,略有弹性,与空白对照组软骨相近；10ng/ml实验组移植软骨色泽偏黄,透明感及弹性均较差。成年兔中,各组差别不明显,但软骨弹性均较幼年软骨差。 2.HE染色：幼年兔中,50ng/ml实验组软骨膜完整,骨原细胞较多,软骨细胞增生活跃；手术对照组及10ng/ml实验组软骨膜结构紊乱,细胞结构少,软骨细胞发生退行性变。成年兔中,50ng/ml实验组软骨膜细胞结构较其余各组多,但4组软骨均存在空泡形成及基质分离现象。 3.透射电镜检查：幼年兔软骨透射电镜表现：在空白对照组及50ng/ml实验组中,可见健康的软骨细胞,软骨与周围组织交界处成纤维细胞少；手术对照组中,可见到坏死的软骨细胞,与周围组织交界处成纤维细胞活跃；10ng/ml实验组中,可见到散在的钙化灶,未发现正常的软骨细胞。成年兔软骨透射电镜表现：空白对照组中,可见软骨与周围组织交界处有成熟的成纤维细胞；手术对照组中,可见软骨与周围组织交界处有坏死的软骨细胞；10ng/ml实验组中,也可见到软骨细胞坏死,但软骨膜细胞正常；50ng/ml实验组中,可见健康的软骨细胞,同时软骨基质增多明显。 4.软骨羟脯氨酸分析：幼年兔：空白对照组与50ng/ml实验组间差别无统计学意义(P0.05),且均大于手术对照组和10ng/ml实验组,差别有统计学意义(P0.05),手术对照组与10ng/ml实验组之间差异无统计学意义(P0.05)。成年兔：各组间差异无统计学意义(P0.05)。结论 1.在幼年兔中,50ng/ml浓度的IGF-Ⅰ凝胶可以保持其正常生理状况下的增殖,和软骨细胞合成细胞外基质的能力,维持移植后的软骨及软骨膜细胞的正常表型。 2. 10ng/ml浓度的IGF-Ⅰ凝胶对幼年兔移植软骨及软骨膜细胞的生长没有促进作用,而凝胶可能影响软骨及软骨膜细胞的正常增殖与代谢。 3.成年兔中,在大体观察、光镜、电镜及生化检查等各个方面,各组之间均无明显差别,提示随年龄增长,软骨中的细胞成分减少,且处于静息状态,软骨及软骨膜细胞对IGF-Ⅰ的敏感性降低。 4.幼年期行自体肋软骨移植的效果优于成年。
[Abstract]:During auricle reconstruction, costal cartilage is widely used for its convenience and convenience for carving. Although autologous cartilage transplantation has no rejection, there are still some adverse effects on the reconstruction of auricle deformation, contracture, and abnormal appearance of the auricle. Insulin-like growth factor - I (IGF- I) is an important growth factor for regulating the synthesis of cartilage cells. First, in vitro, domestic and foreign scholars have proved that it can stimulate cell division and proliferation and enhance the synthesis of cell proteoglycan and type II collagen. However, it has not been reported that the effect of IGF- I on autogenous autogenous costal cartilage has not been reported.
Objective: To evaluate the effect of IGF- I gel on the proliferation and metabolism of autogenous transplantation of costal cartilage.
Materials and methods
Experimental animals and groups: 24 healthy New Zealand white rabbits, young and adult 12, male and female, and male and female, young rabbit 1 month old, weight LKG, adult rabbit 6 month old, weight 2.5kg. They are divided into young group and adult group according to age. Each group is randomly divided into operation control group, 10ng/ml experiment group and 50ng/ml experimental group, 4 rats in each group. The operation is 4. The autogenous costal cartilage was removed and transplanted subcutaneously in the back. After taking out the autogenous costal cartilage in the 10ng/ml experimental group, the 10ng/ml IGF- I gel was mixed with the 0.5ml 10ng/ml IGF- I gel as the cartilage growth factor complex and transplanted subcutaneously into the back. The 50ng/ml experimental group removed the autogenous costal cartilage and mixed with the 0.5ml 50ng/mlIGF- I gel into cartilage growth. After 8 weeks of operation, the costal cartilage growing on the contralateral side was taken as a blank control group.
Gelatin preparation and operation methods: fibrinogen (200mg/ml), thrombin (10u/ml), IGF- I (10,50ng/ml) mixed with IGF- I fibrin gel, now used. Experimental rabbit anaesthesia, after disinfection of the 8,9 rib cartilage, cut into 3 segments, according to different groups directly transplanted in the back subcutaneous or mixed with different concentration gels after transplantation to the back subcutaneous.
Sample collection and results evaluation: after 8 weeks of transplantation, the animals were killed, the costal cartilage was removed and the control group was tested together with the blank control group. The test items included gross observation, HE staining, transmission electron microscopy and cartilage hydroxyproline analysis.
Statistical method: Using Stata 7 statistical software to carry out data statistical processing. For the results of the content of cartilage hydroxyproline in each group, there is no difference between the 4 groups by the single factor variance analysis of the complete random design. The evaluation of the differences between each group is realized by the t test.
Result
1. general observation: 8 weeks after operation, in the young rabbits, the graft cartilage of 50ng/ml experimental group was firmly adhered to the surrounding tissue, and it was not easy to separate. There was no obvious adhesion between the graft cartilage and the surrounding tissue in the operation control group and the 10ng/ml experimental group. The transplantation cartilage of the operation control group and the 50ng/ml experimental group were white, translucent and slightly elastic. In 10ng/ml experimental group, the color of the graft was yellow, and the transparency and elasticity were poor. In adult rabbits, the differences were not obvious, but the cartilage elasticity was worse than that of young ones.
2.HE staining: in young rabbits, the cartilage membrane of the 50ng/ml experimental group was complete, the osteogenic cells were more and the chondrocytes proliferated active. The cartilage membrane structure was disorganized in the operation control group and the 10ng/ml experimental group, the cell structure was less, the chondrocytes were degenerative. In the adult rabbit, the cartilage membrane cell structure of the 50ng/ml experimental group was more than the other groups, but the cartilage of the 4 groups existed in all the other groups. The formation of vacuoles and the separation of matrix.
3. transmission electron microscope examination: the transmission electron microscopy of young rabbit cartilage: in the blank control group and the 50ng/ml experimental group, the healthy chondrocytes and the fibroblasts at the junction of the surrounding tissue were found less. The necrotic chondrocytes could be seen in the operation control group, and the fibroblasts were active at the intersection of the surrounding tissues; in the 10ng/ml experimental group, In the blank control group, there were mature fibroblasts at the junction of the cartilage and surrounding tissue; in the operation control group, there was necrotic cartilage cells at the junction of the cartilage and surrounding tissue; in the 10ng/ml experimental group, the soft tissue could also be seen in the soft tissue. Osteocytes were necrotic, but the chondrocytes were normal. In the 50ng/ml experimental group, healthy chondrocytes were observed, and cartilage matrix increased significantly.
4. cartilage hydroxyproline analysis: young rabbits: there was no statistical difference between the blank control group and the 50ng/ml experimental group (P0.05), and all were larger than the operation control group and the 10ng/ml experimental group, the difference was statistically significant (P0.05), the difference between the control group and the 10ng/ml experimental group was not statistically significant (P0.05). Learning meaning (P0.05).
conclusion
1. in young rabbits, the 50ng/ml concentration of IGF- I gel can maintain its normal physiological condition, and the ability of chondrocytes to synthesize the extracellular matrix, and maintain the normal phenotype of the cartilage and the cartilage membrane cells after transplantation.
2. 10ng/ml concentration of IGF- I gel has no effect on the growth of transplanted cartilage and chondrocytes in young rabbits, but the gel may affect the normal proliferation and metabolism of cartilage and cartilage membrane cells.
In 3. adult rabbits, there were no significant differences in the general observation, light microscopy, electron microscopy and biochemical examination. It was suggested that the cell components in cartilage decreased with age and were resting, and the sensitivity of cartilage and cartilage membrane cells to IGF- I was reduced.
4. the effect of autogenous costal cartilage transplantation in childhood is better than that in adults.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764.9

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