软骨单位与软骨细胞混合移植修复兔膝关节软骨缺损的作用

发布时间：2018-05-02 17:55

本文选题：组织工程 + 种子细胞　；参考：《山西医科大学》2016年硕士论文

【摘要】：背景:关节软骨由于无血管、神经及淋巴通过,其一旦损伤则很难自行修复,已知组织工程技术促进软骨的损伤修复,研究证实:在体外,软骨细胞作为种子细胞在体外培养极易发生去分化;在体内,经由其发育而来的修复组织的表型及力学特性均较正常关节软骨差,而且随着体内修复时间的推移,修复组织显现出现快速退变的特征,制约其临床应用的前景。软骨单位(chondron)被作为关节软骨的基本功能结构,其主要由软骨细胞与细胞周基质(PCM)组成,其中PCM对软骨细胞代谢和力学传导起着重要作用。体外实验证实:软骨单位与软骨细胞以1:1在海藻酸钠凝胶球中共培养,其生物学特性优于其他比例;然而,软骨单位和1:1共培养组对体内软骨缺损修复效果如何并不是很清楚。目的:探讨使用海藻酸钠立体培养的软骨单位、立体共培养的软骨单位与软骨细胞(1:1)移植对兔膝关节软骨缺损修复的作用,并对比分析两者修复效果。方法:1.选取2月龄新西兰兔5只,通过酶解法将膝关节软骨消化成软骨细胞和软骨单位,与1.2%海藻酸钠凝胶混合,分别制备成密度为5.8×106/mL的软骨细胞、软骨单位以及软骨细胞和软骨单位(1:1)混悬液,最后使用模具分别制成内含单纯软骨细胞、单纯软骨单位和软骨细胞与软骨单位(1:1)共培养的三种海藻酸钠凝胶球(大小约25μL/个);2.选取4月龄新西兰兔45只(4.0±0.2 Kg),通过取双侧股骨滑车内外侧髁连线中点行直径为4mm,深度为3mm全层软骨缺损,随机分为3组,将软骨细胞、软骨单位与软骨单位和软骨细胞(1:1)混合体植入缺损处;3.分别于植入后6、12、24周,通过mri评估、大体观察、he染色、番红o染色、免疫组化染色、rt-pcr检测以及tunel原位检测凋亡状况,以此来综合判断缺损修复情况。结果:1.mriroberts评分显示:自6周至24周,三组评分均呈增高趋势;12周时共培养组较软骨细胞组和软骨单位组均明显增高(p0.05);24周时共培养组较软骨单位组和软骨细胞组增高显著(p0.05)。2.ii型胶原免疫组化:自6周到24周,软骨单位组和共培养组其表达量呈增多趋势;在6周和12周软骨单位组其表达量均高于共培养组,而24周其共培养组高于软骨单位组。3.wakitani组织修复评分显示:随着时间递增,软骨单位组及共培养组评分均呈逐渐降低趋势,且12周与6周相比有统计学意义(p0.05);各时间组共培养组均明显低于软骨单位组(p0.05)。4.rt-pcr显示:6周至24周,共培养组中col-ii,sox-9mrna表达量均逐渐增多,软骨单位组col-iimrna表达量逐渐增多;6周至12周,软骨单位组和共培养组col-x,mmp-13mrna表达量呈降低趋势,24周时两者均增高,但共培养组较软骨单位组增高幅度较小;6周和12周软骨单位组col-ii及sox-9均高于共培养组,而24周两指标共培养组均高于软骨单位组;三个时间点内,col-x及mmp-13共培养组均低于软骨单位组。5.tunel细胞凋亡检测显示:自6周至24周,软骨细胞组细胞凋亡率呈增高趋势,且24周较12周增高有统计学意义(p0.05);软骨单位组细胞凋亡率有增高趋势;自6周至12周共培养组细胞凋亡率明显降低(p0.05),而24周较12周共培养组细胞凋亡率增高显著(p0.05);在三个时间点共培养组细胞凋亡率均低于软骨单位组和软骨细胞组。结论:1.软骨单位与软骨细胞(1:1)共培养作为种子细胞,早期对缺损组织有好的修复作用,晚期可延缓修复组织退化。2.软骨单位具有延缓软骨细胞凋亡的作用。
[Abstract]:Background: it is difficult to repair the articular cartilage because of no blood vessel, nerve and lymph, and it is difficult to repair itself once the injury is damaged. It is known that tissue engineering technology promotes the repair of cartilage damage. The study confirms that chondrocytes are easily differentiated in vitro as seed cells in vitro; in vivo, the phenotype and force of the repair of tissue through its development in the body. The characteristics of the study were worse than that of the normal articular cartilage, and with the time of repair in the body, the characteristics of rapid degeneration of the repair tissue appeared and the prospect of its clinical application was restricted. The cartilage unit (chondron) was used as the basic functional structure of articular cartilage, which was mainly composed of chondrocytes and cell Zhou Jizhi (PCM), and PCM was used for chondrocytes. Metabolism and mechanical conduction play an important role. In vitro experiments have proved that chondrocytes and chondrocytes are cultured with 1:1 in sodium alginate gel ball, and their biological characteristics are superior to other proportions; however, how the cartilage defect repair effect of cartilage unit and 1:1 co culture group is not very clear. The effects of cartilage unit, co cultured cartilaginous unit and chondrocyte (1:1) transplantation on the repair of articular cartilage defect of the knee in rabbits were studied and compared and analyzed. Methods: 1. 2 month old New Zealand rabbits were selected to digest the articular cartilage of the knee into chondrocytes and cartilaginous units by enzymolysis and mixed with 1.2% sodium alginate gel. The chondrocytes, cartilaginous cells and chondrocytes and cartilage units (1:1) were not prepared with a density of 5.8 x 106/mL. Finally, three kinds of sodium alginate gel balls (size 25 mu L/) co cultured with pure cartilage unit and chondrocyte and cartilage unit (1:1) were made by using molds, respectively; 2. selected 4 month old new West. 45 rabbits (4 + 0.2 Kg) were randomly divided into 3 groups by taking the middle point of the internal and external condyle of the bilateral femur trochlear and the depth of 3mm full layer cartilage defect into 3 groups. The cartilage cells, cartilage units and chondrocytes (1:1) were implanted into the defect. 3. points were compared to 6,12,24 weeks after the implantation, and were evaluated by MRI, gross observation, he staining, O staining, immunohistochemical staining, RT-PCR detection and TUNEL in situ detection of apoptosis were used to determine the defect repair. Results: the scores of the three groups were increased from 6 to 24 weeks, and at 12 weeks, the co culture group was significantly higher than that of the cartilage and cartilage units (P0.05), and the co culture group at 24 weeks. The cartilage unit group and cartilage cell group increased significantly (P0.05).2.ii collagen immunohistochemical staining: the expression of cartilage unit group and co culture group increased from 6 to 24 weeks, and the expression of cartilage unit group in 6 and 12 weeks was higher than that of the co culture group, and the co culture group was higher than the.3.wakitani tissue repair score of the cartilage unit group at the 24 week. With the increase of time, the score of cartilage unit group and co culture group decreased gradually, and the 12 weeks compared with 6 weeks was statistically significant (P0.05). All time group co culture groups were significantly lower than the cartilage unit group (P0.05).4.rt-pcr display: 6 weeks to 24 weeks, the co culture group of col-ii, sox-9mrna expression increased gradually, cartilage unit group col-iimrna The expression amount increased gradually. The expression of col-x and mmp-13mrna in the cartilage unit group and co culture group decreased in 6 weeks to 12 weeks, and both increased at 24 weeks, but the increase in the co culture group was smaller than that of the cartilage unit group. The col-ii and Sox-9 in the cartilage unit group were higher than that of the co culture group at 6 and 12 weeks, and the two index co culture group was higher than the cartilage unit group at the 6 and 12 weeks. Three time points, col-x and MMP-13 co culture group were lower than the cartilage unit group.5.tunel cell apoptosis detection showed that from 6 weeks to 24 weeks, the apoptosis rate of chondrocyte group increased, and 24 weeks higher than 12 weeks (P0.05), apoptosis rate of cartilage unit group increased; from 6 to 12 weeks, Co culture group cell apoptosis The rate of apoptosis was significantly lower (P0.05), while the apoptosis rate of cells in the 24 week group was significantly higher than that in the 12 week group (P0.05). The apoptosis rate in the co culture group at three time points was lower than that of the cartilage unit group and the chondrocyte group. Conclusion: 1. cartilage unit and chondrocyte (1:1) co culture as seed cells, early repair of the defect tissue and late delay can be extended. .2. cartilage units with delayed repair of tissue degradation can retard chondrocyte apoptosis.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R687.4

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