腺病毒介导ChM-I基因抑制BMSCs体内构建的软骨血管化的实验研究

发布时间：2018-01-27 01:12

本文关键词： 软骨组织工程 BMSCs ChM-I 血管化腺病毒　出处：《西北大学》2010年硕士论文　论文类型：学位论文

【摘要】： 研究背景：骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)是软骨组织工程中倍受关注的种子细胞,具有很高的研究意义和广泛的应用前景。但应用BMSCs构建的软骨组织经体内长期移植后,会发生一系列表型不稳定现象,比如丧失表达Ⅱ型胶原的能力,这些严重地影响利用BMSCs进行软骨缺损的长期修复效果。这一系列表型变化现象类似于软骨内骨化的发生过程,即软骨组织先出现肥大、钙化现象,并伴随有血管的侵入,随着体内移植时间的增长血管化程度逐渐增加,渐渐矿化形成骨组织。该过程中血管化的侵入是诱导软骨向骨转变的关键步骤。然而正常的软骨组织在促血管发生的微环境中,仍可持续保持无血管的状态及稳定的软骨表型,经研究发现,其中存在的一种特异性的生长因子——软骨调节素-1(Chondromodulin-I, ChM-I),具有强效的抑制血管发生的功能。我们推测BMSCs在分化为软骨的过程中ChM-I不表达或者表达很低,从而才会导致血管侵入、软骨表型丢失现象的发生。目前,对BMSCs及其分化形成的软骨中ChM-Ⅰ是否表达尚无文献报道。因此,我们从ChM-I基因在BMSCs和软骨细胞中是否存在表达差异出发,研究ChM-I对BMSCs体内构建的软骨血管化的抑制作用,分析其在软骨表型丢失过程的影响。实验目的：观察软骨细胞对BMSCs体内构建软骨的血管化的抑制作用,对比抗血管生成因子ChM-I的基因在两种细胞间的表达差异,并探讨ChM-I抑制BMSCs体内构建软骨血管化的作用,为改善和提高BMSCs来源的软骨组织的表型稳定性提供新的思路。实验方法和结果： 1. BMSCs与软骨细胞以不同比例(3：1,1：1,1：3)混合,接种至珊瑚材料上,移植到裸鼠皮下,同时分别以单纯BMSCs和软骨细胞为阴性和阳性对照组,体内培养1-2个月后,观察构建的组织工程软骨的血管化程度,并分析抗血管生成因子ChM-I在不同组中的表达分布。大体观察和组织学结果可见,单纯BMSCs构成的组织中血管化程度很高,而且有少量成骨出现。相反,含有软骨细胞的各实验组均可形成血管化程度极低的软骨组织,在软骨细胞含量很少(25%)时也可保持很低的血管化水平。免疫荧光结果显示,ChM-I在阳性对照组及各个实验组都有不同程度的表达分布,而单纯BMSCs构成的组织中没有。 2.用RT-PCR的方法比较ChM-I基因在BMSCs和关节软骨细胞之间的表达差异。结果显示ChM-I在BMSCs中几乎不表达,此外,两种细胞之间Ⅱ型胶原和蛋白聚糖的表达也存在显著的差异。 3.获得兔ChM-I基因克隆,构建重组腺病毒Ad5-ChM-I。用RT-PCR的方法从兔的关节软骨组织中获得了ChM-I基因克隆,通过测序验证了其正确性,并成功的构建了高滴度的重组腺病毒Ad5-ChM-I。 4.转染重组腺病毒Ad5-ChM-I的BMSCs与珊瑚材料复合物移植到裸鼠皮下1-2个月后,观察体内构建的软骨组织的血管化的程度,分析软骨表型的变化。大体观察和组织学结果可见,转染Ad5-ChM-I的BMSCs在体内形成的软骨组织的血管化程度很低,免疫荧光结果显示,ChM-I在转染后的BMSCs形成的组织中有一定的表达分布。结论：研究结果表明,ChM-I基因在BMSCs和软骨细胞中确实存在显著的表达差异,BMSCs中几乎不表达ChM-I,软骨细胞及其特异性表达的ChM-I均可以抑制BMSCs体内构建的软骨组织的血管化发生,阻止其向骨的表型发生转变,在一定程度上稳定了软骨表型。因此,我们可以将腺病毒介导的ChM-I基因修饰的BMSCs作为新型的种子细胞,用于构建稳定的无血管侵入的组织工程软骨,或者用于软骨缺损修复之中,达到长期的治疗效果。
[Abstract]:Research background:
Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) is the focus of seed cells in cartilage tissue engineering, has high research value and broad application prospect. But the application of BMSCs construction of the cartilage after long-term in vivo transplantation can produce a series of phenotypic instability, such as the loss of the ability to express type II collagen, which seriously affected the long-term effect of the repair of cartilage defect by BMSCs. During this series of phenotypic changes similar to the phenomenon of endochondral ossification, namely cartilage tissue appeared hypertrophy, calcification and vascular invasion with, with the growth of vascularization in vivo transplantation time gradually increased gradually the mineralization of bone tissue formation. In the process of vascular invasion is a key step to induce cartilage bone transformation. However, the normal cartilage tissue microenvironment in neovascularization in, Still keep the sustainable and stable state of avascular cartilage phenotype, the study found that the presence of a specific growth factor -- chondromodulin -1 (Chondromodulin-I, ChM-I), a potent inhibition of vascular function. We speculate that the occurrence of BMSCs in the process of differentiation into cartilage in ChM-I expression or expression is very low thus, can lead to vascular invasion, occurrence of cartilage phenotype loss. At present, there is no report about whether the expression of ChM- 1 on the formation of BMSCs and differentiation of cartilage. Therefore, we from the ChM-I gene in BMSCs and chondrocytes in the existence of differences in expression of the inhibitory effect of ChM-I on cartilage vascularization in vivo construction of BMSCs the analysis, lost in the cartilage phenotype process.
Objective:
To observe the inhibitive effect of chondrocytes on chondrogenesis of BMSCs in vivo vascular contrast, anti angiogenic factor ChM-I gene in two cell differential expression and inhibitory effect of ChM-I on BMSCs in vivo cartilage angiogenesis in construction, to provide new ideas for improving and enhancing the phenotypic stability of cartilage tissue derived from BMSCs.
Experimental methods and results:
1. BMSCs and chondrocytes in different proportions (3:1,1:1,1:3) mixed inoculation to coral material, transplanted into nude mice, were pure BMSCs and cartilage cells as negative control group and positive control group cultured in vivo after 1-2 months, the degree of vascularization of tissue engineering cartilage construction observation, and analysis of the distribution of anti angiogenic factor the expression of ChM-I in different groups. The general observation and vascular tissue visible, highly pure BMSCs tissues, and have a small amount of bone. On the contrary, each experimental group can contain chondrocytes in the cartilage formation of blood vessels is extremely low, rarely in the cartilage cell content (25%) also can maintain a very low level of vascularization. Immunofluorescence results showed that ChM-I has a distribution of different degrees of expression in the positive control group and the experimental group, and a simple BMSCs organization.
2., the expression difference of ChM-I gene between BMSCs and articular chondrocytes was compared by RT-PCR. Results showed that ChM-I was almost not expressed in BMSCs, and there was significant difference in the expression of type II collagen and proteoglycan between two kinds of cells.
3. the rabbit ChM-I gene was cloned, and the recombinant adenovirus Ad5-ChM-I. was constructed. The ChM-I gene clone was obtained from rabbit articular cartilage tissue by RT-PCR. The correctness of the gene was verified by sequencing, and the recombinant adenovirus Ad5-ChM-I. with high titer was successfully constructed.
BMSCs and coral composites 4. transfected with recombinant adenovirus Ad5-ChM-I transplanted into nude mice after 1-2 months, the construction of cartilage tissue in vivo vascularization, changes of cartilage phenotype. The gross observation and tissue vascularization of cartilage tissue, Ad5-ChM-I transfected BMSCs is formed in the body is low immunofluorescence results showed that the formation of ChM-I in transfected BMSCs tissues have a certain distribution of expression.
Conclusion:
The results showed that the ChM-I gene indeed significant differential expression in BMSCs and chondrocytes, BMSCs almost no expression of ChM-I, ChM-I and chondrocyte specific expression could inhibit cartilage tissue BMSCs in vivo construction of vascularization, prevent the changes to the table type bone, to a certain extent, the stability of the cartilage phenotype. Therefore, we can use the ChM-I gene modified adenovirus mediated BMSCs as a new source of seed cells for cartilage tissue engineering, without vascular invasion to construct stable, or for the repair of cartilage defects, to achieve the long-term therapeutic effect.

【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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