hTGF-β1转染MSCs与hBMP-2转染髓核细胞共植入重建椎间盘髓核

The principal change of intervertebral disc degeneration included two points, one was the reduction of the quantity and function of nucleus pulposus cells(NPCs), the other was the degression of collagen protein Ⅱ and proteoglycans in the stroma. It could not increase the quantity of the NPCs or augment NPCs' function as soon as raise the content of collagen protein Ⅱ and proteoglycans in the stroma to transplant bone marrow stromal cells (MSCs) or NPCs alone in long-term. In our research, we devise to rebuild the nucleus pulposus by transplanting Ab-hBMP-2-transfected NPCs mixed with Ab-hTGF-β1-transfected MSCs. In earlier stage, MSCs and NPCs were activated by cell factor including BMP-2 and TGF-β1. MSCs and NPCs both proliferated and produced collagen protein Ⅱ and proteoglycans. And MSCs was differentiating to NPCs under the excitation of cells immediate contact; in later stage, proliferatied NPCs and nucleus pulposus like cells come from MSCs produced collagen protein Ⅱ and proteoglycans. These two kinds of cells collaborated to rebuilding the nucleus pulposus for long term. In our study, we would uses the technique such as fluorescence resonance energy transfer (FRET) and Total internal reflection fluorescence (TIRF) of the laser scanning confocal microscopy, flow cytometer, mass chromatographic analysis, MRI, histology observation, vitodynamics testing. We would investigate the relationship of the content discrepancy of hBMP-2and hTGFβ1 in cells or stroma and the phenotype of the cells connect with cell gap junction interception experiment, in order to find the potential signal molecule and to reveal the molecular mechanism of signal transduction between the two kinds of cells. We would also observe the result of the rebuilding nucleus pulposus after the mixed cells transplantation in animal model. The numerical data of our research would confirm that it was valid to rebuild the nucleus pulposus by transplanting Ab-hBMP-2-transfected NPCs mixed with Ab-hTGF-β1-transfected MSCs. Looking to the future, it would be a feasible minimally invasive method to reverse intervertebral disc degeneration by transplanting Ab-hTGF-β1-transfected MSCs and Ab-hBMP-2-transfected NPCs into intervertebral space through Intervertebral disc puncture in clinic. These research ideal was a bold essay established in the former research foundation at home and abroad.

椎间盘退行性变的共同表现是髓核细胞数量减少、功能降低和间质中胶原蛋白Ⅱ、蛋白聚糖减少。单独移植MSCs或NPCs不能同时解决中长期细胞和间质两方面的问题。本研究拟设计hTGF-β1转染的MSCs和hBMP-2转染的NPCs联合移植重建髓核；早期局部高效表达的细胞因子激活两种细胞增殖并合成基质，利用细胞直接接触诱导MSCs向NPCs分化；后期依靠增殖的NPCs和MSCs分化的类髓核细胞合成基质成分。以期两种细胞优势互补，达到长期修复髓核的目的。采用激光共聚焦显微镜、流式细胞仪、蛋白质质谱分析等技术，结合细胞连接阻断实验研究不同分组两种细胞及其基质中hBMP-2和hTGFβ1含量与细胞表型差异间的关系；探寻两种细胞间可能存在的其他信号分子和相互刺激诱导的分子机制；MRI、组织学、生物力学观察动物模型髓核重建的生物学效果。为临床细胞学治疗椎间盘退变提供实验依据，为微创治疗椎间盘退变提供新的思路。