ABSTRACT
Objective To investigate the effectiveness of human insulin-like growth factor-Ⅰ(hIGF-Ⅰ) in transfecting adipose-derived mesenchymal stem cells (AdMSCs) and the expression of the transfected genes and find the possibility of differentiation of ADSCs to chondrocytes after hIGF-Ⅰ transfection. Methods AdMSCs were extracted from the fat tissue of the back of rabbits' neck and cultured in vitro by monolayer. Then, the recombinant eukaryotic expression plasmid poDNA3.1 + hIGF-Ⅰwas transfected into AdMSCs by Lipofectamine 2000. The growth of the ADSCs was observed by inverted microscope at 4-72 hours after transfection, at the end of passage and after stable transfection respectively, and the transfection efficiency was calculated. The hIGF-Ⅰ concentration in the supernatant was detected by enzyme-linked immnnosorbent assay (ELISA), the expression of collagen-Ⅱ by immunohistochemistry, the expression of hIGF-Ⅰ mRNA by reverse transcription polymerase chain reaction (RTPCR), the expression of hIGF-Ⅰ protein by Western blot and the cell cycle by flow cytometry. Results AdMSCs were shaped long shuttle and adhered to the disk at the 4th hour. AdMSCs were increased and distributed in cluster at 24th hour. After tranafection, AdMSCs were proliferated actively, with shortened doubling generation time. The concentration of hlGF-I in the supematant increased slowly after transfection and reached peak at 32.45 ng/ml at the 72nd hour. The hIGF-Ⅰ concentration remained at 28.36 ng/ml after stable transfectian. Immunohistochemistry affirmed a large number of collagen-Ⅱ in the endochylema and positive expression of hIGF-Ⅰ mRNA and hIGF-Ⅰ protein. The percentage of AdMSCs at stage S increased after transfection, which was significantly higher than that of AdMSCs without transfection, with statistical difference (P < 0.05). Conclusion After stable transfection, AdMSCs can secrete high concentration of hIGF-Ⅰ that can promote cell proliferation and differentiation of AdMSCs to chondrocytes.
ABSTRACT
Objective To study the technical details, clinical results and complication of microendoscopic discectomy (MED) for the treatment of lumbar disc herniation. Methods Ninety cases of lumbar disc herniation were operated upon with MED, and followed up for 24-32 months. Under direct endoscopic manipulation, semi circular excision of the inferior part of lamina, removal of the lateral part of the ligamentum flavum and the medial part of the facet were performed. The annulus fibrosus was circularly excised and the nucleus pulposus was removed. Results After operation, the patients could walk in 1.9 days, resumed their daily activities in (2.4?1.9) weeks and went back to work in (4.3?3.8) weeks. The rate of improvement was 83.1%. Several technical problems could happen in the beginning of using MED, including wrong identification of the vertebral level, difficulty to enter into the spinal canal and to control the bleeding, as well as injury of nerve root, dura mater and facet joint. Conclusion The advantages of MED are removal of the annulus fibrosus, the calcified ligamentum flavum and osteophyte under direct vision. However, sometimes it is difficult to fix the tip of microendoscopic tube on the surface of the lamina. Improvement is needed for the instrument design in order to increase the safety and easier manipulation.