| Background:Neck-shoulder pain and low back pain are mainly due to intervertebral disc degeneration, and lead a heavy burden to patients and their families. Since 1934, Mixter and Barr first time used surgical treatment in sciatica caused by lumbar disc herniation, the surgical treatment has been one of the main treatments to the symptoms caused by disc herniation. However, long-term efficacy of surgical treatment is not sure. As the times progress and technology develop, degeneration of the intervertebral disc has been deeply reserched. the current consensus view is that:As the nucleus anabolic and catabolic imbalance, the biochemical properties of the intervertebral disc are changed, and induce the changes of its pathophysiology, leading to the reduce of proteoglycan and collagen typeâ…¡in degenerated intervertebral disc, and the height of intervertebral disc can't maintain, thereby causing spinal nerve compression and stimulation,then appearing low back pain and low back pain and other symptoms. Although the morphological and biochemical changes of intervertebral disc degeneration are clear, the complex interrelationship between various etiological factors is still not entirely clear, and need in-depth study and exploration.Objective:Through normal human nucleus pulposus cells cultured to observe the changes of nucleus pulposus cells morphological and typeâ…¡collagen in vitro, and investigate different concentrations of BMP-7 and IL-6 acts on cultured human nucleus pulposus cells in typeâ…¡collagen and TIMP-1 gene expression. From the gene level to observe the interact of cytokines in the process of ntervertebral disc degeneration. providing a base on gene therapy and organizations engineering study of intervertebral disc degeneration.Method:1,human intervertebral disc nucleus pulposus were separated and digested to obtain a single cell and complete the primary cell inoculated culture 2,the control group and experimental group [IL-6 (100ng/mL); IL-6 (100ng/mL)+BMP-7 (100ng/mL); IL-6 (100ng/mL)+BMP-7(1000ng/mL); BMP-7 (1000ng/mL] were established when the primary cell fusion reach to the third generation.3,observed the cell morphous changes using inverted microscopy and HE staining,and detected the expression of typeâ…¡Collagen by immunohistochemical staining.4,semi-quantitative detected the gene expression levels of typeâ…¡-collagen and TIMP-1 in vitro cultured human nucleus pulposus cells with different concentrations of BMP-7 and IL-6 using the RT-PCR.5,analysis the data by the spss13.0 statistical software.Results:1,using the inverted phase contrast microscope to observe cells continuously, after 10 days, more than 90% cells were adherent, and primary cells were mostly spindle-shaped, with protruding pseudopodium; After passage by the trypsin digestion, the cells adherent growth rate accelerated, but the fourth generation of cells adhesion and growth began to slow.2,HE staining cells were mostly spindle-shaped, with protruding pseudopodia, nuclei were round or oval around the nucleus, many vacuoles were seen in the cytoplasm. Typeâ…¡collagen immunohistochemical staining showed positive results.3,Using reverse transcription-polymerase chain reaction (RT-PCR) semi-quantitative detection, typeâ…¡collagen and TIMP-1 band gray value analysis of variance,â…¡collagen:the F value is 148.058, P=0.000, TIMP-1:the F value is 69.995, P=0.000, showed the differences between five groups had the significance. By LSD-t test, results showed that compared with the 1,3,4,5 groups, IL-6 group had significantly differences, P<0.05; the 1,3 and 4,5 groups respectively were significantly different, P<0.05; the 1 group compared with the 3 had little difference, P> 0.05; the 4 group and 5 group compared with no difference, P> 0.05.Conclusion:The experiment provides a simple and effective method on the culture of disc nucleus pulposus; As the cells repeatedly passaged, nucleus pulposus cells gradually degenerated; BMP-7 can confront the role of IL-6 and positive regulate the gene expression of disc cells typeâ…¡collagen and TIMP-1, suggesting that BMP-7 may have a reverse effect in the clinical treatment of intervertebral disc degeneration. |
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