| Intervertebral disc degeneration (IVDD) disease could induce intervertebral disc hernia, degenerative instability of lumbar vertebrae, degenerative scoliosis and collateral low back pain; etc. IVDD is the common disease in orthopedics surgery, it is characterized by decrease of proteoglycans (PGs) and water, transformation of collagens, increasing activity of catabolic enzymes, releasing inflammatory factors and increasing cell apoptosis, among these factors, the inflammatory reaction and cell apoptosis play the important roles in IVDD. The concrete causes of IVDD were still unknown, many researchers investigated and found that the IVDD is related with biomechanics, nutrition, metabolism, molecular biology, aging and genetic factors.Intervertebral disc (IVD) consists of two independent anatomical parts:outer layer is annulus fibrosus (AF), which contains amount of collagen and present circles shape; inner layer is nucleus pulposus (NP), which contains amount of proteoglycans and aggrecan and present gelatin shape. IVD links the vertebral body by cartilage end plate. The AF structure makes the IVD the tensile resistance, and the NP structure makes IVD the pressure resistance. Though the concrete mechanisms and pathological changes were not obvious, many evidences showed that the IVD degeneration began in NP tissue. Nucleus pulposus cells (NPCs) are a type of cartilage-like cells. NPCs are the only cells type in NP, whose effects are important in secreting and maintaining of extracellular matrix (ECM).Many researchers investigated and found that, reduced metabolism, increased cell apoptosis and cell differentiation of NPCs were the main causes in NP degeneration. In the NP tissue of patients with IVD degeneration, the synthesis of extracellular matrix (ECM) is reduced, especially the collagen type-2and aggrecan; the viability of many kinds of matrix catabolic enzymes and their inhibitors were changed, such as matrix metalloproteinase-3(MMP-3) increased, tissue inhibitor of metalloproteinase-1(TIMP-1) reduced. The expression of nitric oxide (NO) and bone morphogenetic protein-2(BMP-2) were also changed. Another important appearance of NP degeneration is increased apoptosis of NPCs, especially the cysteine containing aspartate specific progeases-3(caspase-3) and Bcl-2play key roleS in NPCs apoptosis.Carboxymethylated chitosan (CMCS) is a kind of novel derivative of chitosan, apart from the characteristics of chitosan, the CMCS also have the well water solubility, this water solubility makes the CMCS has a wide usage extent. There are many studies indicated that CMCS has the protective role on degeneration of cartilage. The effects of CMCS on NPCs were still unknown; the aim of this research is to investigate the effects of CMCS on degeneration of NPCs and its probable mechanisms.Part1:Effects of carboxymethylated chitosan on cell proliferation and secretion of extracellular matrix in cultured NPCsObjective:The purpose of this study was to observe the effects of CMCS on proliferation and extracellular matrix secretion of NPCs in vitro, and the probable mechanisms in those processes.Methods:Eight three-months age Sprague Dawley (SD) rats were used to gathering the NPCs. The rats were put to death eagerly. The thoracolumbar spine was extracted and prepared for separation of nucleus pulposus tissue and NPCs culture. Cultured cells were identified by immunohistochemistry of collagen type-2and aggrecan. The NPCs were randomly divided into seven groups. The CMCS was added into each cell group. The final concentrations were as following:0,10,50,100,200,500and1000μg/mL, after24hour treatment, the proliferation of NPCs were assessed by cell counting kit-8(CCK-8). The growth and cell phenotype were observed by inverted microscope after addition of different concentrations of CMCS, and the messenger RNA (mRNA) expression of proliferating cell nuclear antigen (PCNA), collagen type-2and aggrecan were detected by using real time polymerase chain reaction (eeal-time PCR) anaysis.Results:The result of immnohistochemistry showed that cultured cells could secrete collagen type-2and Aggrecan. Cultured cells are the cartilage-derived cells. The cell counting kit-8(CCK-8) assay results indicated that the CMCS in range of50～1000μg/mL could significantly induce the proliferation of NPCs, the maximum response was observed at200μg/mL CMCS treated NPCs. The real-time PCR analysis results indicated that CMCS in different concentrations could induce the secretion and expression of PCNA, collagen type-2and aggrecan in NPCs, especially in200μg/mL, there is the significant difference compared with control group (P<0.05).Conclusion:This study shows that CMCS has a stimulative effect on proliferation of cultured NPCs. This effect is based on the alteration of PCNA systhesis. CMCS also could induce the synthesis and secretion of ECM such as collagen type-2and aggrecan in cultured NCs.Part2:Effects of carboxymethylated chitosan on expression of MMPs, TIMPs and cytokines in cultured NPCsObjective:To observe the effects of CMCS on matrix metalloproteinases-3(MMP-3), tissue inhibitors of metalloproteinase-1(TIMP-1), cytokines such as bone morphogenetic protein-2(BMP-2) and inducible nitric oxide synthase (iNOS) in cultured NPCs.Methods:Eight three-months age Sprague Dawley (SD) rats were used to gathering the NPCs. The rats were put to death eagerly. The thoracolumbar spine was extracted and prepared for separation of nucleus pulposus tissue and NPCs culture. The NPCs were induced to degeneratated cells by cell passaging, the fifth passage of cultured NPCs was used as the degenerated model of NPCs. NPCs were randomly divided into five groups, the CMCS was added into each group, the final concentrations of CMCS were as following:50,100and200μg/mL, then cultured under the5%CO2in air for24hours. The proliferation of NPCs was assessed by CCK-8analysis, the mRNA and protein expression of MMP-3, TIMP-1, BMP-2and iNOS were detected by using real-time PCR.Results:The CCK-8results and real-time PCR results indicated that five times of cell passaging could decrease the proliferation and synthesis of collagen type-2in NPCs. CMCS in concentrations of50～200μg/mL could increase the cell proliferation and secretion of collagen type-2. Real-time PCR and Western blot analysis indicated that CMCS in range of50～200μg/mL could decrease the expression of MMP-3and iNOS mRNA. Meanwhile, CMCS in range of50～200μg/mL could increase the expression of TIMP-1and BMP-2mRNA in cultured NPCs. There was the significant difference compared with control group (P<0.05).Conclusion:These results indicated that CMCS could inhibit MMPs and iNOS in degenerated NPCs, and facilitate the TIMPs and BMP-2in degenerated NPCs.Part3:Protective effects of carboxymethylated chitosan on sodium nitroprusside induced apoptosis of NPCs and its mechanisms Objective:Apoptosis plays the important role in degeneration of nucleus pulposus. This study was designed to investigate the effect of CMCS on apoptosis in cultured NPCs in vitro. The NPCs apoptosis were induced by sodium nitroprusside (SNP) as the indicated methods. The protective effects of CMCS on SNP induced apoptosis in cultured NPCs were investigated.Methods:Eight three-months age Sprague Dawley (SD) rats were used to gathering the NPCs. The rats were put to death eagerly. The thoracolumbar spine was extracted and prepared for separation of nucleus pulposus tissue and NPCs culture. The NPCs were treated by SNP. The final concentrations of SNP were lmM,2mM and3mM. NPCs were stained by both Annexin-V and PI. The apoptotic rate of NPCs treated by SNP was assessed by flow cytometry (FCM) analysis. Meanwhile, the CMCS in concentrations of50,100and200μg/mL was added into NPCs culture medium, the apoptotic model of NPCs was identified by FCM assay, the effect of CMCS on apoptotic rate of SNP induced NPCs was also determined by FCM assay. The real-time PCR and Western blot analysis were used to determine the expression of caspase-3and Bcl-2mRNA and protein, respectively. Hoechst33342staining was used to assess the nuclear alteration of apoptotic NPCs. The Rhodamine123(Rhol23) fluorescence staining was used to assess the mitochondrial membrane potential (ΔΨm) in NPCs. The synthesis and secretion of collagen type-2and aggrecan mRNA in NPCs under the apoptotic environment were determined by real-time PCR analysis.Results:The FCM assay results indicated that SNP in range of1to3mM could induce apoptosis of NPCs with concentration dependence. CMCS in concentrations of50to200μg/mL has the protective effects on SNP induced apoptosis in NPCs. The real-time PCR and Western blot analysis results indicated that CMCS could reduce the expression of caspase-3mRNA induced by SNP, and increase the expression of Bcl-2mRNA induced by SNP. Hoechst33342staining results showed that CMCS could decrease the nuclear fragment of apoptotic NPCs induced by SNP. Rhodamine123staining results indicated that CMCS could protect the impairment of mitochondrial membrane potential in cultured NPCs induced by SNP. The decline of synthesis and secretion of collagen type-2and aggrecan mRNA induce by CMCS was protected by CMCS with concentration dependence.Conclusion:. Those results indicated that CMCS has the protective effect on the SNP induced apoptosis in NPCs, this effect was fulfilled by changing the activation of caspase-3, Bcl-2and mitochondrial membrane potential. |
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