Background The most significant biological change in intervertebral disc degeneration is the decrease of chondrocyte specific gene and protein expression of Sox9 and collagen type Ⅱ. Interleukin-1 （IL-1） is not exp...Background The most significant biological change in intervertebral disc degeneration is the decrease of chondrocyte specific gene and protein expression of Sox9 and collagen type Ⅱ. Interleukin-1 （IL-1） is not expressed in the normal intervertebral disc tissue but increases in the degenerated intervertebral disc tissue. This suggests that IL-1 may play a role in regulation of the expression of Sox9 and collagen type Ⅱ. Methods Human intervertebral disc cells were isolated and cultured. Sox9 and collagen type Ⅱ expression during treatment with IL-1, with or without the nuclear factor-κB （NF-κB） activity inhibitor curcumin, were detected by using reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting, and the activity of the NF-κB signaling pathway was detected by the electrophoretic mobility shift assay （EMSA）. Results IL-1 lowered the mRNA level and protein expression of Sox9 and collagen type Ⅱ in the cultured intervertebral disc cells in a dose dependent manner （P 〈0.05）, and this effect was attenuated by curcumin. Curcumin alone had no effect on Sox9 and collagen type Ⅱ expression （P 〉0.05）. IL-1 at concentrations of 0.1 ng/ml, 1 ng/ml and 10 ng/ml could stimulate the activity of NF-κB in the intervertebral disc cells in a dose dependent manner （P 〈0.05） that was inhibited by curcumin. Conclusions We demonstrated the previously unknown function of IL-1 in inhibiting Sox9 and collagen type Ⅱ via NF-κB in the intervertebral disc cells. This inhibition can be attenuated by curcumin, which is an effective NF-κB activity inhibitor.展开更多
Background Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration.This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs a...Background Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration.This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor （CTGF） and tissue inhibitor of metalloprotease-1（TIMP-1） expression mediated by adeno-associated virus （AAV） on collagen type Ⅱ and proteoglycan levels.The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.Methods Rhesus monkey lumbar intervertebral disc nucleus pulposus cells （NPCs） were isolated by enzyme digestion,cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection （MOl） of 106.The expression of collagen type Ⅱ and proteoglycan was measured using RT-PCR and Western blotting.The synthetic rate of proteoglycan was measured using 35S incorporation.Results Rhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1,rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected.Compared to the control,CTGF promoted the synthesis of collagen type Ⅱ and proteoglycan.TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type Ⅱ.Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type Ⅱ.Conclusions Single gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan.CTGF expression can also enhance collagen type Ⅱ protein synthesis.Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type Ⅱ to levels greater than transduction of a single gene alone.Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.展开更多
目的研究人骨形态蛋白(human bone morphogenetic protein-2,hBMP-2)对体外培养人腰椎间盘髓核细胞Ⅱ型胶原和aggrecan的影响。方法人退变髓核细胞体外分离培养,通过免疫组织化学鉴定椎间盘细胞。利用ELISA法检测对照组和不同剂量hBMP-2...目的研究人骨形态蛋白(human bone morphogenetic protein-2,hBMP-2)对体外培养人腰椎间盘髓核细胞Ⅱ型胶原和aggrecan的影响。方法人退变髓核细胞体外分离培养,通过免疫组织化学鉴定椎间盘细胞。利用ELISA法检测对照组和不同剂量hBMP-2(10ng/ml和100ng/ml)组Ⅱ型胶原和aggrecan的表达水平。用RT-PCR检测细胞Ⅱ型胶原和aggrecan的mRNA表达水平。结果加入hBMP-2后Ⅱ型胶原和aggrecan表达增加,并存在剂量依赖关系(P<0.01)。在第6天RT-PCR结果显示hBMP-2组aggrecan和Ⅱ型胶原mRNA的表达高于对照组,并且随剂量增高aggrecan和Ⅱ型胶原mRNA的表达量逐渐增加。结论hBMP-2可促进体外培养人腰椎间盘细胞合成代谢,Ⅱ型胶原和aggrecan表达量增加,并存在剂量依赖关系,提示hBMP-2可能对退变椎间盘具有修复功能。展开更多
基金The study was supported by a grant from the National Natural Science Foundation of China （No. 2004NSFC30471741）.
文摘Background The most significant biological change in intervertebral disc degeneration is the decrease of chondrocyte specific gene and protein expression of Sox9 and collagen type Ⅱ. Interleukin-1 （IL-1） is not expressed in the normal intervertebral disc tissue but increases in the degenerated intervertebral disc tissue. This suggests that IL-1 may play a role in regulation of the expression of Sox9 and collagen type Ⅱ. Methods Human intervertebral disc cells were isolated and cultured. Sox9 and collagen type Ⅱ expression during treatment with IL-1, with or without the nuclear factor-κB （NF-κB） activity inhibitor curcumin, were detected by using reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting, and the activity of the NF-κB signaling pathway was detected by the electrophoretic mobility shift assay （EMSA）. Results IL-1 lowered the mRNA level and protein expression of Sox9 and collagen type Ⅱ in the cultured intervertebral disc cells in a dose dependent manner （P 〈0.05）, and this effect was attenuated by curcumin. Curcumin alone had no effect on Sox9 and collagen type Ⅱ expression （P 〉0.05）. IL-1 at concentrations of 0.1 ng/ml, 1 ng/ml and 10 ng/ml could stimulate the activity of NF-κB in the intervertebral disc cells in a dose dependent manner （P 〈0.05） that was inhibited by curcumin. Conclusions We demonstrated the previously unknown function of IL-1 in inhibiting Sox9 and collagen type Ⅱ via NF-κB in the intervertebral disc cells. This inhibition can be attenuated by curcumin, which is an effective NF-κB activity inhibitor.
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 30471750）.
文摘Background Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration.This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor （CTGF） and tissue inhibitor of metalloprotease-1（TIMP-1） expression mediated by adeno-associated virus （AAV） on collagen type Ⅱ and proteoglycan levels.The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.Methods Rhesus monkey lumbar intervertebral disc nucleus pulposus cells （NPCs） were isolated by enzyme digestion,cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection （MOl） of 106.The expression of collagen type Ⅱ and proteoglycan was measured using RT-PCR and Western blotting.The synthetic rate of proteoglycan was measured using 35S incorporation.Results Rhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1,rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected.Compared to the control,CTGF promoted the synthesis of collagen type Ⅱ and proteoglycan.TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type Ⅱ.Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type Ⅱ.Conclusions Single gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan.CTGF expression can also enhance collagen type Ⅱ protein synthesis.Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type Ⅱ to levels greater than transduction of a single gene alone.Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.
文摘目的研究人骨形态蛋白(human bone morphogenetic protein-2,hBMP-2)对体外培养人腰椎间盘髓核细胞Ⅱ型胶原和aggrecan的影响。方法人退变髓核细胞体外分离培养,通过免疫组织化学鉴定椎间盘细胞。利用ELISA法检测对照组和不同剂量hBMP-2(10ng/ml和100ng/ml)组Ⅱ型胶原和aggrecan的表达水平。用RT-PCR检测细胞Ⅱ型胶原和aggrecan的mRNA表达水平。结果加入hBMP-2后Ⅱ型胶原和aggrecan表达增加,并存在剂量依赖关系(P<0.01)。在第6天RT-PCR结果显示hBMP-2组aggrecan和Ⅱ型胶原mRNA的表达高于对照组,并且随剂量增高aggrecan和Ⅱ型胶原mRNA的表达量逐渐增加。结论hBMP-2可促进体外培养人腰椎间盘细胞合成代谢,Ⅱ型胶原和aggrecan表达量增加,并存在剂量依赖关系,提示hBMP-2可能对退变椎间盘具有修复功能。
