颈椎突出椎间盘对后纵韧带骨化形成机制的研究

发布时间：2018-08-27 06:03

【摘要】：实验背景：后纵韧带骨化症(ossification of the posterior longitudinal ligament, OPLL)是发生在脊柱后纵韧带中的异位骨化,压迫脊髓和神经根而导致其功能受损的一种疾病。其发病机制至今不明确。后纵韧带增厚及骨化与突出的椎间盘是导致颈椎椎管狭窄常见的原因,在临床上,我们经常发现椎间盘的突出和后纵韧带的增厚、骨化是同时存在的,但是两者之间是否具有相关性,目前尚未见报道。目的：本实验旨在：在体外,研究髓核细胞分泌的细胞因子对后纵韧带细胞增殖、细胞毒性及成骨分化能力的影响,探讨颈椎退变突出的椎间盘对后纵韧带骨化(OPLL)发展的作用,为后纵韧带骨化症的诊治提供新的理论依据。方法：1. 骨化后纵韧带细胞培养及传代通过影像学资料(X、CT及MRI)选取后纵韧带骨化合并颈椎间盘突出的患者10例,通过颈椎前路椎体次全切手术获取骨化的后纵韧带(标本采集经患者和家属同意),剔除骨化的部分,保留未骨化的韧带,用组织块原代培养法行颈椎后纵韧带成纤维细胞体外培养并传代,收集第三代细胞用于实验。2.髓核细胞的培养及传代通过影像学资料(X、CT及MRI)选取颈椎间盘突出的患者1例,通过颈椎前路椎间盘切术获取突出的髓核组织(标本采集经患者和家属同意),利用酶解法(Ⅱ型胶原酶消化)提取髓核细胞,行原代培养并传传代,收集第三代细胞用于实验。3. 建立实验分组建立实验组及对照组,通过Transwell建立后纵韧带细胞和髓核细胞共培养体系。实验组：OPLL细胞接种于Transwell六孔板下室,髓核细胞接种与Transwell六孔板上室,中间隔以孔径为0.4μm的聚酯膜；对照组：OPLL细胞种板于普通六孔板。4.酶联免疫法(enzyme-linked immunosorbent assay, ELISA)测细胞因子实验组及对照组于37℃、5% C02、饱和湿度的培养箱培养两天后,收集实验组及对照组细胞培养液,2000rpm离心20分钟,取上清液于EP管中待测,按ELISA试剂盒操作方法分别检测IL-1a, IL-6、 PGE2及TNF-a含量。5. MTT比色法测细胞毒性实验组及对照组于37℃、5% C02、饱和湿度的培养箱培养两天后, 加入MTT液,继续在37℃、5% C02、饱和湿度的培养箱培养4小时,小心吸去每孔内的培养液, 加入DMSO,酶标仪测OD490值。6. EdU法测细胞增殖实验组和对照组于37℃、5% C02、饱和湿度的培养箱培养2天后,吸除培养液,加入含EdU溶液的培养基(1000：1的比例稀释),行EdU标记,在孵箱中继续培养4小时。吸除培养基,磷酸盐缓冲液清洗3遍,固定液固定细胞,依次用Apollo染色液及Hoechst染料对DNA染色。于荧光显微镜下镜检,计数细胞增殖率(红色细胞核数／总细胞核数)。7. RT-PCR法检测mRNA的表达使用Trizol提取总RNA;用逆转录试剂盒反应体系将所得RNA转录成cDNA,此过程分两步进行：首先去除基因组DNA,其次进行反转录反应;将cDNA加入PCR反应体系,进行PCR扩增,使用2-△△Ct值分析mRNA相对表达。8. Von Kossa染色法及ALP染色法检测成骨活性实验组和对照组于37℃、5% C02及饱和湿度的培养箱培养48h后,采用ALP染色试剂盒(钙钴法),分别对实验组和对照组OPLL细胞进行碱性磷酸酶染色。实验组和对照组于37℃、5% C02及饱和湿度的培养箱培养48h后,采用Von Kossa染色试剂盒操作方法对实验组和对照组染色。9. 统计分析应用SPSS 11.5统计软件对数据进行分析,用t检验方法比较组间不同处理的差异,P0.05为统计具有差异性。结果：1.细胞毒性MTT法检测共培养体系对后纵韧带细胞细胞毒性发现：实验组OD490均较对照组OD490高,对照组OD49o值为0.25±0.06,实验组OD49o为0.28±0.09,差异有统计学意义(P0.05)。2.细胞增殖荧光显微镜下荧光激发、拍摄及合成图片示：实验组中伴有DNA新合成的红色细胞核数明显多于对照组,在4小时内,实验组中OPLL细胞增殖率为14.30±2.67%,对照组中OPLL细胞增殖率为2.21±0.64%,差异有统计学意义(P0.05)。3. Von Kossa染色及ALP染色在Von Kossa染色中,实验组中有明显的钙结节沉淀,而对照组中钙结节沉淀几乎不可见；A LP染色中,实验组中染色阳性的细胞数明显较对照组多。4. RT-PCR检测Ⅰ、Ⅺ型胶原及骨钙素mRNA的表达与对照组相比,实验组中Ⅰ型胶原、Ⅺ型胶原及骨钙素mRNA的表达均上调,差异具有统计学意义(P0.05)。5. ELISA法测炎症因子与OPLL组及NP组相比,OPLL+NP组上清液中IL-1a 、 TNF-a 、 PGE2的含量明显增加,差异有统计学意义(P0.05)。各组上清液中IL-6含量低于最低检测标准6.25pg/ml。结论：在体外,髓核细胞能够促进OPLL细胞的增殖,提高OPLL细胞的成骨分化能力并且无明显的细胞毒性。颈椎突出椎间盘促进后纵韧带骨化的发展,髓核组织分泌的细胞因子对后纵韧带骨化起了重要作用。
[Abstract]:BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) is a disease that occurs in the posterior longitudinal ligament of the spine, which is heterotopic ossification, compressing the spinal cord and nerve roots and causing impairment of its function. The pathogenesis of OPLL is still unclear. Objective: To study the effect of cytokines secreted by nucleus pulposus cells on the proliferation and fineness of posterior longitudinal ligament cells in vitro. Objective: To investigate the effect of cervical intervertebral disc degeneration and protrusion on the development of ossification of posterior longitudinal ligament (OPLL) and provide new theoretical basis for the diagnosis and treatment of OPLL. Methods: 1. Cell culture and passage of ossified posterior longitudinal ligament (POL) were studied by X-ray, CT and MRI. Ten patients with cervical disc herniation underwent anterior subtotal resection of the cervical vertebra to obtain ossified posterior longitudinal ligament (specimens were collected with the consent of the patient and his family members). The ossified part was removed and the non-ossified ligament was retained. The fibroblasts of the cervical posterior longitudinal ligament were cultured and subcultured in vitro with tissue block primary culture method. The third generation cells were collected and used in the experiment. 1 patient with cervical disc herniation was selected by X-ray, CT and MRI. The prominent nucleus pulposus tissue was obtained by anterior cervical discectomy (with the consent of the patient and his family members). The nucleus pulposus cells were extracted by enzymatic hydrolysis (collagenase type II digestion). The nucleus pulposus pulposus cells were primary cultured and subcultured. The experimental group: OPLL cells were inoculated in the subcompartment of Transwell six-hole plate, the nucleus pulposus cells were inoculated in the subcompartment of Transwell six-hole plate, and the polyester membrane with a pore size of 0.4 micron was used in the middle of the compartment. LL cell culture medium was collected from the experimental group and the control group after two days of incubation in a saturated humidity incubator. The supernatant was centrifuged for 20 minutes at 2 000 rpm. The supernatant was collected and tested in EP tube. METHODS IL-1a, IL-6, PGE2 and TNF-a contents were measured by MTT colorimetric assay. The cytotoxicity of the experimental group and the control group was measured by MTT colorimetric assay at 37 C, 5% C02. After two days of incubation in saturated humidity incubator, MTT solution was added, and then incubated in 37 C, 5% C02, saturated humidity incubator for 4 hours. The culture medium in each hole was carefully sucked away, DMSO was added, and OD490 was measured by enzyme-labeling apparatus. EdU method was used to measure cell proliferation in the experimental group and the control group at 37, 5% C02, saturated humidity incubator for 2 days, then the culture medium was sucked, the medium containing EdU solution was added (1000:1 diluted), and the cells were labeled with EdU for 4 hours in the incubator. DNA was stained with LLO staining solution and Hoechst dye. Cell proliferation rate (red cell nucleus / total cell nucleus) was counted under fluorescence microscope. 7. Total RNA was extracted by Trizol and transcribed into cDNA by reverse transcription kit reaction system. The process was carried out in two steps: first, genomic DNA was removed, and the total RNA was extracted. 8. Von Kossa staining and ALP staining were used to detect the osteogenic activity of the experimental group and the control group. After incubated at 37 C, 5% C02 and saturated humidity for 48 hours, ALP staining kit (Ca-Co method) was used for the experimental group and the control group, respectively. The OPLL cells in the control group were stained with alkaline phosphatase. The experimental group and the control group were cultured at 37 C, 5% C02 and saturated humidity incubator for 48 hours. Von Kossa staining kit was used to stain the OPLL cells in the experimental group and the control group. 9. Results: 1. The cytotoxicity of the co-culture system to the posterior longitudinal ligament cells was detected by cytotoxicity MTT assay. The results showed that the OD490 of the experimental group was higher than that of the control group, and the OD49o of the control group was 0.25 [0.06], and the OD49o of the experimental group was 0.28 [0.09], the difference was statistically significant (P 0.05). 2. The number of newly synthesized red cell nuclei with DNA in the experimental group was significantly higher than that in the control group. Within 4 hours, the proliferation rate of OPLL cells in the experimental group was 14.30 (+ 2.67%) and that of OPLL cells in the control group was 2.21 (+ 0.64%). The difference was statistically significant (P 0.05). 3. Von Kossa staining and ALP staining were detected in Von Kossa staining. The number of positive cells in the experimental group was significantly higher than that in the control group. 4. The expression of collagen type I, collagen type VII and osteocalcin mRNA in the experimental group was significantly higher than that in the control group. Compared with OPLL group and NP group, the levels of IL-1a, TNF-a and PGE2 in the supernatant of OPLL+NP group were significantly increased (P 0.05). The levels of IL-6 in the supernatant of each group were lower than the minimum detection standard of 6.25 pg/ml. Conclusion: In vitro, nucleus pulposus cells can be detected. The cervical disc herniation promotes the ossification of the posterior longitudinal ligament, and the cytokines secreted by the nucleus pulposus play an important role in the ossification of the posterior longitudinal ligament.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R681.5

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