VDR及EMP-1在人退变腰椎间盘髓核组织中的表达研究

发布时间：2018-08-01 12:32

【摘要】：目的:通过免疫组织化学及实时荧光定量PCR等方法检测维生素D受体(vitamin D receptor,VDR)及上皮膜蛋白1(Epithelial membrane protein1,EMP-1)在人退变腰椎间盘髓核组织中的表达,探讨在人类腰椎间盘髓核组织中表达变化及在退变中的作用机制,为预防及治疗椎间盘退变引起的椎间盘退变性疾病提供新的思路。方法:(1).取材均来自川北医学院附属医院骨科2016年1月—2016年8月的23例手术后腰椎间盘髓核标本,分为对照组和实验组。对照组诊断为腰椎爆裂性骨折并行前路手术,共8例,其中男性5例,女性3例,平均年龄28.38±6.84岁,既往健康,排除其他慢性疾病,经影像学证实无明显椎间盘退变(Pfirrmann分级标准分级为I~II级)。实验组诊断为腰椎间盘突出症患者,共15例,其中男性10例,女性5例,平均年龄43.60±5.82岁,既往健康,排除其他慢性疾病,经影像学证实存在明显椎间盘退变(Pfirrmann分级标准分级为III~Ⅴ级),且有明显腰腿痛患者。以上均通过伦理委员会,患者本人及家属同意病签字。(2).同一患者的腰椎间盘髓核组织分为两块,一块用4%中性甲醛固定,常规脱水石蜡包埋,待HE染色和免疫组织化学检查。另一块椎间盘髓核组织保存在冻存管内存放于-80°C冰箱,后期行荧光定量PCR检测。实验数据应用SPSS 19.0统计软件处理,统计作图应用GraphPad Prism 6.0软件。计量资料采用均数标准差(sx±)表示,两样本均数的比较采用t检验,P0.05为差异具有统计学意义,计数资料采用率进行描述,统计推断采用χ2检验或Fisher确切概率法,P0.05为差异具有统计学意义。结果:(1).he染色及髓核细胞计数:对照组髓核组织在he染色切片中表现为髓核细胞分布较均匀,细胞呈卵圆形,细胞周围有不规则的陷窝,细胞多孤立存在于细胞陷窝内。实验组髓核细胞胞质中可见大量空泡,髓核细胞肥大,多个肥大的髓核细胞聚集在一起,呈现髓核细胞的“巢状”结构。髓核细胞在基质中分布很不均匀,主要表现为呈“巢状”结构的髓核细胞团多出现在椎间盘裂痕的周围。对髓核细胞进行计数,对照组髓核细胞平均数为:174.58±10.23(个/单位视野100x),实验组为:88.64±3.16(个/单位视野100x),对照组与实验组髓核组织中的髓核细胞数相比,实验组中髓核细胞数较对照组明显减少,差异有统计学意义(p0.05)。(2).免疫组织化学:测得vdr在对照组和实验组阳性细胞率分别为25%和86.67%;emp-1在对照组和实验组阳性细胞率分别为12.5%和93.33%;结果表明实验组中vdr和emp-1的表达情况明显强于对照组(p0.05)。(3).实时荧光定量pcr法检测:可见各椎间盘髓核组织标本中的基因扩增曲线、熔解曲线良好,未出现明显杂峰和主峰的异常增宽。vdr和emp-1mrna在实验组中的表达均明显高于对照组(p0.05)。结论:(1).人退变腰椎间盘髓核组织中髓核细胞数量明显减少;(2).退变腰椎间盘髓核组织中vdr及emp-1的表达明显升高;(3).腰椎间盘髓核组织中vdr及emp-1表达的升高可能是导致退变腰椎间盘中髓核细胞增殖与凋亡失衡的原因之一,同时也是引起腰椎间盘退变的重要机制之一。
[Abstract]:Objective: to detect the expression of vitamin D receptor (vitamin D receptor (VDR) and upper membrane protein 1 (Epithelial membrane protein1, EMP-1) in the lumbar intervertebral disc nucleus of human degeneration by immunohistochemistry and real-time fluorescence quantitative PCR, and to explore the expression changes in the human lumbar intervertebral disc nucleus and the mechanism of action in the degeneration of human lumbar intervertebral disc. To provide new ideas for the prevention and treatment of degenerative disc degeneration caused by intervertebral disc degeneration. Methods: (1) 23 cases of lumbar disc nucleus pulposus from the Department of orthopedics of Affiliated Hospital of Chuanbei Medical College from January 2016 to August 2016 were collected and divided into the control group and the experimental group. The control group was diagnosed as a parallel anterior approach to the burst fracture of the lumbar spine. A total of 8 cases, including 5 male and 3 female, with an average age of 28.38 + 6.84 years old, were previously healthy and excluded other chronic diseases. There were no obvious intervertebral disc degeneration (Pfirrmann grading standard I~II). The experimental group was diagnosed as lumbar intervertebral disc herniation, with 15 cases, including 10 men and 5 women, with an average age of 43.60 + 5.82 years old. To health and to eliminate other chronic diseases, it was confirmed by imaging that there was an obvious disc degeneration (Pfirrmann grading standard III~ V), and patients with lumbago and leg pain were obvious. All of these were approved by the ethics committee, the patient and the family agreed to sign the disease. (2) the lumbar disc nucleus of the same patient was divided into two pieces, one with 4% neutral formaldehyde. Fixed, dehydrated paraffin embedded, HE staining and immunohistochemical examination. The other intervertebral disc nucleus was stored in the cryopreservation tube in -80 / C refrigerator, and the fluorescence quantitative PCR was detected at the later stage. The experimental data were treated with SPSS 19 statistical software, and the statistical mapping was applied to the GraphPad Prism 6 software. The measurement data used the mean standard deviation (s). X +) indicated that the comparison of the average number of two samples was compared with t test. The difference was statistically significant in P0.05, and the use rate of the count data was described. The statistical inference was made by x 2 test or Fisher exact probability. The difference was statistically significant. Results: (1).He staining and nucleus pulposus count: the control group was displayed in HE staining section. The nucleus pulposus cells are evenly distributed, the cells are oval, and there are irregular lacunae around the cells. The cells are mostly isolated in the cell lacunae. In the experimental group, a large number of vacuoles are found in the cytoplasm of the nucleus pulmedulla cells, the nucleus cells of the medullary nucleus are hypertrophic, and the nucleus cells of the nucleus pulmedes are gathered together, and the nucleus pulposus cells are divided into the matrix. The distribution of the nucleus pulmedullary cell mass appeared mostly around the disc fissure. The nucleus pulposus cells were counted. The average number of nucleus pulmedullary cells in the control group was 174.58 + 10.23 (100x per unit field of vision), the experimental group was 88.64 + 3.16 (100x / single visual field), the control group and the nucleus pulposus nucleus of the experimental group. Compared with the control group, the number of nucleus pulposus cells in the experimental group was significantly lower than that in the control group (P0.05). (2). Immunohistochemistry: the positive cell rates of VDR in the control and experimental groups were 25% and 86.67%, respectively, and the positive cell rates of EMP-1 in the control and experimental groups were 12.5% and 93.33%, respectively. The results showed that in the experimental group, the VDR and EMP-1 were in the experimental group. The expression was obviously stronger than that of the control group (P0.05). (3). Real time fluorescence quantitative PCR method was used to detect the gene amplification curve in the tissue specimens of intervertebral discs. The fusion curve was good, the abnormal widening of the main peaks and the abnormal widening of.Vdr and emp-1mrna in the experimental group were obviously higher than those of the control group (P0.05). Conclusion: (1) the degenerative lumbar vertebra The number of nucleus pulmedullary cells in the nucleus pulposus of intervertebral disc decreased significantly, (2) the expression of VDR and EMP-1 in the degenerative lumbar intervertebral disc increased obviously; (3) the increase of VDR and EMP-1 in the lumbar intervertebral disc nucleus may be one of the causes of the imbalance of the proliferation and apoptosis of the nucleus pulmedulo cells in the degenerative lumbar intervertebral disc, and also the cause of the lumbar intervertebral disc degeneration. One of the important mechanisms of change.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.53

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