髓核细胞炎性因子分泌特性的实验研究

发布时间：2018-05-17 07:36

本文选题：腰椎间盘突出 + 软骨细胞簇　；参考：《天津医科大学》2016年硕士论文

【摘要】：第一部分LDH患者椎间盘组织中炎性相关因子表达的一般特点目的:明确不同类型腰椎间盘突出症(LDH)患者间盘组织的病理学特点,归纳出炎性相关因子在间盘组织中表达分布情况,探讨间盘组织中炎性因子的来源及其可能病理生理学意义。方法:选取天津医院脊柱外科于2014年12月到2015年9月所收治、确诊并接受手术治疗的LDH患者26例。根据临床症状体征、影像学检查(CT、MRI)结果及术中所见纤维环的完整性将所选病例分为两组:(1)损伤疝出型椎间盘突出组(R组)(2)退变突出型椎间盘突出组(NR组)。采用HE染色观察不同分型间盘组织的病理学特点,通过免疫组化、Westernblot等方法检测IL-17、IL-23、NLRP3、Caspase-1等炎性相关因子在间盘组织中的表达分布情况,进而归纳出炎性相关因子在组织中表达的一般特点。结果:两种类型LDH患者的间盘组织均表现出退变征象,但损伤疝出组病理表现更为复杂、退变更加明显,主要表现为:组织裂隙明显增多,纤维排列更加紊乱、疏松,甚至断裂;组织边缘常可见纤维素样坏死、粘液样变性、滑膜样化生、钙结晶沉积、新生血管长入、炎性细胞浸润和软骨细胞簇形成等现象。其中细胞增生和软骨细胞簇形成部位间盘退变也较为明显。免疫组化可知IL-17、IL-23、NLRP3、Caspase-1等在两种类型的间盘组织中均有表达,且损伤疝出型表达含量高于退变突出型;各炎性相关因子在软骨样的髓核细胞中均可见阳性表达,且在组织裂隙周围的髓核细胞中、软骨细胞簇中、浸润的炎性细胞周围及新生血管周围阳性表达结果尤为明显。结论:1.两种类型LDH患者的间盘组织病理学差异较为明显,提示可能有更多的机制参与间盘组织损伤疝出后的病理表现。增生细胞和软骨细胞簇部位组织退变程度明显,提示软骨细胞簇形成在间盘病理变化中可能发挥着一定的作用。2.间盘细胞能够分泌炎性相关因子,且在间盘细胞增生处和软骨细胞簇中表达呈强阳性,提示间盘细胞可能与浸润的免疫细胞一起在间盘突出后诱发的炎症过程中发挥着一定的作用。第二部分体外培养人髓核细胞炎性相关因子分泌特性的实验研究目的:腰腿疼痛是间盘源性相关疾病的主要症状,炎症作为疼痛的主要诱因,在其中可能发挥着重要的作用。近来研究发现炎症的主要介质炎性因子在病变的间盘中表达升高,支持上述假说,但其来源尚有一定的争论,人间盘细胞是否能分泌炎性因子及其分泌特点尚鲜有研究报道,本部分拟对此进行初步的探究。方法:取退变突出型LDH患者术中摘除的间盘组织,仔细分离培养后获得髓核细胞,采用P2代进行后续实验研究。采用番红O、甲苯胺蓝、瑞士姬姆萨染色及Ⅱ型胶原免疫组化染色等技术对髓核细胞进行鉴定。对髓核细胞进行饥饿处理或脂多糖(LPS)刺激一段时间后(4h、16h、24h、48h),采用RT-PCR法检测炎性相关因子IL-1β、IL-12、IL-17、IL-23、TNF-α、EBI3、IL-37的表达情况,同时采用ELISA方法对髓核细胞培养液中IL-1β、IL-17、TNF-α的蛋白含量进行检测验证。结果:经鉴定所培养细胞为髓核细胞,通过PCR发现基础状态下髓核细胞中不仅有IL-1β、IL-12、IL-17、IL-23、TNF-α等炎性因子的表达,也有抗炎因子EBI3(IL-35)、IL-37的表达,且经饥饿或LPS刺激后其表达量较基础状态下明显增高。采用ELISA方法从蛋白角度对IL-1β、IL-17、TNF-α在细胞培养液的含量进行检测,也呈现出相似的表达特点。结论:人髓核细胞不仅能够表达多种炎性因子,也能够表达抗炎因子。在饥饿或LPS刺激后其表达量升高,且呈一定的时间依赖性。由于这些炎性相关因子在间盘基质合成与分解、血管生成、神经内生长、炎症进展、疼痛致敏等方面发挥着重要的作用,对这些炎性相关因子分泌及作用机制的认识有助于我们了解间盘的生物学特性并为干预或治疗间盘相关疾病提供提供新的思路和理论依据。
[Abstract]:Part 1 the general characteristics of the expression of inflammatory factors in the intervertebral disc tissue of LDH patients: to clarify the pathological characteristics of intervertebral disc tissue in different types of lumbar intervertebral disc herniation (LDH), and to summarize the distribution of inflammatory factors in the intervertebral disc tissue, and explore the source of inflammatory factors in the intervertebral disc and their possible pathophysiology. Methods: 26 patients with LDH in Tianjin Hospital from December 2014 to September 2015 were selected and treated with surgical treatment. According to the symptoms and signs, the results of CT (MRI) and the integrity of the fibrous ring during the operation, the selected cases were divided into two groups: (1) the degeneration of herniated herniation group (group R) (2) degeneration. The protruding intervertebral disc herniation group (group NR). The pathological features of different types of intervertebral disc tissues were observed by HE staining. The expression and distribution of inflammatory related factors such as IL-17, IL-23, NLRP3 and Caspase-1 in the intervertebral disc were detected by immunohistochemistry and Westernblot, and the general characteristics of the inflammatory related factors in the tissue were summarized. Results: the intervertebral disc tissues of the two types of LDH patients showed signs of degeneration, but the pathological features of the injured hernia group were more complex, and the degeneration was more obvious. The main manifestations were: the tissue fissure increased obviously, the fiber arrangement was more disorder, loosely and even broken, and the fibrous necrosis, mucoid degeneration, synovial metaplasia and calcium crystallization were often seen on the edge of the tissue. Deposition, neovascularization, inflammatory cell infiltration, and chondrocyte cluster formation, among which cell proliferation and cartilage cell cluster forming part of disc degeneration are also obvious. IL-17, IL-23, NLRP3, Caspase-1, and other two types of intervertebral disc tissues are observed by immunohistochemistry, and the expression content of the damaged hernia type is higher than that of the degenerative protruding type. All the inflammatory factors were positive in the chondroid nucleus pulposus cells, and in the nucleus pulposus cells around the tissue fissure, the positive expression of the infiltrating inflammatory cells and around the neovascularization was particularly obvious in the chondrocyte clusters. Conclusion: the pathological changes in the intervertebral disc of the 1. types of LDH patients were more obvious, suggesting that the intervertebral disc of the two types of patients were more obvious. There are more mechanisms to participate in the histopathological manifestations of intervertebral tissue injury after hernia. The degree of degeneration is obvious in the tissue of proliferative and chondrocytes, suggesting that the formation of chondrocyte clusters may play a role in the pathological changes of intervertebral disc. The.2. intervertebral disc cells can secrete inflammatory factors, and at the proliferation and cartilage of the intervertebral disc cells. The expression in the cluster is strongly positive, suggesting that the intervertebral disc cells may play a role in the inflammatory process induced by the intervertebral disc herniation with the infiltrating immune cells. Second the experimental study on the secretion characteristics of the inflammatory related factors in human medullary cells in vitro As the main cause of pain, it may play an important role in it. Recent studies have found that inflammatory factors in the main mediators of inflammation are expressed in the disks, which support the hypothesis, but there is still a debate on whether the source of human disc cells can secrete inflammatory factors and their secretions. Methods: the intervertebral disc tissue extirpated in the LDH patients with degenerative protrusion was taken, the nucleus pulposus cells were obtained after careful separation and culture, and the P2 generation was used for subsequent experimental study. The nucleus pulposus cells were identified by using red O, toluidine blue, Swiss Giemsa staining and type II collagen immunohistochemical staining. After a period of starvation or LPS stimulation (4h, 16h, 24h, 48h), the expression of inflammatory related factors, IL-1 beta, IL-12, IL-17, IL-23, TNF- alpha, EBI3, was detected by RT-PCR method. The cultured cells were nucleus pulposus cells. Through PCR, the expression of IL-1 beta, IL-12, IL-17, IL-23, TNF- alpha and other inflammatory factors, such as EBI3 (IL-35) and IL-37, were also expressed in the basal state of the nucleus pulposus, and the expression of the nucleus was significantly higher than that in the basic state after starvation or LPS stimulation. 7, TNF- alpha was detected in cell culture, and also showed a similar expression. Conclusion: human medullary nucleus cells can not only express a variety of inflammatory factors, but also express anti-inflammatory factors. The expression of the cells is increased after starvation or LPS stimulation and is dependent on a certain time. Decomposition, angiogenesis, internal nerve growth, inflammatory progression, and pain sensitization play an important role. The understanding of the secretions and mechanisms of these inflammatory factors will help us to understand the biological characteristics of the intervertebral disc and provide new ideas and theoretical basis for the intervention or treatment of intervertebral disc related diseases.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R681.5

【参考文献】