【作者】 王涛；

【导师】 周初松；

【作者基本信息】 南方医科大学 ， 外科学（骨外科）（专业学位）， 2023， 硕士

【摘要】 研究背景强直性脊柱炎(ankylosing spondylitis,AS)是一种持续发展的慢性炎症性疾病,其典型的临床表现包括骶髂关节炎、脊柱僵硬并后凸畸形及髋膝关节屈曲挛缩等,严重影响到脊柱的稳定性和功能。发病多在15～40岁人群当中,是青壮年致畸、致残的重要原因之一。然而,该病发病机制至今尚未研究清楚。大量研究表明:强直性脊柱炎患者体内持续慢性炎症状态引起机体免疫系统紊乱。如何调节强直性脊柱炎进展过程中异常的炎症反应,是针对强直性脊柱炎治疗相关研究的关键科学问题。本课题组前期研究发现:AS患者体内单核-巨噬细胞相比正常人具有更高水平的焦亡触发级联反应释放大量促炎因子,引起机体出现持续的慢性炎症反应。如何降低单核-巨噬细胞焦亡的发生、减少促炎因子释放、缓解炎症状态,将为AS治疗提供新方向。有研究报道:雷帕霉素可以激活细胞的自我保护机制,在维持细胞稳定、机体生长发育以及疾病发生等生命活动中发挥积极作用。由此我们假设雷帕霉素可以缓解巨噬细胞焦亡来减轻炎症反应。研究目的构建稳定的巨噬细胞焦亡模型,探究雷帕霉素缓解巨噬细胞焦亡的机制。研究方法通过脂多糖(LPS)+尼日利亚菌素(Nigericin,Nig)处理巨噬细胞建立焦亡模型,以Western blot检测cleaved Casp-1和GSDMD-N表达水平,用ELISA检测TNF-α、IL-1β分泌水平,通过检测LDH释放量及荧光染色反映conrol组和LPS+Nig组之间焦亡变化情况,据此选择理想建模条件;设置使用雷帕霉素预处理的梯度浓度,以此选择最佳药物处理条件;以前述相同方法检测control组、LPS+Nig组及RAPA+LPS+Nig组间焦亡相关指标;通过转录组测序表征巨噬细胞表达谱,获得阴性对照组(C组)、阳性对照组(T)和雷帕霉素组(R)的差异表达特征,通过GO功能富集分析和KEGG通路富集分析差异表达mRNA富集情况,筛选目的基因并通过qRT-PCR进行验证。根据上述筛出的因子CCL2,使用外源性CCL2干预验证其在本研究中的作用,按照上述相同方法检测 control 组、LPS+Nig 组、RAPA+LPS+Nig 组和 RAPA+CCL2+LPS+Nig组焦亡变化情况。研究结果经LPS+Nig处理巨噬细胞后,可以观察到显著的巨噬细胞焦亡。进一步摸索合适浓度及时间梯度,确定以500nM LPS处理6小时后,以10nM Nig刺激2小时的方法来建立稳定的巨噬细胞焦亡模型。在诱导巨噬细胞焦亡模型前,我们选取不同浓度梯度雷帕霉素处理细胞,结果表明:常规时间一定浓度范围内的雷帕霉素刺激对巨噬细胞死亡率没有明显影响,经雷帕霉素预处理的巨噬细胞更少发生焦亡。转录组测序结果显示CCL2在阴性对照组(C组)、阳性对照组(T)和雷帕霉素组(R)间具有显著差异(P<0.05),且qRT-PCR结果以及蛋白表达水平与转录组测序结果基本一致,表明雷帕霉素可能通过调控CCL2表达来影响巨噬细胞焦亡的发生。当外源性CCL2加入雷帕霉素组后,很大程度上消减了雷帕霉素对巨噬细胞焦亡的缓解作用。结论雷帕霉素可以通过抑制CCL2分泌减少巨噬细胞焦亡。更多还原

【Abstract】 BackgroundAnkylosing spondylitis(AS)is a chronic inflammatory disease with persistent development.Its typical clinical manifestations include sacroilioarthritis,spinal stiffness with kyphosis and flexion contracture of the hip and knee joint,which seriously affect the stability and function of the spine.It is one of the important causes of teratogenesis and disability in young adults that the incidence is mostly among people aged 15～40.However,the pathogenesis of this disease has not been studied clearly.So far,a large number of studies have shown that the persistent chronic inflammatory state in patients with ankylosing spondylitis causes the disorder of the immune system.How to regulate the abnormal inflammatory reaction during the progression of ankylosing spondylitis is a key scientific issue for the research on the treatment of ankylosing spondylitis.Our previous study found that monocyte-macrophages in AS patients had a higher level of pyroptosis-triggered cascade reaction than that in normal ones,which released a large number of pro-inflammatory factors and caused persistent chronic inflammatory reaction.How to reduce the occurrence of pyroptosis in monocyte-macrophages and the release of pro-inflammatory factors and relieve inflammatory state will provide a new direction for AS treatment.It has been reported that rapamycin can activate the self-protection mechanism of cells and play an active role in maintaining stable cell growth and evolution,disease occurrence and other life activities.Therefore,we hypothesized that rapamycin can alleviate the inflammatory reaction by alleviating the pyroptosis of macrophages.ObjectTo establish a stable model of pyroptosis in macrophage,then explore the mechanism of rapamycin relieving pyroptosis of macrophages.MethodsAfter build the model of pyroptosis with LPS+Nigericin treatment,Using Western blot to detect the expressions of cleaved Casp-1 and GSDMD-N,using ELISA to detect the secretions of TNF-α and IL-1β,and different conditions of pyroptosis between control group and LPS+Nig group were detected by LDH release and fluorescent staining,thereunder,the ideal modeling conditions were selected;The gradient concentration in normal time of pretreatment with rapamycin were set in order to select the optimal conditions for drug treatment;Pyroptosis related indexes among the control,LPS+nig and Rapa+LPS+nig groups were detected by the same method as described previously;Using transcriptome sequencing technology to characterize the expression profiles and find out the differential expression characteristics from different treatment groups.Screen the target genes through GO function enrichment analysis and KEGG pathway enrichment analysis and verify them by qRT-PCR.According to the screened factor above,finally,we use exogenous CCL2 to verify its role in this study.The changes of pyroptosis between control group,LPS+Nig group,RAPA+LPS+Nig group and RAPA+CCL2+LPS+Nig group were detected according to the same method mentioned above.ResultsAfter LPS+Nigericin treatment,the pyroptosis in macrophages can be observed significantly.In the further study,we selected 10nM Nigericin to stimulate for 2 hours to establish the pyroptosis model of macrophages via concentration and time gradient.Before inducing the model,we selected different concentration and time gradient of rapamycin to treat the cells.The results showed that within a certain range,the concentration and time of rapamycin stimulation had no obvious effect on the mortality of macrophages.The macrophages pretreated with rapamycin were less likely to die of pyroptosis;The results of transcriptome sequencing showed that CCL2 was significantly different(P<0.05)between negative control group(C group),positive control group(T)and rapamycin group(R),and the results of qRT-PCR and protein expression level were consistent with those of transcriptome sequencing,which indicated that rapamycin might affect the occurrence of pyroptosis by regulating CCL2 expression.When exogenous CCL2 was added to rapamycin group,the relieving effect of rapamycin on focal death of macrophages was greatly reduced.ConclusionOur research shows that rapamycin can reduce pyroptosis of macrophages by inhibiting CCL2 secretion.更多还原