LRP1在免疫性血小板减少症患者中的表达及机制初探
发布时间：2019-04-26 00:19
【摘要】：研究目的:明确LRP1(Low-density lipoprotein receptor-related protein-1,LRP1)在原发性免疫性血小板减少症(Primary immune thrombocytopenia,ITP)中的表达情况。初步探索LRP1在ITP中的作用机制,并观察地塞米松(dexamethasone,DXM)对ITP患者LRP1表达情况的影响。研究方法:采集105例ITP患者及30例健康成人外周枸橼酸钠抗凝6ml,利用密度梯度离心的方法获得PBMC(Peripheral Blood Mononuclear Cell),经过总RNA提取后反转录制备cDNA,以GAPDH作为管家基因,采用实时荧光定量PCR(Taqman探针法)检测LRP1 mRNA的相对表达水平。比较正常对照组及ITP患者外周血PBMC中LRP1 mRNA相对表达水平的差异性,探索LRP1与ITP发生发展的可能相关性。利用生物信息学(Immusort数据库)分析外周血细胞中LRP1的表达情况,采用流式细胞术检测外周血PBMC中单核细胞、淋巴细胞以及中性粒细胞膜上和膜内LRP1蛋白的表达情况。分析外周血细胞中LRP1的表达情况。接下来,利用流式细胞检测外周血Th17及Treg细胞比例,同时使用实时荧光定量PCR(SYBR Green法)检测ITP患者和正常对照组外周血Th17和Treg细胞分泌的相关细胞因子mRNA的相对表达情况。在体外将ITP患者PBMC与si LRP1共孵育12h、24h后,采用实时荧光定量PCR(SYBR Green法)检测Th17和Treg细胞分泌的相关细胞因子mRNA表达水平。采集ITP患者新鲜外周血,磁珠法分离单核细胞,体外加入siLRP1孵育24h后,利用荧光定量PCR检测IKK-NF-?B通路相关分子mRNA的相对表达水平,并应用流式细胞术检测单核细胞NF-?B的磷酸化水平。分析LRP1对Th17/Treg细胞功能的影响,探索LRP1在ITP发病机制中的可能调控作用。最后,对20例接受DXM治疗的新诊断和慢性ITP患者进行随访研究3-6个月,观察其疗效及LRP1mRNA表达情况的变化,分离14例病情缓解患者的治疗前外周血PBMC,使用不同浓度激素处理24h后,利用荧光定量PCR检测PBMC中LRP1 mRNA表达的情况,观察激素对LRP1表达水平的影响。结果:与正常对照组相比,ITP患者PBMC中LRP1 mRNA表达量显著降低,两组差异具有统计学意义。LRP1蛋白主要表达于单核细胞上,并且膜内与膜上均有表达,与正常对照组相比,ITP患者单核细胞膜上和膜内的LRP1表达均显著下降。ITP患者外周血Th17细胞比例增高,且Th17细胞相关细胞因子的表达也显著增高,而Treg细胞比例下降,且Treg细胞相关细胞因子的表达也显著降低。si LRP1处理ITP患者PBMC后,与培养基刺激组相比,Th17细胞相关细胞因子表达上升,而Treg细胞相关细胞因子表达下降。siLRP1刺激ITP患者单核细胞后,与正常对照组相比,IKK、NF-?B mRNA的相对表达水平上升,且NF-?B的磷酸化水平升高。经DXM治疗病情缓解的14例患者LRP1 mRNA表达水平较治疗前上升,差异具有统计学意义。体外激素刺激ITP缓解患者的治疗前PBMC,随着刺激激素浓度的增加,LRP1 mRNA的表达水平升高,呈现剂量依赖性。结论:ITP患者中LRP1 mRNA表达水平明显下降,LRP1参与了ITP的发病机制。LRP1主要表达于单核细胞,可能通过负向调控单核细胞的IKK-NF-?B信号通路,进而对Th17/Treg细胞功能产生影响,参与了ITP的发展。DXM治疗可以正调控LRP1的表达,且这种作用呈现剂量依赖性。
[Abstract]:Objective: To study the expression of LRP1 (Low-density lipotein receptor-related protein-1, LRP1) in primary immune thrombocytopenic purpura (ITP). The mechanism of LRP1 in ITP was first explored and the effect of dexamethasone (DXM) on the expression of LRP1 in ITP was observed. Methods: A total of 105 ITP patients and 30 healthy adult peripheral blood donors were collected for 6ml, and the peripheral blood Mononomar Cell was obtained by density gradient centrifugation. The cDNA was prepared by reverse transcription after total RNA extraction, and GAPDH was used as the housekeeping gene. The relative expression level of LRP1 mRNA was detected by real-time fluorescence quantitative PCR (Taqman probe method). To compare the difference of the relative expression level of LRP1 mRNA in peripheral blood mononuclear cells (PBMC) of the normal control group and the ITP patients, the possible correlation between the development of LRP1 and ITP was explored. The expression of LRP1 in peripheral blood cells was analyzed by using bioinformatics (Immucor database), and the expression of LP1 in peripheral blood mononuclear cells (PBMC), lymphocytes and neutrophils was detected by flow cytometry. The expression of LRP1 in peripheral blood cells was analyzed. Next, the relative expression of the related cytokine mRNA in the peripheral blood Th17 and Treg cells in the ITP patients and the normal control group was detected by flow cytometry using the real-time fluorescence quantitative PCR (SYBR Green method). In vitro, PBMCs of ITP patients were incubated with si LRP1 for 12 h and 24 h, and the expression level of relevant cytokines in Th17 and Treg cells was detected by real-time fluorescence quantitative PCR (SYBR Green method). The relative expression levels of the related molecular mRNA of IKK-NF-? B pathway were detected by fluorescence quantitative PCR after 24 h incubation with siLRP1, and the phosphorylation of NF-? B was detected by flow cytometry. The effect of LRP1 on the function of Th17/ Treg cells was analyzed and the possible regulatory effects of LRP1 in the pathogenesis of ITP were explored. Finally,20 patients with newly diagnosed and chronic ITP treated with DXM were followed up for 3-6 months to observe the changes of the therapeutic effect and the expression of LRP1mRNA. The expression of LRP1 mRNA in PBMC was detected by fluorescence quantitative PCR, and the effect of the hormone on the expression level of LRP1 was observed. Results: Compared with the normal control group, the expression of LP1 mRNA in PBMCs of ITP patients decreased significantly, and the difference between the two groups was of statistical significance. The LP1 protein was mainly expressed on the monocytes, and the expression of LRP1 in the membrane and the membrane was significantly decreased in ITP patients compared with the normal control group. The proportion of Th17 cells in peripheral blood of ITP patients was increased, and the expression of Th17 cell-related cytokines was also significantly increased, and the ratio of Treg cells decreased, and the expression of the related cytokines in Treg cells was also significantly reduced. After the treatment of PBMCs in ITP patients with si LRP1, the expression of the associated cytokines in Th17 cells increased compared to the medium stimulation group, while the expression of the relevant cytokines in Treg cells decreased. The relative expression level of IKK, NF--? B mRNA was increased compared with the normal control group, and the phosphorylation level of NF-? B was increased after siLRP1 stimulated the ITP patient's monocytes. The expression of LRP1 mRNA in 14 patients with remission by DXM was higher than that before treatment, and the difference was of statistical significance. In vitro, the expression level of LRP1 mRNA was increased and the dose-dependent was present with the increase of the concentration of the stimulating hormone. Conclusion: The expression level of LRP1 mRNA in ITP patients decreased significantly, and LRP1 was involved in the pathogenesis of ITP. LRP1 is mainly expressed in the monocytes, which can regulate the IKK-NF-B signaling pathway of the monocytes in the negative direction, and further influence the function of Th17/ Treg cells, and is involved in the development of ITP. DXM therapy may be regulating the expression of LRP1 and this effect presents a dose-dependent manner.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R558.2
本文编号：2465586
[Abstract]:Objective: To study the expression of LRP1 (Low-density lipotein receptor-related protein-1, LRP1) in primary immune thrombocytopenic purpura (ITP). The mechanism of LRP1 in ITP was first explored and the effect of dexamethasone (DXM) on the expression of LRP1 in ITP was observed. Methods: A total of 105 ITP patients and 30 healthy adult peripheral blood donors were collected for 6ml, and the peripheral blood Mononomar Cell was obtained by density gradient centrifugation. The cDNA was prepared by reverse transcription after total RNA extraction, and GAPDH was used as the housekeeping gene. The relative expression level of LRP1 mRNA was detected by real-time fluorescence quantitative PCR (Taqman probe method). To compare the difference of the relative expression level of LRP1 mRNA in peripheral blood mononuclear cells (PBMC) of the normal control group and the ITP patients, the possible correlation between the development of LRP1 and ITP was explored. The expression of LRP1 in peripheral blood cells was analyzed by using bioinformatics (Immucor database), and the expression of LP1 in peripheral blood mononuclear cells (PBMC), lymphocytes and neutrophils was detected by flow cytometry. The expression of LRP1 in peripheral blood cells was analyzed. Next, the relative expression of the related cytokine mRNA in the peripheral blood Th17 and Treg cells in the ITP patients and the normal control group was detected by flow cytometry using the real-time fluorescence quantitative PCR (SYBR Green method). In vitro, PBMCs of ITP patients were incubated with si LRP1 for 12 h and 24 h, and the expression level of relevant cytokines in Th17 and Treg cells was detected by real-time fluorescence quantitative PCR (SYBR Green method). The relative expression levels of the related molecular mRNA of IKK-NF-? B pathway were detected by fluorescence quantitative PCR after 24 h incubation with siLRP1, and the phosphorylation of NF-? B was detected by flow cytometry. The effect of LRP1 on the function of Th17/ Treg cells was analyzed and the possible regulatory effects of LRP1 in the pathogenesis of ITP were explored. Finally,20 patients with newly diagnosed and chronic ITP treated with DXM were followed up for 3-6 months to observe the changes of the therapeutic effect and the expression of LRP1mRNA. The expression of LRP1 mRNA in PBMC was detected by fluorescence quantitative PCR, and the effect of the hormone on the expression level of LRP1 was observed. Results: Compared with the normal control group, the expression of LP1 mRNA in PBMCs of ITP patients decreased significantly, and the difference between the two groups was of statistical significance. The LP1 protein was mainly expressed on the monocytes, and the expression of LRP1 in the membrane and the membrane was significantly decreased in ITP patients compared with the normal control group. The proportion of Th17 cells in peripheral blood of ITP patients was increased, and the expression of Th17 cell-related cytokines was also significantly increased, and the ratio of Treg cells decreased, and the expression of the related cytokines in Treg cells was also significantly reduced. After the treatment of PBMCs in ITP patients with si LRP1, the expression of the associated cytokines in Th17 cells increased compared to the medium stimulation group, while the expression of the relevant cytokines in Treg cells decreased. The relative expression level of IKK, NF--? B mRNA was increased compared with the normal control group, and the phosphorylation level of NF-? B was increased after siLRP1 stimulated the ITP patient's monocytes. The expression of LRP1 mRNA in 14 patients with remission by DXM was higher than that before treatment, and the difference was of statistical significance. In vitro, the expression level of LRP1 mRNA was increased and the dose-dependent was present with the increase of the concentration of the stimulating hormone. Conclusion: The expression level of LRP1 mRNA in ITP patients decreased significantly, and LRP1 was involved in the pathogenesis of ITP. LRP1 is mainly expressed in the monocytes, which can regulate the IKK-NF-B signaling pathway of the monocytes in the negative direction, and further influence the function of Th17/ Treg cells, and is involved in the development of ITP. DXM therapy may be regulating the expression of LRP1 and this effect presents a dose-dependent manner.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R558.2
【参考文献】
相关期刊论文 前2条
1 Dong Zheng;Chen-Song Huang;Shao-Bin Huang;Chao-Xu Zheng;;Laparoscopic splenectomy for primary immune thrombocytopenia:Current status and challenges[J];World Journal of Gastrointestinal Endoscopy;2016年17期
2 徐瑞容,顾静,陆璐,陈秀芳;再障和特发性血小板减少性紫癜患者血浆血小板生成素含量研究[J];南通医学院学报;2003年04期
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