LRP1在免疫性血小板減少癥患者中的表達(dá)及機(jī)制初探
發(fā)布時(shí)間：2019-04-26 00:19
【摘要】：研究目的:明確LRP1(Low-density lipoprotein receptor-related protein-1,LRP1)在原發(fā)性免疫性血小板減少癥(Primary immune thrombocytopenia,ITP)中的表達(dá)情況。初步探索LRP1在ITP中的作用機(jī)制,并觀察地塞米松(dexamethasone,DXM)對(duì)ITP患者LRP1表達(dá)情況的影響。研究方法:采集105例ITP患者及30例健康成人外周枸櫞酸鈉抗凝6ml,利用密度梯度離心的方法獲得PBMC(Peripheral Blood Mononuclear Cell),經(jīng)過總RNA提取后反轉(zhuǎn)錄制備cDNA,以GAPDH作為管家基因,采用實(shí)時(shí)熒光定量PCR(Taqman探針法)檢測(cè)LRP1 mRNA的相對(duì)表達(dá)水平。比較正常對(duì)照組及ITP患者外周血PBMC中LRP1 mRNA相對(duì)表達(dá)水平的差異性,探索LRP1與ITP發(fā)生發(fā)展的可能相關(guān)性。利用生物信息學(xué)(Immusort數(shù)據(jù)庫)分析外周血細(xì)胞中LRP1的表達(dá)情況,采用流式細(xì)胞術(shù)檢測(cè)外周血PBMC中單核細(xì)胞、淋巴細(xì)胞以及中性粒細(xì)胞膜上和膜內(nèi)LRP1蛋白的表達(dá)情況。分析外周血細(xì)胞中LRP1的表達(dá)情況。接下來,利用流式細(xì)胞檢測(cè)外周血Th17及Treg細(xì)胞比例,同時(shí)使用實(shí)時(shí)熒光定量PCR(SYBR Green法)檢測(cè)ITP患者和正常對(duì)照組外周血Th17和Treg細(xì)胞分泌的相關(guān)細(xì)胞因子mRNA的相對(duì)表達(dá)情況。在體外將ITP患者PBMC與si LRP1共孵育12h、24h后,采用實(shí)時(shí)熒光定量PCR(SYBR Green法)檢測(cè)Th17和Treg細(xì)胞分泌的相關(guān)細(xì)胞因子mRNA表達(dá)水平。采集ITP患者新鮮外周血,磁珠法分離單核細(xì)胞,體外加入siLRP1孵育24h后,利用熒光定量PCR檢測(cè)IKK-NF-?B通路相關(guān)分子mRNA的相對(duì)表達(dá)水平,并應(yīng)用流式細(xì)胞術(shù)檢測(cè)單核細(xì)胞NF-?B的磷酸化水平。分析LRP1對(duì)Th17/Treg細(xì)胞功能的影響,探索LRP1在ITP發(fā)病機(jī)制中的可能調(diào)控作用。最后,對(duì)20例接受DXM治療的新診斷和慢性ITP患者進(jìn)行隨訪研究3-6個(gè)月,觀察其療效及LRP1mRNA表達(dá)情況的變化,分離14例病情緩解患者的治療前外周血PBMC,使用不同濃度激素處理24h后,利用熒光定量PCR檢測(cè)PBMC中LRP1 mRNA表達(dá)的情況,觀察激素對(duì)LRP1表達(dá)水平的影響。結(jié)果:與正常對(duì)照組相比,ITP患者PBMC中LRP1 mRNA表達(dá)量顯著降低,兩組差異具有統(tǒng)計(jì)學(xué)意義。LRP1蛋白主要表達(dá)于單核細(xì)胞上,并且膜內(nèi)與膜上均有表達(dá),與正常對(duì)照組相比,ITP患者單核細(xì)胞膜上和膜內(nèi)的LRP1表達(dá)均顯著下降。ITP患者外周血Th17細(xì)胞比例增高,且Th17細(xì)胞相關(guān)細(xì)胞因子的表達(dá)也顯著增高,而Treg細(xì)胞比例下降,且Treg細(xì)胞相關(guān)細(xì)胞因子的表達(dá)也顯著降低。si LRP1處理ITP患者PBMC后,與培養(yǎng)基刺激組相比,Th17細(xì)胞相關(guān)細(xì)胞因子表達(dá)上升,而Treg細(xì)胞相關(guān)細(xì)胞因子表達(dá)下降。siLRP1刺激ITP患者單核細(xì)胞后,與正常對(duì)照組相比,IKK、NF-?B mRNA的相對(duì)表達(dá)水平上升,且NF-?B的磷酸化水平升高。經(jīng)DXM治療病情緩解的14例患者LRP1 mRNA表達(dá)水平較治療前上升,差異具有統(tǒng)計(jì)學(xué)意義。體外激素刺激ITP緩解患者的治療前PBMC,隨著刺激激素濃度的增加,LRP1 mRNA的表達(dá)水平升高,呈現(xiàn)劑量依賴性。結(jié)論:ITP患者中LRP1 mRNA表達(dá)水平明顯下降,LRP1參與了ITP的發(fā)病機(jī)制。LRP1主要表達(dá)于單核細(xì)胞,可能通過負(fù)向調(diào)控單核細(xì)胞的IKK-NF-?B信號(hào)通路,進(jìn)而對(duì)Th17/Treg細(xì)胞功能產(chǎn)生影響,參與了ITP的發(fā)展。DXM治療可以正調(diào)控LRP1的表達(dá),且這種作用呈現(xiàn)劑量依賴性。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R558.2
本文編號(hào)：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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R558.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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,顧靜,陸璐,陳秀芳;再障和特發(fā)性血小板減少性紫癜患者血漿血小板生成素含量研究[J];南通醫(yī)學(xué)院學(xué)報(bào);2003年04期
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