磷脂類(lèi)代謝產(chǎn)物S1P、LPA對(duì)于血小板功能的影響
發(fā)布時(shí)間：2018-09-17 08:23
【摘要】：血小板在執(zhí)行生理性止血作用的同時(shí),在心血管疾病、炎癥、腫瘤形成、肺組織纖維化等許多重要的病理進(jìn)程中扮演非常重要的角色。目前在臨床上,輸注血小板是預(yù)防和緩解血小板減少癥或者功能障礙的重要治療措施。但部分患者反復(fù)輸注血小板后會(huì)出現(xiàn)血小板輸注無(wú)效。如何避免血小板輸注無(wú)效的發(fā)生和血小板的浪費(fèi),提高血小板治療效果,是目前輸血醫(yī)學(xué)界面臨的一個(gè)重要的課題。為了研究SIP、LPA與血小板儲(chǔ)存損傷之間的聯(lián)系,本文檢測(cè)并分析單采血小板震蕩保存過(guò)程中發(fā)生的一系列形態(tài)和功能的變化以及1-磷酸鞘氨醇(S1P)和溶血磷脂酸(LPA)的釋放規(guī)律,意在為臨床血小板輸注無(wú)效的預(yù)防和治療提供新的思路和方法。我們發(fā)現(xiàn),隨著血小板保存時(shí)間的延長(zhǎng),血漿中S1P的含量逐漸增加,通過(guò)直接添加S1P維持血漿中一定濃度S1P后,發(fā)現(xiàn)血小板儲(chǔ)存損傷相關(guān)指標(biāo)優(yōu)于對(duì)照組：活化率和凋亡率的增加明顯受到抑制(P0.05),血小板凋亡率從15%降到5%、活化率從55%下降到43%。聚集率從5%增加到23%,低滲休克反應(yīng)從11%增加到25%、形變能力從3%增加到7%),而體外添加LPA維持血漿中LPA一定濃度,對(duì)于這些指標(biāo)影響并不顯著(第5天時(shí),P0.05)。體外直接添加二甲基鞘氨醇(DMS)有效地抑制鞘氨醇激酶活性,減少了S1P的生成,此時(shí)血小板儲(chǔ)存損傷顯著增加：第5天時(shí),保存期內(nèi)血小板凋亡率從9%增加到14%、活化率從43%增加到60%,活化率和凋亡率有顯著增加(P0.05),聚集率從28%下降到5%,低滲休克反應(yīng)從30%下降到18%、形變能力從8%下降到2%；此時(shí)添加S1P恢復(fù)血漿中的S1P的含量后,活化率和凋亡率的增加明顯受到抑制(P0.05),保存期第5天血小板凋亡率從14%降到10%、活化率從51%下降到42%,,聚集率從8%增加到18%,低滲休克反應(yīng)從18%增加到24%、形變能力從2%增加到6%,降低趨勢(shì)也明顯受到抑制(P0.05)。以上結(jié)果表明,在保存期間血小板S1P含量逐漸增加,這可能是因?yàn)檠“逶隗w外儲(chǔ)存期間由于血小板凋亡破裂釋放出活性分子導(dǎo)致更多的血小板活化,進(jìn)而導(dǎo)致血漿中S1P含量增加。體外添加S1P后,血小板的凋亡途徑受到抑制(如抑制Caspase-3活性),細(xì)胞破裂減少,降低了活性分子的釋放,有效地抑制了血小板儲(chǔ)存損傷的發(fā)生,具體的機(jī)制需要進(jìn)一步研究。另外,在這篇文章的附錄1和附錄2中,我們還探討了漢族、維吾爾族CD61基因外顯子10多態(tài)性分析和上海人群中CD36缺失頻率和相關(guān)分子機(jī)制的研究工作。我們?cè)诶肂loodchip對(duì)中國(guó)漢族人群血型基因分型的篩查中,發(fā)現(xiàn)有些個(gè)體的]HPA-6bw無(wú)法判斷分型。由于Bloodchip針對(duì)HPA-6bw基因分型的探針結(jié)合部位位于HPA-6bw型突變位點(diǎn)附近,推測(cè)無(wú)法判斷分型的這些個(gè)體在HPA-6bw型突變位點(diǎn)附近可能存在未知的突變,影響了引物或者探針與模板的結(jié)合。為了研究編碼血小板膜糖蛋白CD61基因的多態(tài)性,探索HPA-6bw血型系統(tǒng)基因檢測(cè)合理引物和探針設(shè)計(jì),本文在第二章節(jié)中分析了CD61基因外顯子10的突變以及這些突變?cè)跐h族、維吾爾族人群中的分布情況,我們隨機(jī)采集149人份漢族和96人份維吾爾族的血液樣本,擴(kuò)增這些樣本CD61基因外顯子10,然后直接測(cè)序分析外顯子10編碼區(qū)基因序列。漢族和維族人群的CD61基因外顯子10除決定HPA-6bw多態(tài)性的SNP(單核苷酸多態(tài)性位點(diǎn))外,漢族和維族人群的CD61基因外顯子10存在3個(gè)SNP(1533AG、1545GA、1529CT),且在漢族和維族人群分布頻率差異甚小,這顯示兩個(gè)民族之間血緣上的關(guān)系比較親近。血小板膜糖蛋白CD36發(fā)生缺失則可產(chǎn)生抗-CD36抗體,導(dǎo)致血小板減少、輸血后紫癜和血小板輸注無(wú)效等輸血不良反應(yīng)。在第三章節(jié)我們篩選1022名健康獻(xiàn)血者,分析血小板和單核細(xì)胞上CD36表達(dá),明確CD36 Ⅰ型缺失和Ⅱ型缺失類(lèi)型,統(tǒng)計(jì)分析等位基因頻率,獲得獻(xiàn)血人群CD36基因突變主要類(lèi)型及分布頻率,發(fā)現(xiàn)新基因突變類(lèi)型,比較中國(guó)與其他報(bào)道人種中CD36分子背景差異。我們擴(kuò)增CD36基因外顯子3-14和兩側(cè)相關(guān)部分內(nèi)含子,直接測(cè)序比對(duì)基因序列有差異的樣本,利用克隆測(cè)序進(jìn)行證實(shí)并確認(rèn)了CD36基因的一些新的突變。結(jié)果顯示,在本研究中CD36 Ⅰ型缺失和Ⅱ型缺失的頻率分別為0.2%和2.0%。中國(guó)上海人群CD36缺失頻率高于歐美國(guó)家,略低于亞洲其他國(guó)家。我們的研究結(jié)果顯示,有13個(gè)突變類(lèi)型,其中8個(gè)突變已有報(bào)道,另有6種突變未見(jiàn)報(bào)道,它們分別位于3、12、13、14號(hào)外顯子,引起讀碼框移位或者氨基酸突變,最終導(dǎo)致表達(dá)蛋白質(zhì)結(jié)構(gòu)的變化。5號(hào)外顯子中的突變371CT與CD36 Ⅰ型缺失相關(guān),其余的突變與CD36 Ⅱ型缺失相關(guān)。大多數(shù)突變存在于CD36基因的外顯子中,并引起相應(yīng)的氨基酸變換(除了同義突變1008GT)。另外,位于14號(hào)外顯子中的1344insTCTT引起448位氨基酸處的閱讀框移位,導(dǎo)致終止子的缺失。然而,基因水平上的研究還不足以揭示蛋白質(zhì)水平上的分子行為,為探索CD36缺失的分子機(jī)制還需要更多的研究。
[Abstract]:Platelet plays an important role in many important pathological processes, such as cardiovascular disease, inflammation, tumor formation, pulmonary fibrosis and so on. At present, platelet transfusion is an important therapeutic measure to prevent and alleviate thrombocytopenia or dysfunction. Invalid platelet transfusion occurs after platelet transfusion. How to avoid ineffective platelet transfusion and platelet waste and improve the therapeutic effect of platelet transfusion is an important issue facing the blood transfusion medical interface. A series of morphological and functional changes and the release of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) during platelet preservation provide new ideas and methods for the prevention and treatment of platelet transfusion failure. After adding S1P to maintain a certain concentration of S1P in plasma, it was found that platelet storage injury related indicators were superior to the control group: the increase of activation rate and apoptosis rate was significantly inhibited (P 0.05), the platelet apoptosis rate decreased from 15% to 5%, the activation rate from 55% to 43%, the aggregation rate increased from 5% to 23%, the hypotonic shock reaction from 11% to 25%, and the deformability from 15% to 5%, and the activation rate from 55% to 43%. 3% increased to 7%, while LPA supplementation in vitro maintained a certain concentration of LPA in plasma, which had no significant effect on these indicators (P 0.05 on the 5th day). Dimethylsphingosine (DMS) supplementation in vitro effectively inhibited sphingosine kinase activity and decreased S1P production, at which time platelet storage injury increased significantly: on the 5th day, platelet apoptosis during storage period. The activation rate increased from 9% to 14%, the activation rate increased from 43% to 60%, the activation rate and the apoptosis rate increased significantly (P 0.05), the aggregation rate decreased from 28% to 5%, the hypoosmotic shock reaction decreased from 30% to 18%, and the deformability decreased from 8% to 2%; at this time, the activation rate and the apoptosis rate increased significantly (P 0.05) after adding S1P to restore the content of S1P in plasma. The platelet apoptosis rate decreased from 14% to 10%, the activation rate decreased from 51% to 42%, the aggregation rate increased from 8% to 18%, the hypoosmotic shock reaction increased from 18% to 24%, the deformability increased from 2% to 6%, and the decreasing trend was also inhibited (P 0.05). In vitro, platelet apoptosis pathway was inhibited (such as inhibiting Caspase-3 activity), cell rupture was reduced, release of active molecules was decreased, and platelet storage was effectively inhibited. Additionally, in Appendix 1 and 2 of this paper, we also discussed the polymorphisms of CD61 gene exon 10 in Han and Uygur populations and the frequency of CD36 deletion in Shanghai population and the related molecular mechanisms. In genotyping screening, it was found that some individuals]HPA-6bw could not be typed. Since the probe binding site of Bloodchip for HPA-6bw genotyping was located near the HPA-6bw mutation site, it was speculated that there might be unknown mutations near the HPA-6bw mutation site in these individuals, affecting the primers or probes. In order to study the polymorphism of CD61 gene encoding platelet membrane glycoprotein and to explore the rational primer and probe design for gene detection of HPA-6bw blood group system, we analyzed the mutation of exon 10 of CD61 gene and the distribution of these mutations in Han and Uygur population in the second section of this paper. The exon 10 of CD61 gene was amplified from the blood samples of Han and 96 Uygurs, and the sequence of the coding region of exon 10 was analyzed by direct sequencing. There were three SNPs (1533AG, 1545GA, 1529CT), and the frequency of distribution in Han and Uygur population was very small, which indicated that the blood relationship between the two nationalities was close. The absence of platelet membrane glycoprotein CD36 could produce anti-CD36 antibody, resulting in thrombocytopenia, post-transfusion purpura and platelet transfusion inefficiency and other adverse blood transfusion reactions. In chapter 1, we screened 1022 healthy blood donors, analyzed the expression of CD36 on platelets and monocytes, identified the types of CD36 type I and type II deletions, analyzed the allele frequencies, obtained the main types and distribution frequencies of CD36 gene mutations in blood donors, found new gene mutation types, and compared CD36 molecules between China and other reported populations. We amplified the exon 3-14 of the CD36 gene and the related introns on both sides, directly sequenced the samples with different gene sequences, and confirmed some new mutations in the CD36 gene by cloning and sequencing. The results showed that the frequencies of deletion of CD36 type I and deletion of CD36 type II were 0.2% and 2.0% respectively. The frequency of CD36 deletion in Shanghai population was higher than that in Europe and America, but slightly lower than that in other Asian countries. Our results showed that there were 13 types of mutations, 8 of which had been reported, and 6 other mutations were not reported. They were located in exon 3, 12, 13 and 14 respectively, causing frame translocation or amino acid mutation, which eventually led to the expression of protein structure. Mutation 371CT in exon 5 was associated with deletion of CD36I, while the rest was associated with deletion of CD36II. Most of the mutations were found in exons of CD36 gene and resulted in corresponding amino acid transformation (except for synonymous mutation 1008GT). In addition, 1344insTCTT in exon 14 caused the reading frame of 448 amino acids. However, studies at the gene level are not enough to reveal the molecular behavior at the protein level, and more research is needed to explore the molecular mechanism of CD36 deletion.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R446.6;R457.1
本文編號(hào)：2245291
[Abstract]:Platelet plays an important role in many important pathological processes, such as cardiovascular disease, inflammation, tumor formation, pulmonary fibrosis and so on. At present, platelet transfusion is an important therapeutic measure to prevent and alleviate thrombocytopenia or dysfunction. Invalid platelet transfusion occurs after platelet transfusion. How to avoid ineffective platelet transfusion and platelet waste and improve the therapeutic effect of platelet transfusion is an important issue facing the blood transfusion medical interface. A series of morphological and functional changes and the release of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) during platelet preservation provide new ideas and methods for the prevention and treatment of platelet transfusion failure. After adding S1P to maintain a certain concentration of S1P in plasma, it was found that platelet storage injury related indicators were superior to the control group: the increase of activation rate and apoptosis rate was significantly inhibited (P 0.05), the platelet apoptosis rate decreased from 15% to 5%, the activation rate from 55% to 43%, the aggregation rate increased from 5% to 23%, the hypotonic shock reaction from 11% to 25%, and the deformability from 15% to 5%, and the activation rate from 55% to 43%. 3% increased to 7%, while LPA supplementation in vitro maintained a certain concentration of LPA in plasma, which had no significant effect on these indicators (P 0.05 on the 5th day). Dimethylsphingosine (DMS) supplementation in vitro effectively inhibited sphingosine kinase activity and decreased S1P production, at which time platelet storage injury increased significantly: on the 5th day, platelet apoptosis during storage period. The activation rate increased from 9% to 14%, the activation rate increased from 43% to 60%, the activation rate and the apoptosis rate increased significantly (P 0.05), the aggregation rate decreased from 28% to 5%, the hypoosmotic shock reaction decreased from 30% to 18%, and the deformability decreased from 8% to 2%; at this time, the activation rate and the apoptosis rate increased significantly (P 0.05) after adding S1P to restore the content of S1P in plasma. The platelet apoptosis rate decreased from 14% to 10%, the activation rate decreased from 51% to 42%, the aggregation rate increased from 8% to 18%, the hypoosmotic shock reaction increased from 18% to 24%, the deformability increased from 2% to 6%, and the decreasing trend was also inhibited (P 0.05). In vitro, platelet apoptosis pathway was inhibited (such as inhibiting Caspase-3 activity), cell rupture was reduced, release of active molecules was decreased, and platelet storage was effectively inhibited. Additionally, in Appendix 1 and 2 of this paper, we also discussed the polymorphisms of CD61 gene exon 10 in Han and Uygur populations and the frequency of CD36 deletion in Shanghai population and the related molecular mechanisms. In genotyping screening, it was found that some individuals]HPA-6bw could not be typed. Since the probe binding site of Bloodchip for HPA-6bw genotyping was located near the HPA-6bw mutation site, it was speculated that there might be unknown mutations near the HPA-6bw mutation site in these individuals, affecting the primers or probes. In order to study the polymorphism of CD61 gene encoding platelet membrane glycoprotein and to explore the rational primer and probe design for gene detection of HPA-6bw blood group system, we analyzed the mutation of exon 10 of CD61 gene and the distribution of these mutations in Han and Uygur population in the second section of this paper. The exon 10 of CD61 gene was amplified from the blood samples of Han and 96 Uygurs, and the sequence of the coding region of exon 10 was analyzed by direct sequencing. There were three SNPs (1533AG, 1545GA, 1529CT), and the frequency of distribution in Han and Uygur population was very small, which indicated that the blood relationship between the two nationalities was close. The absence of platelet membrane glycoprotein CD36 could produce anti-CD36 antibody, resulting in thrombocytopenia, post-transfusion purpura and platelet transfusion inefficiency and other adverse blood transfusion reactions. In chapter 1, we screened 1022 healthy blood donors, analyzed the expression of CD36 on platelets and monocytes, identified the types of CD36 type I and type II deletions, analyzed the allele frequencies, obtained the main types and distribution frequencies of CD36 gene mutations in blood donors, found new gene mutation types, and compared CD36 molecules between China and other reported populations. We amplified the exon 3-14 of the CD36 gene and the related introns on both sides, directly sequenced the samples with different gene sequences, and confirmed some new mutations in the CD36 gene by cloning and sequencing. The results showed that the frequencies of deletion of CD36 type I and deletion of CD36 type II were 0.2% and 2.0% respectively. The frequency of CD36 deletion in Shanghai population was higher than that in Europe and America, but slightly lower than that in other Asian countries. Our results showed that there were 13 types of mutations, 8 of which had been reported, and 6 other mutations were not reported. They were located in exon 3, 12, 13 and 14 respectively, causing frame translocation or amino acid mutation, which eventually led to the expression of protein structure. Mutation 371CT in exon 5 was associated with deletion of CD36I, while the rest was associated with deletion of CD36II. Most of the mutations were found in exons of CD36 gene and resulted in corresponding amino acid transformation (except for synonymous mutation 1008GT). In addition, 1344insTCTT in exon 14 caused the reading frame of 448 amino acids. However, studies at the gene level are not enough to reveal the molecular behavior at the protein level, and more research is needed to explore the molecular mechanism of CD36 deletion.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R446.6;R457.1
【相似文獻(xiàn)】
相關(guān)期刊論文 前10條
1 李濟(jì)生,趙冉;血小板輸注無(wú)效的原因及預(yù)防[J];實(shí)用醫(yī)技雜志;2004年05期
2 孫曉明,牛愛(ài)軍,尹秋霞,薛煉,胡成進(jìn);血小板相關(guān)抗體與血小板輸注無(wú)效關(guān)系探討[J];中國(guó)輸血雜志;2005年03期
3 劉曉麗;王愛(ài)東;石小寧;;血小板輸注無(wú)效的調(diào)查分析[J];河北北方學(xué)院學(xué)報(bào)(醫(yī)學(xué)版);2007年04期
4 邵媛;葉欣;徐秀章;鄧晶;王嘉勵(lì);陳揚(yáng)凱;丁浩強(qiáng);;92例血小板輸注無(wú)效的原因探討[J];中國(guó)輸血雜志;2011年08期
5 許潔;;血小板輸注無(wú)效癥相關(guān)問(wèn)題探討(附60例報(bào)告)[J];中國(guó)社區(qū)醫(yī)師(醫(yī)學(xué)專(zhuān)業(yè));2012年07期
6 李建國(guó),張漩,邢莉;血小板輸注無(wú)效的臨床分析[J];淮海醫(yī)藥;1999年02期
7 吳玉敏;;臨床血小板輸注無(wú)效原因分析及對(duì)策[J];當(dāng)代醫(yī)學(xué);2014年06期
8 V.M.J.Novotny ,黃海龍;血小板輸注無(wú)效的預(yù)防和處理[J];國(guó)外醫(yī)學(xué).輸血及血液學(xué)分冊(cè);2001年01期
9 謝利群,陳愛(ài)萍;25例臨床診斷血小板輸注無(wú)效診治的分析[J];重慶醫(yī)學(xué);2002年06期
10 王曉珠;血小板輸注無(wú)效及其對(duì)策[J];日本醫(yī)學(xué)介紹;2003年03期
相關(guān)會(huì)議論文 前7條
1 邵科釘;周郁鴻;胡致平;;美羅華治療血小板輸注無(wú)效一例報(bào)道[A];第八屆全國(guó)中西醫(yī)結(jié)合血液病學(xué)術(shù)會(huì)議論文集[C];2007年
2 邵科釘;周郁鴻;胡致平;;美羅華治療血小板輸注無(wú)效一例報(bào)道[A];浙江省中西醫(yī)結(jié)合學(xué)會(huì)血液病專(zhuān)業(yè)委員會(huì)第二次學(xué)術(shù)年會(huì)暨省級(jí)繼續(xù)教育學(xué)習(xí)班資料匯編[C];2007年
3 邵科釘;胡致平;周郁鴻;;美羅華治療血小板輸注無(wú)效一例報(bào)道[A];2007年浙江省血液病學(xué)術(shù)年會(huì)論文匯編[C];2007年
4 李大成;魏天莉;藍(lán)欲曉;周華;張宏;陳悅康;吳國(guó)光;;免疫性血小板輸注無(wú)效患者血小板抗體特異性研究[A];中國(guó)輸血協(xié)會(huì)第四屆輸血大會(huì)論文集[C];2006年
5 王志梅;徐建民;鄒善華;;血漿置換聯(lián)合長(zhǎng)春新堿治療血小板輸注無(wú)效并繼發(fā)免疫性血小板減少一例[A];第三屆全國(guó)血液免疫學(xué)學(xué)術(shù)大會(huì)論文集[C];2003年
6 孫曉明;牛愛(ài)軍;尹秋霞;薛煉;胡成進(jìn);;血小板相關(guān)抗體(PAIgG)的檢測(cè)及其與血小板輸注無(wú)效關(guān)系探討[A];第十屆全軍檢驗(yàn)醫(yī)學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2005年
7 董國(guó)飛;張哲;彭明喜;陳長(zhǎng)水;湯曉嫻;付荊艷;杜勇;高家良;白潔;馬幼麗;錢(qián)伯勇;馬建波;;血小板配型及其臨床應(yīng)用[A];中國(guó)輸血協(xié)會(huì)第四屆輸血大會(huì)論文集[C];2006年
相關(guān)重要報(bào)紙文章 前1條
1 記者 曹斯 通訊員 張學(xué)元;廣州首現(xiàn)CD36抗體引起血小板輸注無(wú)效病例[N];南方日?qǐng)?bào);2011年
相關(guān)碩士學(xué)位論文 前4條
1 田青;血小板輸注無(wú)效的多因素分析[D];山西醫(yī)科大學(xué);2014年
2 伍偉健;血小板輸注無(wú)效檢測(cè)策略的建立及臨床應(yīng)用[D];南方醫(yī)科大學(xué);2014年
3 喬鄭磊;磷脂類(lèi)代謝產(chǎn)物S1P、LPA對(duì)于血小板功能的影響[D];華東師范大學(xué);2015年
4 劉忠;HPA基因型檢測(cè)及臨床應(yīng)用研究[D];第一軍醫(yī)大學(xué);2004年
，本文編號(hào)：2245291
本文鏈接：http://sikaile.net/huliyixuelunwen/2245291.html
最近更新
教材專(zhuān)著
