磷脂类代谢产物S1P、LPA对于血小板功能的影响

发布时间：2018-09-17 08:23

【摘要】：血小板在执行生理性止血作用的同时,在心血管疾病、炎症、肿瘤形成、肺组织纤维化等许多重要的病理进程中扮演非常重要的角色。目前在临床上,输注血小板是预防和缓解血小板减少症或者功能障碍的重要治疗措施。但部分患者反复输注血小板后会出现血小板输注无效。如何避免血小板输注无效的发生和血小板的浪费,提高血小板治疗效果,是目前输血医学界面临的一个重要的课题。为了研究SIP、LPA与血小板储存损伤之间的联系,本文检测并分析单采血小板震荡保存过程中发生的一系列形态和功能的变化以及1-磷酸鞘氨醇(S1P)和溶血磷脂酸(LPA)的释放规律,意在为临床血小板输注无效的预防和治疗提供新的思路和方法。我们发现,随着血小板保存时间的延长,血浆中S1P的含量逐渐增加,通过直接添加S1P维持血浆中一定浓度S1P后,发现血小板储存损伤相关指标优于对照组：活化率和凋亡率的增加明显受到抑制(P0.05),血小板凋亡率从15%降到5%、活化率从55%下降到43%。聚集率从5%增加到23%,低渗休克反应从11%增加到25%、形变能力从3%增加到7%),而体外添加LPA维持血浆中LPA一定浓度,对于这些指标影响并不显著(第5天时,P0.05)。体外直接添加二甲基鞘氨醇(DMS)有效地抑制鞘氨醇激酶活性,减少了S1P的生成,此时血小板储存损伤显著增加：第5天时,保存期内血小板凋亡率从9%增加到14%、活化率从43%增加到60%,活化率和凋亡率有显著增加(P0.05),聚集率从28%下降到5%,低渗休克反应从30%下降到18%、形变能力从8%下降到2%；此时添加S1P恢复血浆中的S1P的含量后,活化率和凋亡率的增加明显受到抑制(P0.05),保存期第5天血小板凋亡率从14%降到10%、活化率从51%下降到42%,,聚集率从8%增加到18%,低渗休克反应从18%增加到24%、形变能力从2%增加到6%,降低趋势也明显受到抑制(P0.05)。以上结果表明,在保存期间血小板S1P含量逐渐增加,这可能是因为血小板在体外储存期间由于血小板凋亡破裂释放出活性分子导致更多的血小板活化,进而导致血浆中S1P含量增加。体外添加S1P后,血小板的凋亡途径受到抑制(如抑制Caspase-3活性),细胞破裂减少,降低了活性分子的释放,有效地抑制了血小板储存损伤的发生,具体的机制需要进一步研究。另外,在这篇文章的附录1和附录2中,我们还探讨了汉族、维吾尔族CD61基因外显子10多态性分析和上海人群中CD36缺失频率和相关分子机制的研究工作。我们在利用Bloodchip对中国汉族人群血型基因分型的筛查中,发现有些个体的]HPA-6bw无法判断分型。由于Bloodchip针对HPA-6bw基因分型的探针结合部位位于HPA-6bw型突变位点附近,推测无法判断分型的这些个体在HPA-6bw型突变位点附近可能存在未知的突变,影响了引物或者探针与模板的结合。为了研究编码血小板膜糖蛋白CD61基因的多态性,探索HPA-6bw血型系统基因检测合理引物和探针设计,本文在第二章节中分析了CD61基因外显子10的突变以及这些突变在汉族、维吾尔族人群中的分布情况,我们随机采集149人份汉族和96人份维吾尔族的血液样本,扩增这些样本CD61基因外显子10,然后直接测序分析外显子10编码区基因序列。汉族和维族人群的CD61基因外显子10除决定HPA-6bw多态性的SNP(单核苷酸多态性位点)外,汉族和维族人群的CD61基因外显子10存在3个SNP(1533AG、1545GA、1529CT),且在汉族和维族人群分布频率差异甚小,这显示两个民族之间血缘上的关系比较亲近。血小板膜糖蛋白CD36发生缺失则可产生抗-CD36抗体,导致血小板减少、输血后紫癜和血小板输注无效等输血不良反应。在第三章节我们筛选1022名健康献血者,分析血小板和单核细胞上CD36表达,明确CD36 Ⅰ型缺失和Ⅱ型缺失类型,统计分析等位基因频率,获得献血人群CD36基因突变主要类型及分布频率,发现新基因突变类型,比较中国与其他报道人种中CD36分子背景差异。我们扩增CD36基因外显子3-14和两侧相关部分内含子,直接测序比对基因序列有差异的样本,利用克隆测序进行证实并确认了CD36基因的一些新的突变。结果显示,在本研究中CD36 Ⅰ型缺失和Ⅱ型缺失的频率分别为0.2%和2.0%。中国上海人群CD36缺失频率高于欧美国家,略低于亚洲其他国家。我们的研究结果显示,有13个突变类型,其中8个突变已有报道,另有6种突变未见报道,它们分别位于3、12、13、14号外显子,引起读码框移位或者氨基酸突变,最终导致表达蛋白质结构的变化。5号外显子中的突变371CT与CD36 Ⅰ型缺失相关,其余的突变与CD36 Ⅱ型缺失相关。大多数突变存在于CD36基因的外显子中,并引起相应的氨基酸变换(除了同义突变1008GT)。另外,位于14号外显子中的1344insTCTT引起448位氨基酸处的阅读框移位,导致终止子的缺失。然而,基因水平上的研究还不足以揭示蛋白质水平上的分子行为,为探索CD36缺失的分子机制还需要更多的研究。
[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.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6;R457.1

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