摘要
2型糖尿病（type2diabetes mellitus，T2DM）是一种由多种基因协同作用、并且受环境因素影响的慢性代谢性疾病，随着病程的延长，其慢性并发症给患者带来了严重的精神和经济负担。因此对2型糖尿病发病机制及其靶向干预的研究一直是研究的重点，具有重要的意义。近年来，随着单核苷酸多态性（singlenucleotide polymorphism，SNP）检测技术的发展，以SNP作为遗传图谱定位糖尿病易感基因的研究已经得到了广泛的关注。目前报道的与2型糖尿病相关的易感基因SNP位点已近百个，但其基因型与临床表型之间的关系尚不十分清楚，明确两者之间的相关性可以很好的为2型糖尿病的早期诊断和临床防治奠定基础。现已建立了很多种检测SNP位点基因型的方法，综合技术的可靠性、操作的简便性、仪器的精密性、筛选的通量性等原因都没能应用于临床检测，因此临床中需要一种成本低廉、操作简单、高通量性等优点的2型糖尿病易感基因SNP位点的检测方法。
连接酶检测反应（ligase detection reaction，LDR）技术是在较高温度下，耐高温的连接酶可以对SNP位点进行识别，当SNP位点上下游探针与待测DNA模板完全互补，并且两条探针之间没有空隙时，进行连接反应；而当基因突变使探针序列与DNA模板存在碱基错配时，连接反应则不能进行。不同SNP基因型所对应的探针长度不同，并且探针末端带有荧光标记，根据DNA测序原理通过荧光扫描扩增片段的长度，不同的SNP基因型会在相应的片段长度处产生荧光发射峰，根据荧光发射峰位置的差异，进行SNP位点基因型的判定。多重LDR技术即是在LDR原理的基础上，在同一检测体系中同时加入多个待测SNP位点的探针，能够同时进行多个SNP位点基因型的检测。虽然目前LDR检测技术的发展应用已经较为成熟，但仍需要DNA测序等步骤，价格较为昂贵，不适用于大规模临床应用推广。
多色荧光编码微球（Multicolor encoded Fluorescent Microspheres，MFMs）的应用发展，为高通量检测技术开辟了更为广阔的空间。与传统有机染料相比，荧光纳米材料发光强度更强、稳定性更高，非常适合应用于生物荧光标记研究，多色荧光编码微球同时还具有生物相容性好、荧光性质优异等特点，作为可与各种生物分子相偶联的标记分子，尤其适用于多种生物大分子的高通量同步分析。特别是随着纳米材料表面修饰及其生物链接技术的成熟，更加促进了其在生物医学领域的进一步应用。应用多色荧光编码微球，可以使寡核苷酸探针在有限的长度范围内拥有更多颜色的组合形式，从而增加了在同一反应体系中同时检测更多SNP位点的能力。
磁性纳米粒子（Magetic nanoparticls，MNP）在生物医学领域的应用范围非常广泛，利用磁性分离技术可以实现对生物分子的快速分离纯化。通过分子修饰技术，可以在磁性纳米粒子表面修饰各种功能基团，使其具有同偶联的生物大分子相同的特性。利用功能化磁性粒子表面配体-受体之间特异性作用，可以快速、有效地实现对DNA产物的纯化，该方法操作简单，不需要凝胶电泳回收等步骤，而且可以在后续实验步骤中随时快速提取纯化的产物。
本课题应用连接酶检测反应技术、磁性纳米微粒高效纯化技术以及荧光编码微球标识技术的结合，对2型糖尿病四种易感基因SNP位点进行了检测，为分子病因学以及临床防治的结合建立了一种新型的检测技术。
本研究分四个部分：1、LDR法检测SLC30A8、CDKN2A/2B、HHEX、TCF7L2基因SNP位点及其与T2DM的关系。应用LDR技术对T2DM患者的4个易感基因SNP即SLC30A8基因rs13266634位点、CDKN2A/2B基因rs10811661位点、HHEX基因rs1111875位点以及TCF7L2基因rs7903146位点基因型进行检测，在中国东北汉族人T2DM患者及正常人群中的分布频率进行分析，并以DNA测序法为标准印证LDR检测结果的准确性，同时探讨SLC30A8、CDKN2A/2B、HHEX和TCF7L2基因多态性与T2DM发病的相关性，以及其与糖代谢、脂代谢及慢性并发症之间的关系。2、磁性纳米微粒及荧光编码纳米微球的合成、修饰及其与DNA的偶联。应用共沉淀法制备了SiO2包覆的Fe3O4纳米粒子，并通过调控聚集体的大小和二氧化硅壳层厚度控制最终粒子的粒径和磁含量，得到SiO2基磁性纳米微球。通过共价偶联法、静电吸附法和聚电解质桥梁法等方法制备了4种染料掺杂的SiO2荧光纳米微球，即多色荧光编码微球。分别在磁性和荧光纳米微球的表面进行羧基修饰并与链霉亲和素偶联，制备成具有与生物大分子连接能力的SiO2基磁性及荧光纳米微球-链霉亲和素复合物。然后将其分别与5’端经生物素修饰的LDR上游探针、3’端经生物素修饰的LDR下游探针进行偶联反应。3、多重PCR-多重LDR法检测SNP位点体系的优化。运用多重PCR和多重LDR的技术原理，根据SLC30A8基因rs13266634位点、CDKN2A/2B基因rs10811661位点、HHEX基因rs1111875位点以及TCF7L2基因rs7903146位点，设计PCR引物及LDR探针，同时以全血基因组DNA为模板优化反应体系，探索最适反应条件，建立一种应用多重LDR法检测T2DM易感基因SNP位点的最佳反应体系。4、基于荧光编码微球标识的T2DM易感基因SNP检测体系的建立。以全血基因组DNA为模板，应用经过优化的多重LDR技术，磁性纳米微粒偶联LDR上游探针，快速、特异的分离出多重LDR扩增产物，去除反应体系中未参加连接反应的下游探针，荧光纳米微球标记LDR下游探针，通过对不同颜色的荧光的接收，有效地提高LDR检测的特异性，增加在同一多重LDR反应体系中同时检测更多SNP位点的能力。建立一种基于纳米材料多重LDR技术的T2DM易感基因SNP检测体系。
本研究得到如下结论：
1、应用LDR技术成功的对113例2型糖尿病和107例健康者的SLC30A8基因rs13266634位点、CDKN2A/2B基因rs10811661位点、HHEX基因rs1111875位点以及TCF7L2基因rs7903146位点基因型进行检测。发现SLC30A8基因rs13266634位点C等位基因与空腹胰岛素分泌不足、胰岛功能衰竭、胆固醇增高、心血管并发症的发病率相关；CDKN2A/2B基因rs10811661位点T等位基因与胰岛β细胞功能受损，胰岛素分泌减少及微血管并发症的发病率相关，HHEX基因rs1111875位点G等位基因与总胆固醇、低密度脂蛋白胆固醇增高及心血管并发症的发病率相关。
2、成功合成了链酶亲和素修饰的SiO2复合磁性纳米微球和多色荧光编码微球，并分别将磁性纳米微球与LDR上游探针成功偶联、多色荧光编码微球与LDR下游探针成功偶联，国内外首次成功制备了用于2型糖尿病易感基因高通量检测的荧光编码微球标记的寡核苷酸探针。
3、优化了多重PCR、多重LDR的反应条件，证明了适当增加反应体系中的MgCl2及dNTP浓度、调整各基因引物使用的比例、降低退火温度可以有效减少多重PCR中非特异性产物的产生、提高PCR产物含量；适当增加上游探针含量比例、减少探针使用量、降低退火温度有利于提高多重LDR的分辨率。为临床中2型糖尿病多个易感基因位点同时检测奠定了技术基础。
4、将多重PCR-多重LDR技术结合磁性纳米微粒高效纯化技术，同时利用4种荧光编码微球标记的寡核苷酸探针对T2DM易感基因位点进行基因型检测，国内外率先证实了多色荧光编码微球可以用于标记寡核苷酸探针进行LDR检测，建立了一种可同时检测4种T2DM易感基因SNP位点基因型的检测方法，为进一步进行T2DM易感基因SNP位点基因型高通量检测奠定了实验基础。
本研究结合了多重LDR检测体系的简便性、高通量性，磁性纳米微粒纯化技术的简便性、可重复性以及多色荧光编码微球的通量性等优点，建立了一种经济、简便、快速、高通量的T2DM易感基因SNP位点基因型的检测方法，可以有效地降低检测成本，适合用于临床推广，可将生物病因学研究与临床防治有效对接。
Type2diabetes mellitus (T2DM) is a chronic metabolic diseases, contributedsynergistically by multiple genes and environmental factors, and its chroniccomplications cause severe mental and financial burden to patients with a longduration. Therefore, the pathogenesis of and targeting interference against T2DM areof great importance and are focued on by T2DM researchers. Recently, with thedevelopment of single nucleotide polymorphism (SNP) technology, identification ofT2DM susceptibility genes has been extensively focused on, and there have beenmore than100SNP sites of T2DM susceptibility genes being reported. However,little is known about the correlation of these genetypes with their phenotypes, thoughthe identification of the correlation can facilitate the early diagnosis and clinicalprevention and treatment of T2DM. Nowadays, there have been multiple methods toidentify the SNPs, however, most of these methods are still on the way to clinicalassay, being limited by inadequate reliability, simplicity of operation, instrumentprecision, throughput of screening. Therefore, a novel method of low cost, simpleoperation and high throughput is in need to identify the SNPs of T2DMsusceptibility genes.
Ligase detection reaction (LDR）is based on the specific binding of thermostableligase to SNP sites under high temperature. If there were a completecomplementation between the upstream, downstream probes and the SNP site,without gap between the two probes, the ligation completes; While the lagation fails,if the probe sequences mismatched the mutated SNP site. The lenghth of probe sequences varies with SNPs of genptypes, as can be identified by the fluorescencescanning of the fluorescence labeled at the end of various probes, basing on theemission peak of various SNP genotypes of various fragment length. Basing on theprinciple of LDR technology, multiple LDR developes, in which, there are multipleprobes targeting multiple SNPs, capable of multiple SNPs identificationsimultaneously. However, the LDR detection technology is restricted to large-scaleclinical application by its high cost, complicated procedures, including DNAsequencing.
The development of Multicolor encoded Fluoresent Microspheres (MFMs) hasopened a novel area for high-throughput detection technology. Compared withconventional organic dyes, fluorescent nano-materials emission is stronger, morestable, therefore, more suitable for the fluorescent biomarker research. Moreover,because of their good biocompatibility, excellent fluorescence properties, MFMs areparticularly suitable for simultaneous high throughput analysis of multiple biologicalmacromolecules, by being coupled to a variety of biological molecules, Especially,the mature of surface modification of nano-materials and bio-link technologiespromote the further application of MFMs in the biomedical field. MFMs can giveoligonucleotide probes unique color, let along their length, thereby facilitating thesimutanuos detection of multiple SNPs in same reaction system.
Magnetic nanoparticles (MNPs) have been widely applicated in the biomedicalfield, the magnetic separation technology can achieve rapid separation andpurification of biomolecules. And the molecular modification technique can couplethe MNPs to various biomolecules via multiple functional groups on MNPs, withoutchanging the characteristics of coupled molecules. Basing the functionalized specificligand-receptor interaction of magnetic nanoparticles, DNA products can be rapidlyand efficiently purified without procedures, such as gel electrophoresis recycling,and the DNA purification can be carried out in any subsequent experiments rapidly.
In present study, we constructed a novel detection method for molecularetiology and for clinical prevention and treatment, basing on LDR, MNPs and MNPs, and then identified SNPs of four susceptibility genes to T2DM via the method.
There are foure parts in this study:
1. We determined the SNPs of SLC30A8, CDKN2A/2B, HHEX, TCF7L2gene, and analyzed the correlation of these SNPs with T2DM. LDR method wasadopted to identify the SNPs of four T2DM susceptability genes of T2DM patients,i.e. the site of rs13266634in SLC30A8, the site of rs10811661in CDKN2A/2B, thesite of rs1111875in HHEX and the site of rs7903146in TCF7L2. Then thedistribution frequency of each site was analyzed in T2DM patients of Han innortheast China. And the acuracy of LDR results were reconfirmed with DNAsequencing. In addition, we also determined the association of the polymorphisms inSLC30A8, CDKN2A/2B, HHEX and TCF7L2gene with the T2DM morbidity, aswell as with the abnormal glucose metabolism, abnormal lipid metabolism andchronic complications.
2. Synthesis, modification and DNA-conjugation of MNPs and MFMs.SiO2-coated Fe3O4nanoparticles were prepared by coprecipitation method, theSiO2-based magnetic nanoparticles were obtained under strict control of the particlesize and magnetic density by regulating the size and thickness of the silica shell to ofthe aggregates. By covalent coupling method, electrostatic adsorption andpolyelectrolyte bridges and other methods, we prepared four kinds of SiO2fluorescence coded nanoparticles with multi-color fluorescence, i.e. MFMs. Then themagnetic and fluorescent nanospheres surface was modified with carboxyl group andcoupled to streptavidin-biotin, and thus these particles were capable to bind tobiological macromolecules to develop SiO2-based magnetic or fluorescentnanoparticles-streptavidin-biotin complex. Thus, these particles were utilized toconjunct to the biotin modified5' end of LDR upstream probe, or the biotin modified3' end of LDR downstream probe.
3. Optimization of multiple PCR-LDR assay for multiple SNPs. According tothe principles of multiple PCR and multiple LDR, PCR primers and LDR probeswere designed targeting the site of rs13266634in SLC30A8, the site of rs10811661 in CDKN2A/2B, the site of rs1111875in HHEX and the site of rs7903146inTCF7L2. The reaction conditions of the reaction system, with whole genomic DNAas template, were optimezed to establish the optimal reaction system for T2DMsusceptable SNPs identification, by multiple LDR.
4. Establishment of the detection system for T2DM susceptable gene SNPs,basing on MFMs.The optimized multiple LDR technique was adopted, with wholeblood genomic DNA as template, magnetic and with nanoparticles coupled upstreamLDR probes, the amplificated product of multiple LDR was rapidly and specificallyisolated from the irrelevant downstream probe. The fluorescent nanospheres coupledLDR downstream probe effectively promoted the detection specificity basing on theabsorbance of the fluorescence with different color, and promoted the detectioncapbility of mutiple SNPs simultaneously in the same reaction system. Thus, weestablished a multi-LDR detection system for T2DM susceptibility gene SNPs,basing on nanomaterials technology.
Research conclusions are as following:
1We have successfully identified the SNP of rs13266634in SLC30A8,rs10811661in CDKN2A/2B, rs1111875in HHEX and rs7903146in TCF7L2, of113cases of T2DM patients and107healthy subjects. And we found that there was anassociation of C allele at rs13266634of SLC30A8gene with fasting insulinsecretion deficiency, Islet function failure, increased cholesterol and increasedincidence of cardiovascular complications. And we also found that there was anassociation of T allele at rs10811661of CDKN2A/2B gene with islet β celldysfunction, Islet function failure, insulin secretion reduction, increased incidence ofmicrovascular complications.
2We have successfully synthesized the streptavidin-biotin-modifiedSiO2-composed magnetic nanospheres and multicolor fluorescence codedmicrospheres. And then we respectively coupled the magnetic nanospheres with theupstream LDR probe, coupled the multicolor fluorescence coded microspheres withthe downstream LDR probe. This is the first successful preparation of the oligonucleotide probes coupled with multicolor fluorescence coded microspheres forthe high throughput detection of T2DM susceptibility genes.
3We have optimized the reaction conditions of multiple PCR and multipleLDR, and we found that non-specific PCR products could be significantly reduced,and the target PCR products could be elevated by appropriately increasing theconcentration of MgCl2and dNTP, adjusting the ratio of the primers pairs of eachgene, by reducing the amount of probe, or decreasing the annealing temperature ofthe reaction system to improving the resolution of multiple LDR. These findingslayed the technical foundation for the simultaneous detection of clinical T2DMsusceptibility gene SNPs.
4We have identified the T2DM susceptability gene SNPs via the combinedusage of the multiple PCR, multiple LDR technology, the high efficient purificationtechnology with MNPs, and the oligonucleotide probes with four kinds of MFMs.We confirmed for the first time, MFMs could be utilezed to label the oligonucleotideprobes for LDR detection, and we established a detection method, simutaneouslyidentifying SNPs of four T2DM susceptability genes, laying the foundation forhigh-throughput detection of T2DM susceptibility gene SNPs.
In present study, we established a cheap, simple, rapid, and high-throughputdetection method for the identification of T2DM susceptability gene SNPs, with thecombination of the simplicity, high-throughput of muliple LDR, the simplicity andrepeatability of MNPs, and the high-throughput of MFMs. It could effectively reducetesting costs, was suitable for clinical application, with an effective combination ofthe biological research and clinical prevention etiology.
引文
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