白细胞介素—6与2型糖尿病发生的研究进展

白细胞介素－6（IL－6）是一种多功能的细胞因子。IL－6的升高可通过激活急性相反应影响2型糖尿病的发病,并可通过与胰岛素、瘦素的相互作用与2型糖尿病的发生相关联。     

猪白细胞介素2与融合白细胞介素4/6基因共表达的免疫效应研究

目的构建猪融合白细胞介素4/6与猪白细胞介素2基因的共表达载体,研究其共表达对小鼠免疫的协同效应。方法以2A自剪接技术首次构建猪融合白细胞介素4/6与白细胞介素2基因的共表达重组质粒,以壳聚糖纳米材料包裹制成纳米颗粒,进行体外转染HEK293细胞,提取总RNA进行RT-PCR分析,最后肌肉注射小鼠进行体内实验并分析。结果成功构建了重组共表达质粒VRIL4/6-2;壳聚糖包裹后转染HEK293细胞,48 h后收集细胞,RT-PCR检测显示目的基因能够在HEK293细胞中高效地转录与表达。小鼠接种实验结果表明,实验组血清中的IgG和IgG2a的含量比对照组显著增加,并且VRIL4/6-2-CS接种组的小鼠体液免疫水平明显增高(P0.05);VRIL4/6-2实验组的CD4+淋巴细胞显著多于其它实验组(P0.05);实验组IL-2、IL-4、IL-6、IL-23基因的表达水平明显高于对照组(P0.05),实验组小鼠外周血白细胞、血小板和血红蛋白的含量均显著高于对照组(P0.05)。结论 VRIL4/6-2共表达质粒能够更好的增强动物体液免疫和细胞免疫机能,可作为提高动物体液免疫和细胞免疫的经济高效安全新型免疫调节剂,具有潜在的重要应用前景。     

白细胞介素8 基因-251A/T 多态与子宫内膜异位症的相关性研究

目的:探讨白细胞介素8基因(IL-8)-251A/T多态性在子宫内膜异位症发生中的作用。方法:采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和焦磷酸测序方法,研究152例子宫内膜异位症患者和134例正常子宫内膜中IL-8基因-251A/T多态性的分布情况。结果:IL-8基因-251A/T多态位点的AA、AT和TT基因型在子宫内膜异位症组中分别为12.5%、46.1%、41.5%,等位基因A和T的频率分别为35.5%、64.5%;在对照组中AA、AT和TT三种基因型分布频率分别为24.6%、41.0%、34.3%,等位基因A和T的频率分别为45.1%、54.9%。IL-8基因-251A/T单核苷酸多态是子宫内膜异位症发病的独立的危险因素(P<0.05);A等位基因携带者患子宫内膜异位症的风险增高(P<0.05)。结论:在中国北方汉族人群中IL-8基因-251A/T单核苷酸多态性与子宫内膜异位症的发病具有相关性,A等位基因是子宫内膜异位症发病重要的遗传学标记。     

可溶性白细胞介素6受体基因在链霉菌的克隆表达研究

以分离提取的ＨｅＬａ细胞总ＲＮＡ为模板,通过ＲＴ－ＰＣＲ反应扩增得到了１０１７ｂｐ的人可溶性白细胞介素６受体（ｓＩＬ－６Ｒ）ｃＤＮＡ片段,将扩增片段克隆到ｐＵＣ１９质粒中进行序列测定。结果证明该片的序列与文献报道的ｓＩＬ－６ＲｃＤＮＡ的序列完全一致,将ｓＩＬ－６ＲｃＤＮＡ与链霉素信号肽ｍｅｌＣｌ的编码序列融合后得到的融合基因ｍｅｌ／ｓＩＬ－６Ｒ克隆到链霉菌质粒ｐＬＪ４５９中,构建成重组表达质粒ｐＬ     

壳聚糖纳米颗粒包裹猪白细胞介素2和融合白细胞介素4/6基因对猪圆环病毒2型疫苗免疫的强化

目的探讨猪Sus scrofa domesticus白细胞介素2(IL-2)和融合白细胞介素4/6(IL-4/6)基因的共表达对仔猪免疫应答的影响,为进一步研制新型免疫调节剂来加强仔猪抵御断奶后多系统衰竭综合征奠定基础。方法将猪IL-2和IL-4/6融合基因的共表达重组质粒,用壳聚糖材料包裹制成纳米颗粒,记作VRIL-4/6-2-CS。将仔猪分为2组,分别肌肉注射VRIL-4/6-2-CS(实验组)和生理盐水(对照组),2组均同时接种猪圆环病毒2型(PCV-2)疫苗,在接种后的第0、7、14、28天采集血样并分析免疫变化。结果实验组仔猪血清中的IgG2a抗体数量和血液中CD4~+、CD8+~T淋巴细胞数量均显著高于对照组(P0.05);同时,实验组仔猪的IL-2、IL-4、IL-6、TNF-α、TLRs(TLR-2,7)、STATs(STAT-1,2,3)基因表达水平也显著高于对照组(P0.05)。尽管2组之间PCV-2特异性抗体的含量差异无统计学意义,但是实验组仔猪的生长速率显著高于对照组(P0.05)。结论 VRIL-4/6-2-CS能促进仔猪对PCV-2疫苗的免疫应答,是一种安全有效的免疫佐剂。     

一种白细胞介素2基因变构体的高效表达与纯化工艺

利用定点诱变技术构建表达质粒pET15b-MhIL-2并将其在大肠杆菌中进行表达发酵的优化研究,高效表达出可溶性的MhIL-2重组蛋白。蛋白经过亲和层析、Thrombin酶切、离子交换层析和凝胶过滤层析纯化,MhIL-2纯度达95%,且MhIL-2比hIL-2具有更强的促进T细胞增殖生物活性。     

eNOS基因第四内含子a/b多态与湖北汉族人原发性高血压而不是2型糖尿病相关亮

目的:探讨内皮型一氧化氮合酶基(eNOS)与湖北汉族人原发性高血压(EH)和2型糖尿病(T2DM)的关系.方法:采用病例一对照设计.分析了657例样本eNOS第四内含子重复序列多态性a/b,测量了身高、体重、腰围、臀围、收缩压、舒张压、空腹血糖,餐后2小时血糖等临床指标.结果:EH病例组eNOS ab+aa基因型和a等住基因频率显著高于EH对照组(基因型:25.3%vs 18.9%,P=0.049;等位基因:13.3%vs9.8%.P=0.045);而T2DM病例组与T2DM对照组的eNOS ab+aa基因型频率没有显著差异(20.2%vs24.1%,P=0.247).单因素Logistic回归分析显示eNOS ab+aa基因型是EH的危险因子(OR=1.623,95%CI 1.053-2.506,P=0.029).多因素回归分析显示,EH的独立风险因素是年龄、体重指数和eNOS基因a/b多态性,而体重指数和腰臀比是T2DM的独立风险因素.结论:eNOS基因a/b多态性是湖北汉族人群EH的一个易感标记,而与T2DM没有相关性.     

双生子多巴胺D3受体基因多态性对儿童体成分的影响

均衡的体成分构成对维持机体的健康状态具有重要作用,体成分受遗传与环境因素的共同影响。多巴胺参与摄食、运动及认知等活动的调节,多巴胺D3受体（DRD3）对多巴胺神经通路起关键调节作用,进而对摄食功能发挥作用,从而可能对体成分产生影响。为了解遗传与环境因素对双生子儿童体成分的影响,并探讨DRD3基因单核苷酸多态性（SNP）与体成分的相关性,对160对4-12岁双生子肱三头肌皮褶厚度（d₁）、肩胛下皮褶厚度（d₂）、髂前上棘位皮褶厚度（d₃）和体质量（m）进行了测量,计算d₄(d₁+d₂)、d₅(d₂/d₁),体脂率（P_f）、瘦体质量（m_l）;从口腔拭子中提取全基因组DNA;通过Amp FISTR Sino filerPlus试剂盒分析确定卵型;采用SNaPshot技术对DRD3基因4个SNP位点进行检测;使用Mx软件估算各指标遗传度;运用广义估计方程模型分析各指标与DRD3基因SNP的相关性。校正年龄效应后,除个别指标（d₃, m_l）外,男女生指标遗传度（h）学龄前期总体偏低,且某些指标（d₂, d₄, P_f, m_l）的遗传度存在一定的性别差异。d₂分别与rs324029、rs226082存在相关（P<0.05）; d₃分别与rs2134655、rs226082存在相关（P<0.05）;d₅分别与rs2134655、rs167771存在相关（P<0.05）;P_f分别与rs226082、rs167771存在相关（P<0.05）;m_l分别与rs2134655、rs226082、rs167771存在相关（P<0.05）。本研究结果表明,遗传和环境因素对儿童体成分发育均有影响,但遗传效应可能存在一定的发育阶段和性别差异;DRD3基因SNPs与儿童的体成分可能存在一定的相关性。     

白细胞介素-1受体相关激酶-2的共价修饰与激酶活性的研究

白细胞介素-1受体相关激酶-2(IRAK2)是调节IL1和Toll样受体信号通路的一个关键性分子,目前对于共价修饰如何调节IRAK2的活性还所知甚少。当与IRAK1共转染时,IRAK2能被共价修饰,在SDS-PAGE分离中呈现出迁移率变慢的多条电泳带。在小鼠骨髓干细胞分化的巨噬细胞(BMMs)中,内源表达的IRAK2在TLR配体刺激下也呈现出类似的共价修饰。而且IRAK2的共价修饰具有磷酸酯酶敏感性,提示大部分为磷酸化修饰。通过体外磷酸激酶活性分析,发现巨噬细胞中表达的IRAK2能在LPS诱导下被激活,成为一个具有激酶活性的调节蛋白。进一步研究发现激酶灭活的IRAK2突变体不能重建IRAK2基因敲除巨噬细胞的功能。通过Western杂交和定量PCR分析,发现IRAK2的激酶活性是介导LPS诱导的信号通路和炎症因子表达所必须的。因此,在LPS诱导下,IRAK2可能被IRAK1进行磷酸化修饰而活化,从而介导下游的信号转导通路、诱导炎症因子的表达。     

Association of Complement C5 Gene Polymorphisms with Proliferative Diabetic Retinopathy of Type 2 Diabetes in a Chinese Han Population

Purpose

To investigate the association of C5 SNPs with proliferative diabetic retinopathy (PDR) of type 2 diabetes (T2D).

Methods

A total of four C5 SNPs including rs2269067, rs7040033, rs1017119 and rs7027797 were genotyped in 400 PDR patients with T2D (cases) and 600 non- proliferative diabetic retinopathy PDR (NPDR) with T2D patients (controls) by using PCR-RFLP method. mRNA expression was examined by real-time PCR. Cytokine production was detected by ELISA.

Results

The frequency of GG genotype of C5 rs2269067 was significantly increased in cases compared with controls (Pc = 3.4×10⁻⁵, OR = 1.87). And C5 mRNA expression was significantly increased in rs2269067 GG cases as compared with CG or CC cases (P = 0.003, P = 0.001, respectively). Moreover, the production of IL-6 was significantly increased in rs2269067 GG cases compared to CG cases or CC cases (P = 0.002, P = 0.001, respectively).

Conclusions

C5 rs2269067 GG genotype confers risk for PDR of T2D in Chinese han population and is associated with an elevated C5 mRNA expression and an increased IL-6 production.     

SLC30A8基因rsl3266634多态性与内蒙古地区汉族人群2型糖尿病的相关性研究

目的：研究内蒙古地区汉族人群SLC30A8（solute carrier family 30,member 8）基因rsl3266634单核苷酸多态性（Single nucleotide polymorphism,SNP）的等位基因和基因型频率分布与2型糖尿病（Type 2 diabetes,T2DM）的相关性。方法：采用等位基因特异性聚合酶链式反应（AS-PCR）,对222例内蒙古地区汉族人（其中T2DM组125例,正常对照NC组97例）rsl3266634进行基因分型。结果：T2DM组中rsl3266634的C等位基因频率、CC基因型频率分别为61.2%和28.4%,均显著高于NC组的53.1%和24.7%（P值均〈0.05）;而T2DM组的TT基因型频率为6.4%,显著低于NC组的18.6%（P〈0.05）。C等位基因携带者患T2DM的风险是T等位基因的1.64倍（OR=1.64,95%CI=1.125-2.402）。结论：SLC30A8基因rsl3266634多态性位点的C等位基因可能是T2DM的风险等位基因,该位点C/T多态性与内蒙古地区汉族人群T2DM具有相关性,可能是内蒙古地区汉族人T2DM的易感基因之一。     

SLC30A8 基因rsl3266634 多态性与内蒙古地区汉族人群2 型糖尿 病的相关性研究

目的:研究内蒙古地区汉族人群SLC30A8(solute carrier family 30,member 8)基因rsl3266634单核苷酸多态性(Single nucleotide polymorphism,SNP)的等位基因和基因型频率分布与2型糖尿病(Type 2 diabetes,T2DM)的相关性。方法:采用等位基因特异性聚合酶链式反应(AS-PCR),对222例内蒙古地区汉族人(其中T2DM组125例,正常对照NC组97例)rsl3266634进行基因分型。结果:T2DM组中rsl3266634的C等位基因频率、CC基因型频率分别为61.2%和28.4%,均显著高于NC组的53.1%和24.7%(P值均<0.05);而T2DM组的TT基因型频率为6.4%,显著低于NC组的18.6%(P<0.05)。C等位基因携带者患T2DM的风险是T等位基因的1.64倍(OR=1.64,95%CI=1.125-2.402)。结论:SLC30A8基因rsl3266634多态性位点的C等位基因可能是T2DM的风险等位基因,该位点C/T多态性与内蒙古地区汉族人群T2DM具有相关性,可能是内蒙古地区汉族人T2DM的易感基因之一。     

Validation of Type 2 Diabetes Risk Variants Identified by Genome-Wide Association Studies in Han Chinese Population: A Replication Study and Meta-Analysis

Background

Several genome-wide association studies (GWAS) involving European populations have successfully identified risk genetic variants associated with type 2 diabetes mellitus (T2DM). However, the effects conferred by these variants in Han Chinese population have not yet been fully elucidated.

Methods

We analyzed the effects of 24 risk genetic variants with reported associations from European GWAS in 3,040 Han Chinese subjects in Taiwan (including 1,520 T2DM cases and 1,520 controls). The discriminative power of the prediction models with and without genotype scores was compared. We further meta-analyzed the association of these variants with T2DM by pooling all candidate-gene association studies conducted in Han Chinese.

Results

Five risk variants in IGF2BP2 (rs4402960, rs1470579), CDKAL1 (rs10946398), SLC30A8 (rs13266634), and HHEX (rs1111875) genes were nominally associated with T2DM in our samples. The odds ratio was 2.22 (95% confidence interval, 1.81-2.73, P<0.0001) for subjects with the highest genetic score quartile (score>34) as compared with subjects with the lowest quartile (score<29). The incoporation of genotype score into the predictive model increased the C-statistics from 0.627 to 0.657 (P<0.0001). These estimates are very close to those observed in European populations. Gene-environment interaction analysis showed a significant interaction between rs13266634 in SLC30A8 gene and age on T2DM risk (P<0.0001). Further meta-analysis pooling 20 studies in Han Chinese confirmed the association of 10 genetic variants in IGF2BP2, CDKAL1, JAZF1, SCL30A8, HHEX, TCF7L2, EXT2, and FTO genes with T2DM. The effect sizes conferred by these risk variants in Han Chinese were similar to those observed in Europeans but the allele frequencies differ substantially between two populations.

Conclusion

We confirmed the association of 10 variants identified by European GWAS with T2DM in Han Chinese population. The incorporation of genotype scores into the prediction model led to a small but significant improvement in T2DM prediction.     

Association of Single Nucleotide Polymorphisms of IL23R and IL17 with Ulcerative Colitis Risk in a Chinese Han Population

Background

Previous studies implicated that IL23R and IL17 genes play an important role in autoimmune inflammation. Genome-wide association studies have also identified multiple single nucleotide polymorphisms (SNPs) in the IL23R gene region associated with inflammatory bowel diseases. This study examined the association of IL23R and IL17A gene SNPs with ulcerative colitis susceptibility in a population in China.

Methodology

A total of 270 ulcerative colitis and 268 healthy controls were recruited for the analyses of 23 SNPs in the IL23R and IL17A regions. Genomic DNA was extracted and analysis of these 23 SNPs using ligase detection reaction allelic (LDR) technology. Genotype and allele associations were calculated using SPSS 13.0 software package.

Principal Findings

Compared to the healthy controls, the variant alleles IL23R rs7530511, and rs11805303 showed a statistically significant difference for ulcerative colitis susceptibility (0.7% vs 3.3%, P = 0.002; 60.4% vs 53.2%, P = 0.0017, respectively). The linkage disequilibrium (LD) patterns of these SNPs were measured and three LD blocks from the SNPs of IL23R and one block from those of IL17A were identified. A novel association with ulcerative colitis susceptibility occurred in haplotypes of IL23R (Block1 H3 P = 0.02; Block2 H2 P = 0.019; Block3 H4 P = 0.029) and IL17A (H4 P = 0.034). Pair-wise analyses showed an interaction between the risk haplotypes in IL23R and IL17A (P = 0.014).

Conclusions

Our study demonstrated that rs7530511, and rs11805303 of IL23R were significantly associated with ulcerative colitis susceptibility in the Chinese population. The most noticeable finding was the linkage of IL23R and IL17A gene region to ulcerative colitis risk due to the gene-gene interaction.     

Obesity-Related Genomic Loci Are Associated with Type 2 Diabetes in a Han Chinese Population

Background and Aims

Obesity is a well-known risk factor for type 2 diabetes. Genome-wide association studies have identified a number of genetic loci associated with obesity. The aim of this study is to examine the contribution of obesity-related genomic loci to type 2 diabetes in a Chinese population.

Methods

We successfully genotyped 18 obesity-related single nucleotide polymorphisms among 5338 type 2 diabetic patients and 4663 controls. Both individual and joint effects of these single nucleotide polymorphisms on type 2 diabetes and quantitative glycemic traits (assessing β-cell function and insulin resistance) were analyzed using logistic and linear regression models, respectively.

Results

Two single nucleotide polymorphisms near MC4R and GNPDA2 genes were significantly associated with type 2 diabetes before adjusting for body mass index and waist circumference (OR (95% CI) = 1.14 (1.06, 1.22) for the A allele of rs12970134, P = 4.75×10⁻⁴; OR (95% CI) = 1.10 (1.03, 1.17) for the G allele of rs10938397, P = 4.54×10⁻³). When body mass index and waist circumference were further adjusted, the association of MC4R with type 2 diabetes remained significant (P = 1.81×10⁻²) and that of GNPDA2 was attenuated (P = 1.26×10⁻¹), suggesting the effect of the locus including GNPDA2 on type 2 diabetes may be mediated through obesity. Single nucleotide polymorphism rs2260000 within BAT2 was significantly associated with type 2 diabetes after adjusting for body mass index and waist circumference (P = 1.04×10⁻²). In addition, four single nucleotide polymorphisms (near or within SEC16B, BDNF, MAF and PRL genes) showed significant associations with quantitative glycemic traits in controls even after adjusting for body mass index and waist circumference (all P values<0.05).

Conclusions

This study indicates that obesity-related genomic loci were associated with type 2 diabetes and glycemic traits in the Han Chinese population.     

中国汉族人群2型糖尿病易感性相关基因多态性

2型糖尿病（type2 diabetes mellitus,T2DM）的发病与多个基因累加效应及多种环境因素相关。已在中国汉族人群中研究过的与T2DM易感性相关的基因多态性包括：全基因组相关研究中的CDKAL1、CDKN2A／B、SLC30A8、IGF2BP2、HHEX、FTO以及KCNQI基因;脂联素基因;核呼吸因子基因;葡萄糖激酶基因;肿瘤坏死因子α基因等。探索这些易感基因可以为人类治疗T2DM起到极大的推动作用。但至今已明确的基因依然很少,国内外的研究结果不尽相同,尚需进一步地深入研究。     

An Exploratory Study of the Association between KCNB1 rs1051295 and Type 2 Diabetes and Its Related Traits in Chinese Han Population

Since the KCNB1 encoding Kv2.1 channel accounts for the majority of Kv currents modulating insulin secretion by pancreatic islet beta-cells, we postulated that KCNB1 is a plausible candidate gene for genetic variation contributing to the variable compensatory secretory function of beta-cells in type-2 diabetes (T2D). We conducted two studies, a case-control study and a cross-section study, to investigate the association of common single-nucleotide polymorphisms (SNPs) in KCNB1 with T2D and its linking traits. In the case-control study, we first examined the association of 20 tag SNPs of KCNB1 with T2D in a population with 226 T2D patients and non-diabetic subjects (screening study). We then identified the association in an enlarged population of 412 T2D patients and non-diabetic subjects (replication study). In the cross-sectional study, we investigated the linkage between the candidate SNP rs1051295 and T2D by comparing beta-cell function and insulin sensitivity among rs1051295 genotypes in a general population of 1051 subjects at fasting and after glucose loading (oral glucose tolerance tests, OGTT) in 84 fasting glucose impaired subjects, and several T2D-related traits. We found that among the 19 available tag SNPs, only the KCNB1 rs1051295 was associated with T2D (P = 0.027), with the rs1051295 TT genotype associated with an increased risk of T2D compared with genotypes CC (P = 0.009). At fasting, rs1051295 genotype TT was associated with a 9.8% reduction in insulin sensitivity compared to CC (P = 0.008); along with increased plasma triglycerides (TG) levels (TT/CC: P = 0.046) and increased waist/hip (W/H) ratio (TT/CC: P = 0.013; TT/TC: P = 0.002). OGTT confirmed that genotype TT exhibited reduced insulin sensitivity by 16.3% (P = 0.030) compared with genotype TC+CC in a fasting glucose impaired population. The KCNB1 rs1051295 genotype TT in the Chinese Han population is associated with decreased insulin sensitivity and increased plasma TG and W/H ratio, which together contribute to an increased risk for T2D.