Klotho基因多态性及蛋白水平与慢性肾脏病动脉硬化相关性研究

发布时间：2018-04-21 04:16

本文选题：Klotho基因 + 单核苷酸多态性　；参考：《青岛大学》2015年博士论文

【摘要】：目的研究慢性肾脏病患者Klotho基因G-395A单核苷酸多态性的分布,探讨该基因多态性位点及蛋白水平与慢性肾脏病及动脉硬化的相关性。方法运用FQ-PCR技术(Taqman探针法)对各研究对象[包括156例慢性肾脏病患者(其中非透析组包括CKD2-3期56例、CKD4-5期60例,血透组40例)和健康对照组(60例)]进行klotho基因G-359A多态性分型,检测klotho蛋白水平,颈动脉超声检查进行动脉硬化分组,比较各组间基因型频率、等位基因频率,分析了CKD患者慢性肾脏病及动脉硬化发生的相关性因素。结果(1)在研究人群中G-395A多态性位点共检测出GG、GA、AA 3种基因型,各组符合Hardy-Weinberg平衡检验,具有群体代表性。(2)慢性肾脏病组G-359A基因频率以及等位基因频率和对照组之间存在差异(P均0.05)。非透析组G-395A基因型分布及等位基因频率与对照组比较差异有统计学意义(P均0.05),血液透析组等位基因频率与对照组等位基因频率存在差异(P0.01)。CKD4-5期GA+AA基因频率及A等位基因频率低于对照组及CKD2-3期组(P均0.05)。(3)CKD2-5期非透析患者动脉硬化组GA+AA基因型频率及A等位基因频率均低于非动脉硬化组(P均0.01),血液透析患者动脉硬化组GA+AA基因型频率及A等位基因频率低于非动脉硬化组(P均0.05)。(4)CKD2-5期非透析患者动脉硬化组与非动脉硬化组患者年龄、糖尿病、磷、i PTH、、e GFR、klotho蛋白差异有统计学意义(P均0.05),基因型间临床指标及kltoho蛋白水平无统计学差异,Logistic回归分析表明在CKD2-5期非透析患者中年龄、糖尿病、e GFR、klotho蛋白、G-395A等位基因(GA+AA)与动脉硬化具有相关性,klotho蛋白与慢性肾脏病的发生具有相关性。(5)血液透析患者组动脉硬化组与非动脉硬化组在年龄、糖尿病、收缩压、hs CRP、FGF23、klotho蛋白方面差异有统计学意义(P0.05),基因型间临床指标及kltoho蛋白水平无统计学差异,Logistic回归分析表明年龄、hs CRP、FGF23、G-395A基因型(GA+AA)与动脉硬化具有相关性。结论(1)在研究人群中存在Klotho基因G-395A多态性。(2)CKD患者血清klotho蛋白水平升高,使慢性肾脏病发生危险度减少,证实血清klotho蛋白水平可能是CKD发生的保护性因素。(3)Klotho基因G-395A位点A等位基因可能是CKD非透析及血透患者动脉硬化发生的遗传保护因素。CKD非透析患者血清klotho蛋白水平的升高,动脉硬化发生的危险度减少,证实血清klotho蛋白水平可能是其动脉硬化发生的保护性因素。(4)年龄、糖尿病、FGF23、e GFR降低是CKD非透析患者动脉硬化发生的危险因素。(5)年龄、hs CRP、FGF23是CKD血透患者动脉硬化发生的危险因素。目的观察慢性肾脏病不同阶段klotho蛋白、FGF23水平的变化,探讨其与矿物质代谢及动脉硬化的关系。方法选取慢性肾脏病患者163例,根据e GFR分组CKD2期23例,CKD3期32例、CKD4期30例,CKD5期78例,CKD5期分为非透析组41例,血透组37例。CKD2-5期非透析患者经颈部彩超检测颈动脉内膜厚度分为动脉硬化组54例,非动脉硬化组72例。另设健康对照组33例。应用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测klotho蛋白及FGF23水平,统计学分析klotho蛋白、FGF23与矿物质代谢及动脉硬化的关系。结果(1)慢性肾脏病非透析患者中,klotho蛋白水平均较对照组降低(P0.01),CKD2期及3期组klotho蛋白水平均高于CKD4、5期组(P0.01);CKD3-5期组患者FGF23、i PTH水平均较对照组升高(P0.01),CKD2期-5期各组FGF23、i PTH水平存在统计学差异(P0.01);相关性分析表明klotho蛋白与FGF23(r=-0.823,P0.01)、钙磷乘积(r=-0.697,P0.01)、血磷(r=-0.785,P0.01)、i PTH(r=-0.692,P0.01)均呈负相关关系,与血钙呈正相关关系(r=-0.739,P0.01)。校正年龄、性别及e GFR后,klotho仍与年龄、e GFR呈独立相关关系。FGF23与i PTH(r=0.813,P0.01)、血磷(r=0.838,P0.01)、钙磷乘积(r=0.794,P0.01)均呈正相关关系,与血钙呈负相关关系(r=-0.675,P0.01)。(2)CKD5期非透析组klotho蛋白高于血液透析患者,有统计学意义(P0.01),FGF23高于血液透析组,无统计学意义(P0.05),i PTH高于血透组有统计学意义(P0.01),血钙、磷均低于血液透析组,有统计学意义(P0.01),钙磷乘积低于血透组(P0.01)。(3)应用LOESS观察CKD2-5期患者(非透析)血清klotho蛋白、FGF23、i PTH、血钙、磷与e GFR的相关性,发现血清klotho蛋白与e GFR呈线性相关关系,e GFR每下降1ml/min/1.73m2,血清klotho下降5.35pg/ml(95%CI5.02-5.68pg/ml,P0.01)。校正年龄后,e GFR每下降1ml/min/1.73m2,血清klotho蛋白降低5.31pg/ml(95%CI5.01-5.64pg/ml,P0.01)。血清FGF23、i PTH、血磷均与e GFR呈非线性相关。在CKD2期血清FGF23水平无明显变化,在CKD2-3期i PTH和血磷变化不显著。当e GFR下降至42ml/min/1.73m2时血FGF23出现显著升高,而血i PTH当e GFR下降至28ml/min/1.73m2时、血磷当e GFR下降至28ml/min/1.73m2时出现升高显著。(4)CKD2-5期非透析患者动脉硬化组年龄、BMI、HGB、FGF23、i PTH高于非动脉硬化组,有统计学意义(P0.05),而血清klotho蛋白明显低于非动脉硬化组,有统计学意义(P0.05)。行Logistic回归分析,结果显示klotho蛋白(OR=0.988,95%CI0.978-0.998,P0.05)和FGF23(OR=1.086,95%CI 1.019-1.056,P0.05)与CKD患者动脉硬化发生具有相关性。结论(1)CKD2-5期非透析患者血清FGF23的升高早于血磷和血i PTH的升高,而血清klotho蛋白在血清FGF23升高之前出现下降,因此推断klotho蛋白可能是CKD矿物质代谢紊乱的早期生物学标记物。(2)Klotho蛋白、FGF23可能是CKD2-5期非透析患者动脉硬化的影响因素,klotho蛋白是保护性因素,而FGF23是独立危险因素。
[Abstract]:Objective to study the distribution of single nucleotide polymorphisms of Klotho gene G-395A in patients with chronic renal disease (CKD), and to explore the correlation between the polymorphic loci and protein levels of the gene and the chronic renal disease and arteriosclerosis. Methods the FQ-PCR technique (Taqman probe) was used to study the subjects [including 156 patients with slow kidney disease (including non dialysate group, CKD2-3). 56 cases, 60 cases of CKD4-5 stage, 40 cases of hemodialysis group and healthy control group (60 cases) were divided into G-359A polymorphism of Klotho Gene, the level of Klotho protein was detected, carotid ultrasonography was used to group the arteriosclerosis group, and the genotype frequency and allele frequency of each group were compared, and the correlation factors of chronic kidney disease and arteriosclerosis in CKD patients were analyzed. Results (1) 3 genotypes of GG, GA, and AA were detected in the G-395A polymorphic loci in the study population. Each group accords with the Hardy-Weinberg balance test. (2) there is a difference between the frequency of G-359A gene and the allele frequency of the chronic kidney disease group and the control group (P all 0.05). The distribution of G-395A genotypes and the allele frequency of non dialysis group G-395A Compared with the control group, the difference was statistically significant (P 0.05). The frequency of allele in hemodialysis group was different from that of control group (P0.01), the frequency of GA+AA gene in.CKD4-5 phase and A allele frequency were lower than that of control group and CKD2-3 group (P 0.05). (3) the frequency of GA+AA genotype and A in non dialysis patients with non dialysis patients, and A and so on. The frequency of the position gene was lower than that in the non arteriosclerosis group (P 0.01). The frequency of GA+AA genotype and the A allele frequency in the arteriosclerosis group were lower than those in the non arteriosclerosis group (P 0.05). (4) the age of the patients with non dialysate and non arteriosclerosis group in the CKD2-5 phase of non dialysis patients, diabetes, phosphorus, I PTH, e GFR, Klotho protein differences were statistically significant Meaning (P 0.05), there was no statistical difference between the clinical index of genotypes and the level of kltoho protein. Logistic regression analysis showed that age, diabetes, e GFR, Klotho protein, G-395A allele (GA+AA) were associated with arteriosclerosis in CKD2-5 phase, and Klotho protein was associated with the occurrence of chronic kidney disease. (5) hemodialysis patients There were significant differences in age, diabetes, systolic pressure, HS CRP, FGF23, Klotho protein in the group of arteriosclerosis and non arteriosclerosis (P0.05). There was no statistical difference between the clinical indexes of the genotype and the level of kltoho protein. Logistic regression analysis showed that age, HS CRP, FGF23, G-395A genotypes (GA+AA) were associated with arteriosclerosis. Conclusion (1) there is a polymorphism of Klotho gene G-395A in the study population. (2) the increase of serum Klotho protein level in CKD patients reduces the risk of chronic renal disease, and the level of serum Klotho protein may be a protective factor for the occurrence of CKD. (3) the A allele of the Klotho gene G-395A site may be the arteriosclerosis of non dialysis and hemodialysis patients in CKD. The genetic protection factor,.CKD non dialysis patients, increased the level of serum Klotho protein, decreased the risk of arteriosclerosis, and confirmed that serum Klotho protein level may be a protective factor for its arteriosclerosis. (4) age, diabetes, FGF23, e GFR decrease is a risk factor for the occurrence of arteriosclerosis in non dialysis patients with CKD. (5) age, H S CRP, FGF23 is a risk factor for arteriosclerosis in CKD hemodialysis patients. Objective To observe the changes of Klotho protein and FGF23 level in different stages of chronic kidney disease, to explore the relationship between the mineral metabolism and arteriosclerosis. Methods 163 patients with chronic kidney disease were selected, according to e GFR group CKD2 stage 23 cases, CKD3 period 32 cases, CKD4 phase 30 cases, CKD5 period 78 cases. Phase D5 was divided into 41 non dialysis patients. 37 non dialysis patients in the hemodialysis group were divided into 54 cases of arteriosclerosis, 72 cases of non arteriosclerosis group, 72 cases of non arteriosclerosis and 33 cases of healthy control group. The level of Klotho protein and FGF23 was detected by enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA), and the statistics of Klotho protein and FGF23 were measured. The relationship between Klotho protein, FGF23 and mineral metabolism and arteriosclerosis was analyzed. Results (1) the level of Klotho protein in non dialysis patients with chronic renal disease was lower than that in the control group (P0.01). The level of Klotho protein in CKD2 and 3 groups was higher than that in the CKD4,5 group (P0.01), and FGF23 and I PTH were all higher in the CKD3-5 group than those in the control group. The levels of FGF23 and I PTH were statistically different in each group (P0.01), and the correlation analysis showed that Klotho protein and FGF23 (r=-0.823, P0.01), calcium and phosphorus product (r=-0.697, P0.01), blood phosphorus (r=-0.785, P0.01) were all negative correlation and positive correlation with blood calcium. Age, e GFR was independent of.FGF23 and I PTH (r=0.813, P0.01), blood phosphorus (r=0.838, P0.01), calcium and phosphorus product (r=0.794, P0.01) were positively correlated, and had a negative correlation with blood calcium (r=-0.675,). (2) the non dialysis group was higher than the hemodialysis patients, which was higher than the hemodialysis group. Significance (P0.05), I PTH was higher than that in hemodialysis group (P0.01), blood calcium and phosphorus were lower than that in hemodialysis group (P0.01), and the product of calcium and phosphorus was lower than that of hemodialysis group (P0.01). (3) the correlation between serum Klotho protein, FGF23, I PTH, blood calcium and phosphorus was observed by LOESS. E GFR decreased 1ml/min/1.73m2 and serum Klotho decreased 5.35pg/ml (95%CI5.02-5.68pg/ml, P0.01). After correction of age, e GFR decreased every 1ml/min/1.73m2, serum Klotho protein reduced 5.31pg/ml. There was no significant change in I PTH and blood phosphorus in CKD2-3 phase. When e GFR dropped to 42ml/min/1.73m2, the blood FGF23 increased significantly, while the blood I PTH decreased to 28ml/min/1.73m2 when e GFR decreased to 28ml/min/1.73m2. The vein sclerosis group had statistical significance (P0.05), while the serum Klotho protein was significantly lower than the non arteriosclerosis group, with statistical significance (P0.05). Logistic regression analysis showed that the Klotho protein (OR=0.988,95%CI0.978-0.998, P0.05) and FGF23 (OR=1.086,95%CI 1.019-1.056, P0.05) were associated with the occurrence of atherosclerosis in the CKD patients. Conclusion (1) The increase of serum FGF23 in non dialysis patients in phase -5 was earlier than that of blood phosphorus and blood I PTH, while serum Klotho protein decreased before serum FGF23 increased. Therefore, it is concluded that Klotho protein may be an early biomarker for the metabolic disorder of CKD. (2) Klotho protein, FGF23 may be the influencing factor of arteriosclerosis in the CKD2-5 phase of non dialysis patients. Klotho protein is a protective factor, while FGF23 is an independent risk factor.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R692;R543.5

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