本文選題：Klotho基因 + 單核苷酸多態(tài)性　； 參考：《青島大學》2015年博士論文

【摘要】：目的研究慢性腎臟病患者Klotho基因G-395A單核苷酸多態(tài)性的分布,探討該基因多態(tài)性位點及蛋白水平與慢性腎臟病及動脈硬化的相關(guān)性。方法運用FQ-PCR技術(shù)(Taqman探針法)對各研究對象[包括156例慢性腎臟病患者(其中非透析組包括CKD2-3期56例、CKD4-5期60例,血透組40例)和健康對照組(60例)]進行klotho基因G-359A多態(tài)性分型,檢測klotho蛋白水平,頸動脈超聲檢查進行動脈硬化分組,比較各組間基因型頻率、等位基因頻率,分析了CKD患者慢性腎臟病及動脈硬化發(fā)生的相關(guān)性因素。結(jié)果(1)在研究人群中G-395A多態(tài)性位點共檢測出GG、GA、AA 3種基因型,各組符合Hardy-Weinberg平衡檢驗,具有群體代表性。(2)慢性腎臟病組G-359A基因頻率以及等位基因頻率和對照組之間存在差異(P均0.05)。非透析組G-395A基因型分布及等位基因頻率與對照組比較差異有統(tǒng)計學意義(P均0.05),血液透析組等位基因頻率與對照組等位基因頻率存在差異(P0.01)。CKD4-5期GA+AA基因頻率及A等位基因頻率低于對照組及CKD2-3期組(P均0.05)。(3)CKD2-5期非透析患者動脈硬化組GA+AA基因型頻率及A等位基因頻率均低于非動脈硬化組(P均0.01),血液透析患者動脈硬化組GA+AA基因型頻率及A等位基因頻率低于非動脈硬化組(P均0.05)。(4)CKD2-5期非透析患者動脈硬化組與非動脈硬化組患者年齡、糖尿病、磷、i PTH、、e GFR、klotho蛋白差異有統(tǒng)計學意義(P均0.05),基因型間臨床指標及kltoho蛋白水平無統(tǒng)計學差異,Logistic回歸分析表明在CKD2-5期非透析患者中年齡、糖尿病、e GFR、klotho蛋白、G-395A等位基因(GA+AA)與動脈硬化具有相關(guān)性,klotho蛋白與慢性腎臟病的發(fā)生具有相關(guān)性。(5)血液透析患者組動脈硬化組與非動脈硬化組在年齡、糖尿病、收縮壓、hs CRP、FGF23、klotho蛋白方面差異有統(tǒng)計學意義(P0.05),基因型間臨床指標及kltoho蛋白水平無統(tǒng)計學差異,Logistic回歸分析表明年齡、hs CRP、FGF23、G-395A基因型(GA+AA)與動脈硬化具有相關(guān)性。結(jié)論(1)在研究人群中存在Klotho基因G-395A多態(tài)性。(2)CKD患者血清klotho蛋白水平升高,使慢性腎臟病發(fā)生危險度減少,證實血清klotho蛋白水平可能是CKD發(fā)生的保護性因素。(3)Klotho基因G-395A位點A等位基因可能是CKD非透析及血透患者動脈硬化發(fā)生的遺傳保護因素。CKD非透析患者血清klotho蛋白水平的升高,動脈硬化發(fā)生的危險度減少,證實血清klotho蛋白水平可能是其動脈硬化發(fā)生的保護性因素。(4)年齡、糖尿病、FGF23、e GFR降低是CKD非透析患者動脈硬化發(fā)生的危險因素。(5)年齡、hs CRP、FGF23是CKD血透患者動脈硬化發(fā)生的危險因素。目的觀察慢性腎臟病不同階段klotho蛋白、FGF23水平的變化,探討其與礦物質(zhì)代謝及動脈硬化的關(guān)系。方法選取慢性腎臟病患者163例,根據(jù)e GFR分組CKD2期23例,CKD3期32例、CKD4期30例,CKD5期78例,CKD5期分為非透析組41例,血透組37例。CKD2-5期非透析患者經(jīng)頸部彩超檢測頸動脈內(nèi)膜厚度分為動脈硬化組54例,非動脈硬化組72例。另設健康對照組33例。應用酶聯(lián)免疫吸附試驗(enzyme-linked immunosorbent assay,ELISA)檢測klotho蛋白及FGF23水平,統(tǒng)計學分析klotho蛋白、FGF23與礦物質(zhì)代謝及動脈硬化的關(guān)系。結(jié)果(1)慢性腎臟病非透析患者中,klotho蛋白水平均較對照組降低(P0.01),CKD2期及3期組klotho蛋白水平均高于CKD4、5期組(P0.01);CKD3-5期組患者FGF23、i PTH水平均較對照組升高(P0.01),CKD2期-5期各組FGF23、i PTH水平存在統(tǒng)計學差異(P0.01);相關(guān)性分析表明klotho蛋白與FGF23(r=-0.823,P0.01)、鈣磷乘積(r=-0.697,P0.01)、血磷(r=-0.785,P0.01)、i PTH(r=-0.692,P0.01)均呈負相關(guān)關(guān)系,與血鈣呈正相關(guān)關(guān)系(r=-0.739,P0.01)。校正年齡、性別及e GFR后,klotho仍與年齡、e GFR呈獨立相關(guān)關(guān)系。FGF23與i PTH(r=0.813,P0.01)、血磷(r=0.838,P0.01)、鈣磷乘積(r=0.794,P0.01)均呈正相關(guān)關(guān)系,與血鈣呈負相關(guān)關(guān)系(r=-0.675,P0.01)。(2)CKD5期非透析組klotho蛋白高于血液透析患者,有統(tǒng)計學意義(P0.01),FGF23高于血液透析組,無統(tǒng)計學意義(P0.05),i PTH高于血透組有統(tǒng)計學意義(P0.01),血鈣、磷均低于血液透析組,有統(tǒng)計學意義(P0.01),鈣磷乘積低于血透組(P0.01)。(3)應用LOESS觀察CKD2-5期患者(非透析)血清klotho蛋白、FGF23、i PTH、血鈣、磷與e GFR的相關(guān)性,發(fā)現(xiàn)血清klotho蛋白與e GFR呈線性相關(guān)關(guān)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)。校正年齡后,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、血磷均與e GFR呈非線性相關(guān)。在CKD2期血清FGF23水平無明顯變化,在CKD2-3期i PTH和血磷變化不顯著。當e GFR下降至42ml/min/1.73m2時血FGF23出現(xiàn)顯著升高,而血i PTH當e GFR下降至28ml/min/1.73m2時、血磷當e GFR下降至28ml/min/1.73m2時出現(xiàn)升高顯著。(4)CKD2-5期非透析患者動脈硬化組年齡、BMI、HGB、FGF23、i PTH高于非動脈硬化組,有統(tǒng)計學意義(P0.05),而血清klotho蛋白明顯低于非動脈硬化組,有統(tǒng)計學意義(P0.05)。行Logistic回歸分析,結(jié)果顯示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)與CKD患者動脈硬化發(fā)生具有相關(guān)性。結(jié)論(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升高之前出現(xiàn)下降,因此推斷klotho蛋白可能是CKD礦物質(zhì)代謝紊亂的早期生物學標記物。(2)Klotho蛋白、FGF23可能是CKD2-5期非透析患者動脈硬化的影響因素,klotho蛋白是保護性因素,而FGF23是獨立危險因素。
[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.

【學位授予單位】：青島大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R692;R543.5

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