基于Klotho基因多態(tài)性對(duì)糖尿病腎病中醫(yī)證候的研究
發(fā)布時(shí)間：2018-08-19 18:18
【摘要】：糖尿病腎病(DN)是糖尿病最嚴(yán)重的微血管并發(fā)癥之一,目前認(rèn)為遺傳背景在DN的發(fā)生發(fā)展中扮演著重要角色。因此,從遺傳學(xué)角度闡述DN及其風(fēng)險(xiǎn)因素對(duì)疾病的診斷和臨床治療均意義重大。目的研究Klotho基因多態(tài)性與2型糖尿病、糖尿病腎病發(fā)病風(fēng)險(xiǎn)及糖尿病腎病患者中醫(yī)癥狀、證候分型之間的關(guān)聯(lián),篩選可能的易感基因型,以期對(duì)潛在的高�；颊呒皶r(shí)進(jìn)行針對(duì)性的防治;在第一部分研究結(jié)果基礎(chǔ)上,進(jìn)一步系統(tǒng)評(píng)價(jià)中醫(yī)藥治療糖尿病腎病的療效及安全性。方法1、本研究共采集分析了178例受試者,均為湖北地區(qū)漢族人群,其中病例組123例(2型糖尿病患者56例,2型糖尿病腎病患者67例),健康對(duì)照組55例。對(duì)受試者進(jìn)行一般資料的采集、中醫(yī)證候?qū)W觀察,運(yùn)用離心柱法提取血液基因組DNA。通過美國(guó)國(guó)家生物信息中心的Gen Bank獲取Klotho基因信息及啟動(dòng)子區(qū)域G-395A及外顯子區(qū)域F352V、C370S的序列。采用PCR聯(lián)合直接測(cè)序技術(shù)檢測(cè)上述位點(diǎn)單核苷酸多態(tài)性(SNP)。SPSS軟件統(tǒng)計(jì)分析Klotho SNP在2型糖尿病、糖尿病腎病人群中的基因型、等位基因分布特征,并探討Klotho SNP與糖尿病,糖尿病腎病的發(fā)病有無相關(guān)性,進(jìn)一步分析糖尿病腎病患者Klotho基因多態(tài)性與主要中醫(yī)癥狀及各證候分型之間的關(guān)系。2、電子檢索外文數(shù)據(jù)庫Pub Med、EMBASE、Cochrane圖書館,及相關(guān)期刊論文、中國(guó)生物醫(yī)學(xué)文獻(xiàn)數(shù)據(jù)庫、萬方數(shù)據(jù)庫、維普數(shù)據(jù)庫,并輔以手工檢索,全面收集溫陽活血利水法治療糖尿病腎病的隨機(jī)對(duì)照臨床研究,檢索時(shí)間截止于2016年4月1日,對(duì)符合標(biāo)準(zhǔn)的文獻(xiàn)采用Rev Man5.3軟件進(jìn)行Meta分析。1、Klotho基因G-395A位點(diǎn)共檢測(cè)出三種基因型,F352V與C370S僅檢測(cè)出兩種基因型。2、G-395A基因型及等位基因分布頻率在病例組和健康對(duì)照組間無統(tǒng)計(jì)學(xué)差異(X2=1.197,P=0.274;X2=1.083 P=0.298);而在2型糖尿病組和糖尿病腎病組間比較,差異有統(tǒng)計(jì)學(xué)意義(X2=5.016,P=0.025,OR=2.475,95%CI:1.108~5.528;X2=5.872,P=0.015,OR=2.404,95%CI:1.166~4.956);糖尿病腎病患者G-395A基因型分布頻率與Mogensen分期無關(guān)(Z=0.123,P0.05);Logistic回歸分析結(jié)果:攜帶A等位基因、高血壓病史、糖尿病病程及糖化血紅蛋白與DN的發(fā)生有統(tǒng)計(jì)學(xué)關(guān)聯(lián)(OR依次為1.774、2.198、1.735、1.306,95%CI依次為1.195-2.635、1.330-3.632、1.183-2.548、1.022-1.671)。3、糖尿病腎病組三種中醫(yī)證候分型間G-395A的基因型及等位基因頻率分布有顯著差別,具有統(tǒng)計(jì)學(xué)意義(X2=8.700,P=0.013;X2=6.591P=0.037);其中GA+AA基因型、A等位基因分布頻率均為:陰陽俱虛型氣陰兩虛型脾腎陽虛型;每?jī)山M基因型GA+AA的分布進(jìn)行比較,氣陰兩虛組與脾腎陽虛組比較差異有統(tǒng)計(jì)學(xué)意義(X2=6.551,P=0.01),陰陽俱虛組與脾腎陽虛組比較差異有統(tǒng)計(jì)學(xué)意義(X2=6.866,P0.01),而氣陰兩虛組與陰陽俱虛組比較,差別無統(tǒng)計(jì)學(xué)意義(X2=0.015,P0.05);每?jī)山MG-395A等位基因頻率分布進(jìn)行比較,氣陰兩虛組與脾腎陽虛組比較差異有統(tǒng)計(jì)學(xué)意義(X2=4.73,P0.05),陰陽俱虛組與脾腎陽虛組比較差異有統(tǒng)計(jì)學(xué)意義(X2=4.86,P0.05),而氣陰兩虛組與陰陽俱虛組比較,差別無統(tǒng)計(jì)學(xué)意義(P0.05)4、不同基因型、攜帶不同等位基因的DN患者水腫輕重程度有差別,差異有統(tǒng)計(jì)學(xué)意義(Z=2.951,P=0.003;Z=2.549,P=0.011),并未發(fā)現(xiàn)G-395A多態(tài)性與乏力、腰膝酸軟、納差脘痞這些癥狀的輕重存在統(tǒng)計(jì)學(xué)關(guān)聯(lián)(P0.05)。結(jié)果5、共納入16個(gè)臨床隨機(jī)對(duì)照試驗(yàn),合計(jì)1107例糖尿病腎病患者。Meta分析結(jié)果顯示:溫陽活血利水法為主的中西醫(yī)結(jié)合試驗(yàn)組治療糖尿病腎病顯效率、總有效率優(yōu)于西醫(yī)常規(guī)對(duì)照組(RR=1.97,95%CI 1.50~2.59,Z=4.86,P0.00001;RR=1.48,95%CI 1.35~1.62,Z=8.58,P0.00001);試驗(yàn)組較對(duì)照組降低血肌酐、尿素氮更為明顯(MD=-32.33,95%CI-58.20~-6.47,Z=2.45,P=0.01;MD=-2.20,95%CI-4.13~-0.26,Z=2.22,P=0.03);升高高密度脂蛋白效果更好(MD=0.16,95%CI 0.09~0.23,Z=4.33,P0.0001);降低膽固醇、甘油三酯、低密度脂蛋白效果更為明顯(MD=-0.82,95%CI-1.28~-0.36,Z=3.49,P=0.0005;MD=-0.40,95%CI-0.53~-0.26,Z=5.95,P0.0001;MD=-0.49,95%CI-0.76~-0.21,Z=3.46,P=0.0005);減少尿蛋白效果更明顯(MD=-0.46,95%CI-0.61~-0.31,Z=6.01,P0.00001);在控制空腹血糖及改善糖化血紅蛋白方面更明顯(MD=-0.34,95%CI-0.57~-0.12,Z=3.00,P=0.003;MD=-0.31,95%CI-0.50~-0.12,Z=3.15,P=0.002);而在降低腫瘤壞死因子-α水平方面療效相當(dāng)(P=0.33)。結(jié)論1、G-395A三種基因型在湖北地區(qū)漢族人群中的分布具有良好的群體代表性,F352V與C370S多態(tài)性較為罕見。2、本課題未發(fā)現(xiàn)G-395A位點(diǎn)多態(tài)性與2型糖尿病的發(fā)病風(fēng)險(xiǎn)存在關(guān)聯(lián)性;Klotho G-395A多態(tài)性與糖尿病腎病病情輕重程度無關(guān);A等位基因的出現(xiàn)可能增加了2型糖尿病患者發(fā)生腎損害的風(fēng)險(xiǎn),攜帶A等位基因可能是湖北地區(qū)漢族人群2型糖尿病患者合并腎臟并發(fā)癥的獨(dú)立危險(xiǎn)因素之一,而雜合型GA及純合突變AA基因型可能是糖尿病腎病的遺傳易感危險(xiǎn)性基因型。同時(shí),糖化血紅蛋白、糖尿病病程、高血壓病史可能是2型糖尿病腎病發(fā)病的獨(dú)立危險(xiǎn)因素。3、GA、AA基因型可能是脾腎陽虛的糖尿病腎病患者的易感基因型;攜帶A等位基因的糖尿病腎病患者可能更易演變?yōu)槠⒛I陽虛,攜帶A等位基因可能是脾腎陽虛型糖尿病腎病的風(fēng)險(xiǎn)因素之一。4、GA、AA基因型可能是水腫程度較重的糖尿病腎病患者的易感基因型;攜帶A等位基因的糖尿病腎病患者可能更易出現(xiàn)水腫加重。5、溫陽活血利水法為主的中西醫(yī)結(jié)合試驗(yàn)組治療糖尿病腎病臨床綜合療效顯著優(yōu)于西醫(yī)常規(guī)治療對(duì)照組。具體表現(xiàn)在不僅能降低血肌酐、尿素氮水平,還能減少尿蛋白、調(diào)節(jié)脂類代謝異常,改善血糖;但在改善炎癥狀態(tài)方面療效相當(dāng)。中西醫(yī)結(jié)合治療安全性較好,值得在臨床推廣應(yīng)用。
[Abstract]:Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes mellitus. It is considered that genetic background plays an important role in the genesis and development of DN. Therefore, it is of great significance to elucidate DN and its risk factors in the diagnosis and clinical treatment of the disease from the genetic perspective. The association between the risk of diabetic nephropathy and the symptoms and syndrome types of diabetic nephropathy patients was studied, and the possible susceptible genotypes were screened for timely targeted prevention and treatment of potential high-risk patients. A total of 178 subjects were collected and analyzed, including 123 cases (56 patients with type 2 diabetes mellitus and 67 patients with type 2 diabetic nephropathy) and 55 healthy controls. The subjects were collected for general information, observed for TCM syndromes, and genomic DNA was extracted by centrifugal column method. Gen Bank of information center obtained Klotho gene information and sequence of promoter region G-395A and exon region F352V, C370S. Single nucleotide polymorphism (SNP) was detected by PCR and direct sequencing. SPSS software was used to analyze the genotype and allele distribution of Klotho SNP in type 2 diabetes mellitus and diabetic nephropathy. To explore the relationship between Klotho SNP and diabetes mellitus and diabetic nephropathy, and to further analyze the relationship between Klotho gene polymorphism and the main symptoms and syndrome types of diabetic nephropathy. Three genotypes of Klotho gene G-395A locus were detected by using Rev Man 5.3 software for meta-analysis. F352V and C370S only detected two genotypes.2, G-395A genotype and allele distribution frequencies were not significantly different between the case group and the healthy control group (X2 = 1.197, P = 0.274; X2 = 1.083 P = 0.298); however, there were significant differences between the type 2 diabetes mellitus group and the diabetic nephropathy group (X2 = 5.016, P = 0.025, OR = 2.475, 95% CI: 1.108-5.528; X2 = 5.083 P = 5.528). 872, P = 0.015, OR = 2.404, 95% CI: 1.166-4.956; G-395A genotype distribution frequency in diabetic nephropathy patients was not associated with Mogensen stage (Z = 0.123, P 0.05); Logistic regression analysis showed that carrying allele A, hypertension history, diabetes duration and glycosylated hemoglobin were statistically associated with DN (OR: 1.774, 2.198, 1.735, 1.306, 95% C, respectively). I was 1.195-2.635, 1.330-3.632, 1.183-2.548, 1.022-1.671). 3. The genotype and allele frequencies of G-395A were significantly different among the three TCM syndromes of diabetic nephropathy group (X2 = 8.700, P = 0.013; X2 = 6.591P = 0.037); and the gene frequencies of GA + AA and A alleles were all: deficiency of both yin and Yang type of Qi and yin. The distribution of GA + AA genotype of deficiency of both spleen and Kidney Yang was compared between the two groups. The difference between deficiency of both qi and Yin and deficiency of spleen and Kidney Yang was statistically significant (X2 = 6.551, P = 0.01). The difference between deficiency of both yin and Yang and deficiency of spleen and Kidney Yang was statistically significant (X2 = 6.866, P 0.01). X2 = 0.015, P 0.05); G-395A allele frequency distribution of each two groups were compared, the difference between Qi-yin deficiency group and spleen-kidney Yang deficiency group was statistically significant (X2 = 4.73, P 0.05), the difference between Yin-yang deficiency group and spleen-kidney Yang deficiency group was statistically significant (X2 = 4.86, P 0.05), and the difference between Qi-yin deficiency group and yin-yang deficiency group was not statistically significant (P 0.05). 4. There was a significant difference in the degree of edema among DN patients with different genotypes and alleles (Z = 2.951, P = 0.003; Z = 2.549, P = 0.011). There was no significant correlation between G-39A polymorphism and the severity of symptoms such as fatigue, lumbar and knee soreness, and abnormal appetite (P 0.05). Results 5, 16 randomized controlled trials were included. A total of 1107 patients with diabetic nephropathy were enrolled in this study. The results of Meta-analysis showed that the experimental group mainly treated with Wenyang Huoxue Lishui was more effective than the control group (RR = 1.97,95% CI 1.50-2.59, Z = 4.86, P 0.00001; RR = 1.48,95% CI 1.35-1.62, Z = 8.58, P 0.00001); and the experimental group decreased the blood and muscle compared with the control group. MD = - 32.33, 95% CI - 58.20 - 6.47, Z = 2.45, P = 0.01; MD = - 2.20, 95% CI - 4.13 - 0.26, Z = 2.22, P = 0.03; MD = - 32.33, 95% CI - 58.20 - 6.47, Z = 2.45, P = 2.45, P = 0.01; MD = - 2.20, 95% CI - 4.13 - 0.26, Z = 2.22, Z = 2.22, P = 0.03); higher HD-HD-LDwas better (MD = 0.16, 95% CI 0.16, 95% CI 0.09-0.09-0.23, Z = 4.33, Z = 4.33, P 0.0001); lower cholestero, triZ = 3.49, P = 0.0005; MD = - 0 40,95% CI-0.53-0.53-0.26,Z=5.95,P=5.95,P 0.00001;MD=-0.49,95% CI-0.76-0.21,Z=3.46,P=0.0005;MD=-0.46,95% CI-0.53-0.53-0.26,Z=5.53-0.25,Z=5.95% CI-0.53-0.25,P=0.000 1;MD=-0.40,95% CI-0.49,95% CI-0.95% CI-0.76-0.76-0.21,Z=3.46,P=0.0005;urinprotein reduction effect was more obvious (MD =-0.46,95% CI-0.46,=-0.31, 95% CI-0.50-0.12, Z = 3.15, P = 0.002) Conclusion 1. The distribution of G-395A genotypes in Hubei Han population is well representative, F352V and C370S polymorphisms are rare. 2. There is no association between G-395A polymorphisms and the risk of type 2 diabetes mellitus. 95A polymorphism is not associated with the severity of diabetic nephropathy; the presence of allele A may increase the risk of kidney damage in type 2 diabetes mellitus; carrying allele A may be an independent risk factor for renal complications in type 2 diabetes mellitus in Hubei Han population, while heterozygous GA and homozygous AA genotype may be. At the same time, glycosylated hemoglobin, duration of diabetes and history of hypertension may be independent risk factors for type 2 diabetic nephropathy. 3, GA and AA genotypes may be susceptible genotypes of diabetic nephropathy patients with spleen and kidney yang deficiency, and diabetic nephropathy patients with allele A may be susceptible. A allele may be one of the risk factors of diabetic nephropathy with spleen-kidney Yang deficiency. 4. GA and AA genotypes may be the susceptible genotypes of diabetic nephropathy with severe edema. Diabetic nephropathy patients with A allele may be more susceptible to edema aggravation. 5. Warming Yang, activating blood circulation and eliminating water therapy is the main method. The experimental group of integrated traditional Chinese and Western medicine is better than the control group in the treatment of diabetic nephropathy. It can not only reduce the levels of serum creatinine and urea nitrogen, but also reduce urinary protein, regulate the abnormal metabolism of lipids and improve blood sugar. But it has the same effect in improving inflammation. Good, worthy of clinical application.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R259;R277.5
本文編號(hào)：2192445
[Abstract]:Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes mellitus. It is considered that genetic background plays an important role in the genesis and development of DN. Therefore, it is of great significance to elucidate DN and its risk factors in the diagnosis and clinical treatment of the disease from the genetic perspective. The association between the risk of diabetic nephropathy and the symptoms and syndrome types of diabetic nephropathy patients was studied, and the possible susceptible genotypes were screened for timely targeted prevention and treatment of potential high-risk patients. A total of 178 subjects were collected and analyzed, including 123 cases (56 patients with type 2 diabetes mellitus and 67 patients with type 2 diabetic nephropathy) and 55 healthy controls. The subjects were collected for general information, observed for TCM syndromes, and genomic DNA was extracted by centrifugal column method. Gen Bank of information center obtained Klotho gene information and sequence of promoter region G-395A and exon region F352V, C370S. Single nucleotide polymorphism (SNP) was detected by PCR and direct sequencing. SPSS software was used to analyze the genotype and allele distribution of Klotho SNP in type 2 diabetes mellitus and diabetic nephropathy. To explore the relationship between Klotho SNP and diabetes mellitus and diabetic nephropathy, and to further analyze the relationship between Klotho gene polymorphism and the main symptoms and syndrome types of diabetic nephropathy. Three genotypes of Klotho gene G-395A locus were detected by using Rev Man 5.3 software for meta-analysis. F352V and C370S only detected two genotypes.2, G-395A genotype and allele distribution frequencies were not significantly different between the case group and the healthy control group (X2 = 1.197, P = 0.274; X2 = 1.083 P = 0.298); however, there were significant differences between the type 2 diabetes mellitus group and the diabetic nephropathy group (X2 = 5.016, P = 0.025, OR = 2.475, 95% CI: 1.108-5.528; X2 = 5.083 P = 5.528). 872, P = 0.015, OR = 2.404, 95% CI: 1.166-4.956; G-395A genotype distribution frequency in diabetic nephropathy patients was not associated with Mogensen stage (Z = 0.123, P 0.05); Logistic regression analysis showed that carrying allele A, hypertension history, diabetes duration and glycosylated hemoglobin were statistically associated with DN (OR: 1.774, 2.198, 1.735, 1.306, 95% C, respectively). I was 1.195-2.635, 1.330-3.632, 1.183-2.548, 1.022-1.671). 3. The genotype and allele frequencies of G-395A were significantly different among the three TCM syndromes of diabetic nephropathy group (X2 = 8.700, P = 0.013; X2 = 6.591P = 0.037); and the gene frequencies of GA + AA and A alleles were all: deficiency of both yin and Yang type of Qi and yin. The distribution of GA + AA genotype of deficiency of both spleen and Kidney Yang was compared between the two groups. The difference between deficiency of both qi and Yin and deficiency of spleen and Kidney Yang was statistically significant (X2 = 6.551, P = 0.01). The difference between deficiency of both yin and Yang and deficiency of spleen and Kidney Yang was statistically significant (X2 = 6.866, P 0.01). X2 = 0.015, P 0.05); G-395A allele frequency distribution of each two groups were compared, the difference between Qi-yin deficiency group and spleen-kidney Yang deficiency group was statistically significant (X2 = 4.73, P 0.05), the difference between Yin-yang deficiency group and spleen-kidney Yang deficiency group was statistically significant (X2 = 4.86, P 0.05), and the difference between Qi-yin deficiency group and yin-yang deficiency group was not statistically significant (P 0.05). 4. There was a significant difference in the degree of edema among DN patients with different genotypes and alleles (Z = 2.951, P = 0.003; Z = 2.549, P = 0.011). There was no significant correlation between G-39A polymorphism and the severity of symptoms such as fatigue, lumbar and knee soreness, and abnormal appetite (P 0.05). Results 5, 16 randomized controlled trials were included. A total of 1107 patients with diabetic nephropathy were enrolled in this study. The results of Meta-analysis showed that the experimental group mainly treated with Wenyang Huoxue Lishui was more effective than the control group (RR = 1.97,95% CI 1.50-2.59, Z = 4.86, P 0.00001; RR = 1.48,95% CI 1.35-1.62, Z = 8.58, P 0.00001); and the experimental group decreased the blood and muscle compared with the control group. MD = - 32.33, 95% CI - 58.20 - 6.47, Z = 2.45, P = 0.01; MD = - 2.20, 95% CI - 4.13 - 0.26, Z = 2.22, P = 0.03; MD = - 32.33, 95% CI - 58.20 - 6.47, Z = 2.45, P = 2.45, P = 0.01; MD = - 2.20, 95% CI - 4.13 - 0.26, Z = 2.22, Z = 2.22, P = 0.03); higher HD-HD-LDwas better (MD = 0.16, 95% CI 0.16, 95% CI 0.09-0.09-0.23, Z = 4.33, Z = 4.33, P 0.0001); lower cholestero, triZ = 3.49, P = 0.0005; MD = - 0 40,95% CI-0.53-0.53-0.26,Z=5.95,P=5.95,P 0.00001;MD=-0.49,95% CI-0.76-0.21,Z=3.46,P=0.0005;MD=-0.46,95% CI-0.53-0.53-0.26,Z=5.53-0.25,Z=5.95% CI-0.53-0.25,P=0.000 1;MD=-0.40,95% CI-0.49,95% CI-0.95% CI-0.76-0.76-0.21,Z=3.46,P=0.0005;urinprotein reduction effect was more obvious (MD =-0.46,95% CI-0.46,=-0.31, 95% CI-0.50-0.12, Z = 3.15, P = 0.002) Conclusion 1. The distribution of G-395A genotypes in Hubei Han population is well representative, F352V and C370S polymorphisms are rare. 2. There is no association between G-395A polymorphisms and the risk of type 2 diabetes mellitus. 95A polymorphism is not associated with the severity of diabetic nephropathy; the presence of allele A may increase the risk of kidney damage in type 2 diabetes mellitus; carrying allele A may be an independent risk factor for renal complications in type 2 diabetes mellitus in Hubei Han population, while heterozygous GA and homozygous AA genotype may be. At the same time, glycosylated hemoglobin, duration of diabetes and history of hypertension may be independent risk factors for type 2 diabetic nephropathy. 3, GA and AA genotypes may be susceptible genotypes of diabetic nephropathy patients with spleen and kidney yang deficiency, and diabetic nephropathy patients with allele A may be susceptible. A allele may be one of the risk factors of diabetic nephropathy with spleen-kidney Yang deficiency. 4. GA and AA genotypes may be the susceptible genotypes of diabetic nephropathy with severe edema. Diabetic nephropathy patients with A allele may be more susceptible to edema aggravation. 5. Warming Yang, activating blood circulation and eliminating water therapy is the main method. The experimental group of integrated traditional Chinese and Western medicine is better than the control group in the treatment of diabetic nephropathy. It can not only reduce the levels of serum creatinine and urea nitrogen, but also reduce urinary protein, regulate the abnormal metabolism of lipids and improve blood sugar. But it has the same effect in improving inflammation. Good, worthy of clinical application.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R259;R277.5
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