基于Klotho基因多态性对糖尿病肾病中医证候的研究
发布时间：2018-08-19 18:18
【摘要】：糖尿病肾病(DN)是糖尿病最严重的微血管并发症之一,目前认为遗传背景在DN的发生发展中扮演着重要角色。因此,从遗传学角度阐述DN及其风险因素对疾病的诊断和临床治疗均意义重大。目的研究Klotho基因多态性与2型糖尿病、糖尿病肾病发病风险及糖尿病肾病患者中医症状、证候分型之间的关联,筛选可能的易感基因型,以期对潜在的高危患者及时进行针对性的防治;在第一部分研究结果基础上,进一步系统评价中医药治疗糖尿病肾病的疗效及安全性。方法1、本研究共采集分析了178例受试者,均为湖北地区汉族人群,其中病例组123例(2型糖尿病患者56例,2型糖尿病肾病患者67例),健康对照组55例。对受试者进行一般资料的采集、中医证候学观察,运用离心柱法提取血液基因组DNA。通过美国国家生物信息中心的Gen Bank获取Klotho基因信息及启动子区域G-395A及外显子区域F352V、C370S的序列。采用PCR联合直接测序技术检测上述位点单核苷酸多态性(SNP)。SPSS软件统计分析Klotho SNP在2型糖尿病、糖尿病肾病人群中的基因型、等位基因分布特征,并探讨Klotho SNP与糖尿病,糖尿病肾病的发病有无相关性,进一步分析糖尿病肾病患者Klotho基因多态性与主要中医症状及各证候分型之间的关系。2、电子检索外文数据库Pub Med、EMBASE、Cochrane图书馆,及相关期刊论文、中国生物医学文献数据库、万方数据库、维普数据库,并辅以手工检索,全面收集温阳活血利水法治疗糖尿病肾病的随机对照临床研究,检索时间截止于2016年4月1日,对符合标准的文献采用Rev Man5.3软件进行Meta分析。1、Klotho基因G-395A位点共检测出三种基因型,F352V与C370S仅检测出两种基因型。2、G-395A基因型及等位基因分布频率在病例组和健康对照组间无统计学差异(X2=1.197,P=0.274;X2=1.083 P=0.298);而在2型糖尿病组和糖尿病肾病组间比较,差异有统计学意义(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);糖尿病肾病患者G-395A基因型分布频率与Mogensen分期无关(Z=0.123,P0.05);Logistic回归分析结果:携带A等位基因、高血压病史、糖尿病病程及糖化血红蛋白与DN的发生有统计学关联(OR依次为1.774、2.198、1.735、1.306,95%CI依次为1.195-2.635、1.330-3.632、1.183-2.548、1.022-1.671)。3、糖尿病肾病组三种中医证候分型间G-395A的基因型及等位基因频率分布有显著差别,具有统计学意义(X2=8.700,P=0.013;X2=6.591P=0.037);其中GA+AA基因型、A等位基因分布频率均为:阴阳俱虚型气阴两虚型脾肾阳虚型;每两组基因型GA+AA的分布进行比较,气阴两虚组与脾肾阳虚组比较差异有统计学意义(X2=6.551,P=0.01),阴阳俱虚组与脾肾阳虚组比较差异有统计学意义(X2=6.866,P0.01),而气阴两虚组与阴阳俱虚组比较,差别无统计学意义(X2=0.015,P0.05);每两组G-395A等位基因频率分布进行比较,气阴两虚组与脾肾阳虚组比较差异有统计学意义(X2=4.73,P0.05),阴阳俱虚组与脾肾阳虚组比较差异有统计学意义(X2=4.86,P0.05),而气阴两虚组与阴阳俱虚组比较,差别无统计学意义(P0.05)4、不同基因型、携带不同等位基因的DN患者水肿轻重程度有差别,差异有统计学意义(Z=2.951,P=0.003;Z=2.549,P=0.011),并未发现G-395A多态性与乏力、腰膝酸软、纳差脘痞这些症状的轻重存在统计学关联(P0.05)。结果5、共纳入16个临床随机对照试验,合计1107例糖尿病肾病患者。Meta分析结果显示:温阳活血利水法为主的中西医结合试验组治疗糖尿病肾病显效率、总有效率优于西医常规对照组(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);试验组较对照组降低血肌酐、尿素氮更为明显(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);降低胆固醇、甘油三酯、低密度脂蛋白效果更为明显(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);减少尿蛋白效果更明显(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红蛋白方面更明显(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);而在降低肿瘤坏死因子-α水平方面疗效相当(P=0.33)。结论1、G-395A三种基因型在湖北地区汉族人群中的分布具有良好的群体代表性,F352V与C370S多态性较为罕见。2、本课题未发现G-395A位点多态性与2型糖尿病的发病风险存在关联性;Klotho G-395A多态性与糖尿病肾病病情轻重程度无关;A等位基因的出现可能增加了2型糖尿病患者发生肾损害的风险,携带A等位基因可能是湖北地区汉族人群2型糖尿病患者合并肾脏并发症的独立危险因素之一,而杂合型GA及纯合突变AA基因型可能是糖尿病肾病的遗传易感危险性基因型。同时,糖化血红蛋白、糖尿病病程、高血压病史可能是2型糖尿病肾病发病的独立危险因素。3、GA、AA基因型可能是脾肾阳虚的糖尿病肾病患者的易感基因型;携带A等位基因的糖尿病肾病患者可能更易演变为脾肾阳虚,携带A等位基因可能是脾肾阳虚型糖尿病肾病的风险因素之一。4、GA、AA基因型可能是水肿程度较重的糖尿病肾病患者的易感基因型;携带A等位基因的糖尿病肾病患者可能更易出现水肿加重。5、温阳活血利水法为主的中西医结合试验组治疗糖尿病肾病临床综合疗效显著优于西医常规治疗对照组。具体表现在不仅能降低血肌酐、尿素氮水平,还能减少尿蛋白、调节脂类代谢异常,改善血糖;但在改善炎症状态方面疗效相当。中西医结合治疗安全性较好,值得在临床推广应用。
[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.
【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259;R277.5
本文编号：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.
【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259;R277.5
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3 姜元吉;益肾通络解毒散治疗消渴肾病（糖尿病肾病DN）脾肾阳虚兼瘀毒证的临床及实验研究[D];长春中医药大学;2007年
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