【摘要】 目的:运用抑制性消减杂交技术构建2型糖尿病肾阴虚证DNA消减文库,为制定2型糖尿病的一、二级康复预防提供理论参考。方法:选择2004-08南方医科大学南方医院内分泌科就诊的汉族2型糖尿病患者1例,符合中医肾虚证诊断标准中关于典型肾阴虚证的诊断,肝肾功能正常,无急慢性感染。以同时期、同性别、年龄相当的自愿者1名作为正常人对照。参照Maxim方法采用硅胶法抽提2例观察对象血白细胞DNA,用RsaI酶切基因组DNA成大小不等的片段,分别与两种不同的接头连接,进行两次消减杂交及两次抑制性聚合酶链反应,将聚合酶链反应产物与U/A载体连接,经蓝白斑筛选及菌液聚合酶链反应筛选出阳性重组质粒,构建糖尿病肾阴虚证DNA消减文库。测定抽提基因组DNA的纯度及含量;基因组DNARsaI酶切产物;消减杂交产物扩增结果;蓝白斑筛选阳性克隆;聚合酶链反应方法筛选重组质粒。结果:用抑制性消减杂交技术筛选出了糖尿病肾阴虚证的差异DNA片段,基因组DNA经过RasI酶切消化后,所得DNA片段主要集中在200～2000bp;消减杂交产物经两次聚合酶链反应扩增后,产物明显增多,内含数条隐约可见的条带,主要集中在0.2～1.5kb;蓝白斑α互补筛选出含有外源插入片段的阳性克隆,结果正向消减杂交产物得到286个克隆,反向消减杂交产物得到262个克隆;随机挑取92个阳性克隆,经聚合酶链反应分析证实86个克隆的质粒内均载有差异性DNA片段,大小为200～1000bp,从而成功构建了糖尿病肾阴虚证特异的DNA消减文库。结论:抑制性消减杂交技术能够快速有效地分离差异DNA片段以构建糖尿病肾阴虚证DNA消减文库,为进一步筛选和克隆糖尿病肾阴虚证相关基因奠定了基础。更多还原

【Abstract】 AIM:To construct the DNA subtractive library of kidney yin deficiency of type 2 diabetic mellitus by using the technique of suppression subtractive hybridization, so as to provide theoretical references for establishing the primary and secondary rehabilitative preventions of type 2 diabetes mellitus.METHODS:A patients with type 2 diabetes mellitus of Han nationality,who was treated in the Department of Endocrinology,Nanfang Hospital of Southern Medical University in August 2004,was involved in the study,The patient was in accordance with the diagnosis of classic kidney yin deficiency in the traditional Chinese medicine diagnostic standards for kidney yin deficiency,had normal liver and kidney functions,and had no acute or chronic infections.Another normal subject of the same gender and age at the same period was taken as the control voluntarily.The blood leucocyte DNA of the two subjects was extracted with the Maxim method by means of silica gel;After Rsa I enzyme restriction into segments of different size,it was connected with bonds of two different kinds respectively for twice subtractive hybridization and twice suppression polymerase chain reaction;The products of polymerase chain reaction was connected with the U/A vector,positive recombinant plasmid was screened by blue white screening system and bacteria liquid polymerase chain reaction,and then the DNA subtractive library of kidney yin deficiency was constructed.The purity and content of extracted genome DNA,products of genome DNA Rsa I enzyme restriction,amplification results of the subtractive hybridization products,positive clone screened by blue white screening system,and the recombinant plasmid screened by polymerase chain reaction were determined.RESULTS:The different DNA segments of diabetic kidney yin deficiency were screened with the technique of suppression subtractive hybridization,after Rsa I enzyme restriction,the obtained genome DNA segments mainly concentrated in 200 to 2000 bp;After amplification of twice polymerase chain reaction,the products of subtractive hybridization were obviously increased,there were several scraps could be seen indistinctly inside,and mainly concentrated in 0.2 to 1.5 kb;Positive clones containing exogenous insert were screened by blue white alpha complementation,and 286 and 262 clones were obtained with forward and backward subtractive hybridization products respectively;Ninety two positive clones were randomly selected,and it was proved by polymerase chain reaction analysis that different DNA segments were observed in the plasmid of 86 clones,the sizes were 200 to 1 000 bp,and the DNA subtractive library of kidney yin deficiency of diabetes mellitus was set up successfully.CONCLUSION:Suppression subtractive hybridization can quickly and efficiently separate different DNA segments to construct the DNA subtractive library of kidney yin deficiency of diabetic mellitus,and it provides a foundation for further screening and cloning the related genes of kidney yin deficiency of diabetic mellitus.更多还原