Fang Ding-zhi, Liu Bing-wen, Shen Tao, Bai Huai, Zhang Chao-liang
Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2004 Mar;35(2):149-53.
To inquire into the mechanism of prothrombotic state (PTS) and the roles of liver therein by constructing a reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS.
The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization. The rat model of PTS was induced by a high-carbohydrate diet. Poly A+ mRNAs were isolated from PTS and control rats, and cDNAs were synthesized from the mRNAs. After digestion by means of Ras I, cDNAs 400-600 bp in size were obtained. For suppression subtractive hybridization, cDNAs from PTS rat were used as Driver and the cDNAs from control rat as Tester. The Tester was divided into two parts and ligated to adaptor 1 and adaptor 2R respectively. After two times of subtractive hybridization and two times of nested PCR, the products of the last PCR amplification were inserted into T/A plasmid vectors to transform the Escherichia coli JM109 cells. The transformed cells were incubated at 37 degrees C overnight on a LB agar plate containing ampicillin (50 micrograms/ml), IPTG and X-gal. The colonies were counted.
78% of the colonies were white and the reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state was successfully constructed.
Prothrombotic state caused by malfunction of homeostasis and fibrinolysis is an important risk factor of cardiovascular disease. Liver plays important roles in the development of PTS, for the majority of the factors in the coagulation as well as fibrinolytic cascades are generated by the liver and secreted into the bloodstream. The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS, successfully constructed in the present study, provides an efficient way to further investigate the mechanism of PTS and the relevant liver functions.
通过构建血栓前状态（PTS）大鼠肝脏差异表达基因的反向消减cDNA文库，探讨PTS的形成机制及肝脏在其中的作用。
采用抑制性消减杂交技术构建PTS大鼠肝脏差异表达基因的反向消减cDNA文库。用高糖饮食诱导大鼠形成PTS模型。从PTS大鼠和对照大鼠中分离出Poly A+ mRNA，并从mRNA合成cDNA。经Ras I酶切后，获得大小为400 - 600 bp的cDNA。在抑制性消减杂交中，将PTS大鼠的cDNA作为驱动子，对照大鼠的cDNA作为检测子。检测子分为两部分，分别与接头1和接头2R连接。经过两次消减杂交和两次巢式PCR后，将最后一次PCR扩增产物插入T/A质粒载体中，转化大肠杆菌JM109细胞。将转化后的细胞在含有氨苄青霉素（50微克/毫升）、IPTG和X - gal的LB琼脂平板上于37℃过夜培养。统计菌落数。
78%的菌落为白色，成功构建了血栓前状态大鼠肝脏差异表达基因的反向消减cDNA文库。
内稳态和纤维蛋白溶解功能失调导致的血栓前状态是心血管疾病的重要危险因素。肝脏在PTS的发生发展中起重要作用，因为凝血和纤维蛋白溶解级联反应中的大多数因子由肝脏产生并分泌到血液中。本研究成功构建的PTS大鼠肝脏差异表达基因的反向消减cDNA文库，为进一步研究PTS的机制及相关肝脏功能提供了有效途径。
