Evaluation of the function and the regeneration ability of the transplanted partial liver using a small amount of hepatocytes obtained by fine needle aspiration biopsy.

利用细针抽吸活检获得的少量肝细胞评价移植部分肝脏的功能和再生能力。

• 批准号: 04557057
• 负责人: TANAKA Koichi
• 金额: $ 8.26万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04557057/
• 关键词: partial liver transplantation; liver function; aspiration cytology; RT-PCR; PT-PCR; 肝再生

To evaluate the liver function and the regeneration ability after clinical living related donor partial liver transplantation, the method for the determination of the amount of the mRNA of albumin in the hepatocytes contained in the small samples obtained by percutaneous fine needle aspiration biopsy was developed using competitive RT-PCR.The primer for site A was selected from the sequence between exon 4 and exon 5 and the primer for site B was selected from the sequence between exon 11 and exon 12 of the rat albumin gene. The primers introduced with a point mutation, which generate a new restriction site, were synthesized for the site A and the site B.The PCR product from the cDNA obtained by reverse transcriptase using these primers with the point mutation was used as the competitor (internal control), which was recognized by electrophoresis after digestion with the specific endonuclease. Total nucleic acids were extracted from the small amount of the samples of rat liver by Iso Quick^<TM>. The cDNAs were translated from mRNAs using Super Script Preamplification Kit^<TM> and amplified with the genomic DNA and the various amounts of the competitor by PCR.The quantitation of the initial amounts of the mRNA for albumin in the sample was achieved by the determination of the ratio of these products by dencitometry. This method may provide a rapid way to quantitate the amount of the mRNA of albumin in the very small amount of the clinical samples obtained by fine needle aspiration biopsy.

评价临床活体亲属部分肝移植术后肝功能及再生能力，使用竞争性RT-PCR，开发了测定通过经皮细针抽吸活组织检查获得的小样品中所含肝细胞中白蛋白mRNA量的方法。位点A的引物选自大鼠白蛋白基因外显子4和外显子5之间的序列，位点B的引物选自大鼠白蛋白基因外显子11和外显子12之间的序列。对于位点A和位点B，合成了引入了点突变的引物，该点突变产生了新的限制性位点。将使用这些具有点突变的引物通过逆转录酶获得的cDNA的PCR产物用作竞争物（内部对照），该竞争物在用特异性内切酶消化后通过电泳识别。用Iso Quick ®从少量的大鼠肝脏样品中提取总核酸<TM>。使用Super Script Preamplification Kit^从mRNA翻译cDNA<TM>，并用基因组DNA和不同量的竞争剂通过PCR扩增。通过密度计测定这些产物的比率来实现样品中白蛋白mRNA的初始量的定量。该方法可为临床细针穿刺活检中微量白蛋白mRNA的定量提供一种快速的方法。

项目成果

K.Tanaka: "Postoperative management based on the Redox theory in living related liver transplantation in children" J.Japanese Society of Pediatr.Surg. 29. 256-263 (1993)

K.Tanaka：“基于氧化还原理论的儿童活体肝移植术后管理”J.日本儿科外科学会。

K.Tanaka: "Liver transplantation in children from living-related donors." Transplantation Proceedings. 25. 1084-1086 (1993)

K.Tanaka：“儿童肝移植来自亲属捐献者。”

K.Ozawa: "Living related douor liver transplantation" KARGER, 250 (1994)

K.Ozawa：“与生活相关的门肝移植”KARGER，250（1994）

S.Fujita: "Living related liver transplantation for biliary atresia" Clinical Transplantation. (in press).

S.Fujita：“胆道闭锁的活体相关肝移植”临床移植。

K.Tanaka: "Complication management in living related liver transplantation" Ame.J.Surg.(in press).

K.Tanaka：“活体相关肝移植的并发症管理”Ame.J.Surg.（出版中）。