胰岛素蛋白质工程;[B9Glu,B10Asp]人胰岛素

用缺口双链ＤＮＡ的定向突变方法分别将胰岛素Ｂ链第９和第１０位的Ｓｅｒ和Ｈｉｓ改变为Ｇｌｕ和Ａｓｐ,获得「Ｂ９Ｇｌｕ,Ｂ１０Ａｓｐ」人胰岛素。其受体结合能力为猪胰岛素的３４．４％,而体内活力与猪胰岛素基本相同     

B8Gly在胰岛素结构模体中的可能作用

在胰岛素结构模体ｎ１－Ｃｙｓ－Ｇｌｙ－Ｘ１０－Ｃｙｓ－ｎ２－Ｃｙｓ－Ｃｙｓ－Ｘ３－Ｃｙｓ－Ｘ８－Ｃｙｓ－ｎ３中,有７个绝对保守的氨基酸残基,只有位于Ｂ８位的是Ｇｌｙ。通过定点突变将其改变为Ａｌａ,得到「Ｂ８Ａｌａ」人胰岛素,其受体结合能力和体内生物活力分别为天然猪胰岛素的２．５％和１０％。「Ｂ８Ａｌａ」人胰岛素和重组人胰岛素的远紫外圆二色谱比较表明,「Ｂ８Ａｌａ」人胰岛素的α－螺旋的相对含量有一家     

高受体结合和低免疫原性的［B1—Ala，B2—Ala］－胰岛素

通过化学半合成从天然猪胰岛素得到［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］胰岛素。这一胰岛素类似物经聚丙烯酰胺凝胶电泳和ＨＰＬＣ鉴定证明是均一的,氨基酸组成与理论值相符生物活性测定结果表明：［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素的体内活力与天然猪胰岛素相同,而与人胎盘细胞膜胰岛素受体的结合能力为天然猪胰岛素的１３２％。这一结果进一步说明胰岛素Ｂ链Ｎ端肽段参子与受体相互作用。此外,［Ｂ１－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素的免疫活性很低,远小于天然猪胰岛素的４％。     

用酵母双杂交系统研究胰岛素样生长因子—1突变体与其结合蛋白 …

为研究胰岛素样生长因子－１（ＩＧＦ１）及其突变体与ＩＧＦ结合蛋白－３（ＩＧＦＢＰ３）的相互作用,针对ＩＧＦ１的第３、４、１５、１６位氨基酸残基,采用定点突变的方法构建了「Ｙ１５Ｌ１６」ＩＧＦ１和「Ｑ３Ａ４Ｙ１５Ｌ１６」ＩＧＦ１。然后分别将ＩＧＦ１／ＩＧＦ１突变体和ＩＧＦＢＰ３ ｃＤＮＡ克隆至酵母表达载体ｐＧＢＴ９和ｐＡＣＴ２中,利用用酵母双杂交技术检测ＩＧＦ１／ＩＧＦ１突变体和ＩＧＦＢＰ３之间的相     

高受体结合和低免疫原性的[Bl—Ala,B2—Ala]—胰岛素

通过化学半合成从天然猪胰岛素得到［Ｂｌ－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素,这一胰岛素类似物经聚丙烯酰胺凝胶电泳和ＨＰＬＣ鉴定证明是均一的,氨基酸组成与理论值相符,生物活性测定结果表明：［Ｂｌ－Ａｌａ,Ｂ２－Ａｌａ］－胰岛素的体内活力与天然猪胰岛素相同,而与人胎盘细胞膜胰岛素受的结合能力为天然猪胰岛素的１３２％。这一结果进一步说明胰岛素Ｂ链Ｎ端肽段参予与受体相互作用,此外,［Ｂｌ－Ａｌａ,Ｂ２－Ａｌａ     

[B3—Lys]—胰岛素的研究：受体结合及生物活性

本文用＾１２５Ｉ－［Ｂ３－Ｌｙｓ］－胰岛素和＾１２５Ｉ－胰岛素研究了［Ｂ３－Ｌｙｓ］－胰岛素和胰岛素与人胎盘细胞膜胰岛素受体结合物性并进行了比较。实验结果表明［Ｂ３－Ｌｙｓ］－胰岛素与ＨＰＭ胰岛素受体结合能力比天然猪胰岛素高。由竞争取代曲线得到的［Ｂ３－Ｌｙｓ］－胰岛素和猪胰岛素的ＩＣ（５０）值分另为０．６５和１．１１ｎｍｏｌ／Ｌ。经Ｓｃａｔｃｈａｒｄ分别得出［Ｂ３－Ｌｙｓ］－胰岛素与ＨＰＭ胰岛素     

胰岛素蛋白质工程：高活性（B10Asp）人胰岛素

用基因定位突变方法将胰岛素Ｂ链第１０位的Ｈｉｓ变为Ａｓｐ,获得高活力胰岛素（Ｂ１０Ａｓｐ）人胰岛素,其受体结合能力和离体生物少分别为猪胰岛素的２６２％和２３５％体内生物活力也明显高于猪胰岛素,它的促细胞生长能力为猪胰岛素的１７４％。     

胰岛素蛋白质工程：高活性［B10Asp］人胰岛素

用基因定位突变方法将胰岛素Ｂ链第１０位的Ｈｉｓ变为Ａｓｐ,获得高活力胰岛素──［Ｂ１０Ａｓｐ］人胰岛素。其受体结合能力和离体生物活力分别为猪胰岛素的２６２％和２３５％;体内生物活力也明显高于猪胰岛素;它的促细胞生长能力为猪胰岛素的１７４％。     

［B3－Lys］－胰岛素的研究：受体结合及生物活性

本文用１２５Ｔ－［Ｂ３－Ｌｙｓ］－胰岛素和１２５Ⅰ－胰岛素研究了［Ｂ３－Ｌｙｓ］－胰岛素和胰岛素与人胎盘细胞膜（ＨＰＭ）胰岛素受体结合特性并进行了比较。实验结果表明［Ｂ３－Ｌｙｓ］－胰岛素与ＥＰＭ胰岛素受体结合能力比天然猪胰岛素高。由竞争取代曲线得到的［Ｂ３－Ｌｙｓ］－胰岛素和猪胰岛素的ＩＣ（５０）值分别为０．６５和１．１１ｎｍｏｌ／Ｌ。经Ｓｃａｔｃｈａｒｄ分析得出［Ｂ３－Ｌｙｓ］－胰岛素与ＨＰＭ胰岛素受体中高亲和位点和低亲和位点结合的亲和常数分别为１．７２×１０９和２．２７×１０６Ｌ／ｍｏｌ,而猪胰岛素分别为１．２６×１０９和１．４７×１０６／ｍｏｌ。促脂肪细胞合成脂肪实验结果表明［Ｂ３－Ｌｙｓ］－胰岛素也同样具有比天然猪胰岛素更高的离体生物活力,ＥＣ（５０）分别为０．１７５和０．３０１ｎｍｏｌ／Ｌ。可见［Ｂ３－Ｌｙｓ］－胰岛素的受体结合能力和高体生物活力都为猪胰岛素的１．７倍。     

内皮素对培养心肌细胞内游离钙浓度的作用

实验用培养新生ＳＤ大鼠心室肌细胞,以Ｆｕｒａ－２／ＡＭ荧光指示剂负载检测收肌细胞内游离钙浓度（「Ｃａ＾２＋」）的变化,探讨内皮素－１（ＥＴ－１）对「Ｃａ＾２＋」ｉ的作用及其机制。结果显示：ＥＴ－１引起心肌细胞「Ｃａ＾２＋」ｉ升高有两个时相,瞬时相持续相。ＥＴ－１诱导的瞬时相「Ｃａ＾２＋」ｉ升高呈浓度依赖性,预先用ＥＴＡ特异性受阻断剂ＢＱ１２３处理,可阻断ＥＴ－１引起的「Ｃａ＾２＋」ｉ升高,揭示上述     

人肝再生增强因子在毕赤酵母中的表达、纯化和生物学活性的研究

肝再生增强因子（ALR）是一类胞源性肝细胞生长因子。为在毕赤酵母中分泌表达人肝再生增强因子（rhALR）,以色谱法分离纯化后进行体外活性研究,构建表达载体pPICZαA- ALR,经电穿孔转入毕赤酵母中,用0.5％甲醇诱导表达;重组酵母培养上清经SDS-PAGE电泳和western blot鉴定后表明, rhALR以分子量为30kD的二聚体为主;定量分析结果表明,重组酵母培养上清中rhALR约占总蛋白的66％,表达量约为40mg/L;经DEAE柱和G75柱纯化后,获得的rhALR纯度大于95％,得率为52％;体外生物学活性实验表明,rhALR能明显促进HepG2、SMMC-7721和NIH-3T3细胞的增殖。     

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac     

肝再生增强因子的cDNA克隆、表达及表达产物的生物活性研究

以肝部分切除后再生肝组织为起始材料,利用RT-PCR扩增出大鼠肝再生增强因子（ALR）,亚克隆于pGEM-T载体,核苷酸序列测定证实为大鼠ALR;将ALRcDNA亚克隆于pBV220质粒,构建了原核表达栽体,并获高效表达菌株,特异表达蛋白占细菌总蛋白的15％,原核表达的ALR在体外缺乏促进大鼠原代培养肝细胞及SMMC-7721肝癌细胞DNA合成的活性,但在体内1／3肝部分切除模型中可刺激肝细胞DNA合成;ALR在生物学活性方面与肝脏刺激物（HSS）存在一定差别,ALR和HSS应是两种不同的活性因子．ALR还具有促肝损伤修复的作用,对其深入研究可能为临床治疗严重肝病提供有效的药物.     

Regulation of polyamine synthesis in human hepatocytes by hepatotrophic factor augmenter of liver regeneration

Different stages of liver regeneration are regulated by a variety of factors such as the liver growth associated protein ALR, augmenter of liver regeneration. Furthermore, small molecules like polyamines were proven to be essential for hepatic growth and regeneration. Therefore, using primary human hepatocytes in vitro we investigated the effect of ALR on the biosynthesis of polyamines. We demonstrated by HPLC analysis that recombinant ALR enhanced intracellular hepatic putrescine, spermidine, and spermine levels within 9-12h. The activation of polyamine biosynthesis was dose dependent with putrescine showing the strongest increase. Additionally, ALR treatment induced mRNA expression of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase, both key enzymes of polyamine biosynthesis. Further, ALR induced c-myc mRNA expression, a regulator of ODC expression, and therefore we assume that ALR exerts its liver regeneration augmenting effects through stimulation of its signalling pathway leading in part to enhanced polyamine synthesis.     

肝部分切除后肝再生增强因子信使核糖核酸（mRNA）表达增强

肝再生增强因子（ＡＬＲ）是一种新的肝增殖刺激因子。本研究选择７０％肝部分切除（ＰＨ）大鼠为模型,观察了ＰＨ后残存肝组织胞浆液促肝细胞增殖活性与ＡＬＲ特异ｍＲＮＡ表达动态变化的关系。发现正常肝组织几无ＡＬＲ ｍＲＮＡ表达,其肝胞质液也无促肝细胞增殖活性;７０％ＰＨ后１２ｈ肝组织ＡＬＲ ｍＲＮＡ表达明显增加,并于术后２４ｈ达高峰,肝胞质液活性也于术后２４ｈ内达高峰,这种ｍＲＮＡ表达－效应的时间关系提示     

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl4-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl₄ in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl₄-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.     

肝再生增强因子研究进展

肝再生增强因子是新近克隆的蛋白质因子,能特异地刺激肝源细胞的增殖,并对ＣＣl4所引起的急性肝衰竭有效治作用。本文综述了肝再生增强因子的发现、基因克隆及组织分布等。目前已开始了该因子的基因工程产品研制,它有望成为一种治疗肝病的新药。     

Augmenter of liver regeneration: Essential for growth and beyond

Liver regeneration is a well-orchestrated process that is triggered by tissue loss due to trauma or surgical resection and by hepatocellular death induced by toxins or viral infections. Due to the central role of the liver for body homeostasis, intensive research was conducted to identify factors that might contribute to hepatic growth and regeneration. Using a model of partial hepatectomy several factors including cytokines and growth factors that regulate this process were discovered. Among them, a protein was identified to specifically support liver regeneration and therefore was named ALR (Augmenter of Liver Regeneration). ALR protein is encoded by GFER (growth factor erv1-like) gene and can be regulated by various stimuli. ALR is expressed in different tissues in three isoforms which are associated with multiple functions: The long forms of ALR were found in the inner-mitochondrial space (IMS) and the cytosol. Mitochondrial ALR (23 kDa) was shown to cooperate with Mia40 to insure adequate protein folding during import into IMS. On the other hand short form ALR, located mainly in the cytosol, was attributed with anti-apoptotic and anti-oxidative properties as well as its inflammation and metabolism modulating effects. Although a considerable amount of work has been devoted to summarizing the knowledge on ALR, an investigation of ALR expression in different organs (location, subcellular localization) as well as delineation between the isoforms and function of ALR is still missing. This review provides a comprehensive evaluation of ALR structure and expression of different ALR isoforms. Furthermore, we highlight the functional role of endogenously expressed and exogenously applied ALR, as well as an analysis of the clinical importance of ALR, with emphasis on liver disease and in vivo models, as well as the consequences of mutations in the GFER gene.