胆红素自由基对大鼠肝细胞损伤作用的研究

用不同自由基源处理的胆红素与鼠肝细胞相互作用,结果表明：胆红素自由基可引起鼠肝细胞脂过氧化,使总谷胱甘肽及ＧＳＳＧ水平明显下降,细胞损伤后,乳酸脱氢酶外漏,上述结果与自由基浓度正相关,而与其种类无关。牛血清蛋白有明显的抑制作用,差示光谱表明：胆红素可能与细胞色素Ｐ－４５０快速形成络合物,根据以上结果,重点讨论了胆红自由基对肝细胞损的化学本质及其与胆结石形成的关系。     

银杏叶提取物对自由基所致脑神经细胞损伤的保护作用研究

从细胞分子水平,采用大脑皮层神经细胞分离技术,自旋标记技术,酶学方法及细胞染色技术探讨自由基对神经细胞的损伤及ＥＧｂ保护作用。结果显示,用自由基攻击的细胞其自旋标记膜的序参数Ｓ,旋转相关时间τｃ及膜蛋白巯基的Ｓ／Ｗ比对照组高（Ｐ＜０．０５）,提示：受自由基攻击的细胞膜,其流动性比正常减低,且蛋白质构象发生了改变。加ＥＧｂ保护后,可减少自由基引起的膜流动性及蛋白质构象改变,起到保护神经细胞的作用。经·ＯＨ攻击的神经细胞,其ＬＤＨ活性及死细胞比率都比对照组高,而ＥＧｂ对此有保护作用。本文结果说明一定浓度ＥＧｂ可保护大脑皮层神经细胞免受自由基的损伤。     

生长抑素对羟自由基损伤的人胃粘膜上皮细胞系GES—1的影响

目的和方法：采用人胃粘膜上皮细胞系ＧＥＳ１细胞传代培养技术,利用Ｆｅ２＋与Ｈ２Ｏ２反应生成的羟自由基（ｈｙｄｒｏｘｙｌｒａｄｉｃａｌ,ＯＨ·）建立细胞损伤模型,探讨ＯＨ·损伤人胃粘膜细胞的机制,观察生长抑素（ｓｏｍａｔｏｓｔａｔｉｎ,ＳＳ）对ＧＥＳ１细胞抗ＯＨ·损伤的影响。结果：（１）ＯＨ·可直接损伤ＧＥＳ１细胞,表现为细胞存活率下降而细胞乳酸脱氢酶（ｌａｃｔａｔｅｄｅｈｙｄｒｏｇｅｎａｓｅ,ＬＤＨ）漏出量增多;预先在细胞培养液中加入ＯＨ·特异性清除剂二甲基亚砜（５ｍｍｏｌ／Ｌ）,可预防该损伤。预先加入ＳＳ（０．０１ｍｇ／Ｌ,０．１ｍｇ／Ｌ,１．０ｍｇ／Ｌ,１０ｍｇ／Ｌ）对细胞存活率和细胞ＬＤＨ漏出量无影响;（２）加入ＯＨ·后,细胞丙二醛（ＭＤＡ）、氧化型谷胱甘肽（ＧＳＳＧ）含量上升而还原型谷胱甘肽（ＧＳＨ）含量下降。以ＳＳ０．１ｍｇ／Ｌ,１．０ｍｇ／Ｌ,１０ｍｇ／Ｌ预处理,可部分减轻上述改变。结论：ＯＨ·可直接损伤ＧＥＳ１细胞,其机制与破坏细胞的巯基稳态及加重细胞的脂质过氧化程度有关;ＳＳ可通过维持细胞的巯基稳态,部分减轻ＯＨ·所致的ＧＥＳ１细胞脂质过氧化程度。     

胆红素自由基对人红细胞的损伤作用

为了进一步证明胆红素自由基对细胞造成的直接损伤,探讨胆红素对细胞表现毒性的机理以及在色素类结石形成中所起的作用,我们以胆红素自由基作用于人红细胞,用TBA法研究了完整人红细胞的脂质过氧化,用SDS-PAGE法研究了红细胞膜的损伤以及用荧光法研究了膜损伤后某些性质的改变。结果表明:胆红素自由基能引起人红细胞膜脂质过氧化,使膜蛋白发生降解,从而直接可测氨基减少,而溶液中游离氨基的含量却相应增加。根据以上事实,重点讨论了胆红素对细胞表现毒性的可能原因以及与色素类结石形成的关系。     

肝细胞生长因子对四氯化碳损伤原代培养大鼠肝细胞的保护作用

本工作采用无血清原代培养大鼠肝细胞法,观察了重组人肝细胞生长因子（ｒ－ｈＨＧＦ）对四氯化碳（ＣＣｌ４）致大鼠肝细胞损伤的保护作用。结果表明,ｒ－ｈＨＧＦ对ＣＣｌ４染毒肝细胞有明显的保护作用。ｒ－ｈＨＧＦ保护组较ＣＣｌ４染毒组细胞存活率显著升高,细胞内丙氨酸转氨酶、钾离子漏出明显降低。结果提示,ｒ－ｈＨＧＦ可减轻ＣＣｌ４对肝细胞膜的损伤,提高细胞膜的结构完整性     

锌对缺血／再灌注肝脏自由基含量和细胞凋亡的影响

目的：观察补锌对缺血再灌注（HIR）大鼠肝脏自由基含量及细胞凋亡的影响。探讨补锌保护肝损伤的机制。方法：用荧光分光光度法测定血清MDA含量;用电子自旋共振法测定肝脏自由基浓度;用流式细胞术检测肝细胞凋亡。结果：HIR组大鼠血清MDA水平和肝自由基产生均增加,补锌后降低;肝脏缺血再灌注后肝细胞凋亡率达到57.72%,补锌后降低40.85%。结论：减少自由基产生和抑制细胞凋亡是锌保护肝缺血再灌注损伤的重要机制。     

金属硫蛋白清除自由基及其对自由基引起的核酸损伤保护作用的研究

通过化学反应体系产生ＯＨ－和Ｏ自由基,采用荧光和化学发光检测体系,比较研究了不同亚型及不同结合金属的金属硫蛋白（ＭＴ）清除自由基能力的大小。结果表明,对于同一亚型,Ｚｎ结合ＭＴ清除自由基的能力大于Ｃｄ结合ＭＴ同一结合金属的ＭＴ,ＭＴ１清除自由基的能力大于ＭＴ２。通过比较ＺｎＭＴ１与谷胱甘肽（ＧＳＨ）及超氧化物歧化酶（ＳＯＤ）清除自由基的能力大小发现,ＺｎＭＴ１清除ＯＨ的能力是ＧＳＨ的１００倍,清除Ｏ自由基的能力分别是ＧＳＨ和ＳＯＤ的２５和０．０１倍。即ＭＴ是一种很好的ＯＨ自由基清除剂。以ＯＨ对核酸（ＤＮＡ）的损伤为例,研究了ＭＴ对核酸损伤的保护作用,其变化规律与上述结果相一致。     

表皮生长因子对肝细胞醋氨酚急性损伤的保护作用

本工作采用无血清培养的小鼠原代肝细胞制备了醋氨急性肝损伤模型,然后利用该模型观察了表皮生长因子（ＥＧＦ）对肝细胞的保护作用。结果如下：（１）小鼠原代肝细胞无血清培养液中加入终深度度为２０ｍｍｏｌ／Ｌ的醋氨酚培养１２－１４ｈ后,培养液中ＧＰＴ和ＧＰＴ的活性明显升高,可作为一种适当的肝细胞损伤。（２）提前１ｈ加入不同剂量（５０,１００,５００,１０００ｎｇ／ｍｌ）的ＥＧＦ可减轻醋氨酚相起的肝细胞损伤。     

肝细胞表面去唾液酸糖蛋白受体的流式细胞分析

建立肝细胞表面去唾液酸糖蛋白受体（ASGPR）的流式细胞分析方法（FCM）,对正常及损伤鼠肝细胞、肝癌细胞（BEL-7402）表面的ASGPR作同步比较分析.以异硫氰酸荧光素标记的新半乳糖白蛋白（FITC-NGA）为ASGPR的特异性配体,以培养的正常肝细胞（L-02）为靶细胞,建立肝细胞表面ASGPR的FCM.测定并计算正常及损伤鼠肝细胞,BEL-7402细胞与同一浓度的FITC-NGA同步反应后的平均荧光强度（MIF）值.FITC-NGA与L-02细胞表面ASGPR趋近饱和结合的浓度为0.4 mg/L,该浓度下正常及损伤鼠肝细胞,BEL-7402细胞的MIF值分别为228.7、5.81、1.13.该结合可以被至少50倍于FITC-NGA的NGA或10 mmol/L的EDTA完全抑制.FCM能够良好地揭示FITC-NGA同ASGPR之间的受配体结合特性.该方法证实BEL-7402细胞表面几乎没有ASGPR,损伤鼠肝细胞表面ASGPR的数量较正常鼠肝细胞显著减少.     

利用GFP示踪细胞内源性P53活性检测DNA损伤

ＤＮＡ损伤的检测对预防癌症和遗传病等非常重要。采用分子克隆技术,将报告基因—绿色荧光蛋白（ＧＦＰ）置于ＳＶ４０基本启动子调控下,构建成对照载体ｐＳＶ－ＧＦＰ。在ＳＶ４０基本启动子上游插入寡核苷酸Ｐ５３ＲＥ,构建成示踪载体ｐ５３ＲＥ－ＧＦＰ。转染ＮＩＨ３Ｔ３细胞,以ＧＦＰ示踪细胞内源性Ｐ５３的转录激活活性。紫外线照射或Ｈ２Ｏ２处理转化细胞使ＤＮＡ损伤,诱导细胞内源性Ｐ５３的表达。用激光扫描共聚焦成像系统（ＬＳＣＩＳ）对细胞进行红、绿、蓝三色光融合成像,并测定ＧＦＰ经４８８ｎｍ激发后发出的绿色荧光光密度,验证ＧＦＰ示踪Ｐ５３的特异性。ｐ５３ＲＥ－ＧＦＰ转化细胞３Ｔ３－ＲＥＧ经紫外线照射或Ｈ２Ｏ２处理后,ＧＦＰ的表达增高,处理后１ｈｒ光密度即达到最高水平,随后逐渐降低。血清“饥饿”—非ＤＮＡ损伤处理的３Ｔ３－ＲＥＧ细胞,以及经紫外和Ｈ２Ｏ２处理的对照载体ｐＳＶ－ＧＦＰ转化细胞３Ｔ３－ＳＶＧ,ＧＦＰ的表达无明显增强。实验表明：ＧＦＰ示踪内源性Ｐ５３转录激活活性用于检测ＤＮＡ损伤有很高的灵敏度和特异性,适宜推广应用。     

Monoclonal antibodies to cytochrome P-450 immunopurify a 45-kDa protein from a human lymphoblastoid cell line

Monoclonal antibodies (MAbs) to rat liver cytochromes P-450 have previously been used for successful immunopurification of cytochromes P-450 from animal tissues. We now report application of this MAb-based immunopurification technique to the human lymphoblastoid AHH-1 cell line. Immunopurification carried out with 3 different MAbs each yielded a 45-kDa polypeptide. The purified protein contains an MAb-specific epitope present on cytochromes P-450, and may therefore be a human cytochrome P-450.     

Preparation of catalytically active cytochromes P-450 by antigen exchange on monoclonal antibody based immunoadsorbents

Catalytically active cytochromes P-450 have been prepared by monoclonal antibody (MAb) directed immunopurification using an antigen-exchange technique. Immunoaffinity-purified cytochromes P-450 that require denaturants for efficient desorption from the immunoaffinity matrix, although significantly lacking in catalytic activity, were found to retain epitopic structural integrity as probed by radioimmunoassay using MAbs to 3-methylcholanthrene and phenobarbital-induced rat liver cytochromes P-450. These denatured cytochromes P-450 were capable of displacing from the immunoaffinity matrices epitopically related cytochromes P-450 that retained aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities. Such epitope-specific exchange of denatured for native antigen on a solid-phase matrix containing a MAb may be generally applicable to preparation of proteins with the retention of activity.     

Secondary structure prediction of liver microsomal cytochrome P-450; proposed model of spatial arrangement in a membrane

The secondary structure of rabbit liver microsomal cytochrome P-450 LM2, rat liver microsomal cytochromes P-450b and P-450e (phenobarbital-inducible), and rat liver microsomal cytochromes P-450c, P-450d (3-methylcholanthrene-inducible) was predicted by a combination of methods (i) identifying the transmembrane parts of integral membrane proteins, and (ii) statistically predicting the secondary structure of globular proteins. The results are similar for all phenobarbital-inducible enzymes and make it possible to construct two structural models with seven or four transmembrane alpha-helices. The cytochromes of the second group obviously form a second structural family with four membrane-spanning alpha-helices. In both cases, a large ectodomain with several consecutive alpha-helices, which may provide the heme-binding pocket, is exposed out of the membrane.     

Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.     

Use of monoclonal antibody probes against rat hepatic cytochromes P-450c and P-450d to detect immunochemically related isozymes in liver microsomes from different species

Nine distinct monoclonal antibodies raised against purified rat liver cytochrome P-450c react with six different epitopes on the antigen, and one of these epitopes is shared by cytochrome P-450d. None of these monoclonal antibodies recognize seven other purified rat liver isozymes (cytochromes P-450a, b, and e-i) or other proteins in the cytochrome P-450 region of "Western blots" of liver microsomes. Each of the monoclonal antibodies was used to probe "Western blots" of liver microsomes from untreated, or 3-methylcholanthrene-, or isosafrole-treated animals to determine if laboratory animals other than rats possess isozymes immunochemically related to cytochromes P-450c and P-450d. Two protein-staining bands immunorelated to cytochromes P-450c and P-450d were observed in all animals treated with 3-methylcholanthrene (rabbit, hamster, guinea pig, and C57BL/6J mouse) except the DBA/2J mouse, where no polypeptide immunorelated to cytochrome P-450c was detected. The conservation of the number of rat cytochrome P-450c epitopes among these species varied from as few as two (guinea pig) to as many as five epitopes (C57BL/6J mouse and rabbit). The relative mobility in sodium dodecyl sulfate-gels of polypeptides immunorelated to cytochromes P-450c and P-450d was similar in all species examined except the guinea pig, where the polypeptide related to cytochrome P-450c had a smaller Mr than cytochrome P-450d. With the use of both monoclonal and polyclonal antibodies, we were able to establish that purified rabbit cytochromes P-450 LM4 and P-450 LM6 are immunorelated to rat cytochromes P-450d and P-450c, respectively.     

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain.

Polyclonal antibodies to the major beta-naphthoflavone (BNF)-inducible form of cytochrome P-450 (P450IA) and to the major phenobarbitone (PB)-inducible form (P450IIB) have been used to quantify the contribution of these subfamilies to the total amount of cytochrome P-450 in rat livers and rat hepatocyte cultures treated with PB, BNF and metyrapone for 24 and 72 h. The P450IA and IIB subfamilies were not detectable (less than 5 pmol/mg of microsomal protein) in the livers of control rats, but administration of BNF resulted in the P450IA subfamily comprising more than 80% of the total hepatic cytochrome P-450. Administration of PB and metyrapone to rats did not elevate the level of this subfamily but elevated the levels of the P450IIB subfamily to 60% and 30% respectively of the total. Thus metyrapone is a ''PB-like'' inducer. However, in contrast with their effects in vivo, treatment with PB and metyrapone of rat hepatocytes did not elevate the proportion of the P450IIB subfamily relative to that in untreated cells but rather, like BNF, increased the P450IA subfamily. This would account for the ability of metyrapone to produce in hepatocyte culture, like BNF, a pronounced induction of ethoxyresorufin O-de-ethylase activity, but it does not account for why of all inducers studied only metyrapone can maintain the total cytochrome P-450 content of cultured hepatocytes, or the activity of ethylmorphine N-demethylase. This activity is generally considered to be associated with the P450IIB subfamily, but the lack of effect of metyrapone on this subfamily in hepatocyte culture must suggest that metyrapone is able to prevent the loss of the total amount of the cytochrome by increasing the expression of other cytochromes P-450.     

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).     

Immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa

Previously, we described two olfactory-specific cytochromes P-450: rat cytochrome P-450olf1 (IIG1), identified by cDNA cloning, and bovine cytochrome P-450olf2 (IIA), identified by peptide microsequencing of a transmembranal polypeptide (p52). Here we describe the preparation of polyclonal antisera against peptide sequences of these proteins and their use in the immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa. Immunoreactivities related to both enzymes are found in the subepithelial Bowman's glands of olfactory mucosa. Practically no immunoreactivity was found in other rat tissues, including liver, lung, kidney and respiratory mucosa. In addition, double-labeling experiments demonstrated that cytochromes P-450olf1 and P-450olf2 are present in the same population of Bowman's glands. The olfactory-specific localization of cytochromes P-450olf1 and P-450olf2 is consistent with a role for these enzymes in the modification or clearance of odorants from the chemosensory tissue.     

Separation and analysis of immunochemical properties of multiple forms of cytochrome P-450 from the rat liver

Four cytochromes P-450 induced by phenobarbital (PB-1--PB-4) and two cytochromes P-450 induced by S-methylcholanthrene (MC-1, MC-2) were purified to electrophoretic homogeneity from rat liver microsomes. The purification procedure involved sequential chromatography on n-aminooctyl-Sepharose 4B, DEAE-Sephacel and hydroxylapatite columns. The spectral and immunochemical properties of the cytochromes P-450 were estimated. All, but MC-1, cytochromes P-450 were found to exist in a low spin state. Using the Ouchterlony double diffusion method, it was shown that all cytochromes P-450 under study can be divided into two groups, i. e., PB-1--PB-2 and PB-3--PB-4, sharing common antigenic determinants inside the groups. High performance liquid chromatography of PB-3 and MC-2 on anion-exchangers yielded two additional peaks from the PB-induced major cytochrome P-450 PB-3 and three peaks from the MC-induced major cytochrome P-450 MC-2. The multiplicity of cytochrome P-450 forms is discussed.