Sex Differences in Long Chain Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway.Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [(14)C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more (14)C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [(14)C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells.In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [(14)C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [(14)C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified.The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to those of male FABP. In contrast, the concentration of FABP, per milligram cytosolic protein, was 44% greater in female liver than in male, as indicated by measurement of [(14)C]oleate binding and of 280 nm OD in the FABP fraction of 105,000 g supernate after gel filtration chromatography.These experiments demonstrate profound sex differences in hepatocyte utilization of long chain fatty acids at concentrations within and below the physiological range, and suggest that these are attributable at least in part to corresponding differences in cytosolic FABP concentration. At higher FFA concentrations, sex differences in hepatocyte FFA utilization are virtually eliminated, suggesting that under these conditions, differences in FABP concentration are not rate determining. Sex differences in hepatic lipoprotein production may largely reflect these important differences in the initial stages of hepatocyte FFA utilization.     

Molecular Dynamics Simulation of Ligand Dissociation from Liver Fatty Acid Binding Protein

The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized “portal region” could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques.     

慢性丙型肝炎并脂肪肝血清A-FABP与病毒及代谢的相关分析

目的 探讨慢性丙型肝炎(chronic hepatitis C,CHC)并脂肪肝(fatty liver,FL)血清脂肪细胞型脂肪酸结合蛋白(A-FABP)水平与病毒和代谢因素的相关性.方法 选择118例初次就诊且未行抗病毒治疗的CHC,根据是否伴FL分为FL组55例和非FL组63例.检测两组A-FABP水平,并比较其水平与丙型肝炎病毒、血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、体重指数(BMI)、空腹血糖(FPG)、胰岛素水平(FINS)、甘油三酯(TG)、胆固醇(CHO)及胰岛素抵抗指数(HO-MA-IR)等的相关性.结果 FL组TG较非FL组明显升高,FINS、HOMA-IR及A-FABP血清水平明显低于非FL组(P＜0.05).相关性及回归分析表明,A-FABP同BMI、FPG、FINS、HOMA-IR明显呈正相关,并且同TG水平明显负相关,FINS是A-FABP水平的独立相关因素(r2=0.830,P＜0.01).结论 CHC并发FL与患者血清A-FABP水平下降密切相关,与病毒因素不相关,且FINS是此类患者A-FABP水平变化的独立相关因素.     

人心肌型脂肪酸结合蛋白(H-FABP)在大肠杆菌中的表达和纯化

目的获得重组人心肌型脂肪酸结合蛋白,为进一步开发应用奠定基础。方法根据已报道的心肌型脂肪酸结合蛋白(H-FABP)基因序列,采用RT-PCR的方法获得心肌型脂肪酸结合蛋白基因。将所得的PCR产物插入原核表达载体pQE30中,得重组质粒(pQE-H-FABP)并转化大肠杆菌(E.coli)M15,通过IPTG诱导表达出带有六个组氨酸的融合蛋白。经镍固定金属亲和层析纯化。结果序列分析表明:H-FABP基因成熟肽编码区含有396bp,编码132个氨基酸;与GenBank(NM004012)中已报道的H-FABP分离物的核苷酸序列有99·9%同源性。经IPTG诱导表达,SDS-PAGE电泳和免疫印迹分析显示,表达的融合蛋白占菌体蛋白总量的27%,分子质量约为15kDa并与商品化的H-FABP单抗(Clone6B6)呈特异性反应。经Ni 2-NTAagarose纯化获得SDS-PAGE电泳下单一条带。结论在大肠杆菌中获得了人心肌型脂肪酸结合蛋白的高效表达,为研究其生物学功能和制备单克隆抗体奠定了基础。     

Effect of Protein Concentration and Binding on Antibiotic Assays

Assay curves, using a disk diffusion method for the antibiotics gentamicin and cefazolin, were prepared with: saline, saline plus 10% serum, and ascitic, synovial, cerebrospinal, and pleural fluids. The curves were compared with a standard curve prepared with pooled human serum. The pH, total protein, glucose, blood urea nitrogen, sodium, potassium, calcium, phosphorus, chloride, CO₂ content, uric acid, cholesterol, bilirubin, serum glutamic oxalacetic transaminase, CPK, LDH, and alkaline phosphatase were determined and compared for all fluids. Measurements for cefazolin levels were falsely elevated in those fluids with low protein content when serum was used as a reference standard. There was a linear inverse relationship between the protein content of the fluids and the cefazolin level with serum as the standard for the assay of this highly protein-bound antibiotic. No discrepancies were observed in the assay curves for gentamicin, an antibiotic known not to be bound by serum proteins.     

心型脂肪酸结合蛋白检测对早期心肌损伤辅助诊断的临床价值

目的探讨心型脂肪酸结合蛋白(H-FABP)检测对早期心肌损伤辅助诊断的临床价值。方法选择2014年5月至2015年7月确诊为心肌损伤68例患者作为患者组,分为不稳定型心绞痛组(A组)、非ST段抬高心肌梗死组(B组)、ST段抬高心肌梗死组(C组),3个小组,选择同期进行健康体检且健康者25例作为健康对照组。检测所有研究对象的血清H-FABP、缺血修饰清蛋白(IMA)、肌红蛋白(MYO)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)浓度。结果与健康对照组比较,患者组患者在入院早期(3~6h)和中晚期(6~12h)2次检测血清HFABP、IMA、MYO、CK、CK-MB的浓度均明显升高,差异有统计学意义(P0.05),但A、B、C 3组间H-FABP浓度差异并无统计学意义(P0.05);A、B、C 3组患者在入院早期检测血清H-FABP、IMA、MYO的阳性率均超过80.00%,明显高于CK和CK-MB的阳性率,差异有统计学意义(P0.05),但血清H-FABP、IMA、MYO在3组间的阳性率差异无统计学意义(P0.05);A、B、C 3组患者在入院中晚期检测该5项指标,阳性率均超过80.00%,5项指标阳性率分别两两比较,差异均无统计学意义(P0.05)。早期和中晚期H-FABP检测结果与确诊结果的Kappa一致性分析显示,结果分别为0.80(P0.05)和0.95(P0.05)。结论 H-FABP对心肌损伤患者的早期诊断具有一定的临床价值,较高的灵敏度和特异度适用于心肌损伤的筛查,能有效降低漏诊率和误诊率。     

血尿酸与代谢相关脂肪性肝病患者肝功能异常的相关性及因果研究

目的 研究血尿酸(UA)与代谢相关脂肪性肝病(MAFLD)患者肝功能异常的相关性及因果关系。方法 纳入于四川大学华西医院就诊的MAFLD患者98例。用Spearman相关和多元Logistic回归分析UA与丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)间的相关性。用双向孟德尔随机化研究分析UA与ALT、AST之间的因果关系。结果 UA与ALT、AST呈正相关,相关系数分别为0.56(P<0.001)和0.46(P=0.01)。高UA组相较于低UA组ALT(OR=3.58,P=0.003)、AST(OR=3.24,P=0.005)水平增加。正向因果中,UA每升高1标准差(SD),ALT增加0.07SD(P=0.002),AST增加0.06SD(P=0.03)。而在反向因果中,尚未发现ALT、AST与UA间相关(P>0.05)。结论 UA可导致ALT、AST水平升高,是MAFLD患者肝功能受损潜在的危险因素。     

Glucagon and Insulin Binding to Liver Membranes in a Partially Nephrectomized Uremic Rat Model

To investigate the role of glucagon and insulin receptor binding in the glucagon hypersensitivity and insulin resistance which characterize the glucose intolerance of uremia, liver plasma membranes were prepared from control rats (blood urea nitrogen [BUN] 15+/-1 mg/100 ml, creatinine 0.7+/-0.2 mg/100 ml), and from 70% nephrectomized rats (BUN 30+/-2 mg/100 ml, creatinine 2.2+/-0.2 mg/100 ml), and from 90% nephrectomized rats (BUN 46+/-3 mg/100 ml, creatinine 4.20+/-0.7 mg/100 ml), 4 wk after surgery. As compared to controls, the 90% nephrectomized rats had significantly higher levels of plasma glucose (95+/-4 vs. 125+/-11 mg/100 ml), plasma insulin (28+/-9 vs. 52+/-11 muU/ml), and plasma glucagon (28+/-5 vs. 215+/-18 pg/ml). Similar, but less marked, elevations were observed in the 70% nephrectomized animals.In liver plasma membranes from nephrectomized rats, specific binding of (125)I-glucagon was increased by 80-120%. Furthermore, glucagon (2 muM)-stimulated adenylate cyclase activity in nephrectomized rats was twofold higher than in controls. In contrast, fluoridestimulated adenylate cyclase activity was similar in both groups of rats. In marked contrast to glucagon binding, specific binding of (125)I-insulin to liver membranes from nephrectomized rats was reduced by 40-50% as compared to controls. Data analysis suggested that the changes in both glucagon and insulin binding are a consequence of alterations in binding capacity rather than changes in affinity. Liver plasma membranes from nephrectomized rats degraded (125)I-glucagon and (125)I-insulin to the same extent as control rats.THESE RESULTS DEMONSTRATE THAT: (a) the 70 and 90% nephrectomized rats simulate the hyperglycemia, hyperinsulinemia, and hyperglucagonemia observed in clinical uremia; (b) in these animals specific binding of glucagon to liver membranes is increased and is accompanied by higher glucagon-stimulated adenylate cyclase activity; and (c) specific binding of insulin is markedly decreased. These findings thus provide evidence of oppositely directed, simultaneous changes in glucagon and insulin receptor binding in partially nephrectomized rats. Such changes may account for the hypersensitivity to glucagon and may contribute to resistance to insulin observed in the glucose intolerance of uremia.     

视黄醇结合蛋白4检测在营养性疾病及肝肾损害检测中的临床应用

视黄醇结合蛋白（retinol binding protein,RBP）是一种分泌型视黄醇结合蛋白,主要合成于肝脏,广泛分布于人体血液、尿液等体液中的视黄醇（VitA）运载蛋白,在协助 VitA发挥生理功能中起到关键作用[1]。最新研究表明：RBP4是一种新的脂肪细胞因子,参与胰岛素抵抗和2型糖尿病的发生,与糖尿病肾病、营养性疾病等的发展存在着一定的相关性。这一发现使得 RBP4更加受到人们的重视。该文就 RBP4的生理功能,在肝肾疾病、糖尿病等各类疾病方面的应用以及新型临床检测方法作一综述。     

脓毒症患者血清肠型脂肪酸结合蛋白、二胺氧化酶水平检测对早期肠组织损伤及预后的评估价值

目的　探讨血清肠型脂肪酸结合蛋白（ I-FABP）,二胺氧化酶（ DAO）对脓毒症患者早期肠组织损伤及预后的评估价值。方法　选择 2019年 4~12月于同济大学附属同济医院急诊科收治的 80例脓毒症患者及 40例同期健康体检者（对照组）。脓毒症患者又根据急性胃肠损伤分级 (AGI)分为非 AGI组（n=35）,AGI组（n=45）。根据入院 28天内死亡情况,将患者分为存活组（ n=51）与死亡组（ n=29）。比较各组患者血清 I-FABP,DAO及其他临床特征,采用 logistic回归分析脓毒症患者 28天内预后的危险因素。结果　与对照组比较,非 AGI组和 AGI组患者 I-FABP（20.28±3.37, 26.15±4.67μg/L vs 17.16±2.44 μg/L）,DAO（2.49±0.63, 3.28±0.87mmol/L vs 1.31±0.34 mmol/L）水平升高（ F=65.92, 94.24, P<0.05）;与非 AGI组比较, AGI组 I-FABP,DAO水平亦显著升高（ P<0.05）。I-FABP,DAO与 CRP,D-乳酸呈显著正相关（ r=0.415,0.477,0.426和 0.465,均 P<0.05）,而与 TNF-α,IL-6无明显相关性（ P>0.05）。与存活组比较,死亡组年龄较大, MV时间明显延长, CRP,PCT,APACHE II评分, SOFA评分, AGI评分, I-FABP,DAO显著升高（ t=2.27~11.21,均 P<0.05）。多因素 logistic回归分析显示,APACHE II评分（ OR=3.13,95%CI:1.67~4.48）, AGI分级（ OR=2.36,95%CI:1.38~3.49）,I-FABP（OR=1.73,95%CI:1.24~2.51）,DAO（OR=1.42,95%CI:1.13~1.84）均是 28天内死亡的独立危险因素（ χ2=9.37~20.67,均 P<0.05）。结论　脓毒症患者血清 I-FABP和 DAO明显升高,与 CRP和 D-乳酸间有良好相关性,可反映早期肠组织损伤,有效预测脓毒症患者预后。     

Ethnic and Sex Differences in Fatty Liver on Cardiac Computed Tomography: The Multi-Ethnic Study of Atherosclerosis

ObjectiveTo describe ethnic and sex differences in the prevalence and determinants of fatty liver in a multiethnic cohort.Patients and MethodsWe studied participants of the Multi-Ethnic Study of Atherosclerosis who underwent baseline noncontrast cardiac computed tomography between July 17, 2000, and August 29, 2002, and had adequate hepatic and splenic imaging for fatty liver determination (n=4088). Fatty liver was defined as a liver/spleen attenuation ratio of less than 1. We compared the prevalence and severity of fatty liver, in 4 ethnicities (white, Asian, African American, and Hispanic), and the factors associated with fatty liver in each ethnicity, stratifying by obesity and metabolic syndrome. Multivariable ordinal logistic regression was used to determine the effect of cardiometabolic risk factors on the prevalence of fatty liver in different ethnicities.ResultsThe prevalence of fatty liver varied significantly by ethnicity (African American, 11%; white, 15%; Asian, 20%; and Hispanic, 27%; P<.001). Although African Americans had the highest prevalence of obesity, a smaller percentage of obese African Americans received a diagnosis of fatty liver than did other ethnicities (African American, 17%; white, 31%; Asian, 37%; and Hispanic 39%; P<.001). Hispanics had the highest prevalence of fatty liver, including the obese and metabolic syndrome population. An increase in insulin resistance predicted a 2-fold increased prevalence of fatty liver in all ethnicities after multivariable adjustment.ConclusionAfrican Americans have a lower prevalence and Hispanics have a higher prevalence of fatty liver than do other ethnicities. There are distinct ethnic variations in the prevalence of fatty liver even in patients with the metabolic syndrome or obesity, suggesting that genetic factors may play a substantial role in the phenotypic expression of fatty liver.     

Developmental Pattern of a Serum Binding Protein for Multiplication Stimulating Activity in the Rat

The concentration of multiplication stimulating activity (MSA), an insulinlike growth factor (IGF), is high in fetal rat serum. We now report that MSA is exclusively associated wth an albumin-size binding protein in fetal rat serum; the growth hormone-dependent, gamma globulin-size binding protein, which predominates in the older animal, is absent from fetal rat serum. When ¹²⁵I-MSA was incubated with fetal rat serum and then gel filtered on Sephadex G-200, specific radioactivity eluted in the void volume (peak I) and the albumin region (peak III); by contrast, specific radioactivity eluted mainly in the gamma globulin region (peak II) in adult rat serum. Pools of the Sephadex G-200 fractions were chromatographed on Sephadex G-50, in 1 M acetic acid, to separate the binding protein from IGF activity. Analysis of IGF activity by chick embryo fibroblast bioassay, competitive protein binding assay, and MSA by radioimmunoassay revealed that all the IGF activity and MSA in fetal rat serum resided in peak III. Measurement of MSA binding capacity of the stripped binding protein by Scatchard analysis demonstrated that the majority of binding capacity also was found in peak III in fetal rat serum; most of MSA binding capacity was in peak II in adult rat serum. In fetal rat sera, in addition to the peak III binding protein, which is the major carrier of endogenous MSA, there is a component in peak I capable of specifically binding ¹²⁵I-MSA. This component elutes as a single species from a Sepharose-6B column. As MSA associated with peak III gradually declined in early neonatal life, peak II-associated IGF activity measured by chick embryo fibroblast bioassay showed a rise of activity with a peak at 5 d of neonatal life, a nadir at 20 d, with an increase again to attain adult levels. These studies demonstrate that the MSA binding protein in the fetus is different from the growth hormone-dependent binding protein in adult life.     

Difference in Hepatic Metabolism of Long- and Medium-Chain Fatty Acids: the Role of Fatty Acid Chain Length in the Production of the Alcoholic Fatty Liver

Replacement of dietary triglycerides containing long-chain fatty acids (LCFA) by triglycerides containing medium-chain fatty acids (MCFA) markedly reduced the capacity of alcohol to produce fatty liver in rats. After 24 days of ethanol and MCFA, the increase in hepatic triglycerides was only 3 times that of controls, whereas an 8-fold rise was observed after ethanol and LCFA. The triglyceride fatty acids that accumulated in the liver after feeding of ethanol with MCFA contained only a small percentage of the MCFA; their composition also differed strikingly from that of adipose lipids.To study the mechanism of the reduction in steatosis, we compared oxidation to CO(2) and incorporation into esterified lipids of (14)C-labeled chylomicrons or palmitate-(14)C (representing LCFA), and of octanoate-(14)C (as MCFA) in liver slices and isolated perfused livers, in the presence or absence of ethanol. Ethanol depressed the oxidation of all substrates to CO(2); MCFA, however, was much more oxidized and reciprocally much less esterified than LCFA, with a 100-fold difference in the ratio of esterified lipid-(14)C to (14)CO(2). Furthermore, in hepatic microsomal fractions incubated with alpha-glycerophosphate, octanoate was much less esterified than palmitate. This propensity of MCFA to oxidation rather than esterification represents a likely explanation for the reduction in alcoholic steatosis upon replacement of dietary LCFA by MCFA.     

Evaluation of Non-alcoholic Fatty Liver Disease Using Acoustic Radiation Force Impulse Imaging Elastography in Rat Models

The aim of this study is to evaluate the utility of acoustic radiation force impulse (ARFI) elastography for assessing hepatic fibrosis stage and non-alcoholic fatty liver disease (NAFLD) severity, as well as the relationship among hepatic histologic changes using shear wave velocity (SWV). Animal models with various degrees of NAFLD were established in 110 rats. The right liver lobe was processed and embedded in a fabricated gelatin solution (porcine skin). Liver mechanics were measured using SWV induced by acoustic radiation force. Among the histologic findings, liver elasticity could be used to differentiate normal rats from rats with simple steatosis (SS) as well as distinguish SS from non-alcoholic steatohepatitis (NASH), with areas under the receiver operating characteristic curves (AUROC) of 0.963 (95% confidence interval = 0.871–0.973) and 0.882 (95% confidence interval = 0.807–0.956), respectively. For NAFLD rats, the diagnostic performance of ARFI elastography in predicting significant fibrosis (F ≥ 2) had an AUROC of 0.963. For evaluating steatosis severity, we found a progressive increase in ARFI velocity proportional to steatotic severity in NAFLD rat models, but we observed no significant differences for steatotic severity after excluding the rats with fibrosis. ARFI elastography may be used to differentiate among degrees of severity of NAFLD and hepatic fibrotic stages in NAFLD rat models.     

2型糖尿病合并非酒精性脂肪性肝病患者血浆脂肪细胞特异性脂肪酸结合蛋白水平的分析

目的研究2型糖尿病（type 2diabetes mellitus,T2DM）合并非酒精性脂肪性肝病（non-alcoholic fattyliver disease,NAFLD）患者血浆脂肪细胞特异性脂肪酸结合蛋白（adipocyte-specific fatty acid-binding protein,A-FABP）的水平及其相关因素。方法 2009年10月～2010年10月选取T2DM合并NAFLD组（A组）60例,未合并NAFLD组56例（B组）为研究对象。测定体质量指数（body mass index,BMI）,检测血脂、糖化血红蛋白（hemoglobinA1c,HbA1c）等生化指标。放射免疫法测定空腹胰岛素（fasting insulin,FINS）,空腹C肽水平（fasting C-peptide,FCP）,计算胰岛素抵抗指数（homeostasis model of assessment-insulin resistance,HOMA-IR）、胰岛素敏感指数（insulin sensitivity index,ISI）,测定A-FABP、C反应蛋白（C-reaction protein,CRP）及肿瘤坏死因子-α（tumor necrosisfactor-α,TNF-α）。结果与B组患者相比,A组患者其血浆A-FABP水平、BMI、腰围、腰臀比、丙氨酸氨基转移酶、门冬氨酸氨基转移酶、CRP、TNF-α、FCP、FINS、总胆固醇、甘油三酯、Ln（HOMA-IR）升高,Ln（ISI）降低,差异有统计学意义（P〈0.05）;两组HbA1c差异无统计学意义（P〉0.05）。A-FABP水平变化与TNF-α、HOMA-IR、CRP呈正相关,与ISI呈负相关。结论 T2DM伴NAFLD中,A-FABP升高与胰岛素抵抗是并存的,且存在明显相关关系,二者在疾病的发生发展中均可能具有重要的作用。     

Acoustic Impedance Analysis with High-Frequency Ultrasound for Identification of Fatty Acid Species in the Liver

Acoustic properties of free fatty acids present in the liver were studied as a possible basis for non-invasive ultrasonic diagnosis of non-alcoholic steatohepatitis. Acoustic impedance was measured for the following types of tissue samples: Four pathologic types of mouse liver, five kinds of FFAs in solvent and five kinds of FFAs in cultured Huh-7 cells. A transducer with an 80-MHz center frequency was incorporated into a scanning acoustic microscopy system. Acoustic impedance was calculated from the amplitude of the signal reflected from the specimen surface. The Kruskal–Wallis test revealed statistically significant differences (p < 0.01) in acoustic impedance not only among pathologic types, but also among the FFAs in solvent and in cultured Huh-7 cells. These results suggest that each of the FFAs, especially palmitate, oleate and palmitoleate acid, can be distinguished from each other, regardless of whether they were in solution or absorbed by cells.     

大鼠肝硬化形成早期的红细胞电泳率和红细胞膜胆固醇含量改变的研究

目的探求在大鼠肝硬化形成的早期,红细胞电泳率和红细胞膜胆固醇含量的变化情况.方法将Wistar大鼠随机分成对照组和肝硬化组,其中肝硬化组又分成2、5、7、10周组,每组10只.肝组织行H-E和天狼猩红染色,下腔静脉取血检测红细胞电泳率和红细胞膜胆固醇的含量.结果在本实验中,肝硬化7w鼠的肝组织中有大量的胶原纤维增生并开始形成假小叶.对照组和2w肝硬化组间的红细胞电泳率的差异不明显(P>0.05),而自给药的第5周起出现明显的增加;肝硬化2w鼠红细胞膜胆固醇含量明显升高,至第5周开始降至正常水平以下.结论本大鼠肝硬化模型的红细胞电泳率和红细胞膜胆固醇含量在第5周后出现明显的异常,比形态学上出现假小叶等典型的肝硬化表现约提前了2周时间,为临床肝硬化的早期诊断治疗提供实验依据.     

Effects of Parathyroid Hormone on Skeletal Muscle Protein and Amino Acid Metabolism in the Rat

Because prominent skeletal muscle dysfunction and muscle wasting are seen in both chronic uremia and in primary hyperparathyroidism, and because markedly elevated parathyroid hormone levels occur in both disorders, potential effects of parathyroid hormone on skeletal muscle protein, amino acid, and cyclic nucleotide metabolism were studied in vitro using isolated intact rat epitrochlearis skeletal muscle preparations. Intact bovine parathyroid hormone and the synthetic 1-34 fragment of this hormone stimulated the release of alanine and glutamine from muscle of control but not from chronically uremic animals. This stimulation was dependent upon the concentration of parathyroid hormone added: At 10⁵ ng/ml parathyroid hormone increased alanine release 84% and glutamine release 75%. Intracellular levels of alanine and glutamine were not altered by parathyroid hormone. Increasing concentrations of the 1-34 polypeptide decreased [³H]leucine incorporation into protein of muscles from both control and uremic animals. Using muscles from animals given a pulse-chase label of [guanido-¹⁴C]arginine in vivo, parathyroid hormone increased the rate of loss of ¹⁴C label from acid-precipitable protein during incubation and correspondingly increased the rate of appearance of this label in the incubation media. Parathyroid hormone increased muscle cAMP levels by 140% and cGMP levels by 185%, but had no effect on skeletal muscle cyclic nucleotide phosphodiesterase activities as assayed in vitro. Adenylyl cyclase activity in membrane preparations from control but not uremic rats was stimulated by parathyroid hormone in a concentration-dependent fashion. However, no stimulation of guanylyl cyclase activity was noted by parathyroid hormone, although stimulation by sodium azide was present. Incubation of muscles with added parathyroid hormone produced a diminished responsiveness towards epinephrine or serotonin regulation of amino acid release and cAMP formation in the presence compared to the absence of parathyroid hormone. In the absence of parathyroid hormone, detectable inhibition of alanine and glutamine release was produced by 10⁻⁹ M epinephrine, whereas in the presence of parathyroid hormone (1,000 ng/ml) inhibition of alanine and glutamine release required 10⁻⁶ M or greater epinephrine. Resistance to cyclic AMP action as well as inhibition of cyclic AMP formation by parathyroid hormone was found. Preincubation of rat sarcolemma with 1-34 parathyroid hormone produced a decreased activity of the isoproterenol-stimulable adenylyl cyclase activity but there was no apparent change in the concentration of isoproterenol required for one-half maximal and maximal stimulation of the enzyme.     

大鼠凝血因子Ⅶ EGFP-EGF1融合蛋白的表达及其结合功能初步研究

本研究通过融合蛋白EGFP-EGF1的表达,探讨大鼠凝血因子Ⅶ上EGF1片段与组织因子（TF）的结合功能。用RT-PCR方法自大鼠肝脏组织获得EGF1编码区基因并将其插入EGFP原核表达载体中,构建pET28a-EGFP-EGF1融合蛋白表达载体;将该重组质粒转化大肠杆菌BL21中,用IPTG诱导表达EGFP-EGF1融合蛋白;采用亲和层析方法Ni柱纯化融合蛋白,将融合蛋白作用于经脂多糖刺激表达TF的大鼠血管内皮细胞;通过荧光显微镜及流式细胞术观察和检测融合蛋白与细胞结合情况。结果表明：EGFP-EGF1在转化的大肠杆菌中获得高效表达并成功纯化,SDS-PAGE证实分子量约为36kD。荧光显微镜及流式细胞术检测显示该融合蛋白能与内皮细胞膜上的TF结合。结论：大鼠凝血因子Ⅶ上EGF1区可介导FⅦ与TF特异性结合,从而可能为分子靶向抗栓治疗研究提供部分基础。