大肠杆菌分泌表达重组人肝再生增强因子的纯化、鉴定与活性研究

目的建立一种大肠杆菌分泌表达型重组人肝再生增强因子(rhALR)的纯化工艺,并对产品进行鉴定和活性研究。方法通过沉淀、色谱等方法的条件优化,建立纯化工艺,通过电泳、HPLC、N-末端氨基酸测序、免疫杂交、同位素掺入、全波长扫描等方法对纯化产品进行鉴定和活性测定。结果建立了rhALR的纯化工艺路线,40%(NH4)2SO4沉淀,Butyl-S FF疏水色谱,CM-S FF离子交换色谱,Superdex-75分子筛色谱纯化,重组蛋白质的纯度大于95%。该蛋白质稳定存在的形式为二聚体,其单体的相对分子质量为15 000。该蛋白质结合辅酶黄素腺嘌呤二核苷酸(FAD),对损伤后的肝细胞具有明显的促进作用,明显优于以包涵体形式表达的rhALR。结论从大肠杆菌分泌表达体系能分离纯化得到高纯度、具有生物学功能和活性的rhALR,是潜在的临床治疗肝病药物。     

百令胶囊对肾小管间质纤维化大鼠肾小管上皮-间质转分化的干预作用

目的探讨百令胶囊对小管间质纤维化大鼠小管上皮-间质转分化的干预作用。方法利用腺嘌呤诱导建立肾小管间质纤维化大鼠模型,实验SD大鼠随机分为模型组、干预组、对照组各30只,分别于实验第7周、12周、17周收获动物各10只,进行功能学和肾脏组织病理学检测和观察。利用免疫组织化学对骨形态发生蛋白-7(BMP-7)、转化生长因子-β1(TGF-β1)和α-平滑肌肌动蛋白(-αSMA)在肾小管间质纤维化大鼠中的表达变化进行动态观察及百令胶囊的干预影响。结果实验第7周模型组大鼠即表现出大量蛋白尿、小管损伤、间质的轻度纤维化和炎症细胞浸润(P<0.01);随病程进展,上述病变呈进行性加重(P<0.01)。百令胶囊干预组动物在实验第7周、12周功能学组织学的改善同实验组相比有明显差异(P<0.01),17周后二者无显著差异。免疫组织化学显示百令胶囊12周前能显著上调BMP-7的表达和降低-αSMA和TGF-β1在肾脏小管间质中的表达(P<0.01),12周后这种变化无差异。结论百令胶囊在发病早期通过有效干预上皮-间质转分化达到改善肾脏纤维化的作用,随着肾小管间质纤维化程度的加重,百令胶囊逐渐失去其阻断作用。     

Decreased mitochondrial gene expression in isolated islets of rats injected neonatally with streptozotocin

Summary The aim of the present study was to evaluate the possible role of the expression of the mitochondrial genome for the regulation of insulin production in the pancreatic Beta cell. For this purpose, islets of Langerhans were isolated from adult control rats and rats injected neonatally with streptozotocin and the islet contents of specific mitochondrial DNAs and RNAs together with nuclear-encoded RNAs were determined. The contents of mitochondrial cytochrome b mRNA, the mitochondrial 12 S rRNA and insulin mRNA were all 30–40% lower in islets isolated from the streptozotocin-treated rats as compared to islets from control rats. In contrast, the nuclear mRNA coding for the mitochondrial adenine nucleotide translocator was not decreased in the streptozotocin-treated rats. Contents of mitochondrial DNA, as assessed by the Southern blotting technique, were markedly decreased in the streptozotocin islets. Sequence analysis of mitochondrial DNA from streptozotocin islets and control islets however, did not reveal any differences in nucleotide sequences. In control islets the contents of mitochondrial cytochrome b mRNA increased in response to a high glucose concentration during a 4-h incubation period. Serum deprivation or the addition of theophylline or 4-phorbol 12-myristate 13-acetate failed to affect the cytochrome b mRNA contents in vitro. It is concluded that islets of streptozotocin-treated rats contain low contents of mitochondrial DNA and RNA. Since a lower mitochondrial RNA content may result in a diminished oxidative capacity, it is conceivable that a deficiency of this messenger may contribute to the development of insulin deficiency.     

Two populations of calbindin D28k-immunoreactive neurones in the striatum of the rat

In order to examine the localisation of calbindin D28k in the normal striatum of the rat, immunocytochemistry using monoclonal antibodies was carried out at both the light and electron microscopic levels. As has previously been shown, many striatal spiny neurones were immunopositive, however, a small population of neurones with smooth dendrites and indented nuclei were also identified. At least some of these cells also displayed NADPH-diaphorase activity. These findings indicate that, in common with the primate striatum and grafted striatal tissue in the rat, the normal rat striatum contains at least two populations of neurones that express calbindin D28k.     

中药有效成分对肿瘤恶病质脂耗竭的抑制作用

为探索抑制毒激素-L所诱导的恶病质脂耗竭的有效药物,将中药有效成分次黄嘌吟、尿囊素、腺嘌吟、大叶茜草素及欧琴素—乙等分别与毒激素-L和从大鼠获得的脂细胞一起孵育,并与单纯中药组和单纯毒激素-L组相比较。结果5种中药成分和毒激素-L组的游离脂肪酸的含量明显低于单纯毒激素-L组(P<0.01),提示5种中药成分均可抑制毒激素-L所诱导的脂解反应。而单纯中药组与正常对照组无差异(P>0.05),提示该5种中药成分对正常脂解反应无影响。     

Maintenance of rat liver viability enhanced by cold flushing with oxygenated perfluorochemicals

The possible benefit of oxygenation during initial cold flushing was investigated as a means of improving the quality of liver preservation in rats. In five groups of animals (total 61 experiments), the livers were flushed with different perfusates. Non-oxygenated groups included controls, Collins’ solution alone and Collins’ solution containing perfluorotributylamine (FC-43). In the oxygenated groups, Collins’ solution alone and Collins’ solution containing FC-43 were oxygenated by bubbling. The hepatic ATP level and histopathological changes were used to assess the quality of liver preservation. Oxygenation during the initial cooling process proved to be effective in maintaining energy metabolism and preventing the characteristic microscopic changes of ischemic damage. Oxygenated Collins’ solution containing FC-43 showed a much longer lasting effect compared with oxygenated Collins’ solution alone, without FC-43. Under light microscopy, the integrity of the liver appeared to be well preserved up to eight hours with the former solution. It is concluded that enhanced oxygenation with FC-43 in the initial cold flushing period can improve the quality of liver preservation.     

Architectural alterations of rat hind-limb skeletal muscles immobilized at different lengths

Modifications in architectural profiles of the rat soleus (Sol), medial gastrocnemius (MG), and tibialis anterior (TA) muscles were studied after hind-limb immobilization with an external brace. The muscles were chronically lengthened, shortened, or maintained at approximately resting length (neutral) for 4 weeks, when muscle weight and length, and cross-sectional areas of fibers (F-XSA) and whole muscle were measured. Compared with control muscles, the Sol and MG immobilized in a lengthened position were able to maintain muscle weight better than when immobilized neutrally (approximately 85 versus 55% of control weight) due, in part, to elongation of muscle fibers of the Sol and MG (123 and 111% of control length, respectively). In addition, the F-XSAs of lengthened Sol and MG muscles were 72 and 20% greater, respectively, compared with neutrally fixed muscle fibers. Immobilization in a shortened position resulted in the most extreme muscle atrophy due to significantly shorter fibers (86% of control Sol; 74% of control MG) and reduced F-XSA (42% of control Sol; 42% of control MG). In contrast to the Sol and MG, muscle weight, fiber length, and F-XSA of the TA immobilized with the ankle in extreme plantarflexion were not significantly different from neurtrally fixed muscle. That the TA was unaffected in this position is due presumably to a difference in the anatomic attachment of the distal tendon of this muscle, compared with that of the Sol and MG. These results suggest that for muscle affected by immobilization at extreme joint angles, chronic lengthening is a potent stimulus for elongation of muscle fibers and more normally maintained F-XSA. In contrast, the removal of this stimulus leads to extreme muscle atrophy which is manifested by reductions in both muscle fiber length and F-XSA.     

Abnormal Property of Human Mutant Hypoxanthine-Guanine Phosphoribosyltransferase: Insensitivity of Fibroblast Enzyme to Stabilization against Freezing and Thawing by 5-Phosphoribosyl-1-Pyrophosphate

Abstract. Freezing and thawing of dilute normal human fibroblast suspensions causes partial inactivation of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and adenine phosphoribosyltransferase (APRT). Phosphoribosyl-pyrophosphate (PRPP) stabilizes both phosphoribosyltrans-ferases against this inactivation. Mutant HGPRT enzymes from a patient with the Lesch-Nyhan syndrome and from a gouty patient with partial HGPRT deficiency were similarly inactivated by freezing and thawing, but only the former mutant enzyme could be stabilized by PRPP. The insensitivity of the mutant HGPRT from the patient with partial enzyme deficiency to PRPP stabilization indicates a structural enzyme alteration. The different sensitivity of the two HGPRT mutants to PRPP stabilization reflects the heterogeneity of HGPRT mutations in man.