2004年 · 第117卷第02期

Background To express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins. Methods Bovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from the duodenal mucosa of a bovine obtained at a wholesale market, and then cloned Into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His · Tag affinity chromatography, and its bioactivity was analyzed. Results Compared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DNA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28%of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using a Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 21℃ overnight and shown to have a high autocatalytic cleavage activity. Conclusion The EKL gene from a Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products. Chin Med J 2004; 117(2): 286-290

Background There have been no detailed reports of the three-dimensional structure and the relationship between the external and internal vascularizations observed successively for a long duration in the rat fetus, although many authors have studied the vascular morphology of the developing brain. This study examined the three-dimensional structure of both the external and internal vascularizations of the prenatal rat telencephalon from embryonic days 12 (E12) to 20 (E20). Method A microvascular casting method for scanning electron microscopy (SEM) was used in this study, along with vascular staining using gold-gelatine solution-autometallography (GGS-AMG) after intravascular injection of colloidal gold, as well as hematoxylin-eosin (HE) staining for paraffin embedded specimens. Results In GGS-AMG stains, E16 fetuses had a few short perforating cortical blood vessels(SPCVs); E17 fetuses had long perforating cortico-medullary vessels (LPCVs). Older fetuses had specific patterns of vascular networks in the cortex and the deeper subcortical part of the telencephalon. In the cortex, fine longitudinal blood vessels were connected by transverse channels. The deep telencephalon had fine blood vessels running in all directions. Using SEM, the external vascularization was already visible in E12 fetuses as arborizations of arterial branches, forming a mesh of fine vascular networks covering the telencephalon. A coralliform fine venous plexus was observed in the external vascularization of E16 fetuses. There were ring-like anastomoses and bud-like protrusions in the network of small blood vessels, most likely the angiogenesis of fetal vessels. From E12 to E16, an immature and incomplete internal vascularization began to appear. There were short blood vessels with ballooned terminals branching from the external vascularization. They penetrated the brain tissue to form networks in the superficial layer, comparable to SPCVs. In E17 to E20 fetuses, tortuous venous branches, straight arterial blood vessels, and a fine network of small blood vessels formed the external vascularization. There were fewer arterial than venous branches connecting to the fine networks of small blood vessels. LPCVs were noted at E17, at the time the white matter emerged. They branched from the external vascularization, and perpendicularly penetrated the brain surface, traversing the cortical plate, and entering into the deep brain. At E17, arterial and venous blood vessels could be clearly distinguished in the external vascularization. At E20, the cortex and white matter contained specific arrangements of networks of fine blood vessels, as seen by GGS-AMG staining. Conclusion These findings show that the development of both the external and internal vascularization follows the development of the telencephalon. In particular, the emergence of the cortical plate and white matter on E16 and E17 influence the development of both the internal and the external vascularization. The laminal arrangement of blood vessels was not observed corresponding to the respective laminal neuronal layers. Chin Med J 2004; 117(2): 280-285

Background Ischemia-reperfusion (IR) injury to the liver is still a critical and daunting problem in the field of hepatobiliary surgery. Ischemic preconditioning (IP) of the liver serves as an effective approach against IR injury. This study was to develop a novel procedure that could mimic IP, but might be more feasible than IP during surgery. Methods Eighty-two SD rats were randomly divided into 5 groups. L group (n=21): 0. 4%lidocaine (10 mg/kg) was injected into the hepatoduodenal ligament 10 minutes before a 40-minute hepatic IR. IP group (n=16): a 5-minute ischemia was followed by a 10-minute reperfusion prior to a 40-minute hepatic IR. ILR group (n=15): after a 40-minute ischemia of the liver, 0.4% lidocaine (10mg/kg) was injected into the hepatoduodenal ligament 10 minutes prior to a 40-minute reperfusion of the liver. IR group (n=15): the liver of the rat was subjected to a 40-minute IR. Control group (n=15): 0.9%sodium chloride was injected into the hepatoduodenal ligament without other treatments. The levels of plasma alanine transaminase (ALT) and aspartate transaminase (AST) were determined for each group after treatment. Results The mean concentrations of ALT and AST were (379. 80 ±141. 69) U/L and (606. 05 ±220. 26) U/L for the L group, (334. 64 ±141.94) U/L and (625. 68 ±267. 06) U/L for the IP group,(523. 36 ±170. 35) U/L and (765. 47 ±238. 45) U/L for the ILP group, (524. 29 ±163. 59) U/L and(764. 63 ±246. 79) U/L for the IR group, and (150. 90 ±27. 05) U/L and (298.15 ±47.68) U/L for the control group (standard error of the mean). Conclusion A significant decrease in ALT and AST levels was observed in the L and IP groups when compared to the ILR and IR groups (P<0. 05), but no significant difference in ALT and AST levels was observed in the L group when compared to the IP group (P>0.05). These results suggest that pretreatment with lidocaine injected into the hepatoduodenal ligament prior to IR provides effective protection against subsequent IR injury to the liver. The novel approach of blocking innervation with lidocaine mimics hepatic IP, but is more convenient than IP at the time of liver surgery. Chin Med J 2004; 117(2): 275-279

Background Guided tissue regeneration procedures provide predictable reconstruction of periodontal tissues in the treatment of furcation involvements in animals and huamns. This study was to compare long-term effectiveness of two different types of polylactic acid (PLA) membranes on periodontal regeneration in surgically created class II furcation defects in dogs. Methods Full thickness mucoperiosteal flap was raised on the buccal aspects of the experimental teeth and class II furcation defects having 5 mm vertical dimensions were created on mandibular premolar III and IV on each quadrant. The exposed root surfaces were thoroughly planed and PLA membranes were placed over the experimental defects on both sites. One site received liquid polymer membrane (LPM), and resorbable periodontal mesh (RPM) membranes were applied to the other site. The animals were sacrificed at 7 months after surgery and the specimens were processed for histological evaluation. Results The average length of new attachment formed on the treated roots in both groups ranged from 3.02 mm to 4. 5 mm. Complete bone filling was observed at the furcation srtes. No statistically significant differences were found between two membranes in any of the parameters (P>0.05). Conclusion This study demonstrates favorable regenerative outcomes by the use of two different types of PLA membranes that could be used as alternatives for guided tissue regeneration (GTR). Chin Med J 2004; 117(2): 270-274

Background Damage to the gut barrier often occurs during critical illnesses. In such cases, it is very important to alleviate impairment of the intestinal barrier and protect intestinal barrier function. This study investigated the protective effect of growth hormone on intestinal barrier function in rats under stress. Methods This study consisted of prospective, randomized, and controlled animal experiments. Twenty-five Sprague-Dawley rats served as total parenteral nutrition (TPN) models and were divided into three groups: TPN group, sepsis (Sep) group, and growth hormone (GH) group. Another 8 rats served as normal controls. Each group received different stress stimuli. Rats were fed for 7 days, and samples were taken for examination 24 hours after gavaging with dual saccharides. Results The architecture of the small intestinal mucosa in the Sep group showed the most severe damage among all groups. Nitric oxide levels in blood plasma and immunoglobulin A levels in the intestinal mucosa of the GH group were significantly lower than in the Sep group (P <0. 02). There were no significant changes in CD3 counts and in the CD4/CD8 ratio between the four groups. Dual sugar tests and bacteriological examinations revealed that intestinal permeability and rate of bacterial translocation in the GH group were lower than in the Sep group (P<0.011 respectively). Conclusion Prophylactic treatment with growth hormone can alleviate damage to intestinal barrier function caused by trauma and endotoxemia in rats under stress. Chin Med J 2004; 117(2): 264-269

Background Post-stenting restenosis is a significant clinical problem, involving vascular smooth muscle cells (VSMCs) proliferation and apoptosis. It is reported that c-myc antisense oligodeoxynucleotides (ASODNs) local delivered by catheter can inhibit VSMCs proliferation. This study was designed to assess tissue distribution of c-myc ASODN local delivered using gelatin-coated platinum-iridium (Pt-Ir) stents, and its effect on apoptosis of VSMCs. Methods Gelatin-coated Pt-Ir stents that had absorbed caroboxyfluorescein-5-succimidyl ester(FAM) labeled c-myc ASODNs (550 μg per stent) were implanted into the right carotid arteries of 6rabbits. Tissue samples were obtained at 45 minutes, 2 hours, and 6 hours. Tissue distribution of c-myc ASODNs was assessed by fluorescence microscopy. In addition, 32 rabbits were randomly divided into two groups. Rabbits in the control group (n=16) were implanted with gelatin-coated Pt-Ir stents, and those in the treatment group (n=16) were implanted with gelatin-coated stents that had absorbed c-myc ASODNs. 7, 14, 30, or 90 days (n=4, respectively, for each group) after the stenting procedure, the stented segments were harvested, and histopathological examinations were performed to calculate neointimal area and mean neointimal thickness. The expression of c-myc was assessed using in situ hybridization (ISH) and immunohistochemical methods. Apoptotic VSMCs were detected using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and transmission electron microscope (TEM). Results According to fluorescence microscopic results, FAM-labeled c-myc ASODNs were concentrated in the target vessel media at the 45 minutes time point, and then dispersed to the adventitia. Morphometric analysis showed that neointimal area and mean neointimal thickness increased continuously up to 90 days after stent implantation, but that total neointimal area and mean neointimal thickness were less in the treatment group than in the control group at all time points (P <0. 0001). At day 7 and day 14 after stenting, there were no detectable apoptotic cells in either group. However, apoptotic cells were present in the neointima 30 and 90 days after stenting, and the number of apoptotic cells was less at 30 days than at 90 days. Meanwhile, c-myc ASODNs appeared to induce apoptosis in more cells in the treatment group than that in the control group. Typical apoptotic VSMCs were observable under TEM. The expression of c-myc was positive in the control group and negative or weakly positive in the c-myc ASODN treatment group, according to both ISH and immunohistochemical examination. Conclusion Gelatin-coated Pt-Ir stent mediated local delivery of c-myc ASODNs is feasible. The localization of c-myc ASODN is primarily in the target vessel walls. c-myc ASODNs can inhibit VSMCs proliferation and induce its apoptosis after local delivery in vivo. Chin Med J 2004; 117(2):258-263

Background Basic fibroblast growth factor (bFGF) plays important roles in retina degeneration, light injury, mechanical injury, especially in retina ischemia-reperfusion injury (RIRI). This study was to investigate the therapeutical effect of bFGF on RIRI and its mechanisms. Methods Experimental RIRI was induced by increasing intraocular pressure (IOP) in the eyes of 48rats. These rats were divided into normal control, ischemia-reperfusion and bFGF-treated groups. Histological and ultrastructural changes of in the retina of different groups were observed, and the number of retinal ganglion cells (RGCs) was quantitatively analyzed under microscopy. Apoptotic cells were detected using the TdT-dUTP terminal nick-end labeling (TUNEL) method. The expression of caspase-3 was determined by streptavidin peroxidase (SP) immunohistochemistry. Atomic absorption spectrum method was used to evaluate the intracellular calcium changes. Results At the early stage of retinal ischemia-reperfusion injury, retina edema in the treated group was significantly eliminated compared with the untreated ischemic animals. RGCs in the bFGF-treated group was more than those in the untreated ischemic group during the post-reperfusion stages. In ischemic group, apoptotic cells could be found at 6th hours after reperfusion and reached the peak at 24th hours. At 72th hours no apoptotic cells could be found. The changes in caspase-3 expression had a similar manner. The intracellular calcium of rat retina began to increase at 1th hour, reached the peak at 24 hours, and began to decease at 72th hours. The change of the three markers in the treatment group showed a similar pattern, but they were all relatively less obvious. Conclusion Apoptosis may play a vital role in RIRI. bFGF may has therapeutical effects on RIRI by inhibiting the increase of intracellular calciums and caspase-3 expression. Chin Med J 2004; 117(2): 252-257

Background Previous research indicated that the development of diabetic retinopathy (DR) is closely related to the excessive expression of growth factors. This paper was to study the relationship of DR with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and the retinal vascular pathological change. Methods Fifty-five Wistar rats, weighing 100 -200 g, were selected and randomly divided into four groups: control group (no streptozocin injection, n=10), M1 group (streptozocin induced diabetes for 1 month, n=15), M3 group (streptozocin induced diabetes for 3 months, n=15), and M5 group(streptozocin induced diabetes for 5 months, n=15). In situ hybridization and immunohistochemistry were used to investigate the expressions of bFGF and VEGF on retinal vascular, and retinal vessels were observed by transmission electron microscope. Results There was no difference in the number of pericytes between M1 and control group (P>0.05), but the number of pericytes decreased obviously in M3 and M5 groups compared with the control group (P <0. 01, P<0. 001, respectively). Capillary embolization and non-cell capillary were seen in M5 group. Positive expression of VEGF was found in M5 group using in situ hybridization and immunohistochemistry respectively. Positive expression of bFGF could be seen in M3(78%) and M5group (89%). Most remarkable changes of vessles were observed in M5 group including fragmental thickness, split of basement membrane, swelling and distortion of endothelial cells. Conclusions In retinal vascular of the streptozocin (STZ) rats, there shows the expression of bFGF at the third month and that of VEGF at the fifth month. Chin Med J 2004; 117(2): 247-251

Background Ilexonin A (IA), purified from the Chinese herbal medicine Maodongqing (Ilex pubescens Hook, et Am) has been commonly used in south China to treat thrombotic disorders. In this study, we aimed to study the inhibiting effects and mechanism of IA on von Willebrand factor(vWF)-dependent high shear-induced platelet aggregation. Methods vWF-dependent high shear (10 800 s-1) induced aggregation of platelets obtained from normal donors in the presence or absence of IA was measured by a modified cone-plate viscometer and shear-induced vWF binding was measured by quantitative flowcytometry with monoclonal antibody known to bind exclusively to the C-terminal domain of vWF (LJ-C3) directly labeled with fluorescein isothiocyanate (FITC). P-selectin surface expression was also measured by a similar method with FITC conjugated anti-P-selectin monoclonal antibody (WGA1). Results Shear-induced platelet aggregation was inhibited by IA in a dose-dependent manner. The extent of aggregation decreased from (78. 6 ±4. 6)% in the absence of IA to (36. 5 ±2.1)% in the presence of IA (3.3 mmol/L) (P<0. 0001, n=9) with a high shear rate of 10 800 s-1. vWF binding and P-selectin expression were also inhibited by IA in a dose dependent manner. The number of binding FITC-LJ-C3 molecules increased after exposure of platelet-rich plasma to a high shear rate of 10 800 s-1 for 6 minutes, but this shear-induced increased binding platelet surface vWF moiecules and P-selectin expression can be decreased in the presence of IA. Conclusion vWF binding and vWF mediated platelet activation, aggregation occurring under high shear rate were inhibited by IA. IA may be a unique antithrombotic drug inhibiting the vWF-GP Ibαinteraction, and may thus facilitate drug design targeting arterial thrombosis. Chin Med J 2004; 117(2): 241-246

Background To explore the effect of human osteopontin (hOPN) on the proliferation, transmigration and expression of matrix metallproteinase-2 (MMP-2) and matrix metallproteinase-9 (MMP-9) in osteosarcoma (OS) cells in vitro. Methods The prokaryotic-expression vector of hOPN was produced. hOPN was then subcloned into E. coli BL21 (DE3) cells and purified with ProBond™ Columns. The proliferation, cell cycle and the expression of cyclin A in OS cells were investigated by using MTT assay, flow cytometry and Western blot respectively. The transmigration of OS cells was checked by using transwell cell culture chamber. The micro-pore-filter-membrane system was used to study the chemiotaxis of hOPN to OS cells. The levels of total protein were examined according to Coomassie Brilliant Blue manuals. The expression of MMP-2 and MMP-9 were evaluated by detecting the volume of degradation of gelatin on SDS-PAGE gel. Results The prokaryotic-expression vector of hOPN and purified hOPN protein were achieved hOPN promoted OS cells proliferation in a dose-dependent manner, and stimulated cyclin A expression in OS cells to accelerate cell division cycle. hOPN facilitated the trans-membrane migration of OS cells. hOPN also enhanced the secretion of MMP-2 and MMP-9 in OS cells. Conclusion hOPN could stimulate cyclin A expression in OS cells. hOPN has chemiotaxis to OS cells and increases their transmigration. hOPN enhances the secretion of MMP-2 and MMP-9 in OS cells. Chin Med J 2004; 117(2): 235-240

Purpose The purpose of this study is to review the urine products of bone breakdown as markers of bone resorption and usefulness of urinary hydroxyproline. Data Related researches published in 1985 -2000 were systematically reviewed. Results Bone markers could be used for early diagnosis of bone metabolic diseases. Biochemical markers of bone resorption that reflect osteoclast activity and/or collagen degradation provide a new and potentially important clinical tool for the assessment and monitoring of bone metabolism. Assessment of bone resorption can be achieved with measurement of urinary hydroxylysine glycosides, urinary excretion of the collagen pyridinium cross-links, urinary excretion of type I collagen telopeptide breakdown products (cross-linked telopeptides) and urinary hydroxyproline. Conclusion Urinary hydroxyproline has been in use as a marker of bone resorption, but it lacks sensitivity and specificity. It is a modified aminoacid that is a metabolic product of collagen breakdown. Hydroxyproline may be released either free or with fragments of the collagen molecule attached during bone resorption, and it is also liberated by the breakdown of complement and nonskeletal collagen. Chin Med J 2004; 117(2): 291-295