Objective To investigate the expression of protein gene product 9.5 (PGP9.5) in human gastric cancer, and to find out the relations between its expression and carcinogenesis, invasion and metastasis of gastric cancer. Methods The expression of PGP9.5 was detected by immunohistochemistry (SP) and Western blot in 80 samples of gastric cancer and 8 samples of normal gastric tissues. Results ①Of 80 gastric cancer specimens examined, 56 cases (70.0％) showed staining with PGP9.5 in most tumor cells, whereas no PGP9.5 expression was detected in normal epithelium, which was consistent with the results of Western blot. ②The results of immunohistochemistry revealed that there were significantly correlations between the expression of PGP9.5 and the depth of invasion, the degree of differentiation, and the occurrence of lymph node metastasis (Plt;0.05), respectively; yet, there were no relation between the expression of PGP9.5 and age, gender, histopathologic type and TNM stage (Pgt;0.05). Conclusion PGP9.5 may play an important role in the invasion and metastasis of gastric cancer, and the invasion of gastric cancer could be detected by PGP9.5, which may be a useful molecular marker.
Objective To explore the regulating mechanism of hepatic injury in obstructive jaundice (OJ). Methods①Rat hepatocytes were isolated by in situ collagenase perfusion and primary culture. Hepatocytes were pretreated with various concentrations of protein kinase C (PKC) agonist parsmeae (PMA) and inhibitor chelerythrine for 20 min. After pretreatment, 50 μmol/L glycochenodeoxycholate (GCDC) was added. Cells were next detected by FCM and TUNEL.②Experimental obstructive jaundice was induced by double ligation of the bile duct (BDL), BDL for 3, 7, 14, 21days.We detected apoptotic status in liver with TUNEL and PKC protein in liver with immunohistochemistry method. Results①PMA increased GCDCinduced apoptosis and chelerythrine decreased GCDCinduced apoptosis in a concentrationdependent manner. ②The apoptotic rate of liver was related to time of OJ. Apoptosis index (AI) was the highest in 14day bile duct ligation. The ber PKC expression, the more number of apoptotic cells in OJ.Conclusion PKC takes part in the regulation and the occurrence and progression of hepatic injury in OJ.
Abstract： Objective To study the changes of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) expression of isolated rat hearts after diazoxide preconditioning (DPC), and to explore the possible mechanism of cAMP signaling pathway in myocardial protection by DPC. Methods Isolated working heart Langendorff perfusion models of 40 Wistar rats were set up and were divided randomly into four groups. For the ischemia reperfusion injury(I/R) group (n=10), 30 min of equilibrium perfusion was followed by a 60 min reperfusion of KrebsHenseleit (K-H) fluid. The DPC group (n=10) had a 10 min equilibrium perfusion and two cycles of 5 min of 100 μmol/L diazoxide perfusion followed by a 5 min diazoxidefree period before the 30 min ischemia and the 60 min reperfusion of K-H fluid. The blank control group (control group, n=10) and the Dimethyl Sulphoxide(DMSO) group (n=10) were perfused with the same treatment as in the DPC group except that diazoxide was replaced by natriichloridum and DMSO respectively. The activity of creatine kinase (CK) in coronary outflow, the activity of malonyldialdehyde (MDA) and superoxide dismutase (SOD) in myocardium were detected. And the scope of myocardial infarction and the concentrations of myocardial cAMP and PKA were also assessed. Results Compared with the I/R group, the level of MDA for the DPC group decreased significantly (8.28±2.04 nmol/mg vs. 15.52±2.18 nmol/mg, q=11.761,Plt;0.05), the level of SOD increased significantly (621.39±86.23 U/mg vs. 477.48±65.20 U/mg, q=5.598,Plt;0.05). After a 30 min reperfusion, compared with the I/R group, the content of CK decreased significantly (82.55±10.08 U/L vs. 101.64±19.24 U/L, q=5.598, Plt;0.05) and the infarct size reduced significantly (5.63%±9.23% vs.17.58%±5.76%, q=6.176,Plt;0.05) in the DPC group. The cAMP concentration in the DPC group was much higher than that in the I/R group (0.64±0.07 pmol/g vs. 0.34±0.05 pmol/g, q=14.738,Plt;0.05), and PKA concentration was also much higher than that in the I/R group ［17.13±1.57 pmol/(L·min·mg) vs. 12.85±2.01 pmol/(L·min·mg), Plt;0.05］. However, there were no significant differences between the I/R group, DMSO group and the control group in the above indexs (Pgt;0.05). Conclusion DPC significantly improves the releasing of cAMP and PKA, decreases oxygen free radicals, and relieves myocardial ischemia reperfusion injury. The cAMP signaling pathway may be involved in triggering the process of myocardial protection mechanisms of DPC.
Abstract： Objective To investigate the mechanism of protein kinase C(PKC) in immature myocardial ischemic preconditioning in order to further its clinical applicability. Methods Langendorff perfusion heart models of 24 rabbits were set up and they were randomly divided into 4 groups: ischemic reperfusion group (I/R group), myocardial ischemic preconditioning group (MIP group), chelerythrine group (CLT group) and protein kinase C group (PKC group). The emodynamics, biochemistry and myocardial ultrastructure were observed. Results The heart function recovery and myocardial water content in the MIP and the PKC groups were better than those of the I/R and the CLT groups (Plt;0.01). The adenosine triphosphate (ATP) content, superoxide dismutase activity, mitochondrial Ca2+-ATPase activity and synthesizing ATP activity of mitochondria in the MIP and the PKC groups were significantly higher than those of the I/R and the CLT groups (Plt;0.01). The dehydrogenase and creatine kinase leakage, malondialdehyde content, myocardial cell Ca2+ content and mitochondrial Ca2+ content in the MIP and the PKC groups were significantly lower than those of the I/R and the CLT groups (Plt;0.01). The myocardial ultrastructure injuries in the MIP and the PKC groups were less than that of the I/R and the CLT groups. Conclusion Myocardial ischemic preconditioning plays an important role in protecting immature myocardium, which is probably realized by the activation of PKC.
Objective To separate each protein band from the nerve regeneration conditioned fluid(NRCF）and to study whether there are somenew and unknown neurotrophic factors in the protein bands with a relative molecular mass of 220×103. Methods The silicone nerve regenerationchambers were formed in the sciatic nerve of the 25 New Zealand rabbits (weight,1.8-2.5 kg), and NRCF was taken from it at 1 week after operation. The Nativepolyacrylamide gel electrophoresis (Native-PAGE) was used for separating the proteins from NRCF and detecting the relative molecular mass. The Western blot and ELISA were used to observe whether the protein bands ［220×103 (Band a), (20-40)×103(Band c)］ of NRCF could combine with the antibody of the known antibody of neurotrophic factor (NTF):nerve growth factor(NGF), glial cell-derived neurotrophic factor(GDNF), brainderived neurotrophic factor(BDNF), neurotrophin 3(NT-3)， NT-4, ciliang neurotrophic factor(CNTF). Results Separated by Native-PAGE, NRCF mainly contained two protein bands:Band a had a relative molecular mass about 220×103, and Band c had a relative molecular mass about (20-40)×103. Band a could not combine with the antibodies of the NGF, BDNF, CNTF, and NT-3, but could combine with the antibody of NT-4.Band c could combine with the antibodies of NGF, BDNF, CNTF and NT-3, but could not combine with the antibodies of NT-4 and GDNF. Conclusion The protein bands with a relative molecular mass of 220×103 have ber neurotropic and neurotrophic effects than the protein bands with a relative molecular mass of (20-40)×103, which contains NGF,CNTF, etc. NT-4 just has a weak or no effect on the sympathetic neurone. This indicates that there is a new NTF in the protein bands with a relative molecular mass of 220×103, which only combines with the antibody of NT-4.
Objective To clone the genes of nogo-66 and NEP1-40 from spinal cord of rat and to realize the expression of its protein in vitro. Methods The nogo-66 and NEP1-40 genes were cloned from the spinal cord of juvenil rat by use of RT-PCR techniques, and the objective genes were bonded to T vector through gene coupled action, recombinant plasmid were sequencing, and the genes were cloned into PQE30-GST vector, then the recombinant plasmids were induced by isopropylthiogalactoside(IPTG) to express the proteins. The two proteins were purified by Ni-column and detected by using Westernblot test. Results The Nogo-66 and NEP1-40 genes were successfully cloned from rat, which were 215 bp and 137 bp for each one when add the enzyme site. No gene mutations were detected in the two genes after sequencing. The expression plasmids were cut by the two enzyme (BamH Ⅰ and Hind Ⅲ), the target bands were seen on the results of electrophoresis. The expression plasmids were induced by IPTG and got the purified GST fusion protein nogo-66 and NEP1-40, which relative molecular weight were 33.2×103 and 30.3×103 respectively. The results of Westernblot test confirmed that the antigenicity of the two proteins was precise. Conclusion Nogo-66 and NEP1-40 proteins can be expressed in a high efficiency in vitro using genetic engineering, so it provides a good basis for further research on its function and vaccine for spinal injury.
OBJECTIVE L-arginine is a semiessential dibasic amino acid for humans and animals. This paper aims to investigate the therapeutic effect of L-arginine supplementation on partial-thickness burned patients. METHODS A randomized controlled clinical trial was designed to evaluate the cellular immune function (T cell count, ratio of CD4/CD8, natural killer cell activity and IL-2 level) and protein metabolism (transferrin, prealbium and nitrogen balance) of patients in the experimental group which daily given 15 g arginine and the control group which daily given 25 g glycine. RESULTS The natural killer cell activity and IL-2 production in the experimental group were higher than that of the control group. The suppression of transferrin and prealbium was alleviated and the nitrogen balance was improved in the experimental group. CONCLUSION It suggests that exogenous arginine supplementation is beneficial for recovery of cellular immunity function and protein anabolism in partial-thickness burned patients.
Objective To observe the effect of high glucose on the expression of activating transcription factor 4 (ATF4) in cultured retinal Muuml;ller glia cells. Methods The retinal tissue of Sprague-Dawley (SD) rats was collected, and Muuml;ller cells were isolated and cultured. The glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) of Muuml;ller cells were identified by streptavidin-biotin-peroxidase complex. Cultured rat Muuml;ller cells were divided into control group (5.5 mmol/L glucose), group A (20 mmol/L glucose), group B (30 mmol/L glucose) and group C (40 mmol/L glucose). ATF4 protein expressions in Muuml;ller cells of four groups were measured by Western blot four days after cultured. Results GFAP and GS expressed in more than 95% of Muuml;ller cells. Over 95% of Muuml;ller cells of group A, B and C were positive for GFAP and GS. Western blots indicated that ATF4 protein in group A, B and C increased obviously compared with the control group (q=0.293, 0.754,0.484;P＜0.05). Conclusion High glucose can increase the expression of ATF4 protein and cause endoplasmic reticulum stress in retinal Muuml;ller glia cells in vitro.
Objective To observe the effects of bevacizumab on aquaporin 4 (AQP4) expression in human retinal Muuml;ller cells in vitro under hypoxia. To explored the mechanism of treating retinal edema with bevacizumab. Methods Human Muuml;ller cells were cultured using the enzymatic digestion method. Transmission electron microscopic analysis and immunofluorescence staining identified Muuml;ller cells. With semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the expression of AQP4 mRNA and vascular endothelial growth factor （VEGF） mRNA in Muuml;ller cells cultured under different concentration of COCl2 for different hours were observed. The expression of AQP4 mRNA in Muuml;ller cells cultured using CoCl2 precultured with 200 mu;g/ml bevacizumab was measured. Results More than 95% of primary cells showed positive reaction to glial fibrillary acidic protein, glutamine synthetase, vimentin and alpha;-smooth muscle actin with immunofluorescence staining. Characteristic 8-10 nm intracellular filaments could be seen in the cytoplasm viewed with transmission electron microscopy. The results using RT-PCR showed that CoCl2 increased the AQP4 and VEGF mRNA expression in Muuml;ller cells in a dose and time dependent manner (r=0.952, 0.954;P＜0.05). The expression of AQP4 mRNA in Muuml;ller cells was increased by VEGF (F=12.43,P＜0.05). The expression of AQP4 mRNA was significantly decreased by bevacizumab (F=2 370.37，P＜0.05). Conclusion Bevacizumab can down-regulate the expression of AQP4 mRNA in human Muuml;ller cells under hypoxic conditions partially by VEGF path, which may be a mechanism for treating retinal edema with bevacizumab.
Objective To observe the dynamic expression of nestin and glial fibrilary acidic protein (GFAP) in the development of retina in rats.Methods In 48 Wistar rats, 24 were divided into 8 groups with 3 rats in each according to their age (1 day, 1 week, and 2, 3, 4, 7, 12, and 20 weeks old). The sagittal freezing sections of the eye were made; nestin/glutamine synthetase (GS) and GFAP/GS were stained by immunofluorescence and were observed under the confocal microscope. Total RNA was extracted from 18 rats which were divided into 6 groups according to the age (1 day, 1 week, and 2, 3, 4, and 12 weeks old) with 3 rats in each. The expression of nestin, GAFA and GS mRNA were detected by realtime quantitative reverse transcription polymerase chain reaction (RT-PCR). Müller cells were cultured from postnatal day 7-12 rats; the expression of nestin and GFAP was detected by immunostaining study. Double immunofluorescence was carried out between nestin/GS and GFAP/GS.Results One day after the birth, nestin positive cells were found in the whole retinal neuroblast layers with elongated retinal progenitor cells; the GFAP positive astrocytes were observed in the inner retina. One week after the birth, Müller glial cells expressed GS and nestin but not GFAP; GFAP positive cells localized in the inner retina.Two to 12 weeks after the birth, the expression of nestin in Müller cells decreased and even disappeared; the expression of GFAP in astrocytes didn't change much. The Müller cells expressed nestin but no GFAP in vitro. The expression of nestin and GFAP mRNA in retina was accordant with the results of immunofluorescence staining.Conclusion In the developing retina, the expression of nestin in Müller cells decreases gradually, and no expression of nestin can be found in adult rats; the expression of GFAP can't be observed in Müller cells in neonatal and adult rats.