1.Effects of homocysteine on expression of TNFSF4 gene and interleukin-10 in monocytes cultured in vitro
Chinese Journal of Tissue Engineering Research 2007;0(15):-
BACKGROUND: Inflammatory response is one of potential cellular mechanisms by which hyperhomocysteinemia accelerates atherosclerosis. There are still many details need to be illuminated. TNFSF4 gene and interleukin (IL)-10 are two inflammatory factors related to atherosclerosis. But their correlation with hyperhomocysteinemia is seldom reported. OBJECTIVE: To investigate the effects of homocysteine (Hcy) on expression of TNFSF4 gene and IL-10 in the monocytes cultured in vitro. DESIGN, TIME AND SETTING: A randomized control experiment in vitro was conducted from July to December in 2007 in the Pathophysiology Laboratory of West China School of Preclinical and Forensic Medical Sciences of Sichuan University. MATERIALS: THP-1 monocytes were offered by the Pathophysiology Laboratory of West China School of Preclinical and Forensic Medical Sciences of Sichuan University); Hcy, dexamethasone (Dex) and vitamin D3 (Vit D3) were all purchased from Sigma. METHODS: Cultured THP-1 monocytes were induced to macrophages by 0.1 ?mol/L phorbol myristate acetate, and the differentiated THP-1 macrophages were incubated with 200 ?mol/L Hcy for 24, 48 and 72 hours respectively. Then cell supernatant and lysate were collected as condition medium. Any other differentiated THP-1 macrophages were incubated with a combination of Dex and Vit D3 for 6 days, and then the condition mediums were added to incubate together for 24 hours. Cells were divided into blank control group, 200 ?mol/L Hcy group, Dex+Vit D3 group, 24-hour Hcy+Dex+Vit D3 group, 48-hour Hcy+Dex+Vit D3 group, and 72-hour Hcy+ Dex+Vit D3 group. MAIN OUTCOME MEASURES: The expression level of TNFSF4 mRNA was determined by reverse transcription polymerase chain reaction. The expression level of IL-10 protein was determined by ELISA. RESULTS: Compared with blank control group, no significant difference of TNFSF4 mRNA expression was found in the 24-hour Hcy group, while the expression level was significantly higher in 48-hour and 72-hour Hcy groups. Compared with Dex+Vit D3 group, the IL-10 expression of all Hcy+Dex+Vit D3 groups was significantly lower than that in the Dex+Vit D3 group. CONCLUSION: Hcy may increase TNFSF4 mRNA expression and decrease IL-10 expression in THP-1 macrophages.
2.Effects of homocycteine on methylation status and mRNA expression of TNFSF4 gene in macrophages in vitro
Journal of Third Military Medical University 2003;0(07):-
Objective To investigate the effects of homocysteine (Hcy) on methylation status and mRNA expression of TNF superfamily member 4 (TNFSF4) gene in THP-1 macrophages. Methods Cultured THP-1 monocytes were induced to macrophages by 0.1 ?mol/L PMA treatment for 72 h, then the differentiated THP-1 macrophages were incubated with homocysteine (50-1 000 ?mol/L) for 24, 48 or 72 h. The status of methylation of TNFSF4 gene in THP-1 macrophages was analyzed by methylation specific polymerase chain reaction (MS-PCR).The expression level of TNFSF4 mRNA was determined by RT-PCR. Results The MS-PCR results showed that the unmethylated bands gradually became stronger, and the methylated bands gradually became weaker along with the prolongation of treatment time and the increased Hcy concentrations. The 72-hour treatment with Hcy induced a complete demethylation in the promotor region of TNFSF4 gene, where left the only unmethylated bands. Meanwhile, the TNFSF4 mRNA expression was also increased in a dose-dependent manner. Conclusion Hcy may contribute to atherogenesis by inducing demethylation in the promotion region of the TNFSF4 gene and increasing TNFSF4 expression in THP-1 macrophages.
3.Substitution of Elisa Miriam Reader Vacuum Pump
Shuren ZHOU ; Youlin PEN ; Jianli WANG
Chinese Medical Equipment Journal 2003;0(10):-
Objective To replace the imported vacuum pump with a Chinese-made instrument using a micro-ELISA negative pressure pump,to further extend the life of the machine.Methods Electric aspirator(YB.Dx23D) was used as source of negative pressure to connect with barrels of waste and the vacuum-phase of imported enzyme immunoassay instrument.The circuit was transformed,and the host matched the load,so the host and negative pressure pumps worked simultaneously.Results Compared with other aspirators,the modified electric aspirator YB.Dx23D has low-noise ratio,high negative pressure,vast flow,small volume,and mobile function.Conclusion The modified aspirator YB.Dx23D electric instrument is well used in washing ELISA plates and the effective use of waste water.Meanwhile,it can be applied to other necessary suction negative pressure(vacuum) equipment.
4.Effects of propofol on apoptosis signaling pathways in a rat model of renal ischemia/reperfusion injury
Jin LI ; Lili WANG ; Shuren LI
Chinese Journal of Tissue Engineering Research 2009;13(31):6013-6017
BACKGROUND:Renal ischemia/reperfusion during renal transplant surgery induces the process of apoptosis signaling pathways.Propofol possibly protects kidney from renal ischemia/reperfusion injury.So it is important to investigate propofol's mechanism underlying apoptosis.OBJECTIVE:To investigate the effects of propofol on apoptosis signaling pathways in a rat model of renal ischemia/reperfusion injury and the possible mechanism of action.DESIGN,TIME AND SETTING:An animal experiment,cell morphology observation was performed at the laboratory of the Department of Urinary Surgery,Beijing Friendship Hospital between 2004 and 2005.MATERIALS:A total of 99 male adult outbred Sprague Dawley rats were included in this study.Rabbit anti-rat Bcl-2,Bax,caspase 3,and cytochrome C were produced in Wuhan Boster Bioengineering Co.,Ltd.,China.METHODS:Rats were randomly divided into three groups:control(n = 26),ischemia/reperfusion(n = 35),and ischemia/reperfusion+propofol(n = 38).Rat model of renal ischemia/reperfusion injury was established in each group as follows.Following single intravenous transfusion of 5 mL/kg ringer lactate solution,the right kidney was excised through a median abdominal incision.The left renal pedicle was occluded for 45 minutes using an atraumatic vascular clamp,followed by reperfusion and full-layer suture.At 0,3,12,24,and 72 hours after reperfusion,the left kidney was excised,and simultaneously,rat was sacrificed through bloodletting.In the ischemia/reperfusion+propofol group,propofol(1 mg/kg per minute)was administered from 15 minutes prior to ischemia to 30 minutes after reperfusion,for a total of 75 minutes.In the control and ischemia/reperfusion groups,rats were administered the same amount of ringer lactate solution.MAIN OUTCOME MEASURES:Morphological changes of injured kidney and expression of apoptosis-related protein Bcl-2,Bax,caspase 3 and cytochrome C.RESULTS:Renal ischemia/reperfusion-caused kidney damages could be observed through an optical microscope,and proximal convoluted tubule was severest,followed by distal convoluted and collecting duct,and lastly renal glomerulus.Immunohistochemistry results demonstrated that compared with the control group,Bax,caspase 3,and cytochrome C expression was increased,but no obvious change in Bcl-2 expression was observed in the ischemia/reperfusion group;compared with the control group,Bax,caspase 3 and cytochrome C expression was significantly decreased,but Bcl-2 expression was significantly increased in the ischemia/reperfusion+propofol group(P<0.05).CONCLUSION:Propofol is likely to exhibit protective effects on cellular apoptosis caused by renal ischemia/reperfusion.Propofol inhibits pro-apoptotic protein Bax,caspase 3 and cytochrome C expression but does not produce effects on Bcl-2 expression.The underlying mechanism correlates with apoptosis signaling pathways in mitochondrion.
5.Effect of isoflurane-induced hypotension on the cerebrospiral fluid concentration of S100B protein in patients undergoing clipping of intracranial aneurysm
Ruquan HAN ; Shuren LI ; Baogua WANG
Chinese Journal of Anesthesiology 1994;0(04):-
Objective Recent studies showed that change in plasma and cerebrospinal fluid (CSF) level of S100B protein was closely related to cerebral damage. The aim of this study was to investigate if deliberate hypotension induced by isoflurane can increase the release of S100B protein in CSF during clipping of intracranial aneurysm.Methods Thirty ASA Ⅰ - Ⅱ patients (16 male, 14 female) aged 24-68 yr undergoing elective intracranial aneurysm clipping were randomly divided into two groups : group A deliberate hypotension ( n = 15) and group B control ( n = 15) in which BP was maintained at normal level during operation. In both groups anesthesia was induced with midazolam 0.06 mg? kg-1 , fentanyl 3-5 ?g? kg-1 , propofol 2 mg? kg-1 and vecuronium 0.1 mg ? kg-1 and maintained with 1.2% isoflurane and intermittent intravenous (i. v.) blouses of fentanyl and vecuronium. After tracheal intubation the patients were mechanically ventilated (VT 8-10 ml?kg-1 , RR 12 bpm, I :E = 1:2). PaCO, was maintained at 35-40 mm Hg, In group A deliberate hypotension was induced by increasing the inhaled concentration of isoflurane until BP was reduced by 30 % -40 % of the baseline value. After clipping of aneurysm MAP was restored to baseline level. CSF level of S100B protein was measured before deliberate hypotension and 0, 2, 4 h after aneurysm clipping. Results (1) MAP was decreased from (95 ? 12) mm Hg to (59 ? 5) mm Hg 30 min after deliberate hypotension was started and restored to (75 ? 8) mm Hg 30 min after aneurysm was clipped. In group A both systemic peripheral vascular resistance and myocardial contraction acceleration were decreased but cardiac output and HR remained stable, as compared with those in group B. (2) CSF level of S100B protein was significantly increased 4 h after aneuuysm clipping in both groups but CSF level of S100B protein was significantly higher in group A than that in group B. Conclusion Deliberate hypotension induced by isoflurane increases the release of S100B protein and may worsen cerebral vasospasm and be detrimental to perioperative cerebral protection.
6.Molecular Biological Mechanism of Opioids Induced Hyperalgesia(review)
Qiang WANG ; Shuren LI ; Weihua CUI
Chinese Journal of Rehabilitation Theory and Practice 2010;16(8):707-710
Opioids can activate the pro-nociceptive channel by which the sensitivity to pain has been increased, which is called opioids induced hyperalgesia (OIH). The mechanism of OIH has not been identified, but it is possibly involved in the increased activity of central glutamatergic system, changed function of opioid receptors, roles of peripheral receptors, effects of endogenous neuropeptides, decreased function of inhibitory neurotransmission system, and changed elements in signal transduction pathway, etc.
7.Studies on injury of endothelial cells by homocysteine
Yufang WANG ; Shuren WANG ; Haiyan CHEN ; Zhimei YANG ; Ling GU
Chinese Journal of Pathophysiology 2001;17(3):268-270
AIM: This study is to detail its possible mechanisms that homocysteine (Hcy) induces injury of cultured endothelial cells.METHODS: Hcy in sequential concentrations was added into the cultured human umbilial vein endothelial cells for 24 hours in serum-free medium. The lipid peroxidation, release of LDH, cell total protein content, cell apoptosis and necrosis were assessed. RESULTS: Hcy increased the apoptosis of endothelial cells.In high Hcy concentration the cells also showed obvious injurious and necrotic morphological changes. Lipid peroxidation increased, with LDH releasing up and cell total protein content down, and they showed a positive dose-effect relationship with the Hcy concentration. All the above effects of Hcy was strengthened by low density lipoprotein (LDL) which may suggest synergetic effects of Hcy and LDL.CONCLUSION: Our results indicate that Hcy has a strong oxidizing effect, which may be one of its major mechanism for injury of EC.
9.Effects of matrine on ATP binding cassette transporter A1 and cholesterol in monocyte cells
Yideng JIANG ; Huiping ZHANG ; Jun CAO ; Guizhong LI ; Shuren WANG
Basic & Clinical Medicine 2006;0(09):-
Objective To study the effect of matrine on ATP binding cassette transporter A1 (ABCA1) expression and cholesterol in monocyte derived foam cells. Methods The lipid peroxide in cells was measured by TBARS method; The foam cells were examined by oil red O stain. The accumulation of cholesterol in monocyte was measured by fluorescence spectrophotometric method;ABCA1 mRNA and its protein level were determined by RT-PCR and Western blot, respectively. Results In the homonocytes incubated with PMA and low density lipoprotein (LDL), the intracellular accumulated total cholesterol (TC), free cholesterol (FC), cholesteryl ester (CE) and lipid peroxide increased obviously, and a huge amount of foam cells were found by oil red O stain. This was accompanied by a reduction in the membrane content of ABCA1. matrine alter ABCA1 mRNA abundance, indicating that increased ABCA1 transcription, enhanced mRNA level.Conclusion The mechanism of anti-artherosclerosis by matrine may be related to reduce cholesterol and to increase the level of ABCA1 expression.
10.Expression of Human T Cell Receptor ? Chain Gene in Insect Cell
Yang HAN ; Chunxia ZHOU ; Wenbo MA ; Dongmei WANG ; Shuren ZHANG
Chinese Journal of Cancer Biotherapy 1995;0(02):-
Objective: To clone human T cell receptor (TCR) ? chain gene and express its encoding protein by baculoviral expression system in insect cells. Methods: TCR ? chain cDNA was cloned from normal human peripheral blood mononuclear cells (PBMC) by RT-PCR and inserted into baculoviral transfer vector. This vector was co-transfected with baculovirus into insect sf 9 cells. The recombinant protein expressed was identified by SDS-PAGE and flow cytometry with mouse anti-human ? chain monoclonal antibody. Results: Human TCR ? chain cDNA cloned and inserted into baculoviral vector specifically expressed protein in the insect sf 9 cells, accounting for about 11% of the total protein yield in the supernatant of cell lysate. The molecular weight of the recombinant protein determined by SDS-PAGE was identical to what we anticipated. The insect cells transfected with recombinant baculovirus were demonstrated by intracellular labeling flow cytometry to express ? chain protein. Conclusion: Human T cell receptor ? chain gene was successfully cloned from human PBMC, and its encoding protein was highly expressed in insect cells. The TCR ? chain protein obtained by bioengineering technique is useful for in depth biological function study.