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Effect of Cyclosporine A on Activity and Expression of MMP-2 and MMP-9 in Renal Tubular Epithelial Cells

Zhihui YUE ; Liangzhong SUN ; Yijuan LI ; Ying MO ; Xiaoyun JIANG ; Shumei CHEN

Journal of Sun Yat-sen University(Medical Sciences).2010;31(1):91-94,133.

[Objective] To observe the effect of cyclosporine A (CSA) on the activity and expression of MMP-2 and MMP-9 in renal tubular epithelial cells. [Methods] NRK52E cells were cultured until its reached confluent. Then NRK52E cells were exposed to different concentration of CSA (0, 0.42, 0.84, 4.2, and 8.4 μmoL/L) for 48 h or 72 h respectively. MMP-2 and MMP-9 activities were detected by gelatin zymography. The expression of MMP-2 and MMP-9 mRNA were detected by RT-PCR. [Results] MMP-2 activity and mRNA levels were decreased in a dose dependent manner after exposed to different concentration of CSA for 48 h or 72 h in NRK52E cells. Compared with control (CSA 0 μmoL/L), CSA 0.42, 0.84, 4.2, 8.4 μmoL/L decreased the MMP-2 activities to 27%, 24%, 11%, and 9% respectively; The differences are significant, P<0.05. But the MMP-9 activity and mRNA levels were increased after exposed to CSA for 48 h or 72 h in NRK52E cells. Compared with control group, CSA 4.2 μmoL/L exposure increased MMP-9 activity to 438% in 48 h, and 237% in 72 h; the differences are significant as well, P<0.05. [Conclusion] A dose-dependent decrease in the expression and activity of MMP-2, and the up-regulation of the expression and activity of MMP-9 by CSA in renal epithelial cells may related to CSA associated tubulointerstitial damage.

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Role of TGF-β Expressed by Biliary Epithelial Cells in Pathogenesis of Cholestatic Liver Disease

Yao HE ; Baili CHEN ; Rongping YANG ; Zhirong ZENG ; Ming REN

Journal of Sun Yat-sen University(Medical Sciences).2010;31(1):85-90,99.

[Objective] To investigate the change of liver histology, proliferation of BDEC, and expression of TGF-β in different stages of liver chelestusis. [Methods] ①Rat cholestatic livers were induced by common bile duct ligation (BDL) and separated into 3 groups, namely control group (DO), 7 days after BDL group (DT), and 18 days after BDL group (D18). ② Histological changes of livers in different groups were evaluated based on Knodell HAI score. ③ Real-time PCR was used to detect the expression of TGF-β in liver tissue and isolated BDEC in different groups. ④ Statistically analyzing the correlation between Knedell HAl score and the levels of TGF-β_1 mRNA. ⑤ In vitro study was performed to investigate the effect of TGF-β_1 on an immortalized mouse intrahepatic bililary epithelial cell line (mIBEC). [Results] (I) Knedell HAI score and the proliferation of intrahepatic bile ducts increased as the liver cholestasis aggravated. ② The levels of TGF-β_1, TGF-β_2, and TGF-β_3 mRNA were significantly up-regulated in liver tissues and BDEC as the liver cholestasis aggravated. ③ Positive correlation was found between Knedell fibrotic score and the levels of TGF-β_1 mRNA in liver tissues and BDEC(r=0.9376, P<0.05 and r=0.9682, P < 0.01). ④ In vitro study showed that TGF-β_1 inhibited the proliferation of mIBEC. [Conclusion] ① Liver injury, biliary proliferation, and the levels of TGF-β mRNA expression increased as liver cholestasis aggravated. ② The interaction of TGF-β_1 and BDEC plays an important role in the pathogenesis of BDL induced cholestatic liver disease. ④ Up-regulated expression of TGF-β_1 mRNA in the proliferated BDEC participates in the formation of BDL induced cholestatic liver fibrosis.

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Effects of Hirudin on Ventricular Arrhythmia after Acute Myocardial Infarction in Rats

Jun LIU ; Lilong TANG ; Xinxue LIAO ; Anli TANG ; Chong FENG

Journal of Sun Yat-sen University(Medical Sciences).2010;31(1):50-54,78.

[Objective] To determine the effects and possible mechanism of the thrombin antagonist r-RGD-Hirudin (HIR) on ventricular arrhythmia(VA) after acute myocardial infarction (AMI). [Methods] Seventy adult male Sprague-Dawley rats were randomly subjected to the 10 groups according to duration of left coronary occlusion: HIR 0 min, HIR 5 rain, HIR 10 min, HIR 20 min, HIR 30 min, and normal saline(NS) 0 min, NS 5 min, NS 10 min, NS 20 min, NS 30 min; and the average of every group is 7 rats. Acute myocardial infarction was produced by the occlusion of the left anterior descending coronary artery, then the measurements of arrhythmia and infarction sizing by Evans blue were assessed as well as the expression of three isoforms of inositol 1,4,5-trisphosphate receptors (IP3Rs) mRNA in isehemic myocardium by reverse transeriptase polymerase chain reactions (RT-PCR). [Results] Compared with NS groups, the measurements of VA in HIR were reduced significantly in 5 to 20 minutes after AMI (P<0.05). The incidence of VA was all positive related to the expression of three isoforms of IP3Rs mRNA (P<0.01). Compared with NS groups, the expression of type2,inositol 1,4,5-trisphosphate receptor (IP3R2) mRNA at 10 min and type3, inositol 1,4,5-trisphosphate receptor mRNA (IP3R3) at 10 min and 20 min after AMI were significant decreased (P<0.05) in HIR groups. [Conclusion] The thrombin antagonist r-RGD-Hirudin exerts its myocardial protection against ventricular arrhythmia after acute myocardial infarction possible through IP3R2 and IP3R3 and not typel, inositol 1,4,5-trisphosphate receptor (IP3R1).

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Activation of Muscarinic Cholinergic Receptors Protects Cerebellar Granule Neurons from Dopamine-Induced Apoptosis via ERK Pathway

Xuemin WANG ; Pengxin QIU ; Xingwen SU ; Wenming LI ; Shoujian HUANG ; Guangmei YAN

Journal of Sun Yat-sen University(Medical Sciences).2001;22(3):161-164,169.

【Objective】To investigate the molecular mechanism of dopamine (DA)-induced apoptosis in cultured cerebellar granule neurons (CGNs) and the effect of muscarinic cholinergic receptor (mAChR) agonist carbachol on it.【Methods】The apoptosis of neurons was measured by phase-contrast microscopy,Hoechst 33258 nucleus staining and DNA fragmentation agarose gel electrophoresis.The neuronal viability was measured by fluorescein diacetate (FDA) staining.The activation of extracellular signal-regulated protein kinase (ERK) was determined by Western blot.【Results】Dopamine increases the phosphorylation of ERK and induces apoptosis in CGNs,which is blocked by both carbachol and PD 98059.The protective effect and the inhibiting ERK phosphorylation of carbachol were blocked by atropine.【Conclusion】DA-induced apoptosis in CGNs may be mediated by activation of ERK.Carbachol protects CGNs from DA-induced apoptosis by activating mAChR and subsequent inhibition of activation of ERK.

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An Inhibitor of p38 MAPK Prevents Apoptosis of Cultured Cerebellar Granule Neurons via Inhibiting the Activity of JNK

Mingtao LI ; Wenya WANG ; Juan SUN ; Xiaoli TANG ; Xingwen SU ; Pengxin QIU ; Guangmei YAN

Journal of Sun Yat-sen University(Medical Sciences).2001;22(3):165-169.

【Objective】To study the effect of the specific p38 mitogen-activated protein kinase(p38 MAPK) inhibitor SB203580 on apoptosis of cerebellar granule neurons induced by low potassium.【Methods】Apoptosis was induced by switching the cultured cerebellar granule neurons from a culture medium containing K+ 25 mmol*L-1 to a medium containing K+ 5 mmol*L-1 (cLK).Fragmentation of DNA was analyzed using agarose gel eletrophoresis.SAPK/JNK activity was measured by SAPK/JNK assay kit.【Results】Low potassium resulted in apoptosis as characterized by morphological and biochemical features,but the specific p38 MAPK inhibitor SB203580 improved the survival of cerebellar granule neurons cultured in cLK medium by blocking apoptosis in a concentration-dependent manner.The expression and phosphorylation of c-Jun increased and the activity of c-Jun N-terminal protein kinase (JNK) elevated when cerebellar granule neurons were cultured in cLK medium.But when the cerebellar granule neurons cultured in cLK medium were exposed to 25 μmol*L-1 SB203580,the expression and phosphorylation of c-Jun and the activity of JNK were both decreased evidently.【Conclusions】These results indicate that SB203580 inhibits the activation of JNK and phosphorylation of c-Jun,and therefore protects granule neurons from apoptosis induced by low potassium.

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Cloning and Sequencing of the Human Thalassemic Gene β654

Xiaowu FANG ; Ruiping ZENG ; Bin HU

Journal of Sun Yat-sen University(Medical Sciences).2001;22(3):174-176.

【Objective】In order to establish the foundation for transgenic mouse model,the human thalassemic gene(β654) was cloned and sequenced.【Methods】The human β654 gene was amplified by PCR,and cloned into the plasmid BGT51 in which the human β gene was cut out aforehand.The recombinant plasmid was certified by enzyme-digestion,reverse dot hybridization and sequencing.【Results】A recombinant plasmid was obtained,which contained the human β654 gene in the correct recombinant direction.Sequencing showed that the cloned insert was correct.【Conclusions】The recombinant plasmid constructed is useful for establishing a transgenic mouse.

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Cloning,Expression and Activity Detection of Vascular Endothelial Growth Factor

Zijun LI ; Pingjin HU ; Xiyong YU

Journal of Sun Yat-sen University(Medical Sciences).2001;22(3):177-179.

【Ojective】To clone,express and detect activity of VEGF165.【Methods】Using human heart cDNA library as template,amplified the VEGF gene by PCR.The PCR product was ligated to PUC19 plasmid and sequenced.A eukaryotic expression plasmid AdtrackCMV harbouring VEGF165 was constructed.,then transformed into 293 cells.RNA dot blot and Western blotting were performed to demonstrated whether the tranfermants expressed VEGF165 at mRNA and protein level respectively.Furthermore the bio-activity of VEGF165 was preliminarily detected with Miles test.【Results】Sequence of 582bp VEGF165 cDNA was proved correct by sequencer analysis.The expression of VEGF165 mRNA was identified by RNA dot blot,Western blotting indicated that the molecular weight of VEGF165 protein was 22ku.VEGF165 also is of vascular permeability.【Conclusion】VEGF165 gene has been successfully cloned and expressed,which makes a basis for the further study in vivo.

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Effect of Stent Implantation on the Expression of PCNA and Cyclin E and Apoptosis in Rabbit Vascular Smooth Muscle Cells

Yingmei LIU ; Xuming ZHANG ; Wei WU ; Yingling ZHOU

Journal of Sun Yat-sen University(Medical Sciences).2001;22(3):180-183.

【Objective】To evaluate the impact of stent implantation on proliferation and apoptosis in media vascular smooth muscle cells and to explore the mechanism of restenosis after stent implantation.【Methods】Fifty male New Zealand rabbits were randomized into balloon group and stent group.Control group were set up.The materials were harvested on 3,7,14,28 and 56 day after operation and the following investigation were carried out.① Assessing the expression of proliferating cell nuclear antigen (PCNA) and Cyclin E of media smooth muscle cells with immunohistochemistry;② Analyzing apoptosis of media smooth muscle cells by TUNEL technique.【Results】The expressions of PCNA,Cyclin E and apoptosis in stent and balloon groups were markedly increased compared to control groups.① Stent group induced significant increased expression of PCNA and Cyclin E in the media smooth muscle cells compared to balloon group.On day 7,the positive rates of PCNA and Cyclin E were 24.36±0.55% vs 18.74±1.09% (P<0.01) and 22.65±1.00% vs 17.68±1.10% (P<0.01) respectively;② Stent group induced much more significant apoptosis than balloon group.The highest rate of apoptosis appeared on day 7:12.46±1.13% vs 5.54±0.53% (P<0.01);③By calculating the ratio of positive rates of PCNA to apoptosis and Cyclin E to apoptosis respectively,the ratio of balloongroup was higher than that of stent group.【Conclusion】Stent group induces augmented proliferation and much more significant apoptosis of media smooth muscle cells compared to balloon group.It shows that the severity of restenosis is relieved after stent implantation.

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Effect of Light Weight-bearing Activity on Bone Mineral Density,Histomorphometry and Biomechanics in Ovarietomized Rats

Bailing CHEN ; Weiming LIAO ; Fobao LI ; Ming FU ; Dongliang XU

Journal of Sun Yat-sen University(Medical Sciences).2001;22(3):187-191.

【Objective】To study the effect of light weight-bearing activity on postmenopausal osteoporosis.【Methods】36 female rats were randomly divided into 3 groups:① Sham,② Ovx (ovarietomized),③ Ovx+Im (ovarietomized and immobilized).All the group's maintained daily activity.And because of being immobilized,the right hind limbs of the third group lacked weight-bearing activity.12 weeks after ovarietomy,the BMD (bone mineral density),histomorphometry and biomechanics of the right femurs of rats were measured and analyzed.【Results】Comparing with the Sham group,the Ovx group's right femurs were manifested with the decrease of BMD,TBV (trabecular bone volume),MTT(mean trabecular thickness) and MCT(mean cortex thickness),while the increase of RS(resorption surface) and OS(osteotoid surface).Meanwhile their biomechanic nature declined.But statistically the BMD,MCT and the criteria of mechanical strength were not significant decrease.Otherwise,the Ovx+Im group's right femurs showed more apparent decrease of BMD,TBV,MTT and MCT.And the biomechanic nature was worse.Comparing with the Sham group,the BMD,MCT and the criteria of mechanical strength of the Ovx+Im group were statistically significant decreased.【Conclusion】If maintaining light weight-bearing activity,the ovarietomized rats were able to maintain relatively better bone quality.A lack of light weight-bearing activity wouldcause thedecline of bone quality.Thusthestudy suggested light weight-bearing activity was significantly effective on the prevention and treatment of postmenopausal osteoporosis.

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Relationship Between the Expression of Lymphocyte Function-associated Antigen-1 on Peripheral Lymphocytes and Acute Rejection After Liver Transplantation

Yuyang FU ; Xiaoshun HE ; Jianlin CHEN ; Guihua CHEN ; Jiefu HUANG

Journal of Sun Yat-sen University(Medical Sciences).2001;22(3):192-194.

【Objective】To investigate the relationship between the expression of lymphocyte function-associated antigen-1 (LFA-1) on peripheral lymphocytes and acute rejection after orthotopic liver transplantation (OLTx) in rat.【Methods】Adult male rats were divided into 2 groups.In the non-rejection group,40 SD rats were used as both donors and recipients.In the rejection group,20 Wistar rats were used as donors and 20 SD rats as recipients.Blood samples were collected through the tail vein 1 day before transplantation and on days 1、3、5、7 after OLTx.The expression of LFA-1 (CD11a) on peripheral lymphocytes was analyzed by using indirect immunofluorescent marker-flow cytometry.【Results】The expression level of LFA-1 on peripheral lymphocytes in recipient rats after orthotopic liver transplantation was markedly lower than that before operation (P<0.01);The expression level of LFA-1 on peripheral lymphocytes in rats with acute liver rejection was significantly higher than that in the non-rejection group (P<0.01).【Conclusion】Monitoring the expression level of LFA-1 on peripheral lymphocytes may be helpful to the diagnosis of acute graft rejection.

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