Tumor necrosis factor-alpha increases in vitro blood brain barrier permeability / 中国组织工程研究

BACKGROUND:The levels of interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are increased during infectious brain edema, and are positively relevant to the degree of brain damage. However, whether TNF-α can enhance blood brain barrier (BBB) permeability remains unclear, especially in vitro.OBJECTIVE: To understand the changes and possible mechanism of the BBB permeability induced by TNF-α in vitro.DESIGN: Randomized controlled cell model study in vitro.SETTING:Department of Pediatrics, Xiangya Hospital, Central South University; Department of Biochemistry, Xiangya Medical College, Central South University.MATERIALS: Twenty 7-day-old healthy Sprague-Dawley rats, of clean grade and either gender, were provided by the Animal Center, Xiangya Hospital, Central South University. TNF-α was purchased from sigma Company; DMEM fluid medium and fetal bovine serum were purchased from Hyclone Company; Y-27632 was purchased from Alexis Company,and rabbit anti-human factor Ⅷ -related antigen was purchased from Zymed Company; Mouse anti-rat glial fibrillary acidic protein (GFAP) was purchased from Neomarkers. Other biochemical reagents were imported (Sigma Company).METHODS: This experiment was carried out in the Xiangya Hospital, Central South University between March 2004 and April 2005. Brain microvascular endothelial cells and astrocytes were co-cultured 10 days to set up rat models of BBB in vitro. Then, the cells were divided into 4 groups: model group(BBB models were prepared), TNF-α group ( BBB model incubated with 0.01 g/L TNF-α for 5 hours), Y-27632 pretreated group ( BBB model incubated with 30 μmol/L Y-27632 for 1 hour before 0.01g/L TNF-α challenge ) and Y-27632 control group (BBB models only incubated with Y-27632 as those in the Y-27632 pretreated group). The effect of TNF-α on BBB permeability was observed by detecting the 125 I -BSA, which passed through the inserts at each time point (30,60,120 and 240 minutes) using .γradioimmunoassay counter.MAIN OUTCOME MEASURES: The 125 I -BSA, which passed through the inserts in rat models of BBB at different time points after intervention.RESULTS: The 125 I -BSA, which passed through the inserts in rat models of BBB, was all significantly higher in the TNF-α group than in the other groups at 30, 60, 120 and 240 minutes after intervention, respectively (P ＜ 0.01), and reached the peak at 240 minutes; The 125 I -BSA, which passed through the inserts, was lower in the Y-27632 pre-treated group than in the TNF-α group at 30 and 60 minutes after intervention (P＜ 0.01). There was also significant difference in 125 I -BSA permeation between Y-27632 pretreated group and Y-27632 control group after 120 minutes (P ＜ 0.05).CONCLUSION: TNF-α can increase BBB permeability, and Y-27632 pretreatment can early reverse the effect of TNF-α on BBB permeability.

Polymorphisms and functions of the aldose reductase gene 5' regulatory region in Chinese patients with type 2 diabetes mellitus / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function.</p><p><b>METHODS</b>The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins.</p><p><b>RESULTS</b>Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P < 0.05) respectively, and the frequencies of WT/C (-12) G were 10.5% and 2.5% (P > 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P < 0.025). The relative transcription activities of the wild-type, the C (-12) G and the C (-106) T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors.</p><p><b>CONCLUSION</b>The polymorphisms C (-12) G and C (-106) T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.</p>

Influence of ovariectomy and estrogen replacement on gene expression profile of myocardium in rats / 中国病理生理杂志

AIM:To investigate the influence of ovariectomy and estrogen replacemeot treatment on profile of gene expression in myocardium by cDNA microarray,and to characterize the targeting genes of estrogen.METHODS:cDNA microarray containing 1 400 rat cDNAs was used to study the genes differentially expressed in myocardium between sham(Ⅰ),ovariectomy(Ⅱ,OVX)and estrogen replacement treatment(Ⅲ,OVX+E2)group.Then down-regulated genes in myocardium of OVX rats were further confirmed by RT-PCR.RESULTS:177 genes were differentially expressed in myocardium between sham and OVX rats,with 91 genes up-regulated and 86 genes down-regulated in OVX rats.164 genes were differentially expressed in myocardium between OVX and OVX+E2 rats,with 113 genes up-regulated and 54 genes down-regulated in OVX rats.There were 54 genes differentially expressed in OVX compared to sham and OVX+E2.They are involved in membrane channels and transporters(18),cell receptors(9),intracellular transducers/effectors/modulator(7)and metabolism(6).Most of the genes(45)were down-regulated in OVX rats and up-regulated in OVX+E2 rats.RT-PCR test confirmed the results of cDNA microarray.CONCLUSIONS:Long-term estrogen replacement may influence the expression of genes involved in membrane channels and transporters,cell receptors,intracellular transducers/effectors/modulator and metabolism.Long-term estrogen replacement has some beneficial effects on ionic concentration and cardiac function which partially comes from the results of influence of expression on Na+,K+-ATPase and Na+/H+ exchanger.Estrogen has an inhibitory effect on the expression of dopamine receptor,which partially clarify the myocardial protection of estrogen.

Regulatory mechanism in expression of IRS-1 and 2 by estrogen and high concentration of insulin / 中国病理生理杂志

AIM: To study the molecular mechanism in modulation of expression of insulin receptor substrate-1 and-2 (IRS-1,-2) by estrogen and high concentration of insulin. METHODS: The 5′-regulatory regions of IRS-1 and IRS-2 gene were cloned into the pGL3 plasmid with luciferase reporter, and the clones were transfected into HeLa cells. The cells were incubated with estradiol (1 nmol/L) and high concentration of insulin (100 nmol/L). The relatively transcriptional activity of the 5′-regulatory regions of IRS-1 and IRS-2 gene was detected. RESULTS: It was found that the relatively transcriptional activity of the 5′-modulatory regions of IRS-2 reduced markedly after cells were incubated with 100 nmol/L insulin (P