Value of CD, MPO, Ki-67 and C-MYC Positive Rate in the Pathological Tissues and C-MYC Gene of Patients with T-LBL/ALL for Predicting Prognosis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the value of of CD, MPO, Ki-67, C-MYC positive rates in the pathological tissues and C-MYC gene of patients with T-LBL/ALL for predicting Prognosis.</p><p><b>METHODS</b>Ninty cases of T-LBL/ALL patients in our hospital were selected and included in the T-LBL/ALL group, and 30 cases of lymphnode reactive hyperplasia were selected as control group. Immunohistochemical staining was used to detect the changes of CD, MPO, Ki-67 and C-MYC positive rate in 2 groups, and the changes of C-MYC gene were detected by fluorescence in situ hybridization.</p><p><b>RESULTS</b>In 90 patients with T-LBL/ALL, there were CD1a34 cases (37.8%), CD367 cases (74.4%), epsilon CD347 cases (52.2%), CD785 cases (94.4%), CD1033 cases (36.7%), CD3422 cases (24.4%), CD4348 cases (53.3%), CD45RO46 cases (51.1%), CD9988 cases (97.8%), TDT85 cases (94.4%); and CD23, CD20, and MPO all were negative; Ki-67>80% 47 cases (52.2% cases), Ki-67≤80%, 43 cases (47.8%). In 90 T-LBL/ALL patients, the positive rate of C-MYC (66.7%) was significantly higher than the control group (positive rate 0.0%) (P< 0.05); the Ki-67 index, mediastinal widening of T-LBL/ALL patients and the positive rate of C-MYC positively were correlated (P< 0.05). The overall survival rate (44.0%) of C-MYC negative patients was significantly higher than that of C-MYC positive patients (0.0%). The overall survival rate of C-MYC negative patients was significantly higher than that of C-MYC positive patients (P< 0.05).Ann Arbor staging, LDH, bone marrow involvement, mediastinal widening, Ki-67 positive index, and C-MYC protein expression of patients with T-LBL/ALL did not correlated with increased C-MYC gene breakage and copy number (P> 0.05).</p><p><b>CONCLUSION</b>The overall survival rate of C-MYC positive patients decreases, which positively correlates with Ki-67 positive index and mediastinal width, suggesting that the prognosis of the patients with C-MYC protein expression is poorer.</p>

Mechanisms of decorin inhibiting epithelial-to-mesenchymal transition induced by transforming growth factor beta1 in renal tubular epithelial cells / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the mechanisms of decorin inhibiting epithelial-to-mesenchymal transition (EMT) induced by transforming growth factor beta1 (TGF-beta1) in renal tubular epithelial cells.</p><p><b>METHOD</b>HK-2 cells in vitro were divided into 4 groups: (1) negative control group; (2) decorin group, added with decorin 100 ng/ml ; (3) TGF-beta1 group, added with TGF-beta1 10 ng/ml; (4) decorin and TGF-beta1 group, added with decorin 100 ng/ml and TGF-beta1 10 ng/ml. The protein level of phosphor-ERK, phosphor-PI3K, phosphor-Smad(3) and beta-catenin was detected by Western blotting method. The snail mRNA level was tested by real time-PCR, while the lymphoid enhancer factor-1 (LEF-1) mRNA level was measured by RT-PCR.</p><p><b>RESULTS</b>The snail (2.59 +/- 0.70:1.02 +/- 0.13) and LEF-1 mRNA (1.85 +/- 0.08:0.30 +/- 0.11) were significantly up-regulated, meanwhile the protein level of phosphor-ERK (1.11 +/- 0.09:0.47 +/- 0.07), phosphor-PI3K (14.79 +/- 1.02:2.48 +/- 0.06), phosphor-Smad(3) (0.95 +/- 0.02:0.08 +/- 0.01) and beta-catenin (1.46 +/- 0.20:0.49 +/- 0.05) were significantly increased in TGF-beta1 group compared to control group, while there were no statistically significant difference in all figures between control group and decorin group. The phosphor-ERK protein level (0.58 +/- 0.08) and the snail mRNA level (1.24 +/- 0.03) were significantly down-regulated in TGF-beta1 and decorin group compared to TGF-beta1 group, however there were no statistically significant differences in the level of phosphor-PI3K (15.84 +/- 1.64), phosphor-Smad(3) (0.90 +/- 0.04) and beta-catenin (1.42 +/- 0.09) between these two groups.</p><p><b>CONCLUSION</b>Decorin inhibited EMT induced by TGF-beta1 which may be through blocking the ERK signal transduction pathway.</p>

Resistin Binding Peptide Stimulates Basal Insulin Secretion of RINm5F Insulinoma Cells / 实用儿科临床杂志

Objective A resistin binding peptide (RBP) was selected by phage display in our previous work. Studies had shown that RBP could antagonize the role of resistin on the lipid metabolism and endocrine function of adipose tissue, but whether RBP affects the insulin secretion of pancreatic cells is still unknown. The aim of this study is to assess the effect of RBP on basal insulin secretion in RINm5F insulinoma cells. Methods The cell viability was measured by 3-[4,5-dimethyhhiazol-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay. The supernatants were assayed for insulin content by enzyme linked immunosorbent assay (ELISA). Reverse transcriptase-PCR assay and Western blotting were used to determine the expression of glucose transporter 2 (GLUT2) involved in insulin secretion. Cytosolic Ca2+, the trigger of insulin exocytosis, was analyzed with the fluorescent probe FURA-3/AM. Results RBP did no effect on the cell viability with a concentration of 10-8-10-12mol/L of 2 hours intervention. But it stimulated basal insulin secretion of RINm5F cells, accompanied by up-regulated increased expression of GLUT2 and elevated concentration of cytosolic Ca2+. Conclusion RBP could stimulate basal insulin secretion without affecting the cell viability.

Effects of core proteoglycan on the transdifferentiation of human renal tubular epithelial cell induced by transforming growth factor-beta1 in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>To study the effects of core proteoglycan on the transdifferentiation of human renal tubular epithelial cell induced by transforming growth factor beta1 (TGF-beta1) in vitro.</p><p><b>METHOD</b>The cultured HK-2 cells were divided into six groups: A. negative control group; B. 10 ng/ml TGF-beta1 group; C. 10 ng/ml core proteoglycan treated group; D. 100 ng/ml core proteoglycan treated group; E. 10 ng/ml TGF-beta1 + 10 ng/ml core proteoglycan group; F. 10 ng/ml TGF-beta1 + 100 ng/ml core proteoglycan group. The changes in configuration of HK-2 cells were inspected 48 hours after adding the stimulating factor. At the same time, changes in mRNA of keratin, alpha-smooth muscle actin, vimentin were analyzed.</p><p><b>RESULTS</b>Compared with group A, group B showed great changes in the morphology of cells, most cells converted into spindle shape, like fibroblast; groups E and F, especially group F showed significantly reduced spindle shape cells. Compared with group A, groups C and D had no significant changes in morphology of cells Compared with 10 ng/ml TGF-beta1 group and negative control, the mRNA expression of alpha-smooth muscle actin and vimentin had significant increase, but that of keratin reduced (P < 0.05). However, after combined treatment with TGF-beta1 and core proteoglycan, alpha-smooth muscle actin and vimentin expression were reduced significantly, while expression of keratin was up-regulated. Single core proteoglycan treated group and negative control group had no dramatic differences (P > 0.05).</p><p><b>CONCLUSION</b>TGF-beta1 can induce the transdifferentiation of human renal tubular epithelial cell and core proteoglycan has some inhibitory effect on transdifferentiation of human renal tubular epithelial cell induced by TGF-beta1 in vitro.</p>

Transdifferentiation and collagen-synthesis effects of exogenous connective tissue growth factor on renal tubular epithelial cells in vitro / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effects of recombinal human connective tissue growth factor (rhCTGF) stimulation on epithelial-myofibroblast transdifferentiation (EMT) and collagen-synthesis in human renal tubular epithelial cell line (HK2) in vitro.</p><p><b>METHODS</b>The cultured HK2 cells were stimulated with rhCTGF of 5 ng/mL. The morphological changes were observed under an inverted microscope. The cells were collected at 0, 3, 6, 12, 24 and 48 hrs after rhCTGF stimulation. The expression of E-cadherin,alpha-smooth muscle actin (alpha-SMA), collagen Ialpha1 (Col Ialpha1) and collagen IValpha1 (Col IValpha1) mRNAs were detected by RT-PCR.</p><p><b>RESULTS</b>rhCTGF stimulation changed the HK2 cell appearance from oval to fusiformdown-regulated the E-cadherin mRNA expression and up-regulated alpha-SMA mRNA expression, but had no effects the Col Ialpha1 and Col IValpha1 mRNA expression.</p><p><b>CONCLUSIONS</b>Exogenous CTGF can mediate the EMT but has no collagen-synthesis effects on HK2 cells.</p>

A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor.</p><p><b>METHODS</b>Three cDNA fragments with the same 11 bp 5' sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5' sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5' sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide.</p><p><b>RESULTS</b>Three cDNA fragments with the same 11 bp 5' sequence were found. The TGA stop codon in reading frames of the same 11 bp 5' sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation.</p><p><b>CONCLUSION</b>RBP could effectively rescue the promoted differentiation of resistin overexpressed 3T3-L1 preadipocyte.</p>

Suppressive effects of par-4 antisense oligodeoxynucleotide on up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the uppressive effects of par-4 antisense oligodeoxynucleotide on the up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects.</p><p><b>METHODS</b>Cationic lipid-mediated par-4 antisense oligodeoxynucleotide (par-4-AS-ODN) was transfected into PC12 cells and followed by glutamate for treatment. Mismatch oligodeoxy-nucleotide (MS-ODN) was used as the control. Morphological assessment and evaluation of the anti-apoptosis effects of par-4-AS-ODN on PC12 cells were performed by laser scanning confocal microscopy by double staining of the cells with Hoechest 33258/propidium iodide (Hoe/PI) and flow cytometry respectively. The mRNA and protein levels of calpain 10 and par-4 were measured by RT-PCR and Western blot. Intracellular calcium concentration was determined by using laser scanning confocal microscope with Fura-2/AM as the fluorescent dye.</p><p><b>RESULTS</b>Par-4-AS-ODN repress the increase of par-4 protein in PC12 cell (52.3 +/- 5.0 vs 90.0 +/- 3.2, < 0.01). Par-4-AS-ODN significantly inhibited the apoptosis of PC12 cells induced by glutamate (53% vs 31%, < 0.01). Par-4-AS-ODN significantly suppress the up-regulation of intracellular calcium concentration in PC12 cells induced by glutamate (Rate of fluorescent density: 167.9 +/- 32.4 vs 228.8 +/- 36.8, < 0.01). Par-4-AS-ODN inhibited the increase of calpain 10 mRNA in PC12 cells induced by glutamate (46.3 +/- 3.7 vs 34.8 +/- 2.1, < 0.01).</p><p><b>CONCLUSIONS</b>par-4-AS-ODN enables to inhibit apoptosis of PC12 cells induced by glutamate. The mechanism of the inhibition may be closely related to suppression of the up-regulation of intracellular calcium concentration and calpain transcription expression.</p>

The cloning of human Smac gene and its pro-apoptotic effect on Burkitt's lymphoma cells / 中华儿科杂志

<p><b>OBJECTIVE</b>Second mitochondria-derived activator of caspase (Smac) is a recently identified, novel pro-apoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac promotes activation of caspases by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The objective of the study was to examine the pro-apoptotic effect of human Smac gene on Burkitt's lymphoma Raji cells.</p><p><b>METHODS</b>The full length cDNA of human Smac gene was amplified by reverse transcription-PCR from total RNA of HEK-293 cells. The PCR product was ligated with linearized vector pGEM-T-easy supplied in the TA cloning kit and sequenced. The correct cDNA of full length Smac was subcloned into eukaryocytic expression vector pcDNA3.1/myc-his and transfected into human Burkitt's lymphoma cell line Raji by lipofectamine-mediated transfection. The expression of full length Smac was determined by Western blot. Morphological observation was done with the laser scanning confocal microscope by double staining the Raji cells with Hoechest 33,258 and propidium iodide. Flow cytometry was used to evaluate apoptosis. Relative caspase-3 activity was determined by colorimetric assay.</p><p><b>RESULTS</b>Recombinant eukaryocytic expression vector pcDNA3.1/Smac, which contained full length Smac, was successfully constructed. After pcDNA 3.1/Smac was transfected into human Burkitt's lymphoma Raji cell line for 24 hours, Raji cells showed apparent apoptosis with a percentage of (43.7 +/- 2.5)%, which was higher than that of non-transfected group and free vector-transfected group (P < 0.05). Compared with non-transfected group (0.136 +/- 0.036) and free vector-transfected group (0.138 +/- 0.026), the relative caspase-3 activity of Raji cells transfected by pcDNA3.1/Smac (0.936 +/- 0.041) was significantly enhanced (P < 0.05).</p><p><b>CONCLUSION</b>Transfection and expression of human Smac gene could significantly induce apoptosis of human Burkitt's lymphoma Raji cells. The mechanism is associated with the increase of caspase-3 activity.</p>

Transfection of Lipoxin A4 receptor-like protein gene enhanced the inhibitory effect of Lipoxin A4 on human lung fibroblasts proliferation induced by connective tissue growth factor / 中华儿科杂志

<p><b>OBJECTIVE</b>Lipoxin A(4) is formed by the metabolism of arachidonic acid. Anti-inflammatory and anti-proliferative effect of lipoxin A(4) has been shown in many human diseases. Recently, as a novel high affinity receptor for ligand lipoxin A(4), Lipoxin A(4) receptor-like protein (LRLP) has been identified. Currently close attention is paid to the important contribution of connective tissue growth factor (CTGF) in lung fibrosis. The purpose of the study was to transfect LRLP gene into human lung fibroblasts and investigate the mechanism of its enhancing antagonistic effect of Lipoxin A(4) on human lung fibroblasts proliferation induced by connective tissue growth factor.</p><p><b>METHODS</b>Eukaryocytic expression vector pEGFP/LRLP which contained LRLP and green fluorescence protein fusion gene (GFP) was constructed and transfected into human lung fibroblasts (HLF). After selecting with G418, HLF/LRLP cell clone which stably expressed LRLP/GFP fusion protein was isolated and characterized by the laser scanning confocal microscope. Cultured HLF and HLF/LRLP were stimulated for 24 h with CTGF (1 microg/ml) in the presence and absence of pretreatment of Lipoxin A(4) (10.0 nmol/L) for 30 min. Inhibition of cell proliferation was determined by MTT assay. Cell cycle analysis was performed by flow cytometry. Western blot was used to detect the expression of cyclin D(1) protein. Electrophoretic mobility shift assay (EMSA) was employed to detect the DNA binding activity of STAT(3).</p><p><b>RESULTS</b>(1) HLF/LRLP cell clone which stably expressed LRLP and GFP fusion protein was successfully obtained. (2) Proliferation of HLF and HLF/LRLP was induced by 1 microg/ml CTGF. Pretreatment with 10 nm Lipoxin A(4) inhibited the proliferation of HLF and HLF/LRLP. And the inhibitory rate of HLF/LRLP was significantly higher than that of HLF [(54.1 +/- 4.2)%, (21.2 +/- 3.7)%, P < 0.05]. (3) The flow cytometry analysis showed that compared with HLF, more HLF/LRLP were arrested at G(0)/G(1) phase in the presence of pretreatment of Lipoxin A(4). [(76.3 +/- 3.5)%, (60.8 +/- 2.0)%, P < 0.05]. (4) Ten nmol/L Lipoxin A(4) antagonized CTGF induced increase of cyclin D(1) protein expression in HLF and HLF/LRLP. And its antagonistic effect on HLR/LRLP was stronger than that on HLF (P < 0.05). (5) Ten nmol/L Lipoxin A(4) antagonized CTGF induced increase of STAT(3) DNA binding activity, and its antagonistic effect on HLF/LRLP was more powerful than that on HLF (P < 0.05).</p><p><b>CONCLUSIONS</b>Transfection of Lipoxin A(4) receptor-like protein gene enhanced the inhibitory effect of Lipoxin A(4) on human lung fibroblasts proliferation induced by CTGF. Its mechanism might be related to regulation of cyclin D(1) protein expression and STAT(3) DNA binding activity.</p>

Effects of colchicine on synthesis and excretion of cytokines and extracellular matrix by human renal fibroblasts / 中华儿科杂志

<p><b>OBJECTIVE</b>Renal interstitial fibrosis is the final common pathway leading to end-stage renal failure for progressive renal disease of various types. The present study was undertaken to add to the knowledge on colchicine's antiinflammatory and antifibrotic properties confirmed by both human and experimental studies. As the main effector cells, fibroblasts have a central role in the pathogenesis of renal fibrosis. This study aimed to investigate the effects of colchicine on the synthesis and excretion of cytokines transforming growth factor-beta1 (TGF-beta1), interleukin-1beta (IL-1beta) and extracellular matrix (collagen III, collagen IV) in human renal fibroblast.</p><p><b>METHODS</b>Various concentrations of colchicine (5.0 micromol/L, 10.0 micromol/L, 20.0 micromol/L, 40.0 micromol/L) were used to pretreat human embryo renal fibroblasts for 1 hour which were cultured in vitro and then stimulated by 10.0 microg/ml of lipopolysaccharide (LPS). After 18 hours, these fibroblasts and their supernatant were collected. The expression of TGF-beta1 mRNA, IL-1beta mRNA in the cells was studied by using RT-PCR, and the excretion of TGF-beta1, IL-1beta, collagen III and collagen IV by the fibroblasts was assessed by ELISA respectively.</p><p><b>RESULTS</b>(1) By pure stimulation with 10.0 micro g/ml LPS, the expression of TGF-beta1 mRNA and IL-1beta mRNA of fibroblasts was up-regulated 3 times (66.1 +/- 1.6 vs. 22.3 +/- 2.0, q = 590.5, P = 0.002) and 4.7 times (22.0 +/- 2.2 vs. 4.7 +/- 0.8, q = 106.8, P = 0.009), respectively. The protein excretion of TGF-beta1 and IL-1beta was remarkably increased as well compared with the control group [TGF-beta1: (516 +/- 14) pg/ml vs. (420 +/- 5) pg/ml (q = 80.3, P = 0.012), IL-1beta: (3.4 +/- 0.3) pg/ml vs. (0.3 +/- 0.1) pg/ml (q = 297.9, P = 0.003)]. (2) Colchicine could inhibit the expression of TGF-beta1 mRNA and protein in a dose-dependent manner. IL-1beta mRNA and protein were both up-regulated by colchicine. (3) LPS could not stimulate the excretion of extracellular matrix by fibroblasts, but the excretion of collagen III and collagen IV was down regulated by colchicine in a dose-dependent manner.</p><p><b>CONCLUSION</b>(1) The expression of TGF-beta1 mRNA and the excretion of TGF-beta1 protein in the fibroblasts was significantly suppressed by colchicine, while the expression of IL-1beta mRNA and the excretion of IL-1beta protein were enhanced. (2) Colchicine has significant inhibitory effect on the excretion of extracellular matrix such as collagen III and collagen IV in fibroblasts.</p>

Expression of TSG-6 gene during 3T3-L1 preadipocyte differentiation and regulative role of tumor necrosis factor-alpha / 中华儿科杂志

<p><b>OBJECTIVE</b>Tumor necrosis factor alpha-stimulated gene-6 (TSG-6 gene) differentially expressed in adipose tissue of obese and normal human subjects or rats. To explore the relationship between the differential expression of TSG-6 and adipocyte differentiation, adipogenesis and obesity, the present study aimed to investigate the changes of TSG-6 gene expression during 3T3-L1 preadipocyte differentiation and to analyze the regulative role of TNF-alpha on TSG-6 gene expression in matured 3T3-L1 adipocytes.</p><p><b>METHODS</b>3T3-L1 preadipocytes were cultured in vitro and differentiated into the matured adipocytes. TNF-alpha in different concentrations (0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml) was added into the culture medium of fully differentiated adipocytes (day 10) for various times (0.5 h, 2 h, 6 h, 12 h, 24 h). Total RNA from these adipocytes was extracted and the levels of TSG-6 gene mRNA expression were evaluated by RT-PCR.</p><p><b>RESULTS</b>(1) In preadipocytes, the level of TSG-6 gene mRNA expression remained low. In the presence of dexamethasone (Dex), MIX and insulin, with the 3T3-L1 preadipocytes being differentiated into the matured adipocytes, the level of TSG-6 gene mRNA expression was upregulated and reached the higher level in fully differentiated adipocytes. There is a significant difference between any two detected phases in the levels of TSG-6 gene mRNA expression (P < 0.05), except that the levels of TSG-6 gene mRNA expression did not increase obviously on day 0 to day 2, day 3 to day 5, day 4 to day 6 and day 7 to day 10 (P > 0.05). (2) Treatment of day 10 3T3-L1 adipocytes with TNF-alpha of different concentrations (0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml) resulted in a significant decrease in the level of TSG-6 gene mRNA expression. The inhibition effect of TNF-alpha on TSG-6 gene mRNA expression generally tended to be reinforced with the increasing concentrations of TNF-alpha and the elongation of time course, except for the period of 6 - 24 h after the stimulation of 10.0 ng/ml TNF-alpha. When 0.1 ng/ml TNF-alpha was applied, the level of TSG-6 gene expression decreased by 33.73% at 6 h, 97.39% at 12 h. While 1.0 ng/ml TNF-alpha was used, the level of TSG-6 gene expression decreased by 78.68% at 6 h, which remained until 24 h. At a concentration of TNF-alpha up to 10.0 ng/ml, the level of TSG-6 gene expression decreased by 96.27% at 2 h. TSG-6 gene expression was almost fully inhibited.</p><p><b>CONCLUSION</b>(1) TSG-6 gene may be involved in adipocyte differentiation and adipogenesis. (2) TNF-alpha can downregulate the mRNA expression of TSG-6 gene in matured adipocytes. The inhibitory effect of TNF-alpha on TSG-6 gene expression is generally dose-correlated.</p>

Curcumin inhibited the proliferation and extracellular matrix production of human mesangial cells / 中华儿科杂志

<p><b>OBJECTIVE</b>Glomerulosclerosis is characterized by extracellular matrix accumulative and is often associated with mesangial cell proliferation. Curcumin showed a protective effect on anti-glomerular basement membrane (anti-GBM) nephritis in vivo, although their cellular localization and mechanism of action is still unclear. In this study, a glomerular mesangial cell line derived from fetus was used to determine whether curcumin could inhibit the cell proliferation and alter the extracellular matrix turnover.</p><p><b>METHODS</b>The cell activity was determined with MTT method. Mesangial cells were cultured in vitro and incubated with 0, 3.125, 6.25, 12.5, 25, 50, 100 and 200 micromol/L curcumin. In addition,human mesangial cells were cultured with or without LPS (10 microg/ml) in presence or absence of various concentrations of curcumin (4, 16 and 200 micromol/L), respectively. The supernatant and cells were collected. Then, the levels of the collagen type IV and III protein in the supernatant were determined by using enzyme-linked immunosorbent assay and the IL-1 beta and MCP-1 mRNA in the cells was measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) after subconfluent quiescent mesangial cells were incubated with various concentrations of curcumin for 24 h in vitro.</p><p><b>RESULTS</b>Curcumin at the concentration equal to or over 6.25 micro mol/L was able to inhibit the proliferation of mesangial cells in a dose-dependent manner, the optical density according to the sequential concentrations of curcumin was 0.65 +/- 0.02, 0.62 +/- 0.04, 0.56 +/- 0.01, 0.53 +/- 0.02, 0.51 +/- 0.03, 0.44 +/- 0.05, 0.41 +/- 0.07 and 0.38 +/- 0.06. Without any stimulation, human mesangial cells secreted some collagen type IV and III (10 +/- 9.13 ng/ml and 29.5 +/- 0.58 ng/ml, respectively) and expressed some MCP-1 mRNA, but did not express IL-1 beta mRNA. LPS increased the expression of collagen type IV and III in the culture medium of mesangial cells in vitro [(138.75 +/- 23.23) ng/ml and (38.25 +/- 5.38) ng/ml] and up-regulated the IL-1 beta and MCP-1 mRNA expression [(16.91 +/- 1.68)% and (76.6 +/- 6.59)%]. Yet curcumin could significantly decrease collagen type IV and III in the supernatant of cultured mesangial cells induced by LPS (20.5 +/- 1.00, P < 0.05 and 20.5 +/- 4.12 ng/ml, P < 0.05) and down-regulated the mRNA expression of IL-1 beta and MCP-1 in mesangial cells induced by LPS (P < 0.01).</p><p><b>CONCLUSION</b>Curcumin could inhibit the human mesangial cell proliferation and alter the extracellular matrix turnover, meanwhile it could down-regulate the IL-1 beta and MCP-1 mRNA expression induced by LPS, which may be valuable in decreasing the progression of glomerulosclerosis.</p>

Expression of Calcium/Calmodulin - Dependent Protein Kinase Ⅱ Gene in 3T3 - L1 Preadipocyte Differentiation and Regulation of Timor Necrosis Factor- ? / 实用儿科临床杂志

Objective To investigate the changes of calcium /calmodulin - dependent protein kinase Ⅱ (CAMKⅡD) gene expression in 3T3 - L1 preadipocyte diffe-rentiation and TNF - ? regulation of CAMKⅡD gene expression on matured adipocytes. Methods 3T3 - L1 preadipocytes were cultured and differentiated into adipocytes. The levels of CAMKⅡD gene mRNA expression at various times were evaluated by RT-PCR. Matured adipocytes were interfered with 0.1,1.0,10.0 ?g/L TNF - ? and the levels of CAMKⅡD gene mRNA expression were evaluated. Results The levels of CAMKⅡD gene mRNA expression in 3T3 - L1 adipocytes were significantly down- regulated at first day, compared with those at zero day (P0. 05). CAMKⅡD gene mRNA expression decreased significantly at 12 hours after treatment with 10.0 ?g/L TNF-?, treatment of matured adipocytes with TNF - ? did not have any regulation role on it. Conclusions CAMKⅡD gene is involved in the differentiation of adipocytes and related to the etiology of obesity. The changes in the level of CAMKⅡD gene mRNA expression during 3T3 - L1 preadipocyte differentiation may contribute to the differentiation and adipogenesis of adipocytes. It seems that TNF - ? does not have any regulation role on the expression of CAMKⅡD genes.