ATP-binding cassette transporter Al (ABCAl) is a kind of membrane intergrate protein and may have multiple and diverse functions. It mediates the cellular efflux of phospholipids and cholesterol to lipid-poor apolipoproteinA- Ⅰ (apoA- Ⅰ ) and plays a significant role in high density lipoprotein (HDL) metabolism. Mutations in human ABCAl cause severe HDL deficiencies characterized by the virtual absence of apoA- Ⅰ and HDL and prevalent atherosclerosis. ABCAl expression is highly regulated and implies a variety of molecular actors. All of the nuclear receptors which involve in regulation of ABCAl expression act via the DR4 element in the ABCAl promoter. cAMP up-regulates ABCAl expression by acting both at the transcriptional and translational level. Cytokines have been shown to exert pleiotropic and antinomic effects on ABCAl transcription, In addition to these, some of enzymes and proteins such as protein kinase A, protein kinase CK2, cathepsin D are involved in the regulation of ABCAl expression. The recent progress in the structure, function and regulation of ABCAl transporter is reviewed.

The purpose of this study is to investigate the activity and mechanism of a new anti-tumor agent T03. MTT and colony formation assay were performed to determine anti-proliferation activity of T03 in vitro. Antitumor activity was observed by Renca xenograft model in vivo. The effect of T03 on cell cycle and apoptosis were measured by FCM analysis. Western blotting was performed to investigate the expression level of proteins in HepG2 cell lines treated with T03. T03 had anti-tumor activity by inhibiting tumor cell growth and colony formation in vitro, especially on hepatocellular carcinoma cells (HCC). At the concentration of 10 micromol x L(-1), T03 induced cell apoptosis and cell cycle arrest in HCC. Moreover, it proved that T03 reduced the tumor weight with the rate of 42.30% without any obviously side effect in Renca xenograft model. At the concentration of 2.0 micromol x L(-1), T03 was able to reduce the level of p-c-Raf (Ser259), and thus blocked Raf/MEK/ERK and AKT signaling in HepG2 cell lines. The result suggested that T03 has the potential to inhibit cell proliferation and induce cell apoptosis both in vitro and in vivo, particularly active against HCC, indicating T03 and its analogues may serve as a new anti-cancer drug against hepatocellular carcinoma.

Interests on the effects of cytochrome P450 (CYP450) monooxygenases and epoxyeicosatrienoic acids (EETs) on myocardial ischemic-reperfusion injury has been increased in recent years. The CYP450/EET system may influence the degree of myocardial ischemic-reperfusion injury through "poly-targets", such us oxygen free radical, calcium overload, leukocytes adherence, nitric oxide, ATP-sensitive K+ channels, and mitogen activated protein kinase. The exaggeration or recovery of injury depends on the physical status. Study of factors that affects CYP450/EET, particularly identification of their involvement in cardioprotective signaling and specific roles, will elucidate the mechanisms of myocardial ischemic-reperfusion injury, and find a new way of prevention and treatment. This article will review the relationship between the changes of CYP450/EETs system and myocardial ischemic-reper-
Animals ; Cytochrome P-450 Enzyme System ; metabolism ; Eicosapentaenoic Acid ; metabolism ; Humans ; Mixed Function Oxygenases ; metabolism ; Myocardial Reperfusion Injury ; etiology ; metabolism

Objective To investigate the effects of pioglitazone on glucose metabolism,circulating resistin and adiponectin concentrations,and tissue resistin levels in rats with insulin resistance induced by free fatty acid (FFA).Methods A hyperinsulinaemic-euglycaemic clamp and[3-~3H]-glucose tracing technique were used in awake rats.Insulin-mediated peripheral and hepatic glucose metabolism,plasma resistin and adipenectin levels, resistin levels in tissues were assessed by hyperinsulinaemic-euglycaemic clamp with elevation of FFA by lipid infusion over 4 h in rats pretreated with or without pioglitazone.Results During steady-state of clamp,there was a significant increase in plasma FFA in lipid-infused group(L group)and pioglitazone-pretreated lipid-infused group(P/L group)compared to control rats(P＜0.01).The glucose infusion rate(GIR)in P/L group was significantly reduced as compared with controls[(20.6?0.9 vs 33.6?1.5)mg?kg~(-1)?min~(-1),P＜0.01], whereas the GIR was lower in L group than in P/L group[(12.6?0.8 vs 20.6?0.9)mg?kg~(-1)?min~(-1),P＜0.01].As compared with baseline,the hepatic glucose production(HGP)was significantly suppressed by 85% [(18.3?2.1 vs 2.7+2.4)mg?kg~(-1)?min~(-1) and (17.5?2.6 vs 2.6?1.0)mg?kg~(-1)?min~(-1),both P＜0.01]during the hyperinsulinaemic clamp in control and P/L groups.The suppressive effect of insulin on HGP was significantly blunted in L group[(17.3?2.1 vs 15.8?1.5]mg?kg~(-1)?min~(-1)].The rate of glucose disappearance(G_(Rd))was reduced in L group and P/L group compared with controls(P＜0.01).Baseline plasma resistin level was lower in P/L group than that in the controls[(7.8?1.3 vs 29.1?3.1)?g/L,P＜0.01].After lipid infusion,plasma resistin levels significantly increased in P/L group,but remained lower than that of L group [(18.1?3.8 vs 47.0?2.2)?g/L,P＜0.01].Baseline adiponectin level was higher in P/L group than those in the controls and L groups[(3.9?0.2 vs 2.8?0.1 and 2.6?0.2)mg/L,P＜0.01].After clamp,plasma adiponectin levels were decreased in L group and L/P group compared with baseline(both P＜0.05).In addition, the resistin level in the liver of P/L group decreased compared with the controls and L groups(both P＜0.05), whereas significantly increased in the muscle of L group.Conclusion Lipid infusion induces an acute insulin- resistance in vivo.Pioglitazone pretreatment partly prevents FFA-induced insulin resistance possibly by changing resistin and adiponectin levels.

To test the hypothesis that concentration polarization of atherogenic lipids may occur in the arterial system and play an important role in localization of atherosclerosis, we simulated and measured in vitro the luminal surface concentration of low density lipoprotein (LDL) in local stenosis at the distal end of carotid artery by number simulation and laser scanning confocal microscopy, then we designed carotid stenosis model to test the role of LDL concentration polarization in atherogenesis. The in vitro experiment showed that the luminal surface LDL concentration was higher than the bulk concentration as predicted by the concentration polarization theory. The relative luminal surface LDL concentration changed with the flow velocity and ratio of stenosis. The wall concentration of LDL was highest in the round tube with 40% stenosis at the same velocity, while the wall concentration of LDL was higher when Re was 250 than Re was 500 at the same extent of narrowness. The animal experiment also revealed that general atherogenic plaques obviously occurred at the distal end of local stenosis where concentration polarized. The results strongly support our hypothesis that concentration polarization of lipoproteins occurs in local stenosis at the distal end of carotid artery, and this in turn promotes the localization of atherosclerosis which develops in the arterial system.
Animals ; Atherosclerosis ; physiopathology ; Carotid Stenosis ; physiopathology ; Disease Models, Animal ; Lipoproteins, LDL ; metabolism

OBJECTIVETo develop a rapid and reproducible method for the culture of human fetal hair follicle bulge cells, and observe the plasticity of its differentiation into sebaceous gland in vitro.

METHODSThe bulge cells isolated from fetal human hair follicles by enzymatic digestion (digestion method) and manual microdissection (conventional method) were cultured and passaged respectively, the efficiency and biological features of cells were investigated , the clone forming efficiency was assayed by MTT, and the expression of K19 was further compared by immunocytochemistry (ABC). The morphological change and the expression of EMA of bulge cells were also observed after induction.

RESULTSBy conventional method, 8-10 bulges were harvested in one hour, 40%-50% of their cells were found to adhere to the culture plate after culturing for 48h, and they became confluent after 14 days. In comparison, about 100 bulges were harvested in one hour by digestion method, the adherence efficiency of their cells was 30% after cultivation for 12h and became confluent after 7 days. The cells grew larger with time, with irregular shape and droplets of lipid around the nucleus. The clone forming efficiency of bulge cells cultured by digestion method was (18.2 +/- 2.1) %, which was much higher than that of cells obtained by conventional method[ (12.7 +/- 3.4) %, P < 0.05]. Immunocytochemistry staining showed that positive staining of K19 was observed in most of the bulge cells, with a large amount of brown granules in the cytoplasm.

CONCLUSIONHuman hair follicle bulge cells can be efficiently cultured and multiplied in vitro, and they retained the characteristics of stem cells. And they have the potential to differentiate into sebaceous glands by induction in vitro.

Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Fetal Stem Cells ; cytology ; Hair Follicle ; cytology ; Humans ; Sebaceous Glands ; cytology

Objective To investigate the ostengenie potential of adipose-derived stem cells(AD- SCs)when exposed to adenovirns containing hBMP-2 cDNA(Adv-hBMP-2)and offer a choice of cell source for gene therapy and tissue engineering.Methods Human adipose tissues were obtained from patients who received orthopaedic surgery or liposuction.ADSCs were obtained by digesting the adipose tissues.Firstly,flowcytometric analysis was performed for the confirmation of mesenchymal stem cell ori- gin and the surface markers including CD34,CD44,CD68,CD71,CD90,and CD105.The ADSCs were transfected by Adv-hBMP-2 and the effects were tested in vitro,lmmunoprecipitation and Western blotting and ELISA were performed for confirming BMP gone transduction and its stable expression.The transform of ADSCs was assessed by extracellular ALP staining,intracellular ALP spectrophotometry,von Kossa staining and RT-PCR.In the in vivo experiment ADSC-Adv-hBMP-2 cells were injected into the hind limb of nude mice and analyzed radiographically and histologically.Results ADSCs were successfully isolated from human adipose tissues.The isolated ADSCs expressed CD44,CD71,CD90 and CD105 and CD34 and CD68 were absent.The result confirmed the mesenchymal stem cell origin of the cells.West- ern blotting and ELISA confirmed successful and persistent hBMP-2 production by ADSC-Adv-hBMP-2 cells.Extracellular ALP staining,intracellular ALP spectrophotometry,yon Kossa staining and RT-PCR revealed that ADSCs treated with Adv-hBMP-2 had a tendency of transfering into osteoblast.X-ray and H&E sections from hind limb of nude mice injected with ADSC-Adv-hBMP-2 cells confirmed bone forma- tion at 2 weeks.Conclusions Liposuction aspirates contain abundant ADSCs that can be transduced with hBMP-2 gene,and the tranduced ADSCs differentiate into the osteoblast.ADSCs may be an ideal source of mesenchyme-lineage stem cells for gone therapy and tissue engineering.

The purpose of this study is to investigate the activity and mechanism of a new anti-tumor agent T03. MTT and colony formation assay were performed to determine anti-proliferation activity of T03 in vitro. Antitumor activity was observed by Renca xenograft model in vivo. The effect of T03 on cell cycle and apoptosis were measured by FCM analysis. Western blotting was performed to investigate the expression level of proteins in HepG2 cell lines treated with T03. T03 had anti-tumor activity by inhibiting tumor cell growth and colony formation in vitro, especially on hepatocellular carcinoma cells (HCC). At the concentration of 10 micromol x L(-1), T03 induced cell apoptosis and cell cycle arrest in HCC. Moreover, it proved that T03 reduced the tumor weight with the rate of 42.30% without any obviously side effect in Renca xenograft model. At the concentration of 2.0 micromol x L(-1), T03 was able to reduce the level of p-c-Raf (Ser259), and thus blocked Raf/MEK/ERK and AKT signaling in HepG2 cell lines. The result suggested that T03 has the potential to inhibit cell proliferation and induce cell apoptosis both in vitro and in vivo, particularly active against HCC, indicating T03 and its analogues may serve as a new anti-cancer drug against hepatocellular carcinoma.
Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; Hep G2 Cells ; drug effects ; Humans ; Liver Neoplasms ; pathology ; Signal Transduction ; drug effects ; Xenograft Model Antitumor Assays

OBJECTIVE: To investigate the effect of overexpression of glycosylphosphatidyl-inositol-specific phospholipase D (GPI-PLD) on the biological character of hepatocellular carcinoma cell line HepG2. METHODS: The GPI-PLD gene eukaryon expression vector pcDNA3.1(+)/ GPI-PLD was transiently transfected into HepG2 cell by lipid-media transfection. The untransfected HepG2 and HepG2 transfected with pcDNA3.1(+) were used as controls. After screening with G418, the single clone was obtained. The expression level of GPI-PLD mRNA in HepG2 was identified by reverse transcription polymerase chain reaction (RT-PCR). GPI-PLD activities were analyzed quantitatively by triton-X-114 partition with GPI anchored placental alkaline phosphatase (PLAP) as a substrate. Cell count was used to detect the proliferation of the 3 groups, and complement dependent cytotoxicity (CDC) effects were observed by the staining of trypan blue. Apoptosis cells were analyzed by flow cytometry. Carcinoembryonic antigen (CEA)was detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with HepG2 and pcDNA3.1(+)/HepG2 cell, the levels of GPI-PLD activities and its mRNA from pcDNA3.1(+)/GPI-PLD/HepG2 were increased with almost 2 to 5 times,respectively. The GPI anchored PLAP and CEA released into the medium by GPI-PLD, and the rate of CDC killing on the cells were significantly increased. However, the proliferative capacity was obviously decreased, and the typical apoptosis cells were presented in positive clones and its apoptosis rates were increased significantly. CONCLUSION The stable cell line with overexpression of GPI-PLD has been constructed. The overexpression of GPI-PLD in these cells increases the sensitivity of these cells to CDC killing and impairs the proliferative capacity of cells, and promotes the apoptosis.
Apoptosis ; genetics ; Carcinoma, Hepatocellular ; genetics ; pathology ; Complement Activation ; genetics ; Cytotoxicity, Immunologic ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; pathology ; Phospholipase D ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

BACKGROUNDStudy of the relationship between mast cells and atherosclerosis is mostly dependent on pathological observation and cytology experiments. To investigate the effects of mast cells degranulation on plaque and their possible mechanisms we used apolipoprotein E knockout mice which had been placed perivascular common carotid collar with mast cells degranulator compound 48-80.

METHODSForty apolipoprotein E knockout mice were fed a western-type diet and operated on with placement of perivascular right common carotid collar. Four weeks after surgery, the mice were intraperitoneally injected with compound 48-80 (0.5 mg/kg) or D-Hanks every other day for 4 times. The serum lipids and activity of tryptase were measured. Tissue sections were stained with hematoxylin and eosin. Corresponding sections were stained with toluidine blue and immunohistochemically with antibodies against macrophage-specific antigen, alpha-smooth muscle actin, interleukin-1beta and von Willebrand factor. Simultaneously, basic fibroblast growth factor was detected by in situ hybridization and immunofluorescence.

RESULTSNo pathological change was observed in common carotid non-collar placement but atherogenesis in common carotid collar placement of both groups. There was a significant increase in plaque area ((5.85+/-0.75) x 10(4) vs (0.86+/-0.28) x 10(4) microm(2), P<0.05), the degree of lumen stenosis ((81+/-15)% vs (41+/-12)%, P<0.05), the activity of tryptase in serum ((0.57+/-0.13) U/L vs (0.36+/-0.10) U/L, P<0.05), and the percentage of degranulated mast cells ((80.6+/-17.8)% vs (13.5+/-4.1)%, P<0.05). The expressions of macrophage-specific antigen, alpha-smooth muscle actin, interleukin-1beta, basic fibroblast growth factor and the density of neovessel in plaque were more in the compound 48-80 group than in the control group.

CONCLUSIONSPerivascular common carotid collar placement can promote atherosclerotic plaque formation in apolipoprotein E knockout mice. Compound 48-80 increases plaque area and the degree of lumen stenosis by the mechanism that compound 48-80 promotes proliferation of smooth muscle cells and aggregation of macrophages. Compound 48-80 promotes angiogenesis in plaque. The mechanism is potentially that compound 48-80 increases the expressions of basic fibroblast growth factor mRNA and protein in plaque. Compound 48-80 enhances the expression of interleukin-1beta in plaque.

Animals ; Apolipoproteins E ; genetics ; Atherosclerosis ; chemically induced ; genetics ; metabolism ; pathology ; Carotid Arteries ; drug effects ; pathology ; Fluorescent Antibody Technique ; Immunohistochemistry ; In Situ Hybridization ; In Vitro Techniques ; Male ; Mast Cells ; drug effects ; metabolism ; Mice ; Mice, Knockout ; p-Methoxy-N-methylphenethylamine ; pharmacology