Receptor-mediated endocytosis of low-density lipoprotein by cultured human glomerular cells

Lipoproteins might be involved in the pathogenesis of glomerular damage. Uptake of low-density lipoprotein (LDL) by cultured human glomerular cells has been studied using LDL, labelled with the fluorescent probe 1,1'-dioctadecyl-3,3,3'3'-tetramethyl-indocarbocyanine perchlorate (diI). Cells have been characterised using phase-contrast microscopy, monoclonal antibodies and lectins. Differentiated glomerular epithelial cells, epithelial-like cells and mesangial cells all took up diI-LDL. Uptake was specific for LDL, of high affinity and inhibited by excess unlabelled LDL, heparin and preloading the cells with cholesterol. Binding of diI-LDL to the cell surface was restricted to discrete areas which were arranged in linear arrays on mesangial cells. Endocytosis of surface-bound diI-LDL occurred within 3 min and breakdown of internalised diI-LDL within 30 min. These results indicate that cultured human glomerular cells take up LDL by receptor-mediated endocytosis.     

Metabolism of low-density lipoprotein from patients with diabetic hypertriglyceridemia by cultured human skin fibroblasts

To test whether triglyceride-enriched low-density lipoprotein (LDL) obtained from subjects with diabetic hypertriglyceridemia is metabolized normally by cells, LDL was separated from seven healthy control subjects (fasting plasma glucose [FPG] 91 +/- 10 mg/dl [mean +/- SD], triglyceride [TG] 110 +/- 47 mg/dl), six diabetic normolipidemic patients (FPG 218 +/- 65 mg/dl; TG 139 +/- 75 mg/dl), six diabetic hypertriglyceridemic patients (FPG 214 +/- 71 mg/dl; TG 1915 +/- 1680 mg/dl), and five nondiabetic hypertriglyceridemic patients (FPG 92 +/- 8 mg/dl; TG 2013 +/- 1889 mg/dl). Binding of 125I-labeled LDL from hypertriglyceridemic subjects with and without diabetes to cultured skin fibroblasts was significantly decreased to 74 +/- 19% and 78 +/- 14% of that seen with LDL from normolipidemic nondiabetic subjects and diabetic normolipidemic controls (100 +/- 0%, 101 +/- 25%; P less than 0.005). Unlabeled LDL from hypertriglyceridemic subjects with and without diabetes failed to suppress LDL receptor activity and sterol synthesis from 14C-acetate as efficiently as unlabeled LDL from healthy subjects. The ability of LDL from hypertriglyceridemic subjects, whether diabetic or not, to suppress LDL binding was inversely related to the ratio of triglyceride to protein in LDL (r = 0.71, P less than 0.01) and showed a positive correlation with the LDL cholesterol/protein ratio (0.69, P less than 0.01). Thus, LDL from patients with hypertriglyceridemia, with or without coexistent diabetes, shows impaired binding to LDL receptors and less ability to downregulate LDL receptor activity and sterol synthesis than does LDL from normolipidemic diabetic and nondiabetic subjects. These findings suggest that factors associated with hypertriglyceridemia rather than with diabetes result in altered metabolism of LDL in these disorders.     

Cholesterol uptake by human glioma cells via receptor-mediated endocytosis of low-density lipoprotein

Low-density lipoprotein (LDL) is a carrier of the cholesterol found in human plasma. Cells utilize cholesterol for membrane synthesis by taking up LDL via receptor-mediated endocytosis. In the present study, interactions of LDL with human malignant glioma cell lines (U-251 MG and KMG-5) were investigated biochemically and morphologically. The LDL, labeled with the fluorescent dyes 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) and fluorescein isothiocyanate (FITC), was internalized by both cell processes and cell bodies. Reductive methylation of DiI-labeled LDL, which abolishes the ability of the cell to bind to the LDL receptor, prevented the internalization of the cholesterol moiety of LDL. Cellular binding of 125I-LDL to U-251 MG cells at 4 degrees C revealed the presence of a specific saturable-associated receptor (dissociation constant (Kd) approximately 38 micrograms/ml). Endocytic uptake of 125I-LDL or 3H-cholesterol oleate-labeled LDL (3H-LDL) at 37 degrees C demonstrated the cell-associated 125I-LDL and 3H-LDL increase. The intracellular degradation of protein moiety increased linearly with time. Reductive methylation of 3H-LDL led to a remarkable decrease in the cell-associated cholesterol moiety of LDL. The difference in uptake of the cholesterol moiety of LDL between U-251MG cells and KMG-5 cells showed that the U-251MG cells, which proliferate more actively than KMG-5 cells, take up more of the cholesterol moiety of LDL than do the KMG-5 cells. Thus, LDL cholesterol seems to be endocytosed predominantly via the LDL receptor present on the plasma membrane of malignant glioma cells. In addition, for growth, these cells may require large amounts of the cholesterol moiety of LDL.     

脂蛋白（a）对肾小球系膜细胞的作用

目的探讨脂蛋白（ａ）［Ｌｐ（ａ）］对肾小球系膜细胞（ＧＭＣ）的作用。方法将体外培养的系膜细胞，加脂蛋白（ａ）刺激后，测定细胞生长率和细胞上清中血小板活化因子（ＰＡＦ）、肿瘤坏死因子（ＴＮＦ）、乳酸脱氢酶（ＬＤＨ）及纤维连结蛋白（ＦＮ）水平，并以未经刺激者作对照。结果经脂蛋白（ａ）刺激４８小时，在终浓度５～２０ｍｇ／Ｌ时细胞生长率轻度增加，而在５０～４００ｍｇ／Ｌ时细胞生长率明显下降。刺激１８小时，细胞上清中ＰＡＦ的水平明显高于对照，且与脂蛋白（ａ）浓度呈明显正相关（ｒ＝０．９３７，Ｐ＜０．０１）；Ｌ９２９细胞的杀伤率有所增加，但未达５０％；ＬＤＨ的水平在脂蛋白（ａ）１００ｍｇ／Ｌ以下时有所增加；刺激１８、４８、７２小时后的细胞上清纤维连结蛋白均为阴性。结论脂蛋白（ａ）对系膜细胞体外增殖有低浓度轻度促进、高浓度抑制的双向作用，对细胞ＰＡＦ的产生具有浓度依赖的促进作用，并能增加细胞上清中ＬＤＨ及ＴＮＦ水平，因而可通过多种途径介导肾小球损伤。     

Study of the growth factor requirements of human bone-derived cells: a comparison with human fibroblasts

Recent techniques have been devised for the culture of bone cells derived from human bone explants. These cells, which are thought to represent several stages in the osteoblast lineage, respond to PTH with an increase in cyclic AMP content, and have high basal alkaline phosphatase activity which is increased on exposure to 1,25-dihydroxyvitamin D3 and decreased by PTH. Such characteristics distinguish these cells from fibroblasts. In this study, we demonstrate that human bone-derived cells also differ from fibroblasts in their growth characteristics. Bone-derived cells proliferated in basal medium supplemented with platelet-poor plasma. The rate of proliferation was enhanced by additional supplementation with platelet-derived growth factor (PDGF), and further increased when a combination of growth factors was added (PDGF, TGF-beta and EGF). In contrast, fibroblasts did not proliferate in basal medium supplemented with platelet-poor plasma and the addition of PDGF alone stimulated fibroblast proliferation to the same extent as 10% fetal bovine serum. Supplementation with other growth factors did not further enhance the response of fibroblasts to PDGF. These results emphasize the differences in proliferative responses between human bone-derived cells and human fibroblasts, and indicate that the factors responsible for osseous regeneration in vivo may differ from those factors which regulate repair of soft tissue wounds.     

Receptor mediated uptake of apo B and apo E rich lipoproteins by human glomerular epithelial cells

Various pathological disorders are accompanied by the deposition of lipids into glomerular cells. To gain insight into these disorders, it is essential to know if glomerular cells possess lipoprotein receptors. We therefore characterized the activity of lipoprotein receptors in cultured epithelial cells of the human glomerulus. Podocytes were chosen as they are directly exposed to lipoproteins in pathological states like in glomerular proteinuria (such as, nephrotic syndrome). Isolated human glomeruli (purity greater than 95%) were incubated in buffered RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum at 37 degrees C and 5% CO2. Outgrowing cells were vimentin and keratin positive. Monolayer cultures of human glomerular epithelial cells upon incubation in lipoprotein deficient serum for 48 hours expressed a receptor-dependent uptake of lipoproteins. These cells showed about 10% of the maximal capacity for LDL uptake as compared to fibroblasts; however, the Km values for binding, internalization and degradation were similar in the cultures of glomerular epithelial cells and fibroblasts. The Km values for degradation of LDL, chylomicron remnants, beta-VLDL from cholesterol-fed rabbits and VLDL from familial LCAT-deficiency patients were 14.2, 4.9, 2.9, 4.5 micrograms protein/ml medium, respectively, for glomerular epithelial cells. The avid uptake of 125I-labeled apo E-containing lipoproteins was further substantiated by their poor displacement by a 25-fold excess of unlabeled LDL and their ability to down regulate the apo B,E receptor activity. LDL as well as beta-VLDL were able to suppress the incorporation of 14C acetate into sterols and to stimulate 3H-cholesterylester formation. These experiments show that cultured glomerular epithelial cells express lipoprotein receptor activity. Plasma concentrations of apo E-containing lipoproteins are increased in certain renal diseases (such as, nephrotic syndrome); these lipoproteins could be rapidly removed by glomerular epithelial cells and lead to lipid deposition in glomeruli.     

Stiffening of human skin fibroblasts with age

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in?vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ～60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.     

Novel glomerular lipoprotein deposits associated with apolipoprotein E2 homozygosity

BACKGROUND: Hyperlipoproteinemia is occasionally associated with severe glomerular injury caused by abnormal accumulation of lipid in glomeruli, which occurs in conditions such as lipoprotein glomerulopathy (LPG). This study investigates the cases of two siblings with homozygous apolipoprotein (apo) E2 who show unique histologic features, massive proteinuria, and dysbetalipoproteinemia. METHODS: Histologic studies were performed using renal biopsy specimens. Plasma lipoproteins were extensively characterized. The exons of the apo E genes were sequenced to avoid missing any mutations. RESULTS: Histologically, the siblings' condition resembled LPG by light microscopy studies. Electron microscopy studies revealed large lipoid deposits in the paramesangium, subendothelium, and subepithelium of the glomeruli, which were different from LPG in terms of not forming the layered structure resembling a fingerprint even in large lipoprotein thrombi, and mesangial foam cells. Immunohistochemically, the lipoid deposits contained apo E and apo B. These patients did not have either diabetic nephropathy or other known forms of glomerulonephritis. The sequence of exons of the apo E genes revealed homozygosity for apo E2 in both cases. CONCLUSION: The extensive lipoprotein deposition in glomeruli, which resembles LPG, can also occur in apo E2 homozygous individuals, but in a distinct fashion. Because the two cases were siblings, they may have other shared alleles, in addition to the apo E2 allele, that negatively affect processing of lipoproteins and lead to abnormal accumulation of lipoprotein deposits in glomeruli.     

Low-density lipoprotein uptake and cholesterol accumulation by cultured renal cells.

The uptake of low-density lipoprotein (LDL) and the accumulation of cholesterol were assessed in opossum kidney (OK) and Madin-Darby canine kidney (MDCK) cells. OK and MDCK cells were grown to confluency on Millicell well inserts. The uptake of human LDL across the apical and basolateral surfaces of OK and MDCK cells was assessed by the degradation of internalized (125I)LDL to trichloroacetic acid-soluble products. LDL uptake via the apical surface of OK cells increased linearly with LDL concentration, indicating nonreceptor-mediated uptake. In contrast, LDL uptake via the basolateral surface of OK cells and both apical and basolateral surfaces of MDCK cells followed a saturable pattern. In addition, (125I)LDL bound to the apical membrane of MDCK cells, but not to the apical membrane of OK cells, was displaced by heparin and by excess of unlabeled LDL. Exposure to LDL (100 mg/mL) resulted in an increase in total cholesterol content of OK and MDCK cells (23 and 18%, respectively). Most of the increase in total cholesterol content with LDL exposure resulted from increased free cholesterol content in MDCK cells and esterified cholesterol in OK cells. The differences in cholesteryl ester formation were consistent with the slower rates of (14C) oleate incorporation into cholesteryl ester and lower cholesterol esterifying activity observed in MDCK cells compared with that in OK cells. These results demonstrate that LDL uptake can be receptor or nonreceptor mediated, depending upon the renal cell type and the surface exposed to LDL, and that LDL exposure leads to increased cholesterol content in OK and MDCK cells.     

Monoclonal antibodies to human glomerular cells: a marker for glomerular epithelial cells

A monoclonal antibody, PHM 5, directed against human glomerular epithelial cells, was prepared after immunisation of a mouse with isolated human glomeruli and fusion of spleen cells with a mouse myeloma. Binding of antibody to isolated glomeruli was detected by radioimmune indirect binding assay, and specificity for glomerular epithelial cells was shown using a four-layer peroxidase-antiperoxidase technique applied to epoxy resin sections of the human kidney cortex. PHM 5 appears to detect a human-specific cell surface carbohydrate antigen not previously described, and can be used to identify epithelial cells in renal biopsy sections and in culture outgrowths of isolated glomeruli.     

Downregulation of high-density lipoprotein receptor in human fibroblasts by insulin and IGF-I

High-density lipoprotein (HDL3) particles bind to a cell surface receptor, thereby promoting the efflux of cholesterol from extrahepatic nonsteroidogenic cells. This receptor appears to be upregulated by increased cell cholesterol content and also may be responsive to the growth state of cells. Because insulin can be mitogenic, the effect of insulin on HDL-receptor function was tested. HDL-receptor activity of cholesterol-loaded fibroblasts was inhibited by insulin treatment. Insulin decreased HDL binding in a log-dose fashion (-25% at 67 nM insulin) in association with increases in [3H]thymidine incorporation into DNA. HDL-mediated cholesterol efflux from cholesterol-loaded cells was diminished by insulin treatment of cells in parallel with decreased HDL binding. Insulin induced reciprocal changes in HDL- and low-density lipoprotein (LDL)-receptor activity. In cells in which these receptors were upregulated by varying cell cholesterol content, insulin increased LDL binding (+88%) and decreased HDL binding (-24%). Insulin-like growth factor I (IGF-I, 100 ng/ml) also significantly decreased HDL binding and HDL-mediated cholesterol efflux to a comparable degree. Pooled human serum similarly induced a reduction in HDL binding to its receptor. These results are consistent with the hypothesis that growth factors in general, and insulin and IGF-I in particular, decrease HDL-receptor activity, possibly to promote retention of cholesterol needed for new membrane synthesis during cell proliferation. Such a mechanism could be partly responsible for accumulation of cholesteryl esters in arterial wall cells during atherogenesis in diabetes mellitus.     

Glycerol suppresses proliferation of rat hepatocytes and human HepG2 cells

BACKGROUND: In fulminant hepatic failure (FHF), the ability of surviving hepatocytes to proliferate is diminished. Therefore, it is important that medical therapy cause no further impairment of liver regeneration. In FHF, intracranial hypertension secondary to brain edema is the most common cause of brain injury and death and glycerol is used in some countries to treat this complication. Glycerol has been long known to suppress the growth of various cell types. We therefore decided to examine the effect of glycerol on hepatocyte proliferation in vitro and in vivo in rats subjected to partial (2/3) hepatectomy. Additionally, we investigated the effect of glycerol on the proliferation of HepG2 cells. MATERIALS AND METHODS: Mitogen-induced primary rat hepatocytes were cultured in a hormonally defined Dulbecco's modified Eagle's medium containing increasing amounts of glycerol (0.5, 1.0, 2.0, 4.0%). HepG2 cells were cultured in minimal essential medium/10% FBS. After 2 days, HepG2 cells were exposed to glycerol (1.0-2.0-4.0%) and harvested after 48 h. Control dishes contained no glycerol. Cell proliferation was measured by the incorporation of [(3)H]thymidine and/or bromodeoxyuridine (BrdU). In vivo, Sprague-Dawley rats were subjected to standard partial 2/3 hepatectomy and assigned to intraportal administration of either 400 microl of glycerol or saline. Rats were killed after 1, 2, 3, 5, and 7 days. Liver weight/body weight ratio and BrdU uptake were measured. RESULTS: In all cultures tested, glycerol suppressed the growth of cells in a dose-dependent manner. In vivo, a single intraportal dose of glycerol slowed the liver regenerative response. CONCLUSIONS: This study demonstrated that glycerol has a potent growth-inhibitory effect on hepatocyte proliferation in vivo and in vitro. Remarkably, glycerol inhibited the proliferation of liver cancer cells as well. The results of this study have important clinical implications.     

Whole cell and nuclear androgen uptake in skin fibroblasts from infertile men

In order to reexamine the hypothesis that a high percentage of infertile men with oligo/azoospermia have androgen resistance due to androgen receptor abnormalities, both whole cell and nuclear uptake of [3H]R1881 (a synthetic, nonmetabolizable androgen) were measured in intact, dispersed fibroblasts cultured from pubic skin biopsy specimens of 15 men selected because of infertility associated with varying degrees of oligozoospermia. Eight men had sperm densities less than or equal to 2 X 10(6)/ml; 7 were greater than 2 X 10(6)/ml. Serum levels of FSH and LH were elevated in the severely oligo/azoospermic group, but normal in the other infertile men; concentrations of testosterone, estradiol, and prolactin were normal in both groups. The controls were six normal, age-matched, fertile males. There was no difference in binding capacity or dissociation constant for androgen uptake either into whole cells (3940 +/- 940 [mean +/- SE] sites/cell vs. 4700 +/- 1120 sites/cell, P = NS) or into nuclei (1360 +/- 340 sites/cell vs. 1460 +/- 340 sites/cell, P = NS) of the fibroblasts from the patients vs. the controls, respectively. Furthermore, there was no correlation between patient sperm densities and fibroblast whole cell or nuclear uptake binding capacities. Finally, there was no difference in any androgen binding parameter when only the fibroblasts from the men with severe oligozoospermia or azoospermia were compared with the controls. The authors conclude that the infertility of men with severe testicular germ cell depletion cannot be accounted for by a quantitative androgen receptor abnormality in their pubic skin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoprotein glomerulopathy: glomerular lipoprotein thrombi in a patient with hyperlipoproteinemia

An unusual nephropathy presumably induced by abnormal lipid metabolism is described in a 57-year-old woman who presented with proteinuria and edema. Histology at renal biopsy was characterized by marked dilatation of capillary lumina. Sudan staining and electron microscopy demonstrated lipid droplets occupying the capillary lumina. The patient had no particular clinical symptoms of lipidosis, but hyperlipoproteinemia similar to type III was suggested by lipid profiles. The nephropathy is thought to be an inherited disorder because proteinuria was detected in her sisters and similar renal histology and lipid profile were observed in one of her sisters. This is believed to be the first detailed report of glomerular lipoprotein thrombi.     

Liposome uptake by melanoma: in vitro comparison with hepatocytes

Liposomes are artificially generated vesicles entrapping aqueous solutions within lipid bilayer membranes. A major potential use of liposomes is for the delivery of antineoplastic agents to malignant tissue. However, liposome-cell interactions with both normal and neoplastic cells must be characterized in vitro to identify neoplastic tissues for which this approach may be most applicable in vivo. In this study, mouse melanoma-liposome interactions were examined in vitro. Small, unilamellar vesicle liposomes of three different phospholipid-cholesterol compositions were synthesized incorporating an aqueous phase fluorescent marker. Mouse melanoma had a significantly greater affinity for phosphotidylcholine-cholesterol liposomes than did mouse hepatocytes, as determined by comparing quantities of free intracellular 6-carboxyfluorescein in cells after a 15-min incubation with each of the three different liposome preparations (P less than 0.001). In addition, the efficiency of liposome internalization, calculated as the percentage of total cell-associated 6-carboxyfluorescein present as free, intracellular 6-carboxyfluorescein, was significantly greater for melanoma than for hepatocytes with all three liposome preparations (P less than 0.05). Therefore, in vitro liposome uptake by melanoma depends on lipid characteristics of the liposome preparation. Because melanoma uptake of liposomes appears to be a very efficient process in vitro, liposomes may be a useful vesicle for delivery of antineoplastic agents to melanoma in vivo.     

Receptor binding and tyrosine kinase activation by insulin analogues with extreme affinities studied in human hepatoma HepG2 cells.

The insulin-receptor affinity of five human insulin analogues with one to four amino acid substitutions was measured with human hepatoma cells (HepG2). The binding affinities ranged from 0.05% for AspB25 insulin, 18% for AspB9, GluB27 insulin, 80% for AspB28 insulin, and 327% for AspB10 insulin to 687% for HisA8, HisB4, GluB10, HisB27 insulin relative to human insulin. Binding constants obtained by competition experiments at steady state with [125I]TyrA14-labeled insulin and unlabeled analogues and by kinetic studies with [125I]TyrA14-labeled analogues and insulin gave essentially the same values. The kinetic studies showed that differences in affinity between analogues were due to differences in both dissociation and association rate constants. The affinity for insulinlike growth factor I receptor was low, ranging from less than 0.005% for AspB25 insulin to 0.6% for HisA8, HisB4, GluB10, HisB27 insulin. The potencies of insulin analogues in activation of the tyrosine kinase of solubilized and partially purified insulin receptors from HepG2 cells, measured with the exogenous substrate poly(Glu80-Tyr20), ranked in the same order as the binding affinities, the actual values being somewhat elevated for the high-affinity analogues, however. We conclude that these human insulin analogues are active in insulin-receptor binding and tyrosine kinase stimulation but show wide variation in affinity.     

Effect of estradiol on low density lipoprotein uptake by bovine aortic endothelial cells

The means by which estrogen retards atherosclerosis cannot be explained solely by changes in circulating lipoprotein levels. We studied the effects of 17 beta-estradiol on the binding, incorporation, and degradation of low density lipoprotein (LDL) by cultured bovine aortic endothelial cells (BAEC). Estrogen receptors in the cytoplasm and nucleus of BAEC could be demonstrated by immunofluorescent staining. Estradiol was found not to affect surface binding of LDL to BAEC. However, at physiologic concentrations (50 pg/ml), estradiol did enhance LDL uptake by the BAEC (P less than 0.005). This enhancement was present but somewhat reduced at higher concentrations of estrogen (P less than 0.05). Only approximately 10% of incorporated LDL was trichloroacetic acid soluble, indicating a low rate of LDL degradation. The relative rate of LDL breakdown within the BAEC was not altered by estrogen. These results, showing estrogen stimulation of LDL uptake by the BAEC, do not clarify the protective effect of this hormone. It is speculated that estrogen may augment the cellular clearance of LDL.     

Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells

To determine effects of metabolic abnormalities associated with diabetes mellitus on proliferation of diploid human cells, cultured human skin fibroblasts and arterial smooth-muscle cells were grown in media containing added glucose in the range often seen in diabetic subjects (10 to 30 mM, 180 to 550 mg./dl.). "High" glucose media enhanced proliferation of fibroblasts, with an "optimal" response at about 18 mM (325 mg./dl.). Equimolar sorbitol gave similar results, with the greatest increase in proliferation occurring at about the same concentration as for glucose (1 mM). Since neither equimolar mannitol nor sucrose produced such effects consistently, these results cannot be explained solely on the basis of hyperosmolarity. In contrast, arterial smooth-muscle cells failed to show a consistent growth response in the presence of either added glucose or sorbitol. These results suggest that studies with cultured human cells may be useful in assessment of responses to components of the disordered metabolic milieu of diabetes. Such studies of arterial smooth-muscle cells should also be useful for investigation of the mechanism of atherosclerosis in diabetes.     

Modulation of androgen receptor protein by culture conditions of human skin fibroblasts

Cultures of skin fibroblasts show variation of androgen binding with culture conditions; binding variations are usually avoided by using confluent cultures. In this work, we analysed the effect of cell density and mitogenic agents on the level of androgen receptor (AR) of cultured human skin fibroblasts. Results demonstrated that in cultures of human skin fibroblasts, cellular binding of dihydrotestosterone was higher in cells grown at low than at high cell density. The reduction in binding resulted from a decrease in the number of high affinity receptors and not from a change in receptor affinity. Immunocytochemistry for AR showed greater staining intensity in cells grown at low than at high cell density. Additionally, immunoblot analysis demonstrated more AR protein in low cell density cultures. On the other hand, it was observed that cells grown at low cell density showed diminished androgen binding capacity after 24 h of treatment with insulin-like growth factor (IGF-l), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or granulocyte-colony stimulating factor (G-CSF); this effect of growth factors was not observed in cells grown at high cell density. In conclusion, we found that cell density of cultures and mitogenic agents can regulate AR binding activity in human fibroblasts. While we do not yet know how changes in cell density affect the amount of AR, we conclude that the mechanism could be mediated by activation of the tyrosine kinase pathway, as the effect was reproduced by mitogens.     

损伤相关模式分子对人肝癌HepG2细胞增殖能力的影响

目的 观察在DAMPs诱导下人肝癌HepG2细胞增殖能力的变化.方法 将HepG2细胞分为对照组和实验组（10、20、40、80 μl DAMPs处理的HepG2细胞）;MTT比色法检测HepG2细胞的增殖能力;实时荧光定量PCR法检测IL-6 mRNA表达的变化;Western Blot法检测HepG2细胞IL-6蛋白的表达情况.结果 HepG2细胞随着DAMPs剂量的递增及时间的延长增殖能力逐渐增强,呈现明显的量效-时效关系,在剂量40 μl,作用时间36 h时细胞增殖能力达到最强,差异有统计学意义（P〈0.01）;选取作用时间为36 h,随着DAMPs剂量的递增,实时定量PCR法检测到HepG2细胞IL-6 mRNA分别为95.55±4.47,171.80±6.60,453.30±14.47,610.59±12.70,441.04±18.91,差异有统计学意义（P〈0.01）;Western Blot法检测HepG2细胞IL-6蛋白的表达分别为1.47、2.07、2.74、3.44、3.00,差异有统计学意义（P〈0.01）.结论 DAMPs在一定剂量及时间内促进人肝癌HepG2细胞增殖,并且呈现明显的量效-时效关系.