| IntroductionPeritoneal Dialysis (PD) is an important renal replacement therapy for end - stage renal failure. The longevity of PD depends on the long - term preservation of the Peritoneal Mesothelial Cells ( PMCs ) as a dialytic membrane, As the survival rate of the patients in PD has increased continuously, the remote complications such as the failure of ultrafiltration , peritoneal fibrosis have been emphasized gradully. The patients 'peritonum in long - term PD is continuously exposed to un-physiologic peritoneal dialysate because of its low pH, hyperosmolality and high glucose content. The unphysiologic dialysate resulted in significant morphological changes: these include increase of mesothelial cells volume, loss of microvilli, denudation of PMCs, increased accumulation of extracellular matrix (ECM) under the mesothelial cells. Thicken and diabetic form reduplication of basement membrane of mesothelium and stroma blood vessels of peritoneum. A great deal fibrous tissue appear , form a region with no cells and at last result in peritoneal fibrosis. The changes in the structure of peritoneum may influence its dialysis efficiency, peritoneum function changed markedly: increased permeability, increased glucose absorption, decreased ultra-filtration ( UF). Many studies indicate high concentration glucose ( Glc) may severly damage the structure of mesothelial cells, which is considered to be the primary cause of the decline in UF, dialysis effi-cacy and peritoneal fibrosis. PMCs can produce ECM which play an important role constituting basement membrane and preserving its completion. Histromorphological examination in peritoneal fibrosis indicates the increase of ECM. Therefore it is necessary to study the affect of high glucose on ECM of PMCs and provide a new method to reduce peritoneal fibrosis. The primary culture of PMCs provides the cell model for the research.The accomplished animal experiment showed that Protein Kinase C (PKC) induced increased ECM in primary cultural rat mesangial cells and increased net Glc utilization. This plays an important role in the progression of diabetic nephropathy. During PD, the PMCs are exposed to dialysates containing high concentration of glucose. Does this kind of exposure stimulate the increase of ECM in PMCs?The present study investigated the effect of high glucose concentration and phorbal esters on ECM in primary cultured PMCs and the association between HKs and glucose uptake, which might provide the theories basis for how to decrease ECM and peritoneal fibrosis.Materials1. Experimental animals: Albino male rats of the Sprague Dawley (SD) strain were used.2. Experimental cells: Rat peritoneal mesothelial cells were isolated by primary culture.3. Reagents relating to cell culture and identification.4. Radioimmunoassay kits relating to mesurement of ECM (LN, coll IV)Methods1. Enzymatic disaggregation used for primary culture2. Immunofluorescent staining used for identification3. Observing the regulating effect of each treatment factor4. Radioimmunoassay used for measurement of ECM ( LN, coll IV) in the culture mediumResults1. Glucose induced the increase of ECM (LN, collⅣ) in a dose - dependent manner.1. 1 At concentration of 2. 5 % and 4.25 % Glc for 24 h, the increase in LN were 2.7 and 7.3 times as much as the control group ( P < 0.0005).1. 2 At concentration of 1. 5% , 2. 5 % and 4.25 % Glc for 24 h the increase in coll Ⅳ were 10.7, 14.8 times (P < 0.0005) and 29 (P <0.0001 ) times as much as the control group.2. With the time dijfferent concentration Glc stimulate the increase of the synthesis of LN, collⅣ.3. At concentration of 1μmol/L PMA for 24 h, LN was 7.9 times ( P <0.0005 ) , collⅣ was 32 times ( P < 0.0001 ) as much as the control group.4. Go6976 decreased the synthesis of LN, coll Ⅳ stimulated by PMA. After incubated at concentration of 20μmol/L, Go6976 for 30 min, PMCs were cultured at concentration of 1 μmol/L PMA for 24 h, LN, coll Ⅳ didnt increase obviously comparing to th...
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