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ABSTRACT:Objective To assess the effect of prolonged laparoscopic surgery on peritoneal mesothelial cells and fibrinolysis in humans.Methods We examined prospectively 1 6 consecutive patients who underwent laparoscopic surgery (LAP)and 2 1 patients who underwent conventional open surgery (OP)for high-medium rectal cancer with curative intent.During the procedure,biopsy of the parietal peritoneum was made before operation and at 45 min,90 min,and 120 min after operation.The tissue-type plasminogen activator (tPA)and plasminogen activator inhibitor-1 (PAI-1 )were determined by enzyme-linked immunosorbent assay in peritoneal tissues.The cellular injury was detected by LDH assay.The proliferation was quantified by MTT assay.Results PAI-1 activity in the peritoneal tissue was significantly lower in LAP group than in the OP group.tPA activity decreased after 45min of open surgery,but there was no significant change in the LAP group.With time extension,the LDH activity increased and the proliferation of the mesothelial cells decreased.Conclusion Preservation of a prolonged hypofibrinolytic state by inhibition of PAI-1 up-regulation during LAP may predispose patients to less postoperative peritoneal adhesion. The cellular injury becomes apparent and the proliferation is inhibited during prolonged laparoscopic surgery.
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BACKGROUND: Peritoneal fibrosis is one of the major causes of technical failure in patients on peritoneal dialysis. Epithelial-to-mesenchymal transition (EMT) of the peritoneum is an early and reversible mechanism of peritoneal fibrosis. Human peritoneal mesothelial cells (HPMCs) have their own renin-angiotensin-aldosterone system (RAAS), however, it has not been investigated whether aldosterone, an end-product of the RAAS, induces EMT in HPMCs, and which mechanisms are responsible for aldosterone-induced EMT. METHODS: EMT of HPMCs was evaluated by comparing the expression of epithelial cell marker, E-cadherin, and mesenchymal cell marker, alpha-smooth muscle actin after stimulation with aldosterone (1-100nM) or spironolactone. Activation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS) were assessed by western blotting and 2',7'-dichlorofluororescein diacetate staining, respectively. The effects of MAPK inhibitors or antioxidants (N-acetyl cysteine, apocynin, and rotenone) on aldosterone-induced EMT were evaluated. RESULTS: Aldosterone induced EMT in cultured HPMCs, and spironolactone blocked aldosterone-induced EMT. Aldosterone induced activation of both ERK1/2 and p38 MAPK from 1 hour. Either PD98059, an inhibitor of ERK1/2, or SB20358, an inhibitor of p38 MAPK, attenuated aldosterone-induced EMT. Aldosterone induced ROS in HPMCs from 5 minutes, and antioxidant treatment ameliorated aldosterone-induced EMT. N-acetyl cysteine and apocynin alleviated activation of ERK and p38 MAPK. CONCLUSION: Aldosterone induced EMT in HPMCs by acting through the mineralocorticoid receptor. Aldosterone-induced generation of ROS followed by activation of ERK, and p38 MAPK served as one of the mechanisms of aldosterone-induced EMT of HPMCs.
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Humans , Actins , Aldosterone , Antioxidants , Blotting, Western , Cadherins , Cysteine , Epithelial Cells , p38 Mitogen-Activated Protein Kinases , Peritoneal Dialysis , Peritoneal Fibrosis , Peritoneum , Phosphotransferases , Protein Kinases , Reactive Oxygen Species , Receptors, Mineralocorticoid , Renin-Angiotensin System , SpironolactoneABSTRACT
ObjectiveTo explore the clinical application of pleural mesothelial cell (PMC) determination in the diagnosis of pleural effusion.MethodsPMC of 36 patients with malignant pleural effusion (malignant group) and 24 patients with benign pleural effusion (benign group) were counted to determine the clinical value of PMC in benign and malignant pleural effusion.ResultsIn malignant group,there was no case with PMC disappear,1 case(2.78％) with the percentage of PMC between 0 and 1％,1case(2.78％) between 1％ and 5％,34 cases (94.44％) more than 5％.In benign group,there were 11 cases (45.83％) with PMC disappear,8 cases (33.33％) with the percentage of PMC between 0 and 1％,2 cases (8.33％) between 1％ and 5％,3 cases( 12.50％ ) more than 5％.The percentage of PMC between two groups had significant difference (x2 =43.4069,P ＜ 0.01).95％ confidence intervals of PMC in benign and malignant group respectively were 0.28％ -5.10％ and 5.13％ -10.91％,which did not overlap.The percentage of PMC more than 5％ was defined as the diagnosis standard of identifying benign and malignant pleural effusion,the sensitivity of the diagnosis was 94.44％(34/36),the specificity was 87.50％(21/24) and the accuracy was 91.67％(55/60).ConclusionsPMC determination is a kind of easy,economical and fast way to diagnose benignand malignant pleural effusion with high sensitivity,specificity and accuracy.Therefore,it has reference value in clinical diagnosis.
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Background: The cytological diagnoses of serous effusions are usually made by routine cytomorphology with certainty. However, overlapping cases sometimes exist between reactive mesothelial and adenocarcinoma cells. We tried to evaluate the diagnostic utility of HBME-1 in distinguishing between reactive mesothelial cells and adenocarcinoma in serous effusions. Materials and Methods: Fifty-two cytologic specimens processed by cell-block technique were retrieved from the archive and were assigned to two groups: Group I: 26 effusions containing reactive/benign mesothelial cells; Group II: 26 effusions containing carcinoma cells. Immunostaining with HBME-1 was performed using an Envision technique. The staining intensity of cells, according to proportion of immunoreactive cells, was scored as: 0 (negative), 1+ (<10%), 2+ (10-50%), and 3+ (≥50%); and the predominant staining pattern of positive cells were determined. Statistical analysis and tests of significance were performed using SPSS software. Results: The calculated mean values (in percentile) for stained cells in adenocarcinoma and reactive mesothelial cells were 25.57 and 67.88, respectively ( P = 0.001). Thin membranous, thick membranous, thick and thin membranous, cytoplasmic and combined patterns of staining in adenocarcinoma cells were respectively 4 cases (21.1%), 0 case, 0 case, 8 cases (42.1%), and 7 cases (36.8%), and in reactive mesothelial cells, these were 7 cases (26.9%), 1 case (3.8%), 18 cases (69.2%), 0 case, and 0 case, respectively. The intensity of staining in majority (88.5%) of reactive mesothelial cells was scored 3+, but the distribution varied in the other group. Conclusions: The staining pattern and intensity for HBME-1 is a useful panel for differentiation of adenocarcinoma and mesothelial cells.
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Objective To investigate the role of oxidative stress in the epithelial-to-mesenchymal transdifferentiation (EMT) of peritoneal mesothelial cells in rat model of peritoneal fibrosis and the effect of probucol on peritoneal fibrosis. Methods The rat model of peritoneal fibrosis was induced by 4.25% high glucose peritoneal dialysis fluid (PDF). The rats were randomly divided into 4 groups:the control group, the saline group, the peritoneal fibrosis group, and the probucol group. A 4 hour peritoneal equilibration test (PET) was performed 4 weeks later. The peritoneal function and net ultrafiltration (UF) volume were determined. The level of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in peritoneal tissue were examined. The histology of peritoneal membrane was evaluated by light microscopy. E-cadherin and α-smooth muscle actin (α-SMA) protein expression was evaluated by immunohistochemical method and Western blot.Results The mesothelial cells were detached from peritoneal membrane in peritoneal firbosis rats. Comparing with the control rats, the thickness of visceral peritoneum, the level of MDA, and the-SMA protein expression were increased while the net ultrafiltration volume, the level of GSH-Px and E-cadherin protein expression were decreased in peritoneal firbosis rats. All these changes were reversed in the rats treated with probucol.Conclusion Oxidative stress plays an important role in transdifferentiation of peritoneal mesothelial cell in the peritoneal fibrosis rats. Probucol can improve structure and function of peritoneum, and partially reverse the EMT by reducing the oxidative stress.
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Objective To investigate the role of mitochondrial respiratory chain in the hyperpermeability of human peritoneal mesothelial cells (HPMCs) induced by high glucose peritoneal glucose PDS was also added. Transmesothelial electrical resistance (TER) measurement was examined for detection of permeability damage in HPMCs. Immunostaining and Western blotting analysis were used to detect claudin-1 expression. Mitochondrial superoxide (MitoSOX) Red staining and respiratory chain complexes activities were determined for detection of mitochondrial reactive oxygen species (ROS) production and mitochondrial complexes activities. Results TER was decreased in a time- and concentration-dependent manner after culture with high glucose PDS for was also down-regulated significantly by high glucose PDS (P＜0.01). Complex Ⅲ activity was inhibited (10.8% of control, P＜0.01) accompanied with increased mitochondrial ROS generation.These changes were partially prevented by glutathione. Conclusion Mitochondrial respiratory complex Ⅲ pathway has crucial importance in maintaining TER of HPMCs, which may reveal a valuable target for novel therapies to fight hyperpermeability of peritoneum during the prolonged PD treatment.
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Objective To observe the effect of ulinastatin on the expression of interleukin 15 (IL-15), connective tissue growth factor (CTGF) and malondialdehyde (MDA) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose. Methods RPMCs were isolated, cultured and passaged by trypsin, then identified. The third generation of cultured RPMCs were used in the experiment. RPMCs were divided into normal control group, high glucose (1.5%, 2.5%, 4.25%) for 6 hours and 12 hours, high glucose (2.5%) for 3, 6, 12, 24 hours or ulinastatin (160, 320, 640U/ml) for 12 hours. IL-15 mRNA was detected by real-time PCR. IL-15 and CTGF protein in supernatants was detected by ELISA. MDA protein was detected by TBAS. Results Compared with the control group, the expression of IL-15, CTGF and MDA was significantly increased in the groups stimulated by high glucose (P＜0.05) in dose- and time-dependent manner. Ulinastatin could significantly decrease the expression of IL-15, CTGF and MDA induced by high glucose in dosedependent manner both in protein and gene levels (P＜0.05). Conclusions High glucose can up-regulate the expression of IL-15, CTGF and MDA in RPMCs. Ulinastatin can reverse these changes.
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Objective To explore the effect of transforming growth factor β1 (TGF-β1) on epithelial-mesenchymal transition in rat peritoneal mesothelial cells(RPMCs) and its mechanism.Methods Primary peritoneal mesothelial cells of SP rats were cultured in vitro. After synchronization for 24 h, RPMCs were randomly divided into 2 groups: Group A (control), Group B (TGF-β1, 10 μg/L). RPMCs were stimulated by 10 μg/L TGF-β1 for different time. The mRNA and protein expression levels of E-cadherin, α-smooth muscle actin (α-SMA) and collagenⅠwere measured by RT-PCR and Western blot, respectively. The protein expression level of total RhoA was measured by Western blot. Active RhoA was extracted by Plasma Membrane Protein Extraction Kit, and assessed by Western blot. Results TGF-β1 down-regulated mRNA and protein expression of E-cadherin in RPMCs, and upregulated mRNA and protein expression of α-SMA and CollagenⅠ. TGF-β1 stimulation elicited a robust increase in RhoA activity in a time-dependent manner. RhoA activity peaked at 1 h.Conclusion RPMCs can be transdifferentiated into myofibroblast under the effect of TGF-(β1,)and the mechanism may be related to the activation of RhoA associated signal pathway.
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This study examined the mechanism by which the gastric cancer cells lead to early peritoneal metastasis.HMrSV5 cells,a human peritoneal mesothelial cell line,were co-incubated with the supernatants of gastric cancer cells.Morphological changes of HMrSV5 cells were observed.The cell damage was quantitatively determined by MTT assay.The apoptosis of HMrSV5 cells was observed under transmission electron microscope.Acridine orange/ethidium bromidestained condensed nuclei was detected by fluorescent microscopy and flow cytometry.The expressions of Bcl-2 and Bax was immunochemically evaluated.The results showed that conspicuous morphological changes of apoptosis were observed in HMrSV5 cells 24 h after treatment with the supematants of gastric cancer cells.The supernatants could induce apoptosis of HMrSV5 cells in a time-dependent manner.Th esupematants could up-regulate the expression of Bax and suppress that of Bcl-2 in HMrSV5 cells.These findings demonstrated that gastric cancer cells can induce the apoptosis of HPMCs through supernatants in the early peritoneal metastasis.The abnormal expressions of Bcl-2 and Bax may contribute to the apoptosis.Anti-apoptosis drugs promise to be adjuvant chemotherapeutic agents in the treatment of peritoneal metastasis of gastric cancer.
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Cytologic examination of the body cavity fluid is very important because the specimens represent a significant percentage of nongynecologic samples and this cytologic examination may be the first, best or only chance for making the diagnosis of an underlying malignancy. The purposes of body cavity fluid examination are to correctly identify cancer cells and if possible, to identify the tumor types and primary sites when presented with unknown primary tumor sites. The most important basic differential diagnosis is that of benign and reactive disease vs malignant disease. Reactive mesothelial cells are a consistent population in body cavity fluid, and these are the most versatile cells in the body. Due to the specific environment of the body cavity, the exfoliated reactive mesothelial cells may show significant morphologic overlap with the morphology of cancer cells. With a focus on the differential points between reactive mesothelial cells and metastatic adenocarcinoma cells, the practical diagnostic approaches, the diagnostic clues and the pitfalls to achieve a correct diagnosis are presented in this review.
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Adenocarcinoma , Diagnosis, Differential , Neoplasms, Unknown PrimaryABSTRACT
Objective To investigate the expression of CD40 and intercellular adhesion molecule-1 (ICAM-1) treated with lipopolysaccharide (LPS) in rat peritoneal mesothelial cells(RPMC) and the role of NF-κB signal transduction pathway. Methods RPMCs were harvested from Sprague-Dawley rat peritoneal cavity and maintained under defined in vitro conditions. The cells were exposed respectively to different concentrations of LPS for 12 h or treated with LPS (5 μg/ml) for different time points. To observe the effect of LPS on the expression of CD40 and ICAM-1, the RPMCs were treated with LPS (5 μg/ml) for different time points. To observe the effect of LPS on the expression of NF-κB and p-NF-κB protein, the RPMCs were treated by LPS or pretreated with BAY11-7085 (5 μmol/L or 1 μmol/L ) for 3 h, then treated with LPS for another 3 h, respectively. Expression of CD40 and ICAM-1 mRNA was examined by RT-PCR. Expression of NF-κB and p-NF-κB protein was detected by Western blot. Results Compared with medium control group, stimulation of RPMCs with 1 μg/ml and 5 μg/ml of LPS resulted in a significant increase in the expression of CD40 and ICAM-1 mRNA(P<0.05). 10 μg/ml of LPS had strongest effect on CD40 and ICAM-1 expression compared with that of 1 μg/ml and 5 μg/ml of LPS. Treatment with 5 μg/ml of LPS resulted in time-dependent increase in the gene level of CD40 and ICAM-1, with the peak at 3 h. However, after that time point, the gene level of them was gradually attenuated. Following treatment with LPS (5 μg/ml), the level of p-NF-κB began to increase at 15 min, gradually reached the peak at 1 h, and then decreased. But the level of p-NF-κB at 2 h was still significantly higher than that of medium control. 5 μmol/L of BAY11-7085 decreased significantly the up-regulation of CD40 and ICAM-1 induced by LPS. Conclusion LPS enhanced the expression of CD40 and ICAM-1 on RPMCs in a concentration-dependent and a time-dependent manner. LPS induced expression of CD40 and ICAM-1 depend on the NF-κB signal transduction pathway.
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Objective To investigate the effects of connective tissue growth factor (CTGF) siRNA delivered by pRetro-Super (PRS) retrovirus vector on extracellular matrix and VEGF expression in human peritoneal mesothelial cells (HPMC). Methods Four pairs of oligonucleotides including 64 bp DNA were designed and synthesized in vitro according to siRNA target sequence and PRS retrovirus desire.PRS-CTGF-siRNA1-4 recombinant retrovirus vectors were constructed.The recombinant retrovirus vectors containing CTGF-siRNA were transferred into PT67 packaging cell lines with lipefectamine 2000,then infected HPMC.mRNA expression was determined by semi-quantitative RT-PCR and protein expression was determined by Western blot.Results Both mRNA and protein expressions of CTGF,FN,Col I,laminin (LN) and VEGF were significantly increased in HPMC with 5 μg/L TGF-β1 stimulation (P＜0.01,respectively).CTGF,FN,Col I,LN mRNA and protein and VEGF mRNA expression stimulated by TGF-β1 were significantly decreased in HPMC infected with PRS-CTGF-siRNA1～4 retrovirus vectors (P＜0.01,respectively).The inhibitory rates on CTGF were 69.3%,22.2%,27.4% and 38.8%,respectively (P＜0.01).At the same time,there was also a significant reduction of VEGF protein expression in HPMC infected with PRS-CTGF-siRNA1 vector (P＜0.01).There was no significant difference in HPMC infected with PRS void vector. Conclusion CTGF siRNA delivered by PRS retrovirus vector can effectively inhibit the enhancement of extracellular matrix and VEGF expression stimulated by TGF-β1 in HPMC.
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BACKGROUND: Peritoneal mesothelial cells are the most important intraperitoneal cells quantitatively and have the capability to secret different types of substances. It may therefore be essential to have information on the mesothelial cell mass during peritoneal dialysis. Cancer Antigen 125 (CA125) is a 22KDa glycoprotein which is a clinically useful tumor marker of non-mucinous epithelial ovarian carcinoma. Recently, other cells including pleural and peritoneal mesothelial cell have been proved to express CA125. This study was undertaken to determine whether CA125 can be used as a marker of mesothelial cell mass in clinically stable 39 CAPD patients. METHODS: We checked serum and peritoneal dialysate CA125 level, D/P creatinine and D/Do glucose after 4 hours dwell in 39 stable continuous ambulatory CAPD patients. RESULTS: No statistically significant correlation was seen among the patient's age, sex, serum and dialysate levels of CA125. The dialysate CA125 levels correlated with the duration of CAPD, negatively (r=-0.345, p=0.039) and a significant positive correlation was seen between the duration of CAPD and D/Do glucose at 4 hours (r=0.523, p=0.001). But there were not a correlation between the dialysate CA125 levels and D/P creatinine after 4 hours dwell nor between the dialysate CA125 levels and D/Do glucose after 4 hours dwell. CONCLUSION: Although the duration of CAPD affects CA125 levels in dialysate, no specific alteration in peritoneal membrance transport properties can be detected or predicted by changes in dialysate concentration of CA125. However longitudinal follow-up of changes in concentration of dialysate CA125 may be useful in evaluating mesothelial cell mass in stable CAPD patients.Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea.
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Humans , Creatinine , Follow-Up Studies , Glucose , Glycoproteins , Internal Medicine , Korea , Peritoneal Dialysis , Peritoneal Dialysis, Continuous Ambulatory , Schools, MedicalABSTRACT
Objective:To investigate the effects of Astragalus on the the apoptosis of cultured human peritoneal mesothelium induced by peritoneal dialysis solution(PDS). Methods:Using RPM1640 culture medium as control,PDS group(commercial PDS containing 1.5% glucose without Astragalus),Astragalus group 1(commercial PDS containing 1.5% glucose and 1% Astragalus) and Astragalus group 2(commercial PDS containing 1.5% glucose and 2% Astragalus) were tested to explore the apoptosis of cultured human peritoneal mesothelial cell in each group.The activity of caspase-3,the terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling(TUNEL) and flow cytometry analysis were used in apoptosis analysis in this study. Results: The activity of caspase-3 in the control group was defined as 1,the relative activity of caspase-3 in PDS group、 Astragalus group 1 and Astragalus group 2 were 3.26?0.91,(1.87?)0.43 and 1.67?0.32 respectively.The activity of caspase-3 in PDS group was significantly higher than those in the Astragalus group 1 and Astragalus group 2(P
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Objective: To investigate the effect of Astragalus mongholicus injection(AM) on the secretion and mRNA expression of transforming growth factor((TGF-?1)) and basic fibroblast growth factor(bFGF) in cultured human peritoneal mesothelial cells((HPMC).) Methods: HPMC got from patients underwent surgical operation were cultured in vitro.At first,cells from the third passage were incubated with RPMI1640 culture medium containing 0.1% FBS for 24 hours.Then,they were divided into control group,PDS group,AM group 1,AM group 2 and AM group 3 for testing.Each group was supplemented with equal volum of RPMI1640 culture medium containing 20% FBS.After 24 hour incubating,mitochondrial dehydrogenases activity(MTT assay),the levels of TGF-?1 and bFGF in supernatant of the cell culture and the mRNA expression of TGF-?1 and bFGF were detected.Results: Significant decrease of mitochondrial dehydrogenase activities were observed in PDS group as compared with those in control group and AM groups(P0.05).The levels of TGF-?1 and bFGF in supernatant of the cell culture were significantly lower in control group than those in PDS group.Marked lower levels of TGF-?1 and bFGF were found in AM group 1,AM group 2 and AM group 3 as compared with those in PDS group(P0.05).The mRNA expressions of TGF-?1 and bFGF in PDS group increased significantly as compared with those in control group.And significant mRNA expressions of TGF-?1 and bFGF were found in PDS group when compared with those in AM groups(P
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BACKGROUND: Protein kinase C (PKC)s consist of three groups of isoenzyme; conventional, novel and atypical PKCs. Diacylglycerol (DAG) activates both conventional and novel PKCs, but not atypical PKCs. High glucose-induced fibronection production was shown to be mediated by activation of DAG-sensitive PKCs. In this study, we investigated whether PKC mediates IL-1beta-induced fibronectin mRNA expression, and the subtypes of PKC involved in the process. METHODS: Fibronectin mRNA level and phosphorylated PKC zeta/iota in total cell lysate were measured by Northern blot and Western blot, respectively. RESULTS: Pretreatment of HPMCs with calphostin C, a pan-PKC inhibitor, at doses of 500, 750 and 1, 000 nM caused dose-dependent inhibition of IL- 1beta (1 ng/mL)-induced fibronectin mRNA level. GF109203X, another pan-PKC inhibitor, at doses of 1, 5 and 10 microM also downregulated IL-1beta (1 ng/ mL)-induced fibronectin mRNA level in a dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA), an activator of conventional and novel PKCs, stimulated fibronectin mRNA level at doses of 1, 10 and 100 nM. After prolonged treatment of the cells for 72 hr with PMA, another dose of PMA did not increase fibronectin mRNA level, while IL-1beta (1 ng/mL) still stimulated it. Pretreatment of the cells with 5, 10, 15 and 20 microM of myristoylated PKC zeta/iota pseudosubstrate inhibited IL-1beta (1 ng/mL)-induced fibronectin mRNA level in a dose-dependent manner, while 20 microM of myristoylated PKC [19-27] pseudosubstrate, given as a control, had no effect. Stimulation of fibronectin mRNA level by IL-1beta (1 ng/mL) was completely prevented by 20 microM of my ristoylated PKC zeta/iotapseudosubstrate. IL-1beta (1 ng/ mL) increased phosphorylated PKC zeta/iota, an active form of the enzyme. CONCLUSION: IL-1beta-induced fibronectin production in HPMCs occurs by way of activation of atypical PKCs (PKC zeta/iota).
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Humans , Blotting, Northern , Blotting, Western , Fibronectins , Interleukin-1beta , Protein Kinase C , Protein Kinases , RNA, MessengerABSTRACT
BACKGROUND: In early phase of peritonitis, mononuclear cells as well as polymorphonuclear leukocytes migrate rapidly into peritoneal cavity. For the migration of mononuclear cells, the expression of VCAM-1 on peritoneal mesothelial cells is important. In this study, we investigated the effect of TGF-beta1 on tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta(IL-1beta) induced VCAM-1 expression in the cultured HPMCs. METHODS: HPMCs were cultured in the presence of TNF-alpha, IL-1beta and/or TGF-beta1. VCAM-1 mRNA level was measured by Northern blot. VCAM-1 in total cell lysate and VCAM-1 expressed on cell surface were measured by Western blot and cellular ELISA, respectively. RESULTS: Incubation of the cultured HPMCs with TNF-alpha (10 ng/mL) or IL-1beta (1 ng/mL) caused an increased level of VCAM-1 mRNA, VCAM-1 protein in total cell lysate, and VCAM-1 expressed on cell surface. This stimulatory effects of TNF-alpha or IL- 1beta were inhibited by TGF-beta1 (0.1, 1, 10 ng/mL), dose-dependently. The level of VCAM-1 mRNA, VCAM-1 protein in total cell lysate, and VCAM-1 expressed on cell surface in the unstimulated cells were also inhibited by TGF-beta1 (10 ng/mL). The rate of VCAM-1 mRNA degradation after an application of actinomycin D was not affected by TGF-beta1. CONCLUSION: TGF-beta1 inhibited inflammatory cytokine induced VCAM-1 production and expression in the cultured HPMCs. Treatment of the cells with TGF-beta1 seems to suppress TNF-alpha or IL-1beta induced VCAM-1 mRNA transcription rather than decrease stabilization of VCAM-1 mRNA.
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Humans , Blotting, Northern , Blotting, Western , Dactinomycin , Enzyme-Linked Immunosorbent Assay , Neutrophils , Peritoneal Cavity , Peritonitis , RNA Stability , RNA, Messenger , Transforming Growth Factor beta1 , Tumor Necrosis Factor-alpha , Vascular Cell Adhesion Molecule-1ABSTRACT
BACKGROUND: High glucose in peritoneal dialysis solution has been implicated in the pathogenesis of peritoneal fibrosis. Macrophages in peritoneal cavity seem to participate in the process of peritoneal fibrosis through the production of various cytokines and growth factors. Monocyte chemoattractant protein-1(MCP-1) plays a key role in the recruitment of monocytes toward the peritoneal cavity. Vascular cell adhesion molecule-1(VCAM-1) is assumed to be important in the transmigration of monocytes. MCP-1 and VCAM-1 can be induced by various cytokines and growth factors in human peritoneal mesothelial cells(HPMC). However, effect of high glucose on the expression of MCP-1 and VCAM-1 in HPMC has not been known well. METHODS: Cultured HPMC were conditioned with glucose(5-100mM) or mannitol for varying periods up to 7 days. Cell proliferation and mRNA expression of MCP-1 and VCAM-1 were assessed by MTT assay and Northern blot analysis respectively. MCP-1 protein was measured using ELISA. Chemotactic activity of high glucose-conditioned culture supernant were evaluated by chemotactic assay. Effect of protein tyrosine kinase(PTK) inhibitor on the high glucose-induced MCP-1 mRNA expression was examined. RESULTS: Glucose inhibited the cell proliferation in a time and dose dependent manner. Northern blot analysis showed that high glucose increased the MCP-1 mRNA expression in a time(2-7days) and dose(15-100mM) dependent manner, but not VCAM-1 mRNA expression. MCP-1 protein in cell culture supernant was also increased. Equivalent osmotic concentration of mannitol had no significant effect. High glucose-conditioned supernant had an increased chemotactic activity for monocyte, which was neutralized by specific anti-MCP-1 antibody. PTK inhibitors such as genistein and herbimycin A suppressed the high glucose-induced MCP-1 mRNA expression in a dose dependent manner. CONCLUSION: High glucose induced MCP-1 expression in HPMC partly via pathways involving PTK.
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Humans , Blotting, Northern , Cell Adhesion , Cell Culture Techniques , Cell Proliferation , Cytokines , Enzyme-Linked Immunosorbent Assay , Genistein , Glucose , Intercellular Signaling Peptides and Proteins , Macrophages , Mannitol , Monocytes , Peritoneal Cavity , Peritoneal Dialysis , Peritoneal Fibrosis , Protein-Tyrosine Kinases , RNA, Messenger , Tyrosine , Vascular Cell Adhesion Molecule-1ABSTRACT
Objective To study the ultrastrueture of vestibular membrane of the cochlea in guineapig. Methods The vestibular membrane in guineapig's cochlea was observed under scanning electron microscope. Results The mesothelium on the perilymphatic side was formed by a single thin layer which was intermittently discontinuous. Microvilli were few on the surface of mesothelial cells. Nuclei of the cells were large and oval in shape. A single layer of polygonal epithelial cells were observed on the endolymphatical side. The irregular polygonal epithelial cells were round and oval in shape. Many microvilli were found on this side. The junction between these cells was very tight. Conclusion The vestibular membrane is a barrier between the scala media and scala vestibule and might play a role in ion and fluid homeostasis
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Objective To enhance the diagnosis rate for metastasis adenocarcinoma cells in serous fluids and detect a panel of a monoclonal antibodies for distinguishing adenocarcinoma cells from reactive mesothelial cells in serous fluids. Methods Three marks of low molecular weight cytokeratin (CK LMW ), carcinoembryonic antigens (CEA) and mesothelial cells (MC) were used to immunostain the cells in serous fluids from 50 patients and in peritoneal washing from 14 patients. Results Expression rate of three groups of cells positive from CK LMW , CEA and MC in adenocarcinoma cells was 95.83%, 70.83%, 16.67%, in suspicious cancer cells 100%, 50.00%, negative, in reactive mesothelial cells 33.33%, 33.33%, 66.66%. The four suspicious cases and two reactive cases in primary cytopathological classifiecation should be reclassified as adenocarcinoma cases. The sensitivity, specificity and reliability of CK LMW , CEA and MC were 96.30%, 80.00%, 77.10%; 70.37%, 90 00%, 61 60%; 90.00%, 85.20%, 75.30%, respectively. Conclusion Immunocytochemistry, using a panel of CK LMW , CEA and MC antibodies appears to be adjunct of important value for distinguishing adenocarcinoma cells from reactive mesothelial cells in serous fluids.