Objective To observe the expression of p53, bcl-2 genes, vascular endothelial cell growth factor（VEGF）, basic fibroblast growth factor（bFGF）, insulin-like growth factor-I （IGF-I）, and the receptors of these factors of retinal vascular endothelial cells (VECs) of 1- to 20-week diabetic rats, and the relationship between the expressions and cell cycle arrest.Methods Retinal sections of diabetic rats induced by alloxan were immunohistochemically stained and observed by light microscopy (LM) and electron microscopy (EM). Dot blotting and Western blotting were used to determine the expression of mRNA, proteins of p53 and bcl-2. Results Under LM, immunohistochemical positive expression of p53 and bcl-2 were found on the vessels of ganglion cell layer and inner nuclear layer of retinae of 8- to 20-week diabetic rats; under EM, these substances were observed depositing in VECs. The retinal VECs also expressed VEGF, bFGF, IGF-I and their receptors. There was no positive expression of other cell types in these retinae, all cell types of retinae in control group, or all cells of retinae of diabetic rats with the course of disease of 1 to 6 weeks. The result of dot blotting revealed that retinal tissue of 20-week diabetic rat expressed p53 and bcl-2 mRNA, and the result of Western blotting revealed that they also expressed p53 and bcl-2 proteins. But retinal tissues of control group did not. Positive expression of bax was not found in the retinae in control group or 1- to 20-week diabetic rats. Conclusion p53, bcl-2 may introduce cell cycle arrest of VECs of retinae in 8- to 20-week diabetic rats. High glucose might stimulate the expression of VEGF, bFGF, IGF-I and their receptors, and the growth factors may keep VECs surviving by self-secretion. (Chin J Ocul Fundus Dis，2003，19：29-33)
Objective To observe the effects of δ-opioid receptor agonists D-Ala2-D-Leu5-enkephali （DADLE） on hepatocyte apoptosis and expressions of bcl-2 and caspase-3 in septic rat, and to investigate the possible mechanism by which DADLE protects the liver in sepsis. Methods Sepsis was reproduced in rats by cecum ligation and puncture (CLP). Fifty-four SD rats (either male or female) were randomly divided into CLP group (n=18), DADLE group (n=18) and sham operation (SO) group (n=18). The rats were respectively killed at different time (2 h, 4 h and 6 h after operation). Hepatocyte apoptosis was detected by TdT-mediated dUTP Nick End Labeling (TUNEL). The expressions of bcl-2 and caspase-3 protein were detected by immunohistochemistry. And the changes of pathology in hepatic tissue were detected by light microscope. Results The hepatic pathological lesion of rats in CLP group was obviously serious compared with SO group, while it was obviously improved in DADLE group. The apoptosis index of rat hepatocytes in CLP group significantly increased compared with SO group, and further it was prominent at 4 h （P＜0.01). The apoptosis index of rat hepatocytes at each time of DADLE group was significantly decreased compared with CLP group （P＜0.01）. Expression of caspase-3 protein in liver tissues of CLP group significantly increased compared with SO group （P＜0.01）, while the expression of bcl-2 protein significantly decreased （P＜0.05）. Expression of caspase-3 protein in liver tissues of DADLE group significantly decreased compared with the CLP group （P＜0.01）, while the expression of bcl-2 protein significantly increased （P＜0.05）. There was positive correlation between expression of caspase-3 in liver tissues and apoptosis index of hepatocyte (r=0.83, P＜0.01) and negative correlation between expression of bcl-2 in liver tissues and apoptosis index of hepatocyte (r=-0.65, P＜0.01). Conclusions The findings indicate that δ-opioid receptor agonists DADLE can obviously improve hepatic pathological changes of septic rats. And its protective mechanism contains down regulation of caspase-3 expression, upregulation of bcl-2 expression and thus the apoptosis of hepatocyte is repressed.
【Abstract】ObjectiveTo investigate the proliferation rate of HepG2 cell after multiple thermotherapy and the possible reasons related to it. MethodsAfter HepG2 cell were treaded by ten repeated cycles of heat exposure at 43 ℃ for 80 minutes twice a day, the doubling time of cell was analyzed， and the cell cycle, bcl2 mRNA and bax mRNA were detected. ResultsThe proliferation rate of HepG2 cell which treated with heat speeded up, the percentage of G2 and S in cell cycle increased, the expression of bcl2 mRNA strengthened and the rate of bcl2/bax increased. ConclusionThe speeded proliferation of HepG2 cell after multiple thermotherapy is related to its high percentage of DNA duplicated and dividing cell, strengthened expression of bcl2 mRNA and increased rate of bcl2/bax.
Objective To determine whether lymph node-targeted chemotherapy with carbon nanoparticles absorbing 5-FU affects expressions of bcl-2, bax and caspase-3 in gastric cancer tissues, metastatic lymph nodes and normal gastric mucosa. Methods Twenty-eight patients with gastric cancer in our department were divided into lymph node-targeted chemotherapy (LNTC) group and control group from October 2005 to August 2006. The patients were treated with carbon nanoparticles absorbing 5-FU before operation in LNTC group and those were operated directly in control group. The gastric cancer tissues, metastatic lymph nodes and normal gastric mucosa were collected after operation. The expressions of bcl-2, bax and caspase-3 in those tissues were determined by immunohistochemical technique. Results In LNTC group, the positive expression rate of bcl-2 in gastric cancer tissues and metastatic lymph nodes was significantly lower than those in control group (28.6% vs . 78.6% , 25.0% vs . 70.0% , P ＜ 0.05), the positive expression rate of bax (85.7% vs . 28.6% , 80.0% vs . 30.0% ) and caspase-3 (57.1% vs . 14.3% , 55.0% vs . 15.0% ) in gastric cancer tissues and metastatic lymph nodes was significantly higher than those in control group ( P ＜ 0.05). The positive expression rate of bcl-2, bax and caspase-3 in normal gastric mucosa was not significantly different between two groups ( P ＞ 0.05). Conclusion The lymph node-targeted chemotherapy with carbon nanoparticles absorbing 5-FU can down-regulate the expression of bcl-2 and up-regulate the expression of bax and caspase-3 in gastric cancer tissues and metastatic lymph nodes, and therefore by affecting the expression levels of these apoptosis molecules may be one of the ways to induce tumor cell apoptosis.
Objective The expression of CD15 antigen and oncoprotein bcl-2 in thyroid cancer were examined in order to study the correlation between them. Methods The expression of CD15 and bcl-2 in 50 thyroid cancers, 20 adjacent noncancerous portion, 45 adenoma and 10 normal thyroid tissue were respectively investigated by microwave-LSAB immunohistochemical technique. Results The positive rate of CD15 and bcl-2 in thyroid cancer was 68.0% and 46.0% respectively, which was significantly higher than that in adenoma or adjacent noncancerous (Ｐ＜0.05). The percentage of CD15 and bcl2 positive expression were found to be significantly correlated with the tumor metastasis (Ｐ＜0.05), but not correlated with histological feature. Expression of CD15 was significantly correlated with bcl-2.Conclusion Expression of CD15 and bcl-2 can be regarded as a parameter to evaluate tumor metastasis and prognosis of thyroid cancer.
Objective To probe into the roles of inositol 1, 4, 5-trisphosphate (IP3) and bcl-2 gene expression in inhabiting hepatocellular carcinoma of nude mice by quercetin. Methods Animals with hepatocellular carcinoma in quercetin group were treated with injection peritoneum of quercetin 50 mg/（kg·d ） for 3 weeks, while which in control group were treated with 0.4% DMSO of RPMI 1640 0.05 ml/（g·d）. Then the volume and the weight of tumors were measured, IP3, bcl-2 mRNA and bcl-2 protein were assayed by IP3-［3H］ Birtrak Assay, RT-PCR and Western blot respectively. Results The volume and weight of tumors in quercetin group were lower than those in control group 〔（15.8±10.1） mm3 vs. （52.3±26.5） mm3 in volume, （44.8±10.4） mg vs.（91.3±31.4） mg in weight， P＜0.01〕. Content of IP3 in quercetin group was lower than that in control group 〔（13.4±1.4） pmol/mg prot vs. （35.3±6.6） pmol/mg prot， P＜0.01〕. There was no significant difference in bcl-2 mRNA expression between quercetin group and control group 〔RI （the gray degree multiply area of bcl-2 /the gray degree multiply area of β-actin）： 0.55±0.05 vs. 0.79±0.19， P＞0.05〕， but the expression of bcl-2 protein in quercetin group was lower than that in control group (RI: 1.07±0.12 vs. 6.69±1.80, P＜0.01). Conclusion Quercetin can inhabit the growth of hepatocellular carcinoma tansplanted into liver of nude mice by reducing IP3 production and down-regulating bcl-2 gene expression.
Objective To investigate the effect of renal cell apoptosis induced by obstructive jaundice on the expression of bcl-2 in rats, and to explore the mechanism of renal impairment induced by obstructive jaundice. Methods Thirty-two male SD rats were randomly divided into 2 groups: SO group and BDL group. The rats in SO group received sham operation. Bile ducts of rats in BDL group were ligated. Pathology of kidneys was observed under the microscope. The levels of D-Bil, TBA, GOT, GPT, Cr and BUN in serum and β2-MG in urine were measured. The apoptotic rate of renal cells was calculated by flow cytometry and the forms of DNA fragmentation in renal cells were detected by agarose gel electrophoresis. The expression of inhibitory gene bcl-2 in the renal tissues was detected by immunohistochemistry. Results The color of urine in BDL group became dark yellow in day 2 after operation; The ears, tails and the muscle of abdominal wall and splanchnic organs, such as liver and kidney, also became yellow and swollen in day 7. The D-Bil, TBA, GOT, GPT, BUN of serum and β2 -MG of urine in BDL group were higher than those in SO group (P＜0.05, P＜0.01), and each value (except β2 -MG) in BDL group of 14 d was higher than that in BDL group of 7 d (P＜0.05, P＜0.01), respectively. The result of flow cytometry showed that the apoptotic rate of SO group and BDL (7 d and 14 d) group were (2.10±0.75)%， (18.17±0.86)% and (36.39±2.23)% respectively, there were significantly difference among them (P＜0.05). The expression rate of bcl-2 of renal cell in BDL group of 7 d was higher than that in BDL group of 14 d. Conclusion Obstructive jaundice could induce apoptosis of the renal cells, and activate the expression of bcl-2 of the renal tubular epithelial cells in feedback, which may regulate the process of apoptosis.
Objective To investigate the growth inhibition and the apoptosis inducement effects of polysaccharide of trametes robiniophila murr (PS-T) on human rectal cancer cell line HR8348 and elucidate the possible mechanisms. Methods After treatment with PS-T, the growth inhibition rate of human rectal cancer cell strain HR8348 was studied by MTT method. The apoptotic index was detected by methyl green and pyronine Y staining and by terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling (TUNEL). The bcl-2, bcl-xl, bax, bak and p53 gene expressions of HR8348 cells were examined by immunohistochemical method.Results PS-T induced a dosedependent inhibition of HR8348 cells in vitro. The maximum percentage of growth inhibition was 71.1% by 36 h after administration of PS-T at a concentration of 4.0 mg/ml. There was no significant difference in the inhibitory rate as compared to the positive control (5-FU 10 μg/ml, P gt;0.05). The typical apoptosis phenomenons were observed under the light microscope by both staining methods. The apoptotic index increased parallelling with the increase of concentration of PS-T. When the PS-T concentration was 4.0 mg/ml, the apoptotic index determined by methyl green and pyronine Y staining and by TUNEL method increased rapidly to 0.1620±0.0128 and 0.2612±0.0158, respectively, which was greater than that of the positive control (5-FU 10 μg/ml, Plt;0.05). The expression of bcl-2, bcl-xl, bak and p53 was increased in the PS-Ttreated HR8348 cells by 36 h, while the bax remained unchanged. Expression index of bak/bcl-2 and bak/bcl-xl was increased significantly as compared with the control (Plt;0.05). Conclusion PS-T can significantly inhibit growth and induce apoptosis of human rectal cancer cell strain HR8348 in vitro. The apoptosis induced by PS-T might be related to the increase of the ratio of bak to bcl-2 and to bcl-xl and upregulation of the expression of p53 gene.
Objective To investigate the molecular mechanism of apoptosis in cultured human retinal pigment epithelial (RPE) cells. Methods The growth media of confluency human RPE cells were replaced with a daunoblastinacontaining one at a dose of 180mu;g/L,and the cells were incubated for 12 hr at 37℃.After incubation with the drug,the medium was withdrawn,fresh medium was added and incubation was carried out for an additional 24 hr.Apoptosis was monitored by light microscopy,enzyme linked immunosorbent assay(ELISA)and terminal deoxynucleotidyl transferase mediated biotin-dUTP nick-end labelling(TUNEL)staining.The expression of bax and bcl-2 were evaluated by immuncoytochemical staining with anti-human bcl-2 and bax antibodies. Results After the RPE cells treated with daunoblastina,shrinkage of cytoplasm and nucleus was identified.The ratio of nucleus to cytoplasm was increased.TUNEL staining showed that many cells were positive staining.The amount of apoptotic cells was directly proportional to the drug dose.The integral optical desity values for expression of bax inereased by 22.0%(Plt;0.05), and that of bcl-2 did not change significantly(Pgt;0.05). Conclusions During human RPE cell apoptosis induced by daunoblastina,overexpression of bax or low bcl-2/bax ratio were demonstrated.The results suggest that bax and bcl-2 gene expression could play a role in regulation of RPE cell apoptosis. (Chin J Ocul Fundus Dis, 1999, 15: 153-156)
ObjectiveTo investigate the effect of post-conditioning with fospropofol disodium on hepatic ischemiareperfusion (I/R) and its possible mechanism in rats. MethodsForty-eight Sprague-Dawley rats were randomly divided into four groups, including sham group (S), control group (C), propofol group (P) and fospropofol disodium group (F). According to the different periods after reperfusion, each group was further divided into 2-hour and 4-hour reperfusion subgroups respectively (n=6 in each subgroup), named S2h, C2h, P2h, and F2h subgroups and S4h, C4h, P4h, and F4h subgroups. The livers of rats were reperfused after hepatic ischemia for one hour. In the beginning of reperfusion, normal saline was infused intravenously in group S and group C continuously, propofol was infused intravenously in group P continuously, fospropofol disodium was infused continuously in group F. The blood was sampled at the end of ischemia and reperfusion for assay of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The bcl-2 and bax protein contents in liver tissue were detected by immunohistochemical analysis, and liver samples were stained with hematoxylin-eosine for histological observation and damage degree evaluation by counting the proportion of necrosis cells. ResultsThe activity of ALT and AST, the rate of necrosis cells and the amount of bcl-2 and bax protein after reperfusion in group C, group P and group F were higher than those in group S at matched reperfusion time points (P<0.05). The activity of ALT and AST, the proportion of necrosis cells and bax protein contents decreased in group P and group F, compared with group C at the same reperfusion time points, while the contents of bcl-2 protein were significantly increased (P<0.05). ConclusionFospropofol disodium can alleviate hepatic injury induced by ischemia-reperfusion in rats, in which the bcl-2 and bax protein may play important roles.