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 investigate the effect of subretinal fluid (SRF) with different grades of proliferative vitreoretinopathy (PVR) on bcl-2 oncoprotein expression in retinal pigment epithelium (RPE) cells and fib roblast (FB)． Methods Using immunohistological staining technique and Western-blotting method to detect the expression of bcl-2 protein in RPE cells and FB under the stimulation of SRF． Results The expression levels of bcl-2 increased in both types of cells to a certain extent compared with those of the control group 4 hours after the cells subjected to SRF； 36 hours later, the expression levels of bcl-2 of experimental groups decreased more quickly than those of the control group， and the control group showed relatively higher bcl-2 protein levels at the end of observation． Conclusions SRF may stimulate the e xpression of bcl-2 in RPE cells and FB under culture at early stage, but accel arate the declining of bcl-2 protein levels a certain time after subjected to SRF． (Chin J Ocul Fundus Dis, 2001,17:58-60)
ObjectiveTo observe the expression of Caspase-3, Caspase-8, Bcl-2, Bax in the rat retina of ocular ischemic syndrome (OIS). Methods30 Brown Norway rats were randomly divided into experimental group and control group, 15 rats in each group. The rats in experimental group were established a model through ligating the bilateral common carotid artery. At 3 months after modeling, the retinal thickness and ganglion cell (RGC) density were measured by hematoxylin eosin staining; the expression of Caspase 3, Caspase 8, Bax and Bcl-2 in the retina was measured by quantitative real-time reverse transcription polymerase chain reaction. ResultsThe RGC density in control group and experimental group was 61.97±9.07 and 38.1±5.98, respectively. Compared to the control group, the RGC density was diminished in the experimental group (t=3.059, P < 0.01). A significant decrease was found in the total retina (t=3.036), inner plexiform (t=3.715), inner nuclear (t=3.339) and outer plexiform (t=3.341) thickness (P < 0.05). However, no change of the thickness was evident in the outer nuclear layers (t=2.000, P > 0.05). The levels of protein and RNA expression of Caspase 3, Caspase 8 and Bax in the retina were increased in experimental group (F=17.036, 7.787, 11.431, 11.162, 17.763, 12.183; P < 0.05), while the Bcl-2 expression were decreased (F=10.298, 12.047; P < 0.05). ConclusionsThere is obvious expression of apoptosis-associated proteins in the rat retina of OIS. Caspase 3, Caspase 8 and Bax expression are increased, while Bcl-2 expression are decreased.
【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 investigate the cellular viability and mitochondrial reactive oxygen species (ROS) production of the Müller cells under high glucose condition, and explore the protection role of the 5,6-dihydrocyclopenta-1, 2-dithiole-3-thione (CPDT) on Müller cells. Methods Müller cells from Sprague Dawley rats were divided into 5 groups randomly, including 25 mmol/L normal glucose group (group A) and 65 mmol/L high glucose group (group B). High glucose group with 45, 60, 70 μmol/L CPDT and cultured them 72 hour was set as group C, D and E. Water soluble tetrazolium salt (WST)-8 was used to measure the cellular viability. Flow cytometry was used to measure the active oxygen and apoptosis index. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), Bcl-2 and Bax protein were measured by Western blot. Results Compared with group A, the WST-8 showed that the viability of Müller cells apparently decreased in group B (t=39.59,P＜0.05). Compared with the group B, the viability of Müller cells had changes in group C (t=0.97,P＞0.05), but recovered in group D and E (t=−4.17, −7.52;P＜0.05). Compared with group A, the FCM showed that the mitochondrial ROS levels was higher in group B (t=−30.99,P＜0.05). Compared with group B, the mitochondrial ROS levels were decreased in group D (t=27.68,P＜0.05). Compared with group A, Bax, Nrf2 and HO-1 increased (t=–11.03, –63.17, –11.44;P＜0.05), while the bcl-2 decreased in group B (t=7.861,P＜0.05). Compared with the group B, Nrf2, HO-1 and Bax decreased (t=15.11, 26.59, 6.27;P＜0.05), while the bcl-2 increased in group D (t=−6.53,P＜0.05). Conclusions Under the high glucose, CPDT may reduce the mitochondrial ROS levels and the expression of Nrf2, HO-1 and Bax protein of Müller cells. It may inhibit apoptosis through activating the Nrf2/HO-1 pathway and balancing of level of Bcl-2 protein and mitochondrial ROS.
Objective To investigate the temporal and spatial expression pattern of Caspase3、Bax and Bclxl in NmethylNnitrosourea (MNU) damaged rat retina. Methods Twenty-four 50 dayold female Sprague-Dawley rats (n=24) received single intraperitoneal injection of MNU 40 mg/kg and were examined at 1, 3, 7 and 10 days after MNU treatment (6 rats sacrificed at each timepoint). As control, six rats were injected with saline (5 ml/kg) and sacrificed 3d after injection. Expressions of Caspase-3 and bax and bcl-xl were detected by RTPCR and immunofluorescence assays, photoreceptor cell apoptosis was measured by terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphatedigoxigenin nick-end labeling (TUNEL). Results Animal models were successful established and confirmed by pathological studies. RTPCR results indicated that caspase3 and bax upregulated at 1 d (caspase-3 RA =83.23plusmn;8.11，P= 0.009; bax-RA=72.73plusmn;9.46，P=0.004) and peaked at 3 d （caspase-3 RA=140.48plusmn;18.40，P=0.000；bax-RA=102.36plusmn;13.97，P=0.001）compared with control (caspase-3 RA=62.45plusmn;7.65; bax-RA =46.53plusmn;4.41). Bcl-xl expression increased and peaked at 3d (3d RA=79.83plusmn;5.58, P=0.000 vs control 45.98plusmn;3.06). It was noted that the ratios of bax / bclxl expression at 1 d, 3 d and 7 d after MNU injection were enhanced (1 d 1.15plusmn;0.14, P= 0.143; 3 d 1.28plusmn;0.16, P=0.001; 7 d 1.17plusmn;0.08, P= 0.079, vs control 1.01plusmn;0.09), and at 3 d the ratio reached the peak, whereas at10 d bax / bcl-xl ratio (0.73plusmn;0.07, P= 0.001) was decreased compared with the control. Immunofluorescence assays demonstrated that the changes of bax, bclxl and caspases3 protein expressions coincided with their RTPCR results respectively. The Bax positive cells were detected in the outer nuclear layer; while caspase3 and bclxl positive cells emerged in several layers of retina included the pigment epithelium layer, the photoreceptor cell inner segments, the outer nuclear layer, the outer plexiform layer, the inner plexiform layer and the ganglion cell layer. Photoreceptor cell apoptosis was only detected in the outer nuclear layer and peaked at 3 d in MNU treated rats (AI= 76.97plusmn;5.83, P= 0.000 vs control 0.00 plusmn; 0.00). Conclusions These data suggest that bax and bcl-xl and caspases3 may involve in the MNUinduced rat photoreceptor cell apoptosis.
To elucidate bcl-2 protein expression in hepatic carcinogenetic process and its relationship with apoptotic changes. bcl-2 protein was evaluated immunohistochemically while apoptosis was approached with terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick end labeling (TUNEL) technique in 8 normal livers (NL), 17 liver cirrhosises (LC) and 77 hepatocellular carcinomas (HCC). The results showed that bcl-2 protein was expressed in 3 of 8 NLs(37.5%), 5 of 17 LCs(29.4%) and 7 of 77 HCCs(9.1%) with significant differences between group NL and HCC and between LC and HCC (Ｐ＜0.05). Apoptosis rates of 1.18±0.42%, 4.85±2.78%, 12.89±2.33% in NL, LC, HCC group respectively were demonstrated with significant differences among them (Ｐ＜0.01). Compared the bcl-2 expression with the apoptosis rate in this hepatocarcinogenetic process, reversed trends were presented. Conclusion: bcl-2 expression could be detected in NL, LC and HCC, and its decreasing expression was related to the inhibition attenuation of hepatocellular apoptosis in the process of hepatocarcinogenesis.
Objective To study the inhibitory effect of RNA interference (RNAi) on bcl-2 expression of vascular smooth muscle cells （VSMCs) in rabbit. Methods The expression vector of bcl-2 gene-targeting small interference RNA (pshRNA-bcl-2) was constructed and was transfected into VSMCs by lipofectamine, and the unloaded vector was used as control. The expression of bcl-2 mRNA was identified by RT-PCR and Western blot, respectively. The growth of the transfected VSMCs was examined by MTT. Results The pshRNA-bcl-2 may inhibit the expression of bcl-2 gene at the levels of transcription and translation. There were significant differences (P＜0.01) of the expressions of bcl-2 mRNA between the VSMCs that were transfected with pshRNA-bcl-2 and the ones in plasmid transfected group and control group, respectively. There was a significant difference (P＜0.01) in the growth of VSMCs between the plasmid transfected and the control groups. Conclusion The plasmid containing the small interference RNA of bcl-2 may have an inhibitory effect on the cell growth and endogenous expression of bcl-2 gene at the levels of transcription and translation in VSMCs.
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 study the effect of hypoxia on proliferation of cultured bovine retinal pigment epithelium (RPE) cells and expression of the antiapoptotic protein bcl-2. Methods The bovine RPE cells were cultured under normal and hypoxic chamber respectively. After 24 hours, the proliferation of RPE cells was evaluated by［3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide, MTT］test. At the same time, anti-bcl-2 protein antibody was examined by immuno-histochemistry method. Results The A value in the hypoxia group was higher than that in the normal group after 24 hours (P＜0.05 )in MTT-test. Positive staining for anti-bcl-2 protein antibody was seen in 72.6% cells in hypoxia group and 38.64% in normal group. The positive staining was more obvious near the nucleus, and fine granules scattered in cytoplasm of some cells. Conclusion Hypoxia can stimulate the proliferation of RPE cells and expression of antiapoptotic protein bcl-2. The results indicate that bcl-2 may play an important role in mediating the proliferation activity of RPE cells. (Chin J Ocul Fundus Dis, 2002, 18: 293-295)