Objective To investigate the protective effects of antitumor necrosis factor-α antibody （TNF-αAb） on lung injury after cardiopulmonary bypass （CPB） and their mechanisms. Methods Forty healthy New Zealand white rabbits，weighting 2.0-2.5 kg，male or female，were randomly divided into 4 groups with 10 rabbits in each group. In groupⅠ，the rabbits received CPB and pulmonary arterial perfusion. In group Ⅱ，the rabbits received CPB and pulmonary arterial perfusion with TNF-αAb. In group Ⅲ，the rabbits received CPB only. In group Ⅳ，the rabbits only received sham surgery. Neutrophils count，TNF-α and malondialdehyde （MDA） concentrations of the blood samples from the left and right atrium as well as oxygenation index were examined before and after CPB in the 4 groups. Pathological and ultrastructural changes of the lung tissues were observed under light and electron microscopes. Lung water content，TNF-α mRNA and apoptoticindex of the lung tissues were measured at different time points. Results Compared with group Ⅳ，after CPB，the rabbitsin group Ⅰ to group Ⅲ showed significantly higher blood levels of neutrophils count，TNF-α and MDA（P＜0.05），higherTNF-α mRNA expression，apoptosis index and water content of the lung tissues （P＜0.05），and significantly lower oxyg-enation index （P＜0.05） as well as considerable pathomorphological changes in the lung tissues. Compared with group Ⅱ，after CPB，the rabbits in groups Ⅰ and Ⅲ had significantly higher blood concentrations of TNF-α （5 minutes after aortic declamping，220.43±16.44 pg/ml vs.185.27±11.78 pg/ml，P＜0.05；249.99±14.09 pg/ml vs.185.27±11.78 pg/ml，P＜0.05），significantly higher apoptosis index （at the time of CPB termination，60.7‰±13.09‰ vs. 37.9‰±7.78‰，P＜0.05；59.6‰±7.74‰ vs. 37.9‰±7.78‰，P＜0.05），significantly higher blood levels of neutrophils count and MDA （P＜0.05），significantly higher TNF-α mRNA expression and water content of the lung tissues （P＜0.05），and significantly loweroxygenation index （P＜0.05） as well as considerable pathomorphological changes in the lung tissues. Compared with groupⅠ，rabbits in group Ⅲ had significantly higher above parameters （P＜0.05） but lower oxygenation index （P＜0.05） only at 30 minutes after the start of CPB. Conclusion Pulmonary artery perfusion with TNF-αAb can significantly attenuate inflammatory lung injury and apoptosis of the lung tissues during CPB.
【 Abstract 】 Objective To probe into the role of inositol 1, 4, 5-trisphosphate (IP3) and bax gene expression in apoptosis of HepG2 cells induced by genistein (Gen). Methods HepG2 cells were treated with different concentrations including 20, 40, 60 and 80 μ mol/L Gen as HepG2 cells cultured with 0 μmol/L Gen for 72 h was control; HepG2 cells were treated with 60 μmol/L Gen for 6, 12, 24, 48 and 72 h as HepG2 cells treated with 60 μmol/L Gen for 0 h was control. IP3 content, bax mRNA expression and apoptosis rate were assayed by IP3- ［ 3H ］ Birtrak assay, RT-PCR and flow cytometry, respectively. ResultsHepG2 cells incubated with each concentration of Gen for 72 h ， IP3 content was lower than that of control 〔 (17.7 ± 1.3), (11.2 ± 0.9), (4.9 ± 0.5), (4.8 ± 0.3) pmol/106 cells vs (29.4 ± 0.5) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression (RI which was the gray degree multiply area of bax/the gray degree multiply area of β -actin) was higher than that of control (0.26 ± 0.02, 0.33 ± 0.05, 0.35 ± 0.06, 0.38 ± 0.05 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate was higher than that of control 〔 (10.1 ± 0.9)%, (18.7 ± 1.6)%, (28.7 ± 2.5)%, (27.9 ± 2.0)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. HepG2 cells were incubated with 60 μ mol/L Gen for 6, 12, 24, 48 and 72 h ， IP3 content was lower than that of control 〔 (22.6 ± 0.9), (12.0 ± 1.4), (7.5 ± 0.8), (5.6 ± 0.5), (4.3 ± 0.6) pmol/106 cells vs (29.2 ± 0.6) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression was higher than that of control incubated with 60 μ mol/L Gen for above 12 h (0.25 ± 0.06, 0.29 ± 0.02, 0.30 ± 0.02, 0.35 ± 0.04 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate in groups incubated with 60 μ mol/L Gen for 24, 48 and 72 h was significantly higher than that in control 〔 (7.4 ± 0.5)%, (20.5 ± 2.0)%, (30.7 ± 1.6)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. ConclusionGen induces apoptosis of HepG2 cells by reducing IP3 production and increasing bax gene expression.
To detect the cell density, apoptotic incidence and the expressions of Bax and Caspase-3in human lumbar intervertebral discs, so as to further understand the mechanism of human lumbar intervertebral discdegeneration and provide a new idea for biologic treatment of it in future. Methods From May to December in 2006,30 human lumbar intervertebral discs in experimental group(L2 to S1)were surgically collected from 27 patients undergoing posterior lumbar intervertebral discoidectomy and fusion. All the cases were affirmed by MRI and they never experienced discography, collagenolysis of nucleus pulposus and percutaneous laser disc decompression. The control group consisted of 20 human lumbar intervertebral discs(L2 to S1)harvested from 5 young men without spine-related condition immediately after their accidental death. Apoptotic disc cells were detected by TUNEL and histomorphology, and immunohistochemical staining with SP method was performed to examine the expressions of Bax and Caspase-3 in all specimens. Results HE staining disclosed that the average cell density in control group (17.16 ± 1.22)/HP was higher than that in experimental group (12.41 ± 0.95)/HP (P lt; 0.01). However, TUNEL staining observed that the average TUNEL positive incidence in control group (6.97% ± 0.92%) was lower than that in experimental group (12.59% ± 0.95%), (P lt; 0.01). Immunohistochemical staining with SP method showed that the Bax and Caspase-3 positive incidence of nucleus pulposus in control group (11.02% ± 1.18%, 9.01% ± 1.00%) were lower than those in experimental group (19.29% ± 1.18%, 15.07% ± 0.97%), (P lt; 0.01). The results of the average gray scale value of nucleus pulposus in control group were 187.33 ± 7.88 and 185.68 ± 3.26, respectively, with 124.98 ±6.69 and 160.13 ± 4.37 in experimental group. There was significant difference between the two groups (P lt; 0.01). When thetotal 50 specimens in the two groups were analyzed, TUNEL positive incidence showed significant inverse correlations with their respectively corresponding cell densities (r = － 0.88, r = － 0.93, P lt; 0.01). The Bax and Caspase-3 positive incidence of nucleus pulposus showed significant positive correlation with the TUNEL positive incidence of nucleus pulposus (r = 0.83, r = 0.91, P lt; 0.01). Conclusion The decrease of cell density is involved in the development of human lumbar intervertebral disc degeneration. Bax and Caspase-3 might play a role in disc cell apoptosis in nucleus pulposus of human lumbar intervertebral disc.
ObjectiveTo observe apoptosis and proliferation of choledochus wall epithelial cell and fibrocyte, to understand the effects of apoptosis and proliferation on choledochal cyst development.MethodsThirty two cases of cystic dilatation,35 cases of cylindrical dilatation，and 25 cases of cholangiectasis caused by choledocholith were collected. All specimens were offered by department of hepatobiliarypediatric surgery. The apoptosis related index (bcl2 and bax) and cell proliferation index (PCNA) were detected by the immunohistochemical technique; Apoptosis was detected by TUNEL method. ResultsThere was serious mucosal epithelial cell damage in cystic dilatation group. In cylindrical dilatation group there was a damage similar to that of the cystis dilatation group, but the damage was not serious. In control group there was little damage in the duct wall, but there was a low positive rate of apoptosis of 〔epithelium cell (2.74±1.00)% and fibroblast (2.95±0.87）%〕, and a low bcl2 and bax’s expression rate, and a high PCNA’s expression rate 〔epithelium cell (3.74±1.00)%, fibroblast (3.71±1.77)%〕. There was no obvious difference between cylindrical dilatation group and cystic dilatation group (Pgt;0.05): the PCNA’s expression rate was low 〔(0.99±0.51)% and (0.90±0.38)% respectively〕, the bax expression rate was high in remaining epithelial cell, and the positive rate of bax was apparently higher than that of bcl2 (P＜0.05), the positive rate of the apoptosis cell was high 〔(13.94±4.77)%, (7.51±3.46)%〕; the expression rate PCNA were high 〔(9.91±2.91)%,(9.70±3.18)%〕, and expression rate of bax’s was low in the fibre tissue, the positive rate of bcl2 was markedly higher than that of bax, and the positive rate of the apoptosis cell was low 〔(3.74±2.12)%,(4.46±2.41)%〕. There were no marked difference between the two groups (Pgt;0.05). The expression of bcl2 and bax had marked difference both in cylindrical dilatation group and cystic dilatation group and as compared to control group (P＜0.05). ConclusionApoptosis has certain promoting effect in the course of choledochal cyst formation.
ObjectiveTo study the effects and mechanism of Octreotide to inhibit the proliferation of human gastric cancer cells in vitro. MethodsHuman gastric cancer cell line SGC7901 was treated with Octreotide. Human fibroblast cell line HF and 5FU were used as control. MTT assay and fluorescent microscopy as well as flow cytometry were performed in this study. ResultsOctreotide inhibited the growth of SGC7901 in vitro within certain concentrations. The suppression was quantity dependent but did not occur when up to a certain concentration. There was no difference between Octreotide and 5FU in their inhibition on SGC7901. Octreotide had no effects on normal human fibroblast cell line HF. When SGC7901 was treated with Octreotide, the typical apoptotic bodies were identified by flow cytometry and fluorescent microscopy. ConclusionOctreotide can inhibit the proliferation of human gastric cancer cell line SGC7901 in vitro. The induction of apoptosis by Octreotide might be the primary mechanism.
目的:探讨丙型肝炎病毒非结构蛋白NS4B在原发性肝癌发生中的作用,及其发生机制。方法:设置对照组、空白载体PCXN2组、转染NS4B组。使用脂质体介导法,转染丙型肝炎病毒非结构蛋白重组质粒NS4B进入Chang肝细胞内,并用G418筛选。绘制生长曲线,分别用流式细胞仪检测瞬时表达及稳定表达时肝细胞凋亡率,用均数±标准差表示,统计学分析采用Dunnett t检验及q检验。结果:瞬时表达各组(PCXN2,NS4B)凋亡率分别为:(918±060)%,(445±053)%。稳定表达各组(PCXN2,NS4B)凋亡率分别为:(1575±209)%,(366±034)%。与PCXN2组比较Plt;001。结论:NS4B抑制肝细胞凋亡率,可能导致肝细胞异常增殖,诱导肝癌发生。
Objective To observe expression of Caspase-3 and apoptosis around the prosthesis and explore the relationship of the expression and the apoptosis with the periimplant osteolysis. Methods From April 2001 to August 2006, 16 patients (10 males, 6 females) underwent the revision total hip arthroplasty surgery, who had the primary total hip arthroplasty at the ages of 45-67 years and had the revision total hip arthroplasty at the ages of 55-78 years, with the implantation duration of 7-13 years. According to their preoperative X-ray films andthe findings during the operation, the patients were divided into two groups: theloose/osteolytic group (n=8) and the loose/non-osteolytic group (n=8). The interface tissues were obtained from the peri-implant region in the patients. The synovial samples were taken from another 6 patients (2 males, 4 females; age, 54-68years; illness course, 9-15 years), who underwent the primary total hip arthroplasty for osteoarthritis. These 6 patients were used as controls. The tissues were prepared for the immunohistochemical assays to determine the expression of Caspase-3. The TUNEL assays were performed to quantify the apoptotic cells. The quantitative analysis on the positive cells and the correlation with the presence of the particulate wear debris and the severity of osteolysis were also performed. Results The level of the expression for Caspase-3 and the apoptosis index inthe loose/osteolytic group were significantly increased when compared with those in the loose/non-osteolytic group and the control group (P＜0.01). The polyethylene particles were surrounded by more positive cells than the metal particles. The positive cells were present at a higher level in the tissue sections where the high-wear status was present when compared with the areas where the low-wear status was present (P＜0.05). Conclusion There is a statistical correlation of the Caspase-3 expression to the apoptosis index and to the presence of the particulate wear debris and the severity of osteolysis, which may be one of the key points for the bone reconstruction inhibition and the bone resorption at the boneimplant interface under the stimulation of the wear debris. The apoptosis is involved in the pathogenesis of the aseptic loosening, which is closely related to the signal transportation of Caspase-3.
ObjectiveTo study the expression of apoptosissuppressing gene (bcl2) and apoptosispromoting gene (bax) in colorectal cancerous tissue and transitional mucosas. MethodsColorectal cancerous tissue, transitional mucosas (3 cm from the cancerous tissue) and normal tissue were taken respectively in thirtyone cases. Immunohistochemical technique SP method was used to detect the expression in those tissues. ResultsThe positive expression rate of bcl2 protein in cancerous tissue and transitional mucosa were 64.5%and 60.0% respectively and significantly higher than that in normal tissue (P＜0.05). The positive expression rate of bcl2 protein in normal tissue was 35.0%. The positive expression rate of bax protein in cancerous tissue and transitional mucosa were 45.2%and 45.0% respectively and significantly lower than that in normal mucosa (P＜0.05). The positive expression rate of bax protein in normal tissue was 60.0%. There was no obvious difference in the positive rate of bax and bcl2 protein between cancerous tissue and transitional mucosa (Pgt;0.05). The expression rate of bax and bcl2 protein in colorectal cancer was irrelative to clinical pathological gradation and clinical stage (Pgt;0.05). ConclusionThere is over expression of bcl2 protein and low expression of bax protein in colorectal cancer and transitional mucosa. bcl2 protein and bax protein can affect the generation of colorectal cancer by participating in the regulation of apoptosis. But it is irrelative to clinical pathological gradation and clinical stage. Transitional mucosas should be viewed as precancerous lesion and resected during operation.
ObjectiveTo investigate the effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells.MethodsThe MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/mL FTY720-P (chloroform as solubilizer) in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein (collagen type Ⅰ and collagen type Ⅲ) was detected by immunofluorescence staining; the alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in 2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis.ResultsAfter 48 hours of culture, the cells of 2 groups had grown into slender fusiform at the bottom of the bottle, and there was no significant difference in cell morphology between 2 groups. Immunofluorescence staining showed that the expression of collagen type Ⅰ was positive in the experimental group and weakly positive in the control group; the integrated absorbance (IA) value of the experimental group was 187 600±7 944, which was significantly higher than that of the control group (14 230±1 070) (t=43.680, P=0.001). The expression of collagen type Ⅲ was weakly positive in the experimental group and the control group, and there was no significant difference in IA value between 2 groups (t=1.976, P=0.119). ALP staining and alizarin red staining were positive in the experimental group and negative in the control group. TUNEL staining was positive in the experimental group and negative in the control group; the rate of TUNEL staining positive cells in the experimental group was 35.82%±2.99%, which was significantly higher than that in the control group (2.28%±0.51%) (t=23.420, P=0.002).ConclusionFTY720-P can promote the osteogenic differentiation of MC3T3-E1 cells with speeding up maturation and mineralization of extracellular matrix and affect the apoptosis of the cells.
ObjectiveTo observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H2O2).MethodsHuman retinal Müller cells cultured in vitro were divided into normal control group, model group (H2O2 group) and experimental group (H2O2+NBP group). The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μmol/L H2O2 for 2 h. Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 μmol/L NBP. The normal control group was a conventional cultured cells. Müller cells were identified by immunofluorescence staining. Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes. MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention. Hoechst33258 staining was used to observe the apoptosis. LIVE/DEAD ® cell activity/cytotoxicity kit was used to detect cell viability. Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells. One-way ANOVA combined with Dunnett statistical method were used for data analysis.ResultsHE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group. MTT assay showed that after 24 h and 48 h of NBP intervention, the differences in cell viability between the normal control group and the H2O2 group, the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96, 3.658, 47.58, 20.33; P＜0.001, 0.022). The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced, and the blue fluorescence of the remaining cells was uniform. The LIVE/DEAD ® cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly, and the number of viable cells with green fluorescence decreased significantly. In the H2O2+NBP group, the number of viable cells with green fluorescence increased, and the number of dead cells with red fluorescence decreased. The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced; the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group.ConclusionNBP alleviates H2O2-induced apoptosis of human retinal Müller cells by inhibiting ROS production.