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.
Objective To evaluate the phenomena of apoptosis and its relevant mechanism during ischemia-reperfusion period. Methods The published papers to explore the apoptotic phenomena and its mechanism in organs or tissues which experienced ischemia-reperfusion injury were reviewed. Results Apoptosis was common in ischemia-reperfusioned organ or tissue. The severity of apoptosis was influenced by many factors such as ischemia, hypoxia, oxygen free radials, intracellular free calcium ion overloading, various cytokines, et al; and also was regulated by bcl-2 family, caspase family and NF-κB，et al. Conclusion Apoptosis is a common phenomenum in ischemiareperfusioned organ or tissue which is affected and regulated by various factors.
目的研究依达拉奉影响肝脏缺血再灌注过程中TNF-α的表达情况，探讨依达拉奉对肝脏缺血再灌注损伤的逆转作用。 方法将80只Wistar大鼠编号，根据计算机产生随机数字，前40为一组，后40为一组，分为实验组和对照组2组，建立常温下部分肝缺血再灌注损伤动物模型。 在肝脏缺血再灌注损伤开始前1 h和开始时对实验组大鼠给予依达拉奉注射液10 ml，对照组则给予同等容量的生理盐水。分别于再灌注后0、1、2及4 h测定肝脏脂质过氧化物酶（LPO）和肝脏谷草转氨酶(AST) 浓度； 应用RT-PCR法检测肝组织TNF-α mRNA含量，并测定肝组织和血清中TNF-α水平； 应用TUNEL染色法检测缺血肝组织的细胞凋亡情况。结果再灌注后1、2及4 h，实验组大鼠肝脏LPO及AST浓度均明显低于对照组（Plt;0.001）； 实验组再灌注后1 h时肝组织TNF-α mRNA表达量、肝组织和血清TNF-α含量均明显升高且达峰值，但均明显低于对照组（Plt;0.05）； 再灌注后各时相实验组肝细胞凋亡率明显升高，但均明显低于对照组（Plt;0.05）。 结论依达拉奉能抑制氧化应激反应，从而降低肝缺血再灌注损伤； 并显著减少炎性细胞因子TNF-α的产生，抑制炎性反应的发生，减少肝细胞的凋亡。
Objective To investigate cell cycle as a new tool to evaluate the biocompatibility of biomaterials.Methods The cell cycle and the expression of related genes were analyzed by the methods of immunocytochemistry, protein blotting, RT PCR and flow cytometry. Results The physical properteis, chemical properties and topological properities of biomaterials could not only influence cell cycle of the cells attached onto biomaterials but also affect the expression of related genes of target cells. Conclusion As an important extension of routine proliferation epxeriments, the study of cell cycle control will be great help for us to to study the cell group as an organic society. It revealed the balance between cell proliferation, cell differentiation and apotosis. It is suggested that the study of cell cycle control will play a key role in the research of tissue engineering.
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.
Objective Bone marrow mesenchymal stem cells (BMSCs) transplantation can potentially regenerate the degenerated intervertebral disc, with the underlying regenerating mechanism remaining largely unknown. To investigate the potential of human BMSCs protecting nucleus pulposus cells (NPCs) from oxidative stress-induced apoptosis in a coculturesystem, and to illustrate the possible mechanisms of BMSCs transplantation for intervertebral disc regeneration. Methods BMSCs collected by density gradient centrifugation in Percoll solution were cultured and sub-cultured till passage 3, and the surface molecules of CD34, CD45, and CD13 were identified. NPCs were isolated by collagenase digestion and the chondrocyte l ike phenotype was confirmed by morphologic observation after HE staining, inverted phase contrast microscope, proteoglycan, and collagen type II expression after toluidine blue and immunocytochemistry staining. The 3rd passage BMSCs and the 1st passage NPCs were divided into four groups: group A, NPCs (1 × 106 cells) were cultured alone without apoptosis inducing (negative control); group B, NPCs (1 × 106 cells) were co-cultured with BMSCs (1 × 106 cells) with apoptosis inducing; group C, NPCs (1 × 106 cells) were co-cultured with BMSCs (3 × 105 cells) with apoptosis inducing; group D, NPCs (1 × 106 cells) were cultured alone with apoptosis inducing (positive control). After 3 or 7 days of culture or co-culture, the NPCs in groups B, C, and D were exposed to 0.1 mmol hydrogen peroxide for 20 minutes to induce apoptosis. With DAPI staining cellular nucleus, Annexin-V/propidium iodide staining cellular membrane for flow cytometry analysis, the apoptosis of NPCs in each group was studied both qual itatively and quantitatively. Besides, the changes in Bax/Bcl-2 gene transcription and Caspase-3 protein content, were analyzed with semi-quantitative RT-PCR and Western blot. Results BMSCs were successfully isolated and CD34-, CD45-, and CD13+ were demonstrated; after isolated from degenerated intervertebral discs and sub-cultured, the spindle-shaped 1st passage NPCs maintained chondrocyte phenotype with the constructive expressions of proteoglycan and collagen type II in cytoplasm. DAPI staining showed the nucleus shrinkage of apoptosis NPCs. Co-cultured with BMSCs for 3 days and 7 days, the apoptosis rates of NPCs in groups B (29.26% ± 8.90% and 18.03% ± 2.25%) and C (37.10% ± 3.28% and 13.93% ± 1.25%) were lower than that in group D (54.90% ± 5.97% and 26.97% ± 3.10%), but higher than that of groupA (15.67% ± 1.74% and 8.87% ± 0.15%); all showing significant differences (P lt; 0.05). Besides, semi-quantitative RT-PCR showed Bcl-2 gene transcription up-regulated (P lt; 0.05) and no significant change of Bax (P gt; 0.05); Western blot result showed that the Caspase-3 protein expression of groups B and C was lower than that of group D, and was higher than that of group A; all showing significant differences (P lt; 0.05). Conclusion In a co-culture system without direct cellular interactions, the oxidative stress-induced apoptosis of human NPCs was amel iorated by BMSCs. The enhanced anti-apoptosis abil ity of NPCs preconditioned by co-culturing with BMSCs might come from the decreased Bax/Bcl-2 gene transcription ratio.
ObjectiveTo study the relationship between hepatocellular apoptosis and glycogen contents during hepatic cold preservationreperfusion and its mechanism.MethodsBased on the model of four groups of rabbit livers with different hepatocellular glycogen contents, hepatocellular apoptosis and bax gene expression were observed during hepatic cold preservationreperfusion.ResultsApoptotic hepatocytes were obviously found in 60 minute reperfusing livers subsequent to 9 hour cold storage, and there was significant difference in the numbers of apoptotic hepatocytes among all the groups. In the same time, there was the close relationship between the levels of bax gene expression and the glycogen contents of hepatocytes.ConclusionIntracellular abundant glycogen may significantly depress the hepatocellular apoptosis during hepatic cold preservationreperfusion by decreasing hepatocellular bax gene expression.
Objective To observe the protective effects of ambroxol hydrochloride ( AMB) on rabbit model of acute lung injury ( ALI) induced by oleic acid and explore its mechanisms. Methods The ALI model of rabbit was induced by oleic acid. Twenty-four Japanese white rabbits were divided into three groups randomly, ie. a normal saline group ( NC group) , an ALI group and an ALI plus ambroxol injection group ( AMB group) . The pathological changes and apoptotic index ( AI) in lung tissue, Caspase-3 activity in lung tissue homogenate were observed 6 hours after the intervention. Serum activity of superoxide dismutase ( SOD) and serum levels of malonaldehyde ( MDA) , interleukin-1β( IL-1β) , and tumor necrosis factor-α ( TNF-α) were measured simutanously. Results The pathological injury of lung in the AMB group was milder than that in the ALI group. Both the AI in lung tissue and Caspase-3 activity in homogenate in the AMB group were lower than those in the ALI group significantly ( P lt;0. 01, P lt;0. 05 respectively) , butwere higher than those in the NC group( both P lt; 0. 01) . The activity of SOD in serum measured 6 hours after AMB intervention was higher while the serum levels of MDA, IL-1βand TNF-αin serum were lower ( P lt;0. 01) than those in the ALI group significantly ( all P lt;0. 01) . Conclusions Ambroxol hydrochloride has protective effects on oleic acid-induced acute lung injury. The mechanisms may be related to inhibition of oxidative stress and suppression of cytokines synthesis ( IL-1βand TNF-α) , the activity of the Caspase-3,and the apoptosis of lung tissue.
Objective To investigate the effect of ursolic acid on the proliferation and apoptosis of human osteosarcoma cell line U2-OS and analyze its mechanism. Methods Human osteosarcoma cell line U2-OS was divided into 4 groups, which was cultured with ursolic acid of 0, 10, 20, and 40 μmol/L, respectively. At 0, 24, 48, and 72 hours after being cultured, the cell proliferation ability was detected by cell counting kit 8 (CCK-8). At 48 hours, the effects of ursolic acid on cell cycle and apoptosis of U2-OS cells were measured by flow cytometry. Besides, the expressions of cyclin D1 and Caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot. Results CCK-8 tests showed that the absorbance (A) value of each group was not significant at 0 and 24 hours (P>0.05); but the differences between groups were significant at 48 and 72 hours (P<0.05). Flow cytometry results showed that, with the ursolic acid concentration increasing, the G1 phase of U2-OS cells increased, the S phase and G2/M phase decreased, and cell apoptosis rate increased gradually. There were significant differences between groups (P<0.05). Compared with the 0 μmol/L group, the relative expressions of cyclin D1 mRNA and protein in 10, 20, and 40 μmol/L groups significantly decreased (P<0.05); whereas, there was no significant difference in relative expression of Caspase-3 mRNA between groups (P>0.05). However, with the ursolic acid concentration increasing, the relative expressions of pro-Caspase-3 protein decreased and the relative expressions of activated Caspase-3 increased; there were significant differences between groups (P<0.05). Conclusion Ursolic acid can effectively inhibit the proliferation of osteosarcoma cell line U2-OS, induce the down-regulation of cyclin D1 expression leading to G0/G1 phase arrest, increase the activation of Caspase-3 and promote cell apoptosis.