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.
Objective To further investigate pathologic mechanism of retinal phototrauma. Methods Twenty Wistar rats were divided into control and experimental groups.Their eyes were extracted in 12，24 and 36 hours after light exposure.HE stained retina samples were examined and TDT-mediated dUTP nick end labelling(TUNEL)method was employed to distinguish apoptotic cells. Results After 12-hour light exposure,slight vesiculation was observed in the rod outer segment of the retinas.After 24-hour light exposure,the outer nuclear layer showed predominant fractured and condensed nuclei and fragmented DNA.After 36-hour light exposure,the rod outer and inner segments were lysed and most of the nuclei in the outer nuclear layer were disappeared. Conclusions Apoptosis of photoreceptor cell is one of the important mechanisms which cause experimental retinal photoinjury of rats. (Chin J Ocul Fundus Dis, 1999, 15: 167-169)
ObjectiveTo explore the new gene therapy method for tumor, the recombinant Caspase3 gene (rcaspase3) eukaryotic expression plasmid was constructed by molecular biologic method. MethodsThe eukaryotic expression plasmid pcDNA3.1(+)/rCaspase3 was constructed by rearrangement of the large subunit and small subunit of Caspase3 and it was transfected into pancreatic carcinoma cells(PCⅡ). After being transfected, the expression of rCaspase3 mRNA in pancreatic carcinoma cells was detected by RTPCR and it’s apoptotic activity was detected by FCM. ResultsThe sequence of rCaspase3 showed that the recombinant molecules (rCaspase3) now had its’ small subunit preceding its’ large subunit. After pancreatic carcinoma cells being transfected with the pcDNA3.1(+)/rCaspase3 by liposomes, a 894 bp strap was observed by RTPCR. No strap was found in control groups. A transparent hypodiploid karyotype peak was found by FCM.ConclusionThe plasmid of pcDNA3.1(+)/rCaspase3 has been constructed successfully. rCaspase3 has apoptotic activity and can be used as target gene in gene therapy for pancreatic carcinoma.
目的 探讨HIF-1α和BAK蛋白在胃癌中的表达情况，以及二者在胃癌中的相互关系及作用。方法 应用免疫组化SABC染色法检测80例胃癌组织和20例正常胃组织中的HIF-1α和BAK蛋白的表达情况。结果 胃癌中HIF-lα和BAK蛋白的表达阳性率分别为56.3%（45/80）和67.5%（54/80），而在胃正常组织中分别为5.0%（1/20）和20.0%（4/20），二者在胃癌中的表达显著高于胃正常组织，其差异有统计学意义（P＜0.05）。HIF-1α蛋白表达与胃癌组织的浸润范围、分化程度及淋巴结转移有关（P＜0.05），与临床分期、年龄及性别无关（P＞0.05）；BAK蛋白表达与胃癌浸润及分化程度相关（P＜0.05），与淋巴结转移、临床分期、年龄及性别无关（P＞0.05）。胃癌组织中HIF-1α与BAK蛋白的阳性表达之间呈正相关（列联系数r=0.056，P＜0.05）。结论 HIF-1α与BAK蛋白在胃癌的临床分期及浸润转移中存在关系，这对于研究胃癌的发生和发展，以及对于探索以二者为靶点的抗肿瘤治疗有重要意义。
Objective To study the effect of water soluble chitosan (WSC) on the apoptosis of peritoneal macrophage induced by lipopolysaccharides (LPS), and discuss the mechanism. Methods Peritoneal macrophages were divided to three groups: phosphate buffered saline (PBS) group, LPS group and LPS plus WSC group. At hour 24, apoptosis cell and active caspase-3 were detected by flow cytometry; nitric oxide (NO) was determined with Griess reagent. Results There were more apoptosis cells in the LPS group than the PBS group. The percentage of apoptosis cells was significantly decreased in the LPS plus WSC group than the LPS group. The expression of active caspase-3 and the secretion of NO were also inhibited by WSC after LPS intervention. Conclusion WSC inhibits apoptosis of peritoneal macrophage induced by LPS.
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 explore the effect on apoptotic genes of pancreatic adenocarcinoma cell BxPC-3 from subcutaneous transplantation tumor in nude mice induced by 5-FU and sulfasalazine (SZ).Methods Changes of apoptosis-related genes 〔bcl-2, cyclinD1, Bax and NF-κB (p65)〕 in subcutaneous transplantation tumor treated by 5-FU, SZ alone or both at the levels of mRNA and protein were measured by RT-PCR and Western blot. Results NF-κB (p65) at mRNA relative content and protein expression in subcutaneously heterotopic transplantation tumor treated by 5-FU (7.5, 15 mg/kg), SZ (10, 20 mg/kg) alone or both showed significant difference, except for two subsets in SZ group, respectively, in comparison with each control group (P＜0.01). Meanwhile bcl-2 and cyclinD1 at the levels of mRNA and protein, and Bax protein level were significantly different from each control group (P＜0.01). The above-mentioned indexes were show obvious interaction of both by multiple factor analysis of variance. Conclusion Up-regulated level of Bax, down-regulated levels of bcl-2, cyclinD1 and NF-κB (p65) might be one of apoptotic mechanisms that SZ synergistically enhanced apoptotic effect on pancreatic adenocarcinoma cell BxPC-3 of subcutaneous transplantation tumor in nude mice induced by 5-FU.
OBJECTIVE: To investigate the effect of vasostomy on apoptosis in the male rat spermatogenic cells after vasoligation. METHODS: Model of vasoligation and vasostomy in male rat was established, and then terminal deoxynucleotidyl transferase-mediated dUTP nick labelling technique to detect the apoptosis of spermatogenic cells at 4, 8, 12, 16 weeks after vasostomy. RESULTS: The number of apoptotic cells in vasostomy group was significantly lower than that of vasoligation group since 8 weeks after vasostomy(P lt; 0.05). The number of apoptotic cells in 8 and 12 weeks after vasostomy were significantly higher than that in prevasoligation(P lt; 0.05). 16 weeks after vasostomy, the number of apoptotic cells restored to the level same as that in prevasoligation stage. CONCLUSION: Vasostomy can reverse the apoptosis of spermatogenic cells due to vasoligation.
【Abstract】ObjectiveTo investigate the effect of ischemia-reperfusion （I/R） injury on apoptosis of pancreatic cells in rats with acute pancreatitis(AP). MethodsFifty-four SD rats were randomized into 3 groups: pancreatitis group (n=24), I/R-injury group (n=24) and control group (n=6). The animal model of AP was induced by retrograde injection of 3% sodium taurocholate into biliopancreatic duct in rats. Pancreatic I/R was caused by blocking the inferior splenic artery and removing the clamp after AP induction. At 1 h, 3 h, 6 h and 12 h, groups of rats were sacrificed. A terminal deoxynucleotidyl transferase-mediated dUTP-biotion nick end labeling (TUNEL) was used to detect pancreatic apoptosis, and histological changes of the pancreas were observed. ResultsPancreatic hemorrhage, necrosis were respectively observed in the pancreatitis rats at 6 h and the I/R-injury rats at 1 h. Histological changes of the pancreatitis rats at 1 h and 3 h were only congestion and edema. Apoptoic acinar cells increased after AP induction, the peak respectively appeared at 6 h in the pancreatitis rats and at 3 h in the I/R-injury rats. Compared with the pancreatitis rats, apoptosis index (AI) of the I/Rinjury rats was significantly higher at 1 h and 3 h (P＜0.01, P＜0.05, respectively), but lower at 6 h and 12 h (P＜0.05, P＜0.01, respectively). ConclusionI/R injury can induce conversion of edematous pancreatitis to hemorrhagic necrotizing pancreatitis and apoptosis of acinar cells. Apoptosis may be a beneficial response to pancreatic injury in AP.
ObjectiveTo investigate the in vitro effect of pseudolaric acid B (PAB) on apoptosis of protoscolece cells and its regulatory effects on angiogenesis and cell apoptosis in the the lesion-host microenvironment tissue in vivo, as well as its possible mechanisms, in order to provide a basis for the clinical development of new alternative drugs for Echinococcus multilocularis. MethodsIn vitro experiments: the protoscoleces, vesicles, germinal cells, human foreskin fibroblasts (HFFs) and normal human liver cells were treated with different concentrations of PAB (0, 2.5, 5, 10, 20, 40, 80, 160 and 320 μmol/L) for 7, 5, 5, 5 and 5 days, then evaluated the survival rate of the protoscoleces, the release level of phosphoglucose isomerase (PGI) from the vesicles, the viability of the germinal cells, as well as the viability of HFFs and normal human liver cells. The protoscoleces and vesicles were fixed with 2.5% glutaraldehyde and used for scanning electron microscopy and transmission electron microscopy observation. Animal experiments: the protoscoleces were isolated from the abdominal lesions of the protected gerbils, and then infected 18 C57BL/6J mice by intraperitoneal injection to establish models, dividing into 3 groups with 6 mice in each group. The model group was given 0.3 mL of PBS by gavage daily, the albendazole (ABZ) group was given 0.3 mL ABZ (100 mg/kg) daily by gavage, the PAB group was given 0.3 mL of PAB (40 mg/kg) by gavage daily. After continuous gavage for 6 weeks, the lesion host microenvironment tissue was taken and ELISA was used to detect the expression levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and cysteinyl aspartate specific proteinase3 (caspase3), the expression levels of nitric oxide (NO) was detected using a biochemical detection kit, Western blot was used to detect the expression levels of BCL2-associated X protein (Bax), B-cell lymphoma-2 (Bcl2), caspase3, cleaved-caspase3, VEGF, vascular endothelial growth factor receptor 2 (VEGFR2), phosphatidylinositol 3 kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (AKT) and phosphorylated AKI (p-AKT) protein. ResultsIn vitro experiments: the protoscoleces of Echinococcus multilocularis were cultured with different concentrations of PAB for 7 days in vitro, the protoscoleces of 40, 80, 160 and 320 μmol/L group all died after 6, 4, 2 and 1 day, respectively; PAB exhibited a certain time and concentration dependence on the protoscoleces of Echinococcus multilocularis. After PAB treatment, the release of PGI in culture supernatant of Echinococcus multilocularis gradually increased with the increase of PAB concentration [concentration for 50% of maximal effect value was (24.40±1.42) μmol/L], the vitality of germinal cells was significantly inhibited [half maximal inhibitory concentration value was (15.94±2.55) μmol/L]. PAB had no significant toxicity to mammalian cells. When 20 μmol/L PAB intervention in the protoscoleces for 3 days, the expression levels of Bax and caspase3 proteins were upregulated, while the expression level of Bcl2 protein was downregulated. Animal experiments: compared with the model group, the wet weight of lesions in the PAB and ABZ groups decreased (P<0.01), and the inhibition rates of lesion growth in the PAB and ABZ groups were 91.03% and 74.44%, respectively. The expression of proliferation and angiogenesis indicators (Ki67, CD34, VEGF, VEGFR2, eNOS, NO) were downregulated in the lesion host microenvironment tissues of mice in the ABZ and PAB groups (P<0.05), while the expression of apoptosis related proteins (caspase3, cleaved-caspase3 and Bax) were upregulated and the expression of PI3K/AKT signaling pathway related proteins (p-PI3K and p-AKT) were downregulated (P<0.05). ConclusionPAB has a strong in vitro and in vivo effect against Echinococcus multilocularis, and its mechanism may be related to the inhibition of PI3K/AKT signaling pathway, leading to increased apoptosis and decreased angiogenesis.