ABSTRACT
Objective: To obtain the conditionally replicative adenovirus with triple-targeting Smac overexpression using gene recombination technology, and to explore its effects on the apoptosis and cell cycle progression of MDA-MB-231 cells. Methods: The triple-targeting Smac overexpression vector pShuttle-Egrl-Smac-HRE-hTERT-ElA-ElBp-ElB55K was constructed by gene recombination technology, which was recombined with the skeleton vector pAdEasy in the BJ5183 bacteria (AdEasy-l +) to obtain the conditionally replicative adenovirus CRAd. pE-Smac. After the MDA-MB-231 cells were infected with CRAd. pE-Smac, the cancer cells were mimiced into hypoxic status with chemical reagent CoCl2, then control group, CRAd. pE-Smac group, hypoxia group and CRAd. pE-Smac + hypoxia group were set up; the cells were irradiated with 4 Gy X-rays, and each group was divided into nonirradiation group and irradiation group. The Smac protein expression was detected by Western blotting assay, the apoptotic rates and the percentages of cells at different phases were detected by flow cytometry. Results: The Western blotting results showed that the Smac protein expressions were increased after infection of CRAd. pE-Smac, hypoxia and 4 Gy irradiation, especially in CRAd. pE-Smac + hypoxia+4 Gy irradiation group. The FCM results showed that the apoptotic rates in CARd. pE-Smac, hypoxia, CARd. pE-Smac + hypoxia group were increased compared with control group (P<0. 05 or P<0. 01), and the apoptotic rates of cells irradiated with 4 Gy were significantly increased compared with the unirradiated cells (P<0. 05 or P<0. 01), especially in CRAd. pE-Smac + hypoxia + 4 Gy irradiation group; the percentages of the cells at S and G2/M phases in irradiation groups were significantly increased (P<0. 05 or P<0. 01), which had the similar regularity with the apoptotic change. Conclusion: After the MDA-MB-231 cells are infected with the conditionally replicative adenovirus CRAd. pE-Smac and treated with hypoxia and irradiation, the triple-targeting Smac overexpression can be achieved, and it has the role of promoting the cancer cell apoptosis and inducing the G2/M arrest.
ABSTRACT
Objective:To obtain the conditionally replicative adenovirus with triple-targeting Smac overexpression using gene recombination technology,and to explore its effects on the apoptosis and cell cycle progression of MDA-MB-231 cells.Methods:The triple-targeting Smac overexpression vector pShuttle-Egr1-Smac-HRE-hTERT-E1A-E1Bp-E1B55K was constructed by gene recombination technology,which was recombined with the skeleton vector pAdEasy in the BJ5183 bacteria (AdEasy-1+-) to obtain the conditionally replicative adenovirus CRAd.pE-Smac.After the MDA-MB-231 cells were infected with CRAd.pE-Smac,the cancer cells were mimiced into hypoxic status with chemical reagent CoCl2,then control group,CRAd.pE-Smac group,hypoxia group and CRAd.pE-Smac+-hypoxia group were set up;the cells were irradiated with 4 Gy X-rays,and each group was divided into nonirradiation group and irradiation group.The Smac protein expression was detected by Western blotting assay,the apoptotic rates and the percentages of cells at different phases were detected by flow cytometry.Results:The Western blotting results showed that the Smac protein expressions were increased after infection of CRAd.pE-Smac,hypoxia and 4 Gy irradiation,especially in CRAd.pE-Smac +hypoxia+4 Gy irradiation group.The FCM results showed that the apoptotic rates in CARd.pE-Smac,hypoxia,CARd.pE-Smac + hypoxia group were increased compared with control group (P＜0.05 or P＜0.01),and the apoptotic rates of cells irradiated with 4 Gy were significantly increased compared with the unirradiated cells (P＜0.05 or P＜0.01),especially in CRAd.pE-Smac + hypoxia + 4 Gy irradiation group;the percentages of the cells at S and G2/M phases in irradiation groups were significantly increased (P＜ 0.05 or P＜0.01),which had the similar regularity with the apoptotic change.Conclusion:After the MDA-MB-231 cells are infected with the conditionally replicative adenovirus CRAd.pE-Smac and treated with hypoxia and irradiation,the triple-targeting Smac overexpression can be achieved,and it has the role of promoting the cancer cell apoptosis and inducing the G2/M arrest.
ABSTRACT
Rheumatoid arthritis (RA) is a chronic inflammatory disease. Joint deformity and dysfunction can occur in the late stage of the disease,which is seriously harmful to human health. Anti-inflammatory factors (AIC), as a protective factor, together with pro-inflammatory factors (PIC) play important roles in the pathogenesis and progression of RA. It is widely accepted by the majority of scholars that the decrease of AIC and the increase of PIC in RA can aggravate the systemic and local inflammatory reactions and accelerate the articular cartilage and subchondral bone destruction, resulting in further progress of RA. A new generation of biological therapy for RA targeting at AIC is in the ascendant. Therefore ,it is important to understand the role of AIC in the pathogenesis of RA. From the perspective of the relationship between AIC and RA and the mechanism, this article reviews the research progress in this field, which provides new concepts for the diagnosis and treatment of RA.
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Objective:To investigate the expression of XIAP and Smac in human non-small-cell lung carcinoma (NSCLC) and the relationship with clinical significance and prognosis. Methods:Immunohistochemical staining was performed to determine the ex-pression of X-linked inhibitor of apoptosis protein (XIAP) and second mitochondria-derived activator of caspase (Smac) in 70 cases of NSCLC and 70 cases of non-cancerous adjacent lung tissues. Results:XIAP is mostly present (59/70) in tumor tissues with 16 high ex-pressions, whereas only five high expressions in non-cancerous adjacent lung tissues are observed (52/70). The statistical difference of these two sets of data is significant (Z=-5.484, P0.05). The Kaplan-Meier analysis results show that survival by XIAP and Smac protein in NSCLC has no significant effect (P>0.05). Conclusion:XIAP and Smac are expressed in NSCLC and noncancerous adjacent lung tissues, and the differences in their expression levels is significant. The deterioration of NSCLC results in apoptosis/anti-apoptotic synchronized with tumor cell proliferation. The expression levels of XIAP and Smac in NSCLC are not related with the prognosis.
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Objective To investigate Smac/DIABLO and cytochrome c(cyt-c)mRNA levels in liver tissue of rats with acute hepatic failure treated by microencapsulated hepatocyte.Methods Acute hepatic failure were induced by intraperitoneal injection of D-galactosamine in rats.and the rats were treated with microencapsulated hepatocytes,free hepatocytes and physical saline(contr01),respectively.Smac/DIABLO and cyt-c mRNA in liver tissue was detected by RT-PCR and the mRNA expression levels among three groups were compared.Results Smac/DIABLO and cyt-c mRNA levels in liver tissues of rats with acute hepatic failure were higher than those of normal rata(F=4.345,14.821,47.565,42.178 and 62.961,P<0.05).The peak values of Smac/DIABLO and cyt-C mRNA expressions in free hepatocytes and control groups were at 48 h.while that in microencapsulated hepatoeytes group was at 24 h.Conclusion Smac/DIABLO and cyt-c mRNA expression is an indicator of apoptosis of hepatocytes.