AIM： To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma （HCC） cell line HepG2 and normal liver cell line L0...AIM： To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma （HCC） cell line HepG2 and normal liver cell line L02. METHODS： We constructed the recombinant replication-incornpetent Ad.rnda-7 virus vector and infected it into the human HCC cell line HepG2 and normal liver cell line L02. RT-PCR was performed to detect the rnRNA expressing in cells, by ELISA was used to detect MDA-7/IL-24 protein expression in the culture supernatant. The effect of apoptosis induced by Ad.rnda-7 was confirmed by Hoechst staining and flow cytometry assay with Annexin-V and PI staining. MTT assay was used to determine growth inhibition of HepG2 cells, and cell-cycle and hypodiploidy analyses were performed by flow cytometry. RESULTS： Recombinant replication-defective virus expressing MDA-7/IL-24 was constructed successfully. RTPCR showed that the Ad.rnda-7 could mediate the expression of the exogenous gene MDA-7/IL-24 into HepG2 and L02. The concentration of MDA-7/IL-24 protein in supernatant was 130 pg/mL and 110 pg/mL in Ad.rnda-7-infected L02 and HepG2 ceils, respectively. Ad.mda-7 infection obviously induced apoptosis （from 2.604±0.72% to 33.64±13.2%, P=0.00012） and growth suppression in HepG2 （inhibition ratio IR=68%） and an increase in the percentage of specific cancer cell types at the G2/M phase of the cell cycle （from 6.44% to 32.29%, P〈 0.01）, but not in L02 cells.CONCLUSION： These results confirm selectively induction of apoptosis and growth suppression by the mda-7/ IL-24 gene with replication-incompetent adenovirus vector in human hepatocellular carcinoma cell line HepG2.展开更多
High-risk human papillomavirus(HPV)replication requires deregulation of host DNA damage response(DDR)and inflammatory pathways.DNA topoisomerase 2β(Top2β)was previously shown to promote HPV replication.We investigat...High-risk human papillomavirus(HPV)replication requires deregulation of host DNA damage response(DDR)and inflammatory pathways.DNA topoisomerase 2β(Top2β)was previously shown to promote HPV replication.We investigated whether its paralog Top2α protein acts similarly to the virus.Elevated levels of Top2α are consistently observed in cervical intraepithelial lesions and the related carcinomas,as well as in HPV-positive cell lines.Silencing Top2α with shRNA severely suppresses HPV genome maintenance and amplification,but in a DDR-independent manner.Instead,Top2α facilitates secretion of interleukin(IL)-6 and IL-8,which are necessary for HPV replication.Mechanistically,this manipulation is regulated by toll-like receptor 4(TLR4).Top2α binds to the TLR4 promoter to transcriptionally induce TLR4 expression.Blockade of TLR4 signaling by the specific inhibitor TAK-242 significantly reduces the secreted IL-6/IL-8 levels and HPV replication.Overall,our results reveal a novel role of Top2α to shape the inflammatory microenvironment that benefits HPV replication,making it a promising therapeutic target for HPV-associated diseases.展开更多
目的探讨重组人白细胞介素24(rhIL-24)联合顺铂(DDP)诱导人肺腺癌A549细胞凋亡相关基因的变化。方法用160 ng/m L rhIL-24、3μg/m L DDP及160 ng/m L rhIL-24联合3μg/m L DDP处理A549细胞,应用Genome LabTMGe XP多基因遗传表达分析系...目的探讨重组人白细胞介素24(rhIL-24)联合顺铂(DDP)诱导人肺腺癌A549细胞凋亡相关基因的变化。方法用160 ng/m L rhIL-24、3μg/m L DDP及160 ng/m L rhIL-24联合3μg/m L DDP处理A549细胞,应用Genome LabTMGe XP多基因遗传表达分析系统检测凋亡相关基因Bax、Bcl-2、存活蛋白(survivin)、caspase-3、视网膜母细胞瘤基因(Rb)、p53的mRNA表达水平。结果 rhIL-24蛋白处理的A549细胞上调Bax、caspase-3、Rb的mRNA水平,下调Bcl-2、survivin的mRNA水平,但p53mRNA变化无规律,rhIL-24联合DDP组对A549细胞上述基因表达的影响更明显。结论 rhIL-24蛋白可通过上调Bax、抑癌基因Rb,下调Bcl-2、survivin,激活caspase-3而诱导人肺腺癌A549细胞凋亡。展开更多
目的:寻求增强CIK细胞的细胞毒活性的有效方法.方法:从健康人外周血中提取出单个核细胞,第1天加入IFN-γ,第2天加入IL-1,CD3 mAb,IL-2诱导CIK细胞,另一组与IL-1.CD3 mAb,IL-2同时加入IL-24.杀伤分为4组:未加IL-24培养组、单独IL-24杀伤...目的:寻求增强CIK细胞的细胞毒活性的有效方法.方法:从健康人外周血中提取出单个核细胞,第1天加入IFN-γ,第2天加入IL-1,CD3 mAb,IL-2诱导CIK细胞,另一组与IL-1.CD3 mAb,IL-2同时加入IL-24.杀伤分为4组:未加IL-24培养组、单独IL-24杀伤组、加IL-24培养组、未加IL-24的CIK细胞培养组在杀伤时加入IL-24.细胞计数法测定细胞的增殖、MTT法测定细胞杀伤活性和流式细胞术分析细胞表型.扫描电镜和透射电镜观察CIK细胞对肿瘤细胞的杀伤和肿瘤细胞的改变.结果:未加IL-24培养组CIK细胞增殖高于加IL-24培养组,两者比较有明显差异(126.34±2.14 vs 108.87±1.29,P<0.05).加IL-24培养组细胞各个效靶比杀伤活性均达到90%以上,明显高于其他各组(效靶比为10:1时95.58%±2.21% vs 27.31%±2.69%,8.74%±2.41%,38.65%±21.30%,P<0.05;效靶比为20:1时91.97%±4.21% vs 34.27%±0.85%,11.54%±2.78%,48.32%±11.72%,P<0.05;效靶比为40:1时91.84%±9.28% vs 50.67%±1.30%,23.73%±11.07%,52.89%±12.26%,P<0.05).不同时间加IL-24培养组各个细胞表型与未加IL-24培养组相比没有差别.透射电镜下观察加IL-24培养组凋亡和坏死肿瘤细胞比未加IL-24培养组明显增多.结论:CIK细胞诱导过程中加入IL-24能明显增强其杀伤活性.展开更多
文摘AIM： To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma （HCC） cell line HepG2 and normal liver cell line L02. METHODS： We constructed the recombinant replication-incornpetent Ad.rnda-7 virus vector and infected it into the human HCC cell line HepG2 and normal liver cell line L02. RT-PCR was performed to detect the rnRNA expressing in cells, by ELISA was used to detect MDA-7/IL-24 protein expression in the culture supernatant. The effect of apoptosis induced by Ad.rnda-7 was confirmed by Hoechst staining and flow cytometry assay with Annexin-V and PI staining. MTT assay was used to determine growth inhibition of HepG2 cells, and cell-cycle and hypodiploidy analyses were performed by flow cytometry. RESULTS： Recombinant replication-defective virus expressing MDA-7/IL-24 was constructed successfully. RTPCR showed that the Ad.rnda-7 could mediate the expression of the exogenous gene MDA-7/IL-24 into HepG2 and L02. The concentration of MDA-7/IL-24 protein in supernatant was 130 pg/mL and 110 pg/mL in Ad.rnda-7-infected L02 and HepG2 ceils, respectively. Ad.mda-7 infection obviously induced apoptosis （from 2.604±0.72% to 33.64±13.2%, P=0.00012） and growth suppression in HepG2 （inhibition ratio IR=68%） and an increase in the percentage of specific cancer cell types at the G2/M phase of the cell cycle （from 6.44% to 32.29%, P〈 0.01）, but not in L02 cells.CONCLUSION： These results confirm selectively induction of apoptosis and growth suppression by the mda-7/ IL-24 gene with replication-incompetent adenovirus vector in human hepatocellular carcinoma cell line HepG2.
基金supported by CQMU Program for Youth Innovation in Future Medicine(172020320220090 W0072)to S.H.Chongqing Yuzhong District Basic Research and Frontier Exploration Project(20210128)to S.H.+1 种基金Natural Science Program of Chongqing Science and Technology Commission(2024NSCQ-KJFZZDX)to S.H.National Natural Science foundation of China(NSFC)Young Scientists Fund(82300970).
文摘High-risk human papillomavirus(HPV)replication requires deregulation of host DNA damage response(DDR)and inflammatory pathways.DNA topoisomerase 2β(Top2β)was previously shown to promote HPV replication.We investigated whether its paralog Top2α protein acts similarly to the virus.Elevated levels of Top2α are consistently observed in cervical intraepithelial lesions and the related carcinomas,as well as in HPV-positive cell lines.Silencing Top2α with shRNA severely suppresses HPV genome maintenance and amplification,but in a DDR-independent manner.Instead,Top2α facilitates secretion of interleukin(IL)-6 and IL-8,which are necessary for HPV replication.Mechanistically,this manipulation is regulated by toll-like receptor 4(TLR4).Top2α binds to the TLR4 promoter to transcriptionally induce TLR4 expression.Blockade of TLR4 signaling by the specific inhibitor TAK-242 significantly reduces the secreted IL-6/IL-8 levels and HPV replication.Overall,our results reveal a novel role of Top2α to shape the inflammatory microenvironment that benefits HPV replication,making it a promising therapeutic target for HPV-associated diseases.
文摘目的探讨重组人白细胞介素24(rhIL-24)联合顺铂(DDP)诱导人肺腺癌A549细胞凋亡相关基因的变化。方法用160 ng/m L rhIL-24、3μg/m L DDP及160 ng/m L rhIL-24联合3μg/m L DDP处理A549细胞,应用Genome LabTMGe XP多基因遗传表达分析系统检测凋亡相关基因Bax、Bcl-2、存活蛋白(survivin)、caspase-3、视网膜母细胞瘤基因(Rb)、p53的mRNA表达水平。结果 rhIL-24蛋白处理的A549细胞上调Bax、caspase-3、Rb的mRNA水平,下调Bcl-2、survivin的mRNA水平,但p53mRNA变化无规律,rhIL-24联合DDP组对A549细胞上述基因表达的影响更明显。结论 rhIL-24蛋白可通过上调Bax、抑癌基因Rb,下调Bcl-2、survivin,激活caspase-3而诱导人肺腺癌A549细胞凋亡。
文摘目的:寻求增强CIK细胞的细胞毒活性的有效方法.方法:从健康人外周血中提取出单个核细胞,第1天加入IFN-γ,第2天加入IL-1,CD3 mAb,IL-2诱导CIK细胞,另一组与IL-1.CD3 mAb,IL-2同时加入IL-24.杀伤分为4组:未加IL-24培养组、单独IL-24杀伤组、加IL-24培养组、未加IL-24的CIK细胞培养组在杀伤时加入IL-24.细胞计数法测定细胞的增殖、MTT法测定细胞杀伤活性和流式细胞术分析细胞表型.扫描电镜和透射电镜观察CIK细胞对肿瘤细胞的杀伤和肿瘤细胞的改变.结果:未加IL-24培养组CIK细胞增殖高于加IL-24培养组,两者比较有明显差异(126.34±2.14 vs 108.87±1.29,P<0.05).加IL-24培养组细胞各个效靶比杀伤活性均达到90%以上,明显高于其他各组(效靶比为10:1时95.58%±2.21% vs 27.31%±2.69%,8.74%±2.41%,38.65%±21.30%,P<0.05;效靶比为20:1时91.97%±4.21% vs 34.27%±0.85%,11.54%±2.78%,48.32%±11.72%,P<0.05;效靶比为40:1时91.84%±9.28% vs 50.67%±1.30%,23.73%±11.07%,52.89%±12.26%,P<0.05).不同时间加IL-24培养组各个细胞表型与未加IL-24培养组相比没有差别.透射电镜下观察加IL-24培养组凋亡和坏死肿瘤细胞比未加IL-24培养组明显增多.结论:CIK细胞诱导过程中加入IL-24能明显增强其杀伤活性.
