ABSTRACT
BACKGROUND AND OBJECTIVES: Human mesenchymal stem cells (hMSCs) are attractive candidates for cell therapy and regenerative medicine due to their multipotency and ready availability, but their application can be complicated by the factors such as age of the donors and senescence-associated growth arrest during culture conditions. The latter most likely reflects the fact that aging of hMSCs is associated with a rise in intracellular reactive oxygen species, loss of telomerase activity, decrease in human telomerase reverse transcriptase (hTERT) expression and finally eroded telomere ends. Over-expression of telomerase in hMSCs leads to telomere elongation and may help to maintain replicative life-span of these cells. The aim of this study was to evaluate of the effect of L-carnitine (LC) as an antioxidant on the telomerase gene expression and telomere length in aged adipose tissue-derived hMSCs. METHODS: For this purpose, cells were isolated from healthy aged volunteers and their viabilities were assessed by MTT assay. Quantitative gene expression of hTERT and absolute telomere length measurement were also performed by real- time PCR in the absence and presence of different doses of LC (0.1, 0.2 and 0.4 mM). RESULTS: The results indicated that LC could significantly increase the hTERT gene expression and telomere length, especially in dose of 0.2 mM of LC and in 48 h treatment for the aged adipose tissue-derived hMSCs samples. CONCLUSION: It seems that LC would be a good candidate to improve the lifespan of the aged adipose tissue-derived hMSCs due to over-expression of telomerase and lengthening of the telomeres.
Subject(s)
Humans , Aging , Carnitine , Cell- and Tissue-Based Therapy , Cytochrome P-450 CYP1A1 , Gene Expression , Mesenchymal Stem Cells , Polymerase Chain Reaction , Reactive Oxygen Species , Regenerative Medicine , Telomerase , Telomere , Tissue Donors , VolunteersABSTRACT
The effects of combined RNA interference(RNAi)of human telomerase RNA(hTR)and human telomerase reverse transcriptase(hTERT)genes on telomerase activity in a bladder cancer cell line(BIU-87 cells)were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer(BTCC).Three TR-specific double-stranded small interfering RNAs(siRNAs)and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA.The phTR-siRNA,pbTERT-siRNA,and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells.The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction,and a telomeric repeat amplification protocol was applied to detect telomerase activity.Growth inhibition of BIU-87 cells was measured by MTT assay.Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro.The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,and pRNAT-hTR-Ⅲ+hTERT-Ⅲ in BIU-87 cells.The inhibition efficiency of pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,pRNAT-hTERT-Ⅲ+pRNAT-hTR-Ⅲ was 67% for TERT mRNA,41% for TR mRNA,57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively.The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased,especially in the cells treated with combined RNAi-hTR and-hTERT.Gene chip analysis revealed that 21 genes were down-regulated(ATM,BAX,BCL2,BCL2L1,B1RC5,CD44,CTNNB1,E2F1,JUN,MCAM,MTA1,MYC,NFKB1,NFKBIA.NME4,PNN,PNN,SERPINE1,THBS1,TNFRSF1A,and UCC1).The results indicated that hTR-siRNA and hTERT-siRNA,especially their combination,siRNA hTR+hTERT,specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity.Molecular biological mechanism by which combined siRNA-TR and-TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation ofthe 21 genes.
ABSTRACT
Objective To establish the human telomerase reverse transcriptase (hTERT) gene viral transferring system. Methods By means of liposome mediation, a retroviral vector pLNC-hTERT carrying a selectable marker neomycin resistance genes (Neo r) and a target gene (hTERT) was transferred into the ectropic package cells ?-2, and by using the supernatant of ?-2 cells to infect the amphotropic producer clone PA317, established PA317/hTERT package cell line. In order to get higher-titer virus, the supernatants of ?-2 infected PA317 repeatedly. Human endothelial cells were used to detect the effect of gene transfer. Results We established a PA317/hTERT package cell line which produced high-titer virus. The viral titer produced by PA317 cells was raised 20 times by repeated infection method, and with higher-titer virus the exogenous genes were successfully transferred into endothelial cell. Conclusion This method can be used to establish higher-titer viral transferring system.
ABSTRACT
Aim Telomerase is highly expression in most tumor cells, and it is an ideal target for cancer molecular targeting therapy. It has been proved that wogonin effectively inhibits telomerase activity and tumor cell growth in vitro. The study was to explore the inhibitory effect of wogonin on the growth of tumor and telomerase activity of implanted human ovarian cancer cell line SKOV3 in nude mice. Methods Nude mice with implanted human ovarian cancer cells SKOV3 were randomly divided into five groups, viz. the high dose group of Wogonin(600 mg?kg-1),low dose group of Wogonin(300 mg?kg-1),normal control group, cisplatin therapy group(3 mg?kg-1), and combined therapy group(cisplatin plus wogonin).The weight of nude mice and the volume of tumor were regularly measured. DNA、RNA and protein were extracted from the tumor tissue. The length of telomere was examined by Southern blot. The expression of telomerase hTERT gene was detected by RT-PCR. The telomerase activity was examined by TRAP-PCR-silver staining. Results The wogonin significantly inhibit the growth of tumor when compared with controlled group.The inhibitory rate of high dose group and low dose group were 56.67% (P=0.002) and 38.10%(P=0.019), respectively. The inhibition rate of cisplatin therapy group was 50.83%(P=0.004). The suppress rate of combined group reached 66.9% and higher than any single therapy(P=0.002). The length of telomere in different concentration groups of wogonin was the same as that in the control group.Wogonin inhibited the expression of telomerase gene hTERT and telomerase activity. The inhibition is related to the dose of wogonin. Conclusion Wogonin suppresses the growth and telomerase activity of tumor. The inhibitory effect is related to the dose of wogonin. Combination of wogonin and cisplatin increase the inhibitory rate in nude mice tumor.