| MSCs (mesenchymal stem cells) are multipotential stem cells derived from mesoderm, possessing potent proliferative capacity and self-renewal and multilineage differentiation. MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and neuron-like cells with the controlled conditions and cytokine. MSCs have been considered as the best seed cells in tissue engineering nowadays for the broad applied prospect. It can be used in the research on repair in trauma, cell substitute therapy, tissue engineering and gene therapy, etc. Otherwise MSCs senescence have been reported a lot both in vivo and in vitro, which interfered the applied study on MSCs. Therefor, it is significance to research on MSCs senescence and its mechanisms.Many researchers reported that there must be some relationship between telomeres, telomerase and cell senescence. Telomeres is a special nucleotide repetitive sequences, located at the end of chromosome in eukaryotic cells. Both telomeres and telomerase have important effect in chromosome duplication and chromosome stable maintain. Telomeres located in the terminal end of a chromosome continued shortening accompany with the caryomitotic in progress. When telomeres shorten into some degree, cells stop growing, then come into senescence. Telomerase is a nuclear ribonuclear protein reverse transcriptase, and its main function is catalyzing and synthetizing telomerase segment. It can continue adding to terminal end of chromosome, keeping the natural life of the cells. There are three parts in human telomerase: human telomerase RNA (hTR), telomerase associated protein1 (TP1) and human telomerase catalytic subunit (hTERT). In these three parts, the activate of hTERT genetic transcription is a rate-limiting step of the telomerase. In recent years, some scholars discovered that MSCs can prolong the natural life of rMSCs cultured in vitro after modified with hTERT, showing that telomerase has relationship with MSCs senescence.In order to verify the senescence happening on MSCs in vitro and the function of telomerase in the process of senescence, our experiment based on rMSCs, observed the occurrence of rMSCs senescence during cultured in vitro, and the relationship between rMSCs senescence and telomerase,expecting to provide new theories and experimental bases for researches on rMSCs senescence. This experiment totally can be divided into three parts:1. rMSCs cultured in vitro and the happening of senescencerMSCs of high purity were isolated and obtained by the density gradient centrifugation, adherence selection and monoclone culture system. A standardized platform has been establised for isolation, cultivation, purification and identification of rMSCs. We analyzed the immunological markers of rMSCs in passage 4 by flow cytometry, showing that highly purified rMSCs have been gained. But the signs of rMSCs senescence gradually showed up during cultured in vitro:â‘ Morphology change: Morphology characteristics were observed by inverted microscope and transmission electron microscope. Before passage 5, the morphology of rMSCs are the same, when the cells grows into 80% to 90%, the shape of the cells likes fishes or whirlpool. At passage 7, the body of rMSCs becomes big, rough endoplasmic reticulum enlarge, microvilli desquamate and plenty secondary lysosome show up, all of this shows that rMSCs came to senescence. When it comes into the passage 9, large part of microvilli has gone, the enlargement of the rough endoplasmic reticulum is more obviously. At passage 12, the normal shape of rMSCs disappears, karyopycnosis and heterochromatin gathered obviously can be found.â‘¡The reproductive activity of rMSCs: Growth curve detected by CCK-8 shows rMSCs have powerful reproductive activity before passage 5, but after passage 7, the reproductive activity becomes weaker and weaker until disappeared at passage 12. Cell cycle tested by flow cytometry shows that the ratio of cells in DNA synthesis phase (S phase) and late DNA synthesis phase (G2/M phase) becomes lower and lower following long-time culture, but the number of the cells in stage of latency ( G0/G1 phase) becomes more obviously.When it comes into passage 12 , the number of rMSCs in DNA synthesis phase becomes zero. â‘¢Differentiation potential of rMSCs: The differentiation potential of rMSCs is evaluated by culturing cells in osteogenic and adipogenic media. Osteogenic differentiation is assayed by alkaline phosphatase (ALP) staining which is a specific protein marker of osteoblast. Adipogenic differentiation is assayed by Oil red O staining. A decline in the ability of osteogenic and adipogenic differentiation was observed. Cells in passage 9 exhibited a decrease in the amount of mineralized matrix compared with passage3 and passage 5. Also, adipogenic differentiation shows a similar tendency. rMSCs after passage 10 exhibited a decrease in the number of adipocytes formed compared with passag 8.â‘£Telomere and telomerase of rMSCs: Using the telomeric repeat amplification protocol assay, we found that telomere length of rMSCs in passage 3 is 19.8kbp, it is 16.2kbp at passage 7,and it is 12.5kbp at passage 3. TRAP-ELISA assay is used to detected the telomerase activity of rMSCs in different passages. The results show that rMSCs possess different telomerase activity before passage 9. At passage 3 and passage 5 high telomerase activity can be observed, but it becomes weak in passage 7 rMSCs. At passage 12, we cannot find telomerase activity any more. Suggesting that there are close relationship between telomerase and rMSCs senescence.2. Telomerase and rMSCs senescence:By means of suppressing and overexpressing telomerase hTERT, we expect to observe the usage of telomerase on rMSCs senescence:â‘ Effects on rMSCs senescence after telomerase activity suppressed by RNAi.we transfect the siRNA vector plasmid pGCsi-hTERT which constructed in our laboratory into rMSCs in passage 3, using non-silencing RNAi transfected cells and untransfected cells as control. TRAP-ELISA assay is used to detect the telomerase activity in transfected cells, identifying that telomerase activity had been effectually suppressed. Then, transmission electron microscopy, CCK-8 detection, flow cytometry and apoptosis Hoechst33258 staining are used to detect the effect on rMSCs senescence after telomerase suppressed. The results show that: compared with the control group cells, signs of senescence appeared after telomerase activity suppressed by RNAi. The signs are rough endoplasmic reticulum obvious expansion, a large number of intracytoplasmic vacuoles-like structure appeared, reproductive activity of rMSCs decreased, the ratio of cells in DNA synthesis phase (S phase) and late DNA synthesis phase (G2/M phase) declined, meanwhile, the number of the cell apoptosis increased, suggesting that rMSCs come into senescence after telomerase activity suppressed.â‘¡Effects on rMSCs senescence after telomerase activity overexpressed.we transfect the telomerase overexpression plasmid pCI-neo-hTERT into rMSCs in passage 9, using non-silencing RNAi transfected cells and untransfected cells as control. TRAP-ELISA assay is used to detect the telomerase activity in transfected cells, identifying that telomerase activity had been overexpressed. Then, CCK-8 detection, flow cytometry and apoptosis Hoechst33258 staining are used to detect the effect of telomerase overexpression on rMSCs senescence. The results show that: compared with the control group cells, the reproductive activity of rMSCs transfected increases, the ratio of cells in DNA synthesis phase (S phase) and late DNA synthesis phase (G2/M phase) markedly increases, meanwhile, the number of the cell apoptosis decreased.These two results suggest that telomerase activity can control the occurrence and process of rMSCs senescence.3. Telomerase activity and the PI3K/AKT signal transduction pathway in rMSCs.In order to explore whether there is some relationship between the regulation of telomerase on rMSCs senescence and PI3K/AKT signal transduction pathway, we expect to measure the expression pattern of P-AKT after telomerase suppressing or overexpression. On the other hand, the specific inhibitor LY294002 is used to block the PI3K/AKT signal transduction pathway, so that we can observe the telomerase activity expression in rMSCs culture in vtro.â‘ Effects on expression pattern of P-AKT in rMSCs after telomerase changed.We suppress the telomerase activity of rMSCs in passage 3 by RNAi, and overexpress the telomerase activity of rMSCs in passage 9, then the expression pattern of P-AKT in rMSCs measures by Western blot assay. The results showe that the expression pattern of P-AKT decreases after telomerase suppressed, while the expression pattern of P-AKT enhanceds after telomerase overexpressed.â‘¡Effects on the expression of telomerase in rMSCs after blocking PI3K/AKT signal transduction pathway.CCK-8 detection and Western blot assay are respectively used to analyze cell proliferation and the phosphorylation of AKT, identigying the effects of specific inhibitor LY294002 in rMSCs. Then we analyze the telomerase hTERT expression by Western blot assay, detect telomerase activity by TRAP-ELISA and measure the telomere length by Southern blot. The results showe that after PI3K/AKT signal transduction pathway blocked by LY294002, telomerase hTERT expression decreases, telomerase activity become weak and telomere length become short, indicating that the regulation of telomerase on rMSCs senescence has close relationship with PI3K/AKT signal transduction pathway.In conclusion, our experiments showed the process of rMSCs senescence cultured in vitro, and then we have observed a close relationship bewteen telomerase and rMSCs senescence, that is telomerase can regulate the process of rMSCs senescence. Furthermore, LY294002 is used to block PI3K/AKT signal transduction pathway, and then the relationship bewteen telomerase activity regulation and PI3K/AKT signal transduction pathway can be observed. Our works indicate the regulation of rMSCs proliferation by telomerase through or under PI3K/AKT signal transduction pathway.
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