| Mesenchymal stem cells (MSCs) represent a non-hematopoietic cell population which present in bone marrow with the ability for self-renewal and multilineage differentiation . The differentiation potential of BM derived MSCs has been extensively investigated, who can give rise to cells of connective tissues, such as bone, cartilage, muscle and fat. It is also known to extend beyond cells of a mesodermal layer to nonmesenchymal neuronal and glial cells.MSCs present in most tissues. Recent research has shown that Umbilical cord blood (UCB) is also a source of human MSCs. Exploration of the biological properties of human UCB has demonstrated an existence of new stem cells whose cellular characteristics and multilineage differentiation capability are equivalent to those of BM-derived MSCs. However, it is difficult to study and apply them because of their limited life span.To resolve these problems, the life span of hMSCs from bone marrow can be extended by retroviral transduction of human telomerase reverse transcriptase (hTERT). Many researchers improved that telomerization of hMSC by hTERT over expression maintains the stem cell phenotype of hMSC and it may be a useful tool for obtaining enough number of cells with a stable phenotype for mechanistic studies of cell differentiation and for tissue engineering protocols.To transfect hTERT gene into normal cells, several methods are available, including retrovirus-mediated gene transfer, adenovirus-mediated gene transfer andnon-viral vector including calcium ion sedimentation and electroporation gene transfer. Taking transduction efficiency into account, the retrovirus and adenovirus methods are prefer. Additionally, the retrovirus vector can make the target gene to integrate into the target cell chromosome stably which widely used in the single or multiple gene transformation, such as all kinds of original cells and cells in vivo.In the present study, we want to construct a recombinant retrovirus vector carrying hTERT , then investigated the growth regulatory mechanism of UCBMSCs and attempted to establish UCBMSCs with hTERT (hTERT-MSCs) to overcome their limited life span. For the more we want to detect whether telomerized UCBMSCs line maintained long-term self-renewal and differentiation capacity. Methods:1. MSCs were isolated from human umbilical cord samples, cultured in vitro and passaged weekly.2. The whole cDNA was generated by PCR amplifications from the plasmid pEGFP-hTERT-Cl then the hTERT segments were subcloned into pLNCX2in-frame at the Bgl II and Not I restriction sites.3. These retro viral particles were infected target cells. We selected the stably transduced cells by neomycin with an expanded life span which were designated hTERT-MSCs. Detected the expression of hTERT in mRNA level by RT-PCR and the telomerase activity by TRAP(PCR)-ELISA ways4. Induced hTERT-MSCs with 5-azacytidine to cardiac muscle. Detected the specific marker of myocardiocyte: a-Sarcomeric actin and Connexin-43 by immunocytochemical staining.Results:1. For verification, the constructed plasmids were digested with restrictionendonucleases( Bgl II and Not I ) Two characteristic segments including 6.1kband 3.6kb were got.2. The highest titre of the four cell clones was 8 X 107cfu/L.3. Proved by RT-PCR way, the hTERT-MSCs expressed hTERT in mRNA level.4. Proved by TRAP(PCR)-ELISA way, the telomerase activity of hTERT-MSCs lower than K562 cells.5. Measured by MTT assay the growth kinetics of hTERT-MSCs were higher than those of UCBMSCs.6. The hTERT-MSCs maintained differentiation capacity in vitro. It can be induced to myocardiocyte .Conclusions:1. The hTERT recombinant retrovirus vector was successfully constructed. The hTERT gene can activate the telomerase and prolongs the life-span of cells.2. The hTERT gene didn't influence some type of differentiation potential of MSCs. |
PDF Full Text Request