The Study Of The Immortalization Of Human Bone Marrow Mesenchymal Stem Cells By Transducting Human Telomerase Reverse Transcriptase Gene

Objective: Human bone marrow mesenchymal stem cells (hBMSCs) have the multipotentials of differentiation into various cells,so they are considered as an important source of seed cells in tissue engineering. In vitro, hBMSCs have a limited life, and gradually lose their differentiation potential after several passages,which restrict their scientific and clinical application.The rapid development of transgenic technology makes the establishment of immortalized cells possible.By using the retroviral vector,we were trying to transduct exogenous human telomerase reverse transcriptase(hTERT) gene into hBMSCs in order to establish the immortalized hBMSCs.Methods:The retroviral vector containing hTERT gene (pLEGFP-C1- hTERT) was transfected into packaging cell PT67 through liposomes.Then cell clones positive for the virus were selected by G418.Three cell clones were chose and amplified culture respectively.NIH 3T3 cell lines were used to detect retrovirus titers.hBMSCs were extracted and amplificated. hBMSCs in passage 3(P3) were infected with the highest titer virus supernatant,then selected by G418.Three monoclones were selected and amplified respectively.The hTERT expressions of the transducted hBMSCs were tested by RT-PCR and Western Blot.We investigated whether the transduced hBMSCs(P3) can sustain the normal cellular properties in morphology,cell phenotype and chondrogenic induction.The cells growth curve and the telomerase activity were detected by MTT and TRAP-ELISA.The tumorigenesis of transducted hBMSCs was detected by the plate clone formation assay and subcutaneous tumor formation in nude mice.Results:The expression of hTERT gene can be tested in the level of mRNA and protein in transducted hBMSCs,while weak mRNA transcription of hTERT gene can be tested in untransducted hBMSCs.In the morphology, the transducted hBMSCs were spindle-shaped or fibrocyte-like shape, which as the same as the untransducted hBMSCs.The green fluorescent protein expression can be found in the transducted hBMSCs under fluoroscope. The phenotype of transducted hBMSCs were positive in CD29,CD71,CD106 and negative in CD34,CD45.The transducted hBMSCs maintain their differentiational capability,which was proved by induced into chondrocyte.In vitro the latter can express the type II collagen protein that was cartilage specific marker.The telomerase activity of transducted hBMSCs (hTERT-hBMSCs) was positive.The growth speed of hTERT-hBMSCs was faster than that of hBMSCs by MTT.Up to now,the passage of hTERT-hBMSCs is more than 50.The rates of hTERT-hBMSCs and hBMSCs cells colony formation were 0.83% and 0.67% respectively which were lower than the rate of normal cells.In the control group,the rate of malignant melanoma cells was 57.4%,which was much higher than that of tumor cell colony formation.In nude mice experiments,malignant melanoma cells can form local massess under skin after three weeks.It was proved that the mass was composed of malignant melanoma cells.There was no tumor formation in experimental groups.Conclusions:There is no change in cell morphology,surface marker and differentiation function in transducted hBMSCs.The hTERT gene activates the telomerase and extends the life span of hBMSCs.There is no malignant transformation in hTERT-hBMSCs.These results suggest that hBMSCs transduced with hTERT gene can be considered as a source of seed cells in tissue engineering because their prolonged life span and normal cellular properties in a long-term culture.