Feasible Research On Construction Of Immortalized Chondrocyte By Transfecting With Human Telomerase Reverse Transcriptase (hTERT)

Since 1965, Smith has isolated chondrocytes successfully from mammal, the culture method receive maturity by continually researched and refined in the past 40 years. The highly purified chondrocytes can be obtained by separate chondrocytes from tenacious cartilage. However, development of cartilaginous tissue engineering is obsessed by another unsolved problem, which is how to obtain a lot of seed cells (chondrocyte) possessing normal phenotype. For it can't satify the number of cells to construct engineered cartilage in vitro just depending on self-source chondrocyte. Also, it would appear the senescence and dedifferentiation phenomenon when culture in vitro from the sixth generation to the eighth generation and cell numbers can't be amplificated adequately in vitro. So search the source of seed cells which consistent with the request of cartilage tissue engineering is a foucuse of the cartilage tissue engineering investigative.Normal cells would ingress into the stage of senium after several limited split, stop multiply and die. But tumor cells get ride of this constraint. It shows the ability of ultimate amplification and immortalization. Since Morin found telomerase was in tumor cells at 1989.People pay more attention to the high expression of it. It has been found that the active of telomerase correlates with the life of cells and plays decisive role at the age of sustain the immortalization of cells. Recent years, generous researches indicate that human telomerase reverse transcriptase (hTERT) is the key factor, which restrict the active and the expression of telomerase. For the weak fissionability and the dedifferentiation phenomenon, the utilization of chondrocyte is restricted. Adopting transgenic technology integrate gene fragment which can prolong the life of cells in vitro into expression vector. And then put expression vector shift in chondrocyte .So the seed cells that could passage long term and possessing normal phenotype of chondrocyte can be obtained. In this way, the juvenescene chondrocyte treated by telomerase can solve the problem that insufficient source of seed cells in tissue engineering. However, it plays significant role in pharmaceutical industry and the study of bioresearch and medicine.In this study, we employed the technology of molecular biology and cell engineering, we abstracted RNA from human embryo kidney 293 cell strain, amplificated cDNA gene of hTERT by RT-PCR method and recombinated it to the plasmid of pcDNA3.1/Zeo(+), constructed eukaryotic expression vector pcDNA3.1/Zeo(+)-hTERT. Human telomerase reverse transcriptase (hTERT) gene was introduced into chondrocytes of rabbit by liposome transgenic method. The biological characteristics of the positive clones selected were detected. The expression condition of hTERT and the influence of biological character are investigated. The way and technique to gain a large amount of tissue engineering seed cells are discussed.It is found that the chondrocytes infected with pcDNA3.1/Zeo(+)-hTERT expressed obviously higher detected by the RT-PCR on the RNA level. The morphology of chondrocyte infected with pcDNA3.1/Zeo(+)-hTERT still keep polygon, which is the same as the normal chondrocyte culture in vitro. After toluidine staining, polysaccharide was stained amethyst, small amethyst particles of proteoglycan were well-distributed. It suggested that the transformed cell still possess the function of secreting cartilage correlated matrix. The multiplicate ability of transformed cells can be analysed by measure the cyc of cells. The result shows that the multiplicate ability increased remarkably, the way of cell division is based on diploidy keeping the stabilization of karyotype.After analysing the ability of multiplication of transformed cells by mensurating the cyc of cells, the result shows that the ability increased remarkably, the DNA content testifyied the way of cell division is based on diploidy which is keeping the stabilization of karyotype.The chondrocyte could keep proliferation and physiological function long time in vitro by transgenic technology. It can serve as an excellent tool to study the biochemical and physiological aspect of chondrocyte and can be used as seeding cells in tissue engineering, and it is a potential method applied in engineered cartilage widespreadly utilize in clinic.In our study, chondrocytes are immortalized by the introduction of hTERT gene into chondrocytes derived from rabbit. The experiment undertakes an exploration of the feasibility for telomerase application in the tissue engineering field and a test for the resource of seed cells of cartilage tissue engineering. Meanwhile, this research provides a useful cell model for the investigation of the biological behaviors of chondrocytes in vitro and the mechanism of telomerase action.