| Neural transplantation was regarded as an optimal treatment technology for the neural injuries and neural degeneration diseases due to the limitation of neutral regenerative ability. With the breakthrough of the cloning and stem cells technologies, therapeutic cloning was suggested in the neural transplantation. Neural stem cells or neural cells derived from therapeutic cloning would be one of the most promising seed cells in the neural transplantation, because many side-effects of cells allo-transplantation will be avoid such as immunorejection.Even though therapeutic cloning had a wide range application in the medicine field, it was limited because of the low cloning efficiency. Since the source of the nuclear donor cell was restricted by the age of patient and the position of biopsy, the selection of the donor cell was more important than other factors that influence the cloning efficiency in therapeutic cloning. The nuclear transfer technique in therapeutic cloning was similar to animal cloning, so is the choice of the donor cell. So far, several somatic cells had been used in the research of animal cloning and therapeutic cloning; however, it was seldom reported which donor cell was the most suitable cells for the research. It was well known that the donor cell in G0/G1 did help for the cloning efficiency. Besides this, it was reported that the extent of the cell's differentiation was related to the cloning efficiency: the more undifferential the cell was, the higher efficiency it was. Several evidences suggested that the telomere length of the donor cell affected the one in the offspring from somatic cell cloning. It may be an optimal donor cell that was undifferential, easy required and longertelomere in therapeutic cloning.The telomerase activity was used as a selecting guideline in this research. The telomerase activity was detected by PCR-ELISA assay in several tissues, which could be the source of the nuclear donor cell using in therapeutic cloning, including skin, muscle, brain, bone marrow and testicle of different age rats. The telomerase activity variety with age was analyzed in these tissues. The density gradient separation method was used to isolate bone marrow stromal cells (BMSCs). BMSCs were cultured under different concentration of fetal bovine serum (FBS). We observed the morphologic characters and the changes of cell growth and apoptosis by MTT assay and flow cytometry. Then we identified the part of cells surface antigen. The telomerase activity and the trans-differential function of BMSCs were researched in this study. We studied the effects of the rat BMSCs and seek suitable FBS concentration used in the cells culture. Finally, the fibroblasts and BMSCs of different age rats were isolated and cultured in vitro. The differences among cell growth, telomerase activity and telomere length were investigated between the fibroblasts and BMSCs in different age. We explored the feasibility and advantages of BMSSC used as the donor cell in therapeutic cloning through the comparison between the fibroblasts and BMSSC. Our study will be the basis of the following work to build up the cloning ESCs and neurotransplantation.The results were as folio wings:1. The telomerase activities in skin, muscle, brain, bone marrow and testicle were remarkably related to the rat age. The telomerase activities were present in the skin, muscle and brain tissues in the neonatal rats and not found in the other age groups. The values of telomerase activity in the skin, brain and muscle of the neonatal rat group were 1.016, 0.829 and 0.354. The telomerase activities of bone marrow and testicle were detected in all groups. The values of the activity in bone marrow and testicle were 1.027, 1.116, 0.934, 1.074, 0.923 and 1.635, 1.483, 0.988, 0.946, 0.859 respectively in age ranged from 1 month to 2 years. The telomerase activity decreased in testicle tissues and kept stable in bone marrow with age.2. The figure of BMSCs collected by the density gradient separation method was similar in the beginning of the culture. After cultured in vitro for 10 days BMSCs were divided into two different morphological cells types-type I and type n. Type Icells were located in the bottom of the culture bottles. They had bigger cells bodies and greater cells number than type II cells and arrayed tightly. The circle or oval nuclear and several outshoots were seen in these cells. Type II cells were laid on the surface of type I cells. They had many figures. Most of the figures were long fibroblast-shape but the other forms as round, triangle and multi-angle were also seen. The cells were presented same direction array after cultured for 2 weeks in vitro.3. The proliferation was slow in primary BMSCs. Their double time was 4.4 days. The cells cycle assay showed that 90.8% cells were in Go/Gi phase and 4.4% and 4.9% cells were in S phase and G2/M phase respectively.4. The TERT immunocytochemistry revealed that type II cells were positive and type I ones were negative. The telomerase activity of type I cells, type II cells and BMSCs were assayed by PCR-ELISA method. The results showed the activity was 0.293, 1.322, and 0.651 respectively. The high telomerase activity was presented in type II cells but near none of it in type I cells. Type II cells were displayed nestin positive but type I cells negative after induced by neural stem cells culture media for 10 days. Type II cells expressed some special markers such as CD29 and CD44, but they did not show CD31, CD34 and CD45.5. The morphologic characters of BMSCs were uniform under different FBS concentration in vitro. The number of type I cells was increased more quickly under high FBS concentration than the one under no or low FBS concentration. The number of type II cells was counted in ten HP randomly. The numbers were 98, 92, 95, 92, 83 cells per HP in 1-month to 2-years group respectively. There were not remarkably different among these groups.6. The proliferation of BMSCs was accelerated with the increasing of FBS concentration in the culture medium. The cells' double time from 0% FBS group to 15 % FBS group was 5.71, 3.82, 3.54, 3.65 and 3.23 days respectively. The effects of FBS on BMSCs cell cycle were exhibited that the percentage of Go/Gi phage in 0% FBS group was 92.93%, which is higher than the ones in other groups that were 88.97%, 88.80%, 85.50% and 86.33% respectively. Compared with other groups' 7.97%, 7.63%, 9.07% and 9.07%, the percentage of S phage was only 3.67% in 0% FBS group. The percentage of the G2/M phage was similar among these groups.There was no influence in cell apoptosis under different FBS concentration during short period (10 days).7. The telomerase activity of BMSCs was 0.838, 0.685, 0.676, 0.734 and 0.593 in different FBS concentration group respectively. There was not statistically different among the groups, though a decreasing tendency was showed from the results with the FBS concentration increasing.8. The morphological characters of fibroblasts were similar among all groups, but the proliferation of fibroblasts was decreased with age. The cells' numbers crept from the tissues were different among these groups. The cells were more in the neonatal group and 1 month group than the ones in 1-year group and 2-years group. The cells' growth was slower in 1-year group and 2-years group than others with passage. It was obviously observed after the seventh passage.9. The double time of the first and the tenth passage fibroblasts was 3.22, 3.91, 3.54, 3.54, 3.6, 3.36 days and 3.19, 3.58, 3.56, 3.46, 4.75, 4.66 days respectively from neonatal group to 2-years group. Compared with the first passage fibroblasts, the double time of the 1-year group and 2-years group in the tenth passage cells was prolonged 1.15 and 1.3 days. From neonatal group to 2-years group, the percentage of GO/Gl phage in the first passage fibroblasts was 71.4%, 74.0%, 78.3%, 80.7%, 82.6%, 82.5% and the percentage of S and G2/M phage was 14.4%, 11.5%, 11.4%, 14.2%, 8.6%, 9.6% and 14.2%, 14.5%, 10.3%, 5.1%, 8.8%, 7.9% respectively. The cells in GO/Gl phage increased and the ones in S or G2/M phage decreased with age in the first passage fibroblasts. Compared to the first passage cells, the percentage of GO/Gl phage added 5.5%, 8.8% and the one of S phage and G2/M phage reduced 2.5%, 5.8% and 3%, 3.1%.10. The telomerase activity was not found in the first and the tenth passage fibroblasts in all groups. The telomere TRF length of the first and the tenth passage fibroblasts were assayed in all groups. The results showed that the TRF length was eroded with age. From neonatal group to 2-years group, the TRF length was 24.52, 23.52, 22.87, 22.55, 20.27, 19.85kb in the first passage cells and 19.79, 19.36, 18.84, 18.42, 16.35, 15.85kb in the tenth passage ones. The telomere length was reduced with the passage times was suggested in this study. The TRF length in the tenth passage cells was 4.73, 4.16, 4.03, 4.13, 3.92, 4kb, shorter than the length in the firstpassage in these groups. The shortened length was similar among groups. The telomere length was decreased about 462bp per passage in fibroblasts.11. The morphologic characters of BMSCs were same in different age groups. The proliferation of BMSCs was slow in all groups. From 1-month group to 2-year group, the double time of BMSCs was 3.51, 4.4, 4.22, 5.73, 4.59 days. The percentage of Go/Gi phage was 90.83%, 89.07%, 88.83%, 91.20%, and 87.83%. S phage and G2/M phage was 4.87%, 2.20%, 3.67%, 4.13%, 3.93% and 4.37%, 8.73%, 7.50%, 4.63%, and 8.23% respectively. There were not obviously different among groups.12. From 1-month group to 2 years group, the telomerase activity of BMSCs was 0.651, 0.676, 0.615, 0.815, 0.672 and the telomere TRF length of type II cells was 24.4, 22.56, 23.56, 22.79, 22.65 kb. There were not statistically different in telomere length and telomerase activity among groups.Conclusion: In this study, it was sure that the telomerase activity was not found in mature tissues and that there were some cells expressing stable telomerase activity in bone marrow with age. Because of the biopsy advantages of easy-requirement and little injury from bone marrow, the cell that expressed telomerase activity may be a better nuclear donor cell used in therapeutic cloning.BMSCs were differentiated into two type cells—type I and type II cells according to the difference in the morphological characters. The telomerase activity was high in type II cells that could be induced into nestin positive cells. They were the stem cells that kept undifferential state and had transdifferention ability in bone marrow stromal system. Type II cells in BMSCs were bone marrow stromal stem cells (BMSSC). They were the cytological basic of the stable telomerase activity in bone marrow. FBS could accelerate the proliferation of type I cells in BMSCs, but it did not influence the type II cells. The effects on type II cells differentiation were not found by FBS during a short period.The fibroblasts were the most common cell type used in animal cloning. They were the donor cells that were the most easy to get and used in therapeutic cloning. Because their telomere length was eroded and their proliferation was decreased with age, they would tamper with the cloning efficiency and offspring cells' functions.BMSSC were one of the most well known adult stem cells. They had manyadvantages such as easy required, simple isolated, little injury and abundant source, et al. Type II cells had high telomerase activity and transdifferention ability induced into nestin positive cells and expressed some special markers such as CD29 and CD44, but they did not show CD31, CD34 and CD45; therefore, type II cells in BMSCs were BMSSC. It was easy to differentiate from the type I cells in our culture because of the different morphologic characters. Compared with the fibroblasts, they had the advantages as folio wings:(1) The cloning efficiency was increased because of the undifferential state.(2) Their telomere length could keep longer and stable with age.(3) They were one of the cells resting on Go/Gi phage naturally, so they could avoid the effects on cells' activity by serum starvation.(4) The telomere erosion and mutation in genetic DNA would be avoided due to the inactive proliferation in vivo.(5) Unlike the fibroblasts, the proliferation was not reduced with age. Compared with the fibroblasts, BMSSC would be an optimal donor cell used intherapeutic cloning.
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