| Bone marrow mesenchymal stem cells (BMSCs) are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time, with the capacity to form a variety of tissue-forming cells such as bone, muscle, cartilage, nerve cells, etc. At present, there are reports that BMSCs derived from human or mice could be cultured and transdifferentiated into multilineage cells. However, there were few reports about dairy goat BMSCs, especially about dairy goat BMSCs being able to transdifferentiate into germ cells. Goats are important economic animal, with the production for meat, wool, milk and other economic value. Furthermore, dairy goats farming is one of the advantages of livestock industry. Using stem cells as seed cells in investigation of genetic breeding and transgenic clone is a shortcut in accelerating good quality and high output goat propagation. In order to provide materials for research on development and differentiation of dairy goat cells as well as animal breeding, and animal disease models. we carried out the following experimental work.1. The cells were isolated from dairy goat bone marrow by different adherent culture and identified by morphology, growth curves, flow cytometry, immunocytochemistry and RT-PCR. The positive rates of non-directional and directed differentiation cells were evaluated by immunocytochemical method. The experiment results showed that BMSCs had a fibroblast-like morphology. The markers were identified in MSCs as same as embryonic stem cells (ESCs) such as OCT4, Nanog, C-Myc and TERT. Flow cytometry results were indicated that CD29,CD44,CD166 were positive and CD71,CD45,CD34 were negative. The spontaneously differentiated cells derived from embryoid bodies expressed markers of three embryonic germ layers. BMSCs were induced to differentiate into early neural-like cells by chemical reagents and were positive forβ-ⅢTubulin. Myocardial cells were obtained from BMSCs and appeared myotube-like cells, which were positive for CT3 and cardiacα-actin. Through RA-induction, the differentiated cells expressed the specific markers of pre-meiotic germ cells including VASA, SCP3 and CD49f, the positive rates of which were 35.7 %, 24 % and 14 % respectively. These results demonstrated that typical mesenchymal stem cells that isolated from dairy goat bone marrow possessed the characteristics of multipotent stem cells, and could lay the foundation for further experiments. 2. According to the protocol of transfection reagent of Fugene?HD, Lipofecter and Lipofectamine? 2000, our results show that Lipofectamine? 2000 were the best, with a efficiency of 44.88 %. Electro transfection was proceeded with the voltage model of 400 V/450 V/500 V/550/650 V/750 V, pulse duration of 3 s, pulse frequency of 3 times, pulse nterval of 1 min, which showed that when the voltage was lower than 500V,the higher voltage, the less alive cells. Higher voltage was beneficial to cell transfection, would lead to unexpected differentiation. In conclusion, transfection with reagents of Lipofectamine? 2000 was the best way to accomplish our experiments to transfect pStra8-EGFP into BMSCs.3. Transfection with Lipofectamine? 2000, a series of inducing condition including RA inducing, combination inducing(RA+GE) and GE monolayer inducing were carried out. These results showed that BMSCs expressed Stra8 and EGFP with RA inducing for 7 days. After RA inducing for 14 days, round cells expressed EGFP and sperm-like cells. In these induced cells, Stra8 and Prm1 could be detected in transcriptional level, and GFR-1, Stra8, VASA, SCP3, DZAL and ACR also could be discovered in protein level. Meanwhile, these induced cells retained the same cell cycle as dairy goat spermatogonia, These results showed that BMSCs could be induced to differentiate into spermatogonia in vitro, and had the hope of developing into meiosis anaphase.4. 25 mice with the defect of sperm production were used to transplant BMSCs into testicle by convoluted seminiferous tubule injection. 15 days, 30 days, and 90 days after transplantation, these mouse were executed by removing cervical to obtain testis. Testis from experiment group and control group were checked by weight, developmental stages of spermatogenic cells, and atrophy of convoluted seminiferous tubule. These data suggested that BMSCs could accelerate development and differentiation of sperm cells in sperm production defection mice 30 days after transplant. BMSCs labeled with CM-DIL also confirmed the conclusion in the same experiment, in which BMSCs were located in basal of convoluted seminiferous tubule under immunofluorescence observation. In addition, PCNA, Stra8 and GFRα-1 were expressed except ACR, indicating allograft BMSCs had the potential to differentiate into sperm cells. |
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