Research On Influence Factor Of Isolation, Purification And In Vitro Culture Of Goat Spermatogonial Stem Cell

Spermatogonial stem cells are the sole stem cells of germ cells in male animals, it has always been the international problem and focus of research at long-term culture in vitro. Current research has established the long-term culture system of spermatogonial stem cells in vitro of three species which are mice, rats and hamsters, however other species, especially domestic animals have not obtained ideal culture system. This subject have determined the optimum age of experimental animal through detecting the purification of isolated SSCs;after using mitomycin C and trypsin dealing with three kinds of cells(MEF、STO、Hela), detecting the stability of cell proliferation capacity to filtrate the best preparation condition of feeder layer; using serum and three types of exogenous additives(LBP、VC、6-BA) with different concentration for in vitro culture SSCs, detected of SSCs the number of colony and its proliferation. Ultimately determining the best way of goat SSCs in vitro culture.The research contents and results are as follows:1. The influence of goat ages on the effect of isolation and purification of SSCsCapturing 2-5 month boer goat testis respectively, regarding CDH1、UCHL1 and THY1 as identification of goats SSCs surface phenotype markers, by detection of different months of old goat testicles in a different orientation, m RNA and protein expression of molecular markers. Results showed that 2 months and 5 months of testis in the location expression of CDH1 and UCHL1; the expression quantity of CDH1 m RNA and protein was decreased with the age increased and the expression quantity of 2 months was the highest; the expression quantity of THY1 and UCHL1 was decreased after rising first and reached its highest at three months. This results indicated that 2 to 3 months of was the optimization age of isolation and purification of goat SSCs.2. The influence of treating time of MMC and trypsin on the effect of feeder layerCapturing three cells of MEF、STO and Hela for feeder layer. MEF was divided into six groups. After treated different time(0、1.5h、2h、2.5h、3h、3.5h)by mitomycin C with the concentration of 10μg/m L, every group was divided into 2 groups. One group called treating group was digested by 0.25% trypsin. The other group of no-treating was called no-treating group and detected the proliferation capacity at a different time. Regarding the stability of the proliferation capacity and significant differences between the groups as the basis of evaluation. Processing method as above of STO and Hela cells.The results showed the proliferation capacity of no-treating group was stable, but the proliferation capacity of treating group was not stable, suggesting that treating group was not conducive to the preparation of feeding layer of cells; In the no-treating group, MEF、STO and Hela cells with the time of 1.5h、2.5h and 2.5h treated by mitomycin C, the proliferation of cell was stable, and significant difference with the control group(P<0.05). This results indicated that no trypsin method was better and 10ug/m L mitomycin C treated 1.5 h of MEF cells, 2.5 h of STO cells, 2.5 h of Hela cells was the optimal option.3. The influence of serum concentration on the effect of culturing goat SSCs in vitroBy different serum concentration(0%、1%、2.5%、5%) addition to basis culture system in vitro culture of goat SSCs, regarding the number of SSCs colony and its proliferation capacity as the basis of evaluation. Results showed the number of SSCs colony at 5% serum group significantly higher than other groups during 3d(P<0.05), but the number of colony were greatly reduced with the increasing of time and proliferation decreased obviously. During the 6d and 10 d, 2.5% serum group significantly promoted the proliferation of SSCs and the number of colony was significantly higher than other groups(P<0.05), meanwhile, its proliferation rate was the fastest. This results indicated that the serum with high concentrations was not suitable for SSCs long-term culture in vitro, 2.5% serum culture system is the optimum option in vitro culture system.4. The influence of LBP concentration on the effect of culturing of goat SSCs in vitroBy different LBP concentration(0μg/m L 、 50μg/m L 、 100μg/m L 、 200μg/m L 、300μg/m L) addition to culture system of goat SSCs in vitro culture, regarding the number of SSCs colony and its proliferation capacity as the basis of evaluation. Results showed that the number of colony at 200μg/m L LBP group and 300μg/m L LBP group were significantly lower than the control group(P<0.05)and the number of colony at 100μg/m L LBP group、200μg/m L LBP group and 300μg/m L LBP group was decreased with the increasing of the time. During the culture of 6d and 10 d, the number of colony at 50μg/m L LBP group was the most and the speed of proliferation was the fatested. This results indicated that high concentration LBP inhibited the proliferation of SSCs. 50μg/m L LBP addition for SSCs in vitro culture was the optimal option.5. The influence of VC concentration on the effect of culturing goat SSCs in vitroBy different VC concentration(0μg/m L、10μg/m L、20μg/m L、30μg/m L、40μg/m L)addition to culture system in vitro culture of goat SSCs, regarding the number of SSCs colony and its proliferation capacity as the basis of evaluation. Results showed the trend of the number of colony at each group was falling after rising with the increasing of the time; the number of colony increased with the rising of the concentration, and significant difference between groups at 6d and 10d(P<0.05), the proliferation capacity of 40μg/m L VC group was lowest than other group at 7d and 10d(P<0.05), the proliferation trend of 30μg/m L VC group was gentle, but other groups was falling trend, the number of colony of 30μg/m L VC group was significant higher than lower concentration group(P<0.05). This results indicated that 40μg/m L VC group was not suitable for SSCs long-term culture in vitro, 30μg/m L VC addition for SSCs in vitro culture was the optimal option.6. The influence of 6-BA concentration on the effect of culturing goat SSCs in vitroBy different 6-BA concentration( 0ng/m L 、 25ng/m L 、 50ng/m L 、 75ng/m L 、100ng/m L、125ng/m L)addition to culture system in vitro culture of goat SSCs, regarding SSCs clone number and its proliferation capacity as the basis of evaluation. Results suggested that the number of colony at 100ng/m L 6-BA group were higher than other groups significantly at 6d and 10d(P<0.05), but the proliferation capacity of treatment group were lower than the control group, this suggested that 6-BA was not suitable for SSCs long-term culturation in vitro. This results indicated that 100ng/m L 6-BA addition for SSCs in vitro culture was the optimal option.From what has been discussed above, the best conditions of in vitro culture of goat SSCs in this experiment was selecting 2-3 months goats testicles to separate SSCs, meanwhile, Utilizing the MEF feeder layer by no trypsin digestion treatment and the serum with 2.5% concentration can significantly improve the proliferation capacity of SSCs. Furthermore, adding 50μg/m L LBP or 30μg/m L of VC or 100ng/m L 6-BA respectively can effectively promote the in vitro culture proliferation of goat SSCs, and adding 30μg/m L VC promote the most significant of culture effect.