The Effect Of The Culture In Vitro And Estradiol On Hair Follicles Bcl-2 And Bax Expression Of Cashmere Goat

The hair follicles of cashmere goat is the growing organ of cashmere and wool. They are directly responsible for the quality of cashmere. So it is of great significance to study the elements which influence the growth and declining g of hair follicles for the growth of the wool and hair follicles. As there are various factors which could effect the growth of the hair follicles, it is no good to study single factor. As to this, a culturing model of isolated hair follicles in vitro is built to avoid being effected by the others complex factors in viro. The purpose of research is to study the factors of controlling and affecting the hair follicles growth through looking for the best condition for culturing hair follicles and studying hair follicles biology.First of all, the paper, based on organ culture of cashmere goat's hair follicles in vitro, compared the effects of different culturing medias on hair follicles culture; secondly, tissue and digestion methods are applied to separate, culture, freeze and multiply the dermal papillas; thirdly, characteristic of different hair follicle tissue structure in different stages are observed through culturing isolated cashmere goat's primary follicles and sheep's hair follicles. In addition, the model of culturing cashmere goat'hair follicles in vitro were set up to study the effect of estrogen on biological character of cashmere goat'hair follicles in vitro. At last, from the perspective of the molecular biology piece of cashmere goat's skin which was cultured in vitro were used to study the effects of estrogen on controlling the Bcl-2, Bax genes. The results are as follows:1. With the reference to the improved traditional separation methods and philpott[55] method , the cashmere goat's skin was cut into the pieces with the size about (3-5)mm×10mm (only single hair follicle included) by combining mirco-dissection with incision cutting method. When the width of the hair follicles are appropriate more isolated hair follicles could be got. The hair follicles which had integrated outer root sheath, smooth form of the dermal papilla were selected to culture. At the same time, it was believed that 2-3mm is most appropriate lenth of hair follicle to grow and be observe in order to facilitate observation and measurement, as well as growth.2. After have been successful to culture isolated hair follicles in vitro, it was found that primary follicles in DMEM medium supplement with 10% fetal calf serum were growing slowly and they began to distort early. In addition, they started to stick to the wall of medium about fourth day later. When primary follicle cultured in serum-free DMEM medium, its growth period lengthened distinctly and its morphologic structure kept well, with the same growth rate as that culture in serum-containing DMEM medium between two conditions from second day(p>0.05); The growth speed of primary follicles was accelerated obviously in the first 5 days due to the using of Williams E medium. However , the growth speed of primary follicle in Williams E serum-free and transferring-free medium was slower than it in Williams E serum-free medium, but their survival time was not significantly different. It was proved that transferring can contribute to hair follicle growth and maintain its pattern. The experiment has proved as well that Williams E serum-free medium is more suitable to culture cashmere goat's hair follicle in vitro.3. For the cashmere goat's secondary follicles which were cultured in williams E serum-free medium the average growth rate was 0.03mm/d in first 3 days. It was close to average growth rate(0.056mm/d) of cashmere goat's cashmere at the same stage, but it only maintained a short time.4. Primary follicles which grew in different stages were cultured in vitro. It was found that the average growth rate of hair follicles in November was 0.08mm/d , which was same with that (0.08mm/d) of primary follicles in viro. Furthermore, the max growth rate (0.09mm/d) of growing in vitro was faster than that (0.087mm/d) of growing in body. In contrast to the fact that the activities of the hair follicles have reached the peak in September, the growing situation is even better than that in September. It can be concluded that activities of hair follicle is related to the temperature.5. The combining of the collagenase digestion and the mechanical separation method to separate primary hair dermal papilla of inner Mongolia goats in anagen and cultured them successfully. Observed with inverted microscope, it was fond that the sharp of hair dermal papilla was fibroblast and aggregative behavior characteristic.6. Compare the growth of the isolated primary follicles of the cashmere goat and that of the sheep. The daily average growth speed of the hair follicles in sheep are obviously faster than those of cashmeres. But the steady level could be maintained at the first 3 days, and the speed was similar to that in the body. With the extension of the cultivating, the growth speed of the sheep hair follicles dropped sharply. For the primary follicles, the growing remained stable in the first 9 days. The changes in patterns to be regular. It indicated that the Williams E serum-free medium was helpful to the growth of the hair follicles of both cashmere and sheep with the temperature of 31 Celsius.7. The 17βestradiol failed to facilitate the growth of primary follicles. There was no significant difference in growth speed between the 0.1nmol/L and the control group(P>0.05), which may be caused by the sane viscosities as the biological viscositiese within the body(0.05～0.19nmol/L[244]). At the same time, the growth speed in the first 10 days was a little faster than that of the control group. But the lower part of the hair follicle began to bend at the later period of the cultivating. Its growth were apparently suppressed with the increase of the viscosities of 17-βestradiol(P<0.01). Most of the hair follicles stopped growing and shuntd back when it came to the ninth days.8. By adding different viscosities of 17-βestradiol, the controlling genes Bcl-2,Bax were examined through the method of real time PCR after cultivating 24h,48h,72h,96h,120h respectively. Quantities of the Bcl-2mRNA in different hair follicle groups decreased with the increase of the viscosities of estradiol. It should be noted that the quantities of Bax mRNA in each group had a trend of going up first and then falling down. The estradiol can make the rate of Bcl-2/Bax mRNA drop down, but the there was no significant difference in ratio. It could be concluded that the estradiol can facilitate the contabescence of the hair follicle.