| Hair follicles are a part of skin that included hair shaft, inner root sheath and outer root sheath. In the present study, in vitro cultured the hair follicles isolated from sheep skin, and observed morphological diversification of earlier stage, intermediate stage and later stage cultured hair follicles using inverted microscope. Measured length of hair follicles by ocular micrometer and observe hair follicles histological character, determined influencing factor in cultured hair follicles growth and periodicity regeneration, thereby established a best culture method to hair follicles. Whereafter, added 17-βestradiol in culture medium, and determined the relationship of expression of Bcl-2 and Baxgene that correlated with cells apoptosis in hair follicles with 17-βestradiol.1. Evaluation of growing period hair follicles The whole hair follicle successfully isolated from sheep skin, using microdissection and enzyme digestion methods. The hair follicles evaluated by Histological and immunohistological studies, the hair papilla shown as ellipse construction with integrity hair shaft, inner root sheath and outer root sheath, hair matrix cells are compacted together and shown as onion longitudinal section construction (Fig 2-21～2-23). Therefore, culture the growing period hair follicles, and by way of a study model, investigate the relationship of expression of Bcl-2 and Baxgene with hormones. Non-growing period hair follicles shown blur and non-immobility construction without hair shaft, inner root sheath and outer root sheath. All of the hair follicles used in present study are growing period hair follicles.2. Establishment of in vitro culture sheep hair follicles Culture growing period hair follicles in different environment and different culture medium, and determined a best culture environment as follow. The isolated hair follicles are cultured in non-serum Williams E medium containing L-glutamine (2mmol/l), Hepes (2mmol/l), insulin (10μg/ml), transferring (10μg/ml), hydrocortisone (10μg/ml), sodium selenite (10ng/ml), penicillin (100u/ml), streptomycin (100μg/ml) at saturated humidity, 5% CO2 and 31℃.3. In this culture environment, the hair follicle has sown rapid growing on 1～10 days, and averaged length of growing is 0.36 mm/d. survival rate achieved 87.25%.Generally, the hair follicles can not occurring adherence on 15～20 days, but hair papilla compacted together and hair follicles became slender in later stage of growing. After 22 day, hair follicles happened adherence, epithelial cells emigrated from outer root sheath, growing of hair follicles became slowness, exhibited clearly aapoptosis stage that morphological gradually nigrescented hair bulb to hair shaft and outer root sheath was divergence to forth, but hair follicles was survivorship yet.4. Effect of 17-βestradiol on hair follicles growth After culture hair follicles 1 day, added 17-βestradiol in non serum Williams E medium, investigated the effect of 17-βestradiol on hair follicles growth. Results shown added the17-βestradiol has the function of inhibiting the growth of hair follicles , but in the specific concentration (10-8 mol/l) the growth is better than negative control, the reasion is that this concentration is close to the normal growth concentration. In this time , the in vitro culture hair follicles growth environment is close to the stable condition in which the hair follicles was growing. So the growth is good. 10-10～10-9mol/l and 10-7mol/l 17-βestradiol has the function of inhibiting the growth of hair follicles.5. Effect of 17-βestradiol on expression of Bcl-2 and Baxgene In this study, culture hair follicles with 17-βestradiol, and determined physiological roles of 17-βestradiol on expression of Bcl-2 and Baxgene in hair follicles by real time PCR. (1) Bcl-2 gene expression was shown culture day dependent decrease in 1～6 days culture. Bcl-2 gene expression was most great in 10-8 M cultured hair follicles, lower than the negative control and the tendency graph was like"∧"between 10-7 M and 10-10 M of ESG concentration. The control group significantly compared with others (p<0.01). (2) Bax gene expression was shown similar pattern with Bcl-2 gene, also like"∧"between 10-7 M and 10-10 M of ESG concentration.But the expression was very low. (3) Bcl-2/Bax expression ratio was significantly differed in 1～6 days cultured hair follicles, 10-8 M ESG was very significantly with others (the negative control group included, P<0.01).(4) In this study, expression of Bcl-2 and Baxgene shown similar pattern in 1～6 days cultured hair follicles, Bcl-2/Baxratio coincident with morphology of hair follicles in vitro . The result of the cultured hair follicles in vitro indicate both the Bcl-2 and Baxgene are concerned with apoptosis process.
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