| In this study, dairy cow mammary epithelial cells were cultured, and the cultured cells were used for the study of the effect of Chinese herb medicine transdermal patch and Chinese herb medicine perfusion fluid on dairy cow mammary epithelial cell proliferation, total protein content and β-casein content. The breast tissue was taken from udder aseptically, and a stable dairy cow mammary epithelial cell line was established by using primary culture in vitro, the separation and purification methods. The dairy cow mammary epithelial cells were observed by the inverted microscope for morphological identification. The cell growth curve was drawn and the P-casein content was determined by SDS-PAGE electrophoresis. The Chinese herb medicine transdermal patch and its main single herbal extracts and the Chinese herb medicine perfusion fluid and its main single herbal extracts were prepared in advance, then added into the medium of dairy cow mammary epithelial cell, the final objective was to study the proliferation of dairy cow mammary epithelial cells and the effect of Chinese herb medicine extracts on the total protein content and the β-casein content.The results were as follows:Dairy cow mammary cells were obtained through tissue culture, the dairy cow mammary fibroblast cells could be basically removed by trypsin digestion repeatedly, and the purified dairy cow mammary epithelial cells were obtained. The morphological and physiological function of dairy cow mammary epithelial cells did not change after passaging, so a stable dairy cow mammary epithelial cell line was established finally.The dairy cow mammary epithelial cells were treated with different concentrations of Chinese herb medicine perfusion fluid and its single herbal extracts for 1 day, the maximum OD value was detected at the concentration of 10 mg·mL-1, but the effect of promoting cell proliferation was not significant (P>0.05). The dairy cow mammary epithelial cells were treated with 10 mg·mL-1 of herbal extracts for 6 days, and compared with control group, Herba Violae Philippicae and Radix Astragali Mongolici had no obvious effect on cell proliferation (P>0.05), but Herba Lycopi Hirti and Caulis Spatholobi had obvious effect on cell proliferation on the 3rd day (P＜0.01), while Chinese herb medicine perfusion fluid had obvious effect on cell proliferation on the 5th day (P＜0.01).The dairy cow mammary epithelial cells were treated with different concentrations of Chinese herb medicine perfusion fluid and its single herbal extracts for 1 day, the maximum total protein value was detected at the concentration of 15 mg·mL-1. The dairy cow mammary epithelial cells were treated with 15 mg-mL"1 of herbal extracts for 6 days, and compared with control group, Herba Violae Philippicae had no obvious effect on the total protein content (P>0.05); Herba Lycopi Hirti and Caulis Spatholobi and Chinese herb medicine perfusion fluid had obvious effect on the total protein on the 1st day (P＜0.01), and the total protein content increased over the time; Fructus Chebulae had obvious effect on cell proliferation on the 1st day (P＜0.01), and compared to the total protein content of the 1st day, the total protein content of the 3rd day was significantly decreased (P<0.01), compared to the total protein content of the 3 rd day, the total protein content of the 5th day was significantly increased (P＜0.01).The dairy cow mammary epithelial cells were treated with different concentrations of Chinese herb medicine perfusion fluid and its single herbal extracts for 1 day, the maximum (3-casein content value was detected at the concentration of 10 mg-mL"1. The dairy cow mammary epithelial cells were treated with 10 mg·mL-1 of herbal extracts for 6 days, and compared with control group, Herba Violae Philippicae, Radix Astragali Mongolici, Herba Lycopi Hirti, Fructus Chebulae and Chinese herb medicine perfusion fluid could very significantly promote the (3-casein content on the 1st day (P＜0.01), Caulis Spatholobi could significantly promote the (3-casein content on the 1st day (0.01＜P＜0.05). Compared to the P-casein content of the 1st day, the P-casein content of the 3rd day in all the herbal groups had no obvious difference (P>0.05). Compared to the β-casein conent of the 3rd day, the β-casein conent of the 5th day in the group of Chinese herb medicine perfusion fluid had great difference (P＜0.01), had difference (0.01＜P＜0.05) in the groups of Radix Astragali Mongolici, Herba Lycopi Hirti, Fructus Chebulae, and had no difference in the groups of Herba Violae Philippicae, Caulis Spatholobi. (P>0.05)The dairy cow mammary epithelial cells were treated with different concentrations of Chinese herb medicine transdermal patch and its single herbal extracts for 1 day, the maximum OD value was detected at the concentration of 10 mg·mL-1, but the effect of promoting cell proliferation was not significant (P>0.05). The dairy cow mammary epithelial cells were treated with 10 mg·mL-1 of herbal extracts for 6 days, and compared with control group, Rhizoma Paridis Chonglou had significant effect on cell proliferation on the 5th day (0.0 1＜P＜0.05), Chinese herbalmedicine transdermal patch had very significant effect on cell proliferation on the 5th day (P＜0.01), but Rhizoma Coptidis, Hibiscus mutabilis L, Herba seu Radix Kalimeridis Indicae had no significant effect on cell proliferation (P>0.05).The dairy cow mammary epithelial cells were treated with different concentrations of Chinese herb medicine transdermal patch and its single herbal extracts for 1 day, the maximum total protein value was detected at the concentration of 10 mg·mL-1. The dairy cow mammary epithelial cells were treated with 10 mg·mL-1 of herbal extracts for 6 days, and compared with control group, Rhizoma Paridis Chonglou had obvious effect on cell proliferation on the 5th day (P＜0.01), Rhizoma Coptidis, Herba seu Radix Kalimeridis Indicae and Chinese herb medicine transdermal patch had obvious effect on cell proliferation on the 1st day (P＜0.01), and Hibiscus mutabilis .had obvious effect on cell proliferation on the 3rd day (P＜0.01).The dairy cow mammary epithelial cells were treated with different concentrations of Chinese herb medicine transdermal patch and its single herbal extracts for 1 day, the maximum β-casein content value was detected at the concentration of 10 mg·mL-1, but the effect of promoting β-casein content was not significant (P>0.05). The dairy cow mammary epithelial cells were treated with 10 mg·mL-1 of herbal extracts for 6 days, and compared with control group, Rhizoma Paridis Chonglou, Hibiscus mutabilis L. and Chinese herbal medicine transdermal patch had no significant effect (P>0.05) on the 1st day. Compared to the P-casein content of the 1st day, the P-casein content of the 3rd day in the groups of Rhizoma Paridis Chonglou, Hibiscus mutabilis L. and Chinese herbal medicine transdermal patch had great difference (P＜0.01), compared to the β-casein conent of the 3rd day, the P-casein conent of the 5th day had great difference (P＜0.01). Rhizoma Coptidis, Herba seu Radix Kalimeridis Indicae had no obvious effect on the β-casein content of dairy cow mammary epithelial cells.The results showed that the dairy cow mammary epithelial cells proliferation and secretion of total protein and β-casein could be significantly promoted by adopting appropriate concentration of herbal extracts. The experiment indicated that Chinese herb medicine perfusion fluid and Chinese herb medicine transdermal patch cauld repair the damaged breast cells, so as to improve the symptoms of mastitis in dairy cows. |
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