Studies On The Effects Of Extracts Of Semen Vaccariae, Astragalus Membranaceus On The Proliferation Rules And Secretion Function Of Cow Mammary Epithelail Cells Cultured In Vitro

The aim of this study is to reveal the regulation mechanisms of the effects of Semen Vaccariae,Astraglus membranaceus on the proliferation rules and secretion function of cow mammary epithelial cells cultured in vitro.Firstly,a cow mammary epithelial cells (CMEC) culture system was established,and based on the optimized culture of CMEC with normal physiological functions,experiments were conducted to select the herbal medicines which have the ability to increase the number of CMEC by increasing growth rate or longevity,and detected their perfected reaction concentrations and reaction time.Based on the selected results,the effects of Semen Vaccariae and Astraglus membranaceus on the biology characteristics of CMEC and their underlying regulation mechanisms were studied.The studies included five sections presented as follows:1 Establishment of a cow mammary epithelial cell line cultured in vitroTo establish an in vitro culture system in which cow mammary epithelial cells(CMEC) grow well,the mammary tissue clumps from Holstein cows in lactation period at adulthood were cultured to harvest epithelial cells.The cell morphological varieties,cell population doubling time,and the cell growth curve were observed and investigated.The epithelial character of CMEC was authenticated using immunity histochemistry technique;the lactation function of the cells was detected by SDS-PAGE and western-blot.The results showed that the cow MEC grew well and proliferated vigorously in this culture system;The celluar skeletal protein18 expressed positively,the electrophoresis and western blot analysis of the products of the cells showed that the cells cultured had a function of expressing caseins.2 The comparative study on stimulative effects of crude exacts from seven herbal medicines on the proliferation of CMECTo study and compare the effects of seven traditional medicine crude extracts on the proliferation of CMEC,and to investigate the mechanisms,Astragalus membranaceu, Semen Vaccariae,Angelica Sinensis,Linguisticum chuanxiong Hort,trichosanthis,fructus et semen,Rhaponticum Uniflorum,Himalayan Teasel Root were selected as experimental drugs,the effects of crude extracts in different concentrations on the proliferation of the second -third passage cells of CMEC were observed by MTT assay.The results showed that: The herbs of Semen Vaccariae(SV),Astragalus membranaceus(AM) and the commixture of SV and AM(SV+AM) demonstrated proliferative activities in CMEC.The highest potency was detected in SV(10 mg/mL),AM(20 mg/mL) and SV+AM(10 mg/mL+20 mg/mL) compared with the control group(p＜0.01),moreover the commixture of SV+AM (10 mg/mL+20 mg/mL) developed the best role.3 The preliminary study on the mechanisms of the effects of Semen Vaccariae,Astraglus membranaceus on the proliferation and differentiation of CMECTo investigate the mechanisms of the effects of Semen Vaccariae(SV),Astragalus membranaceus(AM)on the proliferation and differentiation of CMEC,the second-third passage cells of CMEC were selected as experimental subjects,flow cytometer was used to observe the changes of cell cycle of CMEC after being cultured with SV(10 mg/mL),AM (20 mg/mL) and SV+AM(10 mg/mL+20 mg/mL),and the expression of CyclinD1 and p27 at protein level were detected by using immunohistochemical staining techniques.The results showed that:(1) Compared with the control group,in the cells of the three treatment groups of SV(10 mg/mL),AM(20 mg/mL) and SV+AM(10 mg/mL+20 mg/mL),flow cytomery analysis showed a most significant increase number of cells in S phase(p＜0.01), and a promotion in CMEC's changing from G1 phase to S phase;(2) The positive expression rate of CyclinD1 was increased and p27 expression was decreased in the groups treated with SV(10 mg/mL)(p＜0.01),AM(20 mg/mL)(p＜0.01),and the commixture of SV and AM(10 mg/mL+20 mg/mL)(p＜0.01),compared with the control group.Moreover the commixture of SV+AM(10 mg/mL+20 mg/mL) developed the best role.These results suggested that:Physiological concentrations of aqueous extracts of SV,AM may promote CMEC proliferation via increasing the expression of CyclinD1 and decreasing the expression of p27,thereby changing the cell proliferation cycle,could stimulate the growth and differentiation of CMEC.4 The effects of Semen Vaccariae,Astraglus membranaceus on the secretion performance of CMEC cultured in vitroTo study and compare the effects of Semen Vaccariae(SV),Astragalus membranaceus (AM)on the secretion perfermance of CMEC,the second-third passage cells of CMEC were selected as experimental subjects,changes of ultra-structural morphology,total content and compositions of amino acids in CMEC's lysate,and total content of proteins in CMEC's lysate were observed after CMEC being cultured with SV(10 mg/mL),AM(20 mg/mL) and SV+AM(10 mg/mL+20 mg/mL) for 48 h,respectively.The results showed that:after being incubated with herbal medicines for 48 h,compared with the control groups,(1) the CMEC's ultra-structural morphology were remolded,exhibiting more characteristics of secretion function and differentiation signs;(2) the total contents of amino acids in CMEC's lysate were most significantly increased(p＜0.01),the total contents of free amino acids in CMEC's lysate were more or most significantly increased(SV(10 mg/mL),SV+AM(10 mg/mL+20 mg/mL),p＜0.01;AM(20 mg/mL),p＜0.05);(3) the total contents of proteins in CMEC's lysate were also increased obviously(SV(10 mg/mL),SV+AM(10 mg/mL +20 mg/mL),p＜0.01;but,AM(20 mg/mL),p＞0.05).These results suggested that:Physiological concentrations of aqueous extracts of SV,AM may promote the synthesis of milk proteins via remolding the ultra- structural morphology of CMEC,stimulating the absorption of free amino acids in CMEC.5 The effects of Semen Vaccariae,Astraglus membranaceus on the synthesis of caseins and lactoferrin in CMECTo study and compare the effects of Semen Vaccariae(SV),Astragalus membranaceus (AM)on the synthesis of caseins and lactoferrin in CMEC,the second-third passage cells of CMEC were selected as experimental subjects,the expression of a_s1-casein,β-casein, lactoferrin mRNA in CMEC's lysate were detected by using RT-PCR,the total contents of a_s1-casein,β-casein,lactoferrin in CMEC's lysate were detected by using ELISA.The results showed that:(1) Compared with the control group,the expression of a_s1-casein mRNA in the CMEC's lysate of the groups of SV(10 mg/mL),SV+AM(10 mg/mL+20 mg/mL)were most significantly increased(p＜0.01),the expression of a_s1-casein mRNA in the CMEC's lysate of the group of AM(20 mg/mL)was decreased(p＞0.05);the expression ofβ-casein mRNA in the CMEC's lysate of the groups of SV(10 mg/mL),SV+AM(10 mg/mL+20 mg/mL)were more significantly increased(p＜0.05),the expression ofβ-casein mRNA in the CMEC's lysate of the group of AM(20 mg/mL) was increased too,but there was no significant difference(P＞0.05);the expression of lactoferrin mRNA in the CMEC's lysate of the group of SV+AM(10 mg/mL+20 mg/mL)was more significantly increased (p＜0.05),the expression of lactoferrin mRNA in the groups of AM(20 mg/mL),SV(10 mg/mL) were also increased,but there were no significant difference(P＞0.05);(2) Compared with the control group,the total contents of a_s1-casein in CMEC's lysate in the group of SV+AM(10 mg/mL+20 mg/mL) was more higher(p＜0.01),the total contents of a_s1-casein in the groups of AM(20 mg/mL),SV(10 mg/mL) were higher,but there were no significant difference(P＞0.05);the total contents ofβ-casein in CMEC's lysate in the groups of SV+AM(10 mg/mL+20 mg/mL),SV(10 mg/mL)were more higher(p＜0.01), the total contents ofβ-casein in the group of AM(20 mg/mL) was higher,but there was no significant difference(P＞0.05);the total contents of lactoferrin in CMEC's lysate in the groups of SV+AM(10 mg/mL+20 mg/mL),SV(10 mg/mL),AM(20 mg/mL) had the tendency to be higher but had no significant difference(P＞0.05).Collectively,the results accomplished in this work showed that the physiological concentrations of aqueous extracts of SV,AM promoted significantly the synthesis of a_s1-casein,β-casein,lactoferrin in CMEC,which may be closely related with the effects of SV,AM on stimulating the proliferation of CMEC,facilitating the re-mold of ultrastructural morphology in CMEC,enhancing the absorption of free amino acids of CMEC.