【作者】 丁月云；

【导师】 王恬；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2008， 博士

【摘要】 本论文研究了中草药黄芪、王不留行对奶牛乳腺上皮细胞体外增殖和乳蛋白合成的影响,并对其具体机制作了初步探索。首先建立奶牛乳腺上皮细胞的体外培养体系,培养出具有正常生理功能的奶牛乳腺上皮细胞,并以该细胞系为模型,筛选对乳腺细胞体外生长有促增殖效应的中草药,确定其适宜的作用浓度和作用时间,在此基础上,进一步研究中草药王不留行、黄芪对乳腺细胞一系列生物活性的影响及其作用机理。本研究包括五个部分。1奶牛乳腺上皮细胞体外培养体系的建立采用组织块培养法、联合应用差酶消化法和反复贴壁法建立体外奶牛乳腺上皮细胞培养体系,采用透射电镜、免疫组化、免疫印迹等试验技术方法对乳腺上皮细胞特性进行检测。试验结果表明,运用本方法建立的奶牛乳腺细胞系上皮特性及分泌功能特征完备。2七味中草药粗提液对奶牛乳腺上皮细胞促增殖作用的比较研究研究比较七味中草药对奶牛乳腺上皮细胞体外生长的促增殖效应。确定黄芪、王不留行、当归、川芎、栝楼、漏芦和续断粗提物为备筛药物,采用2-3代奶牛乳腺上皮细胞（CMEC）为试验材料,在细胞培养液中添加不同浓度的上述中草药提取液,采用MTT比色法,比较其对细胞增殖的促进作用。试验结果表明,与对照组相比,浓度分别为10 mg/mL的王不留行,20 mg/mL的黄芪,10 mg/mL+20 mg/mL的王不留行黄芪混合物均可极显著地促进乳腺上皮细胞生长（p<0.01）,尤以10 mg/mL+20mg/mL的王不留行黄芪配伍组作用效果最好。3王不留行与黄芪促进奶牛乳腺上皮细胞增殖调控机理的初步研究试验以2-3代奶牛乳腺上皮细胞（CMEC）为试验模型,采用流式细胞仪、免疫组织化学染色法,检测王不留行（10 mg/mL）、黄芪（20 mg/mL）、王不留行黄芪配伍（10 mg/mL+20 mg/mL）对乳腺上皮细胞增殖周期分布、ClycinD1、p27蛋白在乳腺上皮细胞内阳性表达的影响。试验结果表明,与对照组相比,浓度分别为10 mg/mL的王不留行,20 mg/mL的黄芪,10 mg/mL+20 mg/mL的王不留行黄芪配伍均可促进乳腺上皮细胞细胞周期G1/S期转换（p<0.01）,增加细胞周期中S期细胞的数目（p<0.01）,CyclinD1的阳性表达均得到上调（p<0.01）,p27的阳性表达均下降（p<0.01）,尤以王不留行黄芪配伍作用明显。结果提示,黄芪、王不留行促进乳腺细咆的增殖,是通过上调CyclinD1表达、下调p27表达水平,进而加速细胞周期G1/S期的转换完成的。4王不留行与黄芪对奶牛乳腺上皮细胞体外分泌性能的影响本试验研究了中草药黄芪、王不留行对奶牛乳腺上皮细胞体外分泌性能的影响。以2-3代奶牛乳腺上皮细胞（CMEC）为试验模型,研究中草药黄芪、王不留行作用前后细胞内部超微结构的变化、细胞裂解液中氨基酸总浓度及组分的变化规律,以及细胞裂解液中蛋白总浓度的变化。试验结果表明,与空白对照组相比,王不留行（10mg/mL）、黄芪（20 mg/mL）、王不留行与黄芪配伍（10 mg/mL+20 mg/mL）作用于细胞后,细胞内部超微结构发生了重塑,表现出更多功能分泌分化的特征标志;细胞裂解液中氨基酸的总浓度均得到提高（p<0.01）,细胞裂解液中游离氨基酸的总浓度均显著或极显著地提高（王不留行（10 mg/mL）、王不留行黄芪配伍（10 mg/mL+20mg/mL）,p<0.01;黄芪（20 mg/mL）,P<0.05;）;细胞裂解液中蛋白总含量亦有明显的增高,（王不留行（10 mg/mL）、王不留行黄芪配伍（10 mg/mL+20 mg/mL）,p<0.01;黄芪（20 mg/mL）,P>0.05;）。结果提示,中草药黄芪、王不留行可通过对乳腺细胞内部超微结构的重塑、促进乳腺细胞吸收胞外游离氨基酸,使乳腺细胞蛋白合成增多。5王不留行与黄芪对奶牛乳腺上皮细胞合成乳铁蛋白和酪蛋白的影响本试验以2-3代奶牛乳腺上皮细胞（CMEC）为试验模型,应用荧光定量RT-PCR技术研究了中草药王不留行、黄芪对乳腺细胞a_s1-酪蛋白、β-酪蛋白和乳铁蛋白基因表达的影响;应用ELISA竞争抑制性法检测,王不留行、黄芪作用前后乳腺细胞裂解液中a_s1-酪蛋白、β-酪蛋白和乳铁蛋白含量的变化。试验结果表明,与空白对照组相比,王不留行黄芪配伍（10 mg/mL+20 mg/mL）、王不留行（10 mg/mL）均极显著提高a_s1-酪蛋白mRNA的表达（p<0.01）,黄芪（20 mg/mL）组细胞a_s1-酪蛋白mRNA表达稍低于空白对照组,差异不显著（P>0.05）;王不留行黄芪配伍（10 mg/mL+20mg/mL）、王不留行（10 mg/mL）均显著提高β-酪蛋白mRNA的表达（p<0.05）,黄芪（20 mg/mL）组细胞β-酪蛋白mRNA表达稍高于空白对照组,差异不显著（P>0.05）;王不留行黄芪配伍（10 mg/mL+20 mg/mL）显著提高乳铁蛋白mRNA的表达（p<0.05）,王不留行（10 mg/mL）组和黄芪（20 mg/mL）组细胞乳铁蛋白mRNA的表达稍高于空白对照组,差异不显著（P>0.05）。a_s1-酪蛋白合成量的检测结果表明,黄芪（20mg/mL）+王不留行（10 mg/mL）配伍作用效果较明显,其组内乳腺细胞a_s1-酪蛋白合成量显著高于空白对照（P<0.05）;而黄芪（20 mg/mL）组和王不留行（10 mg/mL）组相比较于空白对照组,组内乳腺细胞a_s1-酪蛋白合成量略有增高,差异不显著（P>0.05）。β-酪蛋白合成量的检测结果表明,黄芪（20 mg/mL）+王不留行（10 mg/mL）配伍组和王不留行（10 mg/mL）组细胞β-酪蛋白合成量显著高于空白对照组（P<0.05）,黄芪（20 mg/mL）组细胞β-酪蛋白合成量略高于空白对照组,但差异不显著（P>0.05）。乳铁蛋白合成量的检测结果表明,与空白对照组比较,中草药作用细胞后,乳腺细胞乳铁蛋白合成量均略有增高,但差异不显著（P>0.05）。以上五个试验部分的研究结果表明:中草药黄芪、王不留行促进乳腺细胞a_s1-酪蛋白、β-酪蛋白和乳铁蛋白的合成,其潜在机制与其能够促进乳腺上皮细胞增殖、促进乳腺上皮细胞内部超微结构重塑、促进乳腺上皮细胞吸收胞外游离氨基酸密切相关。更多还原

【Abstract】 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.更多还原