二、三维培养模式对奶牛乳腺上皮细胞泌乳代谢相关基因的影响
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摘要
本文主要进行了奶牛乳腺上皮细胞（BMEC）三维培养体系的建立，在此基础上通过不同的试验条件下（全营养因素处理、单一氨基酸处理、催乳素处理）比较二、三维养模式对奶牛乳腺上皮细胞泌乳代谢相关基因的影响。同时利用优化的培养模式进行支链氨基酸和催乳素影响乳蛋白基因转录的的研究。通过以上研究，为BMEC三维培养体系的建立和支链氨基酸、催乳素调控BMEC乳蛋白基因转录提供一定的理论基础。试验结果如下：
1、BMEC在Matrigel基质胶中可以快速的形成类腺泡结构，而在鼠尾胶中则形成散落的聚集物。且在培养的过程中Matrigel基质胶比鼠尾胶稳定。三维模式下培养时间和接种密度影响BMEC中αs1-酪蛋白、β-酪蛋白、κ-酪蛋白的基因表达量。三维模式下BMEC在培养的第5d形成了完全成熟的腺泡，三维模式下BMEC的适合接种密度为1x106个/mL。
2、在相同的营养物质及激素水平下，三维培养的BMEC三种乳蛋白的基因表达量显著高于二维培养（P＜0.05），三维培养的BMEC通过western blot的方法能够检测到β-酪蛋白，二维培养的BMEC检测不到β-酪蛋白。三维培养的BMEC在形态上呈现的特点与BMEC在体内所呈现的组织学特征更加接近。二维培养模式下添加催乳素BMEC的αs1-酪蛋白和κ酪蛋白基因的表达量显著高于未添加催乳素组（对照组）（P＜0.05），添加催乳素BMEC的α-乳清蛋白的表达量显著低于对照组（P＜0.05），添加催乳素BMEC的β-酪蛋白基因的表达量和对照组间无显著差异（P＞0.05）。三维培养模式下，添加催乳素BMEC的αs1-酪蛋白和κ酪蛋白基因的表达量和对照组间无显著差异（P＞0.05），添加催乳素BMEC的β-酪蛋白，α-乳清蛋白的表达量显著高于对照组（P＜0.05）。
3、在三维培养模式下，添加leu、ile、val使奶牛乳腺上皮细胞αs1-酪蛋白、β-酪蛋白、κ-酪蛋、mTOR、S6K1、4E-BP1、eIF4E基因的表达量显著高于对照组（P＜0.05）。
本试验表明，在奶牛泌乳代谢的研究中，BMEC的三维培养技术优于二维培养。
The main purposes of the experiments was to investigate the effects oftwo-dimensional culture and three-dimensional culture on the gene expression of lactationmetabolism of bovine mammary epithelial cells (BMEC) under different trial conditionwhich included the factors of full nutritional factors, single branched amino acid, hormone.the results were as follows:
1. BMEC in Matrigel collagen could rapidly form acinar-like structure, but it formedscattered aggregates in rattail collagen. Furthermore, Matrigel collagen was more stablethan rattail collagen in culture process.The incubation time and the inoculum density ofthree-dimensional culture influenced the gene expression of αs1-casein, β-casein, andκ-casein in BMEC. BMEC of three-dimensional culture formed full ripe acinar-likestructure at5d after incubation, and the optimum inoculum density was1x106/mL.
2. On the same nutrients and hormones conditions, the gene expression of αs1-casein,β-casein, and κ-casein in BMEC of three-dimensional culture were significantly higherthan that in two-dimensional culture (P＜0.05), and β-casein in three-dimensional cultureof BMEC could be detected by western blot, but it was not two-dimensional culture ofBMEC.The morphological characters in BMEC of three-dimensional culture were moresimilar to BMEC in vivo.Under two-dimensional culture,the gene expression of αs1-caseinand κ-casein with the addition of prolactin was significantly higher than the control (P＜0.05), the gene expression of α-whey protein in BMEC with the addition of prolactin wassignificantly lower than the control (P＜0.05) and there was no significant difference of thegene expression of β-casein between BMEC with the addition of prolactin and control (P＞0.05) and two-dimensional culture.Under three-dimensional culture,there was nosignificant difference of the gene expression of αs1-casein and κ-casein between BMECwith the addition of prolactin and control(P＞0.05)and the gene expression of β-casein andα-whey protein in BMEC with the addition of prolactin was significantly higher than thecontrol (P＜0.05).
3. The gene expression of αs1-casein, β-casein,κ-casein,mTOR, S6K1,4E-BP1, andeIF4E in BMEC by adding leu, ile, and val were significantly higher than the control (P＜0.05)in three-dimensional culture.
The conclusion of the experiments was three-dimensional culture of BMEC was muchbetter than that two-dimensional culture of BMEC as in vitro cultivation technique in studyof lactation metabolism of bovine mammary epithelial cells
1、BMEC在Matrigel基质胶中可以快速的形成类腺泡结构，而在鼠尾胶中则形成散落的聚集物。且在培养的过程中Matrigel基质胶比鼠尾胶稳定。三维模式下培养时间和接种密度影响BMEC中αs1-酪蛋白、β-酪蛋白、κ-酪蛋白的基因表达量。三维模式下BMEC在培养的第5d形成了完全成熟的腺泡，三维模式下BMEC的适合接种密度为1x106个/mL。
2、在相同的营养物质及激素水平下，三维培养的BMEC三种乳蛋白的基因表达量显著高于二维培养（P＜0.05），三维培养的BMEC通过western blot的方法能够检测到β-酪蛋白，二维培养的BMEC检测不到β-酪蛋白。三维培养的BMEC在形态上呈现的特点与BMEC在体内所呈现的组织学特征更加接近。二维培养模式下添加催乳素BMEC的αs1-酪蛋白和κ酪蛋白基因的表达量显著高于未添加催乳素组（对照组）（P＜0.05），添加催乳素BMEC的α-乳清蛋白的表达量显著低于对照组（P＜0.05），添加催乳素BMEC的β-酪蛋白基因的表达量和对照组间无显著差异（P＞0.05）。三维培养模式下，添加催乳素BMEC的αs1-酪蛋白和κ酪蛋白基因的表达量和对照组间无显著差异（P＞0.05），添加催乳素BMEC的β-酪蛋白，α-乳清蛋白的表达量显著高于对照组（P＜0.05）。
3、在三维培养模式下，添加leu、ile、val使奶牛乳腺上皮细胞αs1-酪蛋白、β-酪蛋白、κ-酪蛋、mTOR、S6K1、4E-BP1、eIF4E基因的表达量显著高于对照组（P＜0.05）。
本试验表明，在奶牛泌乳代谢的研究中，BMEC的三维培养技术优于二维培养。
The main purposes of the experiments was to investigate the effects oftwo-dimensional culture and three-dimensional culture on the gene expression of lactationmetabolism of bovine mammary epithelial cells (BMEC) under different trial conditionwhich included the factors of full nutritional factors, single branched amino acid, hormone.the results were as follows:
1. BMEC in Matrigel collagen could rapidly form acinar-like structure, but it formedscattered aggregates in rattail collagen. Furthermore, Matrigel collagen was more stablethan rattail collagen in culture process.The incubation time and the inoculum density ofthree-dimensional culture influenced the gene expression of αs1-casein, β-casein, andκ-casein in BMEC. BMEC of three-dimensional culture formed full ripe acinar-likestructure at5d after incubation, and the optimum inoculum density was1x106/mL.
2. On the same nutrients and hormones conditions, the gene expression of αs1-casein,β-casein, and κ-casein in BMEC of three-dimensional culture were significantly higherthan that in two-dimensional culture (P＜0.05), and β-casein in three-dimensional cultureof BMEC could be detected by western blot, but it was not two-dimensional culture ofBMEC.The morphological characters in BMEC of three-dimensional culture were moresimilar to BMEC in vivo.Under two-dimensional culture,the gene expression of αs1-caseinand κ-casein with the addition of prolactin was significantly higher than the control (P＜0.05), the gene expression of α-whey protein in BMEC with the addition of prolactin wassignificantly lower than the control (P＜0.05) and there was no significant difference of thegene expression of β-casein between BMEC with the addition of prolactin and control (P＞0.05) and two-dimensional culture.Under three-dimensional culture,there was nosignificant difference of the gene expression of αs1-casein and κ-casein between BMECwith the addition of prolactin and control(P＞0.05)and the gene expression of β-casein andα-whey protein in BMEC with the addition of prolactin was significantly higher than thecontrol (P＜0.05).
3. The gene expression of αs1-casein, β-casein,κ-casein,mTOR, S6K1,4E-BP1, andeIF4E in BMEC by adding leu, ile, and val were significantly higher than the control (P＜0.05)in three-dimensional culture.
The conclusion of the experiments was three-dimensional culture of BMEC was muchbetter than that two-dimensional culture of BMEC as in vitro cultivation technique in studyof lactation metabolism of bovine mammary epithelial cells
引文
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