转sfat-1基因乳腺上皮细胞体外培养研究
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摘要
乳腺上皮细胞由于其特有的合成和分泌的生理功能,而成为研究乳腺生物反应器的重要细胞模型。这种细胞模型能真实反映动物乳腺组织生长、发育及泌乳的启动、维持等各项生物学机制,验证构建的乳腺特异性表达载体构建的是否合理有效。乳腺上皮细胞及以此为基础建立的细胞模型,其应用涉及到分子生物学、内分泌学、发育生物学、细胞生物学、生物化学等多个领域,对于研究不同乳蛋白基因的表达及乳腺生物反应器有重要意义。
人体内,ω-6多不饱和脂肪酸与ω-3多不饱和脂肪酸的比例将会直接影响身体健康及某些慢性病的发生。ω-6多不饱和脂肪酸可以经由食物直接获取,也可以由人体合成。但哺乳动物体内缺乏合成ω-3多不饱和脂肪酸的酶,所以,正常状态下,ω-3PUFAs只能经由饮食获取。而在现代饮食结构中,ω-6/ω-3PUFAs的比例严重偏高,直接影响了现代人的身体健康。在这种情况下,人们需要丰富的ω-3多不饱和脂肪酸的来源,从而降低ω-6/ω-3PUFAs的比例,改善人们的膳食结构。fat-1基因恰好可以编码ω-3不饱和脂肪酸脱氢酶,实现ω-6多不饱和脂肪酸到ω-3多不饱和脂肪酸的转变。
本研究所用实验材料来源于屠宰场屠杀的健康泌乳后期的荷斯坦奶牛的乳腺组织,通过对荷斯坦奶牛乳腺上皮细胞培养方法的筛选,建立了具有正常生理功能的奶牛乳腺上皮细胞系,并通过对乳腺上皮细胞的特异性蛋白角蛋白18的免疫荧光染色,对所建立的乳腺上皮细胞进行了鉴定。结果:利用组织块接种法获得里大量的乳腺上皮细胞用于原代培养。以DMEM-F12为基础培养液,在其中添加20%的胎牛血清、胰岛素(1μg/mL)、转铁蛋白(5μg/mL)、孕酮(1μg/mL)、氢化可的松(1μg/mL),乳腺上皮细胞生长状态良好。乳腺上皮细胞与成纤维细胞混合生长,原代培养中,两种不同细胞之间界限明显,但经过传代后,界限不明显。乳腺上皮细胞有不同的细胞形态,多为鹅卵石样及多角形。
利用本实验室已构建的两个乳腺特异性表达载体pCB1F1E(8979bp)和pCB2F1E(9101bp)及两个对照组载体pCB1E(7823bp)、pCB2E(7948bp),转染所建立的乳腺上皮细胞系,获得了转fat-1基因的乳腺上皮细胞。通过PCR鉴定,证明目的基因sfat-1基因已整合入细胞基因组内。
Mammary gland epithelial cells have special functions of synthesis and lactation .These functionsmake them mammary bioreactor harrow cell. Experimental model made from mammary gland epithelialcells could actually response many biology mechanisms ,such as mammoplasia ,development ,the startand maintenance of lactation .The model could also validate if the construction of tissue specificityexpression vector is rational and effective .Mammary gland epithelial cells and the experimental modelsrelate to molecular biology, endocrinology developmental biology , cell biology , biochemistry and andother fields .It is significant to the gland bioreactor ,and the research of the lactoprotein geneexpression .
The ratio ofω-6/ω-3 fatty acid has effects on health and chronic disease.ω-6 PUFAs can beobtained through the food directly ,or synthesized in the human body .Mammals are lack of the enzymethat could synthesizeω-3 PUFAs, soω-3 PUFAs can be obtained through the food only. In moderndiet ,the ratio ofω-6/ω-3 is very high, it have impact on the health of modern people. In this case, it isnecessary to enrich the food supply withω-3 PUFAs to reduce the ratio and improve the nutritionalcomposition of diet. The fat-1 gene encodesω-3 fatty acid desaturase that could convert theω-6 fattyacid to theω-3 fatty acid.
The experimental material of this study was health Holstein cow mammary gland tissue fromslaughterhouse.We used different methods to culture the primary mammary epithelial cells,successfully cultured bovine mammary epithelial cells(BMEC).The established mammary epithelial cellswere identified by immunofluorescence staining. The results showed that the large numbers of mammaryepithelial cells were successfully separated from mammary tissue inoculation. The cells were incubatedwith DMEM-F12 and 20% fetal bovine serum,insulin(1μg/mL),transferring(5μg/mL),pregnendione(1μg/mL),hydrocortisone(1μg/mL).Mammaryepithelial cells and fibroblasts were mixed grown. The boundaries between the two different cells wereclear during primary culture. But the boundaries were not so clear after passage. Mammary epithelialcells have different morphology, most of them were cobblestone and polygon..
Mammary epithelial cells were transferred using vectors constructed in our lab: two mammarygland-specific vectors pCB1F1E (8976bp) pCB2F1E(9101bp),and two negative control vectors pCB1E(7823bp) and pCB2E (7948bp).The positive transgenic cells were obtained The PCR test proved thatfat-1 gene were successfully integrated within the genome.
人体内,ω-6多不饱和脂肪酸与ω-3多不饱和脂肪酸的比例将会直接影响身体健康及某些慢性病的发生。ω-6多不饱和脂肪酸可以经由食物直接获取,也可以由人体合成。但哺乳动物体内缺乏合成ω-3多不饱和脂肪酸的酶,所以,正常状态下,ω-3PUFAs只能经由饮食获取。而在现代饮食结构中,ω-6/ω-3PUFAs的比例严重偏高,直接影响了现代人的身体健康。在这种情况下,人们需要丰富的ω-3多不饱和脂肪酸的来源,从而降低ω-6/ω-3PUFAs的比例,改善人们的膳食结构。fat-1基因恰好可以编码ω-3不饱和脂肪酸脱氢酶,实现ω-6多不饱和脂肪酸到ω-3多不饱和脂肪酸的转变。
本研究所用实验材料来源于屠宰场屠杀的健康泌乳后期的荷斯坦奶牛的乳腺组织,通过对荷斯坦奶牛乳腺上皮细胞培养方法的筛选,建立了具有正常生理功能的奶牛乳腺上皮细胞系,并通过对乳腺上皮细胞的特异性蛋白角蛋白18的免疫荧光染色,对所建立的乳腺上皮细胞进行了鉴定。结果:利用组织块接种法获得里大量的乳腺上皮细胞用于原代培养。以DMEM-F12为基础培养液,在其中添加20%的胎牛血清、胰岛素(1μg/mL)、转铁蛋白(5μg/mL)、孕酮(1μg/mL)、氢化可的松(1μg/mL),乳腺上皮细胞生长状态良好。乳腺上皮细胞与成纤维细胞混合生长,原代培养中,两种不同细胞之间界限明显,但经过传代后,界限不明显。乳腺上皮细胞有不同的细胞形态,多为鹅卵石样及多角形。
利用本实验室已构建的两个乳腺特异性表达载体pCB1F1E(8979bp)和pCB2F1E(9101bp)及两个对照组载体pCB1E(7823bp)、pCB2E(7948bp),转染所建立的乳腺上皮细胞系,获得了转fat-1基因的乳腺上皮细胞。通过PCR鉴定,证明目的基因sfat-1基因已整合入细胞基因组内。
Mammary gland epithelial cells have special functions of synthesis and lactation .These functionsmake them mammary bioreactor harrow cell. Experimental model made from mammary gland epithelialcells could actually response many biology mechanisms ,such as mammoplasia ,development ,the startand maintenance of lactation .The model could also validate if the construction of tissue specificityexpression vector is rational and effective .Mammary gland epithelial cells and the experimental modelsrelate to molecular biology, endocrinology developmental biology , cell biology , biochemistry and andother fields .It is significant to the gland bioreactor ,and the research of the lactoprotein geneexpression .
The ratio ofω-6/ω-3 fatty acid has effects on health and chronic disease.ω-6 PUFAs can beobtained through the food directly ,or synthesized in the human body .Mammals are lack of the enzymethat could synthesizeω-3 PUFAs, soω-3 PUFAs can be obtained through the food only. In moderndiet ,the ratio ofω-6/ω-3 is very high, it have impact on the health of modern people. In this case, it isnecessary to enrich the food supply withω-3 PUFAs to reduce the ratio and improve the nutritionalcomposition of diet. The fat-1 gene encodesω-3 fatty acid desaturase that could convert theω-6 fattyacid to theω-3 fatty acid.
The experimental material of this study was health Holstein cow mammary gland tissue fromslaughterhouse.We used different methods to culture the primary mammary epithelial cells,successfully cultured bovine mammary epithelial cells(BMEC).The established mammary epithelial cellswere identified by immunofluorescence staining. The results showed that the large numbers of mammaryepithelial cells were successfully separated from mammary tissue inoculation. The cells were incubatedwith DMEM-F12 and 20% fetal bovine serum,insulin(1μg/mL),transferring(5μg/mL),pregnendione(1μg/mL),hydrocortisone(1μg/mL).Mammaryepithelial cells and fibroblasts were mixed grown. The boundaries between the two different cells wereclear during primary culture. But the boundaries were not so clear after passage. Mammary epithelialcells have different morphology, most of them were cobblestone and polygon..
Mammary epithelial cells were transferred using vectors constructed in our lab: two mammarygland-specific vectors pCB1F1E (8976bp) pCB2F1E(9101bp),and two negative control vectors pCB1E(7823bp) and pCB2E (7948bp).The positive transgenic cells were obtained The PCR test proved thatfat-1 gene were successfully integrated within the genome.
引文
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