奶山羊乳腺上皮细胞系的建立及初步应用
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摘要
为了建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系，根据已发表的SV40病毒T基因序列设计并合成PCR引物，以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板，用高保真聚合酶链式反应扩增SV40T基因。凝胶电泳结果显示，扩增产物为2.5kb的单一条带，与预计大小相符。序列测定结果显示，所获序列与已发表的SV40 T基因的同源性大于99％。将PCR扩增产物克隆入剔除neo基因的真核表达载体pTarget，经XhoⅠ和NdeⅠ酶切鉴定出SV40 T基因以正确方向插入的重组表达质粒pTarget-LT。用脂质体介导法将线性化的重组表达质粒pTarget-LT转染入奶山羊原代乳腺上皮细胞，经有限稀释和反复传代后获得了转化的细胞克隆。
部分细胞克隆已在体外传30代以上，它们保持正常乳腺上皮细胞的形态特征，在胶原基质上能形成腺泡样结构，但饱和密度和生长速度明显增加。用基因组DNA进行的斑点杂交试验结果证明，转化细胞的基因组中整合有SV40 T基因。用总RNA进行的斑点杂交试验证明，转化细胞的β-酪蛋白基因能被正常转录。用自制的小鼠抗山羊β-酪蛋白免疫血清进行的酶联免疫吸附试验结果表明，转化细胞在催乳素、胰岛素和氢化可的松共同诱导下，能保持原有的分泌内源性β-酪蛋白的能力，其表达量最高达1μg/10~4个细胞。将含有LacZ报告基因的乳腺特异表达基因构件pHC-LacZ和p205CLacZ3分别转染入转化细胞，经酶组化染色试验证明，在催乳素、胰岛素和氢化可的松的诱导下，报告基因能在转化细胞中获得表达。以上试验结果表明，本研究用PCR方法从COS-1细胞基因组中克隆的SV40T基因保持其原有的细胞转化能力，转化的山羊乳腺上皮细胞能在体外长期培养和传代，符合细胞系的基本特点，不仅保持原有的分泌内源性乳蛋白的能力，而且
扬州大学硕士学位论文
可以用于山羊乳腺特异表达基因构件的质量检验。
To establish dairy goat mammary gland epithelial cell lines for evaluation of mammary gland-specific expression gene constructs, a pair of primers were synthesized according to the previously published DNA sequence and used to amplify the SV40 T gene by high fidelity PCR from COS-1 cell genomic DNA. An expected PCR product of 2.5kb was obtained and shown to be 99% identical to that of the previously published SV40 T gene by DNA sequencing. The amplified SV40 T gene was subcloned into eukaryotic expression vector pTarget and the resulting recombinant vector pTarget-LT was used to transfect dairy goat primary mammary gland epithelial cells. Following limited dilution and repeated passage, 18 cell clones were obtained, one of which had been passed for 30 generations in vitro.
The cell clone maintained its original morphology of mammary gland epithelial cells and was able to form glandulous structures on collagen matrix with an increased maturation density and growth speed. Dot blotting analyses showed that the SV40 T gene was integrated into the genomes of the transformed cells and that the endogenous β - casein gene was transcribed following induction with a mixture of prolactin, insulin and hydrocortisone. The hormone-dependent expression of the endogenous β - casein gene was confirmed by ELISA using self-made anti goat β - casein immunoserum with a highest concentration of 1 μ g /ml. Further experiments showed that the established cell line was able to support expression of LacZ report gene cloned into mammary gland-specific expression vectors pHC and p205C3. These data indicate that the amplified SV40 T gene maintained its original capability for cell transformation and that the recombinant vector pTarget -LT was usable for establishment of dairy "goat mammary gland epithelia
l cell lines. The established cell line maintained the original capabilities of differentiation and expression of normal goat mammary gland epithelial
cells and was a useful cell mode for evaluation of mammary gland-specific expression gene constructs.
部分细胞克隆已在体外传30代以上，它们保持正常乳腺上皮细胞的形态特征，在胶原基质上能形成腺泡样结构，但饱和密度和生长速度明显增加。用基因组DNA进行的斑点杂交试验结果证明，转化细胞的基因组中整合有SV40 T基因。用总RNA进行的斑点杂交试验证明，转化细胞的β-酪蛋白基因能被正常转录。用自制的小鼠抗山羊β-酪蛋白免疫血清进行的酶联免疫吸附试验结果表明，转化细胞在催乳素、胰岛素和氢化可的松共同诱导下，能保持原有的分泌内源性β-酪蛋白的能力，其表达量最高达1μg/10~4个细胞。将含有LacZ报告基因的乳腺特异表达基因构件pHC-LacZ和p205CLacZ3分别转染入转化细胞，经酶组化染色试验证明，在催乳素、胰岛素和氢化可的松的诱导下，报告基因能在转化细胞中获得表达。以上试验结果表明，本研究用PCR方法从COS-1细胞基因组中克隆的SV40T基因保持其原有的细胞转化能力，转化的山羊乳腺上皮细胞能在体外长期培养和传代，符合细胞系的基本特点，不仅保持原有的分泌内源性乳蛋白的能力，而且
扬州大学硕士学位论文
可以用于山羊乳腺特异表达基因构件的质量检验。
To establish dairy goat mammary gland epithelial cell lines for evaluation of mammary gland-specific expression gene constructs, a pair of primers were synthesized according to the previously published DNA sequence and used to amplify the SV40 T gene by high fidelity PCR from COS-1 cell genomic DNA. An expected PCR product of 2.5kb was obtained and shown to be 99% identical to that of the previously published SV40 T gene by DNA sequencing. The amplified SV40 T gene was subcloned into eukaryotic expression vector pTarget and the resulting recombinant vector pTarget-LT was used to transfect dairy goat primary mammary gland epithelial cells. Following limited dilution and repeated passage, 18 cell clones were obtained, one of which had been passed for 30 generations in vitro.
The cell clone maintained its original morphology of mammary gland epithelial cells and was able to form glandulous structures on collagen matrix with an increased maturation density and growth speed. Dot blotting analyses showed that the SV40 T gene was integrated into the genomes of the transformed cells and that the endogenous β - casein gene was transcribed following induction with a mixture of prolactin, insulin and hydrocortisone. The hormone-dependent expression of the endogenous β - casein gene was confirmed by ELISA using self-made anti goat β - casein immunoserum with a highest concentration of 1 μ g /ml. Further experiments showed that the established cell line was able to support expression of LacZ report gene cloned into mammary gland-specific expression vectors pHC and p205C3. These data indicate that the amplified SV40 T gene maintained its original capability for cell transformation and that the recombinant vector pTarget -LT was usable for establishment of dairy "goat mammary gland epithelia
l cell lines. The established cell line maintained the original capabilities of differentiation and expression of normal goat mammary gland epithelial
cells and was a useful cell mode for evaluation of mammary gland-specific expression gene constructs.
引文
1 张忠诚，朱士恩等．动物乳腺生物反应器的原理及研究进展．中国畜牧杂志，2000，36(2)：51-53．
2 Lovell-Badge R H et al. Transformation of embryonic stem cells with the human type-Ⅱ collagen gene and its expression in chimeric mice. Biol, 1985, 50: 707-711.
3 Simons JP et al. Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice. Nature, 1987, 328(6130): 530-532.
4 曹阳，刘美华等．乳腺生物反应器的研究现状．大连大学学报，1999，20(6)：71-74．
5 Wilmut I et al. Production of pharmaceutical proteins in milk. Experientia, 1991, 47(9): 905-912.
6 Shamay A et al. Production of the mouse whey acidic protein in transgenic pigs during lactation. Anim Sci, 1991, 69(11): 552-562.
7 Wright G et al. High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep. Bio Technology,1991, 9: 830-834.
8 劳为德，张旭晨．乳腺生物反应器实用化研究现状与问题．生物工程进展，16(4)：38-45．
9 薛京伦，卢大儒．乳腺生物反应器的研究现状．生物技术通报，1998，15(3)：17-20．
10 张克忠，卢大儒等．乳汁中分泌有活性的人凝血因子Ⅸ的转基因羊的研究．科学通报，1998，43(7)：783-784．
11 卢一凡，邓继先等．转基因动物乳腺定位表达重组蛋白质的研究现状．高技术通讯，1998，(1)：54-57，(2)：59-62．
12 孙力．如何提高奶牛生产能力．中国草食动物，1997，3：11．
13 郭建靖，占飞豹．浅谈提高奶山羊产奶量的技术要点．福建畜牧兽医，1998，6：23-24．
14 李震，陈永福．乳腺生物反应器细胞模型建立的问题探讨．农业生物技学报，1997，5(2)：148-152．
15 Wollf J A et al. Direct gene transfer into mouse muscle in vivo. Science, 1990, 247: 1465-1468.
16 卢一凡，邓继先．乳腺直接注射质粒DNA的转基因暂时性表达研究进展．国外医学遗传学分册，1998，21(6)：310-312．
17 LuYi-fan et al. Expression of human G-CSF in mammary gland of mice by injection of plasmid DNA. Developmental and Reproductive Biology, 1997, 2: 29-32.
18 Lai Liang-xue et al. Expression of human tPA in mammary gland of rabbit by injection of plasmid DNA. Developmental and Reproductive Biology, 1999, 8(1): 17-23.
19 成勇，徐少甫等．牛aS1酪蛋白／HBsAg基因在山羊乳腺中暂态表达．扬州大学学报，1998，1(3)：27-31．
20 王贵，卢一凡等．乳腺直接注射重组质粒表达人G-CSF的研究．核农学报，1997，4：221．
21 Ilan N et al. Establishment and initial characterization of the ovine mammary epithelial cell line NISH. In Vitro Cell Dev Biol Anim, 1998, 34(4): 326-332.
22 李震，林爱星等．牛乳腺细胞系BME-L1β-酪蛋白分泌的诱导及人促红细胞生成素(hEPO)的表达．农业生物技术学报，1999，7(1)：69-72．
23 张克忠，邱信芳．乳腺生物反应器的基础研究．载体构建，检测及反应器的建立方法．国外医学遗传学分册，1997，20(6)：314-318．
24 李震，陈永福．乳腺生物反应器细胞模型建立的问题探讨．农业生物技术学报，1997．5(2)：148-152．
25 Reilly C F et al. Heparin prenents vascular smooth muscle cell progression through the C_1 phase of the cell cycle. Biol Chore, 1989, 264: 6990-6995.
26 Evans M I et al. A somatomedin-like peptide hormone is required during the estrogen-mediated induction of ovalburnin gene transcription. Cell, 1981, 25:187.
27 陶谦，黄洪章．SV40与细胞永生性转化．癌症，2001，20(3)：332-334．
28 Fantini J et al. Suramin inhibits proliferation of rat glioma cells and alters N-cam cell surface expression. Int Camser, 1990, 45: 554-561.
29 欧阳五庆．山羊乳腺上皮细胞培养体系的建立及cAMP对该细胞的影响．西北农业大学博士论文，1999．
30 Rockwell G A. Growth of SV40 Balb/c-3T3 cells in serum-free culture medium. Cell culture methods for molecular and cell biology, New York: Alan R. Liss, Inc, 1984, 221-231.
31 解慧淇，杨志明等．SV40与细胞永生化．中国修复重建外科杂志，2000，14(3)：
170-174．
32 苏映军，陈璧等．SV40病毒对体外培养的人角朊细胞的转化．第四军医大学学报，1999，(5)：382-385．
33 Berthon P et al. Single-step transformation of human breast epithelial cells by SV40 large T oncogene. Int Cancer, 1992, 52(1): 92-97.
34 Basolo F et al. Defective interleukin six expression and responsiveness in human mammary cells transformed by an adeno-5/SV40 hybrid virus. Br J Cancer, 1996, 73(11): 1356-1361.
35 Huynh et al. Establishment of bovine mammary epithelial cell line (MAC-T): An in vitro model for bovine lactation. Exp Cell Res, 1991, 197: 191-199.
36 Zavizion B et al. Establishment and characterization of a bovine mammary epithelial cell line with unique properties. In Vitro Cell Dev Biol Anim, 1996, 32: 138-148.
37 Santarelli R et al. SV40 T-antigen induces breast cancer formation with a high efficiency in lactating and virgin WAP-SV-T transgenic animals but with a low efficiency in ovariectomized animals. Oncogene, 1996, 12(3): 495-505.
38 Shibata MA et al. p~(53)-independent apoptosis during mammary tumor progression in C3 (1)/SV40 large T antigen transgenic mice: suppression of apoptosis during the transition from prencoplasia to carcinoma. Cancer Res, 1996, 56(13): 2998-3003.
39 Chang SE et al. Establishment and characterization of SV40-transformed human breast epithelial cell lines. Cancer Res, 1982, 42(5): 2040-2053.
40 Danielson K G et al. Epithelial mouse mammary cell line exhibiting normal morphogenesis in vivo and functional differentiation in vitro. Proc Natl Acad Sci, USA, 1984, 81:3756-3760.
41 Ball R K et al. Prolactin regulation of β-casein gene expression and of a cytosolic 120kD protein in a cloned mouse mammary, epithelial cell line. EMBO, 1988,7: 2089-2093.
42 Schmid E et al. An epithelial cell line with elongated myoid morphology derived from bovine mammary gland. Exp Cell Res, 1983, 146: 309-328.
43 Soriano J V et al. RTGF-B1 induces morphogenesis of branching cords by cloned mammary epithelial cells at subpicomolar concentrations. Biochem Biophys Res Common, 1996, 220: 879-885.
44 Binas B et al. Hormonal induction of functional differentiation and mammary
derived growth inhibitor expression in cultured mouse mammary gland explants. In Vitro Cell Dev Biol, 1992, 28A: 625-634.
45 Gibson C A et al. Establishment and characterization of bovine mammary epithelial cell lines. In Vitro Cell Dev Biol, 1991, 27A(7): 585-594.
46 Zavizion B et al. Subcloning the MAC-T bovine mammary epithelial cell line: morphology, growth properties, and cytogenetic analysis of clonal cells. Dairy Sci, 1995, 78(3): 515-527.
47 Pantschenko A G et al. Establishment and characterization of a caprine mammary epithelial cell line (CMEC). In Vitro Cell Dev Biol Anim, 2000, 36(1): 26-37.
48 金冬雁，黎孟枫等译，分子克隆实验指南(第二版)，科学出版社，1992．
49 Richards J et al. Method for culturing mammary epithelial cells in rat tail collagen gel matrix. Tissue culture methods, 1983, 8:31.
50 Huschtscha LI et al. Limeted and limited growth of SV40 transformed celld from human diploid MRC 5 fibroblasts. Cell Sci, 1983, 63: 77-99.
51 Shay JW et al. The frequency of immortalization of fibroblasts and mammary epithelial cells transfected with SV40 large T antigen. Exp Cell Res, 1993, 209: 45-52.
52 欧阳五庆，钱菊汾．山羊乳腺上皮细胞培养体的建立．中国兽医学报，1999，19(6)：559．
53 Bolander F F et al. Insulin is essential for accumulation of casein mRNA in mouse mammary epithelial cells. Biochim-Biophysical Res Comm, 1979, 91:247.
54 Beverley W et al. Prolactin independent induction of lactalbum in gene expression in mammary gland explants from pregnant BALB/C mice. Biochim Biophysical Res Comm, 1993, 194: 3.
55 Wolfgang D et al. Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat β-casein gene promotor constructs in mammary epithelial cell line. Proc Nail Acad Sci, USA, 1989, 86:104-108.
2 Lovell-Badge R H et al. Transformation of embryonic stem cells with the human type-Ⅱ collagen gene and its expression in chimeric mice. Biol, 1985, 50: 707-711.
3 Simons JP et al. Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice. Nature, 1987, 328(6130): 530-532.
4 曹阳，刘美华等．乳腺生物反应器的研究现状．大连大学学报，1999，20(6)：71-74．
5 Wilmut I et al. Production of pharmaceutical proteins in milk. Experientia, 1991, 47(9): 905-912.
6 Shamay A et al. Production of the mouse whey acidic protein in transgenic pigs during lactation. Anim Sci, 1991, 69(11): 552-562.
7 Wright G et al. High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep. Bio Technology,1991, 9: 830-834.
8 劳为德，张旭晨．乳腺生物反应器实用化研究现状与问题．生物工程进展，16(4)：38-45．
9 薛京伦，卢大儒．乳腺生物反应器的研究现状．生物技术通报，1998，15(3)：17-20．
10 张克忠，卢大儒等．乳汁中分泌有活性的人凝血因子Ⅸ的转基因羊的研究．科学通报，1998，43(7)：783-784．
11 卢一凡，邓继先等．转基因动物乳腺定位表达重组蛋白质的研究现状．高技术通讯，1998，(1)：54-57，(2)：59-62．
12 孙力．如何提高奶牛生产能力．中国草食动物，1997，3：11．
13 郭建靖，占飞豹．浅谈提高奶山羊产奶量的技术要点．福建畜牧兽医，1998，6：23-24．
14 李震，陈永福．乳腺生物反应器细胞模型建立的问题探讨．农业生物技学报，1997，5(2)：148-152．
15 Wollf J A et al. Direct gene transfer into mouse muscle in vivo. Science, 1990, 247: 1465-1468.
16 卢一凡，邓继先．乳腺直接注射质粒DNA的转基因暂时性表达研究进展．国外医学遗传学分册，1998，21(6)：310-312．
17 LuYi-fan et al. Expression of human G-CSF in mammary gland of mice by injection of plasmid DNA. Developmental and Reproductive Biology, 1997, 2: 29-32.
18 Lai Liang-xue et al. Expression of human tPA in mammary gland of rabbit by injection of plasmid DNA. Developmental and Reproductive Biology, 1999, 8(1): 17-23.
19 成勇，徐少甫等．牛aS1酪蛋白／HBsAg基因在山羊乳腺中暂态表达．扬州大学学报，1998，1(3)：27-31．
20 王贵，卢一凡等．乳腺直接注射重组质粒表达人G-CSF的研究．核农学报，1997，4：221．
21 Ilan N et al. Establishment and initial characterization of the ovine mammary epithelial cell line NISH. In Vitro Cell Dev Biol Anim, 1998, 34(4): 326-332.
22 李震，林爱星等．牛乳腺细胞系BME-L1β-酪蛋白分泌的诱导及人促红细胞生成素(hEPO)的表达．农业生物技术学报，1999，7(1)：69-72．
23 张克忠，邱信芳．乳腺生物反应器的基础研究．载体构建，检测及反应器的建立方法．国外医学遗传学分册，1997，20(6)：314-318．
24 李震，陈永福．乳腺生物反应器细胞模型建立的问题探讨．农业生物技术学报，1997．5(2)：148-152．
25 Reilly C F et al. Heparin prenents vascular smooth muscle cell progression through the C_1 phase of the cell cycle. Biol Chore, 1989, 264: 6990-6995.
26 Evans M I et al. A somatomedin-like peptide hormone is required during the estrogen-mediated induction of ovalburnin gene transcription. Cell, 1981, 25:187.
27 陶谦，黄洪章．SV40与细胞永生性转化．癌症，2001，20(3)：332-334．
28 Fantini J et al. Suramin inhibits proliferation of rat glioma cells and alters N-cam cell surface expression. Int Camser, 1990, 45: 554-561.
29 欧阳五庆．山羊乳腺上皮细胞培养体系的建立及cAMP对该细胞的影响．西北农业大学博士论文，1999．
30 Rockwell G A. Growth of SV40 Balb/c-3T3 cells in serum-free culture medium. Cell culture methods for molecular and cell biology, New York: Alan R. Liss, Inc, 1984, 221-231.
31 解慧淇，杨志明等．SV40与细胞永生化．中国修复重建外科杂志，2000，14(3)：
170-174．
32 苏映军，陈璧等．SV40病毒对体外培养的人角朊细胞的转化．第四军医大学学报，1999，(5)：382-385．
33 Berthon P et al. Single-step transformation of human breast epithelial cells by SV40 large T oncogene. Int Cancer, 1992, 52(1): 92-97.
34 Basolo F et al. Defective interleukin six expression and responsiveness in human mammary cells transformed by an adeno-5/SV40 hybrid virus. Br J Cancer, 1996, 73(11): 1356-1361.
35 Huynh et al. Establishment of bovine mammary epithelial cell line (MAC-T): An in vitro model for bovine lactation. Exp Cell Res, 1991, 197: 191-199.
36 Zavizion B et al. Establishment and characterization of a bovine mammary epithelial cell line with unique properties. In Vitro Cell Dev Biol Anim, 1996, 32: 138-148.
37 Santarelli R et al. SV40 T-antigen induces breast cancer formation with a high efficiency in lactating and virgin WAP-SV-T transgenic animals but with a low efficiency in ovariectomized animals. Oncogene, 1996, 12(3): 495-505.
38 Shibata MA et al. p~(53)-independent apoptosis during mammary tumor progression in C3 (1)/SV40 large T antigen transgenic mice: suppression of apoptosis during the transition from prencoplasia to carcinoma. Cancer Res, 1996, 56(13): 2998-3003.
39 Chang SE et al. Establishment and characterization of SV40-transformed human breast epithelial cell lines. Cancer Res, 1982, 42(5): 2040-2053.
40 Danielson K G et al. Epithelial mouse mammary cell line exhibiting normal morphogenesis in vivo and functional differentiation in vitro. Proc Natl Acad Sci, USA, 1984, 81:3756-3760.
41 Ball R K et al. Prolactin regulation of β-casein gene expression and of a cytosolic 120kD protein in a cloned mouse mammary, epithelial cell line. EMBO, 1988,7: 2089-2093.
42 Schmid E et al. An epithelial cell line with elongated myoid morphology derived from bovine mammary gland. Exp Cell Res, 1983, 146: 309-328.
43 Soriano J V et al. RTGF-B1 induces morphogenesis of branching cords by cloned mammary epithelial cells at subpicomolar concentrations. Biochem Biophys Res Common, 1996, 220: 879-885.
44 Binas B et al. Hormonal induction of functional differentiation and mammary
derived growth inhibitor expression in cultured mouse mammary gland explants. In Vitro Cell Dev Biol, 1992, 28A: 625-634.
45 Gibson C A et al. Establishment and characterization of bovine mammary epithelial cell lines. In Vitro Cell Dev Biol, 1991, 27A(7): 585-594.
46 Zavizion B et al. Subcloning the MAC-T bovine mammary epithelial cell line: morphology, growth properties, and cytogenetic analysis of clonal cells. Dairy Sci, 1995, 78(3): 515-527.
47 Pantschenko A G et al. Establishment and characterization of a caprine mammary epithelial cell line (CMEC). In Vitro Cell Dev Biol Anim, 2000, 36(1): 26-37.
48 金冬雁，黎孟枫等译，分子克隆实验指南(第二版)，科学出版社，1992．
49 Richards J et al. Method for culturing mammary epithelial cells in rat tail collagen gel matrix. Tissue culture methods, 1983, 8:31.
50 Huschtscha LI et al. Limeted and limited growth of SV40 transformed celld from human diploid MRC 5 fibroblasts. Cell Sci, 1983, 63: 77-99.
51 Shay JW et al. The frequency of immortalization of fibroblasts and mammary epithelial cells transfected with SV40 large T antigen. Exp Cell Res, 1993, 209: 45-52.
52 欧阳五庆，钱菊汾．山羊乳腺上皮细胞培养体的建立．中国兽医学报，1999，19(6)：559．
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