奶山羊乳腺上皮细胞系的建立及脂肪酸合酶基因的RNA干扰研究
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摘要
脂肪酸合酶(Fatty acid synthase, FAS)是哺乳动物脂肪酸合成过程中的关键酶,在细胞生命活动中参与着极其复杂的代谢调控过程,它与能量贮存、生物膜的结构、信号转导和蛋白质的乙酰化有密切的关系。羊奶中短、中链脂肪酸含量十分丰富,能有效预防人类的某些代谢疾病,因而具有良好的保健功能。山羊乳腺FAS的乙酰/丙二酸单酰基转移酶(Acetyl-CoA and malonyl-CoA transacylases, AT/MT)功能域具有中链硫酯酶活性,推测其对羊奶特殊脂肪酸组成中起重要调控作用。因此研究山羊乳腺FAS在乳腺上皮细胞脂肪酸合成中的作用有利于揭示羊奶短、中链脂肪含量高的分子机理,同时对通过遗传手段调控羊奶中有益脂肪酸含量具有重要的理论及现实意义。
本研究通过活体采集西北农林科技大学萨能羊原种场纯种萨能奶山羊乳腺组织,利用组织块法分离培养奶山羊乳腺上皮细胞,纯化后转染人端粒酶逆转录酶基因,激活细胞端粒酶,以期获得具有无限增值能力的奶山羊乳腺上皮细胞系,为奶山羊乳腺功能基因组学研究奠定基础。同时,以此细胞系为试验材料,利用腺病毒介导的RNA干扰技术沉默FAS基因,研究该基因沉默后细胞中脂肪酸组成的变化及对参与脂代谢调控的重要基因如瘦素受体、脂肪酸结合蛋白等表达的影响,以探讨乳腺FAS基因在脂肪酸合成中的作用。试验结果如下:
(1)采用组织块法分离培养并纯化了奶山羊乳腺上皮细胞,通过脂质体转染法将编码人端粒酶逆转录酶基因的pCIneo-hTERT质粒导入原代培养的乳腺上皮细胞(第9代),经浓度为400μg/mL的G418筛选15天获得了抗性细胞克隆,采用滤纸片法转移、扩增后,hTERT基因稳定转入细胞,端粒酶活性检测呈阳性。细胞生长曲线测定结果表明,转入hTERT基因的细胞增殖能力增强,其生长曲线具有典型的S型特征。免疫细胞化学分析表明,所培养的细胞表达上皮细胞的标志物角蛋白8及上皮膜抗原(EMA),证明所培养细胞是乳腺上皮细胞。利用反转录PCR及Western blotting技术可以检测到细胞表达β-酪蛋白基因,而且加入催乳素后,β-酪蛋白基因表达量较对照组增加6.5倍,表明所培养细胞保持有乳腺细胞的基本功能。细胞在体外连续稳定培养67代,最终在91代时细胞死亡。
(2)根据FAS基因的序列,设计并合成了三对针对该基因不同区域的shRNA及一个阴性对照(shRNA-5544、shRNA-5936、shRNA-6132及shRNA-NC),将模板退火形成双链后,构建了表达这四条shRNA的pENTR/CMV-GFP/U6-shRNA载体,并在HEK293细胞中通过共转染的方法成功筛选出两条有效干扰序列(shRNA-5544及shRNA-5936)。通过同源重组,成功构建了pAd-shRNA-5544, pAd-shRNA-5936, pAd-shRNA-NC三个重组腺病毒载体,并在HEK 293细胞中包装出腺病毒颗粒,病毒经扩增后滴度分别达6×108 PFU/mL、5×108 PFU/mL及6×108 PFU/mL。将腺病毒(MOI=200,表达shRNA-5936)感染乳腺上皮细胞72h后,荧光实时定量及Western blotting结果显示FAS基因表达水平降低74%(与对照相比),脂肪酸测定结果表明FAS基因沉默后,细胞中C10:0、C12:0、C16:0、C18:0含量分别降低42%、42.37%、18.51%、29.3%;C14:0含量增加29.82%;荧光实时定量结果表明瘦素受体(LEPR)基因及肝X受体(LXRΑ)基因表达量分别为对照组的1.71及1.36倍;脂肪细胞型脂肪酸结合蛋白(AFABP)基因及脂蛋白脂酶(LPL)基因表达水平分别下调32%及25%。研究结果表明FAS基因对乳腺上皮细胞中中链脂肪酸具有重要的调控作用。
Fatty acid synthase (FAS), a key enzyme in the process of fatty acid synthesis in mammal, participates in an extremely complicated metabolism regulation in cell life and activities, such as energy storage, forming of structure of biology membrane, signal transduction and protein acetylation. It is known that goat milk is rich in short and medium-chain fatty acid, which is beneficial to prevent some metabolism illness in human being. The Acetyl-CoA and malonyl-CoA transacylases (AT/MT) domain of FAS gene in goat mammary gland shows medium-chain Thioesterase activity, which may play an important role in the specific fatty acid composition of goat milk. So, it is significant to study the function of FAS gene on fatty acid synthesis in goat mammary gland epithelia cell in order to reveal the mechanism of high concentration of short and medium-chain fatty acid in goat milk, and as a result it will show an theoretical and practical meaning in up-regulating beneficial fatty acid in milk by genetic methods.
In this study, Xinong Saanen goat mammary gland tissue was collected for primary epithelial cell culture. Human Telomerase Reverse Transcriptase (hTERT) gene was transfected into the purified primary cell in order to obtain stable goat mammary gland epithelial cell (GMEC) line which will become an excellent materials in gene function research of goat mammary gland. Additionally, we carry out functional silencing of FAS gene in GMEC cell by adenovirus-mediated delivery of short hairpin RNA, and investigate changes of fatty acid composition and several fatty acid metabolism-related genes (such as Leptin receptor, AFABP, et al) after silence of FAS. The results of this research are as follows:
(1) Purified GMEC were obtained by tissue culture. Then the pCIneo-hTERT plasmid encoding hTERT was transfected into purified primary GMEC, followed by G418 screening as a concentration of 400μg/mL. Anti-G418 positive cell clones appreared and the cell clones were transmitted using small filter papper and expanded for continuous culture. After stable transfection, telomerase activity turned to be positive in these transfected cells. Growth curve, all are typical“S”type, shows that transfected cells possessed more powerful proliferation activities. Immunocytochemistry analysis proofed that the cells are GMEC, because keratin 8 and epithelial membrane antigen, both of which are marker of epithelial cell, can be detected. Additionally, we have detected the expression ofβ-casein in the cells by RT-PCR and western blotting. The expression ofβ-casein up-regulated by 6.5 time compared with control after stimulating by prolactin. The transfected cells have stably passed 67 passages, died at 91st passages at last.
(2) According to the FAS sequence, three shRNA sequences target different area of FAS and one negative control (shRNA-5544, shRNA-5936, shRNA-6132 and shRNA-NC) were designed and synthesized. After annealing, shRNA templates were constructed into pENTR/CMV-GFP/U6 vecor. By cotransfecting HEK 293 Cell, the result shows that entry vector expressing shRNA-5544 and shRNA-5936 sequences caused an obvious interference effect. Then we generated three recombinant adenovirus vectors (pAd-shRNA-5544, pAd-shRNA-5936, pAd-shRNA-NC) by LR recombination and adenovirus were successfully packed in HEK 293 Cell. The titer of the adenoviral stock was determined by TCID50 method and respectively reaches 6×108 PFU/mL (Expressing shRNA-5544 sequence), 5×108 PFU/mL (Expressing shRNA-5936 sequence), 6×108 PFU/mL (Expressing shRNA-NC sequence). Then we use adenovirus (MOI=200, Expressing shRNA-5936 sequence) to infect GMEC (72h), and expression of FAS mRNA was reduced by 74% by Real-time PCR and western blotting analysis. Fatty acid analysis shows that the content of C10:0, C12:0, C16:0, C18:0 reduced by 42%, 42.37%, 18.51% and 29.3%; the content of C14:0 improved by 29.82%. The result of quantitative Real-time PCR shows LEPR and LXR gene were up-regulated by 1.71 and 1.36 fold, while AFABP and LXR gene were respectively down-regulated by 32% and 25% after the silence of FAS gene. The study shows that FAS plays an important role in the regulation in medium chain fatty acid synthesis.
本研究通过活体采集西北农林科技大学萨能羊原种场纯种萨能奶山羊乳腺组织,利用组织块法分离培养奶山羊乳腺上皮细胞,纯化后转染人端粒酶逆转录酶基因,激活细胞端粒酶,以期获得具有无限增值能力的奶山羊乳腺上皮细胞系,为奶山羊乳腺功能基因组学研究奠定基础。同时,以此细胞系为试验材料,利用腺病毒介导的RNA干扰技术沉默FAS基因,研究该基因沉默后细胞中脂肪酸组成的变化及对参与脂代谢调控的重要基因如瘦素受体、脂肪酸结合蛋白等表达的影响,以探讨乳腺FAS基因在脂肪酸合成中的作用。试验结果如下:
(1)采用组织块法分离培养并纯化了奶山羊乳腺上皮细胞,通过脂质体转染法将编码人端粒酶逆转录酶基因的pCIneo-hTERT质粒导入原代培养的乳腺上皮细胞(第9代),经浓度为400μg/mL的G418筛选15天获得了抗性细胞克隆,采用滤纸片法转移、扩增后,hTERT基因稳定转入细胞,端粒酶活性检测呈阳性。细胞生长曲线测定结果表明,转入hTERT基因的细胞增殖能力增强,其生长曲线具有典型的S型特征。免疫细胞化学分析表明,所培养的细胞表达上皮细胞的标志物角蛋白8及上皮膜抗原(EMA),证明所培养细胞是乳腺上皮细胞。利用反转录PCR及Western blotting技术可以检测到细胞表达β-酪蛋白基因,而且加入催乳素后,β-酪蛋白基因表达量较对照组增加6.5倍,表明所培养细胞保持有乳腺细胞的基本功能。细胞在体外连续稳定培养67代,最终在91代时细胞死亡。
(2)根据FAS基因的序列,设计并合成了三对针对该基因不同区域的shRNA及一个阴性对照(shRNA-5544、shRNA-5936、shRNA-6132及shRNA-NC),将模板退火形成双链后,构建了表达这四条shRNA的pENTR/CMV-GFP/U6-shRNA载体,并在HEK293细胞中通过共转染的方法成功筛选出两条有效干扰序列(shRNA-5544及shRNA-5936)。通过同源重组,成功构建了pAd-shRNA-5544, pAd-shRNA-5936, pAd-shRNA-NC三个重组腺病毒载体,并在HEK 293细胞中包装出腺病毒颗粒,病毒经扩增后滴度分别达6×108 PFU/mL、5×108 PFU/mL及6×108 PFU/mL。将腺病毒(MOI=200,表达shRNA-5936)感染乳腺上皮细胞72h后,荧光实时定量及Western blotting结果显示FAS基因表达水平降低74%(与对照相比),脂肪酸测定结果表明FAS基因沉默后,细胞中C10:0、C12:0、C16:0、C18:0含量分别降低42%、42.37%、18.51%、29.3%;C14:0含量增加29.82%;荧光实时定量结果表明瘦素受体(LEPR)基因及肝X受体(LXRΑ)基因表达量分别为对照组的1.71及1.36倍;脂肪细胞型脂肪酸结合蛋白(AFABP)基因及脂蛋白脂酶(LPL)基因表达水平分别下调32%及25%。研究结果表明FAS基因对乳腺上皮细胞中中链脂肪酸具有重要的调控作用。
Fatty acid synthase (FAS), a key enzyme in the process of fatty acid synthesis in mammal, participates in an extremely complicated metabolism regulation in cell life and activities, such as energy storage, forming of structure of biology membrane, signal transduction and protein acetylation. It is known that goat milk is rich in short and medium-chain fatty acid, which is beneficial to prevent some metabolism illness in human being. The Acetyl-CoA and malonyl-CoA transacylases (AT/MT) domain of FAS gene in goat mammary gland shows medium-chain Thioesterase activity, which may play an important role in the specific fatty acid composition of goat milk. So, it is significant to study the function of FAS gene on fatty acid synthesis in goat mammary gland epithelia cell in order to reveal the mechanism of high concentration of short and medium-chain fatty acid in goat milk, and as a result it will show an theoretical and practical meaning in up-regulating beneficial fatty acid in milk by genetic methods.
In this study, Xinong Saanen goat mammary gland tissue was collected for primary epithelial cell culture. Human Telomerase Reverse Transcriptase (hTERT) gene was transfected into the purified primary cell in order to obtain stable goat mammary gland epithelial cell (GMEC) line which will become an excellent materials in gene function research of goat mammary gland. Additionally, we carry out functional silencing of FAS gene in GMEC cell by adenovirus-mediated delivery of short hairpin RNA, and investigate changes of fatty acid composition and several fatty acid metabolism-related genes (such as Leptin receptor, AFABP, et al) after silence of FAS. The results of this research are as follows:
(1) Purified GMEC were obtained by tissue culture. Then the pCIneo-hTERT plasmid encoding hTERT was transfected into purified primary GMEC, followed by G418 screening as a concentration of 400μg/mL. Anti-G418 positive cell clones appreared and the cell clones were transmitted using small filter papper and expanded for continuous culture. After stable transfection, telomerase activity turned to be positive in these transfected cells. Growth curve, all are typical“S”type, shows that transfected cells possessed more powerful proliferation activities. Immunocytochemistry analysis proofed that the cells are GMEC, because keratin 8 and epithelial membrane antigen, both of which are marker of epithelial cell, can be detected. Additionally, we have detected the expression ofβ-casein in the cells by RT-PCR and western blotting. The expression ofβ-casein up-regulated by 6.5 time compared with control after stimulating by prolactin. The transfected cells have stably passed 67 passages, died at 91st passages at last.
(2) According to the FAS sequence, three shRNA sequences target different area of FAS and one negative control (shRNA-5544, shRNA-5936, shRNA-6132 and shRNA-NC) were designed and synthesized. After annealing, shRNA templates were constructed into pENTR/CMV-GFP/U6 vecor. By cotransfecting HEK 293 Cell, the result shows that entry vector expressing shRNA-5544 and shRNA-5936 sequences caused an obvious interference effect. Then we generated three recombinant adenovirus vectors (pAd-shRNA-5544, pAd-shRNA-5936, pAd-shRNA-NC) by LR recombination and adenovirus were successfully packed in HEK 293 Cell. The titer of the adenoviral stock was determined by TCID50 method and respectively reaches 6×108 PFU/mL (Expressing shRNA-5544 sequence), 5×108 PFU/mL (Expressing shRNA-5936 sequence), 6×108 PFU/mL (Expressing shRNA-NC sequence). Then we use adenovirus (MOI=200, Expressing shRNA-5936 sequence) to infect GMEC (72h), and expression of FAS mRNA was reduced by 74% by Real-time PCR and western blotting analysis. Fatty acid analysis shows that the content of C10:0, C12:0, C16:0, C18:0 reduced by 42%, 42.37%, 18.51% and 29.3%; the content of C14:0 improved by 29.82%. The result of quantitative Real-time PCR shows LEPR and LXR gene were up-regulated by 1.71 and 1.36 fold, while AFABP and LXR gene were respectively down-regulated by 32% and 25% after the silence of FAS gene. The study shows that FAS plays an important role in the regulation in medium chain fatty acid synthesis.
引文
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