奶牛体细胞的纯化与人COL1α1 cDNA转染及核移植胚胎发育研究
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摘要
乳腺生物反应器(mammary gland bioreactor),或称动物个体乳腺表达系统,是指利用乳腺特异表达的乳蛋白基因调控序列构建表达载体,制作转基因动物,指导特定外源基因在动物乳腺中特异、高效率地表达,以期从分泌乳汁中获得外源活性蛋白。它具有产量高、生产成本低、易于纯化和产品活性高等优势。目前利用转基因乳腺生物反应器生产的药物蛋白有不少已经成功应用于临床治疗等多种相关领域。利用转基因克隆技术可以把对基因筛选的过程提前到细胞水平,保证出生的克隆个体是转基因动物。
在高等动物体内,胶原广泛分布于结缔组织、皮肤、骨骼、内脏的间充质细胞及肌腔、韧带、巩膜等部位,角膜几乎完全由胶原蛋白组成。胶原是结缔组织及其重要的结构蛋白,起支撑器官、保护机体的功能,是决定结缔组织韧性的主要因素。胶原蛋白因具有高生物活性和低免疫原性,在医药、医疗、美容、食品领域被广泛应用。人源性胶原蛋白对人类更为重要和安全,但由于伦理和道德的限制不能直接获得,所以把人的胶原蛋白利用乳腺生物反应器的方法生产十分必要且安全可行。
利用乳腺生物反应器生产人类胶原蛋白,首先要获得人胶原蛋白基因,并把此基因引入有效的乳腺表达载体中。其次把构建的载体通过适当的转基因方法转入靶细胞,从阳性靶细胞中获得可以无限扩增、遗传稳定的单细胞克隆系,并以此细胞为核移植供体细胞,利用核移植技术获得阳性胚胎,以期在此基础上最终获得阳性个体。
为了获得携带有目的基因的乳腺表达载体,并考虑到表达出来的融合蛋白能进行有效提纯及对阳性细胞能进行有效的筛选,实验首先从人的包皮组织中提取mRNA,反转录获得人胶原蛋白cDNA片段,在cDNA的3’端连接上了6×His蛋白分离标签。然后以pBC1质粒为基础质粒,以β-casein启动子为调控元件,通过表达质粒的改造,构建了人Ⅰ型α1胶原cDNA基因乳腺特异性表达载体。最后,再分别将绿色荧光蛋白(EGFP)和新霉素抗性基因(Neor)作为双标记基因引入表达载体,完成了乳腺特异表达载体的改造和构建。合成的cDNA片段经过测序,Blast结果表明,获得的中国人的Ⅰ型α1胶原cDNA基因与Gene Bank中公布的相应基因序列一致。对构建的载体进行了PCR片段扩增分析,结果显示,构建的载体结构完整、无缺失。
为了获得有效转染的单细胞克隆系,实验首先建立了成纤维细胞、卵丘颗粒细胞和输卵管上皮细胞系,并对这些靶细胞进行了脂质体转染和电穿孔转染实验,对转染的阳性细胞进行G418筛选并结合绿色荧光的表达对阳性细胞进行单细胞克隆的扩增培养。结果,用脂质体转染法对三种靶细胞进行转染后,成纤维细胞与卵丘颗粒细胞获得了成功转染,输卵管上皮细胞未能获得成功。之后对输卵管上皮细胞进行了电穿孔转染,通过一系列实验条件优化组合后,最后在90 mOsmo/kg低渗缓冲液、800V电压(2mm电穿孔管)下获得了20.8%的转染效率。通过对三种细胞进行G418耐受检测,得出了一致的检测结果,即三种细胞在G418浓度为800μg/mL左右时,能有效筛选出阳性细胞。获得初步筛选的细胞采用在绿色荧光下定位提取的方法将来自单细胞的阳性克隆细胞群进行了扩大,最后三种靶细胞都得到了遗传背景一致的单细胞克隆系。在实验中还发现,卵丘颗粒细胞在进行小分子量载体转染时能获得比大分子载体好的转染效果,成纤维细胞转基因细胞系的生长速度较非转基因的要慢3d左右。
为了解在核移植中作为供体的细胞在体外培养时的细胞凋亡情况,用流式细胞术在不同时段对成纤维细胞、卵丘颗粒细胞、输卵管上皮细胞和乳腺上皮细胞进行了细胞凋亡的检测。结果发现,细胞在同一代生长周期中,一旦处于接触抑制或饥饿培养过程中,凋亡速度明显加快,细胞随着培养代数的增加,凋亡率也不断增加。四种测试细胞的凋亡趋势是乳腺细胞凋亡最快,其次是输卵管上皮细胞,卵丘颗粒细胞在15代以前比成纤维细胞凋亡率低,但15代之后却高于成纤维细胞。
最后对三种转基因细胞核移植重构胚发育效果进行了比较,发现：1)成纤维细胞不同转基因细胞系之间的胚胎发育率没有显著差异(P>0.05)；2)同种细胞转基因和非转基因细胞作为供体进行核移植重构胚发育,三种转基因细胞的融合率都出现了明显的下降(P<0.05),但在融合后的发育中,转基因和非转基因细胞在卵裂率和桑椹胚/囊胚率上的差异不明显；3)对三种转基因细胞进行平行比较发现,成纤维细胞和卵丘颗粒细胞的融合率明显比输卵管上皮细胞高,而且差异显著(P<0.05)。但在融合后的桑椹胚/囊胚形成率上,成纤维细胞又明显低于卵丘颗粒细胞和输卵管上皮细胞(P<0.05)。上述结果说明：1)同种细胞不同转基因系之间可能有插入位点和拷贝数的差异,但这种差异对核移植重构胚早期发育没有影响；2)转基因过程和筛选过程对供体细胞的细胞膜结构有一定的影响,造成在融合过程中转基因细胞的融合性差,但转基因细胞一旦与卵母细胞融合,其随后的重构胚发育效果与非转基因细胞相似；3)在融合过程中,输卵管上皮细胞的融合相对于成纤维细胞和卵丘细胞困难,融合后,卵丘细胞和输卵管上皮细胞的早期胚胎发育能力较成纤维细胞强。
本文系统地进行了从载体的构建到细胞系的建立、细胞的转染、单细胞克隆系的筛选与扩大到各种转基因细胞的胚胎发育规律的研究,以期为转基因乳腺生物反应器的研究提供有益的参考。
Animal mammary gland bioreactor, also called individual animal mammary gland expression system, is the use of regulatory sequences of milk protein gene to construct expression vectors, produce transgenic animals and guide specific foreign genes expression.It has the advantages of high output, low cost, easy purification and high biological activity et al. Currently transgenic mammary gland bioreactor have been successfully used in clinical treatment and many other related fields. Transgenic cloning technology can screen genetic modified gene at cellular level, ensure that individuals are genetically modified cloned animals.
In higher animals, collagen is widely distributed in connective tissue, such as skin, bones, organs mesenchymal cells, muscle cavity, ligaments and sclera et al. Cornea is entirely made of collagen. Collagen is an important structural proteins, play the function of supportive organ and protect the organism. Because of its high biological activity and low immunogenicity, in the areas of pharmaceutical, medical, beauty product, food industry, collagen have been widely used. Human collagen, in particular procollagen is more important for human to use, but because of ethical and moral constraints, it can not directly access. So, produce human collagen using mammary gland bioreactor approach is necessary, feasible and safe.
Using mammary gland bioreactor to produce human collagen, first is to obtain collagen gene, then transfer this gene into the mammary gland expression vector. Second is to transfer the vector to target cells and obtain the positive target cells that is genetic stability and can be unlimited amplification. At last, using somatic nuclear transfer technology to obtain positive embryos.
For the purpose of obtaining the mammary gland gene expression vector, experiments were firstly extracted mRNA from human foreskin, reverse transcription to obtain one of the collagen protein cDNA fragment. In the 3'end of the cDNA connect a 6×His protein separation tag. Then using pBCl plasmid as based vector, using (3-casein gene promoter as a regulatory element, reconstructed the expression vector that connect with human type I collagen al cDNA gene. Finally, with enhanced green fluorescent protein (EGFP) and neomycin resistance gene (Neo') as a double marker genes into expression vector, completing the transformation of mammary specific expression vector. Synthesized cDNA fragments were sequenced. Blast results showed that the Chinese cloned type I al collagen cDNA gene are very similarity comparision with Gene Bank. The fragments of the vector were amplified by PCR analysis,the result showed that the constructed vector structure was complete and correct.
In order to obtain efficient transfectant of monoclonal cell lines, experiments established fibroblasts, cumulus granulosa cells and oviduct epithelial cell lines. These target cells were carried out liposome transfection and electroporation transfection. The results showed that fibroblasts and cumulus granulosa cells have been successful transfected by the method of liposome transfection, while oviduct epithelial cells is failed. But for the oviduct epithelial cells, after carrid out electroporation transfection, obtained 20.8% of transfection efficiency on the condition of 90 mOsmo / kg hypotonic buffer and 800V voltage electric tension (2mm electroporation tube). The three types of cells use G418 that concentration at 800μg/mL can effectively screen out the positive cells. The primary screened cells use EGFP as marker can further screen out transgenic positive clones that comes from one single cell and the monoclonal cell lines have the same genetic background.
In order to understand the apoptosis of fibroblasts, cumulus granulosa cells, oviductal epithelial cells and mammary epithelial cells that cultured in vitro, the methods of flow cytometry are performed. The results showed that at the same growth cycle, once the cells at the condition of contact inhibition or plateau phase, the process of apoptosis is significantly faster. Also with the increase of cell generations, the apoptosis rate increased. Overall, the rate of apoptosis in mammary cells is the fastest, followed by the oviduct epithelial cells. Cumulus granulosa cells' apoptosis rate was lower than fibroblast before 15 generations, but after 15 generations the other way around.
Finally, using the genetically modified cells as donors conducted nuclear transfer (NT) and the reconstructed embryos were compared. The comparison found that:1) The reconstructed embryonic development was no significant difference (P>0.05) between the different transgenic cell lines of fibroblasts; 2):Transgenic and non-transgenic cells as NT donors, their fusion rate has shown a significant (P<0.05) decrease, but their cleavage rate and morula/blastocyst rate was not significant; 3) Three types of genetically modified cells as NT donors, the results showed that the fusion rate of fibroblasts and cumulus granulosa cells was significantly (P<0.05) higher than the oviduct epithelial cells. However, the morulae/blastocyst formation rate of fibroblasts was significantly lower than cumulus granulosa cells and oviduct epithelial cells after fused (P<0.05). These results indicate that:1) Different transgenic lines of the same type of cells probably have different insertion site and copy number, but the early embryonic development had no significant difference; 2) Transgenic process and the selection process can give rise the membrane structure change to a certain extent and resulting the fusion rate was poorer; 3) In the process of fusion, oviduct epithelial cells was relative difficulty compared with fibroblasts and cumulus cells, but in the process of reconstructed embryonic early development, cumulus cells and oviduct epithelial cells have higher viability than fibroblasts.
This thesis had a series of systemic study that include vector construct, cells transfection, monoclonal cell lines amplification and the genetically modified embryonic development laws. The finally purpose is to provide useful reference for the development of mammary gland bioreactor.
在高等动物体内,胶原广泛分布于结缔组织、皮肤、骨骼、内脏的间充质细胞及肌腔、韧带、巩膜等部位,角膜几乎完全由胶原蛋白组成。胶原是结缔组织及其重要的结构蛋白,起支撑器官、保护机体的功能,是决定结缔组织韧性的主要因素。胶原蛋白因具有高生物活性和低免疫原性,在医药、医疗、美容、食品领域被广泛应用。人源性胶原蛋白对人类更为重要和安全,但由于伦理和道德的限制不能直接获得,所以把人的胶原蛋白利用乳腺生物反应器的方法生产十分必要且安全可行。
利用乳腺生物反应器生产人类胶原蛋白,首先要获得人胶原蛋白基因,并把此基因引入有效的乳腺表达载体中。其次把构建的载体通过适当的转基因方法转入靶细胞,从阳性靶细胞中获得可以无限扩增、遗传稳定的单细胞克隆系,并以此细胞为核移植供体细胞,利用核移植技术获得阳性胚胎,以期在此基础上最终获得阳性个体。
为了获得携带有目的基因的乳腺表达载体,并考虑到表达出来的融合蛋白能进行有效提纯及对阳性细胞能进行有效的筛选,实验首先从人的包皮组织中提取mRNA,反转录获得人胶原蛋白cDNA片段,在cDNA的3’端连接上了6×His蛋白分离标签。然后以pBC1质粒为基础质粒,以β-casein启动子为调控元件,通过表达质粒的改造,构建了人Ⅰ型α1胶原cDNA基因乳腺特异性表达载体。最后,再分别将绿色荧光蛋白(EGFP)和新霉素抗性基因(Neor)作为双标记基因引入表达载体,完成了乳腺特异表达载体的改造和构建。合成的cDNA片段经过测序,Blast结果表明,获得的中国人的Ⅰ型α1胶原cDNA基因与Gene Bank中公布的相应基因序列一致。对构建的载体进行了PCR片段扩增分析,结果显示,构建的载体结构完整、无缺失。
为了获得有效转染的单细胞克隆系,实验首先建立了成纤维细胞、卵丘颗粒细胞和输卵管上皮细胞系,并对这些靶细胞进行了脂质体转染和电穿孔转染实验,对转染的阳性细胞进行G418筛选并结合绿色荧光的表达对阳性细胞进行单细胞克隆的扩增培养。结果,用脂质体转染法对三种靶细胞进行转染后,成纤维细胞与卵丘颗粒细胞获得了成功转染,输卵管上皮细胞未能获得成功。之后对输卵管上皮细胞进行了电穿孔转染,通过一系列实验条件优化组合后,最后在90 mOsmo/kg低渗缓冲液、800V电压(2mm电穿孔管)下获得了20.8%的转染效率。通过对三种细胞进行G418耐受检测,得出了一致的检测结果,即三种细胞在G418浓度为800μg/mL左右时,能有效筛选出阳性细胞。获得初步筛选的细胞采用在绿色荧光下定位提取的方法将来自单细胞的阳性克隆细胞群进行了扩大,最后三种靶细胞都得到了遗传背景一致的单细胞克隆系。在实验中还发现,卵丘颗粒细胞在进行小分子量载体转染时能获得比大分子载体好的转染效果,成纤维细胞转基因细胞系的生长速度较非转基因的要慢3d左右。
为了解在核移植中作为供体的细胞在体外培养时的细胞凋亡情况,用流式细胞术在不同时段对成纤维细胞、卵丘颗粒细胞、输卵管上皮细胞和乳腺上皮细胞进行了细胞凋亡的检测。结果发现,细胞在同一代生长周期中,一旦处于接触抑制或饥饿培养过程中,凋亡速度明显加快,细胞随着培养代数的增加,凋亡率也不断增加。四种测试细胞的凋亡趋势是乳腺细胞凋亡最快,其次是输卵管上皮细胞,卵丘颗粒细胞在15代以前比成纤维细胞凋亡率低,但15代之后却高于成纤维细胞。
最后对三种转基因细胞核移植重构胚发育效果进行了比较,发现：1)成纤维细胞不同转基因细胞系之间的胚胎发育率没有显著差异(P>0.05)；2)同种细胞转基因和非转基因细胞作为供体进行核移植重构胚发育,三种转基因细胞的融合率都出现了明显的下降(P<0.05),但在融合后的发育中,转基因和非转基因细胞在卵裂率和桑椹胚/囊胚率上的差异不明显；3)对三种转基因细胞进行平行比较发现,成纤维细胞和卵丘颗粒细胞的融合率明显比输卵管上皮细胞高,而且差异显著(P<0.05)。但在融合后的桑椹胚/囊胚形成率上,成纤维细胞又明显低于卵丘颗粒细胞和输卵管上皮细胞(P<0.05)。上述结果说明：1)同种细胞不同转基因系之间可能有插入位点和拷贝数的差异,但这种差异对核移植重构胚早期发育没有影响；2)转基因过程和筛选过程对供体细胞的细胞膜结构有一定的影响,造成在融合过程中转基因细胞的融合性差,但转基因细胞一旦与卵母细胞融合,其随后的重构胚发育效果与非转基因细胞相似；3)在融合过程中,输卵管上皮细胞的融合相对于成纤维细胞和卵丘细胞困难,融合后,卵丘细胞和输卵管上皮细胞的早期胚胎发育能力较成纤维细胞强。
本文系统地进行了从载体的构建到细胞系的建立、细胞的转染、单细胞克隆系的筛选与扩大到各种转基因细胞的胚胎发育规律的研究,以期为转基因乳腺生物反应器的研究提供有益的参考。
Animal mammary gland bioreactor, also called individual animal mammary gland expression system, is the use of regulatory sequences of milk protein gene to construct expression vectors, produce transgenic animals and guide specific foreign genes expression.It has the advantages of high output, low cost, easy purification and high biological activity et al. Currently transgenic mammary gland bioreactor have been successfully used in clinical treatment and many other related fields. Transgenic cloning technology can screen genetic modified gene at cellular level, ensure that individuals are genetically modified cloned animals.
In higher animals, collagen is widely distributed in connective tissue, such as skin, bones, organs mesenchymal cells, muscle cavity, ligaments and sclera et al. Cornea is entirely made of collagen. Collagen is an important structural proteins, play the function of supportive organ and protect the organism. Because of its high biological activity and low immunogenicity, in the areas of pharmaceutical, medical, beauty product, food industry, collagen have been widely used. Human collagen, in particular procollagen is more important for human to use, but because of ethical and moral constraints, it can not directly access. So, produce human collagen using mammary gland bioreactor approach is necessary, feasible and safe.
Using mammary gland bioreactor to produce human collagen, first is to obtain collagen gene, then transfer this gene into the mammary gland expression vector. Second is to transfer the vector to target cells and obtain the positive target cells that is genetic stability and can be unlimited amplification. At last, using somatic nuclear transfer technology to obtain positive embryos.
For the purpose of obtaining the mammary gland gene expression vector, experiments were firstly extracted mRNA from human foreskin, reverse transcription to obtain one of the collagen protein cDNA fragment. In the 3'end of the cDNA connect a 6×His protein separation tag. Then using pBCl plasmid as based vector, using (3-casein gene promoter as a regulatory element, reconstructed the expression vector that connect with human type I collagen al cDNA gene. Finally, with enhanced green fluorescent protein (EGFP) and neomycin resistance gene (Neo') as a double marker genes into expression vector, completing the transformation of mammary specific expression vector. Synthesized cDNA fragments were sequenced. Blast results showed that the Chinese cloned type I al collagen cDNA gene are very similarity comparision with Gene Bank. The fragments of the vector were amplified by PCR analysis,the result showed that the constructed vector structure was complete and correct.
In order to obtain efficient transfectant of monoclonal cell lines, experiments established fibroblasts, cumulus granulosa cells and oviduct epithelial cell lines. These target cells were carried out liposome transfection and electroporation transfection. The results showed that fibroblasts and cumulus granulosa cells have been successful transfected by the method of liposome transfection, while oviduct epithelial cells is failed. But for the oviduct epithelial cells, after carrid out electroporation transfection, obtained 20.8% of transfection efficiency on the condition of 90 mOsmo / kg hypotonic buffer and 800V voltage electric tension (2mm electroporation tube). The three types of cells use G418 that concentration at 800μg/mL can effectively screen out the positive cells. The primary screened cells use EGFP as marker can further screen out transgenic positive clones that comes from one single cell and the monoclonal cell lines have the same genetic background.
In order to understand the apoptosis of fibroblasts, cumulus granulosa cells, oviductal epithelial cells and mammary epithelial cells that cultured in vitro, the methods of flow cytometry are performed. The results showed that at the same growth cycle, once the cells at the condition of contact inhibition or plateau phase, the process of apoptosis is significantly faster. Also with the increase of cell generations, the apoptosis rate increased. Overall, the rate of apoptosis in mammary cells is the fastest, followed by the oviduct epithelial cells. Cumulus granulosa cells' apoptosis rate was lower than fibroblast before 15 generations, but after 15 generations the other way around.
Finally, using the genetically modified cells as donors conducted nuclear transfer (NT) and the reconstructed embryos were compared. The comparison found that:1) The reconstructed embryonic development was no significant difference (P>0.05) between the different transgenic cell lines of fibroblasts; 2):Transgenic and non-transgenic cells as NT donors, their fusion rate has shown a significant (P<0.05) decrease, but their cleavage rate and morula/blastocyst rate was not significant; 3) Three types of genetically modified cells as NT donors, the results showed that the fusion rate of fibroblasts and cumulus granulosa cells was significantly (P<0.05) higher than the oviduct epithelial cells. However, the morulae/blastocyst formation rate of fibroblasts was significantly lower than cumulus granulosa cells and oviduct epithelial cells after fused (P<0.05). These results indicate that:1) Different transgenic lines of the same type of cells probably have different insertion site and copy number, but the early embryonic development had no significant difference; 2) Transgenic process and the selection process can give rise the membrane structure change to a certain extent and resulting the fusion rate was poorer; 3) In the process of fusion, oviduct epithelial cells was relative difficulty compared with fibroblasts and cumulus cells, but in the process of reconstructed embryonic early development, cumulus cells and oviduct epithelial cells have higher viability than fibroblasts.
This thesis had a series of systemic study that include vector construct, cells transfection, monoclonal cell lines amplification and the genetically modified embryonic development laws. The finally purpose is to provide useful reference for the development of mammary gland bioreactor.
引文
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