人血小板生成素基因表达和调控的初步研究
摘要
TPO是新近发现的一个十分重要的造血细胞生长和发育的调控因子，它能特异性
促进巨核细胞增殖、分化及血小板成熟，因而在临床上治疗由于血小板减少而引发的各
类疾病中具有重要的应用价值。为了研究TPO内含子和5'非翻译区对其表达的影响，
拟利用RT-PCR和长距离PCR技术从中国人胎肝组织中分别扩增全长人TPOcDNA，6.2kb
的TPO基因组DNA以及TPO基因中不同的内含子。首先，以胎肝mRNA为模板，应用RT-PCR
技术分两段扩增并克隆了人全长约1.0kb的TPO cDNA分子，序列分析表明：该TPO cDNA
分子中缺失编码Leu~(133)～GIy~(137)的15个碱基。资料表明，这种缺失体存在于正常的人体
内，是由于TPO mRNA的不同剪接造成。为此，参照已发表的TPO cDNA序列，人工合成
包含上述缺失核苷酸序列的DNA片段，重新获得全长TPO cDNA。其次，利用常规PCR
和长距离PCR技术，从胎肝组织中分别克隆了6.2kb的TPO基因组和TPO基因组中的内
含子Ⅰ，内含子Ⅱ～Ⅳ和内含于Ⅴ，全序列分析表明，已扩增的TPO产物中，所有编码
序列，全部内含子/外显子剪切部位序列同文献报道一致，在内含子中有几个碱基同文
献报道不一致，这可能由于个体差异或突变造成。
为了在细胞和动物个体水平上研究TPO基因5'非翻译区和不同内含子对TPO表
达的影响，在已获得了全长人TPOcDNA、TPO基因组DNA以及TPO基因中不同内含子的
基础上，构建了以CMV、大鼠WAP、山羊β-casein基因调控的各类TPO真核表达质粒
（共16种），并经脂质体转染，在COS-1细胞、HC-11细胞上获得了瞬间表达。结果表
明：在转染48小时后，各类TPO重组质粒的表达顺序为：TPO gDNA＜TPO intronⅠ＜
△TPO intronⅠ＜TPOcDNA＜TPO intron Ⅴ。这一结果提示：①在TPO基因组中，最后
一个内含子可显著提高TPO的表达水平，表明其可能含有特殊的增强序列；②在TPO基
因组中可能存在抑制TPO高水平表达的结构元件，造成TPO基因组在细胞中不表达或低
水平表达。
根据上述细胞水平上的表达结果，选用门种以大鼠 PAP、卜casein基因调控的
Top重组表达质粒通过显微注射法制备了转基因小鼠。通过PCR和／或Southern印迹筛
选己整合TPO基因的阳性转基因鼠，通过高灵敏度ELISA方法检测泌乳期阳性转基因鼠
乳汁中N 含量，结果表明：①在以WAP／TPO融合基因制备的6种转基因鼠中，外源基
因平均的整合率为10～15％，有5只雌性转基因阳性鼠乳汁中有人P 的表达，表达
水平为400pg／ml～2000pg／ml，其中以含有TPO最后一个内含子的转基因鼠表达水平最C
高，其表达量分别为 2307pg加l和 1307pg／ml。②在以 D－casein／TPO融合基因制备的
5种TPO转基因鼠中，外源基因的平均整合率为3～23％，有7只雌性转基因阳性鼠乳汁
中有人w 的表达，表达水平为＊／ml～146ng／ml，其中以含有P 最后一个内含子
的转基因鼠表达水平最高，其表达量为 146 ng／ml。这一结果进一步证明：①在TPO
基因组中，最后一个内含子可显著提高TPO的表达水平，表明其可能含有特殊的增强序
列：②在TPO基因组中可能存在抑制TPO高水平表达的结构元件，造成TPO基因组在细
胞中不表达或低水平表达。③p－casein基因调控序列可指导TPO基因在转基因动物乳
腺中高水平表达。
Thrombopoietin(TPO),also known as c-mplligand, is a key regulator of the
proliferation and maturation of megakaryocytes as well as platelet production.
In order to study on regulational role of TPO gene 5 UTR and intron on TPO
expression, TPO eDNA, TPO genomic DNA and all intron of TPO gene were isohted
by PCR and LD-PCR techniques. Sequencing analysis indicates that all coding
sequence, exon/intron splice site are the same with as. that reported by
Sohma, Foster and Gurrey.
To study the role of 5UTR and intron in expression and regulation of TPO
gene,a number of expression vector for TPO gene fused to the regulatory
elements of the CMV. rat WAP gene and goat β-casein gene were constructed.
These expression vector were introduced into cultured cos-1 cells and HC-11
cells for transient expression of TPO gene using lipofectin method. The
supernatant of cells transfeetion with above gene at 48h was analysized by
sandwich ELISA, the results showed that (a)the expression level of the
recombinant plasmids containing CMV-TPO fusion gene was the highest in cos-1
cells; (b) the expression level of the recombinant plasmids containing β-casein
TPO fusion gene was the highest in HC-11 cells; (c)the trend of TPO gene
expression level was TPO intron v> TPOcDNA>ATPO intron I> TPO intron I> TPO
3
gLINA in two cell lines. These results suggested that 5IJTR of TPO gene may
inhibit expression of TPO gene and intron of TPO gene may play a different role
in the expression and regulation of TPO gene.
The WAP/TPO and β-casein/TPO fusion gene were also microinjected into
fertilized eggs of mouse to generate transgenic mice. The total of 546 young
mice (F0) were produced , 1 of them were eaten by their mothers. 3?8% integration
rate of Fa individuals was estimated based on PCR and/or southern blot.
The human TPO was detectable in milk of the 5 lactating transgenic mice
containing WAP/TPO fusion gene, the expression level is approximately
400-2000pg/ml and the level in transgenic mice containing IAP-TPO intronV-TPO
cDNA fusion gene are the highest. The milk of 7 lactating transgenic mice
containing P 梒asein/TPO fusion gene also was analysed, the expression level
of human TPO is approximately lng?46ng/ml and the level in transgenic mice
containing P-TPO intronV-TPO cDNA fusion gene are also the highest in all
transgenic mice detected. These resultes suggested that (a)the last intron of
TPO gene can improve expression level of TPO gene; (b) 5~UTR of TPO gene may
inhibit expression of TPO gene; (c) the regulatory elemenit of goat β-casein gene
can specifically control the high-expression of TPO gene in mammary gland of
transgenic animal.
促进巨核细胞增殖、分化及血小板成熟，因而在临床上治疗由于血小板减少而引发的各
类疾病中具有重要的应用价值。为了研究TPO内含子和5'非翻译区对其表达的影响，
拟利用RT-PCR和长距离PCR技术从中国人胎肝组织中分别扩增全长人TPOcDNA，6.2kb
的TPO基因组DNA以及TPO基因中不同的内含子。首先，以胎肝mRNA为模板，应用RT-PCR
技术分两段扩增并克隆了人全长约1.0kb的TPO cDNA分子，序列分析表明：该TPO cDNA
分子中缺失编码Leu~(133)～GIy~(137)的15个碱基。资料表明，这种缺失体存在于正常的人体
内，是由于TPO mRNA的不同剪接造成。为此，参照已发表的TPO cDNA序列，人工合成
包含上述缺失核苷酸序列的DNA片段，重新获得全长TPO cDNA。其次，利用常规PCR
和长距离PCR技术，从胎肝组织中分别克隆了6.2kb的TPO基因组和TPO基因组中的内
含子Ⅰ，内含子Ⅱ～Ⅳ和内含于Ⅴ，全序列分析表明，已扩增的TPO产物中，所有编码
序列，全部内含子/外显子剪切部位序列同文献报道一致，在内含子中有几个碱基同文
献报道不一致，这可能由于个体差异或突变造成。
为了在细胞和动物个体水平上研究TPO基因5'非翻译区和不同内含子对TPO表
达的影响，在已获得了全长人TPOcDNA、TPO基因组DNA以及TPO基因中不同内含子的
基础上，构建了以CMV、大鼠WAP、山羊β-casein基因调控的各类TPO真核表达质粒
（共16种），并经脂质体转染，在COS-1细胞、HC-11细胞上获得了瞬间表达。结果表
明：在转染48小时后，各类TPO重组质粒的表达顺序为：TPO gDNA＜TPO intronⅠ＜
△TPO intronⅠ＜TPOcDNA＜TPO intron Ⅴ。这一结果提示：①在TPO基因组中，最后
一个内含子可显著提高TPO的表达水平，表明其可能含有特殊的增强序列；②在TPO基
因组中可能存在抑制TPO高水平表达的结构元件，造成TPO基因组在细胞中不表达或低
水平表达。
根据上述细胞水平上的表达结果，选用门种以大鼠 PAP、卜casein基因调控的
Top重组表达质粒通过显微注射法制备了转基因小鼠。通过PCR和／或Southern印迹筛
选己整合TPO基因的阳性转基因鼠，通过高灵敏度ELISA方法检测泌乳期阳性转基因鼠
乳汁中N 含量，结果表明：①在以WAP／TPO融合基因制备的6种转基因鼠中，外源基
因平均的整合率为10～15％，有5只雌性转基因阳性鼠乳汁中有人P 的表达，表达
水平为400pg／ml～2000pg／ml，其中以含有TPO最后一个内含子的转基因鼠表达水平最C
高，其表达量分别为 2307pg加l和 1307pg／ml。②在以 D－casein／TPO融合基因制备的
5种TPO转基因鼠中，外源基因的平均整合率为3～23％，有7只雌性转基因阳性鼠乳汁
中有人w 的表达，表达水平为＊／ml～146ng／ml，其中以含有P 最后一个内含子
的转基因鼠表达水平最高，其表达量为 146 ng／ml。这一结果进一步证明：①在TPO
基因组中，最后一个内含子可显著提高TPO的表达水平，表明其可能含有特殊的增强序
列：②在TPO基因组中可能存在抑制TPO高水平表达的结构元件，造成TPO基因组在细
胞中不表达或低水平表达。③p－casein基因调控序列可指导TPO基因在转基因动物乳
腺中高水平表达。
Thrombopoietin(TPO),also known as c-mplligand, is a key regulator of the
proliferation and maturation of megakaryocytes as well as platelet production.
In order to study on regulational role of TPO gene 5 UTR and intron on TPO
expression, TPO eDNA, TPO genomic DNA and all intron of TPO gene were isohted
by PCR and LD-PCR techniques. Sequencing analysis indicates that all coding
sequence, exon/intron splice site are the same with as. that reported by
Sohma, Foster and Gurrey.
To study the role of 5UTR and intron in expression and regulation of TPO
gene,a number of expression vector for TPO gene fused to the regulatory
elements of the CMV. rat WAP gene and goat β-casein gene were constructed.
These expression vector were introduced into cultured cos-1 cells and HC-11
cells for transient expression of TPO gene using lipofectin method. The
supernatant of cells transfeetion with above gene at 48h was analysized by
sandwich ELISA, the results showed that (a)the expression level of the
recombinant plasmids containing CMV-TPO fusion gene was the highest in cos-1
cells; (b) the expression level of the recombinant plasmids containing β-casein
TPO fusion gene was the highest in HC-11 cells; (c)the trend of TPO gene
expression level was TPO intron v> TPOcDNA>ATPO intron I> TPO intron I> TPO
3
gLINA in two cell lines. These results suggested that 5IJTR of TPO gene may
inhibit expression of TPO gene and intron of TPO gene may play a different role
in the expression and regulation of TPO gene.
The WAP/TPO and β-casein/TPO fusion gene were also microinjected into
fertilized eggs of mouse to generate transgenic mice. The total of 546 young
mice (F0) were produced , 1 of them were eaten by their mothers. 3?8% integration
rate of Fa individuals was estimated based on PCR and/or southern blot.
The human TPO was detectable in milk of the 5 lactating transgenic mice
containing WAP/TPO fusion gene, the expression level is approximately
400-2000pg/ml and the level in transgenic mice containing IAP-TPO intronV-TPO
cDNA fusion gene are the highest. The milk of 7 lactating transgenic mice
containing P 梒asein/TPO fusion gene also was analysed, the expression level
of human TPO is approximately lng?46ng/ml and the level in transgenic mice
containing P-TPO intronV-TPO cDNA fusion gene are also the highest in all
transgenic mice detected. These resultes suggested that (a)the last intron of
TPO gene can improve expression level of TPO gene; (b) 5~UTR of TPO gene may
inhibit expression of TPO gene; (c) the regulatory elemenit of goat β-casein gene
can specifically control the high-expression of TPO gene in mammary gland of
transgenic animal.
引文
1) Kuter DJ, Future directions with platelet growth factors. Seminars in Hematology, 2000,37(2) :41-49．
2) Chang MS, McNinch J, Basu RJ, et al.,Cloning and characterization of the human megakaryocyte growth and development factor (MGDF) gene. J Biol Chem, 1995,270:511-514．
3) Kato T, Matsumoto A, Ogamik, et al., Native thrombopoietin: structure and function. STEM CELLS, 1998,16:322-328．
4) Tahara T, Usukik, Sato H, et al., A sensitive sandwich ELESA for measuring thrombopoietin in human serum: serum thrombopoietin levels in healthy volunteers and in patients with haemopoietic disorders. Br J Haematol, 1996,93:783-788．
5) Hanke J, Alternative splicing of human genes: more the rule than the exception? Trends Genet, 1999,15:389-390．
6) Graveley BR, Alternative splicing: increasing diversity in the proteomic world. Trends Genet, 2001,17:100-107．
7) Ewing B, Green P, Analysis of expressed sequencetags ind: cates 35,000 human genes. Nat Genet, 2000,25:232-234．
8) Gurney AL, Kuarg WT, Xie MH, et al., Genomic structure, chromosomal localization and conserved alternative splice forms of thrombopoietin Blood, 1995,85:981-988．
9) Sohma Y, Akahori H, Seki N, et al., Molecular cloning and chromosomal
localization of the human thrombopoietin gene. FEBS Lett, 1994,353:57-61.
10) Foster DC, Sprecher CA, Crant FJ, et al., Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization. Proc Natl Acad Sci USA, 1994, 91:13023-13027.
11) 王晓斌，刘国仰，有关内含子功能研究的新进展。中华医学遗传学杂志，2000，17(3)：211-212。
12) Kamura I, Handa H, Hamasaki N, et al., Characterization of the human thrombopoietin gene promoter. J Biol Chem, 1997, 272:11361-11368.
13) Ghilardi BN, Wiestner A, Skoda RC, Thrombopoietin production is inhibited by a translational mechanism. Blood, 1998, 92:4024-4030.
14) 曾溢滔，上海：上海教育出版社，2000，111-112．
15) Kato T, Protein characteristics of thrombopoietin. STEM CELLS 1996,14:139-147.
16) Colman A Production of proteins in the milk of transgenic livestock: problems, solutions, and success.Am J Clin Nutr, 1996, 63:6395-645S.
17) Rudopph NS Biopharmaceutical production in transgenic livestock. Trends Biotech, 1999, 17:367-374.
18) Dale TC, Krnacik MJ, Rosen JM, et al., High-level expression of the rat whey acidic protein gene is mediated by elements in promoter and 3′ untranslated vegion. Mole Cell-Biol ,1992, 12:905-914.
19) Chen L-H, and M J, Bissell A novel regalatory mechanism for whey acidic protein gene expression. Cell Regul, 1989,1:45-54.
20) Gordon JW, Scangos GA, Plotkin J, et al., Genetic transformation of mouse embryos by microinjection of purified DNA Proc Nat Acod Sci USA, 1986, 77:7380-4.
21) Palmiter RD, Brinster R1, Hammer RE,et al., Dramatic growth of mice that develop from eggs microinjected with metallothioneingrowth hormone fusion genes. Nature, 1982, 300:611-615
22) 薛京伦，卢大儒．乳腺生物反应器研究现状 生物技术通报，1998，3：17-20
23) Rudopph NS Biopharmaceutical production in transgenic livestock. Trends Biotech, 1999, 17:367-374
24) 卢一凡，田馇，邓继先 等人GCSF基因组克隆及其在转基因小鼠乳腺中表达研究 遗传学报
1999, 26: 281-287
25) Ebert K M,Paul D ,Barry CA et al .,Induction of human tissue plasminogen activator in the mammary gland of transgenic goats BIO/TECHNOLOGY 1994,12:699-702
26) Bonifer C,Developmental regulation of eukaryotic gene loci.TIG, 2000,16(7) :310-315．
27) Giraldo P.Montoliu 1 Size matters:use of YACs,BACs and PACs in transgenic animals Transgenic Research 2001,10:83-103
28) Garrick D, Fiering S, Martin DLK ,et al., Repeatinduced gene silencing in mammals. Nature Gen ,1998,18:56-59．
29) Houdebine LM and Attal J Internal ribosome entry sites (IRESs):reality and use. Transgenic Res, 1999,8:157-177．
30) Ghilardi BN, Wiestoer A, Skoda RC, Thrombopoietin production is inhibited by a translational mechanism. Blood ,1998, 92:4024-4030．
2) Chang MS, McNinch J, Basu RJ, et al.,Cloning and characterization of the human megakaryocyte growth and development factor (MGDF) gene. J Biol Chem, 1995,270:511-514．
3) Kato T, Matsumoto A, Ogamik, et al., Native thrombopoietin: structure and function. STEM CELLS, 1998,16:322-328．
4) Tahara T, Usukik, Sato H, et al., A sensitive sandwich ELESA for measuring thrombopoietin in human serum: serum thrombopoietin levels in healthy volunteers and in patients with haemopoietic disorders. Br J Haematol, 1996,93:783-788．
5) Hanke J, Alternative splicing of human genes: more the rule than the exception? Trends Genet, 1999,15:389-390．
6) Graveley BR, Alternative splicing: increasing diversity in the proteomic world. Trends Genet, 2001,17:100-107．
7) Ewing B, Green P, Analysis of expressed sequencetags ind: cates 35,000 human genes. Nat Genet, 2000,25:232-234．
8) Gurney AL, Kuarg WT, Xie MH, et al., Genomic structure, chromosomal localization and conserved alternative splice forms of thrombopoietin Blood, 1995,85:981-988．
9) Sohma Y, Akahori H, Seki N, et al., Molecular cloning and chromosomal
localization of the human thrombopoietin gene. FEBS Lett, 1994,353:57-61.
10) Foster DC, Sprecher CA, Crant FJ, et al., Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization. Proc Natl Acad Sci USA, 1994, 91:13023-13027.
11) 王晓斌，刘国仰，有关内含子功能研究的新进展。中华医学遗传学杂志，2000，17(3)：211-212。
12) Kamura I, Handa H, Hamasaki N, et al., Characterization of the human thrombopoietin gene promoter. J Biol Chem, 1997, 272:11361-11368.
13) Ghilardi BN, Wiestner A, Skoda RC, Thrombopoietin production is inhibited by a translational mechanism. Blood, 1998, 92:4024-4030.
14) 曾溢滔，上海：上海教育出版社，2000，111-112．
15) Kato T, Protein characteristics of thrombopoietin. STEM CELLS 1996,14:139-147.
16) Colman A Production of proteins in the milk of transgenic livestock: problems, solutions, and success.Am J Clin Nutr, 1996, 63:6395-645S.
17) Rudopph NS Biopharmaceutical production in transgenic livestock. Trends Biotech, 1999, 17:367-374.
18) Dale TC, Krnacik MJ, Rosen JM, et al., High-level expression of the rat whey acidic protein gene is mediated by elements in promoter and 3′ untranslated vegion. Mole Cell-Biol ,1992, 12:905-914.
19) Chen L-H, and M J, Bissell A novel regalatory mechanism for whey acidic protein gene expression. Cell Regul, 1989,1:45-54.
20) Gordon JW, Scangos GA, Plotkin J, et al., Genetic transformation of mouse embryos by microinjection of purified DNA Proc Nat Acod Sci USA, 1986, 77:7380-4.
21) Palmiter RD, Brinster R1, Hammer RE,et al., Dramatic growth of mice that develop from eggs microinjected with metallothioneingrowth hormone fusion genes. Nature, 1982, 300:611-615
22) 薛京伦，卢大儒．乳腺生物反应器研究现状 生物技术通报，1998，3：17-20
23) Rudopph NS Biopharmaceutical production in transgenic livestock. Trends Biotech, 1999, 17:367-374
24) 卢一凡，田馇，邓继先 等人GCSF基因组克隆及其在转基因小鼠乳腺中表达研究 遗传学报
1999, 26: 281-287
25) Ebert K M,Paul D ,Barry CA et al .,Induction of human tissue plasminogen activator in the mammary gland of transgenic goats BIO/TECHNOLOGY 1994,12:699-702
26) Bonifer C,Developmental regulation of eukaryotic gene loci.TIG, 2000,16(7) :310-315．
27) Giraldo P.Montoliu 1 Size matters:use of YACs,BACs and PACs in transgenic animals Transgenic Research 2001,10:83-103
28) Garrick D, Fiering S, Martin DLK ,et al., Repeatinduced gene silencing in mammals. Nature Gen ,1998,18:56-59．
29) Houdebine LM and Attal J Internal ribosome entry sites (IRESs):reality and use. Transgenic Res, 1999,8:157-177．
30) Ghilardi BN, Wiestoer A, Skoda RC, Thrombopoietin production is inhibited by a translational mechanism. Blood ,1998, 92:4024-4030．