Wnt-1的生物信息学分析及其对人表皮干细胞分化与增殖的影响的研究
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摘要
表皮干细胞是皮肤组织特异性干细胞,位于表皮基底层,在维持表皮更新,保持细胞恒定等方面起着重要作用,它也是皮肤及其附属器官发生、修复、改建的关键性源泉。表皮干细胞的增殖与分化不仅为自身基因所预先设置,还受到干细胞“壁龛”(Niche)的调控。Wnt家族是干细胞“壁龛”中重要的组成部分,参与细胞分化、细胞极性、细胞迁移以及细胞增殖等过程。其中,Wnt-1在神经上皮的发育、乳腺癌的发生等上皮性组织的发育与分化的过程中扮演着重要的角色。Wnt-1的表达与皮肤及其附属器的发育在时相上一致:Wnt-1在胚胎时期表达活跃,而成年时多处于相对静止状态;人表皮干细胞于胎龄24周时形成成熟的腺体,而在成年人的创口愈合处,则不能再生为任何腺体,提示表皮及其附属腺体的发育与Wnt-1的表达有很大的关系。基于此,我们在本课题里进行了Wnt-1影响人表皮干细胞分化与增殖的相关研究。
在本课题中,我们基于“结构决定功能”的思想,首先以生物信息学的手段和方法,对Wnt-1进行氨基酸序列分析、二级结构预测和理化性质分析,以期获得对Wnt-1的分子水平的了解。在研究Wnt-1对人表皮干细胞的分化与增殖的影响时,我们借助了重组腺病毒基因导入技术将Wnt-1导入到人表皮干细胞内。这是因为Wnt-1是一种不稳定蛋白,在生理条件下极易降解,添加外源性Wnt-1蛋白至细胞培养基中,难以维持其浓度的稳定,且会增加组间误差和组内误差。在人表皮干细胞内表达Wnt-1蛋白前后,我们检测了其增殖能力、标记性分子、基质金属蛋白酶MMP-2和MMP-7浓度、细胞内Ca~(2+)浓度等多项指标,以研究Wnt-1对人表皮干细胞分化与增殖的影响。
第一部分Wnt-1的生物信息学分析
一、目的:全面分析Wnt-1蛋白的生物信息学特征
二、方法:首先,从NCBI上摘录Wnt家族的全部氨基酸序列,以Phylip和Treeview软件绘制该家族的系统进化树;然后,以ExPASY网站提供的生物信息学分析模块和本地Anthprot生物信息学分析软件对Wnt-1的氨基酸组成成分、等电点、信号肽切割位点、滴定曲线等进行分析;以GOR4、HNN、SOPMA三种方法预测Wnt-1的二级结构;以ExPASY网站相关模块分析Wnt-1的亲水性、极性以及折返系数;最后对这些结果进行比对和分析。
三、结果:Wnt家族中,Wnt-4、Wnt-11和Wnt-16单独存在,其他的成员均成对存在;Wnt-1与Wnt-6配对,二者亲缘关系最近。Wnt-1整体略带正电荷;不稳定指数为62.61,为一种不稳定蛋白;整体亲水性指数为-0.347,为一种疏水性蛋白;等电点约在9.0附近;信号肽为一条含27个氨基酸的短肽。GOR4、HNN、SOPMA三种二级结构预测方法均认为Wnt-1的第25～50、112～172、222～238、272～360位区域主要由不规则卷曲结构和伸展结构所构成。亲水性、极性以及折返系数分析结果均提示:第59～69、89～102、150～160、175~195、209～220、335~342位区域的量化指标高于阈值。
四、结论:Wnt-1与Wnt-6配对存在;Wnt-1在生理条件下不稳定;Wnt-1蛋白第150～160和335~342两个区域既符合功能结构域的空间构成要素,又符合理化构成要素,最有可能成为其功能结构域。
第二部分Wnt-1重组腺病毒的构建
一、目的:构建Wnt-1重组腺病毒颗粒
二、方法:从其他研究者取得已经构建好的,经测序证实序列无误的重组穿梭质粒pAdTrack-CMV/Wnt-1,将其线性化后在BJ5183细菌中和腺病毒骨架质粒pAdEasy-1重组,构建成重组腺病毒质粒pAdEasy-1/Wnt-1;使用脂质体转染试剂盒将重组腺病毒质粒pAdEasy-1/Wnt-1导入QBI-293A细胞中,借助该细胞中提供的反式补偿,产生具有普遍感染能力的pAdEasy-1/ Wnt-1重组腺病毒颗粒。最后,对这些病毒颗粒进行大量扩增,以满足下一步实验之用。
三、结果:重组腺病毒质粒pAdEasy-1/Wnt-1构建完成后,以Pac I酶切后,可见一条远大于15Kb的明亮条带和一条约4.5Kb的条带,与理论值完全吻合,证明重组质粒构建成功;以该质粒转染QBI-293A细胞后,可观察到绿色荧光蛋白的表达,证明产生了携带Wnt-1重组穿梭质粒的腺病毒颗粒;经大量扩增,最终获得足够的pAdEasy-1/Wnt-1重组腺病毒颗粒。
四、结论:Wnt-1重组腺病毒颗粒构建成功。
第三部分Wnt-1对人表皮干细胞分化与增殖的影响的研究
一、目的:研究Wnt-1对人表皮干细胞分化与增殖的影响
二、方法:首先,以IV型胶原快速粘贴法获得人表皮干细胞,并借助重组腺病毒技术将Wnt-1基因导入人表皮干细胞;随后观察绿色荧光蛋白的表达,并以RT-PCR法检测Wnt-1 mRNA在人表皮干细胞中的存在;最后以CASYexcell系统检测人表皮干细胞的增殖能力,以免疫组化法检测角蛋白K5、K6、K7、K8、K10、K18、K19以及CD44、CEA、ER、PR等标记性分子的表达,以ELISA法检测细胞培养基中基质金属蛋白酶MMP-2和MMP-7的浓度,以激光共聚焦技术检测细胞内Ca~(2+)浓度。
三、结果:分离获取的人表皮干细胞在接种后第4天进入快速生长期,第8天时到达平台期;Wnt-1基因导入人表皮干细胞后,可观察到绿色荧光蛋白的表达,RT-PCR也检测到了Wnt-1mRNA的存在,故以双重标准证明了Wnt-1基因在人表皮干细胞中的存在与表达;CASYexcell系统检测后发现,人表皮干细胞直径无明显变化,而细胞增殖则明显加强;人表皮干细胞角蛋白K10的表达由阴性变为阳性,K18的表达增强,K19的阳性表达减弱,说明其已经发生了部分转化;培养基中的MMP-2、MMP-7的浓度均显著增高,说明人表皮干细胞获得了降解细胞外基质的分子基础;人表皮干细胞内Ca~(2+)浓度明显升高,使其获得脱离干细胞“壁龛”的趋势。
四、结论:Wnt-1具有诱使人表皮干细胞向腺样上皮细胞分化的趋势。
Epidermal stem cells are a kind of tissue specificity stem cells which are located in basal layer of epidermis. They are important to epidermal renewal, and they are also the main source for generation, reparation and rebuilding of skin and its appendant organ. The differentiation and proliferation of epidermal stem cells is determined not only by the genomes inside themselves, but also by the environment around the stem cells, this environment is also called“Niche”. Wnt family is one of the most important parts of“Niche”. They take part in the process of cell differentiation, polarity, migration and proliferation. Wnt-1 displays a very important role in development and differentiation of epithelium tissue, such as neuroepithelial development and breast cancer generation. Wnt-1 expression concords to development of skin and its appendant organ in phase: Wnt-1 expressed actively in embryo period, but rested in adult; human epidermal stem cells assembled in primary epidermal ridges in 16th week, and formed matured glandular organs in 24th week, but, no any glandular organ reproductions were observed in adult. These demonstrated that, development of skin and its appendant organ relates to Wnt-1. So, we here studied the influence of Wnt-1 to the differentiation and proliferation of epidermal stem cells.
In this topic, based on the thought of“function is determined by structure”, we first analyzed the amino acids sequence, secondary structure and physico-chemical property of Wnt-1 to get an allround recognize of this protein. And then, using recombinant adenovirus gene introduction technology, we expressed Wnt-1 in epidermal stem cells. This is because Wnt-1 is a kind of unstable protein and is very easy to be degradated. When Wnt-1 were added in the culture medium of epidermal stem cells, it is difficult to maintain the concentrationof Wnt-1, and to result in causing inter class and intra class errors. At the last, the proliferation, marker molecular, and matrix metalloproteinase-2 and 7, [Ca~(2+)]i were detected to analysis the influence of Wnt-1 to differentiation and proliferation of epidermal stem cells.
First Part, Bioinformatics Analysis for Wnt-1 Protein
1. Objective: To get an allround recognize with bioinformatics characters of Wnt-1 protein.
2. Methods: Phyletic evolution scheme of Wnt family was deawed by Phylip and Treeview softwares. Amino acid composition, isoelectric point, molecular weight, signal peptide, cleavage site and titration curve were analysis by related modules from ExPASy and Anthroprot software; Secondary structure of Wnt-1 were predicted by GOR4, HNN and SOPMA. Hydrophilicity, polarity and refractivity were analysized by related modules from ExPASy.
3. Results: Wnt-16, Wnt-4 and Wnt-11 exist solitarily in their family. Wnt-1 conjugates with Wnt-6 for it has similar amino acids sequences to Wnt-6; Wnt-1 is classed to be a kind of positive charged protein and a kind of unstable protein; its theoretical isoelectric point (pI) is about 9; the signal peptide of Wnt-1is a short peptide including 27 amino acids. Regions 50-60, 112-172, 222-238, 272-360 were recognized as random coil and (or) extended structure region of Wnt-1 by GOR4, HNN and SOPMA. Regions 50-60, 112-172, 222-238, 272-360 have the values higher than threshold in hydrophilicity, polarity and refractivity analysis.
4. Conclusion: Wnt-1 conjugate with Wnt-6, it is unstable in physiological condition; regions 150-160 and 335-342 accord to both spatial structure element and physico-chemical property element, have the most possibility to be the functional domain of Wnt-1 protein.
Second Part, Construction of Wnt-1 Recombinant Adenovirus
1. Objective: To construct Wnt-1 recombinant adenovirus particles.
2. Methods: The recombinant shuttle plasmids which include Wnt-1 gene named pAdTrack-CMV/Wnt-1 were got from another researcher. These plasmids were linearized and cotransduced into Ecoli. BJ5183 with adenovirus frame plasmids to construct recombinant adenovirus plasmids pAdTrack-CMV/Wnt-1. Then, pAdTrack-CMV/Wnt-1 were linearized by PacI and transferred into QBI-293A cells to form recombinant adenovirus particles. Finally, those adenovirus particles were amplificated to a great quantity for use later.
3. Results: Recombinant adenovirus plasmids pAdEasy-1/Wnt-1 could be digested by restriction enzyme PacI to two specificity straps, the posterior is far great than 15Kb and the anterior is about 4.5Kb, in coincidence with theory. 36 hours after QBI-293A cells were transduced with recombinant adenovirus plasmids pAdEasy-1/Wnt-1, green fluorescent could be observed. It means that recombinant adenovirus particles were generated correctly. Finally, we got enough recombinant adenovirus particles amplification to be used for next experiment.
4. Conclusion: Wnt-1 recombinant adenovirus particles were constructed correctly.
Third Part, Study on Influence of Wnt-1 to Differentiation and proliferation of Epidermal Stem Cells
1. Objective: To investigate the influence of Wnt-1 to differentiation and proliferation of epidermal stem cells.
2. Methods: The epidermal stem cells were got from foreskin of health child, and were infected by recombinant adenovirus particles which take along with Wnt-1 gene. Then, the expression of green fluorescent was observed; the existing of Wnt-1 mRNA were detected by RT-PCR. Finally, the quantity and diameter of those cells were measured by CASYexcell system; the expression of K5、K6、K7、K8、K10、K18、K19、CD44、CEA、ER and PR were detected by immunohistochemistry; the concentration of matrix metalloproteinase-2 and 7 in cell culture medium were measured by ELISA; [Ca~(2+)]i was dected by confocal laser scanning microscope.
3. Results: Epidermal stem cells began to proliferate quickly after cultivated for about 4 days, and reach the platform for about 9 days. After epidermal stem cells were infected by recombinant adenovirus particles who take along with Wnt-1 gene, green fluorescent could be observed. Existing of Wnt-1 mRNA could be detected by RT-PCR. CASYexcell system showed that, those infected cells proliferated more quickly, but their diameter had not changed. Keratin 10 expressing became positive, keratin 18 expression enhanced significantly, keratin 19 expression attenuated significantly. ELISA showed that MMP-2、MMP-7 expression in cell culture medium became positive. Confocal laser scanning microscope showed that [Ca~(2+)]i raised enormously.
4. Conclusion: Wnt-1 has the ability to induce human epidermal stem cells different to glandular epithelium-like cells.
在本课题中,我们基于“结构决定功能”的思想,首先以生物信息学的手段和方法,对Wnt-1进行氨基酸序列分析、二级结构预测和理化性质分析,以期获得对Wnt-1的分子水平的了解。在研究Wnt-1对人表皮干细胞的分化与增殖的影响时,我们借助了重组腺病毒基因导入技术将Wnt-1导入到人表皮干细胞内。这是因为Wnt-1是一种不稳定蛋白,在生理条件下极易降解,添加外源性Wnt-1蛋白至细胞培养基中,难以维持其浓度的稳定,且会增加组间误差和组内误差。在人表皮干细胞内表达Wnt-1蛋白前后,我们检测了其增殖能力、标记性分子、基质金属蛋白酶MMP-2和MMP-7浓度、细胞内Ca~(2+)浓度等多项指标,以研究Wnt-1对人表皮干细胞分化与增殖的影响。
第一部分Wnt-1的生物信息学分析
一、目的:全面分析Wnt-1蛋白的生物信息学特征
二、方法:首先,从NCBI上摘录Wnt家族的全部氨基酸序列,以Phylip和Treeview软件绘制该家族的系统进化树;然后,以ExPASY网站提供的生物信息学分析模块和本地Anthprot生物信息学分析软件对Wnt-1的氨基酸组成成分、等电点、信号肽切割位点、滴定曲线等进行分析;以GOR4、HNN、SOPMA三种方法预测Wnt-1的二级结构;以ExPASY网站相关模块分析Wnt-1的亲水性、极性以及折返系数;最后对这些结果进行比对和分析。
三、结果:Wnt家族中,Wnt-4、Wnt-11和Wnt-16单独存在,其他的成员均成对存在;Wnt-1与Wnt-6配对,二者亲缘关系最近。Wnt-1整体略带正电荷;不稳定指数为62.61,为一种不稳定蛋白;整体亲水性指数为-0.347,为一种疏水性蛋白;等电点约在9.0附近;信号肽为一条含27个氨基酸的短肽。GOR4、HNN、SOPMA三种二级结构预测方法均认为Wnt-1的第25～50、112～172、222～238、272～360位区域主要由不规则卷曲结构和伸展结构所构成。亲水性、极性以及折返系数分析结果均提示:第59～69、89～102、150～160、175~195、209～220、335~342位区域的量化指标高于阈值。
四、结论:Wnt-1与Wnt-6配对存在;Wnt-1在生理条件下不稳定;Wnt-1蛋白第150～160和335~342两个区域既符合功能结构域的空间构成要素,又符合理化构成要素,最有可能成为其功能结构域。
第二部分Wnt-1重组腺病毒的构建
一、目的:构建Wnt-1重组腺病毒颗粒
二、方法:从其他研究者取得已经构建好的,经测序证实序列无误的重组穿梭质粒pAdTrack-CMV/Wnt-1,将其线性化后在BJ5183细菌中和腺病毒骨架质粒pAdEasy-1重组,构建成重组腺病毒质粒pAdEasy-1/Wnt-1;使用脂质体转染试剂盒将重组腺病毒质粒pAdEasy-1/Wnt-1导入QBI-293A细胞中,借助该细胞中提供的反式补偿,产生具有普遍感染能力的pAdEasy-1/ Wnt-1重组腺病毒颗粒。最后,对这些病毒颗粒进行大量扩增,以满足下一步实验之用。
三、结果:重组腺病毒质粒pAdEasy-1/Wnt-1构建完成后,以Pac I酶切后,可见一条远大于15Kb的明亮条带和一条约4.5Kb的条带,与理论值完全吻合,证明重组质粒构建成功;以该质粒转染QBI-293A细胞后,可观察到绿色荧光蛋白的表达,证明产生了携带Wnt-1重组穿梭质粒的腺病毒颗粒;经大量扩增,最终获得足够的pAdEasy-1/Wnt-1重组腺病毒颗粒。
四、结论:Wnt-1重组腺病毒颗粒构建成功。
第三部分Wnt-1对人表皮干细胞分化与增殖的影响的研究
一、目的:研究Wnt-1对人表皮干细胞分化与增殖的影响
二、方法:首先,以IV型胶原快速粘贴法获得人表皮干细胞,并借助重组腺病毒技术将Wnt-1基因导入人表皮干细胞;随后观察绿色荧光蛋白的表达,并以RT-PCR法检测Wnt-1 mRNA在人表皮干细胞中的存在;最后以CASYexcell系统检测人表皮干细胞的增殖能力,以免疫组化法检测角蛋白K5、K6、K7、K8、K10、K18、K19以及CD44、CEA、ER、PR等标记性分子的表达,以ELISA法检测细胞培养基中基质金属蛋白酶MMP-2和MMP-7的浓度,以激光共聚焦技术检测细胞内Ca~(2+)浓度。
三、结果:分离获取的人表皮干细胞在接种后第4天进入快速生长期,第8天时到达平台期;Wnt-1基因导入人表皮干细胞后,可观察到绿色荧光蛋白的表达,RT-PCR也检测到了Wnt-1mRNA的存在,故以双重标准证明了Wnt-1基因在人表皮干细胞中的存在与表达;CASYexcell系统检测后发现,人表皮干细胞直径无明显变化,而细胞增殖则明显加强;人表皮干细胞角蛋白K10的表达由阴性变为阳性,K18的表达增强,K19的阳性表达减弱,说明其已经发生了部分转化;培养基中的MMP-2、MMP-7的浓度均显著增高,说明人表皮干细胞获得了降解细胞外基质的分子基础;人表皮干细胞内Ca~(2+)浓度明显升高,使其获得脱离干细胞“壁龛”的趋势。
四、结论:Wnt-1具有诱使人表皮干细胞向腺样上皮细胞分化的趋势。
Epidermal stem cells are a kind of tissue specificity stem cells which are located in basal layer of epidermis. They are important to epidermal renewal, and they are also the main source for generation, reparation and rebuilding of skin and its appendant organ. The differentiation and proliferation of epidermal stem cells is determined not only by the genomes inside themselves, but also by the environment around the stem cells, this environment is also called“Niche”. Wnt family is one of the most important parts of“Niche”. They take part in the process of cell differentiation, polarity, migration and proliferation. Wnt-1 displays a very important role in development and differentiation of epithelium tissue, such as neuroepithelial development and breast cancer generation. Wnt-1 expression concords to development of skin and its appendant organ in phase: Wnt-1 expressed actively in embryo period, but rested in adult; human epidermal stem cells assembled in primary epidermal ridges in 16th week, and formed matured glandular organs in 24th week, but, no any glandular organ reproductions were observed in adult. These demonstrated that, development of skin and its appendant organ relates to Wnt-1. So, we here studied the influence of Wnt-1 to the differentiation and proliferation of epidermal stem cells.
In this topic, based on the thought of“function is determined by structure”, we first analyzed the amino acids sequence, secondary structure and physico-chemical property of Wnt-1 to get an allround recognize of this protein. And then, using recombinant adenovirus gene introduction technology, we expressed Wnt-1 in epidermal stem cells. This is because Wnt-1 is a kind of unstable protein and is very easy to be degradated. When Wnt-1 were added in the culture medium of epidermal stem cells, it is difficult to maintain the concentrationof Wnt-1, and to result in causing inter class and intra class errors. At the last, the proliferation, marker molecular, and matrix metalloproteinase-2 and 7, [Ca~(2+)]i were detected to analysis the influence of Wnt-1 to differentiation and proliferation of epidermal stem cells.
First Part, Bioinformatics Analysis for Wnt-1 Protein
1. Objective: To get an allround recognize with bioinformatics characters of Wnt-1 protein.
2. Methods: Phyletic evolution scheme of Wnt family was deawed by Phylip and Treeview softwares. Amino acid composition, isoelectric point, molecular weight, signal peptide, cleavage site and titration curve were analysis by related modules from ExPASy and Anthroprot software; Secondary structure of Wnt-1 were predicted by GOR4, HNN and SOPMA. Hydrophilicity, polarity and refractivity were analysized by related modules from ExPASy.
3. Results: Wnt-16, Wnt-4 and Wnt-11 exist solitarily in their family. Wnt-1 conjugates with Wnt-6 for it has similar amino acids sequences to Wnt-6; Wnt-1 is classed to be a kind of positive charged protein and a kind of unstable protein; its theoretical isoelectric point (pI) is about 9; the signal peptide of Wnt-1is a short peptide including 27 amino acids. Regions 50-60, 112-172, 222-238, 272-360 were recognized as random coil and (or) extended structure region of Wnt-1 by GOR4, HNN and SOPMA. Regions 50-60, 112-172, 222-238, 272-360 have the values higher than threshold in hydrophilicity, polarity and refractivity analysis.
4. Conclusion: Wnt-1 conjugate with Wnt-6, it is unstable in physiological condition; regions 150-160 and 335-342 accord to both spatial structure element and physico-chemical property element, have the most possibility to be the functional domain of Wnt-1 protein.
Second Part, Construction of Wnt-1 Recombinant Adenovirus
1. Objective: To construct Wnt-1 recombinant adenovirus particles.
2. Methods: The recombinant shuttle plasmids which include Wnt-1 gene named pAdTrack-CMV/Wnt-1 were got from another researcher. These plasmids were linearized and cotransduced into Ecoli. BJ5183 with adenovirus frame plasmids to construct recombinant adenovirus plasmids pAdTrack-CMV/Wnt-1. Then, pAdTrack-CMV/Wnt-1 were linearized by PacI and transferred into QBI-293A cells to form recombinant adenovirus particles. Finally, those adenovirus particles were amplificated to a great quantity for use later.
3. Results: Recombinant adenovirus plasmids pAdEasy-1/Wnt-1 could be digested by restriction enzyme PacI to two specificity straps, the posterior is far great than 15Kb and the anterior is about 4.5Kb, in coincidence with theory. 36 hours after QBI-293A cells were transduced with recombinant adenovirus plasmids pAdEasy-1/Wnt-1, green fluorescent could be observed. It means that recombinant adenovirus particles were generated correctly. Finally, we got enough recombinant adenovirus particles amplification to be used for next experiment.
4. Conclusion: Wnt-1 recombinant adenovirus particles were constructed correctly.
Third Part, Study on Influence of Wnt-1 to Differentiation and proliferation of Epidermal Stem Cells
1. Objective: To investigate the influence of Wnt-1 to differentiation and proliferation of epidermal stem cells.
2. Methods: The epidermal stem cells were got from foreskin of health child, and were infected by recombinant adenovirus particles which take along with Wnt-1 gene. Then, the expression of green fluorescent was observed; the existing of Wnt-1 mRNA were detected by RT-PCR. Finally, the quantity and diameter of those cells were measured by CASYexcell system; the expression of K5、K6、K7、K8、K10、K18、K19、CD44、CEA、ER and PR were detected by immunohistochemistry; the concentration of matrix metalloproteinase-2 and 7 in cell culture medium were measured by ELISA; [Ca~(2+)]i was dected by confocal laser scanning microscope.
3. Results: Epidermal stem cells began to proliferate quickly after cultivated for about 4 days, and reach the platform for about 9 days. After epidermal stem cells were infected by recombinant adenovirus particles who take along with Wnt-1 gene, green fluorescent could be observed. Existing of Wnt-1 mRNA could be detected by RT-PCR. CASYexcell system showed that, those infected cells proliferated more quickly, but their diameter had not changed. Keratin 10 expressing became positive, keratin 18 expression enhanced significantly, keratin 19 expression attenuated significantly. ELISA showed that MMP-2、MMP-7 expression in cell culture medium became positive. Confocal laser scanning microscope showed that [Ca~(2+)]i raised enormously.
4. Conclusion: Wnt-1 has the ability to induce human epidermal stem cells different to glandular epithelium-like cells.
引文
1.林京玉,丁日高,应翔宇.表皮干细胞增殖与分化的分子调控研究进展.军事医学科学院院刊, 2003, 27: 308-311.
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8. Eisen, MB, Spellman, P, Brown, PO, et al. Cluster analysis and display of genome-wide expression patterns. Proceedings of the National Academy of Sciences, 1998, 95: 14863-14868.
9.庄立岩,郭应禄,张志文. Wnt基因与生物发育和肿瘤发生.生物化学与生物物理进展, 1999, 26: 117-121
10. Deleage G, Combet C, Blanchet C, Geourjon, C. ANTHEPROT: An integrated protein sequence analysis software with client/server capabilities. Computer in biology and medicine, 2001, 31: 259-267
11. Deleage G, Clerc FF, Roux B. Anthprot-IBM PC and apple macintosh versions. Computer applications in the biosciences, 1989, 5: 159-160
12. Deleage G, Clerc FF, Roux B, Gautheron DC. ANTHEPROT: a package for proteinsequence analysis using a microcomputer. Comput Appl Biosci. 1988, 3: 351-356.
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1.林京玉,丁日高,应翔宇.表皮干细胞增殖与分化的分子调控研究进展.军事医学科学院院刊, 2003, 27: 308-311.
2. He TC, Zhou S, DaCosta CT, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998; 95:2509-2514.
3. Tan R, Li C, J iang S, et al. A novel and simple method for construction of recombinant adenoviruses. Nucleic Acids Res 2006; 34: 89-90.
4. Marini FC, 3rd, Yu Q, Wickham T, et al. Adenovirus as a gene therapy vector for hematopoietic cells [J].Cancer Gene Ther.2000, 7:816-825.
5. Romero EL, Morilla MJ and Bakas LS. Lipid vectors. New strategies for gene therapy [J]. Medicina, 2001, 61: 205-214.
6. Turturro F. Recombinant adenovirus-mediated cytotoxic gene therapy of lymphoproliferative disorders: is CAR important for the vector to ride? [J].Gene Ther, 2003, 10: 100-104.
7. Simonato M, Manservigi R, Marconi P, et al.Gene transfer into neurones for the molecular analysis of behaviour: focus on herpes simplex vectors [J].Trends Neurosci, 2000, 23(5): 183-190.
8. Zhang HG, Xie J, Yang P, et al.Adeno-associated virus production of soluble tumor necrosis factor receptor neutralizes tumor necrosis factor alpha and reduces arthritis[J].Hum Gene Ther.2000,11(17):2431-2442.
9. Bett AJ, Haddara W, Prevec L, et al. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci USA. 1994; 91: 8802-8806.
10. ZengM, Smith SK, Siegel F, et al. AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination[J]. J Biotechniques, 2001, 31(2):260-262.
11.梁杰,何海填,彭智,等.人白介素-4基因重组腺病毒载体的构建与鉴定.中国实用医药, 2007, 2(33): 46-48.
12. Kamen A, Henry O. Development and optimization of an adenovirus production process [J]. J GeneMed, 2004, 6: 184-192.
13.陈力,张心灵,李银平,等.小鼠角质细胞生长因子重组腺病毒载体的构建.中国危重病急救医学, 2007, 19(10): 610-613.
14.袁晓梅,何勇,雷萍. pAd2SIG腺病毒质粒构建及其在MCF27细胞中的表达.医学分子生物学杂志, 2007, 4 (5) : 414-418.
15.孙朝晖,修波,崔志强.腺病毒表达载体pAdEasy-1/pAdTrack-CMV-Shh2N的构建及表达.中华神经外科疾病研究杂志, 2006, 5 (3): 240-243.
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5. Gasteiger E., Hoogland C., Gattiker A., et al. Protein identification and analysis tools on the ExPASy Server. The Proteomics Protocols Handbook, Humana Press, 2005: 571-607
6. Bjellqvist, B., Hughes, G.J., et al. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis, 1993, 14: 1023-1031.
7. Bjellqvist, B., Basse, B., Olsen, E. et al. Reference points for comparisons of two-dimensional maps of proteins from different human cell types defined in a pH scale where isoelectric points correlate with polypeptide compositions. Electrophoresis, 1994, 15: 529-539.
8. Eisen, MB, Spellman, P, Brown, PO, et al. Cluster analysis and display of genome-wide expression patterns. Proceedings of the National Academy of Sciences, 1998, 95: 14863-14868.
9.庄立岩,郭应禄,张志文. Wnt基因与生物发育和肿瘤发生.生物化学与生物物理进展, 1999, 26: 117-121
10. Deleage G, Combet C, Blanchet C, Geourjon, C. ANTHEPROT: An integrated protein sequence analysis software with client/server capabilities. Computer in biology and medicine, 2001, 31: 259-267
11. Deleage G, Clerc FF, Roux B. Anthprot-IBM PC and apple macintosh versions. Computer applications in the biosciences, 1989, 5: 159-160
12. Deleage G, Clerc FF, Roux B, Gautheron DC. ANTHEPROT: a package for proteinsequence analysis using a microcomputer. Comput Appl Biosci. 1988, 3: 351-356.
13. Garnier J, Gibrat J-F, Robson B. GOR secondary structure prediction method version IV. Methods in Enzymology, 1996, 266: 540-553.
14. Guermeur, Y. Combinaison de Classifieurs Statistiques, Application a la Prediction de la Structure Secondaire des Proteines. Ph.D. thesis, UniversitéParis, 1997.
15. Geourjon C, Deleage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci, 1995, 11: 681-684.
16. Hopp TP, Woods KR. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci USA. 1981, 78:3824-3828
1.林京玉,丁日高,应翔宇.表皮干细胞增殖与分化的分子调控研究进展.军事医学科学院院刊, 2003, 27: 308-311.
2. He TC, Zhou S, DaCosta CT, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998; 95:2509-2514.
3. Tan R, Li C, J iang S, et al. A novel and simple method for construction of recombinant adenoviruses. Nucleic Acids Res 2006; 34: 89-90.
4. Marini FC, 3rd, Yu Q, Wickham T, et al. Adenovirus as a gene therapy vector for hematopoietic cells [J].Cancer Gene Ther.2000, 7:816-825.
5. Romero EL, Morilla MJ and Bakas LS. Lipid vectors. New strategies for gene therapy [J]. Medicina, 2001, 61: 205-214.
6. Turturro F. Recombinant adenovirus-mediated cytotoxic gene therapy of lymphoproliferative disorders: is CAR important for the vector to ride? [J].Gene Ther, 2003, 10: 100-104.
7. Simonato M, Manservigi R, Marconi P, et al.Gene transfer into neurones for the molecular analysis of behaviour: focus on herpes simplex vectors [J].Trends Neurosci, 2000, 23(5): 183-190.
8. Zhang HG, Xie J, Yang P, et al.Adeno-associated virus production of soluble tumor necrosis factor receptor neutralizes tumor necrosis factor alpha and reduces arthritis[J].Hum Gene Ther.2000,11(17):2431-2442.
9. Bett AJ, Haddara W, Prevec L, et al. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci USA. 1994; 91: 8802-8806.
10. ZengM, Smith SK, Siegel F, et al. AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination[J]. J Biotechniques, 2001, 31(2):260-262.
11.梁杰,何海填,彭智,等.人白介素-4基因重组腺病毒载体的构建与鉴定.中国实用医药, 2007, 2(33): 46-48.
12. Kamen A, Henry O. Development and optimization of an adenovirus production process [J]. J GeneMed, 2004, 6: 184-192.
13.陈力,张心灵,李银平,等.小鼠角质细胞生长因子重组腺病毒载体的构建.中国危重病急救医学, 2007, 19(10): 610-613.
14.袁晓梅,何勇,雷萍. pAd2SIG腺病毒质粒构建及其在MCF27细胞中的表达.医学分子生物学杂志, 2007, 4 (5) : 414-418.
15.孙朝晖,修波,崔志强.腺病毒表达载体pAdEasy-1/pAdTrack-CMV-Shh2N的构建及表达.中华神经外科疾病研究杂志, 2006, 5 (3): 240-243.
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