小鼠腭裂相关基因mcprl的分布及真核转染的初步研究
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摘要
mcpr1基因是本室的李鑫博士等借助分了生物学的消减杂交技术从维甲酸诱导的C57BL／6N近交系小鼠腭裂组织中克隆到的一个新基因，于2002年初被GenBank收录，登录号为AY074887。国际基因命名委员会批准将获得的新基因命名为mcpr1(Mouse Cleft Palate—Related genel)。经Genbank的ORF工具分析推测第200到598碱基之间有完整的编码132个氨基酸的开放读框。同时克隆到的差异表达基因还有fau基因等。mcpr1基因是未知功能基因，而fau基因与腭裂发生的关系也未有报道，所以，在本研究工作中，我们对这两个基因进行了初步的表达分布的实验研究，并且构建了acpr1基因的荧光真核表达载体，建立并筛选出了稳定表达融合蛋白的COS7-MCPR1细胞株，对其功能做了初步的研究分析。本研究分为七个方面：
1．从出生后C57BL／6N近交系小鼠的多种脏器组织：腭部，脑，心脏，肝脏，肺脏，脾脏，肠组织中，提取总RNA，利用RT-PCR技术，结果发现mcpr1和fau基因在多种正常组织中均有表达。这些基因在正常组织中的广泛性表达提示它们在这些组织的正常发育中有着非常重要的作用；
2．应用原位杂交的方法检测mcpr1和fau基因在与颅面部发育相关的外胚间充质细胞中的表达情况。原位杂交结果显示，mcpr1和fau基因的mRNA在体外培养的腭突未分化外胚间充质细胞的胞浆内有阳性表达，提示基因在腭突的正常发育中有着重要的生物学意义；
3．应用原位杂交的方法检测mcpr1和fau基因在维甲酸诱导的小鼠腭裂模型和正常小鼠的腭突部位的表达差异。原位杂交结果显示，mcpr1和fau基因的mRNA在正常胚胎小鼠的腭突组织与由维甲酸诱导的腭裂胚胎小鼠的腭突组织在12天到15天均表达，用图像分析方法获得基因在两组中表达的灰度值，进行统计学分析。由统计学结果可知，与正常组相比较，腭裂组中基因mRNA的表达显著性增加。
第四军医大学硕十学位论文
4.应用基因重组技术，通过尸CR方法，扩增mcprl基因的外显子片段，
使之克隆入荧光真核表达载体pEGFP一N3的多克隆位点中。并且通过酶
切鉴定，PCR鉴定，测序，显示已经成功构建了m叩r1基因的荧光真核表
达载体，解决了mcprl基因转染的准备工作，为下一步研究mcPrl基因
的功能奠定了基础，为探讨该基因与愕裂的关系提供了必要的条件。
5.将成功构建的真核表达载体用脂质体2000瞬时转染到COS7细胞系中
进行蛋白的亚细胞定位。本实验结果显示，转染细胞在转染后24小时已
经可以观察到在细胞浆中有绿色荧光的表达，但是在48小时，表达最为
强烈。
6.在这一部分实验中，通过G418筛选出了稳定表达融合蛋白的
COS7一MCPRI细胞株。转染的细胞可以观察到绿色荧光蛋白的表达，并且
应用SDS一pAGE也进行了相应的鉴定。稳定表达蛋白的细胞株的成功构建，
为进一步制备MCPRI蛋白的抗体，研究MCPRI蛋白的功能，探讨其在鳄
裂中的致病机理奠定基础。
7.应用MTT方法检测COS7一MCPRI细胞株上清液中的MCPRI融合蛋白对
外胚间充质细胞增殖分化的影响。结果数据显示，MCPRI融合蛋白抑制了
外胚间充质细胞的增殖。所以推测本蛋白可能分泌到上清液中发挥功能，
这与生物学软件的分析结果一致。但是，这需要进一步的工作来验证这
一点，并且进而明确对细胞分化的具体影响。
mcprl gene (mouse cleft palate-related gene 1) is a novel gene (Gene bank access number:AY074887) .which was screened by PCR subtractive hybridization method, and highly expressed in mouse cleft palate tissues induced by retinoic acid. On the basis of sequence, mcprl was predicted encoding a protein comprised of 132 amino acids. But we still know few about the distribution and function of this gene/protein, one of the obscles is lack of MCPR1 protein and specific antibody, fau gene is also one of the different expressing genes. The purpose of present study was to detect the expression of these two genes, analysis their basic function and explore the relationship between genes and cleft palate. It will be helpful to prevent the common craniofacial congenital malformations if mechanism of the cleft palate developing is understood well. The studies include seven parts:
1. This study was undertaken to detect the expression of the mcprl and fau gene in palatal process, brain, heart, liver, lung tissues etc. of normal C57BL/6N strain mouse. The mRNA was extracted respectively directly from these tissues and detectd the expression level by RT-PCR. The result showed that genes were expressed in many tissue of the normal mouse. So it was concluded that the extensive expression of genes in tissue was significant in development of normal tissue.
2. The object of this study was to observe the expression of the mcprl and fau gene in cells derived from undifferentiating ectodermic palatal mesenchyme of C57BL/6N strain mouse. The expression of mcprl and fau gene in the cells was tested by in situ
hybridization. The result showed that genes were expressed in undifferentiating ectodermic palatal mesenchyme cells of the normal mouse. So it was concluded that there was the significance of mcprl and fau gene during developing of the palatal process in C57BL/6N strain mouse.
3. The purpose of this investigation was to observe the expression of mcprl and fau gene in the formation of cleft palate of mice and to explore the relationship between genes and the formation of cleft palate. The expression of genes in embryonic palatine process tissues in the experimental group induced by retinoic acid and control group was detected by in situ hybridization. The data showed that the expressional level of genes was higher in the experimental group than in the control group. It suggested that the genes played an important role to maintain the growth and development of tissues of mice. The high level expression of such genes was related to the occurrence of cleft palate.
4. The aim of this study was to amplify the mcprl gene and construct a high effective expressing vector containing mcprl cDNA. mcprl encoding region cDNA gene was amplified by PCR from the plasmid T-easy/ mcprl, then PCR product was inserted into a high effective fluorescent eukaryotic expressed vector pEGFP桸3. The positive recombinant was identified by PCR analysis, EcoRI and BamHI restriction analysis, and Sequence analysis. The result showed that a 400bp DNA fragment was amplified from the recombinant. Sequence analysis and restriction digest demonstrated that the mcprl gene was successfully inserted into pEGFP桸3 plasmid. So the fluorescent eukaryotic expressed vector pEGFP桸3/mcprl were successfully reconstructed. It would be useful for further studing the function of mcprl gene.
5. In this study an attempt was made to express pEGFP-N3- mcpr1 fusion protein and to examine intercellular localization of MCPR1.
pEGFP-N3- mcpr1 expression plasmid was transfected into COS7 by lipofectin reagent. After 48 hours, the location of the fusion protein in cells was observed under the fluorescent microscope. So it was concluded that MCPR1 localizes in the cytoplasm. The work provides the basis for study of the biochemical and physiological functions of mcprl.
6. In this part, by stable transfection technique, the open reading fream of mcpr1 were sucessfully introduced into continous cell line COS7. After 1 month of G418 treatment, all survived cells and it' s decedents(for a
1．从出生后C57BL／6N近交系小鼠的多种脏器组织：腭部，脑，心脏，肝脏，肺脏，脾脏，肠组织中，提取总RNA，利用RT-PCR技术，结果发现mcpr1和fau基因在多种正常组织中均有表达。这些基因在正常组织中的广泛性表达提示它们在这些组织的正常发育中有着非常重要的作用；
2．应用原位杂交的方法检测mcpr1和fau基因在与颅面部发育相关的外胚间充质细胞中的表达情况。原位杂交结果显示，mcpr1和fau基因的mRNA在体外培养的腭突未分化外胚间充质细胞的胞浆内有阳性表达，提示基因在腭突的正常发育中有着重要的生物学意义；
3．应用原位杂交的方法检测mcpr1和fau基因在维甲酸诱导的小鼠腭裂模型和正常小鼠的腭突部位的表达差异。原位杂交结果显示，mcpr1和fau基因的mRNA在正常胚胎小鼠的腭突组织与由维甲酸诱导的腭裂胚胎小鼠的腭突组织在12天到15天均表达，用图像分析方法获得基因在两组中表达的灰度值，进行统计学分析。由统计学结果可知，与正常组相比较，腭裂组中基因mRNA的表达显著性增加。
第四军医大学硕十学位论文
4.应用基因重组技术，通过尸CR方法，扩增mcprl基因的外显子片段，
使之克隆入荧光真核表达载体pEGFP一N3的多克隆位点中。并且通过酶
切鉴定，PCR鉴定，测序，显示已经成功构建了m叩r1基因的荧光真核表
达载体，解决了mcprl基因转染的准备工作，为下一步研究mcPrl基因
的功能奠定了基础，为探讨该基因与愕裂的关系提供了必要的条件。
5.将成功构建的真核表达载体用脂质体2000瞬时转染到COS7细胞系中
进行蛋白的亚细胞定位。本实验结果显示，转染细胞在转染后24小时已
经可以观察到在细胞浆中有绿色荧光的表达，但是在48小时，表达最为
强烈。
6.在这一部分实验中，通过G418筛选出了稳定表达融合蛋白的
COS7一MCPRI细胞株。转染的细胞可以观察到绿色荧光蛋白的表达，并且
应用SDS一pAGE也进行了相应的鉴定。稳定表达蛋白的细胞株的成功构建，
为进一步制备MCPRI蛋白的抗体，研究MCPRI蛋白的功能，探讨其在鳄
裂中的致病机理奠定基础。
7.应用MTT方法检测COS7一MCPRI细胞株上清液中的MCPRI融合蛋白对
外胚间充质细胞增殖分化的影响。结果数据显示，MCPRI融合蛋白抑制了
外胚间充质细胞的增殖。所以推测本蛋白可能分泌到上清液中发挥功能，
这与生物学软件的分析结果一致。但是，这需要进一步的工作来验证这
一点，并且进而明确对细胞分化的具体影响。
mcprl gene (mouse cleft palate-related gene 1) is a novel gene (Gene bank access number:AY074887) .which was screened by PCR subtractive hybridization method, and highly expressed in mouse cleft palate tissues induced by retinoic acid. On the basis of sequence, mcprl was predicted encoding a protein comprised of 132 amino acids. But we still know few about the distribution and function of this gene/protein, one of the obscles is lack of MCPR1 protein and specific antibody, fau gene is also one of the different expressing genes. The purpose of present study was to detect the expression of these two genes, analysis their basic function and explore the relationship between genes and cleft palate. It will be helpful to prevent the common craniofacial congenital malformations if mechanism of the cleft palate developing is understood well. The studies include seven parts:
1. This study was undertaken to detect the expression of the mcprl and fau gene in palatal process, brain, heart, liver, lung tissues etc. of normal C57BL/6N strain mouse. The mRNA was extracted respectively directly from these tissues and detectd the expression level by RT-PCR. The result showed that genes were expressed in many tissue of the normal mouse. So it was concluded that the extensive expression of genes in tissue was significant in development of normal tissue.
2. The object of this study was to observe the expression of the mcprl and fau gene in cells derived from undifferentiating ectodermic palatal mesenchyme of C57BL/6N strain mouse. The expression of mcprl and fau gene in the cells was tested by in situ
hybridization. The result showed that genes were expressed in undifferentiating ectodermic palatal mesenchyme cells of the normal mouse. So it was concluded that there was the significance of mcprl and fau gene during developing of the palatal process in C57BL/6N strain mouse.
3. The purpose of this investigation was to observe the expression of mcprl and fau gene in the formation of cleft palate of mice and to explore the relationship between genes and the formation of cleft palate. The expression of genes in embryonic palatine process tissues in the experimental group induced by retinoic acid and control group was detected by in situ hybridization. The data showed that the expressional level of genes was higher in the experimental group than in the control group. It suggested that the genes played an important role to maintain the growth and development of tissues of mice. The high level expression of such genes was related to the occurrence of cleft palate.
4. The aim of this study was to amplify the mcprl gene and construct a high effective expressing vector containing mcprl cDNA. mcprl encoding region cDNA gene was amplified by PCR from the plasmid T-easy/ mcprl, then PCR product was inserted into a high effective fluorescent eukaryotic expressed vector pEGFP桸3. The positive recombinant was identified by PCR analysis, EcoRI and BamHI restriction analysis, and Sequence analysis. The result showed that a 400bp DNA fragment was amplified from the recombinant. Sequence analysis and restriction digest demonstrated that the mcprl gene was successfully inserted into pEGFP桸3 plasmid. So the fluorescent eukaryotic expressed vector pEGFP桸3/mcprl were successfully reconstructed. It would be useful for further studing the function of mcprl gene.
5. In this study an attempt was made to express pEGFP-N3- mcpr1 fusion protein and to examine intercellular localization of MCPR1.
pEGFP-N3- mcpr1 expression plasmid was transfected into COS7 by lipofectin reagent. After 48 hours, the location of the fusion protein in cells was observed under the fluorescent microscope. So it was concluded that MCPR1 localizes in the cytoplasm. The work provides the basis for study of the biochemical and physiological functions of mcprl.
6. In this part, by stable transfection technique, the open reading fream of mcpr1 were sucessfully introduced into continous cell line COS7. After 1 month of G418 treatment, all survived cells and it' s decedents(for a
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