基因BC023882發(fā)育學(xué)表達(dá)模式、亞細(xì)胞定位及其對細(xì)胞周期的影響
發(fā)布時(shí)間：2018-08-26 16:54
【摘要】： 研究背景 前期課題“高密度cDNA基因芯片篩選小鼠腦發(fā)育、損傷相關(guān)基因的研究”利用含小鼠發(fā)育文庫cDNA 7680條的高密度基因芯片,對發(fā)育和損傷模型中表達(dá)差異的基因進(jìn)行篩選。結(jié)果發(fā)現(xiàn)發(fā)育過程中高表達(dá)基因560個(gè),其中過度表達(dá)有34個(gè)。這些基因功能廣泛,涉及轉(zhuǎn)錄調(diào)節(jié)、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞代謝、免疫調(diào)節(jié)、細(xì)胞骨架、DNA復(fù)制修復(fù)、細(xì)胞粘附等17類。其中一個(gè)功能未知的過度表達(dá)基因BC023882引人注目,它在胚胎發(fā)育和生后早期小鼠的腦組織存在高表達(dá),其中P0 d時(shí)相點(diǎn)Cy5/Cy3比值(Ratio值)為7.6038,遠(yuǎn)遠(yuǎn)高于2.0000的基因表達(dá)差異篩選標(biāo)準(zhǔn)。提示基因BC023882可能在小鼠早期胚胎中樞神經(jīng)系統(tǒng)的發(fā)育過程中起到重要作用。研究方法與結(jié)果 1.基因BC023882的發(fā)育學(xué)表達(dá)模式及腦區(qū)定位。 (1)半定量RT-PCR檢測基因BC203882在發(fā)育小鼠各個(gè)階段的表達(dá)情況。分別取小鼠胚胎期時(shí)相點(diǎn)(E14、E19 d)及生后發(fā)育時(shí)相點(diǎn)(P1、P7、P14、P30、P60 d)的小鼠組織。其中胚胎期組織包括腦(brain)和胚體(body),而生后組織包括腦、肝、腎、心、肺和小腸。分別提取上述組織的總RAN,利用RT-PCR技術(shù)檢測基因023882在上述組織中的表達(dá)。我們通過RT-PCR實(shí)驗(yàn)觀察到基因BC023882的mRNA呈階段性地表達(dá)于腦組織,P1 d達(dá)到高峰,P60 d表達(dá)消失;其mRNA也少量表達(dá)于胚體組織,具體定位有待進(jìn)一步實(shí)驗(yàn)確定;另外P1 d肝組織也有微弱表達(dá),隨后迅速消失,而腎臟、肺、小腸和心臟等組織均未檢測到該基因表達(dá)。 (2)用原位雜交技術(shù)檢測基因BC023882的mRNA在不同腦區(qū)中的定位。根據(jù)基因BC023882的特異性序列設(shè)計(jì)RT-PCR引物,擴(kuò)增后構(gòu)建在含T7啟動(dòng)子的pcDNA3.1(-)載體中,標(biāo)記cRNA探針。利用該基因探針對各實(shí)驗(yàn)組腦組織進(jìn)行原位雜交。通過組織原位雜交實(shí)驗(yàn)觀察:基因BC023882的mRNA的最早出現(xiàn)在E14 d的胚胎腦組織大腦皮層,包括運(yùn)動(dòng)皮層、軀體感覺皮層、扣帶皮質(zhì)和梨狀皮質(zhì);P1 d時(shí)雜交陽性細(xì)胞出現(xiàn)范圍更廣,如大腦皮層,內(nèi)囊、胼胝體、視前區(qū)諸核團(tuán)等等;雜交信號(hào)
[Abstract]:Background "screening of mouse brain development and damage related genes by high-density cDNA gene chip" using a high-density gene chip containing cDNA 7680 in mouse development library. The differentially expressed genes in developmental and injury models were screened. The results showed that there were 560 overexpression genes, 34 of which were overexpressed. These genes have a wide range of functions, including transcriptional regulation, signal transduction, cellular metabolism, immunomodulation, cytoskeleton DNA replication and repair, cell adhesion, and so on. One of the unknown overexpression genes, BC023882, was highly expressed in the brain of embryonic and early postnatal mice. The P0 day phase point Cy5/Cy3 ratio (Ratio) was 7.6038, which was much higher than the differential screening criterion of gene expression of 2.0000. The results suggest that gene BC023882 may play an important role in the development of central nervous system in early embryonic mice. Methods and results 1. (1) Semi-quantitative RT-PCR was used to detect the expression of gene BC203882 in various stages of development mice. The tissues of mouse embryonic phase (E14, E19 d) and postnatal developmental phase (P1P7, P14, P30, P60 d) were taken respectively. Embryonic tissues include brain (brain) and embryonic body (body), while postnatal tissues include brain, liver, kidney, heart, lung and small intestine. Total RAN, was extracted from the above tissues to detect the expression of gene 023882 by RT-PCR technique. Through RT-PCR experiments, we observed that the mRNA of gene BC023882 was periodically expressed at the peak of P1d in brain tissue and disappeared at P60 day, and its mRNA was also expressed in embryoid tissue. The specific location of mRNA was to be determined by further experiments. In addition, there was also a weak expression in P1d liver tissue, and then disappeared rapidly, while the kidney, lung, No expression of the gene was detected in small intestine and heart. (2) in situ hybridization technique was used to detect the localization of mRNA of gene BC023882 in different brain regions. According to the specific sequence of gene BC023882, RT-PCR primers were designed and amplified into pcDNA3.1 (-) vector containing T7 promoter and labeled with cRNA probe. The gene probe was used for in situ hybridization of brain tissue in each experimental group. By tissue in situ hybridization, the mRNA of gene BC023882 was first found in the cerebral cortex of embryonic brain on day 14, including motor cortex, somatosensory cortex, cingulate cortex and piriform cortex. Such as cerebral cortex, internal capsule, corpus callosum, preoptic nuclei, etc.; hybridization signals
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329
[Abstract]:Background "screening of mouse brain development and damage related genes by high-density cDNA gene chip" using a high-density gene chip containing cDNA 7680 in mouse development library. The differentially expressed genes in developmental and injury models were screened. The results showed that there were 560 overexpression genes, 34 of which were overexpressed. These genes have a wide range of functions, including transcriptional regulation, signal transduction, cellular metabolism, immunomodulation, cytoskeleton DNA replication and repair, cell adhesion, and so on. One of the unknown overexpression genes, BC023882, was highly expressed in the brain of embryonic and early postnatal mice. The P0 day phase point Cy5/Cy3 ratio (Ratio) was 7.6038, which was much higher than the differential screening criterion of gene expression of 2.0000. The results suggest that gene BC023882 may play an important role in the development of central nervous system in early embryonic mice. Methods and results 1. (1) Semi-quantitative RT-PCR was used to detect the expression of gene BC203882 in various stages of development mice. The tissues of mouse embryonic phase (E14, E19 d) and postnatal developmental phase (P1P7, P14, P30, P60 d) were taken respectively. Embryonic tissues include brain (brain) and embryonic body (body), while postnatal tissues include brain, liver, kidney, heart, lung and small intestine. Total RAN, was extracted from the above tissues to detect the expression of gene 023882 by RT-PCR technique. Through RT-PCR experiments, we observed that the mRNA of gene BC023882 was periodically expressed at the peak of P1d in brain tissue and disappeared at P60 day, and its mRNA was also expressed in embryoid tissue. The specific location of mRNA was to be determined by further experiments. In addition, there was also a weak expression in P1d liver tissue, and then disappeared rapidly, while the kidney, lung, No expression of the gene was detected in small intestine and heart. (2) in situ hybridization technique was used to detect the localization of mRNA of gene BC023882 in different brain regions. According to the specific sequence of gene BC023882, RT-PCR primers were designed and amplified into pcDNA3.1 (-) vector containing T7 promoter and labeled with cRNA probe. The gene probe was used for in situ hybridization of brain tissue in each experimental group. By tissue in situ hybridization, the mRNA of gene BC023882 was first found in the cerebral cortex of embryonic brain on day 14, including motor cortex, somatosensory cortex, cingulate cortex and piriform cortex. Such as cerebral cortex, internal capsule, corpus callosum, preoptic nuclei, etc.; hybridization signals
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329
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