【摘要】：肝臟是人體內(nèi)多種物質(zhì)代謝的主要器官,具有重要的生理功能。4-6月孕齡的人胎肝還是造血、免疫系統(tǒng)干祖細(xì)胞的主要來源,其中可能存在大量具有重要功能的新基因。本室通過胎肝大規(guī)模cDNA測序已經(jīng)建立了22周孕齡人胎肝基因表達(dá)圖譜,同時正在進(jìn)行從胎肝中大規(guī)模發(fā)掘重要功能新基因的探索,包括PACT、HPO、CKIP-1、NPDC-1、LMBP等。 基因打靶技術(shù)是一種通過同源重組按預(yù)期方式對靶基因進(jìn)行定點(diǎn)修飾而改變生物活體遺傳信息,進(jìn)而觀察相應(yīng)基因功能的實驗手段。應(yīng)用此技術(shù),我們共構(gòu)建成功了上述5種基因的打靶載體,并將打靶載體導(dǎo)入小鼠胚胎干細(xì)胞,篩選獲得了PACT、HPO、CKIP-13種基因的中靶細(xì)胞。各自的基因型均已經(jīng)過PCR和Southern印跡法鑒定。通過囊胚顯微注射技術(shù),我們獲得了PACT、HPO、CKIP-13種基因的嵌合體小鼠。其中HPO、CKIP-1這2種基因的嵌合體小鼠不能將突變等位基因遺傳至子代。而PACT嵌合體小鼠中的突變等位基因可以遺傳至子代,獲得了可存活、可繁殖、表型正常的PACT雜合體小鼠。將PACT雜合體小鼠自交,在子代中未發(fā)現(xiàn)PACT純合體小鼠。根據(jù)野生型與雜合型小鼠數(shù)目約1∶2的比例,推測其可能在胚胎期純合致死。 我們對PACT雜合體小鼠自交子代做了大量的胚胎期追蹤分析,發(fā)現(xiàn)PACT純合體基因敲除小鼠在胚胎期6.5至7.5天死亡,胚胎發(fā)育停滯,原腸形成失敗。這揭示了PACT在高等動物早期胚胎發(fā)育過程中不可或缺的重要性。通過RT-PCR顯示,7.5天的PACT純合體基因敲除胚胎中,p53的靶基因p21、Mdm2的mRNA水平顯著升高。因為PACT在體外實驗中可以強(qiáng)烈的抑制p53的轉(zhuǎn)錄活性,所以我們推測PACT的缺失可能導(dǎo)致p53轉(zhuǎn)錄活性的失控,其下游基因過度表達(dá)而導(dǎo)致胚胎期致死效應(yīng)。目前,更進(jìn)一步的表型分析和死亡機(jī)制研究正在進(jìn)行中。
[Abstract]:The liver is the main organ of the metabolism of various substances in human body and has important physiological function. The fetal liver of 4-6 months gestational age is still hematopoietic, the main source of stem progenitor cells of immune system, and there may be a large number of new genes with important function. The gene expression map of human fetal liver at 22 weeks of gestation was established by large-scale cDNA sequencing of fetal liver in our laboratory. At the same time, the discovery of new genes of important function from fetal liver, including PACT,HPO,CKIP-1,NPDC-1,LMBP and so on, was carried out on a large scale. Gene targeting is an experimental method to modify the genetic information of living organisms by homologous recombination and modify the target gene in the expected way, and then observe the function of the corresponding gene. Using this technique, we successfully constructed the targeting vectors of the five genes mentioned above, and introduced the target vectors into mouse embryonic stem cells to screen the middle target cells of the PACT,HPO,CKIP-13 gene. Their genotypes have been identified by PCR and Southern blotting. By microinjection of blastocyst, chimeric mice with PACT,HPO,CKIP-13 gene were obtained. The chimeric mice of the two HPO,CKIP-1 genes could not inherit the mutant alleles to their offspring. The mutant alleles in PACT chimeric mice could be inherited to their offspring, and PACT heterozygotes with viable, reproductive and normal phenotypes were obtained. No PACT homozygous mice were found in the offspring of PACT heterozygote mice. According to the proportion of wild and heterozygous mice about 1:2, it is inferred that the mice may die in homozygote at embryonic stage. We have done a lot of embryo tracing analysis on PACT heterozygote mice. We found that PACT homozygous gene knockout mice died from 6.5 to 7.5 days of embryo stage, embryo development stagnated and protointestinal formation failed. This reveals the importance of PACT in the early embryonic development of higher animals. RT-PCR showed that the mRNA level of p53 target gene p21 Mdm2 was significantly increased in 7.5 day PACT homozygous knockout embryos. Because PACT can strongly inhibit the transcriptional activity of p53 in vitro, we speculate that the deletion of PACT may lead to the loss of p53 transcriptional activity, and the overexpression of the downstream gene may lead to the lethal effect of embryo. Further studies on phenotypic analysis and death mechanisms are currently under way.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：Q789

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 尹秀山;CKIP-1基因的敲除小鼠模型建立及其在骨骼、免疫系統(tǒng)中的生理功能研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2009年

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本文編號：2362861