本文選題：心肌細(xì)胞 + 敲除小鼠�。� 參考：《南京醫(yī)科大學(xué)》2013年碩士論文

【摘要】：背景與目的：PP2A磷酸酶家族是哺乳動(dòng)物體內(nèi)最大的絲/蘇氨酸磷酸酶之一。其結(jié)構(gòu)是一種異源三聚體，由一個(gè)結(jié)構(gòu)亞單位A，一個(gè)催化亞單位C和一個(gè)調(diào)節(jié)亞單位B構(gòu)成，調(diào)節(jié)亞單位B決定了PP2A全酶復(fù)合物底物的特異性，在高等真核生物中，A和C兩個(gè)亞單位都分別有α和β兩種亞型。所有亞單位的組合造就了多樣的PP2A全酶，它們可能有不同的底物特異性，不同的亞細(xì)胞定位，以及不同的組織特異性。PP2A調(diào)控多種生物進(jìn)程，包括DNA復(fù)制，致癌的轉(zhuǎn)化，腫瘤抑制，營養(yǎng)感知，細(xì)胞周期進(jìn)程，RNA轉(zhuǎn)錄，凋亡和RNA剪切等。為研究PP2A在心肌細(xì)胞中的功能，我們使用了Myh6-creERT2工具鼠和ppp2caflox/flox鼠交配，來建立ppp2ca的條件型敲除小鼠模型。 方法：利用他莫西芬誘導(dǎo)cre重組酶表達(dá)的Myh6-creERT2小鼠與PP2ACα條件敲除小鼠交配，通過剪腳趾標(biāo)記并抽提基因組DNA，利用特定引物PCR鑒定基因型，經(jīng)過兩代交配獲得Myh6-creERT2；PP2ACαflox/flox的小鼠。在小鼠五周齡時(shí)，注射他莫西芬誘導(dǎo)cre酶表達(dá)，PCR驗(yàn)證cre的重組效率。小鼠心臟彩超檢測敲除小鼠心功能，HE染色觀察敲除小鼠心肌細(xì)胞的變化。 結(jié)果：Myh6-creERT2；PP2ACαflox/flox雜合小鼠心臟內(nèi)，PCR檢測Myh6-creERT2小鼠表達(dá)的cre，可以在小鼠心臟中特異性敲除PP2ACα。小鼠心臟彩超，發(fā)現(xiàn)敲除小鼠的心室擴(kuò)張，射血分?jǐn)?shù)降低，心臟衰竭，并在彩超后三日死亡。病理切片結(jié)果顯示敲除小鼠心肌細(xì)胞與野生型小鼠心肌細(xì)胞細(xì)胞大小與數(shù)量相近。 結(jié)論：小鼠心肌細(xì)胞中特異性敲除PP2ACα，會(huì)導(dǎo)致心室舒張，心功能降低，，并最終導(dǎo)致小鼠死亡，但是心肌細(xì)胞的形態(tài)變化不大，推測小鼠心衰產(chǎn)生與心臟功能收到影響相關(guān)。 背景與目的：Cre/loxP重組酶系統(tǒng)是一種已被廣泛運(yùn)用的有效的基因工程的工具。我們構(gòu)建了CDllb-Cre轉(zhuǎn)基因小鼠，可用于在髓系細(xì)胞內(nèi)特異性敲除基因。Cre重組酶受髓系細(xì)胞內(nèi)特異性表達(dá)的啟動(dòng)子CDllb啟動(dòng)表達(dá)。 方法：我們通過顯微注射構(gòu)建轉(zhuǎn)基因小鼠，并用PCR鑒定小鼠的基因型，篩選出攜帶Cre基因的小鼠。之后，RT-PCR分析轉(zhuǎn)基因小鼠體內(nèi)cre的表達(dá)。我們進(jìn)一步用自己構(gòu)建的轉(zhuǎn)基因小鼠與報(bào)告小鼠系Rosa26交配，獲得CDllb-Cre/Rosa26雜合小鼠，PCR與組織學(xué)分析CDllb-Cre LacZ陽性成年小鼠，X-gal染色小鼠各個(gè)不同器官，驗(yàn)證β-半乳糖苷酶活性，cre酶僅在部分組織的細(xì)胞中特異性表達(dá)，可用于鑒定小鼠在髓系細(xì)胞內(nèi)特異性表達(dá)Cre重組酶的活性。 結(jié)果：在體外，轉(zhuǎn)基因成年小鼠顯示了cre-依賴的β-半乳糖苷酶活性。我們建立的轉(zhuǎn)基因小鼠家系，cre重組酶在體外的重組功能被證實(shí)。 結(jié)論：我們構(gòu)建的CDllb-Cre轉(zhuǎn)基因小鼠可用與條件敲除型小鼠交配，用于髓系細(xì)胞內(nèi)的基因功能分析。
[Abstract]:Background & AIM: PP2A phosphatase family is one of the largest serine / threonine phosphatase in mammals. Its structure is an heterotrimer, consisting of a structural subunit A, a catalytic subunit C and a regulatory subunit B, which determines the specificity of the substrate of PP2A whole enzyme complex. In higher eukaryotes, subunits A and C have two subtypes, 偽 and 尾, respectively. The combination of all subunits creates a variety of PP2A holozymes, which may have different substrate specificity, different subcellular localization, and different tissue specificity. PP2A regulates a variety of biological processes, including DNA replication, carcinogenic transformation. Tumor inhibition, nutritional perception, cell cycle progression, RNA transcription, apoptosis and RNA shearing. In order to study the function of PP2A in cardiomyocytes, we used Myh6-creERT2 tool mice to mate with ppp2caflox/flox mice to establish the conditioned knockout mouse model of ppp2ca. Methods: Myh6-creERT2 mice expressed by tamoxifen were used to mate with PP2AC 偽 conditioned knockout mice. The specific primers were used to identify the genotypes of Myh6-creERT2 mice and PP2AC 偽 flox/flox were obtained by specific primer PCR, and Myh6-creERT2PP2AC 偽 flox/flox was obtained after two generations of mating. At the age of five weeks, tamoxifen was injected into mice to induce the expression of cre. The recombinant efficiency of cre was verified by PCR. Cardiac function of knockout mice was detected by color Doppler ultrasound and HE staining was used to observe the changes of cardiac myocytes in knockout mice. Results the specific knockout of PP2AC 偽 in the heart of Myh6-creERT2 mice was detected by PCR. Murine heart color Doppler ultrasound showed ventricular dilatation, decreased ejection fraction, heart failure, and died 3 days after CDFI. The results of pathological section showed that the size and quantity of cardiac myocytes in knockout mice and wild type mice were similar. Conclusion: the specific knockout of PP2AC 偽 in mouse cardiomyocytes may lead to ventricular relaxation, decrease of cardiac function, and eventually lead to death in mice. However, the morphology of cardiac myocytes does not change much, suggesting that heart failure in mice is related to the influence of heart function. Background and objective: CrerloxP recombinant enzyme system is an effective genetic engineering tool that has been widely used. We constructed CDllb-CRE transgenic mice, which can be used to activate the expression of CDllb in myeloid cells with specific knockout gene. CRE recombinant enzyme was specifically expressed in myeloid cells. Methods: the transgenic mice were constructed by microinjection. The genotypes of the transgenic mice were identified by PCR, and the mice carrying CRE gene were screened out. The expression of cre in transgenic mice was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). We further used the self-constructed transgenic mice to mate with the report mouse line Rosa26, and obtained different organs of CDllb-Crer / Rosa26 hybrid mice by PCR and histology analysis of CDllb-CRE LacZ positive adult mice. It was verified that 尾 -galactosidase activity was expressed specifically in some tissue cells, which could be used to identify the activity of CRE recombined enzyme expressed specifically in myeloid cells of mice. Results: in vitro, transgenic adult mice showed cre- dependent 尾-galactosidase activity. The recombinant function of our transgenic mouse pedigree was confirmed in vitro. Conclusion: our constructed CDllb-Cre transgenic mice can mate with conditional knockout mice for gene function analysis in myeloid cells.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R363;Q78

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