PP2ACα在心脏功能的研究及CD11b-cre转基因小鼠的构建

发布时间：2018-06-22 20:30

本文选题：心肌细胞 + 敲除小鼠　；参考：《南京医科大学》2013年硕士论文

【摘要】：背景与目的：PP2A磷酸酶家族是哺乳动物体内最大的丝/苏氨酸磷酸酶之一。其结构是一种异源三聚体，由一个结构亚单位A，一个催化亚单位C和一个调节亚单位B构成，调节亚单位B决定了PP2A全酶复合物底物的特异性，在高等真核生物中，A和C两个亚单位都分别有α和β两种亚型。所有亚单位的组合造就了多样的PP2A全酶，它们可能有不同的底物特异性，不同的亚细胞定位，以及不同的组织特异性。PP2A调控多种生物进程，包括DNA复制，致癌的转化，肿瘤抑制，营养感知，细胞周期进程，RNA转录，凋亡和RNA剪切等。为研究PP2A在心肌细胞中的功能，我们使用了Myh6-creERT2工具鼠和ppp2caflox/flox鼠交配，来建立ppp2ca的条件型敲除小鼠模型。方法：利用他莫西芬诱导cre重组酶表达的Myh6-creERT2小鼠与PP2ACα条件敲除小鼠交配，通过剪脚趾标记并抽提基因组DNA，利用特定引物PCR鉴定基因型，经过两代交配获得Myh6-creERT2；PP2ACαflox/flox的小鼠。在小鼠五周龄时，注射他莫西芬诱导cre酶表达，PCR验证cre的重组效率。小鼠心脏彩超检测敲除小鼠心功能，HE染色观察敲除小鼠心肌细胞的变化。结果：Myh6-creERT2；PP2ACαflox/flox杂合小鼠心脏内，PCR检测Myh6-creERT2小鼠表达的cre，可以在小鼠心脏中特异性敲除PP2ACα。小鼠心脏彩超，发现敲除小鼠的心室扩张，射血分数降低，心脏衰竭，并在彩超后三日死亡。病理切片结果显示敲除小鼠心肌细胞与野生型小鼠心肌细胞细胞大小与数量相近。结论：小鼠心肌细胞中特异性敲除PP2ACα，会导致心室舒张，心功能降低，，并最终导致小鼠死亡，但是心肌细胞的形态变化不大，推测小鼠心衰产生与心脏功能收到影响相关。背景与目的：Cre/loxP重组酶系统是一种已被广泛运用的有效的基因工程的工具。我们构建了CDllb-Cre转基因小鼠，可用于在髓系细胞内特异性敲除基因。Cre重组酶受髓系细胞内特异性表达的启动子CDllb启动表达。方法：我们通过显微注射构建转基因小鼠，并用PCR鉴定小鼠的基因型，筛选出携带Cre基因的小鼠。之后，RT-PCR分析转基因小鼠体内cre的表达。我们进一步用自己构建的转基因小鼠与报告小鼠系Rosa26交配，获得CDllb-Cre/Rosa26杂合小鼠，PCR与组织学分析CDllb-Cre LacZ阳性成年小鼠，X-gal染色小鼠各个不同器官，验证β-半乳糖苷酶活性，cre酶仅在部分组织的细胞中特异性表达，可用于鉴定小鼠在髓系细胞内特异性表达Cre重组酶的活性。结果：在体外，转基因成年小鼠显示了cre-依赖的β-半乳糖苷酶活性。我们建立的转基因小鼠家系，cre重组酶在体外的重组功能被证实。结论：我们构建的CDllb-Cre转基因小鼠可用与条件敲除型小鼠交配，用于髓系细胞内的基因功能分析。
[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, 伪 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 伪 conditioned knockout mice. The specific primers were used to identify the genotypes of Myh6-creERT2 mice and PP2AC 伪 flox/flox were obtained by specific primer PCR, and Myh6-creERT2PP2AC 伪 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 伪 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 伪 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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R363;Q78

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