慢病毒介导RNA干扰技术制备骨桥蛋白基因沉默小鼠模型
发布时间：2018-08-04 19:34
【摘要】：第一部分靶向骨桥蛋白基因的shRNA慢病毒表达载体的构建与鉴定 [摘要]目的构建针对骨桥蛋白(Osteopontin, OPN)基因的短发夹RNA (short hairpin RNA,shRNA)序列,合成pSicoR-GFP-shRNA慢病毒重组体,筛选对OPN基因表达抑制效率最佳的shRNA序列。方法参照OPN GeneBank上的序列设计合成shRNA-A,及不针对C57小鼠任何基因的阴性对照序列shRNA-NC,参考相关文献合成针对骨桥蛋白基因的shRNA-B。将合成的shRNA插入到线性化的pSicoR-GFP慢病毒载体中；然后转染E.coli JM109感受态细胞,对长出的菌落进行PCR鉴定,PCR鉴定阳性的克隆再进行测序。鉴定序列无误后,将之与pCMV-VSV-G和pCMV-dR8.91辅助质粒包装,然后共转染293FT细胞,进行慢病毒的包装及滴度测定,控制滴度在1×109TU/ml以上。用构建好的慢病毒感染小鼠肝细胞,检测不同shRNA序列对OPN基因表达的抑制效果,筛选最佳shRNA序列。结果经RT-PCR和Western-Blot检测发现：靶向OPN基因的shRNA慢病毒表达载体转染小鼠肝细胞后,序列shRNA-A/B均可有效抑制小鼠肝细胞OPN基因的表达,其中shRNA-A序列效果最佳(可达80%-90%)。结论成功构建靶向OPN基因的shRNA慢病毒表达载体,其转染小鼠肝细胞后可显著抑制OPN基因的表达,这为进一步体内抑制试验奠定了基础。 第二部分C57小鼠肝脏骨桥蛋白基因沉默动物模型的建立 [摘要]目的将包装好的慢病毒经尾静脉注射到C57小鼠体内,抑制其肝脏骨桥蛋白(Osteopontin,OPN)基因的表达,建立OPN基因沉默小鼠动物模型。方法取30只C57小鼠随机平均分为：空白对照组(Blank control,BC):经尾静脉注射灭菌PBS液200u1；阴性对照组(Negative control,NC):经尾静脉注射200ul pSicoR-GFP-shRNA-EC慢病毒液；实验组(Experimental group,EG):通过尾静脉注射200ul pSicoR-GFP-shRNA-A的慢病毒液。注射后第72h、96h、7d、14d,各组随机取2只小鼠断颈处死。收集肝脏,一部分立即用OCT包埋剂进行包埋,冰冻切片检测肝脏慢病毒转染效果：另一部分行qRT-PCR及Western-Blot检测OPN基因表达水平。结果与空白对照组、阴性对照组相比,实验组OPN基因mRNA及蛋白表达水平明显下降,抑制效率在70%-80%之间；空白对照组与阴性对照组之间无明显差别。结论利用RNA干扰技术,借助慢病毒载体,可以成功制备小鼠肝脏OPN基因沉默动物模型。 第三部分骨桥蛋白基因沉默C57小鼠肝脏胆固醇代谢相关基因表达的检测 [摘要]目的研究骨桥蛋白(Osteopontin, OPN)基因表达被抑制后,C57小鼠肝脏胆固醇代谢及转运相关基因表达的变化,为进一步研究OPN在胆固醇结石发生过程中与肝脏胆固醇代谢相关基因的关系提供技术支持。方法通过RT-PCR及Western-Blot检测肝脏胆固醇代谢及转运相关基因HMG-CoA还原酶、CYP-7α1、ABCG5/8的mRNA及蛋白表达的变化。结果RT-PCR结果显示：OPN表达受抑制后,肝脏胆固醇代谢相关基因HMG-CoA还原酶、CYP-7α1、ABCG5/8的mRNA含量无明显变化,Western-Blot结果与之相符。结论通过该部分实验,我们熟练掌握RT-PCR、Western-Blot法检测HMG-CoA还原酶、CYP-7α1、 ABCG5/8mRNA及蛋白表达的变化。抑制C57小鼠肝脏OPN基因表达后,HMG-CoA还原酶、CYP-7α1、ABCG5/8的表达未产生明显变化。通过进一步查阅文献,我们认为产生这一现象的原因可能是：1.OPN是在致病因素导致机体代谢紊乱状态的前提下高表达,然后对相关基因的转录和翻译发挥调节作用的；2.OPN表达水平变化对相关基因表达的影响可能需要较长的时间(约2月)才能表现出来。 总结,本课题利用慢病毒介导的RNA干扰技术,成功制备出C57小鼠肝脏OPN低表达模型,为进一步研究OPN在胆固醇结石形成过程中对胆固醇代谢的调节作用及机制提供了有力的研究工具。通过第三部分的研究,不仅为后续实验奠定了方法学基础,而且提示我们：进一步的研究须对OPN低表达C57小鼠饲以高脂、高胆固醇饮食,构建胆固醇结石模型进行进一步探讨。
[Abstract]:Part I construction and identification of shRNA lentiviral expression vector targeting osteopontin gene
[Abstract] [Abstract] objective to construct a short hairpin RNA (short hairpin RNA, shRNA) sequence for Osteopontin (OPN) gene, to synthesize pSicoR-GFP-shRNA lentivirus recombinant, and to screen for the best shRNA sequence for the inhibition efficiency of OPN gene expression. The negative control sequence shRNA-NC was used to synthesize the shRNA-B. of the osteopontin gene into the linearized pSicoR-GFP lentivirus vector by synthesizing the osteopontin gene, and then transfected into the E.coli JM109 receptive cells to identify the grown colonies by PCR, and then the PCR identified positive clones were sequenced. After the identification of the sequence, the identification sequence would be unmistakable. It was packed with pCMV-VSV-G and pCMV-dR8.91 auxiliary plasmids, then co transfected 293FT cells, carried out the package and titer of the lentivirus, controlled the titer above 1 x 109TU/ml. The inhibitory effect of different shRNA sequences on the expression of OPN gene was detected by the constructed lentivirus, and the best shRNA sequence was screened. The result was RT-PCR and Weste. Rn-Blot detection found that after transfection of shRNA lentivirus expression vector targeting OPN gene into mouse hepatocytes, sequence shRNA-A/B could effectively inhibit the expression of OPN gene in mouse hepatocytes, of which the shRNA-A sequence was best (up to 80%-90%). Conclusion the shRNA Lentivirus Expression Vector of the target OPN gene was successfully constructed. After transfecting the mouse hepatocytes, the gene could be transfected into mouse hepatocytes. It significantly inhibited the expression of OPN gene, which laid a foundation for further in vivo inhibition test.
The second part is the establishment of animal model of osteopontin gene silencing in C57 mice.
[Abstract] [Abstract] objective to inject the packaged lentivirus into C57 mice to inhibit the expression of Osteopontin (OPN) gene in the liver and establish a mouse model of OPN gene silencing. Methods 30 C57 mice were randomly divided into blank control group (Blank control, BC): sterilizing PBS liquid 200u1 through tail vein; negative The control group (Negative control, NC): 200ul pSicoR-GFP-shRNA-EC lentivirus solution was injected into the tail vein, and the experimental group (Experimental group, EG) was injected with the lentivirus solution of 200ul pSicoR-GFP-shRNA-A through the tail vein. After the injection, 2 mice were randomly taken off the neck. Embedding, frozen section detection of liver lentivirus transfection effect: another branch qRT-PCR and Western-Blot detection of OPN gene expression level. Results compared with the blank control group, negative control group, the experimental group OPN gene mRNA and protein expression level decreased significantly, the inhibition efficiency was between 70%-80%; blank control group and negative control group was not clear. Conclusion RNA interference technology and lentivirus vector can successfully prepare mouse liver OPN gene silencing animal model.
The third part of osteopontin gene silencing is related to the detection of liver cholesterol metabolism related gene expression in C57 mice.
[Abstract] [Abstract] Objective To study the changes of cholesterol metabolism and transport related gene expression in the liver of C57 mice after the expression of Osteopontin (OPN) gene was suppressed, in order to further study the technical support for the relationship between OPN and the cholesterol metabolism related genes during the process of cholesterol gallstone. The method was detected by RT-PCR and Western-Blot. Liver cholesterol metabolism and transport related genes HMG-CoA reductase, CYP-7 alpha 1, ABCG5/8 mRNA and protein expression changes. Results RT-PCR results showed that the expression of OPN was inhibited, the cholesterol metabolism related genes of the liver HMG-CoA reductase, CYP-7 a 1, ABCG5/8 mRNA content did not change, Western-Blot results coincide. Conclusion through this conclusion In some experiments, we have mastered RT-PCR and Western-Blot methods to detect the changes of HMG-CoA reductase, CYP-7 alpha 1, ABCG5/8mRNA and protein expression. After inhibiting the OPN gene expression in the liver of C57 mice, the expression of HMG-CoA reductase, CYP-7 alpha 1, ABCG5/8 has not changed obviously. We think that the cause of this phenomenon may be caused by further consulting the literature. It is: 1.OPN is highly expressed on the premise that pathogenic factors lead to metabolic disorders of the body, and then regulate the transcription and translation of related genes; the effect of 2.OPN expression level on related gene expression may take a long time (about February) to manifest.
In this paper, we successfully prepared the C57 mouse liver OPN low expression model by using lentivirus mediated RNA interference technology. It provides a powerful research tool for the further study of the regulation and mechanism of OPN on cholesterol metabolism during the formation of cholesterol stones. The third part study not only laid a methodology for the follow-up experiment. The basis for further study is to further study the model of OPN low expression C57 mice with high fat, high cholesterol diet and construction of cholesterol gallstones.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332
本文编号：2164928
[Abstract]:Part I construction and identification of shRNA lentiviral expression vector targeting osteopontin gene
[Abstract] [Abstract] objective to construct a short hairpin RNA (short hairpin RNA, shRNA) sequence for Osteopontin (OPN) gene, to synthesize pSicoR-GFP-shRNA lentivirus recombinant, and to screen for the best shRNA sequence for the inhibition efficiency of OPN gene expression. The negative control sequence shRNA-NC was used to synthesize the shRNA-B. of the osteopontin gene into the linearized pSicoR-GFP lentivirus vector by synthesizing the osteopontin gene, and then transfected into the E.coli JM109 receptive cells to identify the grown colonies by PCR, and then the PCR identified positive clones were sequenced. After the identification of the sequence, the identification sequence would be unmistakable. It was packed with pCMV-VSV-G and pCMV-dR8.91 auxiliary plasmids, then co transfected 293FT cells, carried out the package and titer of the lentivirus, controlled the titer above 1 x 109TU/ml. The inhibitory effect of different shRNA sequences on the expression of OPN gene was detected by the constructed lentivirus, and the best shRNA sequence was screened. The result was RT-PCR and Weste. Rn-Blot detection found that after transfection of shRNA lentivirus expression vector targeting OPN gene into mouse hepatocytes, sequence shRNA-A/B could effectively inhibit the expression of OPN gene in mouse hepatocytes, of which the shRNA-A sequence was best (up to 80%-90%). Conclusion the shRNA Lentivirus Expression Vector of the target OPN gene was successfully constructed. After transfecting the mouse hepatocytes, the gene could be transfected into mouse hepatocytes. It significantly inhibited the expression of OPN gene, which laid a foundation for further in vivo inhibition test.
The second part is the establishment of animal model of osteopontin gene silencing in C57 mice.
[Abstract] [Abstract] objective to inject the packaged lentivirus into C57 mice to inhibit the expression of Osteopontin (OPN) gene in the liver and establish a mouse model of OPN gene silencing. Methods 30 C57 mice were randomly divided into blank control group (Blank control, BC): sterilizing PBS liquid 200u1 through tail vein; negative The control group (Negative control, NC): 200ul pSicoR-GFP-shRNA-EC lentivirus solution was injected into the tail vein, and the experimental group (Experimental group, EG) was injected with the lentivirus solution of 200ul pSicoR-GFP-shRNA-A through the tail vein. After the injection, 2 mice were randomly taken off the neck. Embedding, frozen section detection of liver lentivirus transfection effect: another branch qRT-PCR and Western-Blot detection of OPN gene expression level. Results compared with the blank control group, negative control group, the experimental group OPN gene mRNA and protein expression level decreased significantly, the inhibition efficiency was between 70%-80%; blank control group and negative control group was not clear. Conclusion RNA interference technology and lentivirus vector can successfully prepare mouse liver OPN gene silencing animal model.
The third part of osteopontin gene silencing is related to the detection of liver cholesterol metabolism related gene expression in C57 mice.
[Abstract] [Abstract] Objective To study the changes of cholesterol metabolism and transport related gene expression in the liver of C57 mice after the expression of Osteopontin (OPN) gene was suppressed, in order to further study the technical support for the relationship between OPN and the cholesterol metabolism related genes during the process of cholesterol gallstone. The method was detected by RT-PCR and Western-Blot. Liver cholesterol metabolism and transport related genes HMG-CoA reductase, CYP-7 alpha 1, ABCG5/8 mRNA and protein expression changes. Results RT-PCR results showed that the expression of OPN was inhibited, the cholesterol metabolism related genes of the liver HMG-CoA reductase, CYP-7 a 1, ABCG5/8 mRNA content did not change, Western-Blot results coincide. Conclusion through this conclusion In some experiments, we have mastered RT-PCR and Western-Blot methods to detect the changes of HMG-CoA reductase, CYP-7 alpha 1, ABCG5/8mRNA and protein expression. After inhibiting the OPN gene expression in the liver of C57 mice, the expression of HMG-CoA reductase, CYP-7 alpha 1, ABCG5/8 has not changed obviously. We think that the cause of this phenomenon may be caused by further consulting the literature. It is: 1.OPN is highly expressed on the premise that pathogenic factors lead to metabolic disorders of the body, and then regulate the transcription and translation of related genes; the effect of 2.OPN expression level on related gene expression may take a long time (about February) to manifest.
In this paper, we successfully prepared the C57 mouse liver OPN low expression model by using lentivirus mediated RNA interference technology. It provides a powerful research tool for the further study of the regulation and mechanism of OPN on cholesterol metabolism during the formation of cholesterol stones. The third part study not only laid a methodology for the follow-up experiment. The basis for further study is to further study the model of OPN low expression C57 mice with high fat, high cholesterol diet and construction of cholesterol gallstones.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332
【参考文献】
相关期刊论文 前1条
1 陈进宏,蔡端,张延龄;胆汁成石趋势及成石性质多因素判断程序的研制及其临床意义[J];肝胆胰外科杂志;1999年03期
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