慢病毒介導(dǎo)RNA干擾技術(shù)制備骨橋蛋白基因沉默小鼠模型
發(fā)布時(shí)間：2018-08-04 19:34
【摘要】：第一部分靶向骨橋蛋白基因的shRNA慢病毒表達(dá)載體的構(gòu)建與鑒定 [摘要]目的構(gòu)建針對(duì)骨橋蛋白(Osteopontin, OPN)基因的短發(fā)夾RNA (short hairpin RNA,shRNA)序列,合成pSicoR-GFP-shRNA慢病毒重組體,篩選對(duì)OPN基因表達(dá)抑制效率最佳的shRNA序列。方法參照OPN GeneBank上的序列設(shè)計(jì)合成shRNA-A,及不針對(duì)C57小鼠任何基因的陰性對(duì)照序列shRNA-NC,參考相關(guān)文獻(xiàn)合成針對(duì)骨橋蛋白基因的shRNA-B。將合成的shRNA插入到線性化的pSicoR-GFP慢病毒載體中；然后轉(zhuǎn)染E.coli JM109感受態(tài)細(xì)胞,對(duì)長出的菌落進(jìn)行PCR鑒定,PCR鑒定陽性的克隆再進(jìn)行測序。鑒定序列無誤后,將之與pCMV-VSV-G和pCMV-dR8.91輔助質(zhì)粒包裝,然后共轉(zhuǎn)染293FT細(xì)胞,進(jìn)行慢病毒的包裝及滴度測定,控制滴度在1×109TU/ml以上。用構(gòu)建好的慢病毒感染小鼠肝細(xì)胞,檢測不同shRNA序列對(duì)OPN基因表達(dá)的抑制效果,篩選最佳shRNA序列。結(jié)果經(jīng)RT-PCR和Western-Blot檢測發(fā)現(xiàn)：靶向OPN基因的shRNA慢病毒表達(dá)載體轉(zhuǎn)染小鼠肝細(xì)胞后,序列shRNA-A/B均可有效抑制小鼠肝細(xì)胞OPN基因的表達(dá),其中shRNA-A序列效果最佳(可達(dá)80%-90%)。結(jié)論成功構(gòu)建靶向OPN基因的shRNA慢病毒表達(dá)載體,其轉(zhuǎn)染小鼠肝細(xì)胞后可顯著抑制OPN基因的表達(dá),這為進(jìn)一步體內(nèi)抑制試驗(yàn)奠定了基礎(chǔ)。 第二部分C57小鼠肝臟骨橋蛋白基因沉默動(dòng)物模型的建立 [摘要]目的將包裝好的慢病毒經(jīng)尾靜脈注射到C57小鼠體內(nèi),抑制其肝臟骨橋蛋白(Osteopontin,OPN)基因的表達(dá),建立OPN基因沉默小鼠動(dòng)物模型。方法取30只C57小鼠隨機(jī)平均分為：空白對(duì)照組(Blank control,BC):經(jīng)尾靜脈注射滅菌PBS液200u1；陰性對(duì)照組(Negative control,NC):經(jīng)尾靜脈注射200ul pSicoR-GFP-shRNA-EC慢病毒液；實(shí)驗(yàn)組(Experimental group,EG):通過尾靜脈注射200ul pSicoR-GFP-shRNA-A的慢病毒液。注射后第72h、96h、7d、14d,各組隨機(jī)取2只小鼠斷頸處死。收集肝臟,一部分立即用OCT包埋劑進(jìn)行包埋,冰凍切片檢測肝臟慢病毒轉(zhuǎn)染效果：另一部分行qRT-PCR及Western-Blot檢測OPN基因表達(dá)水平。結(jié)果與空白對(duì)照組、陰性對(duì)照組相比,實(shí)驗(yàn)組OPN基因mRNA及蛋白表達(dá)水平明顯下降,抑制效率在70%-80%之間；空白對(duì)照組與陰性對(duì)照組之間無明顯差別。結(jié)論利用RNA干擾技術(shù),借助慢病毒載體,可以成功制備小鼠肝臟OPN基因沉默動(dòng)物模型。 第三部分骨橋蛋白基因沉默C57小鼠肝臟膽固醇代謝相關(guān)基因表達(dá)的檢測 [摘要]目的研究骨橋蛋白(Osteopontin, OPN)基因表達(dá)被抑制后,C57小鼠肝臟膽固醇代謝及轉(zhuǎn)運(yùn)相關(guān)基因表達(dá)的變化,為進(jìn)一步研究OPN在膽固醇結(jié)石發(fā)生過程中與肝臟膽固醇代謝相關(guān)基因的關(guān)系提供技術(shù)支持。方法通過RT-PCR及Western-Blot檢測肝臟膽固醇代謝及轉(zhuǎn)運(yùn)相關(guān)基因HMG-CoA還原酶、CYP-7α1、ABCG5/8的mRNA及蛋白表達(dá)的變化。結(jié)果RT-PCR結(jié)果顯示：OPN表達(dá)受抑制后,肝臟膽固醇代謝相關(guān)基因HMG-CoA還原酶、CYP-7α1、ABCG5/8的mRNA含量無明顯變化,Western-Blot結(jié)果與之相符。結(jié)論通過該部分實(shí)驗(yàn),我們熟練掌握RT-PCR、Western-Blot法檢測HMG-CoA還原酶、CYP-7α1、 ABCG5/8mRNA及蛋白表達(dá)的變化。抑制C57小鼠肝臟OPN基因表達(dá)后,HMG-CoA還原酶、CYP-7α1、ABCG5/8的表達(dá)未產(chǎn)生明顯變化。通過進(jìn)一步查閱文獻(xiàn),我們認(rèn)為產(chǎn)生這一現(xiàn)象的原因可能是：1.OPN是在致病因素導(dǎo)致機(jī)體代謝紊亂狀態(tài)的前提下高表達(dá),然后對(duì)相關(guān)基因的轉(zhuǎn)錄和翻譯發(fā)揮調(diào)節(jié)作用的；2.OPN表達(dá)水平變化對(duì)相關(guān)基因表達(dá)的影響可能需要較長的時(shí)間(約2月)才能表現(xiàn)出來。 總結(jié),本課題利用慢病毒介導(dǎo)的RNA干擾技術(shù),成功制備出C57小鼠肝臟OPN低表達(dá)模型,為進(jìn)一步研究OPN在膽固醇結(jié)石形成過程中對(duì)膽固醇代謝的調(diào)節(jié)作用及機(jī)制提供了有力的研究工具。通過第三部分的研究,不僅為后續(xù)實(shí)驗(yàn)奠定了方法學(xué)基礎(chǔ),而且提示我們：進(jìn)一步的研究須對(duì)OPN低表達(dá)C57小鼠飼以高脂、高膽固醇飲食,構(gòu)建膽固醇結(jié)石模型進(jìn)行進(jìn)一步探討。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R-332
本文編號(hào)：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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R-332
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陳進(jìn)宏,蔡端,張延齡;膽汁成石趨勢及成石性質(zhì)多因素判斷程序的研制及其臨床意義[J];肝膽胰外科雜志;1999年03期
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