重組人端粒酶逆轉(zhuǎn)錄酶基因表達載體的構(gòu)建及檢測端粒長度新方法初探
發(fā)布時間：2018-09-11 13:07
【摘要】：第一章人端粒酶催化亞基(hTERT)的研究現(xiàn)狀和研究進展(文獻綜述) 人端粒酶逆轉(zhuǎn)錄酶(human telomerase reverse transcriptase,hTERT)基因是端粒酶的催化亞基,負責在染色體末端添加端粒重復序列,在維持細胞永生化中起重要作用。為探索過表達外源性hTERT基因轉(zhuǎn)染對細胞生物學作用,本研究以人臍靜脈血管內(nèi)皮細胞(hUVEC)為研究對象,通過真核轉(zhuǎn)染及病毒載體介導的基因轉(zhuǎn)移技術(shù)促進hTERT基因在hUVEC細胞中過表達,觀察其對hUVEC細胞增殖作用和端粒酶活性的影響�，F(xiàn)綜合近年來的文獻,對人端粒酶催化亞基的研究現(xiàn)狀作一簡要綜述。 第二章過表達人端粒酶逆轉(zhuǎn)錄酶促進人臍靜脈血管內(nèi)皮細胞增殖 目的:構(gòu)建含人端粒酶逆轉(zhuǎn)錄酶(hTERT)基因的真核表達載體并轉(zhuǎn)染人臍靜脈血管內(nèi)皮細胞(hUVEC),探索轉(zhuǎn)染后hTERT基因的表達及對細胞功能和生長的影響。 方法:攜帶人端粒酶逆轉(zhuǎn)錄酶(hTERT)基因的重組質(zhì)粒(pEGFP-C1-hTERT)是利用已有的PCI-neo-hTERT和pEGFP-C1質(zhì)粒,通過酶切連接后,DNA測序驗證了重組質(zhì)粒pEGFP-C1-hTERT的準確性。將pEGFP-C1-hTERT真核表達載體通過脂質(zhì)體2000轉(zhuǎn)染到hUVEC中,通過RT-PCR、免疫組化、PCR-ELISA法和MTT檢測細胞端粒酶基因表達和活性變化與細胞生長增殖情況。 結(jié)果:所構(gòu)建pEGFP-C1-hTERT真核表達載體結(jié)構(gòu)正確并能夠在真核細胞中表達。轉(zhuǎn)染后細胞可見報告基因GFP的表達;MTT實驗可見轉(zhuǎn)染hTERT基因組72 h后細胞增殖速度快于未轉(zhuǎn)染組及空載體組;RT-PCR、免疫組化及TRAP-PCR-ELISA法檢測轉(zhuǎn)染后的細胞,結(jié)果發(fā)現(xiàn)hTERT mRNA的表達、端粒酶活性均明顯增強。 結(jié)論:本研究成功構(gòu)建了pEGFP-C1-hTERT真核表達載體并能夠在真核細胞中表達,過表達的hTERT基因提高了血管內(nèi)皮細胞端粒酶的活性和增殖能力,初步證實端粒酶活性與細胞增殖活性密切相關(guān),為進一步構(gòu)建永生化細胞系奠定基礎(chǔ)。 第三章構(gòu)建攜帶重組人端粒酶逆轉(zhuǎn)錄酶第三代慢病毒載體及其病毒包裝鑒定 目的:構(gòu)建攜帶人端粒酶逆轉(zhuǎn)錄酶(hTERT)基因的慢病毒表達載體及探索高滴度第三代慢病毒包裝體系的建立,并觀察hTERT基因調(diào)控表達。 方法:用內(nèi)切酶將hTERT基因從已有質(zhì)粒PCI-neo-hTERT上切下,插入慢病毒載體pCDH-copGFP中構(gòu)建慢病毒表達質(zhì)粒pCDH-hTERT,通過雙酶切鑒定、DNA測序分析驗證人端粒酶逆轉(zhuǎn)錄酶基因(hTERT)片段的準確性后,將pCDH- hTERT、pCDH-PACK-GAG、pCDH-PACK-REV和VSV-G共轉(zhuǎn)染包裝細胞293T,濃縮上清并測定病毒滴度獲得重組慢病毒,并進行PCR及293T中hTERT蛋白的表達鑒定重組慢病毒的包裝。重組慢病毒再感染靶細胞hUVEC,通過檢測標記蛋白-綠色熒光蛋白、hTERT蛋白表達和端粒酶活性進一步驗證pCDH-hTERT在細胞中表達。 結(jié)果:pCDH-hTERT攜帶正確hTERT基因,將其與包裝質(zhì)粒共轉(zhuǎn)染293T細胞能產(chǎn)生重組病毒;病毒基因組PCR證實hTERT基因插入,感染后293T可檢測到hTERT蛋白的表達;目的基因hTERT能被重組慢病毒高效地轉(zhuǎn)導入靶細胞并穩(wěn)定表達,熒光顯微鏡下可直接觀察GFP;RT-PCR法、Western blotting法及TRAP-PCR-ELISA法能檢測感染后的細胞,結(jié)果發(fā)現(xiàn)hTERT mRNA的表達、hTERT蛋白的表達及端粒酶活性明顯增強。 結(jié)論:本研究成功構(gòu)建了第三代慢病毒表達載體pCDH-hTERT,并獲得高效的重組慢病毒,將外源hTERT基因轉(zhuǎn)導入靶細胞重建端粒酶活性,為構(gòu)建永生化細胞系奠定基礎(chǔ)。 第四章廣西長壽物種端粒長度的檢測與納米金標記的巰基寡聚核苷酸探針快速靈敏檢測端粒長度新方法的初探 目的:應用Southern雜交技術(shù)對不同年齡段不同長壽物種端粒長度進行檢測,從而了解Southern雜交對端粒長度檢測的利弊,評估其是否適用于其他長壽物種的檢測,探討建立檢測針對不同物種端粒長度新方法的必要性,并初步探索利用納米金技術(shù)建立快速靈敏檢測端粒長度新方法可行性。 方法:選擇廣西常見的長壽物種草龜、眼鏡蛇作為研究對象,應用Southern雜交技術(shù)進行檢測不同年齡段不同長壽物種端粒長度,對檢測端粒長度進行比較研究。以已知三種不同長度的端粒重復片段為標準品,初步探索將納米金的共振散射效應和納米金標記核酸探針結(jié)合起來建立一種測定端粒長度的新方法。通過制備粒徑約10納米的金納米顆粒,用于標記針對人端粒重復序列的共振散射光譜探針5’-(CCCTAA)5(CH2)3SH-3’,對標記納米金生物探針上寡核苷酸連接及與樣品端粒雜交反應體系條件進行優(yōu)化,探索利用納米金技術(shù)建立快速靈敏檢測端粒長度新方法的可行性。 結(jié)果:所應用Southern雜交技術(shù)進行檢測不同年齡段不同長壽物種端粒長度特異性及靈敏性均不高,各組間比較均無統(tǒng)計學意義。并成功完成標記納米金生物探針上寡核苷酸的連接,通過優(yōu)化了雜交反應體系中緩沖溶液的pH、AussDNA濃度、NaCl濃度、超聲波輻照時間等四個主要反應條件的影響,雜交后信號波動較大,但仍未找到適合的雜交的反應條件,尚未能建立起穩(wěn)定的納米金探針檢測端粒長度的新方法。 結(jié)論:Southern雜交技術(shù)采用針對人端粒重復片段特定探針,不適合應用于其他長壽物種的檢測,故需建立快速靈敏檢測針對不同物種端粒長度新方法。新方法中標記好納米金生物探針與樣品端粒雜交反應體系是實驗的一大難題,如何解決和建立納米金生物探針與樣品端粒雜交反應體系將是后續(xù)的實驗的首要解決問題。
[Abstract]:Chapter one: research status and research progress of human telomerase catalytic subunit (hTERT) (literature review)
Human telomerase reverse transcriptase (hTERT) gene is a catalytic subunit of telomerase, which is responsible for adding telomere repeats at the end of chromosome and plays an important role in maintaining cell immortality. To explore the effect of exogenous hTERT gene transfection on cell biology, this study was conducted in human umbilical vein. Dermal cells (hUVEC) were studied to promote the overexpression of hTERT gene in hUVEC cells by eukaryotic transfection and virus vector-mediated gene transfer. The effects of hTERT gene on the proliferation and telomerase activity of hUVEC cells were observed.
Second chapter overexpression of human telomerase reverse transcriptase promotes proliferation of human umbilical vein endothelial cells
AIM: To construct an eukaryotic expression vector containing human telomerase reverse transcriptase (hTERT) gene and transfect it into human umbilical vein endothelial cells (hUVEC) to investigate the expression of hTERT gene and its effect on cell function and growth.
METHODS: Recombinant plasmid carrying human telomerase reverse transcriptase (hTERT) gene (pEGFP-C1-hTERT) was constructed by using the existing plasmids PCI-neo-hTERT and pEGFP-C1. DNA sequencing confirmed the accuracy of the recombinant plasmid pEGFP-C1-hTERT. Epidemic histochemistry, PCR-ELISA and MTT assay were used to detect the expression and activity of telomerase gene and cell proliferation.
Results: The constructed eukaryotic expression vector of pEGFP-C1-hTERT was constructed correctly and could express in eukaryotic cells.GFP expression was observed in the transfected cells.MTT assay showed that the proliferation rate of the transfected cells was faster than that of the untransfected and empty vectors 72 hours after transfection.RT-PCR, immunohistochemistry and TRAP-PCR-ELISA were used to detect the transfected cells. It was found that the expression of hTERT mRNA and telomerase activity were significantly enhanced.
Conclusion: pEGFP-C1-hTERT eukaryotic expression vector was successfully constructed and expressed in eukaryotic cells. Overexpression of hTERT gene enhanced telomerase activity and proliferation ability of vascular endothelial cells. It was preliminarily confirmed that telomerase activity was closely related to cell proliferation activity and laid a foundation for further construction of immortalized cell lines.
The third chapter is to construct a third generation lentiviral vector carrying recombinant human telomerase reverse transcriptase and its viral packaging identification.
Objective: To construct lentiviral expression vector carrying human telomerase reverse transcriptase (hTERT) gene and explore the establishment of high titer third generation lentiviral packaging system, and to observe the regulation of hTERT gene expression.
METHODS: The hTERT gene was cut from the existing plasmid PCI-neo-hTERT by endonuclease and inserted into the lentiviral vector pCDH-copGFP to construct the lentiviral expression plasmid pCDH-hTERT. The accuracy of the fragment of human telomerase reverse transcriptase gene (hTERT) was verified by double enzyme digestion and DNA sequencing analysis. Then pCDH-hTERT, pCDH-PACK-GAG, pCDH-PACK-REV and VSV-G were co-constructed. The recombinant lentivirus was obtained by transfection of 293T into packaging cells, concentration of supernatant and determination of viral titer. The recombinant lentivirus was identified by PCR and expression of hTERT protein in 293T. Expression in cells.
Results: pCDH-hTERT carried the correct hTERT gene and transfected it with the packaging plasmid into 293T cells to produce recombinant virus. Viral genome PCR confirmed that hTERT gene was inserted into 293T cells and the expression of hTERT protein was detected after infection. The target gene hTERT could be transfected into target cells efficiently and stably expressed by recombinant lentivirus, and could be directly expressed under fluorescence microscope. GFP, RT-PCR, Western blotting and TRAP-PCR-ELISA were used to detect the expression of hTERT mRNA, hTERT protein and telomerase activity.
CONCLUSION: The third generation lentiviral expression vector pCDH-hTERT was successfully constructed and highly efficient recombinant lentiviruses were obtained. The foreign hTERT gene was transfected into target cells to reconstruct telomerase activity and lay a foundation for the construction of immortalized cell lines.
CHAPTER IV DETECTION OF TERMINAL LENGTH IN LONG-LIFE SPECIES OF GUANGXI PROVINCE
Objective: To investigate the advantages and disadvantages of Southern hybridization for telomere length detection in different age groups of longevity species, to assess whether it is suitable for other longevity species, to explore the necessity of establishing a new method for telomere length detection in different species, and to explore the use of Na. It is feasible to establish a fast and sensitive method for telomere length detection.
METHODS: The telomere lengths of common longevity turtles and cobras in Guangxi were measured by Southern hybridization technique, and the telomere lengths of different longevity species in different ages were compared. The resonance scattering of gold nanoparticles was preliminarily explored using three telomere repeats with different lengths as standard samples. A new method for determining telomere length was developed by combining the effect with gold nanoparticle labeled nucleic acid probes. The gold nanoparticles with a diameter of about 10 nm were prepared to label the resonance scattering probe 5'-(CCCTAA) 5 (CH2) 3SH-3', to connect the oligonucleotides on the labeled gold nanoprobe and to the end of the sample. The conditions of particle hybridization reaction system were optimized to explore the feasibility of establishing a new method for rapid and sensitive detection of telomere length by nanogold technology.
Results: The specificity and sensitivity of telomere length in different age groups were not high, and there was no significant difference among groups. The ligation of oligonucleotides on the labeled gold nanoprobe was successfully completed. The pH of buffer solution, AussDNA concentration, NaC in the hybridization reaction system were optimized. L concentration, ultrasonic irradiation time and other four main reaction conditions, the signal fluctuated greatly after hybridization, but no suitable hybridization reaction conditions have been found, and no stable nano-gold probe has been established to detect telomere length.
CONCLUSION: Southern hybridization technique using specific probes for human telomere repeat fragments is not suitable for other long-lived species, so it is necessary to establish a new method for rapid and sensitive detection of telomere length for different species. It will be the first problem to establish the reaction system of gold nanoprobe and telomere hybridization.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346
，
本文編號：2236768
[Abstract]:Chapter one: research status and research progress of human telomerase catalytic subunit (hTERT) (literature review)
Human telomerase reverse transcriptase (hTERT) gene is a catalytic subunit of telomerase, which is responsible for adding telomere repeats at the end of chromosome and plays an important role in maintaining cell immortality. To explore the effect of exogenous hTERT gene transfection on cell biology, this study was conducted in human umbilical vein. Dermal cells (hUVEC) were studied to promote the overexpression of hTERT gene in hUVEC cells by eukaryotic transfection and virus vector-mediated gene transfer. The effects of hTERT gene on the proliferation and telomerase activity of hUVEC cells were observed.
Second chapter overexpression of human telomerase reverse transcriptase promotes proliferation of human umbilical vein endothelial cells
AIM: To construct an eukaryotic expression vector containing human telomerase reverse transcriptase (hTERT) gene and transfect it into human umbilical vein endothelial cells (hUVEC) to investigate the expression of hTERT gene and its effect on cell function and growth.
METHODS: Recombinant plasmid carrying human telomerase reverse transcriptase (hTERT) gene (pEGFP-C1-hTERT) was constructed by using the existing plasmids PCI-neo-hTERT and pEGFP-C1. DNA sequencing confirmed the accuracy of the recombinant plasmid pEGFP-C1-hTERT. Epidemic histochemistry, PCR-ELISA and MTT assay were used to detect the expression and activity of telomerase gene and cell proliferation.
Results: The constructed eukaryotic expression vector of pEGFP-C1-hTERT was constructed correctly and could express in eukaryotic cells.GFP expression was observed in the transfected cells.MTT assay showed that the proliferation rate of the transfected cells was faster than that of the untransfected and empty vectors 72 hours after transfection.RT-PCR, immunohistochemistry and TRAP-PCR-ELISA were used to detect the transfected cells. It was found that the expression of hTERT mRNA and telomerase activity were significantly enhanced.
Conclusion: pEGFP-C1-hTERT eukaryotic expression vector was successfully constructed and expressed in eukaryotic cells. Overexpression of hTERT gene enhanced telomerase activity and proliferation ability of vascular endothelial cells. It was preliminarily confirmed that telomerase activity was closely related to cell proliferation activity and laid a foundation for further construction of immortalized cell lines.
The third chapter is to construct a third generation lentiviral vector carrying recombinant human telomerase reverse transcriptase and its viral packaging identification.
Objective: To construct lentiviral expression vector carrying human telomerase reverse transcriptase (hTERT) gene and explore the establishment of high titer third generation lentiviral packaging system, and to observe the regulation of hTERT gene expression.
METHODS: The hTERT gene was cut from the existing plasmid PCI-neo-hTERT by endonuclease and inserted into the lentiviral vector pCDH-copGFP to construct the lentiviral expression plasmid pCDH-hTERT. The accuracy of the fragment of human telomerase reverse transcriptase gene (hTERT) was verified by double enzyme digestion and DNA sequencing analysis. Then pCDH-hTERT, pCDH-PACK-GAG, pCDH-PACK-REV and VSV-G were co-constructed. The recombinant lentivirus was obtained by transfection of 293T into packaging cells, concentration of supernatant and determination of viral titer. The recombinant lentivirus was identified by PCR and expression of hTERT protein in 293T. Expression in cells.
Results: pCDH-hTERT carried the correct hTERT gene and transfected it with the packaging plasmid into 293T cells to produce recombinant virus. Viral genome PCR confirmed that hTERT gene was inserted into 293T cells and the expression of hTERT protein was detected after infection. The target gene hTERT could be transfected into target cells efficiently and stably expressed by recombinant lentivirus, and could be directly expressed under fluorescence microscope. GFP, RT-PCR, Western blotting and TRAP-PCR-ELISA were used to detect the expression of hTERT mRNA, hTERT protein and telomerase activity.
CONCLUSION: The third generation lentiviral expression vector pCDH-hTERT was successfully constructed and highly efficient recombinant lentiviruses were obtained. The foreign hTERT gene was transfected into target cells to reconstruct telomerase activity and lay a foundation for the construction of immortalized cell lines.
CHAPTER IV DETECTION OF TERMINAL LENGTH IN LONG-LIFE SPECIES OF GUANGXI PROVINCE
Objective: To investigate the advantages and disadvantages of Southern hybridization for telomere length detection in different age groups of longevity species, to assess whether it is suitable for other longevity species, to explore the necessity of establishing a new method for telomere length detection in different species, and to explore the use of Na. It is feasible to establish a fast and sensitive method for telomere length detection.
METHODS: The telomere lengths of common longevity turtles and cobras in Guangxi were measured by Southern hybridization technique, and the telomere lengths of different longevity species in different ages were compared. The resonance scattering of gold nanoparticles was preliminarily explored using three telomere repeats with different lengths as standard samples. A new method for determining telomere length was developed by combining the effect with gold nanoparticle labeled nucleic acid probes. The gold nanoparticles with a diameter of about 10 nm were prepared to label the resonance scattering probe 5'-(CCCTAA) 5 (CH2) 3SH-3', to connect the oligonucleotides on the labeled gold nanoprobe and to the end of the sample. The conditions of particle hybridization reaction system were optimized to explore the feasibility of establishing a new method for rapid and sensitive detection of telomere length by nanogold technology.
Results: The specificity and sensitivity of telomere length in different age groups were not high, and there was no significant difference among groups. The ligation of oligonucleotides on the labeled gold nanoprobe was successfully completed. The pH of buffer solution, AussDNA concentration, NaC in the hybridization reaction system were optimized. L concentration, ultrasonic irradiation time and other four main reaction conditions, the signal fluctuated greatly after hybridization, but no suitable hybridization reaction conditions have been found, and no stable nano-gold probe has been established to detect telomere length.
CONCLUSION: Southern hybridization technique using specific probes for human telomere repeat fragments is not suitable for other long-lived species, so it is necessary to establish a new method for rapid and sensitive detection of telomere length for different species. It will be the first problem to establish the reaction system of gold nanoprobe and telomere hybridization.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346
，
本文編號：2236768
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