本文選題：hTERT基因 + 慢病毒　； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：[目的]建立含有hTERT基因的重組慢病毒載體,進(jìn)一步鑒定構(gòu)建的載體是否正確。擴(kuò)增慢病毒顆粒的數(shù)量,并檢測(cè)濃縮后的病毒滴度,以便用于下一步的研究——基因修飾大鼠EPCs。[方法]采用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription-polymerase chain reaction, RT-PCR)的方法獲取hTERT基因的全長(zhǎng)片段,并大量擴(kuò)增。AgeⅠ和NheⅠ內(nèi)切酶處理GV341質(zhì)粒使其在特定酶切位點(diǎn)斷裂,從而線性化。在交換酶的作用下,將獲得的hTERT基因片段與線性化GV341質(zhì)粒交換連接,產(chǎn)生重組質(zhì)粒。將這些重組質(zhì)粒導(dǎo)入制備好的感受態(tài)細(xì)胞(由大腸桿菌制備)中,在含有氨芐青霉素的培養(yǎng)基中培養(yǎng)擴(kuò)增。采用聚合酶鏈反應(yīng)(polymerase chain reaction, PCR)鑒定單克隆菌落中含有的質(zhì)粒,有hTERT基因表達(dá)即為陽(yáng)性克隆。檢測(cè)這些初步鑒定為陽(yáng)性菌落中質(zhì)粒的核苷酸序列,并將結(jié)果與hTERT基因序列進(jìn)行比較,兩者的核苷酸序列完全相同,表示成功構(gòu)建了hTERT基因重組質(zhì)粒。大量擴(kuò)增且純化hTERT基因重組質(zhì)粒和相應(yīng)的輔助質(zhì)粒(pHelper1.0和2.0).在轉(zhuǎn)染試劑和Opti-MEM的輔助下,三種質(zhì)粒一起轉(zhuǎn)染293T細(xì)胞,合成病毒顆粒。2天后,上清濾去細(xì)胞殘?jiān)?離心超濾獲取濃縮的病毒液。制備好的病毒液處理293T細(xì)胞,使病毒轉(zhuǎn)染293T細(xì)胞后,利用含適當(dāng)濃度的篩選藥物(嘌呤霉素)的培養(yǎng)基孵育,在顯微鏡下監(jiān)測(cè)細(xì)胞的狀態(tài),計(jì)算病毒的滴度。[結(jié)果]核苷酸序列檢測(cè)結(jié)果顯示質(zhì)粒與hTERT基因的序列吻合,構(gòu)建的重組質(zhì)粒中的目的基因序列完全正確。利用293T細(xì)胞合成了大量的慢病毒顆粒,利用嘌呤霉素篩選處理,確定濃縮后病毒的最終滴度為2×108TU/ml。[結(jié)論]我們成功建立了含有hTERT基因的重組慢病毒載體,并且合成量較大且濃度高,可順利的用于我們的下一步研究—-hTERT基因修飾EPCs。[目的]將hTERT重組慢病毒載體轉(zhuǎn)染大鼠EPCs,探討外源性hTERT基因在大鼠EPCs內(nèi)的表達(dá)及對(duì)大鼠EPCs的影響。[方法]采用密度梯度離心法原代分離培養(yǎng)大鼠EPCs,細(xì)胞免疫熒光方法檢測(cè)表面標(biāo)志CD31、CD34、CD133和血管內(nèi)皮生長(zhǎng)因子受體-2(vascular endothelial growth factor receptor-2, VEGFR-2)在EPCs中的表達(dá),Western blot法檢測(cè)內(nèi)皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)的表達(dá)。在特定的誘導(dǎo)培養(yǎng)基條件下將大鼠EPCs向平滑肌細(xì)胞和脂肪細(xì)胞定向分化。當(dāng)感染復(fù)數(shù)(multiplicity of infection,MOI)為50時(shí),將hTERT重組慢病毒載體或陰性對(duì)照病毒載體轉(zhuǎn)染大鼠EPCs,得到EPCs-hTERT或EPCs-control。利用嘌呤霉素篩選轉(zhuǎn)染后的大鼠EPCs得到陽(yáng)性克隆。免疫熒光法、Western blot及Real-time PCR法檢測(cè)hTERT及大鼠端粒酶逆轉(zhuǎn)錄酶(rat telomerase reverse transcriptase, rTERT)在EPCs-hTERT中的表達(dá)。端粒重復(fù)序列擴(kuò)增(telomeric repeat amplification protocol, TRAP)法檢測(cè)EPCs-hTERT的端粒酶活性。為進(jìn)一步了解hTERT過(guò)表達(dá)對(duì)大鼠EPCs的影響,細(xì)胞計(jì)數(shù)試劑盒(cell counting kit-8, CCK-8)法及5-乙炔基-2'-脫氧尿嘧啶核苷(5-ethynyl-2'-deoxyuridine,EdU)法檢測(cè)EPCs-hTERT的增殖能力。二氯二氫熒光素二乙酸酯(dichloro-dihydro-fluorescein diacetate, DCFH-DA)熒光探針檢測(cè)EPCs-hTERT內(nèi)的ROS水平。將EPCs-hTERT在氧化應(yīng)激條件下培養(yǎng),CCK-8法檢測(cè)EPCs-hTERT的抗氧化應(yīng)激能力。[結(jié)果]原代分離培養(yǎng)出大鼠EPCs。大鼠EPCs表達(dá)CD31、CD34、CD133和VEGFR-2,陽(yáng)性率分別為(96.8±0.5)%、(97.2±1.4)%、(92.8±5.3)%和(91.5±3.7)%。Western blot可檢測(cè)到大鼠EPCs內(nèi)eNOS的表達(dá)。多向誘導(dǎo)分化鑒定結(jié)果顯示,大鼠EPCs可定向誘導(dǎo)分化為脂肪細(xì)胞(油紅O染色陽(yáng)性)及平滑肌細(xì)胞(α-平滑肌肌動(dòng)蛋白免疫熒光染色陽(yáng)性)。慢病毒轉(zhuǎn)染后,EPCs-hTERT內(nèi)的hTERT基因在nRNA和蛋白水平的表達(dá)顯著升高,并對(duì)rTERT的表達(dá)無(wú)影響。免疫熒光檢測(cè)可見(jiàn)EPC-hTERT內(nèi)的TERT蛋白表達(dá)于細(xì)胞核及細(xì)胞漿內(nèi),同時(shí)flag蛋白表達(dá)于細(xì)胞漿內(nèi),說(shuō)明hTERT表達(dá)于細(xì)胞漿內(nèi)。TRAP端粒酶活性檢測(cè)結(jié)果顯示,EPCs-hTERT的端粒酶活性顯著升高。CCK-8法和EdU法結(jié)果顯示,EPCs-hTERT的增殖能力顯著強(qiáng)于EPCs和陰性對(duì)照病毒轉(zhuǎn)染的EPCs (EPCs-control)。 EPCs-hTERT內(nèi)ROS水平較低,且在氧化應(yīng)激條件下培養(yǎng),EPCs-hTERT的存活率顯著高于EPCs和EPCs-control。[結(jié)論]成功分離并培養(yǎng)大鼠EPCs。hTERT基因重組慢病毒轉(zhuǎn)染后,EPCs-hTERT端粒酶活性升高,且具有較強(qiáng)的增殖能力和抗氧化應(yīng)激能力。EPCs-hTERT對(duì)糖尿病ED大鼠勃起功能的作用及機(jī)制研究[目的]探討陰莖海綿體內(nèi)注射EPCs-hTERT是否能夠改善糖尿病ED大鼠勃起功能,并對(duì)可能機(jī)制進(jìn)行探索。[方法]利用腹腔內(nèi)注射鏈脲佐菌素(60mg/kg)的方法建立大鼠糖尿病模型,并進(jìn)一步采用阿撲嗎啡(apomorphine, APO)實(shí)驗(yàn)篩選糖尿病ED大鼠。將30只SD雄性大鼠分為5組：正常對(duì)照組、糖尿病ED組、采用EPCs治療的糖尿病ED組(EPCs組)、采用EPCs-control治療的糖尿病ED組(EPCs-control組)、采用EPCs-hTERT治療的糖尿病ED組(EPCs-hTERT組)。2周后電刺激海綿體神經(jīng)檢測(cè)每組的勃起功能,并計(jì)算海綿體內(nèi)壓(intracavernous pressure, ICP)與平均動(dòng)脈壓(mean artery pressure, MAP)的比值。免疫熒光法檢測(cè)陰莖組織內(nèi)平滑肌含量和移植的EPCs的存活量。采用Masson's染色檢測(cè)海綿體內(nèi)纖維的含量。TUNEL法檢測(cè)海綿體內(nèi)細(xì)胞凋亡情況。Western blot法檢測(cè)海綿體內(nèi)轉(zhuǎn)化生長(zhǎng)因子-β1(transforming growth factor-β1, TGF-β1)/Smad2/3通路、Bcl-2、Bax、eNOS、磷酸化eNOS (phospho-eNOS, p-eNOS)和神經(jīng)元型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)的表達(dá)情況。分別采用ELISA法和硝酸鹽還原法測(cè)定大鼠陰莖海綿體組織中cGMP濃度和細(xì)胞內(nèi)NO濃度。[結(jié)果]經(jīng)EPCs和EPCs-control治療后,糖尿病ED大鼠的勃起功能顯著改善。EPCs-hTERT組的ICP/MAP比值顯著高于EPCs和EPCs-control組。免疫熒光檢測(cè)發(fā)現(xiàn)陰莖組織內(nèi)的平滑肌含量及EPCs-hTERT存活量明顯多于EPCs或EPCs-control組。Masson's染色顯示EPCs-hTERT組的海綿體內(nèi)纖維含量顯著降低,且TGF-β1/Smad2/3通路的表達(dá)顯著降低。與EPCs和EPCs-control組相比,EPCs-hTERT組陰莖海綿體內(nèi)凋亡水平及Bax/Bcl-2比值均較低。EPCs-hTERT組的海綿體內(nèi)NO、cGMP含量和eNOS、p-eNOS及nNOS表達(dá)水平顯著高于糖尿病ED組。[結(jié)論]EPCs-hTERT可顯著改善糖尿病ED大鼠的勃起功能,其機(jī)制可能為EPCs-hTERT在陰莖海綿體內(nèi)存活量增多,并進(jìn)一步減輕陰莖海綿體纖維化、降低組織內(nèi)細(xì)胞凋亡水平和增加NO合成。
[Abstract]:[Objective] to establish a recombinant lentivirus vector containing hTERT gene to further identify the correctness of the constructed vector, to amplify the number of lentivirus particles, and to detect the virus titer after concentration, so as to use the reverse transcriptase chain reaction (reverse transcription-polymerase C) for the next step of the study of the gene modified rat EPCs.[method] The Hain reaction, RT-PCR) method obtained the full length of the hTERT gene, and expanded the GV341 plasmid with.Age I and Nhe I endonucleases to make the GV341 plasmids broken and linearized. Under the action of the exchange enzyme, the obtained hTERT gene fragment was linked with the linear GV341 particles and produced the recombinant plasmids. The prepared receptive cells (prepared by E. coli) were cultured and amplified in the medium containing ampicillin. Polymerase chain reaction (PCR) was used to identify the plasmids contained in the monoclonal colony, and the expression of hTERT gene was positive, which was identified as the plasmid in the positive colony. The nucleotide sequence was compared with the hTERT gene sequence. The nucleotide sequences of the two were the same, and the recombinant plasmid of hTERT gene was successfully constructed. The recombinant plasmid of hTERT gene was amplified and purified, and the corresponding auxiliary plasmids (pHelper1.0 and 2) were amplified and purified. The transfection of three plasmids together with the aid of the transfected test agent and Opti-MEM was 293. T cells, when the virus particles were synthesized for.2 days, the supernatant filtered out the residue of the cells and obtained the concentrated viral fluid by centrifuge ultrafiltration. The prepared viral fluid was used to treat 293T cells. After transfecting the virus to 293T cells, the virus was incubated with the medium containing the appropriate concentration of the selected drug (purinomycin), and the cell status was monitored under the microscope, and the virus titer was calculated. Nucleotide sequence detection results showed that the sequence of plasmid and hTERT gene was identical. The sequence of target gene in the recombinant plasmid was completely correct. A large number of lentivirus particles were synthesized by 293T cells, and purinamycin was screened by purinamycin. The final titer of the virus was determined to be 2 x 108TU/ml.[conclusion.] we successfully established the hTER The recombinant lentivirus vector of T gene, which has high synthesis and high concentration, can be successfully used in our next study - -hTERT gene modification EPCs.[Objective] to transfect hTERT recombinant lentivirus vector to rat EPCs, and to explore the expression of exogenous hTERT gene in rat EPCs and the effect on rat EPCs. [method] the density gradient centrifugation was used. The expression of CD31, CD34, CD133 and vascular endothelial growth factor receptor -2 (vascular endothelial growth factor receptor-2, VEGFR-2) in the EPCs were detected by cell immunofluorescence in the primary culture of rat EPCs. Rat EPCs was directed to smooth muscle cells and adipocytes under specific inducible medium. When the complex number of infection (multiplicity of infection, MOI) was 50, hTERT recombinant lentivirus vector or negative control virus vector was transfected into rat EPCs to obtain EPCs-hTERT or EPCs-control. using purinomycin to filter the rat EP after transfection. Cs positive clones were obtained. Immunofluorescence, Western blot and Real-time PCR were used to detect the expression of hTERT and rat telomerase reverse transcriptase (rat telomerase reverse transcriptase, rTERT) in EPCs-hTERT, and the telomeric repeat sequence amplification method was used to detect the activity of telomerase. The effect of hTERT overexpression on EPCs in rats was investigated. Cell count Kit (cell counting kit-8, CCK-8) method and 5- acetylene deoxyuridine (5-ethynyl-2'-deoxyuridine, EdU) method were used to detect EPCs-hTERT proliferation. Two chlorine two fluorescein two acetate (dichloro-dihydro-fluorescein) fluorescent probe The level of ROS in EPCs-hTERT was detected. EPCs-hTERT was cultured under oxidative stress and CCK-8 method was used to detect the antioxidant stress of EPCs-hTERT. [results] EPCs expressed CD31, CD34, CD133 and VEGFR-2 in EPCs. rats. The positive rates were (96.8 + 0.5)%, (97.2 + 1.4)%, (92.8 + 5.3)% and (91.5 + 3.7)%.Western. The expression of eNOS in the rat EPCs was detected. The results of multiple induction differentiation showed that the rat EPCs could be induced to differentiate into adipocytes (oil red O staining positive) and smooth muscle cells (alpha smooth muscle actin immunofluorescence staining positive). After lentivirus transfection, the expression of hTERT gene in EPCs-hTERT increased significantly in nRNA and protein levels. There was no effect on the expression of rTERT. The immunofluorescence detection showed that the TERT protein in EPC-hTERT was expressed in the nucleus and cytoplasm, and the flag protein was expressed in the cytoplasm. The results showed that the expression of hTERT in the cytoplasm of.TRAP telomerase activity showed that the telomerase activity of EPCs-hTERT was significantly increased by.CCK-8 method and EdU method, EPCs-hTER. The proliferation ability of T was significantly stronger than that of EPCs (EPCs-control) transfected with EPCs and negative control virus. The level of ROS in EPCs-hTERT was lower, and the survival rate of EPCs-hTERT was significantly higher than that of EPCs and EPCs-control.[in oxidative stress conditions. The survival rate of EPCs-hTERT was successfully separated and cultured in rat EPCs.hTERT gene recombinant lentivirus transfection, EPCs-hTERT telomerase activity was found. A study on the effect and mechanism of.EPCs-hTERT on the erectile function of diabetic ED rats. [Objective] to investigate whether the injection of EPCs-hTERT in the cavernous body of the penis can improve the erectile function of diabetic ED rats and explore the possible mechanism. The diabetic rat model was established by 60mg/kg, and the apomorphine (APO) experiment was used to screen the diabetic ED rats. 30 SD male rats were divided into 5 groups: normal control group, diabetic group ED, EPCs treated diabetes ED group (EPCs group), EPCs-control treated diabetic ED group (EPCs-control group). The erectile function of each group was detected by electrical stimulation of the cavernous nerve in group ED group (group EPCs-hTERT) treated with EPCs-hTERT after.2 weeks, and the ratio of intracavernous pressure (ICP) to the mean arterial pressure (mean artery pressure, MAP) was calculated. The content of smooth muscle in the penis tissue and the survival of the transplanted EPCs were measured by immunofluorescence Masson's staining was used to detect the content of fibrous in cavernous body by.TUNEL method to detect the cell apoptosis in cavernous body..Western blot method was used to detect the /Smad2/3 pathway of TGF - beta 1 (transforming growth factor- beta 1, TGF- beta 1), Bcl-2, Bax, eNOS, phosphorylation and nitric oxide synthase The expression of Ronal nitric oxide synthase, nNOS). ELISA and nitrate reduction were used to determine the concentration of cGMP and intracellular NO in the tissue of the corpus cavernosum of the rats. [results] the erectile function of the ED diabetic rats was significantly improved after the treatment of EPCs and EPCs-control. Trol group. The immunofluorescence test found that the content of smooth muscle and the survival of EPCs-hTERT in the penis tissues were more than those of the group EPCs or EPCs-control,.Masson's staining showed that the fibrous content of the sponge in the EPCs-hTERT group decreased significantly, and the expression of the TGF- beta 1/Smad2/3 pathway decreased significantly. Compared with the EPCs and EPCs-control groups, the EPCs-hTERT group of the penis sponge was compared. The level of apoptosis and the ratio of Bax/Bcl-2 in the body were higher than that in the cavernous body of the low.EPCs-hTERT group. The levels of NO, cGMP and eNOS, p-eNOS and nNOS were significantly higher than those of the diabetic ED group. [conclusion]EPCs-hTERT can significantly improve the erectile function of the diabetic ED rats. The mechanism may be the increase in the amount of memory in the corpus cavernosum of EPCs-hTERT. Cavernous fibrosis can reduce cell apoptosis and increase NO synthesis.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R587.2;R698

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6 陳昭典;;勃起功能和功能障礙:的若干進(jìn)展-2[A];浙江省中醫(yī)藥學(xué)會(huì)2008年不孕不育與性醫(yī)學(xué)研討會(huì)暨繼續(xù)教育學(xué)習(xí)班資料匯編[C];2008年

7 楊為民;饒可;杜廣輝;王少剛;劉繼紅;葉章群;;血脂異常對(duì)男性勃起功能影響的流行病學(xué)研究[A];湖北省性學(xué)會(huì)第二屆第二次學(xué)術(shù)年會(huì)論文集[C];2005年

8 呂伯東;;陰莖海綿體平滑肌細(xì)胞病變與勃起功能早衰的關(guān)系探討[A];2005年浙江省男科學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2005年

9 饒可;杜廣輝;楊為民;劉繼紅;王少剛;;血脂異常與男性勃起功能的相關(guān)性研究[A];21世紀(jì)男科學(xué)——中華醫(yī)學(xué)會(huì)第五次全國(guó)男科學(xué)學(xué)術(shù)會(huì)議論文集[C];2004年

10 趙啟群;孟旭輝;薛s，

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