人端粒酶逆转录酶基因修饰的内皮祖细胞改善糖尿病大鼠的勃起功能及机制研究

发布时间：2018-04-30 05:26

  本文选题：hTERT基因 + 慢病毒　； 参考：《华中科技大学》2016年博士论文

【摘要】：[目的]建立含有hTERT基因的重组慢病毒载体,进一步鉴定构建的载体是否正确。扩增慢病毒颗粒的数量,并检测浓缩后的病毒滴度,以便用于下一步的研究——基因修饰大鼠EPCs。[方法]采用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)的方法获取hTERT基因的全长片段,并大量扩增。AgeⅠ和NheⅠ内切酶处理GV341质粒使其在特定酶切位点断裂,从而线性化。在交换酶的作用下,将获得的hTERT基因片段与线性化GV341质粒交换连接,产生重组质粒。将这些重组质粒导入制备好的感受态细胞(由大肠杆菌制备)中,在含有氨苄青霉素的培养基中培养扩增。采用聚合酶链反应(polymerase chain reaction, PCR)鉴定单克隆菌落中含有的质粒,有hTERT基因表达即为阳性克隆。检测这些初步鉴定为阳性菌落中质粒的核苷酸序列,并将结果与hTERT基因序列进行比较,两者的核苷酸序列完全相同,表示成功构建了hTERT基因重组质粒。大量扩增且纯化hTERT基因重组质粒和相应的辅助质粒(pHelper1.0和2.0).在转染试剂和Opti-MEM的辅助下,三种质粒一起转染293T细胞,合成病毒颗粒。2天后,上清滤去细胞残渣,离心超滤获取浓缩的病毒液。制备好的病毒液处理293T细胞,使病毒转染293T细胞后,利用含适当浓度的筛选药物(嘌呤霉素)的培养基孵育,在显微镜下监测细胞的状态,计算病毒的滴度。[结果]核苷酸序列检测结果显示质粒与hTERT基因的序列吻合,构建的重组质粒中的目的基因序列完全正确。利用293T细胞合成了大量的慢病毒颗粒,利用嘌呤霉素筛选处理,确定浓缩后病毒的最终滴度为2×108TU/ml。[结论]我们成功建立了含有hTERT基因的重组慢病毒载体,并且合成量较大且浓度高,可顺利的用于我们的下一步研究—-hTERT基因修饰EPCs。[目的]将hTERT重组慢病毒载体转染大鼠EPCs,探讨外源性hTERT基因在大鼠EPCs内的表达及对大鼠EPCs的影响。[方法]采用密度梯度离心法原代分离培养大鼠EPCs,细胞免疫荧光方法检测表面标志CD31、CD34、CD133和血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2, VEGFR-2)在EPCs中的表达,Western blot法检测内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)的表达。在特定的诱导培养基条件下将大鼠EPCs向平滑肌细胞和脂肪细胞定向分化。当感染复数(multiplicity of infection,MOI)为50时,将hTERT重组慢病毒载体或阴性对照病毒载体转染大鼠EPCs,得到EPCs-hTERT或EPCs-control。利用嘌呤霉素筛选转染后的大鼠EPCs得到阳性克隆。免疫荧光法、Western blot及Real-time PCR法检测hTERT及大鼠端粒酶逆转录酶(rat telomerase reverse transcriptase, rTERT)在EPCs-hTERT中的表达。端粒重复序列扩增(telomeric repeat amplification protocol, TRAP)法检测EPCs-hTERT的端粒酶活性。为进一步了解hTERT过表达对大鼠EPCs的影响,细胞计数试剂盒(cell counting kit-8, CCK-8)法及5-乙炔基-2'-脱氧尿嘧啶核苷(5-ethynyl-2'-deoxyuridine,EdU)法检测EPCs-hTERT的增殖能力。二氯二氢荧光素二乙酸酯(dichloro-dihydro-fluorescein diacetate, DCFH-DA)荧光探针检测EPCs-hTERT内的ROS水平。将EPCs-hTERT在氧化应激条件下培养,CCK-8法检测EPCs-hTERT的抗氧化应激能力。[结果]原代分离培养出大鼠EPCs。大鼠EPCs表达CD31、CD34、CD133和VEGFR-2,阳性率分别为(96.8±0.5)%、(97.2±1.4)%、(92.8±5.3)%和(91.5±3.7)%。Western blot可检测到大鼠EPCs内eNOS的表达。多向诱导分化鉴定结果显示,大鼠EPCs可定向诱导分化为脂肪细胞(油红O染色阳性)及平滑肌细胞(α-平滑肌肌动蛋白免疫荧光染色阳性)。慢病毒转染后,EPCs-hTERT内的hTERT基因在nRNA和蛋白水平的表达显著升高,并对rTERT的表达无影响。免疫荧光检测可见EPC-hTERT内的TERT蛋白表达于细胞核及细胞浆内,同时flag蛋白表达于细胞浆内,说明hTERT表达于细胞浆内。TRAP端粒酶活性检测结果显示,EPCs-hTERT的端粒酶活性显著升高。CCK-8法和EdU法结果显示,EPCs-hTERT的增殖能力显著强于EPCs和阴性对照病毒转染的EPCs (EPCs-control)。 EPCs-hTERT内ROS水平较低,且在氧化应激条件下培养,EPCs-hTERT的存活率显著高于EPCs和EPCs-control。[结论]成功分离并培养大鼠EPCs。hTERT基因重组慢病毒转染后,EPCs-hTERT端粒酶活性升高,且具有较强的增殖能力和抗氧化应激能力。EPCs-hTERT对糖尿病ED大鼠勃起功能的作用及机制研究[目的]探讨阴茎海绵体内注射EPCs-hTERT是否能够改善糖尿病ED大鼠勃起功能,并对可能机制进行探索。[方法]利用腹腔内注射链脲佐菌素(60mg/kg)的方法建立大鼠糖尿病模型,并进一步采用阿扑吗啡(apomorphine, APO)实验筛选糖尿病ED大鼠。将30只SD雄性大鼠分为5组：正常对照组、糖尿病ED组、采用EPCs治疗的糖尿病ED组(EPCs组)、采用EPCs-control治疗的糖尿病ED组(EPCs-control组)、采用EPCs-hTERT治疗的糖尿病ED组(EPCs-hTERT组)。2周后电刺激海绵体神经检测每组的勃起功能,并计算海绵体内压(intracavernous pressure, ICP)与平均动脉压(mean artery pressure, MAP)的比值。免疫荧光法检测阴茎组织内平滑肌含量和移植的EPCs的存活量。采用Masson's染色检测海绵体内纤维的含量。TUNEL法检测海绵体内细胞凋亡情况。Western blot法检测海绵体内转化生长因子-β1(transforming growth factor-β1, TGF-β1)/Smad2/3通路、Bcl-2、Bax、eNOS、磷酸化eNOS (phospho-eNOS, p-eNOS)和神经元型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)的表达情况。分别采用ELISA法和硝酸盐还原法测定大鼠阴茎海绵体组织中cGMP浓度和细胞内NO浓度。[结果]经EPCs和EPCs-control治疗后,糖尿病ED大鼠的勃起功能显著改善。EPCs-hTERT组的ICP/MAP比值显著高于EPCs和EPCs-control组。免疫荧光检测发现阴茎组织内的平滑肌含量及EPCs-hTERT存活量明显多于EPCs或EPCs-control组。Masson's染色显示EPCs-hTERT组的海绵体内纤维含量显著降低,且TGF-β1/Smad2/3通路的表达显著降低。与EPCs和EPCs-control组相比,EPCs-hTERT组阴茎海绵体内凋亡水平及Bax/Bcl-2比值均较低。EPCs-hTERT组的海绵体内NO、cGMP含量和eNOS、p-eNOS及nNOS表达水平显著高于糖尿病ED组。[结论]EPCs-hTERT可显著改善糖尿病ED大鼠的勃起功能,其机制可能为EPCs-hTERT在阴茎海绵体内存活量增多,并进一步减轻阴茎海绵体纤维化、降低组织内细胞凋亡水平和增加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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R698

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