構(gòu)建攜帶人GFAP啟動(dòng)子和p27基因的腺病毒載體及其對(duì)膠質(zhì)增生的影響
發(fā)布時(shí)間：2019-01-07 20:36
【摘要】：目的：構(gòu)建攜帶人GFAP啟動(dòng)子和p27基因的腺病毒表達(dá)載體和僅含人p27基因的對(duì)照載體。 方法：通過質(zhì)粒抽提、瓊脂糖凝膠電泳、酶切、連接、轉(zhuǎn)化等多種基因工程技術(shù),構(gòu)建和鑒定真核表達(dá)載體pDC315-GFAP-p27-EGFP和對(duì)照pDC315-p27-EGFP;利用腺病毒包裝系統(tǒng)AdMax和293細(xì)胞包裝重組腺病毒表達(dá)載體Ad-GFAP-p27-EGFP和Ad-p27-EGFP,用酶切、PCR法鑒定重組腺病毒表達(dá)載體。 結(jié)果：經(jīng)酶切鑒定和測(cè)序,成功鑒定真核表達(dá)載體pGFAP-IRES2-EGFP-p27;經(jīng)酶切鑒定和PCR鑒定,成功構(gòu)建真核表達(dá)載體pDC315-GFAP-p27-EGFP和對(duì)照pDC315-p27-EGFP;經(jīng)包裝后,Ad-p27-EGFP可見EGFP表達(dá)和細(xì)胞致病效應(yīng)(CPE),而Ad-GFAP-p27-EGFP出現(xiàn)CPE,但EGFP表達(dá)微弱。 結(jié)論：利用腺病毒包裝系統(tǒng)AdMax成功構(gòu)建了構(gòu)建攜帶人GFAP啟動(dòng)子和p27基因的腺病毒表達(dá)載體和僅含人p27基因的對(duì)照載體,為進(jìn)一步研究中樞神經(jīng)系統(tǒng)疾病引起的膠質(zhì)增生的治療提供了基因治療的策略。 目的：明確重組腺病毒載體Ad-GFAP-p27-EGFP和Ad-p27-EGFP對(duì)體內(nèi)外星形膠質(zhì)細(xì)胞增殖的影響。 方法：在體外,用重組腺病毒載體Ad-GFAP-p27-EGFP和Ad-p27-EGFP感染純化培養(yǎng)的星形膠質(zhì)細(xì)胞,通過免疫熒光化學(xué)、Western blot、 MTT和流式細(xì)胞術(shù)等觀察星形膠質(zhì)細(xì)胞感染腺病毒后外源基因EGFP、p27的表達(dá)情況以及對(duì)星形膠質(zhì)細(xì)胞細(xì)胞周期和增殖的影響；重組腺病毒載體Ad-GFAP-p27-EGFP感染神經(jīng)元和血管內(nèi)皮細(xì)胞,檢測(cè)GFAP啟動(dòng)子的特異性。 在體內(nèi),用重組腺病毒載體Ad-GFAP-p27-EGFP和Ad-p27-EGFP感染大鼠MCAO模型,通過免疫熒光化學(xué)、Western blot和行為學(xué)NSS評(píng)分,觀察體內(nèi)灶周腦組織p27和VEGF的表達(dá)情況、星形膠質(zhì)細(xì)胞增生、神經(jīng)元存活情況和行為學(xué)改變。 結(jié)果：重組腺病毒載體Ad-GFAP-p27-EGFP和Ad-p27-EGFP體外感染星形膠質(zhì)細(xì)胞后可見星形膠質(zhì)細(xì)胞均有EGFP的表達(dá),并且p27表達(dá)水平明顯增高,對(duì)星形膠質(zhì)細(xì)胞的細(xì)胞周期和增殖有抑制作用；重組腺病毒載體Ad-GFAP-p27-EGFP感染神經(jīng)元和血管內(nèi)皮細(xì)胞,只見到十分微弱的EGFP的表達(dá)；在體實(shí)驗(yàn)中,重組腺病毒載體Ad-GFAP-p27-EGFP和Ad-p27-EGFP感染大鼠MCAO模型,均可抑制星形膠質(zhì)細(xì)胞的增生,但是Ad-GFAP-p27-EGFP組存活的神經(jīng)元和新生血管的數(shù)目增多,改善了大鼠的NSS評(píng)分。 結(jié)論：重組腺病毒載體Ad-GFAP-p27-EGFP可以有效抑制反應(yīng)性膠質(zhì)細(xì)胞增生。 目的：制備改良的三維硫酸肝素-膠原蛋白生物支架并評(píng)估硫酸肝素-膠原蛋白生物支架與嗅鞘細(xì)胞的生物相容性。 方法：經(jīng)過改良的干燥冷凍程序,制作三維的硫酸肝素-膠原蛋白生物支架并用掃描電鏡進(jìn)行支架三維結(jié)構(gòu)的檢測(cè)；將改良的三維硫酸肝素-膠原蛋白生物支架與嗅鞘細(xì)胞在體外建立三維共培養(yǎng)體系,在第2、6、10和14天時(shí)用MTT法檢測(cè)細(xì)胞增殖活性；將嗅鞘細(xì)胞用CFSE標(biāo)記后以適宜的濃度種植于支架上,通過熒光顯微鏡和掃描電鏡直接追蹤和觀察細(xì)胞在支架上的粘附和生長(zhǎng)情況。 結(jié)果：改良的三維硫酸肝素-膠原蛋白生物支架具有三維立體的微管結(jié)構(gòu),可以促進(jìn)嗅鞘細(xì)胞的粘附、增殖以及細(xì)胞突起的生長(zhǎng)。并且在三維培養(yǎng)體系中,嗅鞘細(xì)胞的形態(tài)呈現(xiàn)出更具有活性的雙極紡錘形,細(xì)胞的突起具有一致延伸方向。 結(jié)論：改良的三維硫酸肝素-膠原蛋白生物支架與嗅鞘細(xì)胞在體外具有很好的生物相容性。因此,改良的三維硫酸肝素-膠原蛋白生物支架可能可以作為細(xì)胞載體,與嗅鞘細(xì)胞組成三維復(fù)合物應(yīng)用于神經(jīng)組織工程。
[Abstract]:Objective: To construct an adenovirus expression vector carrying human GFAP promoter and p27 gene and a control vector containing only human p27 gene. Methods: The recombinant adenovirus expression vector pDC315-GFAP-p27-EGFP and control pDC315-p27-EGFP were constructed and identified by a variety of genetic engineering techniques such as plasmid extraction, agarose gel electrophoresis, enzyme digestion, ligation and transformation. Ad-GFAP-p27-EGFP and Ad-p27-EGFP were expressed by recombinant adenovirus expression vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP. identification of recombinant adenovirus expression vector by PCR Results: The true nuclear expression vector pGFAP-IRES2-EGFP-p27 was successfully identified by enzyme-cut identification and sequencing. The true nuclear expression vector pDC315-GFAP-p27-EGFP and the control pDC315-p27-EGFP were successfully constructed by enzyme-cut identification and PCR identification. The expression of EGFP and the cytopathogenic effect (CPE) of Ad-p27-EGFP were observed after the packaging. Conclusion: AdMax successfully constructed an adenovirus expression vector carrying human GFAP promoter and p27 gene and a control vector containing only human p27 gene, and provided a gene therapy for further study of the treatment of gliosis caused by central nervous system diseases. Objective: To clarify the effect of recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP on the proliferation of astrocytes in vitro. Methods: In vitro, the cultured astrocytes were infected by recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP. Expression of EGFP and p27 and the effect on cell cycle and proliferation of astrocytes; recombinant adenovirus vector Ad-GFAP-p27-EGFP infected neurons and vascular endothelial cells to detect GFAP promoter In vivo, the expression of p27 and VEGF, the expression of p27 and VEGF, the proliferation of astrocyte and the survival of the neurons were observed by using recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP in the rat MCAO model. Results: Ad-GFAP-p27-EGFP and Ad-p27-EGFP were transfected with Ad-p27-EGFP in vitro. It was found that the recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP were infected by Ad-GFAP-p27-EGFP and Ad-p27-EGFP. The number of surviving neurons and new vessels in the Ad-GFAP-p27-EGFP group increased, and the rats were improved. Conclusion: The recombinant adenovirus vector Ad-GFAP-p27-EGFP can effectively inhibit the reaction. Objective: To prepare the modified three-dimensional heparin-collagen biological scaffold and to evaluate the heparin-collagen biological scaffold and the olfactory system. The method comprises the following steps: a three-dimensional heparin-collagen biological scaffold is prepared through an improved drying and freezing process, and the three-dimensional structure detection is carried out by using a scanning electron microscope; and the modified three-dimensional heparin-collagen biological scaffold and the sniffer cells are in vitro establishing a three-dimensional co-culture system, and detecting the cell proliferation activity by the MTT method on the days 2, 6, 10 and 14; and planting the sniffer cells on the support with a proper concentration after the FSE marker is labeled with the CFSE, and directly tracking and observing the cell on the support through the fluorescence microscope and the scanning electron microscope. Results: The modified three-dimensional heparin-collagen scaffold has three-dimensional and three-dimensional microtubule structure, which can promote the adhesion and increase of the olfactory cells. and in a three-dimensional culture system, the morphology of the olfactory cells exhibits a more active, double-polar, spindle-shaped, cell-like shape, Conclusion: The modified three-dimensional heparin-collagen biological scaffold and the olfactory receptor are in the body. therefore, the improved three-dimensional heparin-collagen biological scaffold can be used as a cell carrier,
【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】：2012
【分類號(hào)】：R318.08;R741
本文編號(hào)：2404131
[Abstract]:Objective: To construct an adenovirus expression vector carrying human GFAP promoter and p27 gene and a control vector containing only human p27 gene. Methods: The recombinant adenovirus expression vector pDC315-GFAP-p27-EGFP and control pDC315-p27-EGFP were constructed and identified by a variety of genetic engineering techniques such as plasmid extraction, agarose gel electrophoresis, enzyme digestion, ligation and transformation. Ad-GFAP-p27-EGFP and Ad-p27-EGFP were expressed by recombinant adenovirus expression vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP. identification of recombinant adenovirus expression vector by PCR Results: The true nuclear expression vector pGFAP-IRES2-EGFP-p27 was successfully identified by enzyme-cut identification and sequencing. The true nuclear expression vector pDC315-GFAP-p27-EGFP and the control pDC315-p27-EGFP were successfully constructed by enzyme-cut identification and PCR identification. The expression of EGFP and the cytopathogenic effect (CPE) of Ad-p27-EGFP were observed after the packaging. Conclusion: AdMax successfully constructed an adenovirus expression vector carrying human GFAP promoter and p27 gene and a control vector containing only human p27 gene, and provided a gene therapy for further study of the treatment of gliosis caused by central nervous system diseases. Objective: To clarify the effect of recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP on the proliferation of astrocytes in vitro. Methods: In vitro, the cultured astrocytes were infected by recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP. Expression of EGFP and p27 and the effect on cell cycle and proliferation of astrocytes; recombinant adenovirus vector Ad-GFAP-p27-EGFP infected neurons and vascular endothelial cells to detect GFAP promoter In vivo, the expression of p27 and VEGF, the expression of p27 and VEGF, the proliferation of astrocyte and the survival of the neurons were observed by using recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP in the rat MCAO model. Results: Ad-GFAP-p27-EGFP and Ad-p27-EGFP were transfected with Ad-p27-EGFP in vitro. It was found that the recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP were infected by Ad-GFAP-p27-EGFP and Ad-p27-EGFP. The number of surviving neurons and new vessels in the Ad-GFAP-p27-EGFP group increased, and the rats were improved. Conclusion: The recombinant adenovirus vector Ad-GFAP-p27-EGFP can effectively inhibit the reaction. Objective: To prepare the modified three-dimensional heparin-collagen biological scaffold and to evaluate the heparin-collagen biological scaffold and the olfactory system. The method comprises the following steps: a three-dimensional heparin-collagen biological scaffold is prepared through an improved drying and freezing process, and the three-dimensional structure detection is carried out by using a scanning electron microscope; and the modified three-dimensional heparin-collagen biological scaffold and the sniffer cells are in vitro establishing a three-dimensional co-culture system, and detecting the cell proliferation activity by the MTT method on the days 2, 6, 10 and 14; and planting the sniffer cells on the support with a proper concentration after the FSE marker is labeled with the CFSE, and directly tracking and observing the cell on the support through the fluorescence microscope and the scanning electron microscope. Results: The modified three-dimensional heparin-collagen scaffold has three-dimensional and three-dimensional microtubule structure, which can promote the adhesion and increase of the olfactory cells. and in a three-dimensional culture system, the morphology of the olfactory cells exhibits a more active, double-polar, spindle-shaped, cell-like shape, Conclusion: The modified three-dimensional heparin-collagen biological scaffold and the olfactory receptor are in the body. therefore, the improved three-dimensional heparin-collagen biological scaffold can be used as a cell carrier,
【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】：2012
【分類號(hào)】：R318.08;R741
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 張建祥;胡薇薇;陳忠;;對(duì)膠質(zhì)疤痕所致神經(jīng)再生障礙的治療進(jìn)展[J];浙江大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2009年06期
，本文編號(hào)：2404131
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