tPA-VEGF165双基因静电纺丝涂层机械瓣膜的构建及体外实验研究

发布时间：2018-04-12 07:38

  本文选题：基因治疗 + 静电纺丝　； 参考：《华中科技大学》2011年博士论文

【摘要】：[目的]：通过克隆从模板中获得人组织型纤溶酶原激活因子(tPA)和人血管内皮细胞生长因子165 (VEGF165)目的基因片段,然后将其分别插入真核表达载体pIRES质粒的两个多克隆位点,以构建携带目的基因的两个载体质粒pIRES-VEGF165-tPA和pIRES-tPA-VEGF165。 [材料和方法]：按照人tPA基因的CDS序列(NM_000930.3)和人VEGF165基因的序列(NM_001025368)设计引物,引物分别包含内切酶EcoR I和Xba I酶切位点,以pcDNA3.1-Myc-His B(-)/tPA质粒和cDNA文库为模板,PCR扩增获得目的基因片段,琼脂糖凝胶电泳检测并切胶回收DNA；分别用内切酶EcoR I和Xba I将pIRES质粒线性化,然后把tPA和VEGF165目的基因片段重组入多克隆位点,并调换插入顺序,分别获得pIRES-VEGF165-tPA和pIRES-tPA-VEGF165两个质粒：所构建的表达载体质粒转化感受态细胞DH5α,筛选单克隆,菌落PCR检测目的基因,然后将阳性重组质粒送检测序。 [结果]：琼脂糖凝胶电泳结果表明PCR扩增目的基因,获得大小约为1721bp和1151bp的DNA,与人tPA和VEGF165基因序列相符合；菌落PCR表明pIRES-VEGF165-tPA和pIRES-tPA-VEGF165两个质粒转化感受态细胞均可得到阳性转化子,而他们的测序结果表明载体质粒中所包含的基因序列与Pubmed中其对应的CDS序列相比,插入方向正确,tPA基因中的CTG(编码Leu)同义突变为CTA(编码Leu), VEGF165基因中同样也仅出现数个同义突变,均不会影响蛋白表达。 [结论]：成功构建出了携带人组织型纤溶酶原激活因子(tPA)和人血管内皮细胞生长因子165(VEGF165)目的基因片段的两个载体质粒pIRES-VEGF165-tPA和pIRES-tPA-VEGF165。 第二部分：tPA-VEGF165真核表达载体的体外转染实验 [目的]：通过体外转染实验,检测所构建的真核表达载体pIRES-VEGF165-tPA和pIRES-tPA-VEGF165质粒能否转染内皮细胞,并检测目的基因在转录,翻译,以及蛋白分泌和功能方面的情况,同时比较pIRES-VEGF165-tPA和pIRES-tPA-VEGF165质粒在转染和蛋白表达方面有无区别。 [材料和方法]：转染试剂Attractene Transfection Reagent介导pIRES-VEGF165-tPA和pIRES-tPA-VEGF165质粒体外瞬时转染人脐静脉内皮细胞(EA.hy926),同时,以表达绿色荧光蛋白的pGFP质粒与载体质粒按照1：1的比例共转染EA.hy926细胞,以绿色荧光蛋白作为示踪标志,计算转染后12h,24h和48h瞬时转染效率,设置转染空pIRES质粒和空白PBS作为对照；荧光实时定量RT-PCR检测各组细胞中tPA和VEGF165的mRNA相对含量,Western blot检测细胞中的蛋白表达情况；提取转染后细胞上清,ELISA检测上清液中所含有的分泌蛋白tPA和VEGF165浓度,并用MTT试验和tPA活性检测试剂盒评价蛋白功能。 [结果]：通过计算转染后绿色荧光下阳性细胞的比率发现,转染后约24h,阳性细胞比率最高,随后逐渐减少,pIRES-VEGF165-tPA和pIRES-tPA-VEGF165质粒转染效率分别约为15.6±3.1%和16.3±2.9%,两组之间差异无统计学意义(P0.05)：荧光实时定量RT-PCR和Western blot检测发现转染pIRES-VEGF165-tPA和pIRES-tPA-VEGF165质粒组细胞中VEGF165和tPA的mRNA相对含量及蛋白含量明显高于转染pIRES空质粒和空白对照组,差异有统计学意义(P0.05),转染pIRES-tPA-VEGF165质粒组细胞中tPA基因的mRNA相对含量和蛋白含量高于转染pIRES-VEGF165-tPA组,差异有统计学意义(P0.05)：而VEGF165基因方面两组间差异无统计学意义(P0.05); ELISA, MTT试验以及tPA蛋白活性检测结果提示转染pIRES-VEGF165-tPA和pIRES-tPA-VEGF165质粒组细胞上清中VEGF165和tPA的蛋白含量及功能明显高于转染pIRES空质粒和空白对照组,差异有统计学意义(P0.05),而转染pIRES-VEGF165-tPA和pIRES-tPA-VEGF165质粒组间差异无统计学意义(P0.05)。 [结论]：所构建的pIRES-VEGF165-tPA和pIRES-tPA-VEGF165质粒能够在体外转染内皮细胞,并指导合成、分泌有功能活性的tPA和VEGF165蛋白,尽管转染pIRES-tPA-VEGF165质粒组细胞内目的基因的mRNA和蛋白含量略高,但不能因此认定它与pIRES-VEGF165-tPA质粒在功能上存在差异。 第三部分：交联明胶微球静电纺丝涂层涤纶材料的制备及相关理化性质的研究 [目的]：以交联明胶微球作为缓释载体,通过静电纺丝技术将其与涤纶材料有机结合,制备出能够长期释放质粒DNA的复合材料,并检测该材料在结构稳定性、细胞相容性、载药率等理化性质方面的特点,以期将该材料用于构建新型机械瓣膜。 [材料和方法]：将交联明胶微球和10%聚乙烯醇(PVA)溶液按照不同的质量容积比配制成电纺液,设置电压,喷头距离等参数进行纺丝,涤纶补片置于接收装置内接受纺丝纤维,最终制得超细纤维涂层的复合涤纶材料,扫描电镜检测表面形态；将涂层涤纶材料小块置入蒸馏水中,与摇床上振摇检测材料成分的结构稳定性；MTT试验检测涂层涤纶材料对EA.hy926细胞增殖的影响；按照质粒/涂层涤纶材料不同质量比将两者混合,通过测量结合后溶液中剩余质粒的量来计算涂层涤纶材料载药率,同时也测量单纯涤纶材料以及单纯的纺丝涂层材料的载药率作为对比；最后将结合有质粒的涂层涤纶材料置PBS溶液中,37℃下以30rpm的速度持续振摇,间断提取溶液样本,以在260nm处的吸光度值作为相对含量,绘制材料缓释质粒的曲线。 [结果]：交联明胶微球和PVA溶液的质量容积比在1/100之内时,室温下,电压16-20kV,接收距离10-12cm,注射速度2-4ml/h电纺可以获得均一的超细纤维毡,交联明胶微球散布与纤维之间,并随着微球质量的增加,分布密度也增加；在30-200rpm的速度振摇下涂层涤纶材料各部分结合紧密,结构完整未见明显脱落现象；MTT试验证实涂层涤纶补片干预组细胞与单纯Dacron补片干预组细胞,以及空白对照组细胞间增殖速度相当,差异无统计学意义(P0.05)：当质粒与涂层涤纶材料质量比在1：100左右时,材料的载药率达到最大值75%左右,随后进一步加大投入的质粒量,并不能增加载药率；根据缓释曲线可见涂层涤纶材料可以在体外逐渐释放质粒,前2天释放速度较快,从第8天开始减慢,第12天以后释放的总的质粒的量呈减少趋势。 [结论]：携带质粒的交联明胶微球可以通过静电纺丝技术同涤纶材料进行结合,该复合材料在流体剪切力的作用下可以保持结构完整,它的相容性好对细胞增殖无明显影响,能够携带质粒DNA并且在体外逐步释放,可以用于构建新型机械瓣膜。 第四部分：tPA-VEGF165双基因交联明胶微球静电纺丝涂层涤纶材料的体外转染实验 [目的]：通过体外实验,检测携带质粒的交联明胶微球静电纺丝涂层涤纶材料对细胞的转染效率以及是否能够分泌相关目的蛋白。 [材料和方法]：将携带质量比为1：1的pIRES-tPA-VEGF165质粒和pGFP质粒的涂层涤纶材料浸入完全细胞培养基中,获取其在不同时段的浸提液,然后加入微泡造影剂Sono Vue辅以频率1MHz,强度2 W/cm2的超声照射60sec,介导对EA.hy926细胞的瞬时转染,24h后通过计数绿色荧光的阳性细胞计算转染效率；超声微泡介导pIRES-tPA-VEGF165质粒单独转染EA.hy926细胞,并设置转染空pIRES质粒和空白PBS作为对照,24h后ELISA检测细胞上清中tPA和VEGF165蛋白含量。 [结果]：转染pIRES-tPA-VEGF165/pGFP质粒组细胞在6天内均有阳性细胞,其中前两天转染效率较高,约20%,随后有所下降,对照组无阳性细胞出现：ELISA结果提示转染了pIRES-tPA-VEGF165质粒的各组细胞上清每个时段均能检出tPA和VEGF165蛋白,整体趋势与细胞转染效率相似,而且其相对含量明显高于同时段的对照组,差异有统计学意义(P0.05)。转染pIRES空质粒和空白PBS对照组细胞有少量tPA表达,两组之间差异无统计学意义(P0.05),未检出VEGF165蛋白分泌。 [结论]：携带质粒的交联明胶微球静电纺丝涂层材料在超声微泡的作用下,能够在体外持续转染内皮细胞,并分泌相应的目的蛋白。
[Abstract]:Objective : To construct two vector plasmids pIRES - VEGF165 and pIRES - 1 - VEGF165 , which carry the target gene , by cloning the gene fragments of human tissue type plasminogen activator and human vascular endothelial growth factor 165 ( VEGF165 ) from the template .


The primers were designed according to the CDS sequence ( NM _ 000930 . 3 ) and the sequence of human VEGF165 gene ( NM _ 0025368 ) , respectively . The primers contained EcoR I and Xba I restriction sites , respectively , and pcDNA3.1 - Myc - His B ( - ) and cDNA library were used as templates . The target gene fragment was amplified by PCR , agarose gel electrophoresis was used to detect and cut the DNA ;
The pIRES plasmid was linearized by endonuclease EcoR I and Xba I , and then the gene fragments of the target gene were recombined into the multiple cloning sites , and the insertion sequence was changed to obtain pIRES - VEGF165 and pIRES - 1 - VEGF165 plasmid respectively . The constructed expression vector plasmid transformed competent cell DH5.alpha . , screened the monoclonal antibody and colony PCR to detect the target gene , and then the positive recombinant plasmid was sent to the detection sequence .


The results showed that agarose gel electrophoresis indicated that PCR amplified the target gene and obtained DNA with a size of about 1721bp and bp bp , which was in agreement with the gene sequence of human and VEGF165 .
The positive transformants were obtained by colony PCR , and the results showed that the gene sequence contained in the vector plasmid and the corresponding CDS sequence in Pubmed showed that the insertion direction was correct and CTG ( encoding Leu ) synonymous mutation in the gene was CTA ( encoding Leu ) , and the same mutation in the VEGF165 gene could not affect the expression of protein .


Conclusion : We successfully constructed two vector plasmids pIRES - VEGF165 - and pIRES - 1 - VEGF165 which carry human tissue - type plasminogen activator and human vascular endothelial growth factor 165 ( VEGF165 ) .


The second part : In vitro transfection experiment of the eukaryotic expression vector of tissue - VEGF165 eukaryotic expression vector


Objective : To investigate whether the eukaryotic expression vector pIRES - VEGF165 - 1 was transfected into the endothelial cells by in vitro transfection experiments , and the expression of the target gene in transcription , translation , and protein secretion and function were detected .


The transfected cells were transfected into human umbilical vein endothelial cells ( EA . hy926 ) transiently transfected with pGFP plasmid expressing the green fluorescent protein and the vector plasmid in a ratio of 1 : 1 . The transfection efficiency was calculated at 12 h , 24 h and 48 h after transfection . The transfected cells were transfected with pIRES plasmid and blank PBS as control ;
Real - time quantitative RT - PCR was used to detect the mRNA expression in the cells of each group , and Western blot was used to detect the protein expression in the cells .
After extraction , the supernatant of transfected cells was extracted , and the concentrations of the secreted proteins and VEGF165 contained in the supernatant were detected by ELISA , and the function of protein was evaluated by MTT assay and the assay kit .


Results : After transfection , the expression of VEGF165 mRNA and the protein in the transfected pIRES - VEGF165 - and pIRES - 1 - VEGF165 plasmid were significantly higher than that in the transfected pIRES - VEGF165 - 1 and pIRES - VEGF165 - VEGF165 plasmid , and the difference was statistically significant ( P0.05 ) . The difference was statistically significant ( P0.05 ) .


Conclusion : The pIRES - VEGF165 - PA and pIRES - GM - VEGF165 plasmid constructed can be transfected into endothelial cells in vitro and direct the synthesis and secretion of the functionally active and VEGF165 proteins , although the mRNA and protein content of the target gene in the transfected pIRES - 1 - VEGF165 plasmid group was slightly higher , but it was not considered to be functionally different from the pIRES - VEGF165 - .


The third part : the preparation and related physical and chemical properties of the crosslinked gelatin microsphere electrostatic spinning coating polyester material


Objective : To prepare a composite material capable of releasing plasmid DNA for a long time by using cross - linked gelatin microspheres as a sustained - release carrier , and to prepare a composite material capable of releasing plasmid DNA for a long time , and to detect the characteristics of the material in the aspects of structural stability , cell compatibility , drug loading rate and so on , with a view to using the material to construct a novel mechanical valve .


The preparation method comprises the following steps of : preparing cross - linked gelatin microspheres and 10 percent of polyvinyl alcohol ( PVA ) solution according to different mass - volume ratios to prepare spinning solution , setting parameters such as voltage and spray head distance ;
putting the small block of the coating terylene material into distilled water , and shaking and detecting the structural stability of the material component on the shaking bed ;
Effect of coated polyester material on proliferation of EA . hy926 cells by MTT assay
according to the different mass ratio of the plasmid / coating terylene material , the drug loading rate of the coating polyester material is calculated by measuring the amount of the remaining plasmids in the combined solution , and simultaneously the drug loading rate of the pure polyester material and the pure spinning coating material is also measured as a comparison ;
Finally , the coated polyester material with plasmid was placed in PBS solution at 37 鈩，

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