本文選題：基因治療 + 靜電紡絲　； 參考：《華中科技大學(xué)》2011年博士論文

【摘要】：[目的]：通過克隆從模板中獲得人組織型纖溶酶原激活因子(tPA)和人血管內(nèi)皮細(xì)胞生長因子165 (VEGF165)目的基因片段,然后將其分別插入真核表達(dá)載體pIRES質(zhì)粒的兩個多克隆位點(diǎn),以構(gòu)建攜帶目的基因的兩個載體質(zhì)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)設(shè)計(jì)引物,引物分別包含內(nèi)切酶EcoR I和Xba I酶切位點(diǎn),以pcDNA3.1-Myc-His B(-)/tPA質(zhì)粒和cDNA文庫為模板,PCR擴(kuò)增獲得目的基因片段,瓊脂糖凝膠電泳檢測并切膠回收DNA；分別用內(nèi)切酶EcoR I和Xba I將pIRES質(zhì)粒線性化,然后把tPA和VEGF165目的基因片段重組入多克隆位點(diǎn),并調(diào)換插入順序,分別獲得pIRES-VEGF165-tPA和pIRES-tPA-VEGF165兩個質(zhì)粒：所構(gòu)建的表達(dá)載體質(zhì)粒轉(zhuǎn)化感受態(tài)細(xì)胞DH5α,篩選單克隆,菌落PCR檢測目的基因,然后將陽性重組質(zhì)粒送檢測序。 [結(jié)果]：瓊脂糖凝膠電泳結(jié)果表明PCR擴(kuò)增目的基因,獲得大小約為1721bp和1151bp的DNA,與人tPA和VEGF165基因序列相符合；菌落PCR表明pIRES-VEGF165-tPA和pIRES-tPA-VEGF165兩個質(zhì)粒轉(zhuǎn)化感受態(tài)細(xì)胞均可得到陽性轉(zhuǎn)化子,而他們的測序結(jié)果表明載體質(zhì)粒中所包含的基因序列與Pubmed中其對應(yīng)的CDS序列相比,插入方向正確,tPA基因中的CTG(編碼Leu)同義突變?yōu)镃TA(編碼Leu), VEGF165基因中同樣也僅出現(xiàn)數(shù)個同義突變,均不會影響蛋白表達(dá)。 [結(jié)論]：成功構(gòu)建出了攜帶人組織型纖溶酶原激活因子(tPA)和人血管內(nèi)皮細(xì)胞生長因子165(VEGF165)目的基因片段的兩個載體質(zhì)粒pIRES-VEGF165-tPA和pIRES-tPA-VEGF165。 第二部分：tPA-VEGF165真核表達(dá)載體的體外轉(zhuǎn)染實(shí)驗(yàn) [目的]：通過體外轉(zhuǎn)染實(shí)驗(yàn),檢測所構(gòu)建的真核表達(dá)載體pIRES-VEGF165-tPA和pIRES-tPA-VEGF165質(zhì)粒能否轉(zhuǎn)染內(nèi)皮細(xì)胞,并檢測目的基因在轉(zhuǎn)錄,翻譯,以及蛋白分泌和功能方面的情況,同時比較pIRES-VEGF165-tPA和pIRES-tPA-VEGF165質(zhì)粒在轉(zhuǎn)染和蛋白表達(dá)方面有無區(qū)別。 [材料和方法]：轉(zhuǎn)染試劑Attractene Transfection Reagent介導(dǎo)pIRES-VEGF165-tPA和pIRES-tPA-VEGF165質(zhì)粒體外瞬時轉(zhuǎn)染人臍靜脈內(nèi)皮細(xì)胞(EA.hy926),同時,以表達(dá)綠色熒光蛋白的pGFP質(zhì)粒與載體質(zhì)粒按照1：1的比例共轉(zhuǎn)染EA.hy926細(xì)胞,以綠色熒光蛋白作為示蹤標(biāo)志,計(jì)算轉(zhuǎn)染后12h,24h和48h瞬時轉(zhuǎn)染效率,設(shè)置轉(zhuǎn)染空pIRES質(zhì)粒和空白PBS作為對照；熒光實(shí)時定量RT-PCR檢測各組細(xì)胞中tPA和VEGF165的mRNA相對含量,Western blot檢測細(xì)胞中的蛋白表達(dá)情況；提取轉(zhuǎn)染后細(xì)胞上清,ELISA檢測上清液中所含有的分泌蛋白tPA和VEGF165濃度,并用MTT試驗(yàn)和tPA活性檢測試劑盒評價蛋白功能。 [結(jié)果]：通過計(jì)算轉(zhuǎn)染后綠色熒光下陽性細(xì)胞的比率發(fā)現(xiàn),轉(zhuǎn)染后約24h,陽性細(xì)胞比率最高,隨后逐漸減少,pIRES-VEGF165-tPA和pIRES-tPA-VEGF165質(zhì)粒轉(zhuǎn)染效率分別約為15.6±3.1%和16.3±2.9%,兩組之間差異無統(tǒng)計(jì)學(xué)意義(P0.05)：熒光實(shí)時定量RT-PCR和Western blot檢測發(fā)現(xiàn)轉(zhuǎn)染pIRES-VEGF165-tPA和pIRES-tPA-VEGF165質(zhì)粒組細(xì)胞中VEGF165和tPA的mRNA相對含量及蛋白含量明顯高于轉(zhuǎn)染pIRES空質(zhì)粒和空白對照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),轉(zhuǎn)染pIRES-tPA-VEGF165質(zhì)粒組細(xì)胞中tPA基因的mRNA相對含量和蛋白含量高于轉(zhuǎn)染pIRES-VEGF165-tPA組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)：而VEGF165基因方面兩組間差異無統(tǒng)計(jì)學(xué)意義(P0.05); ELISA, MTT試驗(yàn)以及tPA蛋白活性檢測結(jié)果提示轉(zhuǎn)染pIRES-VEGF165-tPA和pIRES-tPA-VEGF165質(zhì)粒組細(xì)胞上清中VEGF165和tPA的蛋白含量及功能明顯高于轉(zhuǎn)染pIRES空質(zhì)粒和空白對照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而轉(zhuǎn)染pIRES-VEGF165-tPA和pIRES-tPA-VEGF165質(zhì)粒組間差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 [結(jié)論]：所構(gòu)建的pIRES-VEGF165-tPA和pIRES-tPA-VEGF165質(zhì)粒能夠在體外轉(zhuǎn)染內(nèi)皮細(xì)胞,并指導(dǎo)合成、分泌有功能活性的tPA和VEGF165蛋白,盡管轉(zhuǎn)染pIRES-tPA-VEGF165質(zhì)粒組細(xì)胞內(nèi)目的基因的mRNA和蛋白含量略高,但不能因此認(rèn)定它與pIRES-VEGF165-tPA質(zhì)粒在功能上存在差異。 第三部分：交聯(lián)明膠微球靜電紡絲涂層滌綸材料的制備及相關(guān)理化性質(zhì)的研究 [目的]：以交聯(lián)明膠微球作為緩釋載體,通過靜電紡絲技術(shù)將其與滌綸材料有機(jī)結(jié)合,制備出能夠長期釋放質(zhì)粒DNA的復(fù)合材料,并檢測該材料在結(jié)構(gòu)穩(wěn)定性、細(xì)胞相容性、載藥率等理化性質(zhì)方面的特點(diǎn),以期將該材料用于構(gòu)建新型機(jī)械瓣膜。 [材料和方法]：將交聯(lián)明膠微球和10%聚乙烯醇(PVA)溶液按照不同的質(zhì)量容積比配制成電紡液,設(shè)置電壓,噴頭距離等參數(shù)進(jìn)行紡絲,滌綸補(bǔ)片置于接收裝置內(nèi)接受紡絲纖維,最終制得超細(xì)纖維涂層的復(fù)合滌綸材料,掃描電鏡檢測表面形態(tài)；將涂層滌綸材料小塊置入蒸餾水中,與搖床上振搖檢測材料成分的結(jié)構(gòu)穩(wěn)定性；MTT試驗(yàn)檢測涂層滌綸材料對EA.hy926細(xì)胞增殖的影響；按照質(zhì)粒/涂層滌綸材料不同質(zhì)量比將兩者混合,通過測量結(jié)合后溶液中剩余質(zhì)粒的量來計(jì)算涂層滌綸材料載藥率,同時也測量單純滌綸材料以及單純的紡絲涂層材料的載藥率作為對比；最后將結(jié)合有質(zhì)粒的涂層滌綸材料置PBS溶液中,37℃下以30rpm的速度持續(xù)振搖,間斷提取溶液樣本,以在260nm處的吸光度值作為相對含量,繪制材料緩釋質(zhì)粒的曲線。 [結(jié)果]：交聯(lián)明膠微球和PVA溶液的質(zhì)量容積比在1/100之內(nèi)時,室溫下,電壓16-20kV,接收距離10-12cm,注射速度2-4ml/h電紡可以獲得均一的超細(xì)纖維氈,交聯(lián)明膠微球散布與纖維之間,并隨著微球質(zhì)量的增加,分布密度也增加；在30-200rpm的速度振搖下涂層滌綸材料各部分結(jié)合緊密,結(jié)構(gòu)完整未見明顯脫落現(xiàn)象；MTT試驗(yàn)證實(shí)涂層滌綸補(bǔ)片干預(yù)組細(xì)胞與單純Dacron補(bǔ)片干預(yù)組細(xì)胞,以及空白對照組細(xì)胞間增殖速度相當(dāng),差異無統(tǒng)計(jì)學(xué)意義(P0.05)：當(dāng)質(zhì)粒與涂層滌綸材料質(zhì)量比在1：100左右時,材料的載藥率達(dá)到最大值75%左右,隨后進(jìn)一步加大投入的質(zhì)粒量,并不能增加載藥率；根據(jù)緩釋曲線可見涂層滌綸材料可以在體外逐漸釋放質(zhì)粒,前2天釋放速度較快,從第8天開始減慢,第12天以后釋放的總的質(zhì)粒的量呈減少趨勢。 [結(jié)論]：攜帶質(zhì)粒的交聯(lián)明膠微球可以通過靜電紡絲技術(shù)同滌綸材料進(jìn)行結(jié)合,該復(fù)合材料在流體剪切力的作用下可以保持結(jié)構(gòu)完整,它的相容性好對細(xì)胞增殖無明顯影響,能夠攜帶質(zhì)粒DNA并且在體外逐步釋放,可以用于構(gòu)建新型機(jī)械瓣膜。 第四部分：tPA-VEGF165雙基因交聯(lián)明膠微球靜電紡絲涂層滌綸材料的體外轉(zhuǎn)染實(shí)驗(yàn) [目的]：通過體外實(shí)驗(yàn),檢測攜帶質(zhì)粒的交聯(lián)明膠微球靜電紡絲涂層滌綸材料對細(xì)胞的轉(zhuǎn)染效率以及是否能夠分泌相關(guān)目的蛋白。 [材料和方法]：將攜帶質(zhì)量比為1：1的pIRES-tPA-VEGF165質(zhì)粒和pGFP質(zhì)粒的涂層滌綸材料浸入完全細(xì)胞培養(yǎng)基中,獲取其在不同時段的浸提液,然后加入微泡造影劑Sono Vue輔以頻率1MHz,強(qiáng)度2 W/cm2的超聲照射60sec,介導(dǎo)對EA.hy926細(xì)胞的瞬時轉(zhuǎn)染,24h后通過計(jì)數(shù)綠色熒光的陽性細(xì)胞計(jì)算轉(zhuǎn)染效率；超聲微泡介導(dǎo)pIRES-tPA-VEGF165質(zhì)粒單獨(dú)轉(zhuǎn)染EA.hy926細(xì)胞,并設(shè)置轉(zhuǎn)染空pIRES質(zhì)粒和空白PBS作為對照,24h后ELISA檢測細(xì)胞上清中tPA和VEGF165蛋白含量。 [結(jié)果]：轉(zhuǎn)染pIRES-tPA-VEGF165/pGFP質(zhì)粒組細(xì)胞在6天內(nèi)均有陽性細(xì)胞,其中前兩天轉(zhuǎn)染效率較高,約20%,隨后有所下降,對照組無陽性細(xì)胞出現(xiàn)：ELISA結(jié)果提示轉(zhuǎn)染了pIRES-tPA-VEGF165質(zhì)粒的各組細(xì)胞上清每個時段均能檢出tPA和VEGF165蛋白,整體趨勢與細(xì)胞轉(zhuǎn)染效率相似,而且其相對含量明顯高于同時段的對照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。轉(zhuǎn)染pIRES空質(zhì)粒和空白PBS對照組細(xì)胞有少量tPA表達(dá),兩組之間差異無統(tǒng)計(jì)學(xué)意義(P0.05),未檢出VEGF165蛋白分泌。 [結(jié)論]：攜帶質(zhì)粒的交聯(lián)明膠微球靜電紡絲涂層材料在超聲微泡的作用下,能夠在體外持續(xù)轉(zhuǎn)染內(nèi)皮細(xì)胞,并分泌相應(yīng)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，

本文編號：1738810