攜帶tPA真核表達(dá)載體PLGA納米�！曃⑴輳�(fù)合體的構(gòu)建及體外實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-08-14 17:30
【摘要】：目的：構(gòu)建攜帶有組織型纖溶酶原激活因子目的片段的真核表達(dá)載體pIRES-tPA-Dsred Express2。驗(yàn)證該質(zhì)粒轉(zhuǎn)染人臍靜脈內(nèi)皮細(xì)胞系EA.hy926是否高表達(dá)組織型纖溶酶原激活因子，其產(chǎn)物蛋白是否具有生物學(xué)活性。 方法：勾取tPA基因的CDS序列，通過(guò)基因工程技術(shù)將其插入pIRES-DsredExpress2質(zhì)粒，構(gòu)建攜帶有人組織型纖溶酶原激活因子目的片段的真核表達(dá)載體pIRES-tPA-Dsred Express2。將構(gòu)建成功的真核表達(dá)載體pIRES-tPA-Dsred Express2用脂質(zhì)體轉(zhuǎn)染試劑Lipofectamine LTX轉(zhuǎn)染人臍靜脈內(nèi)皮細(xì)胞系EA.hy926。應(yīng)用逆轉(zhuǎn)錄實(shí)時(shí)熒光定量PCR檢測(cè)轉(zhuǎn)染后48小時(shí)，細(xì)胞內(nèi)目的蛋白mRNA含量；Western Blot檢測(cè)轉(zhuǎn)染后相關(guān)蛋白表達(dá)情況；應(yīng)用ELISA技術(shù)，檢測(cè)細(xì)胞培養(yǎng)上清中tPA含量；應(yīng)用酶促反應(yīng)檢測(cè)細(xì)胞培養(yǎng)上清中人組織型纖溶酶原激活因子活性。觀(guān)察轉(zhuǎn)染pIRES-tPA-Dsred Express2載體后人臍靜脈內(nèi)皮細(xì)胞系EA.hy926對(duì)于人組織型纖溶酶原激活因子及其相關(guān)蛋白分泌及其活性隨時(shí)間的變化關(guān)系。 結(jié)果：通過(guò)將tPA目的基因片段插入pIRES-Dsred Express2質(zhì)粒，成功構(gòu)建了真核表達(dá)載體pIRES-tPA-Dsred Express2。體外轉(zhuǎn)染人臍靜脈內(nèi)皮細(xì)胞系EA.hy926后48小時(shí)可以觀(guān)察到細(xì)胞可激發(fā)紅色熒光，轉(zhuǎn)染效率為（20.7±4.2）%。逆轉(zhuǎn)錄實(shí)時(shí)定量PCR檢測(cè)顯示pIRES-tPA-Dsred Express2質(zhì)粒組其tPA，，Dsred的mRNA相對(duì)含量分別為769.21±35.11及1164.26±82.85，顯著高于對(duì)照組。WesternBlot檢測(cè)顯示，目的蛋白tPA及紅色熒光蛋白Dsred含量顯著增高。細(xì)胞上清人組織型纖溶酶原激活因子含量及人組織型纖溶酶原激活因子活性檢測(cè)顯示pIRES-tPA-Dsred Express2質(zhì)粒組（4.73±0.02） ng/hour·(105cells)，活性為(9.48±0.12)IU/hour·(105cells)，顯著高于對(duì)照組。轉(zhuǎn)染pIRES-tPA-Dsred Express2后內(nèi)皮細(xì)胞上清中的人組織型纖溶酶原激活因子濃度及活性在24小時(shí)為最高，而后呈現(xiàn)下降趨勢(shì)。人組織型纖溶酶原激活因子活性變化受纖溶酶原激活物抑制劑-1影響與其濃度變化方式不同。 結(jié)論：成功構(gòu)建了攜帶有人組織型纖溶酶原激活因子的真核表達(dá)載體pIRES-tPA-Dsred Express2。體外轉(zhuǎn)染驗(yàn)證其可正確指導(dǎo)合成及分泌組織型纖溶酶原激活因子，表現(xiàn)出顯著纖溶活性，為后續(xù)實(shí)驗(yàn)奠定了實(shí)驗(yàn)基礎(chǔ)。 目的：通過(guò)超聲微泡介導(dǎo)的基因治療技術(shù)將攜帶有組織型纖溶酶原激活因子基因的質(zhì)粒pIRES-tPA-DsRed-Express2-脂質(zhì)體復(fù)合體體外轉(zhuǎn)染內(nèi)皮細(xì)胞，使內(nèi)皮細(xì)胞高表達(dá)組織型纖溶酶原激活因子，觀(guān)察超聲介導(dǎo)的基因治療技術(shù)對(duì)于內(nèi)皮細(xì)胞質(zhì)粒轉(zhuǎn)染的效果。 方法：薄膜水化制備全氟丙烷超聲微泡。使用全氟丙烷微泡介導(dǎo)pIRES-tPA-DsRed-Express2-脂質(zhì)體復(fù)合體轉(zhuǎn)染人臍靜脈內(nèi)皮細(xì)胞系EA.hy926細(xì)胞。通過(guò)熒光計(jì)數(shù)，計(jì)算細(xì)胞轉(zhuǎn)染效率。應(yīng)用逆轉(zhuǎn)錄實(shí)時(shí)熒光定量PCR檢測(cè)轉(zhuǎn)染后48小時(shí)，細(xì)胞的目的蛋白mRNA含量；應(yīng)用ELISA技術(shù)，檢測(cè)上清中tPA含量及活性。 結(jié)果：制備的全氟丙烷超聲微泡平均大小為（3.5±1.4）μm。微泡濃度為（3.3±1.2）×108/ml。zeta電位為（-2.2±1.5）mV。制備后的12小時(shí)內(nèi)性質(zhì)穩(wěn)定。轉(zhuǎn)染后48小時(shí)，超聲微泡介導(dǎo)組（UM+LTX）轉(zhuǎn)染效率為（27.3±3.6）%，Lipofectamine LTX轉(zhuǎn)染組（LTX）轉(zhuǎn)染效率為（20.6±2.0）%。逆轉(zhuǎn)錄實(shí)時(shí)熒光定量PCR檢測(cè)顯示UM+LTX組及LTX組，目的蛋白tPA的mRNA的相對(duì)含量分別為（953.15±92.77）和（721.32±68.31），具有顯著性差異。熒光蛋白Dsred的mRNA相對(duì)含量分別為（1191.22±109.31）和（1092.15±102.71）。UM+LTX組其細(xì)胞上清中的tPA含量及tPA活性均較LTX顯著升高。 結(jié)論：成功制備了含有全氟丙烷的超聲微泡。通過(guò)超聲微泡介導(dǎo)質(zhì)粒-脂質(zhì)體復(fù)合體轉(zhuǎn)染內(nèi)皮細(xì)胞EA.hy926，進(jìn)一步提高了質(zhì)粒-脂質(zhì)體復(fù)合體轉(zhuǎn)染內(nèi)皮細(xì)胞的轉(zhuǎn)染效率，從而提高目的蛋白組織型纖溶酶原激活因子的表達(dá)分泌量，提高了纖溶活性。 目的：構(gòu)建攜帶有可表達(dá)組織型纖維溶酶激活因子質(zhì)粒的PLGA納米粒-超聲微泡復(fù)合體。檢測(cè)其理化性質(zhì)，體外緩釋方式，細(xì)胞毒效應(yīng)及其在超聲介導(dǎo)下對(duì)于細(xì)胞攝取納米粒的影響。 方法：應(yīng)用雙次乳化法制備攜帶有pIRES-tPA-DsRed Express2質(zhì)粒的PLGA納米粒；應(yīng)用薄膜水化超聲法制備陽(yáng)離子超聲微泡；兩者通過(guò)靜電吸附的方式形成PLGA納米粒-超聲微泡復(fù)合體。檢測(cè)PLGA納米粒的載藥量，PLGA納米粒-超聲微泡復(fù)合體質(zhì)粒載量；檢測(cè)納米粒及納米粒-超聲微泡復(fù)合體細(xì)胞毒性；檢測(cè)PLGA納米粒及PLGA納米粒-超聲微泡復(fù)合體體外緩釋質(zhì)粒的方式；檢測(cè)其在超聲輻照介導(dǎo)的微泡解構(gòu)時(shí)介導(dǎo)體外細(xì)胞吞噬納米粒的能力。 結(jié)果：自制的納米粒其粒徑為（217.2±2.2）nm，zeta電位為（-15.24±0.83）mV。微泡平均大小為（3.2±1.5）μm，Zeta電位為（13.66±2.05）mV。微泡濃度為（4.3±1.1）×108/ml。納米粒-超聲微泡復(fù)合體其平均大小為（4.6±1.7）μm。zeta電位為（2.23±1.45）mV。濃度為（3.0±1.3）×108/ml。1mgPLGA納米粒載有質(zhì)粒（42.3±2.1）μg。1ml納米粒-超聲微泡復(fù)合體中帶有質(zhì)粒（20.5±2.7）μg。納米粒及納米粒-超聲微泡復(fù)合體均表現(xiàn)為在起始段短時(shí)內(nèi)質(zhì)�？焖籴尫�，后呈現(xiàn)穩(wěn)定釋放的趨勢(shì)。到達(dá)第7天時(shí)，釋放量達(dá)到其總量的（57±3）%。納米粒及納米粒-超聲微泡復(fù)合體均未見(jiàn)明顯細(xì)胞毒性效應(yīng)，僅最高濃度組見(jiàn)輕度的細(xì)胞活性減少。PLGA納米粒組及PLGA納米粒-超聲微泡復(fù)合體組均可見(jiàn)大量的納米粒被吞噬。PLGA納米粒-超聲微泡復(fù)合體組在超聲輻照介導(dǎo)下具有更高的細(xì)胞攝取效率。 結(jié)論：所構(gòu)建的納米粒-超聲微泡復(fù)合體顯示出較好的緩釋效果，較低的細(xì)胞毒性，在超聲輻照介導(dǎo)下可增強(qiáng)納米粒吞噬效率。
[Abstract]:AIM: To construct an eukaryotic expression vector pIRES-tPA-Dsred Express2 carrying the target fragment of tissue plasminogen activator (TPA). To verify whether the plasmid transfected human umbilical vein endothelial cell line EA.hy926 overexpresses TPA and whether the product protein has biological activity.
METHODS: CDS sequence of tPA gene was cloned and inserted into pIRES-Dsred Express2 plasmid by genetic engineering technology to construct eukaryotic expression vector pIRES-tPA-Dsred Express2 carrying the target fragment of human tissue plasminogen activator. Tamine LTX transfected human umbilical vein endothelial cell line EA.hy926.Reverse transcription real-time fluorescence quantitative PCR was used to detect the content of target protein mRNA 48 hours after transfection; Western Blot was used to detect the expression of related protein after transfection; ELISA was used to detect the content of tPA in cell culture supernatant; and enzyme-catalyzed reaction was used to detect the content of tPA in cell culture supernatant. The secretion and activity of human tissue plasminogen activator and its related proteins by human umbilical vein endothelial cell line EA.hy926 after transfection with pIRES-tPA-Dsred Express2 vector were observed.
Results: The eukaryotic expression vector pIRES-tPA-Dsred Express2 was successfully constructed by inserting the target gene fragment of tPA into pIRES-Dsred Express2 plasmid. After transfection into human umbilical vein endothelial cell line EA.hy926 in vitro, red fluorescence was observed 48 hours after transfection, and the transfection efficiency was (20.7 4.2)%. Reversed transcription real-time quantitative PCR showed that pIRE could be transfected into human umbilical vein Endo The relative contents of tPA and Dsred mRNA in S-tPA-Dsred Express 2 plasmid group were 769.21 (+ 35.11) and 1164.26 (+ 82.85) respectively, which were significantly higher than those in the control group. Western Blot assay showed that the contents of tPA and red fluorescent protein Dsred were significantly increased. The activity of pIRES-tPA-Dsred Express2 plasmid group (4.73.02) ng/hour ((105 cells) was significantly higher than that of control group (9.48.12) IU/hour ((105 cells). After transfection of pIRES-tPA-Dsred Express2, the concentration and activity of human tissue plasminogen activator in the supernatant of endothelial cells were the highest at 24 hours, and then decreased. The change of human tissue plasminogen activator activity is affected by plasminogen activator inhibitor-1 in a different way than its concentration.
CONCLUSION: The eukaryotic expression vector pIRES-tPA-Dsred Express2 carrying human tissue plasminogen activator was successfully constructed. The results of in vitro transfection showed that it could guide the synthesis and secretion of tissue plasminogen activator and showed significant fibrinolytic activity, which laid the experimental foundation for the follow-up experiments.
Objective: To investigate the effect of ultrasound-mediated gene therapy on endothelial cells by transfecting plasmid pIRES-tPA-DsRed-Express2-liposome complex carrying tissue plasminogen activator gene into endothelial cells. Plasmid transfection.
METHODS: Perfluoropropane ultrasound microbubbles were prepared by membrane hydration. The pIRES-tPA-DsRed-Express2-liposome complex was used to transfect human umbilical vein endothelial cell line EA.hy926 cells. RNA content; ELISA technology was applied to detect the content and activity of tPA in supernatant.
Results: The average size of perfluoropropane ultrasound microbubbles was (3.5 +1.4) micron. The concentration of microbubbles was (3.3 +1.2)*108/ml. Zeta potential was (-2.2 +1.5) mV. The transfection efficiency was (27.3 +3.6)% in UM + LTX group and (20.6 +2.0)% in Lipofectamine LTX transfection group within 12 hours after transfection. Reverse transcription real-time fluorescence quantitative PCR assay showed that the relative content of tPA mRNA in the supernatant of UM+LTX group and LTX group was (953.15+92.77) and (721.32+68.31), respectively. The relative content of fluorescent protein Dsred mRNA was (1191.22+109.31) and (1092.15+102.71), respectively. Sex was significantly higher than that of LTX.
CONCLUSION: Perfluoropropane-containing ultrasound microbubbles were successfully prepared. The transfection efficiency of plasmid-liposome complex into endothelial cells EA.hy926 was further improved by ultrasound microbubbles, and the expression and secretion of tissue-type plasminogen activator (TIPA) of the target protein were increased. Dissolve activity.
OBJECTIVE: To construct PLGA nanoparticles-ultrasound microbubble complexes with plasmids expressing tissue-type plasmin activator (TIPA) and to investigate its physicochemical properties, in vitro sustained release mode, cytotoxic effect and its effect on cell uptake of nanoparticles under ultrasound.
METHODS: PLGA nanoparticles with plasmid pIRES-tPA-DsRed Express 2 were prepared by double emulsification method, cationic ultrasound microbubbles were prepared by film hydration ultrasonic method, and PLGA nanoparticles-ultrasound microbubbles were formed by electrostatic adsorption. The drug loading of PLGA nanoparticles and PLGA nanoparticles-ultrasound microbubbles composite particles were measured. The cytotoxicity of nanoparticles and nanoparticles-ultrasound microbubble complex, the mode of slow-release plasmid of PLGA nanoparticles and PLGA nanoparticles-ultrasound microbubble complex in vitro, and the ability of extracellular cells to phagocytosis nanoparticles during ultrasound-induced microbubble decomposition were examined.
Results: The size of the nanoparticles was 217.2 (+ 2.2) nm, the zeta potential was (- 15.24 (+ 0.83) mV. The average size of the microbubbles was (3.2 (+ 1.5) microbubbles, and the Zeta potential was (13.66 (+ 2.05) mV. The concentration of the microbubbles was (4.3 (+ 1.1) * 108 / ml. The average size of the nanoparticles-ultrasonic microbubbles was (4.6 (+ 1.7) microbubbles. 1 mg PLGA nanoparticles were loaded with plasmids (42.3 There was no significant cytotoxic effect in both nanoparticles and ultrasound microbubble complexes, but only slight decrease in cell activity in the highest concentration group. A large number of nanoparticles were phagocytized in both PLGA nanoparticles and PLGA nanoparticles-ultrasound microbubble complexes. PLGA nanoparticles-ultrasound microbubble complexes had higher cell uptake under ultrasound irradiation. Take efficiency.
CONCLUSION: The nanoparticles-ultrasound microbubbles composite exhibits better sustained release effect and lower cytotoxicity, and can enhance the phagocytic efficiency of nanoparticles under ultrasound irradiation.
【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(lèi)號(hào)】：R392
本文編號(hào)：2183591
[Abstract]:AIM: To construct an eukaryotic expression vector pIRES-tPA-Dsred Express2 carrying the target fragment of tissue plasminogen activator (TPA). To verify whether the plasmid transfected human umbilical vein endothelial cell line EA.hy926 overexpresses TPA and whether the product protein has biological activity.
METHODS: CDS sequence of tPA gene was cloned and inserted into pIRES-Dsred Express2 plasmid by genetic engineering technology to construct eukaryotic expression vector pIRES-tPA-Dsred Express2 carrying the target fragment of human tissue plasminogen activator. Tamine LTX transfected human umbilical vein endothelial cell line EA.hy926.Reverse transcription real-time fluorescence quantitative PCR was used to detect the content of target protein mRNA 48 hours after transfection; Western Blot was used to detect the expression of related protein after transfection; ELISA was used to detect the content of tPA in cell culture supernatant; and enzyme-catalyzed reaction was used to detect the content of tPA in cell culture supernatant. The secretion and activity of human tissue plasminogen activator and its related proteins by human umbilical vein endothelial cell line EA.hy926 after transfection with pIRES-tPA-Dsred Express2 vector were observed.
Results: The eukaryotic expression vector pIRES-tPA-Dsred Express2 was successfully constructed by inserting the target gene fragment of tPA into pIRES-Dsred Express2 plasmid. After transfection into human umbilical vein endothelial cell line EA.hy926 in vitro, red fluorescence was observed 48 hours after transfection, and the transfection efficiency was (20.7 4.2)%. Reversed transcription real-time quantitative PCR showed that pIRE could be transfected into human umbilical vein Endo The relative contents of tPA and Dsred mRNA in S-tPA-Dsred Express 2 plasmid group were 769.21 (+ 35.11) and 1164.26 (+ 82.85) respectively, which were significantly higher than those in the control group. Western Blot assay showed that the contents of tPA and red fluorescent protein Dsred were significantly increased. The activity of pIRES-tPA-Dsred Express2 plasmid group (4.73.02) ng/hour ((105 cells) was significantly higher than that of control group (9.48.12) IU/hour ((105 cells). After transfection of pIRES-tPA-Dsred Express2, the concentration and activity of human tissue plasminogen activator in the supernatant of endothelial cells were the highest at 24 hours, and then decreased. The change of human tissue plasminogen activator activity is affected by plasminogen activator inhibitor-1 in a different way than its concentration.
CONCLUSION: The eukaryotic expression vector pIRES-tPA-Dsred Express2 carrying human tissue plasminogen activator was successfully constructed. The results of in vitro transfection showed that it could guide the synthesis and secretion of tissue plasminogen activator and showed significant fibrinolytic activity, which laid the experimental foundation for the follow-up experiments.
Objective: To investigate the effect of ultrasound-mediated gene therapy on endothelial cells by transfecting plasmid pIRES-tPA-DsRed-Express2-liposome complex carrying tissue plasminogen activator gene into endothelial cells. Plasmid transfection.
METHODS: Perfluoropropane ultrasound microbubbles were prepared by membrane hydration. The pIRES-tPA-DsRed-Express2-liposome complex was used to transfect human umbilical vein endothelial cell line EA.hy926 cells. RNA content; ELISA technology was applied to detect the content and activity of tPA in supernatant.
Results: The average size of perfluoropropane ultrasound microbubbles was (3.5 +1.4) micron. The concentration of microbubbles was (3.3 +1.2)*108/ml. Zeta potential was (-2.2 +1.5) mV. The transfection efficiency was (27.3 +3.6)% in UM + LTX group and (20.6 +2.0)% in Lipofectamine LTX transfection group within 12 hours after transfection. Reverse transcription real-time fluorescence quantitative PCR assay showed that the relative content of tPA mRNA in the supernatant of UM+LTX group and LTX group was (953.15+92.77) and (721.32+68.31), respectively. The relative content of fluorescent protein Dsred mRNA was (1191.22+109.31) and (1092.15+102.71), respectively. Sex was significantly higher than that of LTX.
CONCLUSION: Perfluoropropane-containing ultrasound microbubbles were successfully prepared. The transfection efficiency of plasmid-liposome complex into endothelial cells EA.hy926 was further improved by ultrasound microbubbles, and the expression and secretion of tissue-type plasminogen activator (TIPA) of the target protein were increased. Dissolve activity.
OBJECTIVE: To construct PLGA nanoparticles-ultrasound microbubble complexes with plasmids expressing tissue-type plasmin activator (TIPA) and to investigate its physicochemical properties, in vitro sustained release mode, cytotoxic effect and its effect on cell uptake of nanoparticles under ultrasound.
METHODS: PLGA nanoparticles with plasmid pIRES-tPA-DsRed Express 2 were prepared by double emulsification method, cationic ultrasound microbubbles were prepared by film hydration ultrasonic method, and PLGA nanoparticles-ultrasound microbubbles were formed by electrostatic adsorption. The drug loading of PLGA nanoparticles and PLGA nanoparticles-ultrasound microbubbles composite particles were measured. The cytotoxicity of nanoparticles and nanoparticles-ultrasound microbubble complex, the mode of slow-release plasmid of PLGA nanoparticles and PLGA nanoparticles-ultrasound microbubble complex in vitro, and the ability of extracellular cells to phagocytosis nanoparticles during ultrasound-induced microbubble decomposition were examined.
Results: The size of the nanoparticles was 217.2 (+ 2.2) nm, the zeta potential was (- 15.24 (+ 0.83) mV. The average size of the microbubbles was (3.2 (+ 1.5) microbubbles, and the Zeta potential was (13.66 (+ 2.05) mV. The concentration of the microbubbles was (4.3 (+ 1.1) * 108 / ml. The average size of the nanoparticles-ultrasonic microbubbles was (4.6 (+ 1.7) microbubbles. 1 mg PLGA nanoparticles were loaded with plasmids (42.3 There was no significant cytotoxic effect in both nanoparticles and ultrasound microbubble complexes, but only slight decrease in cell activity in the highest concentration group. A large number of nanoparticles were phagocytized in both PLGA nanoparticles and PLGA nanoparticles-ultrasound microbubble complexes. PLGA nanoparticles-ultrasound microbubble complexes had higher cell uptake under ultrasound irradiation. Take efficiency.
CONCLUSION: The nanoparticles-ultrasound microbubbles composite exhibits better sustained release effect and lower cytotoxicity, and can enhance the phagocytic efficiency of nanoparticles under ultrasound irradiation.
【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(lèi)號(hào)】：R392
【共引文獻(xiàn)】
相關(guān)期刊論文 前6條
1 黃健宏;王茂生;黃健;;心臟瓣膜置換術(shù)后機(jī)械瓣功能不全再次手術(shù)[J];當(dāng)代醫(yī)學(xué);2011年26期
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