本文關(guān)鍵詞：組織型纖溶酶原激活劑基因逆轉(zhuǎn)錄病毒載體靶向溶栓效應(yīng)　出處：《華中科技大學》2008年博士論文　論文類型：學位論文

  更多相關(guān)文章： 逆轉(zhuǎn)錄病毒科 組織型纖溶酶原激活劑 遺傳載體 包裝細胞 ECUV304 心肌細胞 tPA 逆轉(zhuǎn)錄病毒科 組織型纖溶酶原激活劑 基因治療 血栓

【摘要】： 第一部分pLEGFP-N1-tPA逆轉(zhuǎn)錄病毒載體構(gòu)建、鑒定及包裝細胞PT67/pLEGFP-N1-tPA純系培育 目的構(gòu)建含人組織型纖溶酶原激活劑(tissue-type plasminogen activator, tPA)基因增強型綠色熒光蛋白(enhanced green fluorescent protein, EGFP)基因逆轉(zhuǎn)錄病毒載體及產(chǎn)高滴度病毒的純包裝細胞系。 方法用PCR方法擴增目的基因tPA,定向克隆入逆轉(zhuǎn)錄病毒載體(pLEGFP-N1)中,酶切反應(yīng)、PCR及DNA測序鑒定重組逆轉(zhuǎn)錄病毒載體pLEGFP-N1-tPA;在基因轉(zhuǎn)染試劑SofastTM介導(dǎo)下將pLEGFP-N1-tPA轉(zhuǎn)入包裝細胞PT67,經(jīng)相應(yīng)的抗生素篩選、標記、顯微移出,培育出全EGFP純包裝細胞系。用NIH3T3細胞測定病毒滴度。 結(jié)果經(jīng)限制性酶切分析、DNA序列分析、EGFP的表達證實pLEGFP-N1-tPA中含有tPA基因。轉(zhuǎn)染有pLEGFP-N1-tPA的純PT67細胞產(chǎn)病毒滴度為1×10~7CFU/ml。 結(jié)論成功構(gòu)建了pLEGFP-N1-tPA載體和產(chǎn)高滴度病毒純包裝細胞系,為tPA局部靶向治療的理想人工心瓣的研究和應(yīng)用奠定了實驗基礎(chǔ)。 第二部分pLEGFP-N1-tPA感染ECUV304和心肌細胞,ECUV304/ pLEGFP-N1-tPA建純系 目的觀察pLEGFP-N1-tPA感染ECUV304和心肌細胞后,ECUV304/pLEGFP-N1-tPA純系上清和感染后心肌細胞上清在體外血栓模型中溶栓情況及tPA表達情況。 方法pLEGFP-N1-tPA感染ECUV304細胞,單個強表達EGFP的ECUV304細胞→成簇→顯微移出→培育→成純系。建體外血漿平板血栓模型(用人全血),純系ECUV304/pLEGFP-N1-tPA培養(yǎng)上清加入模型中,設(shè)對照組,觀察溶解血栓情況,同時椐蚓激酶活性算出tPA活性。ELISA測出tPA含量。Western blot檢測tPA。原代1-4d乳鼠心肌細胞培養(yǎng),感染pLEGFP-N1-tPA,培養(yǎng)上清加入血漿平板血栓模型,設(shè)對照組,觀察溶解血栓情況,算出tPA活性,測出tPA含量。Western blot檢測tPA。 結(jié)果轉(zhuǎn)導(dǎo)有pLEGFP-N1-tPA的內(nèi)皮細胞和心肌細胞培養(yǎng)細胞上清在體外血栓模型中有大的溶解圈。ECUV304/pLEGFP-N1-tPA純系上清tPA活性是502.16u/10~6 cells/24h,tPA含量是716.27ng/10~6 cells/24h,Western blot檢出外源tPA條帶。心肌細胞/pLEGFP-N1-tPA上清tPA活性是464.27u/10~6 cells/24h,tPA含量是607.34ng/10~6 cells/24h,Western blot亦檢出外源tPA條帶。 結(jié)論成功建立ECUV304/pLEGFP-N1-tPA純系,純系上清有明顯溶栓作用,tPA活性及含量均高,有外源tPA條帶。心肌細胞/pLEGFP-N1-tPA上清有明顯溶栓作用,tPA活性及含量均高,有外源tPA條帶。 第三部分在體研究特氟隆(Dacron)片移植入兔下腔靜脈,Dacron片周圍局部轉(zhuǎn)導(dǎo)pLEGFP-N1-tPA 目的探討組織型纖溶酶原激活劑(tPA)基因局部轉(zhuǎn)導(dǎo)對特氟隆(Dacron)片(與機械瓣瓣環(huán)同質(zhì))的溶血栓作用。 方法70只兔建立下腔靜脈內(nèi)Dacron片植入血栓模型,隨機分為pLEGFP-N1-tPA治療組(n=30)、pLEGFP-N1空載體對照組(n=20)、空白對照組(n=20),局部基因轉(zhuǎn)導(dǎo),于術(shù)后2d、75d各組一半動物取材(6個亞組A1、B1、C1、A2、B2、C2),聚光共聚焦(confocal)觀察所取靜脈增強型綠色熒光蛋白(EGFP)表達,體視鏡和電鏡觀察Dacron片表面血栓情況,免疫印跡(Western blot)、血漿平板和酶聯(lián)免疫吸附實驗(enzyme linked immunosorbent assay, ELISA)分別檢測所取靜脈tPA表達、溶栓、活性及含量變化。 結(jié)果術(shù)后2d和75d取材,治療組所取靜脈,confocal均觀察到多而強的EGFP表達,pLEGFP-N1對照組也有EGFP表達,但空白對照組無EGFP表達。70個Dacron片加未植入組10個Dacron片共80個,在體視鏡(×160倍)和電鏡(×500倍)下觀察,未植入組和治療組Dacron片表面均無血栓,對照組表面均有血栓。治療組所取靜脈Western blot檢測均有外源tPA表達,對照組未檢測到。血漿平板顯示:治療組均有大的溶解圈,有明顯溶栓作用,對照組沒有;根據(jù)蚓激酶計算出的纖溶活性顯示:治療組術(shù)后2d和75d所取靜脈tPA活性分別為529.62±9.05u/g、537.50±12.45u/g,兩時間點比較,差異顯著性(P0.05)。術(shù)后2d和75d治療組、兩對照組中6個亞組所取靜脈tPA含量分別為(A_1 737.64±13.19)、(B_1 29.88±5.61)、(C_1 28.71±5.49)、(A_2 742.87±10.56)、(B_2 32.03±6.26)、(C_2 31.34±5.63)ng/g,治療組明顯高于對照組(P0.01);治療組中兩時間點比較,tPA含量無顯著差異(P0.05)。 結(jié)論pLEGFP-N1-tPA局部基因轉(zhuǎn)導(dǎo)能有效阻止外源移植物表面血栓形成,為研究和應(yīng)用tPA基因瓣膜奠定了基礎(chǔ)。
[Abstract]:The first part of pLEGFP-N1-tPA retroviral vector construction, identification and culture of PT67/pLEGFP-N1-tPA pure line culture
Objective to construct a retroviral vector containing tissue-type plasminogen activator (tPA) gene enhanced green fluorescent protein (enhanced green fluorescent protein, EGFP) gene and a pure package cell line producing high titer virus.
Methods tPA gene was amplified by PCR, cloned into retroviral vector (pLEGFP-N1), enzyme digestion and DNA sequencing, PCR pLEGFP-N1-tPA recombinant retroviral vector; gene transfection reagent in SofastTM mediated pLEGFP-N1-tPA into PT67 packaging cells by corresponding antibiotic screening, marking, micro removed, cultivate full EGFP pure packaging cell line. The virus titer was determined by NIH3T3 cells.
Results restriction endonuclease analysis, DNA sequence analysis and EGFP expression confirmed that pLEGFP-N1-tPA contained tPA gene. The titer of pure PT67 cells transfected with pLEGFP-N1-tPA was 1 * 10~7CFU/ml..
Conclusion pLEGFP-N1-tPA vector and highly titre virus pure packaging cell line were successfully constructed, which laid an experimental foundation for the research and application of tPA ideal targeted artificial heart valve.
The second part of pLEGFP-N1-tPA is infected with ECUV304 and cardiomyocytes, and the pure line of ECUV304/ pLEGFP-N1-tPA
Objective To observe thrombolysis and tPA expression in pure thrombolytic system and supernatant of myocardium after infection of ECUV304 and cardiomyocytes by pLEGFP-N1-tPA in vitro thrombus model of ECUV304/pLEGFP-N1-tPA.
Methods pLEGFP-N1-tPA infection of ECUV304 cells, a strong expression of EGFP in ECUV304 cells, and to nurture and remove the micro clusters into pure lines. Built in vitro plasma plate thrombosis model (with human blood), pure ECUV304/pLEGFP-N1-tPA culture supernatant was added into the model, the control group, observation of thrombus dissolution, at the same time according to the activity of LK calculate the tPA activity of.ELISA tPA.Western blot to detect tPA. content measured in primary cultured rat myocardial cells infected with 1-4d, pLEGFP-N1-tPA, supernatant into plasma plate thrombosis model, control group, observation of dissolving thrombus, calculate the activity of tPA, content of tPA.Western blot tPA. detection measure
Results transduction of endothelial cells and myocardial cells pLEGFP-N1-tPA cell culture supernatant in vitro thrombosis model in active dissolution circle.ECUV304/pLEGFP-N1-tPA pure tPA is 502.16u/10~6 supernatant cells/24h, tPA content is 716.27ng/10~6 cells/24h, Western blot in the detection of exogenous tPA bands. The myocardial cells of /pLEGFP-N1-tPA supernatant tPA activity is 464.27u/10~6 cells/24h, tPA 607.34ng/10~6 cells/24h Western content. Blot detection of exogenous tPA bands.
Conclusion ECUV304/pLEGFP-N1-tPA pure line was successfully established, pure line supernatant had obvious thrombolytic effect, tPA activity and content were high, and exogenous tPA band. Cardiomyocyte /pLEGFP-N1-tPA supernatant had obvious thrombolytic effect, tPA activity and content were high, and exogenous tPA band.
In the third part of the body of Teflon (Dacron) graft into the rabbit inferior vena cava, Dacron around the local transfer of pLEGFP-N1-tPA
Objective to investigate the tissue plasminogen activator (tPA) gene transduction on local Teflon (Dacron) tablets (with mechanical valve ring homogeneous) thrombolytic effect.
Methods 70 rabbits were established in Dacron implanted inferior vena cava thrombosis model, were randomly divided into pLEGFP-N1-tPA treatment group (n=30), pLEGFP-N1 empty vector control group (n=20), control group (n=20), local gene transduction, postoperative 2D, 75D groups were half animal (6 sub groups A1, B1, C1 A2, B2, C2), spotlight, confocal (confocal) to observe the vein of enhanced green fluorescent protein (EGFP) expression, body mirror and electron microscopic observation of surface thrombosis Dacron slices, immunoblotting (Western blot), plasma panel and enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) were detected respectively. The expression of tPA in vein, thrombolysis, changes of activity and content.
Results after 2D and 75D treatment group were taken from vein, confocal were observed and the strong expression of EGFP, pLEGFP-N1 groups had EGFP expression, but the control group the expression of EGFP.70 Dacron tablets and non implanted group 10 Dacron tablets a total of 80, in the stereoscope (x 160) and the electric mirror (x 500) observed under the non implanted group and treatment group Dacron surface showed no thrombosis, the surface of the control group were taken venous thrombosis. The treatment group Western blot detection showed that exogenous tPA expression was not detected in control group. The plasma panel display: the treatment group had a large circle of dissolution, obvious thrombolysis no, the control group; calculated according to Lumbrokinase fibrinolytic activity showed that the treatment group postoperative 2D and 75D venous tPA activity were 529.62 + 9.05u/g, 537.50 + 12.45u/g, two time points, significant difference (P0.05). Group 2D and 75D after treatment, two and 6 in control group a subgroup of the T vein The content of PA was (A_1 737.64 + 13.19), (B_1 29.88 + 5.61), (C_1 28.71 + 5.49), (A_2 742.87 + 10.56), (B_2 32.03 32.03 6.26), (C_2 31.34, 31.34) ng/g, the treatment group was significantly higher than that of the control group (P0.01), and the tPA content in the treatment group had no significant difference (P0.05).
Conclusion pLEGFP-N1-tPA local gene transduction can effectively prevent the formation of external graft surface thrombosis, which lays the foundation for the study and application of tPA gene valve.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R346

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