發(fā)布時(shí)間：2018-06-25 20:47

  本文選題：高分子脂質(zhì)體 + 基因轉(zhuǎn)染　； 參考：《天津醫(yī)科大學(xué)》2012年博士論文

【摘要】：目的： 對(duì)載質(zhì)粒DNA的高分子脂質(zhì)體進(jìn)行性能分析,優(yōu)化其轉(zhuǎn)染條件；觀察其細(xì)胞毒性及轉(zhuǎn)染不同細(xì)胞的轉(zhuǎn)染效率；研究高分子脂質(zhì)體作為基因載體在氧誘導(dǎo)視網(wǎng)膜病變動(dòng)物模型中對(duì)視網(wǎng)膜新生血管的抑制作用,體現(xiàn)其獨(dú)特的穿膜能力和緩釋功能,為將來(lái)制備靶向性藥物/基因共載多功能給藥系統(tǒng)提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。 方法： 1.課題組自行合成羧甲基殼聚糖十八烷基季銨鹽(OQLCS/chol)納米粒子,將穿膜肽(Tat)與其表面的功能基團(tuán)連接制備出兼具跨膜、長(zhǎng)循環(huán)功能的高分子脂質(zhì)體,并對(duì)其表征進(jìn)行檢測(cè)。研究該高分子脂質(zhì)體對(duì)質(zhì)粒DNA在不同質(zhì)量比、不同作用時(shí)間及血清存在與否條件下的包載效能,制定最優(yōu)的轉(zhuǎn)染條件；采用高速離心法進(jìn)行體外釋放動(dòng)力學(xué)實(shí)驗(yàn),繪制體外釋放曲線(xiàn)；利用CCK-8法檢測(cè)不同濃度高分子脂質(zhì)體在不同時(shí)間分別對(duì)視網(wǎng)膜色素上皮(RPE)細(xì)胞的毒性,找到適于細(xì)胞轉(zhuǎn)染的安全濃度；在優(yōu)化條件下轉(zhuǎn)染RPE細(xì)胞,同時(shí)以商品化的Lipofectamine2000做對(duì)照,比較兩者的轉(zhuǎn)染效率。 2.建立缺氧條件下RPE細(xì)胞模型,轉(zhuǎn)染48時(shí)后收集細(xì)胞上清通過(guò)ELISA法檢測(cè)各組VEGF量的變化,細(xì)胞爬片后行免疫組織化學(xué)染色分析各組VEGF蛋白的表達(dá),采用Real-time PCR檢測(cè)各組RPE細(xì)胞內(nèi)VEGF mRNA的變化。 3.采用改良的Smith's法建立氧誘導(dǎo)視網(wǎng)膜病變C57BL/6J小鼠模型,共95只,另有27只小鼠于正常氧環(huán)境中飼養(yǎng)。P11天時(shí)行小鼠玻璃體腔內(nèi)注藥,不同時(shí)間點(diǎn),于正常打藥組中隨機(jī)選取兩只小鼠行視網(wǎng)膜鋪片,熒光顯微鏡下觀察GFP的表達(dá)情況；各組中抽取兩只小鼠FITC-dextran心臟灌注后行視網(wǎng)膜鋪片觀察視網(wǎng)膜血管形態(tài)的變化；分別于各組隨機(jī)抽取兩只小鼠,行HE染色,統(tǒng)計(jì)突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核數(shù)；于各組隨機(jī)抽取的兩只小鼠做5gm冰凍切片,DAPI核染后熒光顯微鏡下觀察,以反映高分子脂質(zhì)體攜帶質(zhì)粒DNA的穿膜能力。 結(jié)果： 1.納米粒平均粒度為134nm,Zeta電位為+39.64mV,包載質(zhì)粒后為236nm。透射電鏡下納米粒呈大小均勻的圓形粒子。體外釋放實(shí)驗(yàn)顯示載質(zhì)粒DNA的高分子脂質(zhì)體在體外最初5天為突釋相,約有70%的質(zhì)粒釋放；此后至第14天呈穩(wěn)態(tài)緩釋。該高分子脂質(zhì)體毒性較低,最高安全濃度為20ug/mL。 2.通過(guò)電泳阻滯實(shí)驗(yàn),篩選出高分子脂質(zhì)體與質(zhì)粒DNA質(zhì)量比≥2：1時(shí)、混合時(shí)間為30-40min、無(wú)血清存在的培養(yǎng)液為最優(yōu)轉(zhuǎn)染條件；并以此條件轉(zhuǎn)染RPE、Hela及RF/16,對(duì)RPE的轉(zhuǎn)染率約為69%,對(duì)Hela細(xì)胞的轉(zhuǎn)染效率約為52%,對(duì)RF/16轉(zhuǎn)染效率約為80%。 3.對(duì)缺氧后的RPE轉(zhuǎn)染后觀察,高分子脂質(zhì)體組和Lipo組均能有效地將質(zhì)粒DNA轉(zhuǎn)染入細(xì)胞并表達(dá)。48小時(shí)后收集細(xì)胞上清,行ELISA法檢測(cè)各組VEGF量,高分脂質(zhì)體組和Lipo組中VEGF含量較模型組明顯降低,差異均有顯著性(P0.01),其中Lipo組抑制程度略較高分脂質(zhì)體組大,但差異不具有顯著性(P=0.10)。72小時(shí)后行細(xì)胞免疫組化顯示細(xì)胞內(nèi)VEGF的變化,Lipo組和高分脂質(zhì)體組對(duì)VEGF表達(dá)產(chǎn)生明顯的抑制作用,胞漿內(nèi)棕色著染明顯淡薄,OD值相比高分子脂質(zhì)體組略低于Lipo組(P=0.443)。Real-time PCR結(jié)果顯示轉(zhuǎn)染后48小時(shí),高分脂質(zhì)體組和Lipo組VEGF mRNA的變化與ELISA結(jié)果相似。 4.成功建立了氧誘導(dǎo)視網(wǎng)膜病變小鼠模型。玻璃體腔內(nèi)注射后可見(jiàn)注藥后第1天視網(wǎng)膜內(nèi)即有GFP表達(dá),至第6天達(dá)到高峰,第11天時(shí)在高分子脂質(zhì)體組仍能看到GFP表達(dá),Lipo組不明顯。FITC(?)心臟灌注后顯示視網(wǎng)膜無(wú)灌注區(qū)及新生血管范圍在高分子脂質(zhì)體組及Lipo組明顯改善,效果相當(dāng)。HE染色后行突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核數(shù),結(jié)果表明P17天時(shí)高分子脂質(zhì)體組及Lipo組均能有效抑制新生血管生成,P22天時(shí),高分子脂質(zhì)體效果仍能顯現(xiàn),新生血管內(nèi)皮細(xì)胞核數(shù)較Lipo組少,差異具有顯著性(P0.01)。冰凍切片顯示,注射后第1天,在高分子脂質(zhì)體組及Lipo組均可以觀察到玻璃體腔內(nèi)有GFP的表達(dá),高分子脂質(zhì)體組有部分于視網(wǎng)膜表面表達(dá)；注射后第6天,兩組的表達(dá)量均較高,且GFP表達(dá)位于RPE層附近；注射后第11天,在高分子脂質(zhì)體組切片中仍能找到GFP的表達(dá),但在Lipo組已很難找出。行VEGF的Western Blot檢測(cè),玻璃體腔內(nèi)注射后6天,高分子脂質(zhì)體組與Lipo組抑制效果相當(dāng)(P=0.092)；P22時(shí)高分子脂質(zhì)體組較Lipo組抑制明顯,差異有顯著性(P0.05)。Real-timePCR結(jié)果與western blot結(jié)果相似。 結(jié)論： 1.自行制備的Tat化的高分子脂質(zhì)體O-羧甲基殼聚糖十八烷基季銨鹽/膽固醇具有粒徑均勻、分散性好、Zeta電位較高、細(xì)胞毒性較小的特點(diǎn),體外釋放能在較短時(shí)間達(dá)到治療濃度,后續(xù)可緩慢平穩(wěn)釋放。 2.該高分子脂質(zhì)體能攜帶基因很好的轉(zhuǎn)染RPE細(xì)胞、Hela細(xì)胞及RF/16細(xì)胞,轉(zhuǎn)染效率與商品化的Lipofectamine2000相當(dāng)。 3.該高分子脂質(zhì)體攜帶治療基因可以減少視網(wǎng)膜新生血管的生成,且具有顯著的穿膜能力及緩釋功能,在體內(nèi)可維持較長(zhǎng)時(shí)間的治療效果,具有臨床應(yīng)用的開(kāi)發(fā)前景。
[Abstract]:Objective:
To analyze the performance of the high molecular liposomes carrying plasmid DNA, optimize the transfection conditions, observe the cytotoxicity and transfection efficiency of different cells, and study the inhibition effect of polymer liposome as gene carrier on retinal neovascularization in the oxygen induced retinopathy animal model, reflecting its unique membrane ability and delay. It provides experimental basis and theoretical basis for the preparation of targeted drug / gene co loading multifunctional drug delivery system in the future.
Method:
1. the 1. subject group syntheses carboxymethyl chitosan eighteen alkyl quaternary ammonium salt (OQLCS/chol) nanoparticles, and connects the membrane peptide (Tat) with its surface functional groups to prepare polymer liposomes with cross membrane and long circulation function, and detect its characterization. The high sub liposomes have different effects on the plasmid DNA in different mass ratio. The optimal transfection conditions were established under the conditions of whether there was or not in the presence of serum and serum, and the release curves were plotted in vitro by high speed centrifugation, and the CCK-8 method was used to detect the toxicity of different concentration of polymer liposomes to retinal pigment epithelium (RPE) cells at different time. The transfection efficiency of RPE cells was compared with that of commercial Lipofectamine2000.
2. the RPE cell model was established under the condition of hypoxia, and the cell supernatant was collected by ELISA to detect the changes of VEGF in each group after 48 time transfection. The expression of VEGF protein in each group was analyzed by immunohistochemical staining, and the changes of VEGF mRNA in each group of RPE cells were detected by Real-time PCR.
3. the modified Smith's method was used to establish the C57BL/6J mice model of oxygen induced retinopathy. There were 95 mice in the normal oxygen environment and 27 mice were fed in the glass cavity of the normal oxygen environment. At different time points, two mice were randomly selected in the normal drug group, and the expression of GFP was observed under the fluorescence microscope. In each group, two mice were selected to observe the changes of retinal vascular morphology after FITC-dextran heart perfusion. Two mice were randomly selected in each group. HE staining was performed to break through the number of vascular endothelial nuclei of the inner boundary membrane of the retina; two mice were randomly selected from each group for 5gm frozen section, and the DAPI nucleus was stained with fluorescence. Microscopic observation was carried out to reflect the penetrating ability of plasmid DNA carrying polymer liposomes.
Result:
The average particle size of 1. nanoparticles was 134nm, the Zeta potential was +39.64mV, and the loaded plasmid was 236nm. transmission electron microscope with uniform round particles. The release experiment in vitro showed that the high molecular liposomes containing plasmid DNA were released in the first 5 days in vitro, about 70% of the plasmids released. The toxicity of plastids was low, and the highest safety concentration was 20ug/mL.
2. through the electrophoretic block test, the mixing time was 30-40min when the mass ratio of high molecular liposomes and plasmid DNA was more than 2:1, and the culture solution without serum was the optimal transfection condition, and RPE, Hela and RF/16 were transfected on this condition, the transfection rate to RPE was about 69%, the transfection efficiency of Hela cells was about 52%, and the efficiency of RF/16 transfection was about 80%..
3. after transfection of RPE after hypoxia, both high molecular liposome group and Lipo group could effectively transfect plasmid DNA into cells and express cell supernatant after.48 hours. ELISA method was used to detect VEGF quantity in each group. The content of VEGF in the liposome group and Lipo group was significantly lower than that in the model group. The difference was significant (P0.01), in which the Lipo group was inhibited. The difference was not significant (P=0.10), but the difference was not significant (P=0.10).72 hours after cell immuno histochemistry showed the changes of intracellular VEGF, Lipo and high fat liposomes group had obvious inhibitory effect on VEGF expression, and the brown staining in the cytoplasm was obviously weak, and the OD value was slightly lower than the Lipo group (P=0.443).Real-time compared with the polymer liposome group. PCR results showed that the changes of VEGF mRNA in high liposomes and Lipo groups were similar to those of ELISA at 48 hours after transfection.
4. a mouse model of oxygen induced retinopathy was successfully established. After intravitreal injection, GFP expression was found in the retina first days after injection, and reached the peak at sixth days. At eleventh days, the expression of GFP could still be seen in the polymer liposome group. The Lipo group was not significantly.FITC (?) after heart perfusion, the area of retinal perfusion and the range of neovascularization were shown. The effect of high molecular liposome group and Lipo group was obviously improved. The result was that after.HE staining, the number of vascular endothelial nuclei was broken through the inner boundary membrane of the retina. The results showed that both the high molecular liposomes group and the Lipo group could effectively inhibit the formation of the neovascularization at P17 days, and the effect of the polymer liposomes still appeared at P22 days, and the number of the nucleus of the neovascular endothelial cells was more than that of the Lipo group. The difference was significant (P0.01). The frozen section showed that the expression of GFP in the vitreous body could be observed in the high molecular liposomes group and the Lipo group first days after the injection. The high molecular liposome group was partly expressed on the retina surface, and the expression of the two groups was higher on the sixth day after injection, and the expression of GFP was near the RPE layer; after the injection, the expression was in the RPE layer. On the 11 day, the expression of GFP could still be found in the high molecular liposomes group, but it was difficult to find out in the Lipo group. Western Blot detection of VEGF and the 6 days after intravitreal injection, the inhibition effect of the polymer liposome group and the Lipo group was equal (P=0.092), and the high molecular liposome group was significantly more inhibited than the Lipo group when P22, and the difference was significant (P0.05).Real-timePC. The results of R were similar to that of Western blot.
Conclusion:
1. the Tat carboxymethyl chitosan eighteen alkyl quaternary ammonium salt / cholesterol prepared by ourselves has the characteristics of homogeneous particle size, good dispersibility, high Zeta potential and less cytotoxicity. In vitro release can reach the treatment concentration in a short time, and the subsequent release can be slowly and smoothly released.
2. the liposome can carry well transfected RPE cells. The transfection efficiency of Hela cells and RF/16 cells is comparable to that of commercialized Lipofectamine2000 cells.
3. the polymer liposomes carrying the therapeutic gene can reduce the formation of retinal neovascularization, and have significant membrane ability and sustained release function. It can maintain the therapeutic effect for a long time in the body, and has the prospect of clinical application.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R774.1

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