本文選題：烷基化聚乙烯亞胺 + 三油酸甘油酯�。� 參考：《復(fù)旦大學(xué)》2008年博士論文

【摘要】：高效、安全的基因遞送載體的研究是目前基因治療研究的主要難點(diǎn)之一。非病毒基因遞送載體由于安全、易組裝等特點(diǎn)備受?chē)?guó)內(nèi)外學(xué)者的關(guān)注。本課題結(jié)合磷脂、聚乙烯亞胺(Polyethylenimine,PEI)以及油酸甘油酯的特點(diǎn),制備了聚陽(yáng)離子納米脂質(zhì)載體(Polycation nanostructured lipid carrier,PNLC),用于基因遞送的研究。 本文研究?jī)?nèi)容包括烷基化聚乙烯合成,PNLC的制備及組分的篩選;PNLC作為綠色熒光蛋白報(bào)告基因pEGFP-N2和蟲(chóng)熒光素酶報(bào)告基因pGL3-luc遞送載體的研究;以SPC-A1細(xì)胞為模型,采用加入特異性抑制劑的方法,考察PNLC/DNA復(fù)合物的細(xì)胞內(nèi)吞途徑和轉(zhuǎn)運(yùn)機(jī)制;采用活細(xì)胞顯微成像技術(shù)觀(guān)察PNLC/DNA復(fù)合物進(jìn)入細(xì)胞以及細(xì)胞內(nèi)轉(zhuǎn)運(yùn)的過(guò)程。 本文將溴化十四烷、溴化十六烷和溴化十八烷分別與聚乙烯亞胺(PEI600Da,PEI1200Da和PEI1800Da)反應(yīng),合成了27種烷基化聚乙烯亞胺,采用乳化溶劑蒸發(fā)法制備PNLC,并對(duì)制備工藝進(jìn)行了優(yōu)化,制備工藝優(yōu)化為:超聲功率200W,超聲時(shí)間5min,分散介質(zhì)為雙蒸水。 PNLC由烷基化聚乙烯亞胺、油相和磷脂三種成分組成。烷基化聚乙烯亞胺為上述合成的27種不同的烷基修飾的聚乙烯亞胺;油相為油酸甘油酯、中鏈甘油三酯M812、三肉豆蔻酸甘油酯、三棕櫚酸甘油酯和辛酸癸酸甘油酯M818;磷脂為卵磷脂EPC和二油酰磷脂酰乙醇胺DOPE。采用乳化溶劑蒸發(fā)法制備PNLC,測(cè)定其粒徑和zeta電位,與pEGFP-N2質(zhì)粒結(jié)合形成PNLC/DNA復(fù)合物,轉(zhuǎn)染SPC-A1細(xì)胞,采用熒光分光光度法測(cè)定SPC-A1細(xì)胞中表達(dá)的綠色熒光蛋白的熒光強(qiáng)度,以SPC-A1細(xì)胞中表達(dá)的綠色熒光蛋白的熒光強(qiáng)度為指標(biāo),分別篩選PNLC的各項(xiàng)組成。 選擇油相為三油酸甘油酯、磷脂為卵磷脂EPC,考察烷基化聚乙烯亞胺對(duì)PNLC基因轉(zhuǎn)染效率的影響。結(jié)果表明PNLC中烷基化聚乙烯亞胺為十六烷基化聚乙烯亞胺時(shí),與三油酸甘油酯和卵磷脂組成的PNLC轉(zhuǎn)染效率較高,而且當(dāng)烷基化聚乙烯亞胺為十六烷基化PEI1200(1:6)即P_(12)C_(16)(1:6)時(shí),PNLC在SPC-A1細(xì)胞中轉(zhuǎn)染pEGFP-N2后,細(xì)胞中表達(dá)的綠色熒光蛋白的熒光強(qiáng)度與陽(yáng)離子脂質(zhì)體Lipofectamine~(TM)2000相當(dāng)。因此,選擇PNLC處方中烷基化聚乙烯亞胺為P_(12)C_(16)(1:6)。 選擇烷基化聚乙烯亞胺為P_(12)C_(16)(1:6),磷脂為卵磷脂EPC,考察油相對(duì)PNLC基因轉(zhuǎn)染的影響。結(jié)果表明當(dāng)油相為油酸甘油酯和中鏈甘油三酯M812時(shí),制備的PNLC在SPC-A1細(xì)胞中轉(zhuǎn)染pEGFP-N2質(zhì)粒后,細(xì)胞中表達(dá)的綠色熒光蛋白的熒光強(qiáng)度較高。因此,選擇PNLC的油相為中鏈甘油三酯M812和油酸甘油酯(三油酸甘油酯、二油酸甘油酯和單油酸甘油酯)。 選擇烷基化聚乙烯亞胺為P_(12)C_(16)(1:6),油相為中鏈甘油三酯M812、三油酸甘油酯、二油酸甘油酯和單油酸甘油酯,考察磷脂的種類(lèi)(卵磷脂EPC或者二油酰磷脂酰乙醇胺DOPE)以及油相/磷脂的比值對(duì)PNLC基因轉(zhuǎn)染的影響。結(jié)果表明當(dāng)油相成分為油酸甘油酯時(shí),PNLC在SPC-A1細(xì)胞中轉(zhuǎn)染質(zhì)粒pEGFP-N2后,細(xì)胞中表達(dá)的綠色熒光蛋白的熒光強(qiáng)度高于陽(yáng)離子脂質(zhì)體Lipofectamine~(TM)2000。PNLC處方中加入油酸甘油酯后,細(xì)胞中表達(dá)的綠色熒光蛋白的熒光強(qiáng)度提高。PNLC處方優(yōu)化結(jié)果如下:在由P_(12)C_(16)(1:6)、油酸甘油酯和卵磷脂EPC組成的PNLC中,P_(12)C_(16)(1:6)與卵磷脂的摩爾比值為0.1,三油酸甘油酯與卵磷脂EPC的摩爾比值為1,二油酸甘油酯與卵磷脂EPC的質(zhì)量比值為2,單油酸甘油酯與卵磷脂EPC的摩爾比值為6;在由P_(12)C_(16)(1:6)、油酸甘油酯和磷脂DOPE組成的PNLC中,P_(12)C_(16)(1:6)與磷脂DOPE的摩爾比值為0.1,三油酸甘油酯與磷脂DOPE的摩爾比值為0.8,二油酸甘油酯與磷脂DOPE的質(zhì)量比值為0.75,單油酸甘油酯與磷脂DOPE的摩爾比值為1.5。上述六種組成PNLC在SPC-A1細(xì)胞和CHO細(xì)胞中轉(zhuǎn)染質(zhì)粒pEGFP-N2,流式細(xì)胞儀測(cè)定其轉(zhuǎn)染效率,結(jié)果表明,在SPC-A1細(xì)胞和CHO細(xì)胞中,由P_(12)C_(16)(1:6)、三油酸甘油酯或者二油酸甘油酯、卵磷脂組成的PNLC轉(zhuǎn)染效率與陽(yáng)離子脂質(zhì)體Lipofectamine~(TM)2000相當(dāng),而由P_(12)C_(16)(1:6)、單油酸甘油酯和卵磷脂組成的PNLC轉(zhuǎn)染效率顯著高于陽(yáng)離子脂質(zhì)體Lipofectamine~(TM)2000;由P_(12)C_(16)(1:6)、油酸甘油酯和磷脂DOPE組成的PNLC轉(zhuǎn)染效率顯著高于陽(yáng)離子脂質(zhì)體Lipofectamine~(TM)2000。PNLC中磷脂為DOPE時(shí)的基因轉(zhuǎn)染效率較高。在含有10%血清的條件下,PNLC仍具有較高的基因轉(zhuǎn)染效率。 選擇由P_(12)C_(16)(1:6)、三油酸甘油酯、DOPE組成的PNLC(PTD),由P_(12)C_(16)(1:6)、二油酸甘油酯和DOPE組成的PNLC(PDD),由P_(12)C_(16)(1:6)、單油酸甘油酯和卵磷脂EPC組成的PNLC(PME),在SPC-A1細(xì)胞和CHO細(xì)胞中轉(zhuǎn)染蟲(chóng)熒光素酶報(bào)告基因pGI3-luc,結(jié)果表明在SPC-A1細(xì)胞中,三種組成的PNLC的轉(zhuǎn)染效率顯著高于陽(yáng)離子脂質(zhì)體Lipofectamine~(TM)2000,而且在含有10%血清的條件下具有較高的轉(zhuǎn)染效率;在CHO細(xì)胞中,PTD和PDD組成的PNLC的轉(zhuǎn)染效率顯著高于陽(yáng)離子脂質(zhì)體Lipofectamine~(TM)2000,而且在含有10%血清的條件下,均具有較高的轉(zhuǎn)染效率,但是PME組成的PNLC的轉(zhuǎn)染效率非常低。 選擇PTD、PDD和PME組成的PNLC,以SPC-A1細(xì)胞為模型,采用加入特異性抑制劑的方法,考察PNLC/DNA復(fù)合物進(jìn)入細(xì)胞的內(nèi)吞途徑以及細(xì)胞內(nèi)的轉(zhuǎn)運(yùn)機(jī)制。分別加入網(wǎng)格蛋白內(nèi)吞途徑抑制劑氯丙嗪和陷穴小泡內(nèi)吞途徑Filipin,測(cè)定PNLC/DNA復(fù)合物的內(nèi)吞情況以及蟲(chóng)熒光素酶報(bào)告基因在細(xì)胞內(nèi)的表達(dá)情況。結(jié)果表明在SPC-A1細(xì)胞中,加入氯丙嗪后,PNLC/DNA復(fù)合物的內(nèi)吞降低,細(xì)胞中表達(dá)的luciferase活性降低了近90%,而加入Filipin后,PNLC/DNA復(fù)合物的內(nèi)吞和細(xì)胞中表達(dá)的luciferase的活性均沒(méi)有顯著性的變化。因此推斷PNLC/DNA復(fù)合物通過(guò)網(wǎng)格蛋白介導(dǎo)的內(nèi)吞途徑進(jìn)入細(xì)胞。為了證明這一推斷,分別對(duì)PNLC/DNA復(fù)合物和細(xì)胞內(nèi)溶酶體用熒光探針進(jìn)行標(biāo)記,用激光共聚焦顯微鏡觀(guān)察發(fā)現(xiàn)PNLC/DNA復(fù)合物進(jìn)入細(xì)胞后與溶酶體的重合,因此,PNLC/DNA復(fù)合物主要是通過(guò)網(wǎng)格蛋白介導(dǎo)的內(nèi)吞途徑進(jìn)入細(xì)胞。 細(xì)胞內(nèi)微管和摩托蛋白對(duì)細(xì)胞內(nèi)的膜泡轉(zhuǎn)運(yùn)具有非常重要的作用,加入微管解聚劑nocodazole和微管穩(wěn)定劑paclitaxel,動(dòng)力蛋白抑制劑原釩酸鈉(SOV)和驅(qū)動(dòng)蛋白Eg5抑制劑monastrol,測(cè)定SPC-A1細(xì)胞內(nèi)表達(dá)的luciferase的活性,考察微管和摩托蛋白對(duì)于PNLC/DNA復(fù)合物在SPC-A1細(xì)胞內(nèi)轉(zhuǎn)運(yùn)的影響。結(jié)果表明SPC-A1細(xì)胞中加入微管解聚劑nocodazole和微管穩(wěn)定劑paclitaxel后,細(xì)胞中幾乎沒(méi)有l(wèi)uciferase表達(dá);加入原釩酸鈉后細(xì)胞中的luciferase的活性提高了50%,加入monastrol后PTD和PDD組成的PNLC轉(zhuǎn)染后luciferase活性降低,而PME組成的PNLC轉(zhuǎn)染后luciferase活性沒(méi)有顯著性變化,同時(shí)加入原釩酸鈉和monastrol后,細(xì)胞內(nèi)表達(dá)的luciferase活性均發(fā)生不同程度的降低。這些結(jié)果表明,微管和摩托蛋白對(duì)于PNLC/DNA復(fù)合物在細(xì)胞內(nèi)的轉(zhuǎn)運(yùn)具有非常重要的影響。 為了進(jìn)一步考察PNLC/DNA復(fù)合物進(jìn)入細(xì)胞的過(guò)程以及細(xì)胞內(nèi)的轉(zhuǎn)運(yùn)情況,選擇PME組成的PNLC,PNLC用綠色熒光探針FITC標(biāo)記,質(zhì)粒DNA用紅色熒光探針TM-Rhodamine進(jìn)行標(biāo)記,用激光共聚焦顯微鏡跟蹤PNLC/DNA復(fù)合物進(jìn)入細(xì)胞以及在細(xì)胞內(nèi)轉(zhuǎn)運(yùn)的過(guò)程。觀(guān)察結(jié)果表明PNLC能夠壓縮質(zhì)粒DNA形成PNLC/DNA復(fù)合物,內(nèi)吞進(jìn)入細(xì)胞后,通過(guò)早期內(nèi)吞泡-晚期內(nèi)吞泡-溶酶體的路徑轉(zhuǎn)運(yùn)到細(xì)胞核核周?chē)鷧^(qū)域,烷基化聚乙烯亞胺和質(zhì)粒DNA均可轉(zhuǎn)運(yùn)進(jìn)入細(xì)胞核。
[Abstract]:The research of efficient and safe delivery vector for gene delivery is one of the main difficulties in the research of gene therapy. The non viral gene delivery carrier has attracted much attention from domestic and foreign scholars because of its safety and easy assembly. This subject has prepared polycationic nanoscale with the characteristics of phospholipid, Polyethylenimine, PEI and oleate. Polycation nanostructured lipid carrier (PNLC) is used for the study of gene delivery.
This article includes the synthesis of alkylated polyethylene, the preparation of PNLC and the screening of components, PNLC as the green fluorescent protein reporter gene pEGFP-N2 and the pGL3-luc delivery carrier of the luciferase reporter gene, and the method of adding specific inhibitors to SPC-A1 cells to investigate the endocytosis pathway of the PNLC/DNA complex. And the transport mechanism. The PNLC/DNA complex was introduced into the cells and the intracellular transport process was observed by living cell microscopy.
In this paper, brominated fourteen alkane, brominated sixteen alkane and brominated eighteen alkane were reacted with polyethyleneimine (PEI600Da, PEI1200Da and PEI1800Da) to synthesize 27 kinds of alkylated polyethyleneimine, and PNLC was prepared by emulsion solvent evaporation method. The preparation process was optimized. The preparation process was optimized by ultrasonic power 200W, ultrasonic time 5min, dispersion mediator. The quality is double steamed water.
PNLC consists of three kinds of alkylated polyethyleneimine, oil phase and phospholipid. Alkylated polyethyleneimine is the 27 different alkyl modified polyethyleneimine synthesized above; oil phase is oleic acid glyceride, medium chain triglyceride M812, three myrisate glyceride, three palmitate glyceride and octanoic acid glyceride M818; phosphatidylcholine is phosphatidylcholine EPC And two oil acyl phosphatidyl ethanolamine DOPE. was prepared by emulsion solvent evaporation method to prepare PNLC, determine its particle size and zeta potential, combine with pEGFP-N2 plasmid to form PNLC/DNA complex, transfect SPC-A1 cells, and use fluorescence spectrophotometry to determine the fluorescence intensity of green fluorescent protein expressed in SPC-A1 cells, and the green fluorescence expressed in SPC-A1 cells. The fluorescence intensity of protein was used as an index to screen the components of PNLC.
The effect of alkylated polyethylenimide on the transfection efficiency of PNLC gene was investigated by selecting oil as three oleate and phosphatidylcholine EPC. The results showed that when alkylated polyethyleneimine was sixteen alkylated polyethyleneimine in PNLC, the transfection efficiency of PNLC with three oleate and lecithin was higher than that of alkylated polyethyleneimine. When sixteen alkylated PEI1200 (1:6), P_ (12) C_ (16) (1:6), PNLC was transfected with pEGFP-N2 in SPC-A1 cells, the fluorescence intensity of the green fluorescent protein expressed in the cells was equivalent to the cationic liposome Lipofectamine~ (TM) 2000. Therefore, the alkylated polyethyleneimine was selected as P_ (12) (16) in the PNLC prescription.
Alkylated polyethyleneimine was selected as P_ (12) C_ (16) (1:6) and phospholipid as lecithin EPC. The effect of oil on the transfection of PNLC gene was investigated. The results showed that when the oil phase was oleate and medium chain triglyceride M812, the PNLC in SPC-A1 cells was transfected with pEGFP-N2 plasmids, and the fluorescence intensity of the green fluorescent protein expressed in the cells was higher. The oil phase of PNLC was selected as medium chain triglyceride M812 and glyceryl oleate (three glyceryl oleate, two oleic acid glyceride and monoglyceride).
Alkylated polyethyleneimine was selected as P_ (12) C_ (16) (1:6), oil phase was triglyceride M812, three oleate, two oleate and monoglyceride, and the effects of phosphatidylcholine (phosphatidylcholine EPC or two oleoyl ethanolamine DOPE) and oil phase / phospholipid ratio on PNLC gene transfection were investigated. The fluorescence intensity of the green fluorescent protein expressed in the cell was higher than that of the cationic liposome Lipofectamine~ (TM) 2000.PNLC. The fluorescence intensity of the green fluorescent protein expressed in the cell was improved by the Lipofectamine~ (TM) 2000.PNLC formulation. The fluorescence intensity of the green fluorescent protein expressed in the cell was improved by the.PNLC formulation. The results were as follows: in P_, the results were as follows: in P_, the results of the fluorescence intensity of the green fluorescent protein expressed in the cells were optimized as follows: in P_, the results were as follows: in P_, the fluorescence intensity of the green fluorescent protein expressed in the cells was as follows: in P_ (12) C_ (16) (16) (1:6), glycerol glyceride and lecithin EPC in PNLC, P_ (12) C_ (16) (1:6) and lecithin ratio 0.1, three olein glyceride and lecithin EPC, 1, two olein glyceride and phosphatidylcholine EPC ratio is 2, the molar ratio of glyceryl glyceride and lecithin EPC is 6; in P_ (12) C_ (16), The molar ratio of P_ (12) C_ (16) (12) C_ (16) (1:6) to phospholipid DOPE is 0.1, the ratio of three oleate to phospholipid DOPE is 0.8, the ratio of two oleate glyceride to phospholipid DOPE is 0.75, and the molar ratio of glycerol to phospholipid DOPE is 1.5. above SPC-A1 cells and six components PNLC. The transfection efficiency of O cells was detected by flow cytometry, and the transfection efficiency was measured by flow cytometry. The results showed that in SPC-A1 cells and CHO cells, P_ (12) C_ (16) (1:6), three oleate or two oleate, the PNLC transfection efficiency of lecithin was equivalent to that of cationic liposome Lipofectamine~ (TM) 2000, while P_ (12) C_ (16), monoglyceride PNLC transfection efficiency of ester and lecithin was significantly higher than that of cationic liposome Lipofectamine~ (TM) 2000; P_ (12) C_ (16) (1:6), oleate glyceride and phospholipid DOPE were significantly higher transfection efficiency than cationic liposome Lipofectamine~ (TM) 2000.PNLC, the gene transfection efficiency was higher when phosphor lipid was DOPE, which contained 10% serum conditions. At the same time, PNLC still has high gene transfection efficiency.
P_ (12) C_ (16) (16), three oleate, DOPE, PNLC (PTD), PNLC (PDD) consisting of P_ (12) C_ (16) (1:6), two oleate and DOPE, was composed of 12 (16), glycerol monoglyceride and lecithin. In 1 cells, the transfection efficiency of three components of PNLC was significantly higher than that of cationic liposome Lipofectamine~ (TM) 2000, and had higher transfection efficiency under the conditions of 10% serum. In CHO cells, the transfection efficiency of PNLC, which was composed of PTD and PDD, was significantly higher than that of cationic liposome Lipofectamine~ (TM) 2000, and in the 10% serum containing strips. The transfection efficiency is high, but the transfection efficiency of PNLC composed of PME is very low.
The PNLC, composed of PTD, PDD and PME, was used as a model of SPC-A1 cells. The endocytosis pathway of PNLC/DNA complex into cells and the mechanism of intracellular transport were investigated by adding specific inhibitors. The internal endocytosis pathway, chlorpromazine and trapping vesicle endocytic pathway Filipin, were added to the PNLC/DNA complex. Endocytosis and the expression of the luciferase reporter gene in the cells showed that the endocytosis of PNLC/DNA complex was reduced after adding chlorpromazine in SPC-A1 cells, and the activity of luciferase in the cells was reduced by nearly 90%. After adding Filipin, the activity of the endocytosis of PNLC/DNA complex and the expression of luciferase in the cells had not been found. Significant changes. Therefore, it is inferred that the PNLC/DNA complex enters cells through the endocytic pathway mediated by the gridin. In order to prove this inference, the PNLC/DNA complex and the intracellular lysosomes are labeled with fluorescence probes, and the coincidence of the PNLC/DNA complex to the lysosome after the entry of the complex is observed by laser confocal microscopy. The PNLC/DNA complex mainly enters the cell through the clathrin mediated endocytosis pathway.
Intracellular microtubules and motor proteins have a very important role in the transport of membrane vesicles in cells, including microtubule depolymerization agent nocodazole and microtubule stabilizer paclitaxel, SOV and Eg5 inhibitor Monastrol, the activity of luciferase in SPC-A1 cells, microtubules and MOS The effect on the transport of PNLC/DNA complex in SPC-A1 cells showed that there was almost no luciferase expression in the cells after the addition of microtubule depolymerization agent nocodazole and microtubule stabilizer paclitaxel, and the activity of luciferase in the cells after adding sodium vanadate was increased by 50%. After Monastrol, PTD and PDD made up of PNLC transfection after Monastrol. The activity of luciferase was reduced, and the activity of luciferase was not significantly changed after the transfection of PME, and the luciferase activity expressed in cells was reduced in varying degrees after adding sodium vanadate and Monastrol. These results showed that microtubules and motorproteins were very important for the transport of PNLC/ DNA complex in cells. Influence.
In order to further investigate the process of PNLC/DNA complex entering cells and the transport of cells, the PNLC of PME was selected, the PNLC was labeled with green fluorescent probe FITC, the plasmid DNA was marked with the red fluorescent probe TM-Rhodamine, and the laser confocal microscope was used to track the entry of PNLC/DNA complex and the process of transport in the cell. The results show that PNLC can compress plasmids DNA to form PNLC/DNA complex, and after endocytosis enters the cell, it can be transported to the nucleus around the nucleus through the pathway of the early endocytic vesicle and late endocytic vesicle lysosome, and the alkylated polyethyleneimine and plasmid DNA can be transported into the nucleus.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R346

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 ;Poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine]: A Non-viral Polymer with Potential for DNA Delivery[J];Chinese Chemical Letters;2004年01期

2 王幽香,沈家驄;谷胱甘肽響應(yīng)的非病毒基因傳遞體系研究[J];高等學(xué)�；瘜W(xué)學(xué)報(bào);2005年04期

3 蔣序林;;AQUEOUS SEC ANALYSIS OF CATIONIC POLYMERS AS GENE CARRIERS[J];Chinese Journal of Polymer Science;2011年04期

4 丁凡;邵增務(wù);;骨肉瘤基因治療載體研究進(jìn)展[J];國(guó)際骨科學(xué)雜志;2007年06期

5 李經(jīng)忠,王青青;非病毒載體的研究現(xiàn)狀[J];國(guó)外醫(yī)學(xué)(分子生物學(xué)分冊(cè));2002年05期

6 李經(jīng)忠,王青青;新型非病毒載體聚乙烯亞胺體內(nèi)應(yīng)用的研究進(jìn)展[J];國(guó)外醫(yī)學(xué).藥學(xué)分冊(cè);2004年01期

7 李經(jīng)忠,王青青;陽(yáng)離子多聚物轉(zhuǎn)基因載體的研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2002年05期

8 陳鋒,胡曉年;非病毒載體在肝癌基因治療中的應(yīng)用[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2005年04期

9 ;Synthesis and characterization of PEGylated comb-like cationic polymer by polycondensation and ATRP[J];Chinese Chemical Letters;2012年07期

10 孫平;杜紅;高利龍;朱蔚璞;李曉東;沈之荃;;可還原降解梳形陽(yáng)離子聚合物的合成[J];高分子學(xué)報(bào);2012年08期

相關(guān)博士學(xué)位論文 前10條

1 要e，

本文編號(hào)：2099145