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研究生:許樂仁
研究生(外文):Paul Yueh-Jen Hsu
論文名稱:重組腺相關病毒在糖尿病基因治療之研究
論文名稱(外文):Gene therapy for type 1 diabetes mellitus by recombinant adeno-associated virus
指導教授:楊雅雯楊雅雯引用關係
指導教授(外文):Ya-wun Yang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:81
中文關鍵詞:基因治療重組腺相關病毒胰島素糖尿病
外文關鍵詞:insulingene therapyDiabetes mellitusrAAV
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胰島素依賴性糖尿病,肇因於胰島β細胞遭受自體免疫破壞,而造成胰島素分泌缺乏所引起的內分泌失調疾病。為了治療此一疾病所進行的葡萄糖反應性胰島素基因治療,主要目標就是依生理訊號變化而控制胰島素產量,以改善病人的生活品質。在本研究之中將利用重組腺相關病毒作為基因載體,以研究在體外及體內試驗中受葡萄糖調控的胰島素生產情形。
重組腺相關病毒載體rAAV-rIns1-hInsM2-ΔEGFP,是將一段在前胰島素轉化酶認識位進行點突變為furin酵素可切割的人類胰島素基因置於rat insulin I (rIns I)驅動子的下游,並接著一段由人類巨細胞病毒早期驅動子(cytomegalovirus immediate early;CMVIE)所控制的強化型螢光蛋白(enhanced green fluorescent protein;EGFP)基因。為了觀察轉殖基因的調控表現,將人類肝癌細胞株Huh 7以重組腺相關病毒轉染後48小時,在培養基中加入不同濃度的葡萄糖(0至25 mM)刺激24小時後,收集上清液並處理之後,利用放射性同位素免疫分析法測定人類胰島素的分泌量,可發現經rAAV轉染的Huh 7細胞會隨葡萄糖濃度調控胰島素分泌。而加入dbcAMP、forskolin或是theophylline會增加胰島素的產量,在以2 mM dbcAMP或是20 μM forskolin處理後,會比只加入葡萄糖刺激增加大約三倍的胰島素分泌量。
為了要觀察在活體中接受rAAV轉染後人類胰島素的表現量,我們以streptozotocin (STZ)注射C57BL/6J鼠以製造糖尿病模式鼠。而為了要增加重組腺相關病毒在活體中的轉染效率,我們以陽離子聚合物polyethylenimine (PEI)和rAAV混合以形成複合物,此一複合物可以增加rAAV在Huh 7細胞上的EGFP基因表現率。經過流式細胞儀分析之後,發現最佳條件為2 µg/ml。而PEI25K對細胞的毒性利用MTT法測定,發現具有濃度相依性。利用PEI與rAAV共混形成複合物,再投與至小鼠肝臟實質細胞,可以產生較單獨給予rAAV時高的胰島素產量,並顯著改善糖尿病所造成的高血糖症。為了要探討葡萄糖在接受rAAV-PEI治療的小鼠上的影響,我們在小鼠的腹腔注射每公斤體重1.5 g的葡萄糖,並且每三十分鐘由尾靜脈採血以測定血糖值變化,及血中人類胰島素的含量,發現其血中人類胰島素的含量會隨血糖變化而調控,在接受葡萄糖注射後一小時會達到峰值。而為了要探討cAMP在接受rAAV-PEI治療後的糖尿病動物中調控胰島素分泌時所扮演的角色,每隻小鼠都接受每公斤體重50 mg的theophylline並與葡萄糖共同注射後可以發現,theophylline這個cAMP降解酵素cyclic nucleotide phosphodiesterase的抑制劑可以增加胰島素的產量而提高葡萄糖的週轉效率。
為了要釐清PEI在增進rAAV轉染效率的機轉,我們測定Huh 7細胞及rAAV顆粒的表面電位,結果發現兩者的表面電位會隨著PEI的濃度增加而上升。而利用無毒性的細胞表面糖蛋白磺基化(sulfation)抑制劑NaClO3處理後的細胞,會因為去磺基化造成細胞表面電位上升並降低rAAV的黏附率,在利用PEI與rAAV混合所形成的複合物會有較rAAV單獨高的基因轉染率,顯示加入PEI會增加對細胞表面的親合力。利用Cy3在rAAV上進行標定後,將螢光標定的Cy3-rAAV投與細胞並比較加入PEI之後對於胞內螢光強度的變化,經過流式細胞儀的分析,發現加入PEI會將rAAV在細胞內的攝入累積量提高10至100倍,而此一現象也在共軛焦螢光顯微鏡下獲得證實。另外,PEI會造成細胞產生活性氧物系以及產生氧化壓力,而氧化壓力的產生與轉染率增加有關。投與抗氧化劑後可以反轉PEI造成的基因轉值率增加。
總之,本研究證實了藉由重組腺相關病毒作為基因遞送載體時,可在體外及體內形成受到葡萄糖濃度變化調控的胰島素分泌。以PEI包覆形成的複合物會增加rAAV媒介的基因轉染效率而產生較高量的胰島素分泌,並改善糖尿病動物的高血糖症。而造成PEI促進重組腺相關病毒轉染率增加的機轉,與增加rAAV對於細胞表面受器的親合力有關,並會使得細胞攝入較多的病毒顆粒。
Insulin-dependent diabetes mellitus (IDDM) is caused by the lack of insulin secretion, involving autoimmune destruction of insulin-producing pancreatic β-cell. Glucose-responsive insulin gene therapy aimed to control the production of insulin in response to physiological signals. In the present study, recombinant adeno-associated virus (rAAV) was employed as a gene delivery vector to study glucose regulated insulin gene therapy.
Recombinant AAV vector, rAAV-rIns1-hInsM2-ΔEGFP, was constructed to contain a furin-mutated human insulin gene downstream of the rat insulin I promoter, and the enhanced green fluorescent protein (EGFP) gene, driven by the cytomegalovirus immediate early (CMVIE) promoter. To examine regulated transgene expression, Huh 7 human hepatoma cells were transduced by rAAV, followed by treatment with glucose of various concentrations. Human insulin production in the rAAV-transduced cells, determined by radioimmunoassay, was shown to vary with the glucose concentration. Addition of N6, 2’-O-dibutyryladenosine 3’, 5’-cyclicmonophosphate (dbcAMP), forskolin and theophylline increased the insulin secretion. Treatment of rAAV-transduced Huh7 cells with 25 mM glucose and 2 mM dbcAMP, or 20 μM forskolin resulted in an approximate three-fold increase of insulin secretion, compared to the control cells treated with glucose alone.
To investigate the insulin expression after rAAV treatment in vivo, C57BL/6J mice were made diabetic by treatment with streptozotocin (STZ). To enhance the virus transduction efficiency, polyethylenimine (PEI) was incorporated into rAAV to form complexes. Complexation of rAAV with PEI resulted in enhancement of EGFP expression in rAAV-transduced Huh 7 cells. The optimal concentration of PEI for the enhancement of transduction efficiency was determined by flow cytometry to be 2 µg/ml. The cytotoxicity of PEI, assessed by MTT assay, was shown to be concentration-dependent. Administration of rAAV-PEI complexes into the mouse liver parenchyma lead to higher secretion of human insulin, as compared with the control group treated with rAAV alone, and ameliorated hyperglycemia in the STZ-induced diabetic animals. Glucose tolerance test was performed by intraperitoneal administration of 1.5 g per kg body weight of glucose, followed by periodical collection of blood samples from the tail vein of the animals. The production of human insulin was obtained in the rAAV-PEI treated diabetic animals, and was shown to vary with blood glucose concentrations, reaching a peak of 14.35 ± 0.51 µU/ml at 60 minutes after glucose injection. To evaluate the effect of cAMP modulation on insulin production, rAAV-PEI treated diabetic animals were co-administrated with theophylline and glucose. Treatment of rAAV-transduced animals with theophylline resulted in increase of insulin secretion, compared to the control group treated with glucose alone.
To investigate the mechanism of PEI enhancement on rAAV-mediated gene transfer, zeta potential of both Huh7 cells and rAAV particles was determined, and was shown to vary with the PEI concentration. Higher transgene expression was obtained in Huh7 cells after treatment with sodium chlorate, a nontoxic inhibitor of ATP-sulfurylase, indicating higher binding affinity of rAAV-PEI complexes to the cell surface, compared to those cells treated with rAAV alone. To quantitate virus uptake by the cells, rAAV was labeled with the carbocyanine dyes Cy3, followed by flow cytometric analysis. Complexation of rAAV with PEI resulted in an approximately 10 to 100 fold increase of rAAV uptake. The enhancement of binding was further confirmed by confocal fluorescence microscopic analysis. Huh 7 cells were shown to generate reactive oxygen species after treatment with rAAV-PEI complexes. Pretreatment of cells with anti-oxidants reduced the transduction efficiency of rAAV-PEI complexes.
In summary, we have demonstrated in this study that glucose-regulated insulin production can be obtained by rAAV-mediated gene transfer. Incorporation of PEI was shown to enhance the transgene expression, resulted in higher secretion of human insulin, and enhanced amelioration of hyperglycemia in diabetic animals. The enhancement mechanism of PEI was shown to be related to increased binding affinity of rAAV particles to the cell surface, leading to higher uptake of the virus particles.
壹、文獻回顧 1
一、胰島素依賴性糖尿病(Insulin-dependent diabetes mellitus;IDDM) 1
二、基因治療(Gene therapy) 3
三、腺相關病毒(adeno-associated virus; AAV) 6
四、聚乙二烯胺(Polyethylenimine) 8
貳、研究動機與目的 10
參、材料與方法 11
一、細胞培養 11
二、質體 DNA 的製備 11
1.大量備製質體 DNA 與純化 11
三、重組腺相關病毒 12
1. 重組腺相關病毒之建構 12
2. 製造重組腺相關病毒 13
3. 訂定重組腺相關病毒的效價 (titer) 14
四、流式細胞儀之分析 15
1.EGFP報告基因之分析 15
2.PEI對於細胞攝入rAAV量之分析 16
3.活性氧物系(Reactive Oxygen Species; ROS)之分析 17
五、PEI對細胞毒性的測定 17
MTT測定法 17
六、細胞與rAAV顆粒表面電位 (zeta potential)之測定 18
七、動物實驗 19
1.糖尿病模式鼠的建立與重組腺相關病毒的活體基因轉染 19
2.小鼠的血糖值測量 19
3. 以放射性免疫測定法測量基因轉染小鼠的血中人類胰島素值 20
4.組織切片與免疫染色 20
八、重組腺相關病毒與PEI作用後進入細胞途徑之測定 21
1.表面受器heparan sulfate proteoglycan (HSPG)去磺基化(desulfation) 21
2.重組腺相關病毒與PEI複合物進入細胞之時刻(time course)攝影 22
九、細胞中NO含量的測定 23
十、數據分析 23
肆、結果 24
第一部分:有關葡萄糖與二次傳訊者作用於重組腺相關病毒轉染肝細胞株之調控 24
一、葡萄糖會刺激rAAV轉染之肝細胞株分泌人類胰島素且具有濃度相依性 24
二、二次傳訊者(Second messenger)會增加葡萄糖刺激rAAV轉染之肝細胞株分泌人類胰島素 25
第二部分:PEI對於與重組腺相關病毒活體轉染效率之影響 27
一、PEI可以增進rAAV對於人類肝細胞株Huh7的轉染效率 28
二、PEI具有細胞毒性並會造成細胞死亡 28
三、PEI會對rAAV及Huh 7表面電位造成影響 29
四、糖尿病模式鼠接受rAAV-PEI複合物治療後可降低高血糖症 30
五、rAAV-PEI複合物可增加活體轉殖基因表現率 31
六、腹腔注射葡萄糖耐受性試驗(Intraperitoneal glucose tolerance test)及饑餓耐受性試驗(fasting tolerance test) 31
七、螢光免疫組織切片染色 32
第一部分之三、動物實驗 33
第三部分:PEI對於與重組腺相關病毒轉染率提昇機轉之探討 35
一、PEI會增加rAAV對於細胞表面受器的親合力 35
二、PEI在rAAV進入細胞過程的作用 36
三、PEI會產生ROS(Reactive Oxygen Species)造成細胞壓力 37
四、PEI增加rAAV基因產物表現量的效果可被抗氧化劑抑制 38
伍、討論 39
第一部分:關於葡萄糖與二次傳訊者作用於重組腺相關病毒轉染細胞之調控 39
第二部分:關於PEI對於與重組腺相關病毒活體轉染效率之影響 42
陸、結論 45
柒、參考資料 46
捌、圖表 58
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