肽核酸阻斷γ→β珠蛋白基因轉換對γ珠蛋白基因表達的影響.pdf

1、目的：通過肽核酸(peptide nucleic acid，PNA)封閉PYR（polypyrimidine complex）的DNA結合序列（γ-δ基因間序列），人為激活γ-珠蛋白基因的表達，從而探索一條β-地中海貧血基因治療的新途徑。
　　方法：1.肽核酸的設計：本研究一共設計三條肽核酸（1）PYR-PNA，與PYR的DNA結合序列互補結合，用于阻斷γ→β珠蛋白開關；（2）β-PNA，與β-珠蛋白基因轉錄起始區(qū)序列互補結合，用

2、于下調β-珠蛋白基因表達；（3）Scr-PNA，即隨機序列PNA（Scramble PNA），作為隨機對照 PNA。2.肽核酸的轉染效率及細胞毒性試驗：以陽離子脂質體lipofectamine2000為載體，將標記有FITC熒光基團的PYR-PNA轉染K562細胞，在熒光顯微鏡下觀察并計算其轉染效率，采用Cell Counting Kit（CCK-8）檢測其細胞毒性，應用正交試驗篩選出最佳轉染條件，并優(yōu)化稀釋培養(yǎng)基血清濃度，以獲得最優(yōu)的

3、轉染效果。3.肽核酸對γ-珠蛋白基因表達的影響：首先，對于K562細胞，實驗分為3個實驗組和2個對照組，實驗組包括PYR-PNA組，β-PNA組， PYR-PNA+β-PNA組；對照組包括Scr-PNA組，空白對照組（無PNA的脂質體）。根據最佳轉染條件，各組PNA經脂質體介導轉染K562細胞，于轉染后24h、48h和72h，用Real Time-PCR和Western blotting分別在轉錄水平和合成水平檢測各組γ-珠蛋白基因的表

4、達，比較各組γ-珠蛋白基因表達量的差異。
　　結果：1.細胞最佳轉染條件結果：以2.5-3×105/mL的細胞密度接種，每100μl的培養(yǎng)基中加入PNA6.25pmol，PNA與脂質體的體積比為1:3.5，稀釋和孵育脂質體和PNA時不加胎牛血清，在轉染5h后加入50μl胎牛血清，轉染48h效果最好。2.細胞毒性及轉染效率結果：經過條件優(yōu)化，K562細胞轉染效率達到87.2％，細胞存活率（relative growth rate,

5、RGR）為94.1%。3. Real Time-PCR結果：PYR-PNA組轉染后24h、48h和72h，γ-珠蛋白基因表達量比對照組分別增加1.702倍、2.036倍和1.498倍，與隨機對照組比較差異均有統(tǒng)計學意義（P＜0.01）；β-PNA組轉染后24h、48h和72h，γ-珠蛋白基因表達量比對照組分別增加1.374倍、1.436倍和1.132倍，與隨機對照組比較24h差異有統(tǒng)計學意義（ P＜0.05）；.Results:1.Th

6、e optimal transfection conditions of cells: When the cells with the density of2.5-3×105/mL were cultured, the optimized transfection conditions were as follow: each100ul medium added with6.25pmol PNA, the volume proporti

7、on of PNA to liposomes was1:3.5, and the medium used as dilution had no fetal bovine serum,50ul fetal calf serum was added after transfection5h and the effect of48h transfection is best.2. The result of the cytotoxicity

8、and transfection efficiency:After optimization the conditions, the best transfection results were achieved, with a transfection efficiency of87.2% and a relative growth rate(RGR) of94.1%.3.The result of the Real Time-PCR

9、: The PYR-PNA group which compared to the control group that theγ-globin gene’s relative expression was increased1.702 times in24h,2.036 times in48h and1.498 times in72h, by statiscal, all P＜0.01, significant difference;

10、Theβ-PNA group which compared to the control group that theγ-globin gene’s relative expression was increased1.374 times in24h,1.436 times in48h and1.132 times in72h, in24h which compared to the control group by statiscal

11、, P＜0.05, significant difference; The PYR-PNA+β-PNA group which compared to the control group in24h,48h and72h that all were no significant with the P＞0.05;Theβ-PNA group and the PYR-PNA+β-PNA group which compared to the

12、 PYR-PNA group by statiscal, all P＜0.01, significant difference.4.The result of the western blotting: The PYR-PNA group which compared to the control group that theγ-globin’s relative expression was increased1.608 times

13、in24h,2.488 times in48h and1.575 times in72h, by statiscal, all P＜0.01, significant difference. Theβ-PNA group which compared to the control group that theγ-globin relative expression was increased1.330 times in24h and1.

14、422 times in48h and1.259 times in72h,there have significant difference in24h(P＜0.01）and48h（P＜0.05）;The PYR-PNA+β-PNA group which compared to the control group that theγ- globin relative expression was increased1.334 time

15、s in24h and1.784 times in48h and1.177 times in72h, which compared to the control group, by statiscal, in24h and48h（P＜0.01）, significant difference; Theβ-PNA group and the PYR-PNA+β-PNA group which compared to the PYR-PNA