肽核酸阻斷γ→β珠蛋白基因轉(zhuǎn)換對(duì)γ珠蛋白基因表達(dá)的影響.pdf

1、目的：通過肽核酸(peptide nucleic acid，PNA)封閉PYR（polypyrimidine complex）的DNA結(jié)合序列（γ-δ基因間序列），人為激活γ-珠蛋白基因的表達(dá)，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(shè)計(jì)：本研究一共設(shè)計(jì)三條肽核酸（1）PYR-PNA，與PYR的DNA結(jié)合序列互補(bǔ)結(jié)合，用于阻斷γ→β珠蛋白開關(guān)；（2）β-PNA，與β-珠蛋白基因轉(zhuǎn)錄起始區(qū)序列互補(bǔ)結(jié)合，用

2、于下調(diào)β-珠蛋白基因表達(dá)；（3）Scr-PNA，即隨機(jī)序列PNA（Scramble PNA），作為隨機(jī)對(duì)照 PNA。2.肽核酸的轉(zhuǎn)染效率及細(xì)胞毒性試驗(yàn)：以陽離子脂質(zhì)體lipofectamine2000為載體，將標(biāo)記有FITC熒光基團(tuán)的PYR-PNA轉(zhuǎn)染K562細(xì)胞，在熒光顯微鏡下觀察并計(jì)算其轉(zhuǎn)染效率，采用Cell Counting Kit（CCK-8）檢測(cè)其細(xì)胞毒性，應(yīng)用正交試驗(yàn)篩選出最佳轉(zhuǎn)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(zhuǎn)染效果。3.肽核酸對(duì)γ-珠蛋白基因表達(dá)的影響：首先，對(duì)于K562細(xì)胞，實(shí)驗(yàn)分為3個(gè)實(shí)驗(yàn)組和2個(gè)對(duì)照組，實(shí)驗(yàn)組包括PYR-PNA組，β-PNA組， PYR-PNA+β-PNA組；對(duì)照組包括Scr-PNA組，空白對(duì)照組（無PNA的脂質(zhì)體）。根據(jù)最佳轉(zhuǎn)染條件，各組PNA經(jīng)脂質(zhì)體介導(dǎo)轉(zhuǎn)染K562細(xì)胞，于轉(zhuǎn)染后24h、48h和72h，用Real Time-PCR和Western blotting分別在轉(zhuǎn)錄水平和合成水平檢測(cè)各組γ-珠蛋白基因的表

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

5、RGR）為94.1%。3. Real Time-PCR結(jié)果：PYR-PNA組轉(zhuǎn)染后24h、48h和72h，γ-珠蛋白基因表達(dá)量比對(duì)照組分別增加1.702倍、2.036倍和1.498倍，與隨機(jī)對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義（P＜0.01）；β-PNA組轉(zhuǎn)染后24h、48h和72h，γ-珠蛋白基因表達(dá)量比對(duì)照組分別增加1.374倍、1.436倍和1.132倍，與隨機(jī)對(duì)照組比較24h差異有統(tǒng)計(jì)學(xué)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