【作者】 吴新华；

【导师】 卢奕； 罗怡； 王曼； 吴继红；

【作者基本信息】 复旦大学 ， 眼科学， 2009， 博士

【摘要】 前言晶状体后囊膜混浊（posterior capsule opacification,PCO）是白内障术后远期视力下降的主要原因。其发生机理主要与白内障术后赤道部晶状体上皮细胞（1ens epithelial cells,LECs）过度增殖并迁移至后囊膜分化为成纤维细胞有关。临床上通常采用YAG激光后囊膜切开术治疗这一并发症,但该方法会增加视网膜脱离、黄斑囊样水肿及眼内压升高的风险。白内障手术技术的提高、IOL设计和材料的改善、以及各种抗增殖药物的应用,虽然在一定程度上可降低PCO的发生率,但尚未能从根本上解决这一问题。因此,探索新的PCO防治途径具有重要意义。随着分子生物学技术的迅速发展,基因治疗技术逐渐被引入PCO防治研究中。目前PCO基因治疗的研究主要集中于自杀基因、细胞周期调控基因和细胞因子相关基因等。这些方法为.PCO的治疗提供了新思路,但仍存在一些缺陷。RNA干扰（RNA interference,RNAi）作为一种新型的基因沉默技术,在生物基础研究中得到广泛应用。本研究拟应用RNAi抑制Bcl-2基因表达,诱导LECs凋亡,从而实现清除增殖的LECs这一目标。为临床解决白内障术后PCO问题提供方法和依据。聚酞胺一胺型树枝状高聚物（PAMAM dendrimers）作为一种非病毒纳米载体,具备无免疫原性、无遗传毒性、转染效率高、可长期稳定表达等优良特性,在基因治疗领域具有良好的应用前景。本研究采用PAMAM G5介导基因转染,观察其与脂质体转染法间的差异。本研究构建了2条以bcl-2基因为靶标的短发夹状RNA（shRNA）,克隆入质粒PGCsi表达载体,分别用脂质体和PAMAM介导转染体外培养的HLECs,检测转染后BCL2基因表达的变化,观察细胞凋亡的情况。同时比较了PAMAM与脂质体在基因导入方面的差异。第一部分Bcl-2 shRNA表达载体的构建、扩增及鉴定目的构建2条Bcl-2 shRNA表达载体方法从GenBank中选取Bcl-2的基因序列（No.NM₀00633）作为分析序列,在Bcl-2 cDNA上寻找2条符合特征的靶序列（编码区分别为1210—1228,2133—2151）。设计可克隆入PGCsi表达载体的短发夹状RNA（shRNA）,5’端对应于BamHⅠ的酶切位点,在3’端带有T₆序列,对应于HindⅢ的酶切位点,命名为P1、P2;同时采用通用阴性对照,命名为P0。寡核苷酸链经退火、连接入线性化载体PGCsi,转化感受态细菌DH5α,再作扩增和测序鉴定。结果重组质粒经自动基因测序仪测序,结果与设计序列完全相同,所含目的基因序列准确无误,质粒构建成功。质粒扩增后的浓度与纯度均满足基因转染的要求。结论成功构建了2条Bcl-2 shRNA表达载体第二部分脂质体介导BCL-2 shRNA转染诱导晶状体上皮细胞凋亡目的应用RNA干扰技术抑制晶状体上皮细胞bcl-2基因表达,观察诱导晶状体上皮细胞（HLECs）凋亡情况方法脂质体Lipofectamine^TM2000介导BCL-2 shRNA（P1、P2、P0）转染人永生性HLECs系SRA01/04,48h后流式细胞仪检测转染效率,免疫印迹法检测bcl-2蛋白的表达,荧光定量PCR检测bcl-2 mRNA表达,并分别用Annexin V-FITC/PI双染法、Hoechst染色法、免疫印迹法检测caspase-3的表达等方法来检测HLECs的细胞凋亡情况。结果转染48h后,流式细胞仪检测发现,P1、P2、P0组的转染率分别为44.1±1.7%、47.2±1.6%、43.8±2.3%,各组间无统计学差异（p＞0.05）。免疫印迹法和荧光定量PCR表明,P1和P2均可抑制bcl-2蛋白和mRNA的表达,与空白组比较差异具有统计学差异（p＜0.05）。Annexin V-FITC/PI双染法检测得P1和P2组凋亡率分别为42.3±0.7%和45.4±0.9%,与空白对照组比较差异有统计学意义（p＜0.05）。P0组可见极少量细胞凋亡,与空白对照组比较差异无统计学差异（p＞0.05）。Hoechst染色显示P1和P2组HLECs细胞核变小,呈亮蓝色,可见核碎裂,而对照组的核呈均匀的蓝色。免疫印迹法测得P1、P2组细胞活化型caspase-3蛋白的表达较P0组、空白对照组增强。结论脂质体可有效介导BCL-2 shRNA转染HLECs。P1、P2可抑制HLECs的BCL-2基因表达。BCL-2基因表达抑制后,HLECs的细胞凋亡增加。第三部分PAMAM介导Bcl-2 shRNA转染诱导晶状体上皮细胞凋亡目的通过PAMAM G5介导Bcl-2 shRNA表达载体质粒,转染HLECs,观察BCL-2基因表达的情况及对HLECs细胞凋亡的影响。方法制备PAMAM G5/Bcl-2 shRNA复合物（质量比6:1）,转染人永生性HLECs系SRA01/04,同时设脂质体对照组、裸质粒对照组、空白对照组。48h后流式细胞仪检测转染效率,免疫印迹法检测bcl-2蛋白的表达,荧光定量PCR检测bcl-2 mRNA表达,并分别用Annexin V-FITC/PI双染法、Hoechst染色法、免疫印迹法检测caspase-3的表达等方法来检测HLECs的细胞凋亡情况。结果转染48h后,流式细胞仪检测转染率,PAMAM G5组为48.5±1.5%,脂质体组为41.1±1.8%,裸质粒组和空白对照组未见明显转染。PAMAM G5组转染率高于脂质体组,差异具有统计学差异（p＜0.05）。免疫印迹法和荧光定量PCR表明,PAMAM G5组可抑制bcl-2蛋白和mRNA的表达,与空白组比较差异具有统计学差异（p＜0.05）。Annexin V-FITC/PI双染法检测得PAMAM G5组凋亡率为44.7±1.2%,与空白对照组比较差异有统计学意义（p＜0.05）。Hoechst染色显示P1和P2组HLECs细胞核变小,呈亮蓝色,可见核碎裂,而对照组的核呈均匀的蓝色。免疫印迹法测得PAMAM G5组细胞活化型caspase-3蛋白的表达较空白对照组增强。结论PAMAM G5可有效介导Bcl-2 shRNA转染HLECs,转染率高于脂质体。转染后,BCL-2基因表达受抑制,HLECs的细胞凋亡增加。更多还原

【Abstract】 Posterior capsule opacification（PCO）is the most common complication of cataract surgery that causes visual impairment.The main cause of PCO is the proliferation,migration and metaplasia of remnant lens epithelial cells（LECs）after cataract surgery.Although we can use laser capsulotomy to treat this complication,it has some severe complications,such as macular edema and retinal detachment. Approaches for prevention of PCO,such as the improvement of surgical techniques and IOL design,and the use of antiproliferative drugs,have greatly decreased the PCO rate,however,the occurrence is still significant.Therefore,the development of an alternative therapy for preventing PCO is of critical importance.Recently,gene therapy for PCO is making headway,which is focused on suicide genes,cell cycle genes and cytokine genes.Gene therapy provides a novel strategy for the treatment of PCO,however,the present methods have their own drawbacks.RNA interference（RNAi）is one of the most exciting discoveries in the past few years. It is triggered by double-stranded RNA（dsRNA）and causes sequence-specific mRNA degradation of single-stranded target RNAs homologous in response to dsRNA.Since its discovery,RNAi show great potential as gene therapy.We plan to investigate whether apoptosis of LECs can be induced by inhibition of bcl-2 with RNAi.Polyamidoamine（PAMAM）dendrimers have received much attention as a new class of gene carriers over the past few years.PAMAM is a non-virus nanometer-sized carrier with the advantages of high transfection efficiency and long-term stable expression without immunogenicity and genotoxicity.In the present study,we used the fifth generation of PAMAM（PAMAM G5）to transfect LECs and compare its efficiency with liposome,the common non-viral vector of gene therapy.In the present study,we constructed two pairs of bcl-2 shRNA and transfer them to human lens epithelial cells（HLECs）with liposome and PAMAM G5,separately. The expression of bcl-2 and apoptosis of HLECs were investigated,and the transfection efficiency of liposome and PAMAM G5 were compared. PartⅠConstruction of Bcl-2 shRNA expression vectorPurpose To construct two pairs of Bcl-2 shRNA expression vectorsMethods Two pairs of Bcl-2 shRNA expression vectors were designed according to the Bcl-2 sequence in the GeneBank（No.NM₀00633）.The targeting point in the Bcl-2 cDNA was 1210-1228,2133-2151 seperately.Two pairs of oligonucleotides were synthesized,annealed and inserted into plasmid PGCsi to generate shRNA eukaryotic expression vectors,named P1 and P2.Then,they were transformed into competence bacterium DH5α.the reconstructed plasmids were amplified and purified. DNA sequence were confirmed by gene sequenator.Results Gene sequenator showed the inserted DNA sequence were correct completely. The Bcl-2 shRNA expression vector were constructed successfully.Conclusion The Bcl-2 shRNA expression vector were constructed successfully.PartⅡLiposome-mediated inhibition of bcl-2 by shRNA to induce apoptosis in human lens epithelial cellsPurpose To investigate whether apoptosis of human lens epithelial cells（HLECs） can be induced by liposome-mediated inhibition of bcl-2 shRNA.Methods HLECs（SRA01/04）with transfected with Lipofectamine^TM2000 by bcl-2 shRNA P1 and P2.At 48h after transfection,the transfection rate was measured by flow cytometry.The whole cell protein was extracted and the bcl-2 protein level was detected by Western blotting.The bcl-2 mRNA level was detected by real-time PCR. The percentage of HLECs undergoing apoptosis was measured by Annexin V-FITC/PI staining.The nuclear morphology of HLECs was observed by staining with Hoechst 33258.The activity of caspase-3 was analyzed by Western blotting.Results At 48h after transfection,the rate of transfection of P1 and P2 was about 44.1±1.7%and 47.2±1.6%respectively.The protein and mRNA level of bcl-2 was greatly down-regulated.The percentage of HLECs undergoing apoptosis was greatly improved.Hoechst staining showed that bcl-2 shRNA transfected cells were in a bad growth status with nuclear fragmentation.The activity of caspase-3 was greatly improved（P＜0.05）.Conclusion P1 and P2 can both down-regulate the expression of bcl-2,and induce the apoptosis of HLECs.It is feasible to use RNA interference mediated by liposome to induce the apoptosis of HLECs.PartⅢPAMAM-mediated inhibition of bcl-2 by shRNA to induce apoptosis in human lens epithelial cellsPurpose To investigate whether apoptosis of human lens epithelial cells（HLECs） can be induced by PAMAM-mediated inhibition of bcl-2 shRNA.Methods HLECs（SRA01/04）with transfected with PAMAM G5 by bcl-2 shRNA P1.At 48h after transfection,the transfection rate was measured by flow cytometry. The transfection rate mediated by PAMAM and liposome were compared.The whole cell protein was extracted and the bcl-2 protein level was detected by Western blotting. The bcl-2 mRNA level was detected by real-time PCR.The percentage of HLECs undergoing apoptosis was measured by Annexin V-FITC/PI staining.The nuclear morphology of HLECs was observed by staining with Hoechst 33258.The activity of caspase-3 was analyzed by Western blotting.Results At 48h after transfection,the rate of transfection of P1 mediated by PAMAM was about 48.5±1.5%,higher than liposome-mediated group which was about 41.1±1.8%.The protein and mRNA level of bcl-2 was greatly down-regulated. The percentage of HLECs undergoing apoptosis was greatly improved.Hoechst staining showed that bcl-2 shRNA transfected cells were in a bad growth status with nuclear fragmentation.The activity of caspase-3 was greatly improved（P＜0.05）.Conclusion PAMAM-mediated bcl-2 shRNA can down-regulate the expression of bcl-2,and induce the apoptosis of HLECs.It has a higher thansfection rate than liposome.更多还原