【作者】 杨瑾；

【导师】 孙慧敏；

【作者基本信息】 天津医科大学 ， 眼科学， 2010， 博士

【摘要】 目的：用无免疫原性的非病毒载体阳离子聚酰胺-胺型树枝状聚合物(PAMAM-D)纳米颗粒做为载体,介导双自杀基因TK及CD,转染人Tenon’囊成纤维细胞(HTFs),观察其在体外对HTFs的杀伤情况及未转染细胞的“旁观者效应”,进而寻找一种安全、有效抑制青光眼滤过手术后滤过通道瘢痕化的治疗方法。方法：1.采用PCR、酶切、连接等技术融合自杀基因yCD和HSV-TK构建含双自杀基因的基因表达质粒pAcGFP1-Hyg-TK-CD,并用酶切及测序的方法证实其正确性；2.利用增强型绿色荧光蛋白真核表达质粒pEGFP-C1作为报告基因,选择第二第五代PAMAM-D作为基因转移载体,并以脂质体LipofectamineTM 2000为对照转染HTFs细胞,应用透射电镜、动态激光散射和电位分析仪等测定各载体表征；抗核酸酶试验检测载体对基因的保护能力；通过改变质粒DNA的浓度、转染复合物质量比等条件参数,优化PAMAM-D转染方案；3.体外培养HTFs,经细胞形态学、组织学及免疫细胞化学鉴定；以PAMAM-D纳米颗粒为载体,将载有双自杀基因的质粒pAcGFP1-Hyg-TK-CD转染HTFs,分别用RT-PCR、Westen-blot等方法检测鉴定双自杀基因TK、CD在HTFs内的表达；4.MTT法检测5-FC、GCV对HTFs的毒性及自杀基因前药系统对表达自杀基因的HTFs-TK-CD细胞的杀伤效应；选择前药5-FC、GCV最佳的作用药物浓度,使其细胞毒性最小,对细胞的杀伤作用最强；光镜及透射电镜观察前体药物GCV及5-FC作用后HTFs的形态变化,流式细胞仪检测HTFs的凋亡率,并观察双自杀基因的“旁观者效应”结果：1.实验构建的质粒酶切产物琼脂糖凝胶电泳所得片段大小为1617bp,与预期片段一致；测序证实最终质粒与pAcGFP1-Hyg-TK-CD序列一致。2. PAMAM-D纳米颗粒在pH 5-10具有高的缓冲能力,PAMAM-D/DNA转染复合物的平均粒径小于100 nm；且PAMAM-D可以在保持质粒DNA完整性的同时增加其对核酸酶的抵抗力；转染质量比对载体的转染效率影响最明显,G2-PAMAM、G5-PAMAM、Lipid与DNA的最佳转染质量比分别为8：1、2：1、4：1,在最佳质量比条件下转染细胞,G5-PAMAM的转染效率明显高于G2-PAMAM (42.1% vs19.4%,P<0.05)3.成功培养出HTFs细胞,转染双自杀基因的HTFs-TK-CD细胞分别经RT-PCR及Westen-blot检测鉴定,证明分别有双自杀基因TK及CD mRNA及蛋白质在HTFs内的表达；4.采用GCV3μg/ml、5-FC 200μg/ml是本实验选择的最佳前体药物浓度。光镜下未转染自杀基因的HTFs细胞,应用前体药物GCV和/或5-FC后,细胞生长状态和增殖无明显变化；且GCV和5-FC联合用药较二者单独用药无明显差异；转染自杀基因的HTFs细胞,应用前体药物后,与对照组相比细胞生长状态变差,细胞数量减少；且GCV和5-FC联合应用较二者单独应用细胞生长状态和数量变化更明显。透射电镜下表现细胞的凋亡变化。HTFs-TK-CD组加入前体药物GCV 3μg/ml、5-FC 200μg/ml及后,细胞凋亡率分别为11.86%和22.37%,二者联合应用时,凋亡率为33.13%。GCV与5-FC不仅能杀伤转染的HTFs细胞,而且具有“旁观者效应”结论：1.利用基因工程技术,使用CD和TK成功构建了含双自杀基因的基因表达载体pAcGFP1-Hyg-TK-CD,经检测及测序证实其正确性；2. PAMAM-D纳米载体安全低毒,在较大的pH范围内和不同缓冲液条件下能稳定存在且具有DNA保护功能,其转染效率主要取决于PAMAM-D与DNA复合物的质量比,同时受质粒DNA浓度、PAMAM-D代数等因素的影响；3. G5-PAMAM-D纳米颗粒介导HSV-TK/GCV联合CD/5-FC双自杀基因系统成功转染HTFs,并通过RT-PCR及Western blot检测证实得到表达；4.双自杀基因TK、CD转染HTFs细胞后,给予前药5-FC、GCV达到一定浓度时,它们转化而成的细胞毒性物质对HTFs细胞在体外产生杀伤作用,采用GCV3μg/ml、5-FC 200μg/ml是本实验选择的最佳前体药物浓度。且联合应用5-FC+GCV对HTFs细胞在体外产生的杀伤作用强于二者单独应用,并存在“旁观者效应”,为最终提高青光眼手术成功率奠定基础。更多还原

【Abstract】 Objective:Double suicide gene pAcGFP1-Hyg-TK-CD mediated by nanoparticals PAMAM dendrimers which is non-immunogenicity as non-virus delivery to transfect Human Tenon’s capsule fibroblasts (HTFs), and observe it’s killing effect to HTFs and ’bystander effects’, in order to look for a safe and effective method for HTFs inhibition modulates after glaucoma filtration surgery.Methods:1. Construct the expression plasmid vector of pAcGFP 1-Hyg-TK-CD with PCR,enzyme restriction and ligation. The vector was ideniified by enzyme cutting and Sequencing.2. EGFP reporter gene was transfected into HTFs using the second-and fifth-generation PAMAM-D and Lipofectamine 2000 respectively.The surface properties of vectors were evaluated with transmission electronic microscope, dynamic laser scattering and zeta potential analyzer. Detect the protection effect to DNA from DNase digestion. A number of variables for optimal transfer of the transfection complexes were tested, including concentrations of plasmid DNA, weight ratios of DNA:vector and dendrimer generations.3. The HTFs were cultured in vitro,and identified by means of morphological, histological and immunocytochemical methods. Nanoparticals PAMAM-D mediated plasmid pAcGFP1-Hyg-TK-CD with TK and CD was used to deliver HTFs, it was detect the expression of TK and CD in HTFs by means of RT-PCR and Westen-blot respectively.4. Detect cytotoxicity and lethal effec of GCV and 5-FC to HTFs cells by MTT assay. Select the optimal concentration of GCV and 5-FC which are low cytotoxicity and high killing effect to HTFs-TK-CD. To observe the morphology changes of transfected HTFs cells after use GCV and 5-FC by light microscope and transmission electronic microscope. Detect the apoptosis rates of HTFs by flow cytometry,and also detect the "by stander effect’of double suicide gene.Results: 1. We got 1617bp fragments through enzyme cutting.The result of sequencing was equal to the expection.2. Our reserch demonstrated that relatively high buffer capacities of PAMAM-D occurred at PH ranges is from 5 to 10. The average size of DNA/dendrimer complexes was below 100nm, and showed an excellent resistance against DNase I digestion. Variables such as concentrations of plasmid DNA and dendrimer generations influenced the transfection efficiency to some extent, whereas the weight ratios of complexes exhibited the most significant effects on transfection level. Optimal weight ratios of G2-PAMAM、G5-PAMAM、Lipid that DNA/dendrimer complexes are 8:1、2:1、4:1, The transfection efficiency of G5 PAMAM-D was higher than of G2 AMAM-D (42.1% vs 19.4%,P<0.05) at optimal weight ratios.3. The HTFs were cultured successfully in vitro. It is identified by RT-PCR and Western-blot that the mRNA and the protein expression of double suicide gene TK and CD respectively.4. The concentration of GCV 3μg/ml、5-FC 200μg/ml is the optimal choise of prodrugs.The light microscope showed that the growth of untransfected HTFs were in good condition, there were no obvious changes of cell population and cell proliferation, the changes with the combination of GCV and 5-FC were no difference from single prodrugs; however, the growth condition of HTFs which transfected double suicide gene went bad and the cells number decreased, especially treated with both GCV and 5-FC.Transmission electronic microscope shows cell apoptosis phenomenon in HTFs.After use GCV 3μg/ml,5-FC 200μg/ml and GCV 3μg/ml+5-FC 200μg/ml in HTFs-TK-CD group, the apoptosis rate is 11.86%,22.37% and 33.13% respectively. Prodrug of GCV and 5-FC not only could kill the transfected HTFs, but also has’by stander effect’.Conclusion:1. Gene engineering technique were used to construct the expression plasmid vector of pAcGFP1-Hyg-TK-CD with CD and TK. The vector was ideniified by enzyme cutting and Sequencing.2. Nanoparticals PAMAM dendrimers is safe and low cytotoxicity, with relatively large PH ranges and high buffer capacities, it was also shown an excellent protection to DNA. The weight ratios of complexes play a key role in transfection efficiency, variables such as concentrations of plasmid DNA and dendrimer generations influenced the transfection efficiency to some extent.3. Double suicide gene TK and CD mediated by nanoparticals PAMAM-D was transfected HTFs, it was detect the expression of double suicide gene TK and CD in HTFs by means of RT-PCR and Westen-blot respectively4. Prodrugs GCV and 5-FC were used in suitable concentration after TK and CD were transfected the HTFs, The concentration of GCV 3μg/ml、5-FC 200μg/ml is the optimal choise of prodrugs.The prodrug could be trnsfered into cytotoxicity material to kill HTFs in vitro, the killing effect that combination of GCV and 5-FC were stronger than single one, it also has’by stander effect’. We can believe that this study will provide a sound basis for glaucoma filtertion surgery.更多还原