【作者】 郑庆丰；

【导师】 王建军； 应敏刚；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 实验一端粒酶逆转录酶siRNA表达质粒的构建、转化、抽提及鉴定目的：制备可供基因转染的端粒酶逆转录酶siRNA表达质粒。方法：设计靶向hTERT基因干扰序列GCATTCCTGCTCAAGCTGA，合成两条互补的单链DNA，排列次序如下：BamHⅠ酶切位点、正义序列、loop环、反义序列、RNA聚合酶Ⅲ终止子，SalⅠ酶切位点以及HindⅢ酶切位点。合成的寡核苷酸退火；采用BamHⅠ以及HindⅢ双酶切pGenesil-1表达载体，切胶回收线性化载体；退火产物与线性化pGenesil-1质粒表达载体相连接；构建hTERT-siRNA重组质粒。采用CaCl₂法制备感受态菌，将重组质粒转化感受态菌，抽提的重组质粒进行Sa1Ⅰ以及PstⅠ单酶切并行凝胶电泳分析，通过测序鉴定以确定插入的干扰片段的准确性，确认针对hTERT基因的siRNA表达质粒构建成功。用碱裂解法大量抽提，检测质粒的纯度和浓度。结果：BamHⅠ以及HindⅢ双酶切pGenesil-1表达载体，电泳可见约4800bp的线性化的质粒条带。重组质粒可被Sa1Ⅰ酶切出一条约400bp的DNA条带，无法被PstⅠ酶切；阴性对照质粒无法被Sa1Ⅰ酶切，能够被PstⅠ酶切出一条约400bp的DNA条带。测序表明目的序列准确地插到预计的位点。五次质粒大量抽提pGenesil-hTERT的A260／A280均在1.8～2.0间，浓度为1.30μg／μl～1.77μg／μl。结论：设计合成的RNA干扰序列准确地克隆到pGenesil-1表达质粒中，靶向hTERT基因的siRNA表达质粒构建成功。该载体可为深入研究端粒酶在肿瘤细胞中的作用机制提供新的手段。大量抽提的pGenesil-hTERT纯度和浓度均能满足基因转染的要求。实验二纳米羟基磷灰石表面修饰及与DNA的结合目的：应用多聚赖氨酸表面修饰纳米羟基磷灰石，研究纳米羟基磷灰石表面修饰及与DNA结合的可行性。方法：采用超声辅助均相沉淀法制备纳米羟基磷灰石。透射电镜观察纳米粒结构。分别在不同pH环境下进行纳米羟基磷灰石（nHAP）的多聚赖氨酸（PLL）修饰。检测nHAP及nHAP-PLL的Zeta电位。采用离心后测上清液DNA浓度及核酸酶消化实验考察PLL修饰后的nHAP结合、保护DNA的能力。结果：透射电镜下nHAP粒径比较均匀，约（15～20）nm×（60～80）nm左右，分散程度良好。nHAP经Na₂CO₃预处理后在pH 7.4及pH 7.6下可获得较理想的的PLL修饰效果。nHAP的Zeta电位为（—42.4±7.5）mV，nHAP-PLL的Zeta电位为（8.5±1.5）mV，两组比较差异有统计学意义（P＜0.01）。DNA结合及抗核酸酶保护实验显示nHAP-PLL和DNA质量比达20∶1时，nHAP-PLL可有效结合并保护相应DNA。结论：超声辅助均相沉淀法制备的羟基磷灰石为纳米级颗粒，粒径较小且均匀、分散程度良好。纳米羟基磷灰石经Na₂CO3预处理后可以有效地进行多聚赖氨酸修饰。经多聚赖氨酸表面修饰的纳米羟基磷灰石可以有效结合并保护DNA。实验三纳米羟基磷灰石介导人肺癌A549细胞体外基因转染目的：探讨经多聚赖氨酸修饰的纳米羟基磷灰石介导人肺癌A549细胞基因转染的可行性。方法：根据实验二的方法配制nHAP-PLL-DNA复合物。实验分为nHAP-PLL组、脂质体组及对照组，分别配制转染复合物并转染A549细胞。荧光显微镜观察EGFP的表达；流式细胞仪检测pGenesil-1的转染率。结果：nHAP-PLL组和脂质体组均见EGFP表达阳性的A549细胞，对照组未见EGFP表达阳性的A549细胞；nHAP-PLL组pGenesil-1转染A549细胞的转染率为（31.8±1.9）％，与脂质体组的转染率（33.4±3.7）％差异无统计学意义（P＞0.01），对照组未见转染阳性的A549细胞。结论：纳米羟基磷灰石经多聚赖氨酸表面修饰后可介导人肺癌A549细胞体外基因转染，有望成为一种新型的基因转染载体。实验四纳米羟基磷灰石介导靶向端粒酶逆转录酶RNA干扰对人肺癌A549细胞的影响目的：探讨纳米羟基磷灰石介导靶向端粒酶逆转录酶RNA干扰对人肺癌A549细胞的影响方法：根据实验二的方法配制nHAP-PLL-DNA复合物。实验分为nHAP-PLL组、脂质体组、nHAP组及对照组，分别配制转染复合物并转染A549细胞。四唑盐（MTT）法测定细胞生长活力；应用RT-PCR及Western Blot检测各实验组A549细胞hTERTmRNA及蛋白表达，TRAP-ELISA检测端粒酶活性；流式细胞仪检测细胞凋亡率。结果：nHAP-PLL组、脂质体组与nHAP组A549细胞的增殖受到抑制。nHAP-PLL组、脂质体组与nHAP组细胞增殖较对照组差别有统计学意义（P＜0.05）；nHAP-PLL组抑制细胞增殖较脂质体组及nHAP组明显，差别均有统计学意义（P＜0.05）。各组的hTERT mRNA、蛋白表达及端粒酶活性的变化趋势与A549细胞的增殖情况检测结果一致。流式细胞仪检测nHAP-PLL组、脂质体组、nHAP组及对照组细胞凋亡率分别是（28.1±1.4）％，（19.2±1.3）％，（10.9±1.2）％与（0.3±0.2）％。nHAP-PLL组、脂质体组及nHAP组均能诱导A549不同程度细胞凋亡，凋亡率与对照组差别有统计学意义（P＜0.05）；nHAP-PLL组细胞凋亡较脂质体组及nHAP组明显，差别均有统计学意义（P＜0.05）。结论：纳米羟基磷灰石可诱导人肺癌A549细胞凋亡。人肺癌A549细胞端粒酶逆转录酶高表达，端粒酶逆转录酶可成为抑制A549的理想靶点。纳米羟基磷灰石本身具有抑制A549细胞的作用，又能有效地保护DNA并介导A549细胞靶向端粒酶逆转录酶的RNA干扰，使之在肺癌基因治疗领域具有重要的应用价值。更多还原

【Abstract】 ExperimentⅠTransformation, extraction and identification of siRNA expressionplasmid targeting human telomerase reverse transcriptaseObjective: To prepare the siRNA expression plasmid targeting human telomerase reversetranscriptase for gene transfection.Methods: An interference target sequence GCATTCCTGCTCAAGCTGA was designed,which aimed at hTERT gene, and then two strands of oligonucleotides were synthesizedwhich including the sequence above.The sequence of the oligonucleotides contained theparts as follow: the BamHⅠsite, positive-sense sequence, loop sequence, antisensesequence, the terminator of RNA polymeraseⅢ, the Sa1Ⅰsite and the HindⅢsite.Thetwo strands of oligonucleotides formed a double-strand DNA after annealing.ThepGenesil-1 plasmid was digested with BamHⅠand HindⅢ, and then purified and retrievedthe 4800bp linear plasmid by agarose electrophoresis separation.The double-strand DNAwas inserted downstream from the U6 promoter of the linear plasmid.The siRNAexpression vector pGenesil-hTERT was constructed.The susceptible E.coli DH5a whichwas prepared by CaCl₂ method.Then, the recombinant plasmid was transfected into thesusceptible E.coli DH5a, and the positive colonies were chosen.The extracted recombinantwas digested by Sa1Ⅰand PstⅠrespectively, and analyzing the result by agarose gelelectrophoresis and DNA sequencing.With alkaline lysis method, plasmids were extractedon a large scale and measured for purity and quantity with spectrophotometer.Results: A linear plasmid vector strip about 4800bp could been seen through agarose gelelectrophoresis analysis after the pGenesil-1 plasmid had been digested with BamHⅠandHindⅢ.The two plasmids were digested by Sa1Ⅰand PstⅠrespectively.Electrophoresis showed that the recombinant plasmid could be digested by Sa1Ⅰand yield a nearly 400bpfragment but could not be digested by PstⅠ.The negative Control recombinant plasmidcould not be digested by Sa1Ⅰbut could be digested by PstⅠand yield a nearly 400bpfragment.The sequencing identification proved that the designed sequence had insertedinto the predicted site precisely.The OD260/280 ratios of all the extracted plasmids rangedfrom 1.8 to 2.0 and the concentration from 1.30μg/μl to 1.77μg/μl.Conclusions: The synthesized shRNA interference sequence cloned to pGenesil-1expression plasmid precisely, and the siRNA expression plasmid that aimed at hTERT wasconstructed successfully.The pGenesil-hTERT can provide new method to the research ofeffect of telomerase in tumor cell.Purity and quantity of pGenesil-hTERT extracted withalkaline lysis method meet the requirement of gene transfection.ExperimentⅡSurface modification and binding with DNA of hydroxyapatitenanoparticlesObjective: To synthesize hydroxyapatite nanoparticles （nHAP） modified withpoly-L-lysine （PLL）, and investigate the feasibility of surface modification of nHAP andthe capability of nHAP in combining and protecting DNA.Methods: The nHAP were synthesized by the ultrasound-assisted homogeneousprecipitation method.The structure of the nanoparticles was observed under transmissionelectron microscope.The resulting nHAP were modified with PLL in various PH solution.Zeta potential of nHAP and nHAP modified with poly-L-lysine （nHAP-PLL） was detectedrespectively.The nHAP-PLL were tested for the capacity of combining DNA by measuringDNA concentration in supernatant after centrifugation.The capacity of protecting DNA of nHAP-PLL was tested by the experiment of DNA degradation.Results: The synthesized hydroxyapatite particles presented well dispersed particles withevenly distributed sizes of （15～20）nm×（60～80）nm under transmission electronmicroscope.Pretreated by Na₂CO₃, nHAP were effectively modified with PLL in PH7.4and PH7.6 solution.The Zeta potential of nHAP was （-42.4±7.5）mV.The Zeta potentialof nHAP-PLL was （8.5±1.5）mV.The Zeta potential of nHAP-PLL was significantlyhigher than that of nHAP （P<0.01）.DNA binding test showed that nHAP-PLL couldcompletely combine DNA when the weight/weight ratio of nHAP-PLL and DNA was morethan 20:1.The nHAP-PLL/DNA mixtures with weight/weight ratio upwards of 20:1 couldeffectively protect DNA against being digested by DNaseⅠ.Conclusions: Through ultrasound-assisted homogeneous precipitation method, nHAP canbe synthesized with well dispersed particles and evenly distributed sizes.The Na₂CO₃pretreated nHAP can be well surface-modified with Poly-L-lysine and effectively combineand protect DNA.ExperimentⅢHydroxyapatite nanoparticles mediated gene transfection to A549human lung cancer cells in vitroObjective: To investigate the capability of nHAP-PLL as a carrier for gene transfection toA549 human lung cancer cells in vitro.Methods: The nanoplexes of nHAP-PLL-DNA were made according to methods ofexperiment tow.The transfection of pGenesil-hTERT into A549 was divided into threegroups as follows: nHAP-PLL group mediated by hydroxyapatite nanoparticles modifiedwith poly-L-lysine （nHAP-PLL）, liposome group mediated by Lipofectamine and control group.After 48 hours transfection, the expression of EGFP was observed with fluorescentmicroscope.Flow cytometry was used to detect the transfection rate of each group.Results: under fluorescent microscope, the expression of EGFP was detected in A549 cellsof nHAP-PLL group and liposome group while no expression of EGFP was seen in A549cells of control group at 48 hours after transfection.Flow cytometry analyses showed that,the transfection rate of A549 cells was （31.8±1.9）% of nHAP-PLL group and （33.4±3.7）% of liposome group.Compared with the control group, there was significantdifference respectively （P<0.01）, but there was no significant difference of transfection ratebetween the nHAP-PLL group and liposome group （P>0.01）.Conclusion: The nHAP modified with poly-L-lysine can mediate the transfection ofplasmid into A549 human lung cancer cells in vitro and may be used as a potential genecarrier.ExperimentⅣHydroxyapatite nanoparticles mediated human telomerase reversetranscriptase RNA interference of A549 human lung cancer cells in vitroObjective: To investigate the effect of nHAP mediated human telomerase reversetranscriptase RNA interference of A549 human lung cancer cells in vitroMethods: The nanoplexes of nHAP-PLL-DNA were made according to methods ofexperiment tow.The transfection of pGenesil-hTERT into A549 was divided into fourgroups as follows: nHAP-PLL group mediated by hydroxyapatite nanoparticles modifiedwith poly-L-lysine （nHAP-PLL）, liposome group mediated by Lipofectamine, nHAP groupmediated by hydroxyapatite nanoparticles and control group.The growth ability of cellswas assayed with methyl thiazolyl tetrazolium method.The level of hTERT mRNA was examined by RT-PCR.The expression level of hTERT protein was analyzed by Westernblotting.Telomerase activity was detected by TRAP-ELISA.Flow cytometry was used todetect the apoptosis ratio of A549.Results: The proliferation of A549 cells of nHAP-PLL group, liposome group and nHAPgroup were obviously inhibited as compared with the control group （P<0.05）.Theinhibition rate of nHAP-PLL group was more than the other groups.There was a significantdifference of inhibition rate between the nHAP-PLL group compared with the liposomegroup and nHAP group （P<0.05）.The level of hTERT mRNA, hTERT protein andtelomerase activity had similar varietal tendencies with the results of proliferation of eachgroup.Flow cytometry showed the apoptosis ratio of nHAP-PLL group, liposome group,nHAP group and control group was （28.1±1.4）%, （19.2±1.3）%, （10.9±1.2）% and（0.3±0.2）% respectively.There was a significant difference of apoptosis ratio between thenHAP-PLL group, liposome group and nHAP group compared with the control group（P<0.05）.In contrast to the nHAP-PLL group, the liposome group and nHAP groupdemonstrated a significantly lower apoptosis rate （P<0.05）.Conclusion: Hydroxyapatite nanoparticles can induce apoptosis of A549 human lungcancer cells in vitro.A549 cells overexpress human telomerase reverse transcriptase,hTERT may be a target for inhibiting proliferation of A549.Hydroxyapatite nanoparticleshave some characters as follow: inhibiting proliferation of A549, combining and protectingDNA and mediating hTERT RNA interference of A549.Therefore, hydroxyapatitenanoparticles may be a useful material in the field of lung cancer gene therapy.更多还原