载ACEshRNA-PEG壳聚糖纳米粒的制备以及对自发性高血压大鼠的治疗作用
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摘要
目的:
首先制备壳聚糖纳米粒,并且给予PEG表面修饰,分析其相关物理学、生物学特性,以期获得一种缓释的非病毒载体介导系统,其次行培养的大鼠血管内皮细胞转染,观察其转染效率,细胞毒性等,并从分子蛋白水平观察对ACE基因的下调作用,最后以筛选出的合适剂量转染自发性高血压大鼠,观察降压疗效以及对靶器官的影响等生物效应。
方法:
1载ACEshRNA-PEG-壳聚糖纳米粒制备以及生物学特性
1 )应用本实验室前期实验中筛选出的能显著下调ACE基因的靶序列:5’—AACCTAACATGTCAGCCTCTG—3’(基因序列位点3603-3624),由武汉晶赛公司协助合成质粒,采用菌液用碱裂解法大量抽提和纯化重组质粒,随机酶切测序,并检测质粒浓度与纯度。
2)采用离子交联法制备壳聚糖纳米粒, PEG修饰壳聚糖纳米粒,复凝聚法制备不同PH值、体积比以及PEG化的载ACEshRNA壳聚糖纳米。喷金电镜下对各种壳聚糖纳米粒悬液扫描并照相,观察纳米粒与形态。同时使用zeta电位仪测定其颗粒大小、zeta电位值和多分散度。应用凝胶阻滞分析验证载ACEshRNA壳聚糖纳米多聚复合物的形成及电荷性质。紫外分光光度计检测离心后上清液中DNA的含量,计算包埋率,绘制PEG-CS/DNA纳米复合物累积释放曲线,以及分析纳米粒对核酸酶的抗性。
2.载ACEshRNA-PEG-壳聚糖纳米粒对血管内皮细胞转染条件的筛选
1)组织贴块法体外培养大鼠血管内皮细胞,免疫荧光法检测胞浆Ⅷ因子进行鉴定,台盼兰染色检测细胞成活率。
2)分别以较佳配比率(纳米粒:质粒为1:1)结合的CS-DNA纳米复合物组以及PEG-CS-DNA纳米复合物60ul、80ul、100ul、120ul转染血管内皮细胞0-96h,并以相应的裸质粒组以及空白组作为对照。
3)荧光显微镜下观察转染细胞绿色荧光的表达,流式细胞仪检测转染效率,MTT法测定细胞活性。
3 .载ACEshRNA-PEG-壳聚糖纳米粒转染体系抑制大鼠血管内皮细胞ACE表达的研究
1)应用Real-time荧光定量PCR检测各组PEG-CS-DNA纳米复合物转染前后ACE mRNA的表达.并以裸质粒和空白组为对照。
2)于转染前及转染后的24h、48h、72h收集各组细胞,用Western-blot法检测ACE蛋白的表达。
3)于转染前及转染后的72h收集培养液2mL,用酶联免疫方法检测ACE以及Ang-Ⅱ的含量。
4.载ACEshRNA-PEG-壳聚糖纳米粒转染体系介导RNAi对自发性高血压大鼠的影响
1)研究对象为12周龄成年雄性自发性高血压大鼠(SHR)和高血压模型对照鼠,采用尾静脉注射DNA含量为25ug,无菌生理盐水稀释后500ul载ACEshRNA-PEG-壳聚糖纳米复合物, 10天重复给药一次。并以每日灌胃给予洛丁新10mg/日/只作为对照,同时设SHR空白对照组、壳聚糖对照组以及正常血压对照组,分别尾静脉注射500ul无菌生理盐水或相同浓度的壳聚糖纳米液。
2)分别于注射前及注射后每1-3天的相同时间点采用尾袖法在安静环境中测量大鼠清醒状态下尾动脉收缩压(SBP)和心率。
3)荧光定量RT-PCR或Western-blot法检测心肌、主动脉、肾脏ACE mRNA或蛋白的表达,并应用酶联免疫法检测各组血清ACE以及Ang-Ⅱ含量的变化,全自动生化分析仪检测肝肾功能。
4)实验结束时,行左侧颈动脉插管测定左心室功能血流动力学检查,并称取全心(HW)、左室重量(LW),求全心/体重、左心室/体重比值,并且制备心、肾及主动脉组织冰冻切片,荧光显微镜下检测载ACEshRNA-PEG-壳聚糖纳米粒的分布,光镜以及透射电镜行心肌组织学检查。
结果:
1.载ACEshRNA-PEG-壳聚糖纳米粒制备以及生物学特性
1.1经紫外分光光度计检测质粒DNA浓度为0.5ug/ul,纯度为1.88。
1.2各因素对壳聚糖纳米复合物理化性质的影响:(1)壳聚糖纳米粒的粒径随着溶液pH值的升高而增大,当pH值为5.5时的PEG壳聚糖纳米粒平均粒径125.8±5.6nm左右,大小均匀,多分散度最小,分布比较集中,zeta电位为正,有利于与带负电荷的质粒结合。(2)PEG化后也对粒径以及zeta电位无明显影响,但是与壳聚糖相同条件相比,分散度均明显减小,可见更适合制备均匀的纳米粒,利于和质粒结合。(3)壳聚糖与质粒体积比(质量比)为1:1制备的壳聚糖纳米粒平均粒径较小,故通过细胞膜的通透性较好,多分散度较小,分布比较集中, zeta电位也为正值,有利于与带负电荷的质粒结合。
1.3各因素对壳聚糖与质粒结合力的影响:(1)壳聚糖纳米以及PEG化壳聚糖纳米质粒复合物均有效结合质粒,由于中和了质粒所带的负电荷,凝胶电泳时,质粒不出孔,而裸质粒则出孔。(2)PH<7时壳聚糖分子中大部分的氨基带正电荷能与质粒DNA有效地结合质粒不出孔。(3)在体积比为1:1、1:2、1:3时,壳聚糖纳米粒能有效地结合质粒.
1.4各因素对复合物包埋率的影响:(1)pH值为5.5时的壳聚糖纳米复合物的DNA包埋率为最高,为(90.43±3.9)%。(2)各组经PEG表面修饰后包埋率明显增高。(3)当体积比为1:1,1:2时包埋率较高,分别为(88.87±13.2)%与(80.13±12.9)%,两组间无显著差异;之后随着该比例增高,包埋率明显下降。
1.5各因素对复合物体外释放的影响:(1)CS-DNA纳米复合物在PBS溶液pH值为3.5-7.5时释放的情况基本相同,均存在开始阶段的爆破释放,释放曲线前段接近直线,约4天(96小时)后,释放量开始稳定,释放曲线缓慢上升,平稳维持释放8天左右。而纳米粒在pH值为9.0的PBS溶液中快速释放,只释放了4天左右。(2)在固定PH=5.5时,各不同体积比例的载基因复合物PBS溶液释放均存在开始阶段的爆破释放,释放曲线快速上升,约4天(96小时)后,释放量开始稳定,释放曲线缓慢上升,但是释放持续时间随着体积比的增大而缩短,在体积比为1:1时,释放时间最长,平稳维持释放8天左右;体积比为1:5时,释放时间最短,仅维持4天左右。(3)取体积比为1:1,pH=5.5的载ACEshRNA壳聚糖纳米粒经PEG化后,没有影响复合物的体外释放的爆破释放特点,约4天(96小时)后,释放量开始稳定,释放曲线缓慢上升,但是释放持续时间延长至10天左右。
1.6 DNaseⅠ消化实验:(1)PEG化载ACEshRNA壳聚糖纳米在体积比为1:1—1:3均能有效抵抗DNA降解,质粒被完全阻滞于加样孔中,体积比为1:4,1:5的载基因壳聚糖纳米复合物则可以显示条带,说明对核酸酶的保护作用降低。(2)PEG化壳聚糖纳米粒能有效地保护质粒不被DnaseⅠ消化,复合物不出孔,而裸质粒则完被DnaseⅠ消化,加样孔内以及条带消失。(3)PH=3、5.5、7时,均能有效地对质粒DNA有保护核酸酶降解的作用,当PH=11.0时,则可以看到明显的条带,表明对核酸酶的保护作用降低。
2.载ACEshRNA-PEG-壳聚糖纳米粒对血管内皮细胞转染条件的筛选
2.1免疫荧光法鉴定证实培养获得稳定的大鼠血管内皮细胞,细胞存活率达95%以上。
2.2转染效率的测定:(1)用荧光显微镜观察可以看到有绿色荧光的表达,而裸质粒组和空白对照组均未见荧光表达。(2)流式细胞仪检测转染率:1)未经PEG化修饰的壳聚糖质粒纳米粒对内皮细胞的转染率均很低,最高达(26.0±3.9)%,PEG对壳聚糖质粒纳米复合物进行化学修饰后,转染效率比修饰前的转染效率明显有提高,最高达(59.4±5.2)%。2)PEG-CS/DNA纳米复合物的量为100ul,即质粒DNA含量为25ug时转染率最高,在一定的范围内转染率的高低与转染的复合物量呈正比,超出一定的量转染率反而开始下降。3)固定转染的体积时,转染不同时间,随着时间的延长,转染效率逐渐增高,72h达高峰(57.1±7.1),(p<0.05);96小时开始有所下降,但仍显著高于0h(p<0.05)。
2.3各转染条件下载ACEshRNA-PEG-壳聚糖纳米复合物转染细胞毒性的测定:当转染的复合物<100ul时与空白对照组比较差异无统计学意义,说明对细胞无毒性作用;而当复合物的量为120 ul时在转染的24h时细胞生长出现了明显的抑制现象,并且随着时间的延长抑制作用逐渐增大(p<0.05)。
3.载ACEshRNA-PEG-壳聚糖纳米粒转染体系抑制大鼠血管内皮细胞ACE表达的研究
3.1转染前后不同组ACE基因mRNA表达的变化:转染载ACEshRNA-PEG-壳聚糖纳米复合物后24小时细胞ACE mRNA表达水平下降达(14.7±5.9)%,48小时达(53.6±5.4)%,与空白对照组和质粒对照组相比有统计学差异(均P﹤0.05),72小时为(60.1±2.1)%。
3.2转染前后不同组细胞ACE蛋白表达的变化:PEG-CS/DNA组转染后24小时细胞ACE蛋白表达无明显变化,48小时明显降低,与空白对照组和质粒对照组相比有统计学差异(P﹤0.05),72小时更低(P﹤0.01),而空白对照组与质粒对照组转染前后各时间点ACE的蛋白表达无明显变化,与RT-PCR结果一致。
3.3血管内皮细胞培养液中ACE以及Ang-Ⅱ的含量;PEG-CS/DNA组转染72小时后,细胞培养液中的ACE与Ang-Ⅱ含量明显减少,与空白对照组以及裸质粒组相比有显著差别(P<0.05)。可见,载ACEshRNA PEG-CS纳米转染系统不仅可以在分子蛋白水平显著下调ACE基因,而且在功能上也显著减少ACE的含量,降低Ang-Ⅱ的合成。
4.载ACEshRNA-PEG-壳聚糖纳米粒转染体系介导RNAi对自发性高血压大鼠的影响
4.1治疗前后尾动脉压及心率的变化:SHR各组之间干预前尾动脉压差异无统计学意义(P﹥0.05),SHR各组干预前尾动脉压均显著高于正常血压对照组(P﹤0.01)。SHR基因治疗组于首次注射后第3天,尾动脉压显著下降约(22±4)mmHg,与治疗前比较有显著性差异(P﹤0.05),之后下降缓慢,降压作用可持续8天左右,最大降压幅度达33mmHg,于注射后约11天时血压开始回升;第2次注射后,尾动脉压再次明显下降约(24±5)mmHg,降压作用可持续10天左右,血压回升,最大降压幅度约25mmHg。两次注射后最大降压幅度累积达50mmHg。洛丁新治疗组开始使用后第3天血压持续降低,于第13天达最大降压幅度39 mmHg,明显低于基因治疗组最大降压幅度(p<0.05)。随后7天内血压无明显下降。SHR空白对照组和壳聚糖对照组尾动脉压持续升高。正常血压对照组尾动脉压无明显变化。各组大鼠治疗前后心率均无明显变化(P >0.05),
4.2 PEG-CS-EGFP-ACE-shRNA的组织器官分布情况:心脏、主动脉、肾脏组织都可见大量绿色荧光表达,与ACE在体内分布一致,说明载ACEshRNA-PEG-CS纳米载体经尾静脉注射后可分布在富含ACE的组织中。
4.3 PEG-CS-EGFP-ACE-shRNA转染72h后对心,肾,主动脉的ACE功能的下调作用: 1)RT-PCR结果:SHR基因治疗组心肌、主动脉、肾脏的ACE mRNA表达均较SHR空白对照组、SHR壳聚糖对照组、药物治疗组显著降低,差异有统计学意义(P﹤0.05),而与正常血压对照组无统计学差异(P﹥0.05)。说明SHR大鼠在尾静脉注射PEG-CS-EGFP-ACE-shRNA后,能显著降低ACE mRNA的表达。2)Western blot结果:SHR基因治疗组心肌、主动脉、肾脏的ACE蛋白表达均较SHR空白对照组、SHR病毒对照组以及药物治疗组显著降低,差异有统计学意义(P﹤0.05),而与正常血压对照组无统计学差异(P﹥0.05),与RT-PCR结果一致。说明SHR大鼠在尾静脉注射载ACEshRNA-PEG-壳聚糖纳米粒后,随着ACE mRNA的表达降低,相应功能蛋白的表达也随之降低。3)对血清ACE以及Ang-Ⅱ的含量的影响:转染72小时后,SHR基因治疗组ACE以及Ang-Ⅱ显著降低,低于SHR空白对照组、壳聚糖对照组、以及药物治疗组,差异有统计学意义(P﹤0.05),而与正常血压对照组无统计学差异(P﹥0.05)。SHR药物治疗组ACE治疗前后无明显变化,但是Ang-Ⅱ显著降低,证实ACEI类药物可以抑制Ang-Ⅱ的合成。
4.4对左室结构以及功能的影响:1)血流动力学检测左室功能:左室舒张压(LVDP)、左室舒张末压(LVDEP)以及最大舒张速度(- dp/dtmax)在SHR各组均高于正常血压组,其中空白对照组、壳聚糖对照组明显高于药物治疗组以及基因治疗组(P<0.05),可见高血压早期即有舒张功能不全,并且基因治疗组与药物治疗组均可显著改善舒张功能不全,两者相比明显有差别,前者更显著降低LVDP与(- dp/dtmax),说明载ACEshRNA-PEG-壳聚糖纳米复合物能更有效地显著改善高血压时的舒张功能不全。2)对全心/体重、左心室/体重的影响:SHR空白对照组和壳聚糖对照组的全心/体重和左心室/体重显著高于正常血压血压组(P﹤0.05),提示未经治疗的SHR有明显的心肌肥厚现象。药物治疗组和基因治疗组上述指标均显著下降(P﹤0.05),其后者降低更明显,但均未降到正常血压对照组水平,可见与ACEI类药物相比,载ACE-shRNA-PEG-壳聚糖纳米复合物改善心肌肥厚的作用更明显。3)组织学观察:光镜下观察到SHR空白对照组和SHR壳聚糖对照组较正常血压对照组心肌细胞明显肥大,SHR基因治疗组即尾静脉注射载ACE-shRNA-PEG-壳聚糖纳米复合物和药物治疗组均使心肌细胞肥大明显减轻。电镜下观察到:SHR空白对照组和壳聚糖对照组相比, SHR基因治疗组以及药物治疗组心肌细胞核膜完整,肌原纤维清晰,排列较整齐,横纹清楚可见,线粒体无肿胀,但局部可见少量增多,心肌间质未见明显胶原纤维增生,说明载ACE-shRNA-PEG-壳聚糖纳米复合物与ACEI类药物都可以改善SHR心肌超微结构的改变。
4.5对肝肾功能的影响:各组大鼠之间肝肾功能指标无统计学差异(P﹥0.05),表明载ACE-shRNA-PEG-壳聚糖纳米复合物对肝肾功能无影响。
结论
1所制备的载ACE-shRNA-PEG-壳聚糖纳米大小均匀,粒径分布范围较窄。并且筛选出在PH=5.5时,与质粒体积比为1:1时,以PEG壳聚糖纳米粒作为基因载体有良好的DNA包埋率,体外释药反应呈现缓释的特性,并且能有效地抑制核酸酶对质粒DNA的降解,为后期体外转染培养细胞以及体内转染提供了实验基础。
2.经过转染条件的筛选以及对壳聚糖纳米的PEG表面修饰,找到了较佳转染条件:DNA25μg、DNA:CS纳米粒体积比为1:1,转染72小时为较大转染效率。
3.本实验合成的载ACEshRNA-PEG-CS纳米转染体系介导的RNA干扰,在体外培养的大鼠血管内皮细胞中成功发挥了ACE基因表达下调作用以及抑制ACE与Ang-Ⅱ的合成等生物学效应,证明了PEG-CS/DNA转染载体可以有效地将目的基因转入靶细胞并且发挥相应的基因下调以及功能学效应。
4.PEG-CS纳米粒介导的ACE-shRNA经尾静脉注射入自发性高血压大鼠体内后,成功发挥了基因沉默作用,有效抑制了ACE mRNA及相应功能蛋白的表达,与传统长效ACEI类药物相比,降压幅度更大、一次给药可以维持10天左右降压效果,并且显著改善了心肌重构,以及心脏舒张功能。因此,运用PEG-CS纳米粒介导的ACE-shRNA发挥的RNA干扰作用进行降压治疗是一种具用潜力的治疗高血压的新策略。
Objective:
In this study, firstly chitosan nanoparticles were prepared and PEG surface modification was given, then their physics and biological characteristics were analysised to obtain a long release non-viral vector-mediated systems. Secondly we observed its transfection and cells cytotoxicity efficiency by transfected cultured rat vascular endothelial cell in vitro, and researched the downward effect of ACE gene in the molecular and protein levels. Finally we selected the appropriate dose of transfection in spontaneously hypertensive rats to observe the antihypertensive effect as well as target organ protect effect.
Methods:
1. Preparation and biological characteristics of PEG-chitosan nanoparticles containing ACEsh RNA
1) Using the ACE gene target sequence obtained from the laboratory’pre-experiment screened: 5'-AACCTAACATGTCAGCCTCTG-3 ' (gene sequences loci: 3603-3624). The synthesis of plasmid was assisted by Wuhan Jin Sai Corporation. The substantial extraction and purification of recombinant plasmid were obtained by bacteria alkaline lysis method. The Sequencing was detected by random enzyme digestion method and the concentration and purity of plasmid.were detected.
2) To prepare chitosan nanoparticles by ionic crosslinking method and modify them with PEG, then prepare PEG-chitosan nanoparticles containing ACEshRNA by rehabilitation method under the condition of different pH values as well as the volume ratio of chitosan and plamid. The chitosan nanoparticles morphology was observed by Spray gold electron microscopy . At the same time, the particles size、zeta potential value and multi-dispersity were measured by the zeta potential machine. The pDNA / CS complex’poly-formation and charge properties were analysised by Gel retardation method. The supernatant DNA concentration after centrifugation was detected by UV method. Furthermore we calculated embedding rate, got PEG-CS/DNA nanocomposites cumulative release curve and analysised nanoparticles against nuclease digesting.
2. The appropriate conditions of transfecting PEG-chitosan nano Containing ACEshRNA on vascular endothelial cells
1) Rat vascular endothelial cells were cultured by Organize paste method and identified of cytosolic factorⅧby immunofluorescence assay . Cells survival rate were detected by Trypan blue staining method.
2) Vascular endothelial cells were respectively transfected 0-96h at the best allocation ratio (nanoparticle: plasmid 1:1) of CS / DNA nanocomposites combination and PEG-CS/DNA nanocomposites with 60ul, 80ul, 100ul, 120ul, and took the corresponding naked plasmid group and blank group as controls .
3) The expression of green fluorescence of transfected cells was observed with fluorescence microscope. Transfection efficiency was detected by flow cytometry and cell activity was determined by MTT method.
3. The inhibit of ACE expression after PEG-chitosan nano containing ACEshRNA transfected on rat vascular endothelial cells
1) The expression of ACE mRNA was detected with Real-time fluorescence quantitative PCR method before and after transfection in each PEG-CS/DNA nanocomposites group, and naked plasmid and blank group as control.
2) ACE protein expression was detected with Western-blot method in collect cells of each group before and after transfection of 24h, 48h, 72h .
3) The concentration of ACE and Ang-Ⅱwere detected in 2mL culture medium before and after transfection of 72h with Enzyme-linked immunosorbent assay methods.
4. ACEshRNA-PEG-CS nano-vector-mediated RNAi effect in Spontaneously Hypertensive Rats
1) 12-week-old adult male spontaneously hypertensive rats (SHR) and high blood pressure model control mice were studied. 25ug DNA mediated diluted to 500ul with sterile saline mediated by ACEshRNA-PEG-chitosan nanocomposites was injected into rats through tail vein , repeated after 10 days. And given daily gavage Lotensin 10mg / day /animal、blank SHR control group, chitosan group normal blood pressure group as control, respectively tail vein injection of 500ul sterile saline or the same concentration of chitosan nano-liquid.
2) Rat tail artery systolic blood pressure (SBP) and heart rate were measured at the same point respectively before injection and 1-3 days after injection with tail cuff method in quiet environment and awaken condition.
3) The myocardial, aortic, renal ACE mRNA or protein expression were detected by fluorescence quantitative RT-PCR or Western-blot assay. The serum levels of ACE and Ang-Ⅱcontent were detected by enzyme-linked immunosorbent assay . The hepatic and renal function were analyed by automatic biochemical method.
4) At the end of the experiment, the whole heart (HW) and left ventricular weight (LW) were weighted to achieve heart / body weight, left ventricular / body weight ratio. And preparation of heart, kidney and aortic Organize frozen section to observe PEG- CS-EGFP-ACE-shRNA distribution under fluorescence microscope, inspect histology change under light microscopy and transmission electron microscopy.
Results:
1. Preparation and biological characteristics of PEG-chitosan nanoparticles containing ACEsh RNA
1.1 The DNA plasmid concentration was 0.5ug/ul, the purity was 1.88 detected by UV.
1.2 The impact of physical nature of various factors on chitosan nano-composite: (1) the particle size of chitosan nanoparticles increased with the solution pH value increasing. when the pH value is 5.5 , PEG chitosan nanoparticles average diameter is about 125.8±5.6nm with size uniformity, smallest multi-dispersity, more concentrated distribution and positive zeta potential which is benefited to combine of the negatively charged plasmid. (2) After PEG modified, zeta potential of particle size had no significant effect, but compared to the same conditions, the dispersity were significantly reduced, showing more suitable to prepare uniform nanoparticles and combine plasmids. (3) A smaller average chitosan particle size was obtained with 1:1 volume ratio (mass ratio) of chitosan nanoparticles to plasmid , with the character of the better permeability through the cell membrane, smaller dispersity , distributed more concentrate and positive zeta potential, which all benefit to combine the plasmid with negative charge.
1.3 The impact of plasmid-binding capacity of chitosan of various factors: (1) Chitosan nano-and PEG-chitosan nano-plasmid complexes have effective combination of plasmid , and the plasmid negative charge were decreased. When gel electrophoresis, the plasmid stayed in the holes and naked plasmid went out of the hole. (2) When PH <7 most of amino with positively charged of the chitosan molecules can effectively combine plasmid DNA plasmid to make it stay in the hole. (3) At volume ratio of 1:1,1:2,1:3, the chitosan nanoparticles can be effectively combine plasmid.
1.4 The impact of embedding rate of various factors on complex: (1) The DNA-embedded rate of chitosan nano-composite was highest (90.43±3.9)% at pH =5.5.. (2) Embedding rate increased significantly in each group after the surface modification by PEG. . (3) the embedding rate were higher at the volume ratio of 1:1,1:2 , respectively (88.87±13.2)% and (80.13±12.9)%, no significant difference between the two groups; And as the ratio increased embedding rate decreased.
1.5. the impact of vitro release of the compound with various factors : (1) The release of CS / DNA nanocomposites in PBS solution is basically same at pH = 3.5-7.5. There are the beginning stages of the release burst, the release of the preceding curve near a straight line, about after 4 days (96 hours), the stability release began, the release curve risen slowly, maintaining a smooth release of about eight days. And the nanoparticles quickly released in pH = 9.0 PBS solution, only lasted about 4 days. (2) The release curve of different gene complexes with different volume ratio all had the begining burst release when fixed at PH = 5.5 . The release curve rapid increased at the beginning, about after 4 days (96 hours)the stability release started. The duration of the release shortened with volume ratio increasing. At volume ratio 1:1, the duration of release was longest, maintained smoothly release about eight days; at volume ratio 1:5, the release time was shortest, only maintained about four days. (3) The beginging burst release characteristics of chitosan nanoparticles containing ACEshRNA were not enfluenced by PEG modified when fixed volume ratio 1:1 and pH = 5.5 . The release begun to stabilized after about 4 days (96 hours) , the release curve slowly raise, but the release duration was extended to 10 days.
1.6 DNaseⅠdigestion experiments: (1) PEG-CS-ACEshRNA gene complexes can be effective against DNA degradation at the volume ratio 1:1-1:3, and the plasmid was completely blocked into the hole. At the volume ratio of 1:4,1:5 bands can be showed indicating the nuclease protective effect decreased. (2) PEG-chitosan nanoparticles can effectively protect the plasmid from DnaseⅠdigestion. (3) The protection of plasmid DNA nuclease degradation can be observed at PH = 3, 5.5, 7. At pH = 11.0, the obvious bands can be seen, suggesting that the nuclease protective effect decreased.
2. The appropriate conditions of transfecting PEG-chitosan nano Containing ACEshRNA on vascular endothelial cells
2.1 Cultivate stable rat vascular endothelial cells were confirmed by immunofluorescence identification with cell survival rate of over 95%.
2.2 Determination of transfection efficiency: (1) Using fluorescence microscopy we can see there is the expression of green fluorescence, while the naked plasmid group and blank control group were no fluorescent expression. (2) Flow cytometry transfection efficiency: 1) Without PEG-modified chitosan-based nanoparticles on the endothelial cells of plasmid transfection rate is low, up to (26.0±3.9)%, After PEG chemical modification, the transfection efficiency has significantly increased,up to (59.4±5.2)%. 2) The transfection efficiency was highest at the volume of 100ul, that is, the plasmid DNA content of 25ug. At a certain range, transfection efficiency and the amount transfection complex volume was proportional . Instead, transfection efficiency rates begin to decline with the volume of transfection increase 3) At fixed volume of transfection, as time increased, the transfection efficiency gradually increased and reached the peak at 72h (57.1±7.1)%, (p <0.05); It began to decreased at 96 hours , but still was significantly higher than at 0h (p <0.05).
2.3 Determination of cytotoxicity: There was no significant difference compared with blank control group when transfection complexes volume was <100ul ,suggesting there were non-toxic to cells. The cell growth was significantly inhibited at the composite volume was 120 ul after transfected 24h, and with the time prolong inhibition gradually increasing (p <0.05).
3. The inhibit of ACE expression after PEG-chitosan nano containing ACEshRNA transfected on rat vascular endothelial cells
3.1 ACE gene mRNA expression changes before and after transfection in different groups: ACE mRNA expression levels decreased after 24 hours PEG chitosan nanocomposites plasmid transfection cells (14.7±5.9)%, up to (53.6±5.4)% after 48 hours, up to (60.1±2.1)% after 72-hour compared with the blank control group and the plasmid control group (all P <0.05).
3.2 ACE protein expression changes before and after transfection in different groups: protein expression had no significant change in PEG-CS/DNA group at 24 hours after transfection , significantly reduced at 48 hours compared to blank control group and plasmid control group (P <0.05), reduced lower at 72 hours (P <0.01). ACE protein expression was no significant change in the blank control group and the control plasmid group at different time points before and after transfected, according to the RT-PCR results.
3.3 The content of ACE and Ang-Ⅱin Vascular Endothelial Cell culture medium; ACE and Ang-Ⅱwere significantly reduced in culture medium in PEG-CS/DNA group after 72 hours transfection compared to the blank control group and the group of naked plasmid.(P <0.05),suggesting that PEG-CS nano-ACEshRNA transfection system can not only significantly reduced ACE gene at the molecular protein level but also on the function of a significant reduction in ACE content, reducing the synthesis of Ang-Ⅱ.
4. The RNAi effect mediated by PEG-CS-nano containing ACE-shRNA in spontaneously hypertensive rats
4.1 Tail arterial pressure and heart rate changes: The arterial pressure was no significant difference in each SHR group before the intervention (P> 0.05),. arterial pressure in each SHR group were significantly higher than that in normal blood pressure control group before intervention (P <0.01). In SHR gene therapy group at the first 3 days after injection, tail arterial pressure decreased significantly about (22±4) mmHg, there is significant difference compared with before treatment (P <0.05), after that the pressure declined slowly, sustained 8 days, and the biggest pressure drop-down was as 33mmHg. At about 11 days after injection, blood pressure began to rise; after second the injection, tail arterial pressure further significantly dropped about (24±5) mmHg, antihypertensive effect sustained 10 days, then blood pressure began rise, the biggest drop-down rate was about 25mmHg. The largest accumulated drop-down rate after second injection was up to 50mmHg. In Lotensin treatment group the blood pressure started to decrease after 3 days , continued to 13 days up to the maximum drop-down 39 mmHg, significantly lower than the maximum drop-down rate in gene therapy group (p<0.05). Followed by 7 days, no significant drop in blood pressure was observed in Lotensin treatment group. Tail arterial pressure continued to rise in SHR control group and the chitosan control group. In normal blood pressure control group no significant changes in arterial pressure were observed. There was no significant change in heart rate Rats in each group before and after treatment (P> 0.05).
4.2 PEG-CS-EGFP-ACE-shRNA distribution in tissues and organs: The expression of green fluorescent substantial can be found in heart, aorta, kidney tissue, these tissures were known as rich in ACE.
4. 3 The downward effect of ACE features of heart, kidney, aorta:after PEG - CS - EGFP - ACE - shRNA transfection 72h . 1) RT - PCR results: ACE mRNA expression of myocardium, aorta, renal was significantly lower in SHR gene therapy group than other SHR control groups, (P <. 05), but had no statistical difference compared with normal blood pressure control group (P>. 05)., suggesting with PEG - CS - EGFP - ACE - shRNA treatment can significantly reduce the expression of ACE mRNA. 2) Western blot results: ACE protein expression of myocardium, aorta, renal in SHR gene therapy group was significantly lower than those in other SHR control groups (P <. 05), no statistical difference compared to normal blood pressure control group. (P>. 05), according to RT - PCR results. 3) Serum ACE and Ang -Ⅱcontent: At 72 hours after transfection, in SHR ACE gene therapy group , ACE as well as Ang -Ⅱsignificantly reduced, which was lower than other SHR control groups. (P <. 05), had no statistical difference compared with normal blood pressure control group (P >. 05). In SHR drug treatment group, before and after treatment ACE had no significant change, but Ang -Ⅱsignificantly reduced, confirming ACEI drugs can inhibit Ang -Ⅱsynthesis.
4.4 Influence of left ventricular structure and function: 1) Detection of left ventricular hemodynamic function: left ventricular diastolic pressure (LVDP), left ventricular end diastolic pressure (LVDEP) and maximal diastolic velocity (- dp / dtmax) in the each SHR group were higher than normal blood pressure group. Those in the blank control group, chitosan control group were significantly higher than those in the drug treatment group and the gene therapy group (P <. 05), suggesting that in the early stage of hypertension diastolic dysfunction existed. Gene therapy group and drug treatment group can be significantly improved diastolic dysfunction, there is obvious difference between the two groups, the former more significantly reduced LVDP and (- dp / dtmax), suggesting that PEG - CS - ACEshRNA can more significantly improved diastolic dysfunction. 2) The heart / body weight, left ventricular / body weight: the heart / body / weight and left ventricular / body weight was significantly higher in SHR control group and the chitosan control group than those in normal blood pressure blood pressure group (P <. 05), suggesting there is a clear cardiac hypertrophy in untreat SHR groups. The above indexes in SHR Drug treatment group and the gene therapy group were significantly decreased (P <. 05), the latter reduced more obviously, but didn’t fell to normal levels in the control group blood pressure, suggesting compared with the ACEI drugs, PEG - CS - EGFP - ACE - shRNA more obviously reduce cardiac hypertrophy. 3) Histological observation: under light microscopy, myocardial cells hypertrophy were observed in SHR control group and SHR Chitosan control group than those in the normal control group. Myocardial hypertrophy were reduced significantly in SHR gene therapy group and drug treatment group. Observed under electron microscopy: myocardial cell membrane integrity, myofibrillar clear, with a more tidy, clear cross striations can be seen in SHR gene therapy group and the drug treatment group compared to those in SHR control group and chitosan control group. And mitochondria without swelling, a small amount of local increase in myocardial interstitial collagen fibers ,without obvious hyperplasia also can be found in both treatment groups,suggesting that both PEG-CS-EGFP-ACE-shRNA and ACEI drugs can improve the SHR myocardial ultrastructure changes.
4.5 The liver and kidney function: in each group were no significant difference (P> 0.05).It showed that PEG-CS-nanoparticles containing ACE-shRNA had no effect on the liver and kidney functions.
Conclusions
1. We have prepared the PEG -chitosan nanoparticles containing ACEshRNA with uniform size, narrower particle size distribution. Under the condition of PH = 5.5 and the volume ratio 1:1, PEG chitosan nanoparticles as a gene vector have good drug encapsulation rate, a prolong release reaction in vitro release characteristics, and can effectively inhibit the nuclease degradation of plasmid DNA,Which provided a foundation for the later in vitro transfection on cultured cells and in vivo transfection experiments.
2. After compared transfection efficiency rate under different conditions as well as surface modification with PEG we have gotten the appropriate transfection conditions: DNA25μg, DNA: CS nanoparticles volume ratio of 1:1, transfected for 72 hours.
3. The RNA interference mediated by ACEshRNA PEG-CS-nanoparticles played a successful downward biology effect on ACE gene expression ,ACE inhibition and Ang-Ⅱsynthetic in cultured rat vascular endothelial cells, proved ACEshRNA PEG-CS-nanoparticles transfection vector can effectively carry gene into target cell and produce gene downward effect.
4. ACE-shRNA-PEG-CS nanoparticles was injected into spontaneously hypertensive rats through the tail vein, which successfully played a role in gene silencing, effectively inhibited ACE mRNA and the corresponding functional protein expression. Compared to the traditional long-acting drugs ACEI , blood pressure was droped even more, one time drug delivery can maintained antihypertensive effect about 10 days, and significantly improved the myocardial remodeling and cardiac diastolic function. Therefore, the ACE-shRNA- PEG-CS-nanoparticles mediated RNA interference technology is a potential new strategy for the treatment of hypertension.
首先制备壳聚糖纳米粒,并且给予PEG表面修饰,分析其相关物理学、生物学特性,以期获得一种缓释的非病毒载体介导系统,其次行培养的大鼠血管内皮细胞转染,观察其转染效率,细胞毒性等,并从分子蛋白水平观察对ACE基因的下调作用,最后以筛选出的合适剂量转染自发性高血压大鼠,观察降压疗效以及对靶器官的影响等生物效应。
方法:
1载ACEshRNA-PEG-壳聚糖纳米粒制备以及生物学特性
1 )应用本实验室前期实验中筛选出的能显著下调ACE基因的靶序列:5’—AACCTAACATGTCAGCCTCTG—3’(基因序列位点3603-3624),由武汉晶赛公司协助合成质粒,采用菌液用碱裂解法大量抽提和纯化重组质粒,随机酶切测序,并检测质粒浓度与纯度。
2)采用离子交联法制备壳聚糖纳米粒, PEG修饰壳聚糖纳米粒,复凝聚法制备不同PH值、体积比以及PEG化的载ACEshRNA壳聚糖纳米。喷金电镜下对各种壳聚糖纳米粒悬液扫描并照相,观察纳米粒与形态。同时使用zeta电位仪测定其颗粒大小、zeta电位值和多分散度。应用凝胶阻滞分析验证载ACEshRNA壳聚糖纳米多聚复合物的形成及电荷性质。紫外分光光度计检测离心后上清液中DNA的含量,计算包埋率,绘制PEG-CS/DNA纳米复合物累积释放曲线,以及分析纳米粒对核酸酶的抗性。
2.载ACEshRNA-PEG-壳聚糖纳米粒对血管内皮细胞转染条件的筛选
1)组织贴块法体外培养大鼠血管内皮细胞,免疫荧光法检测胞浆Ⅷ因子进行鉴定,台盼兰染色检测细胞成活率。
2)分别以较佳配比率(纳米粒:质粒为1:1)结合的CS-DNA纳米复合物组以及PEG-CS-DNA纳米复合物60ul、80ul、100ul、120ul转染血管内皮细胞0-96h,并以相应的裸质粒组以及空白组作为对照。
3)荧光显微镜下观察转染细胞绿色荧光的表达,流式细胞仪检测转染效率,MTT法测定细胞活性。
3 .载ACEshRNA-PEG-壳聚糖纳米粒转染体系抑制大鼠血管内皮细胞ACE表达的研究
1)应用Real-time荧光定量PCR检测各组PEG-CS-DNA纳米复合物转染前后ACE mRNA的表达.并以裸质粒和空白组为对照。
2)于转染前及转染后的24h、48h、72h收集各组细胞,用Western-blot法检测ACE蛋白的表达。
3)于转染前及转染后的72h收集培养液2mL,用酶联免疫方法检测ACE以及Ang-Ⅱ的含量。
4.载ACEshRNA-PEG-壳聚糖纳米粒转染体系介导RNAi对自发性高血压大鼠的影响
1)研究对象为12周龄成年雄性自发性高血压大鼠(SHR)和高血压模型对照鼠,采用尾静脉注射DNA含量为25ug,无菌生理盐水稀释后500ul载ACEshRNA-PEG-壳聚糖纳米复合物, 10天重复给药一次。并以每日灌胃给予洛丁新10mg/日/只作为对照,同时设SHR空白对照组、壳聚糖对照组以及正常血压对照组,分别尾静脉注射500ul无菌生理盐水或相同浓度的壳聚糖纳米液。
2)分别于注射前及注射后每1-3天的相同时间点采用尾袖法在安静环境中测量大鼠清醒状态下尾动脉收缩压(SBP)和心率。
3)荧光定量RT-PCR或Western-blot法检测心肌、主动脉、肾脏ACE mRNA或蛋白的表达,并应用酶联免疫法检测各组血清ACE以及Ang-Ⅱ含量的变化,全自动生化分析仪检测肝肾功能。
4)实验结束时,行左侧颈动脉插管测定左心室功能血流动力学检查,并称取全心(HW)、左室重量(LW),求全心/体重、左心室/体重比值,并且制备心、肾及主动脉组织冰冻切片,荧光显微镜下检测载ACEshRNA-PEG-壳聚糖纳米粒的分布,光镜以及透射电镜行心肌组织学检查。
结果:
1.载ACEshRNA-PEG-壳聚糖纳米粒制备以及生物学特性
1.1经紫外分光光度计检测质粒DNA浓度为0.5ug/ul,纯度为1.88。
1.2各因素对壳聚糖纳米复合物理化性质的影响:(1)壳聚糖纳米粒的粒径随着溶液pH值的升高而增大,当pH值为5.5时的PEG壳聚糖纳米粒平均粒径125.8±5.6nm左右,大小均匀,多分散度最小,分布比较集中,zeta电位为正,有利于与带负电荷的质粒结合。(2)PEG化后也对粒径以及zeta电位无明显影响,但是与壳聚糖相同条件相比,分散度均明显减小,可见更适合制备均匀的纳米粒,利于和质粒结合。(3)壳聚糖与质粒体积比(质量比)为1:1制备的壳聚糖纳米粒平均粒径较小,故通过细胞膜的通透性较好,多分散度较小,分布比较集中, zeta电位也为正值,有利于与带负电荷的质粒结合。
1.3各因素对壳聚糖与质粒结合力的影响:(1)壳聚糖纳米以及PEG化壳聚糖纳米质粒复合物均有效结合质粒,由于中和了质粒所带的负电荷,凝胶电泳时,质粒不出孔,而裸质粒则出孔。(2)PH<7时壳聚糖分子中大部分的氨基带正电荷能与质粒DNA有效地结合质粒不出孔。(3)在体积比为1:1、1:2、1:3时,壳聚糖纳米粒能有效地结合质粒.
1.4各因素对复合物包埋率的影响:(1)pH值为5.5时的壳聚糖纳米复合物的DNA包埋率为最高,为(90.43±3.9)%。(2)各组经PEG表面修饰后包埋率明显增高。(3)当体积比为1:1,1:2时包埋率较高,分别为(88.87±13.2)%与(80.13±12.9)%,两组间无显著差异;之后随着该比例增高,包埋率明显下降。
1.5各因素对复合物体外释放的影响:(1)CS-DNA纳米复合物在PBS溶液pH值为3.5-7.5时释放的情况基本相同,均存在开始阶段的爆破释放,释放曲线前段接近直线,约4天(96小时)后,释放量开始稳定,释放曲线缓慢上升,平稳维持释放8天左右。而纳米粒在pH值为9.0的PBS溶液中快速释放,只释放了4天左右。(2)在固定PH=5.5时,各不同体积比例的载基因复合物PBS溶液释放均存在开始阶段的爆破释放,释放曲线快速上升,约4天(96小时)后,释放量开始稳定,释放曲线缓慢上升,但是释放持续时间随着体积比的增大而缩短,在体积比为1:1时,释放时间最长,平稳维持释放8天左右;体积比为1:5时,释放时间最短,仅维持4天左右。(3)取体积比为1:1,pH=5.5的载ACEshRNA壳聚糖纳米粒经PEG化后,没有影响复合物的体外释放的爆破释放特点,约4天(96小时)后,释放量开始稳定,释放曲线缓慢上升,但是释放持续时间延长至10天左右。
1.6 DNaseⅠ消化实验:(1)PEG化载ACEshRNA壳聚糖纳米在体积比为1:1—1:3均能有效抵抗DNA降解,质粒被完全阻滞于加样孔中,体积比为1:4,1:5的载基因壳聚糖纳米复合物则可以显示条带,说明对核酸酶的保护作用降低。(2)PEG化壳聚糖纳米粒能有效地保护质粒不被DnaseⅠ消化,复合物不出孔,而裸质粒则完被DnaseⅠ消化,加样孔内以及条带消失。(3)PH=3、5.5、7时,均能有效地对质粒DNA有保护核酸酶降解的作用,当PH=11.0时,则可以看到明显的条带,表明对核酸酶的保护作用降低。
2.载ACEshRNA-PEG-壳聚糖纳米粒对血管内皮细胞转染条件的筛选
2.1免疫荧光法鉴定证实培养获得稳定的大鼠血管内皮细胞,细胞存活率达95%以上。
2.2转染效率的测定:(1)用荧光显微镜观察可以看到有绿色荧光的表达,而裸质粒组和空白对照组均未见荧光表达。(2)流式细胞仪检测转染率:1)未经PEG化修饰的壳聚糖质粒纳米粒对内皮细胞的转染率均很低,最高达(26.0±3.9)%,PEG对壳聚糖质粒纳米复合物进行化学修饰后,转染效率比修饰前的转染效率明显有提高,最高达(59.4±5.2)%。2)PEG-CS/DNA纳米复合物的量为100ul,即质粒DNA含量为25ug时转染率最高,在一定的范围内转染率的高低与转染的复合物量呈正比,超出一定的量转染率反而开始下降。3)固定转染的体积时,转染不同时间,随着时间的延长,转染效率逐渐增高,72h达高峰(57.1±7.1),(p<0.05);96小时开始有所下降,但仍显著高于0h(p<0.05)。
2.3各转染条件下载ACEshRNA-PEG-壳聚糖纳米复合物转染细胞毒性的测定:当转染的复合物<100ul时与空白对照组比较差异无统计学意义,说明对细胞无毒性作用;而当复合物的量为120 ul时在转染的24h时细胞生长出现了明显的抑制现象,并且随着时间的延长抑制作用逐渐增大(p<0.05)。
3.载ACEshRNA-PEG-壳聚糖纳米粒转染体系抑制大鼠血管内皮细胞ACE表达的研究
3.1转染前后不同组ACE基因mRNA表达的变化:转染载ACEshRNA-PEG-壳聚糖纳米复合物后24小时细胞ACE mRNA表达水平下降达(14.7±5.9)%,48小时达(53.6±5.4)%,与空白对照组和质粒对照组相比有统计学差异(均P﹤0.05),72小时为(60.1±2.1)%。
3.2转染前后不同组细胞ACE蛋白表达的变化:PEG-CS/DNA组转染后24小时细胞ACE蛋白表达无明显变化,48小时明显降低,与空白对照组和质粒对照组相比有统计学差异(P﹤0.05),72小时更低(P﹤0.01),而空白对照组与质粒对照组转染前后各时间点ACE的蛋白表达无明显变化,与RT-PCR结果一致。
3.3血管内皮细胞培养液中ACE以及Ang-Ⅱ的含量;PEG-CS/DNA组转染72小时后,细胞培养液中的ACE与Ang-Ⅱ含量明显减少,与空白对照组以及裸质粒组相比有显著差别(P<0.05)。可见,载ACEshRNA PEG-CS纳米转染系统不仅可以在分子蛋白水平显著下调ACE基因,而且在功能上也显著减少ACE的含量,降低Ang-Ⅱ的合成。
4.载ACEshRNA-PEG-壳聚糖纳米粒转染体系介导RNAi对自发性高血压大鼠的影响
4.1治疗前后尾动脉压及心率的变化:SHR各组之间干预前尾动脉压差异无统计学意义(P﹥0.05),SHR各组干预前尾动脉压均显著高于正常血压对照组(P﹤0.01)。SHR基因治疗组于首次注射后第3天,尾动脉压显著下降约(22±4)mmHg,与治疗前比较有显著性差异(P﹤0.05),之后下降缓慢,降压作用可持续8天左右,最大降压幅度达33mmHg,于注射后约11天时血压开始回升;第2次注射后,尾动脉压再次明显下降约(24±5)mmHg,降压作用可持续10天左右,血压回升,最大降压幅度约25mmHg。两次注射后最大降压幅度累积达50mmHg。洛丁新治疗组开始使用后第3天血压持续降低,于第13天达最大降压幅度39 mmHg,明显低于基因治疗组最大降压幅度(p<0.05)。随后7天内血压无明显下降。SHR空白对照组和壳聚糖对照组尾动脉压持续升高。正常血压对照组尾动脉压无明显变化。各组大鼠治疗前后心率均无明显变化(P >0.05),
4.2 PEG-CS-EGFP-ACE-shRNA的组织器官分布情况:心脏、主动脉、肾脏组织都可见大量绿色荧光表达,与ACE在体内分布一致,说明载ACEshRNA-PEG-CS纳米载体经尾静脉注射后可分布在富含ACE的组织中。
4.3 PEG-CS-EGFP-ACE-shRNA转染72h后对心,肾,主动脉的ACE功能的下调作用: 1)RT-PCR结果:SHR基因治疗组心肌、主动脉、肾脏的ACE mRNA表达均较SHR空白对照组、SHR壳聚糖对照组、药物治疗组显著降低,差异有统计学意义(P﹤0.05),而与正常血压对照组无统计学差异(P﹥0.05)。说明SHR大鼠在尾静脉注射PEG-CS-EGFP-ACE-shRNA后,能显著降低ACE mRNA的表达。2)Western blot结果:SHR基因治疗组心肌、主动脉、肾脏的ACE蛋白表达均较SHR空白对照组、SHR病毒对照组以及药物治疗组显著降低,差异有统计学意义(P﹤0.05),而与正常血压对照组无统计学差异(P﹥0.05),与RT-PCR结果一致。说明SHR大鼠在尾静脉注射载ACEshRNA-PEG-壳聚糖纳米粒后,随着ACE mRNA的表达降低,相应功能蛋白的表达也随之降低。3)对血清ACE以及Ang-Ⅱ的含量的影响:转染72小时后,SHR基因治疗组ACE以及Ang-Ⅱ显著降低,低于SHR空白对照组、壳聚糖对照组、以及药物治疗组,差异有统计学意义(P﹤0.05),而与正常血压对照组无统计学差异(P﹥0.05)。SHR药物治疗组ACE治疗前后无明显变化,但是Ang-Ⅱ显著降低,证实ACEI类药物可以抑制Ang-Ⅱ的合成。
4.4对左室结构以及功能的影响:1)血流动力学检测左室功能:左室舒张压(LVDP)、左室舒张末压(LVDEP)以及最大舒张速度(- dp/dtmax)在SHR各组均高于正常血压组,其中空白对照组、壳聚糖对照组明显高于药物治疗组以及基因治疗组(P<0.05),可见高血压早期即有舒张功能不全,并且基因治疗组与药物治疗组均可显著改善舒张功能不全,两者相比明显有差别,前者更显著降低LVDP与(- dp/dtmax),说明载ACEshRNA-PEG-壳聚糖纳米复合物能更有效地显著改善高血压时的舒张功能不全。2)对全心/体重、左心室/体重的影响:SHR空白对照组和壳聚糖对照组的全心/体重和左心室/体重显著高于正常血压血压组(P﹤0.05),提示未经治疗的SHR有明显的心肌肥厚现象。药物治疗组和基因治疗组上述指标均显著下降(P﹤0.05),其后者降低更明显,但均未降到正常血压对照组水平,可见与ACEI类药物相比,载ACE-shRNA-PEG-壳聚糖纳米复合物改善心肌肥厚的作用更明显。3)组织学观察:光镜下观察到SHR空白对照组和SHR壳聚糖对照组较正常血压对照组心肌细胞明显肥大,SHR基因治疗组即尾静脉注射载ACE-shRNA-PEG-壳聚糖纳米复合物和药物治疗组均使心肌细胞肥大明显减轻。电镜下观察到:SHR空白对照组和壳聚糖对照组相比, SHR基因治疗组以及药物治疗组心肌细胞核膜完整,肌原纤维清晰,排列较整齐,横纹清楚可见,线粒体无肿胀,但局部可见少量增多,心肌间质未见明显胶原纤维增生,说明载ACE-shRNA-PEG-壳聚糖纳米复合物与ACEI类药物都可以改善SHR心肌超微结构的改变。
4.5对肝肾功能的影响:各组大鼠之间肝肾功能指标无统计学差异(P﹥0.05),表明载ACE-shRNA-PEG-壳聚糖纳米复合物对肝肾功能无影响。
结论
1所制备的载ACE-shRNA-PEG-壳聚糖纳米大小均匀,粒径分布范围较窄。并且筛选出在PH=5.5时,与质粒体积比为1:1时,以PEG壳聚糖纳米粒作为基因载体有良好的DNA包埋率,体外释药反应呈现缓释的特性,并且能有效地抑制核酸酶对质粒DNA的降解,为后期体外转染培养细胞以及体内转染提供了实验基础。
2.经过转染条件的筛选以及对壳聚糖纳米的PEG表面修饰,找到了较佳转染条件:DNA25μg、DNA:CS纳米粒体积比为1:1,转染72小时为较大转染效率。
3.本实验合成的载ACEshRNA-PEG-CS纳米转染体系介导的RNA干扰,在体外培养的大鼠血管内皮细胞中成功发挥了ACE基因表达下调作用以及抑制ACE与Ang-Ⅱ的合成等生物学效应,证明了PEG-CS/DNA转染载体可以有效地将目的基因转入靶细胞并且发挥相应的基因下调以及功能学效应。
4.PEG-CS纳米粒介导的ACE-shRNA经尾静脉注射入自发性高血压大鼠体内后,成功发挥了基因沉默作用,有效抑制了ACE mRNA及相应功能蛋白的表达,与传统长效ACEI类药物相比,降压幅度更大、一次给药可以维持10天左右降压效果,并且显著改善了心肌重构,以及心脏舒张功能。因此,运用PEG-CS纳米粒介导的ACE-shRNA发挥的RNA干扰作用进行降压治疗是一种具用潜力的治疗高血压的新策略。
Objective:
In this study, firstly chitosan nanoparticles were prepared and PEG surface modification was given, then their physics and biological characteristics were analysised to obtain a long release non-viral vector-mediated systems. Secondly we observed its transfection and cells cytotoxicity efficiency by transfected cultured rat vascular endothelial cell in vitro, and researched the downward effect of ACE gene in the molecular and protein levels. Finally we selected the appropriate dose of transfection in spontaneously hypertensive rats to observe the antihypertensive effect as well as target organ protect effect.
Methods:
1. Preparation and biological characteristics of PEG-chitosan nanoparticles containing ACEsh RNA
1) Using the ACE gene target sequence obtained from the laboratory’pre-experiment screened: 5'-AACCTAACATGTCAGCCTCTG-3 ' (gene sequences loci: 3603-3624). The synthesis of plasmid was assisted by Wuhan Jin Sai Corporation. The substantial extraction and purification of recombinant plasmid were obtained by bacteria alkaline lysis method. The Sequencing was detected by random enzyme digestion method and the concentration and purity of plasmid.were detected.
2) To prepare chitosan nanoparticles by ionic crosslinking method and modify them with PEG, then prepare PEG-chitosan nanoparticles containing ACEshRNA by rehabilitation method under the condition of different pH values as well as the volume ratio of chitosan and plamid. The chitosan nanoparticles morphology was observed by Spray gold electron microscopy . At the same time, the particles size、zeta potential value and multi-dispersity were measured by the zeta potential machine. The pDNA / CS complex’poly-formation and charge properties were analysised by Gel retardation method. The supernatant DNA concentration after centrifugation was detected by UV method. Furthermore we calculated embedding rate, got PEG-CS/DNA nanocomposites cumulative release curve and analysised nanoparticles against nuclease digesting.
2. The appropriate conditions of transfecting PEG-chitosan nano Containing ACEshRNA on vascular endothelial cells
1) Rat vascular endothelial cells were cultured by Organize paste method and identified of cytosolic factorⅧby immunofluorescence assay . Cells survival rate were detected by Trypan blue staining method.
2) Vascular endothelial cells were respectively transfected 0-96h at the best allocation ratio (nanoparticle: plasmid 1:1) of CS / DNA nanocomposites combination and PEG-CS/DNA nanocomposites with 60ul, 80ul, 100ul, 120ul, and took the corresponding naked plasmid group and blank group as controls .
3) The expression of green fluorescence of transfected cells was observed with fluorescence microscope. Transfection efficiency was detected by flow cytometry and cell activity was determined by MTT method.
3. The inhibit of ACE expression after PEG-chitosan nano containing ACEshRNA transfected on rat vascular endothelial cells
1) The expression of ACE mRNA was detected with Real-time fluorescence quantitative PCR method before and after transfection in each PEG-CS/DNA nanocomposites group, and naked plasmid and blank group as control.
2) ACE protein expression was detected with Western-blot method in collect cells of each group before and after transfection of 24h, 48h, 72h .
3) The concentration of ACE and Ang-Ⅱwere detected in 2mL culture medium before and after transfection of 72h with Enzyme-linked immunosorbent assay methods.
4. ACEshRNA-PEG-CS nano-vector-mediated RNAi effect in Spontaneously Hypertensive Rats
1) 12-week-old adult male spontaneously hypertensive rats (SHR) and high blood pressure model control mice were studied. 25ug DNA mediated diluted to 500ul with sterile saline mediated by ACEshRNA-PEG-chitosan nanocomposites was injected into rats through tail vein , repeated after 10 days. And given daily gavage Lotensin 10mg / day /animal、blank SHR control group, chitosan group normal blood pressure group as control, respectively tail vein injection of 500ul sterile saline or the same concentration of chitosan nano-liquid.
2) Rat tail artery systolic blood pressure (SBP) and heart rate were measured at the same point respectively before injection and 1-3 days after injection with tail cuff method in quiet environment and awaken condition.
3) The myocardial, aortic, renal ACE mRNA or protein expression were detected by fluorescence quantitative RT-PCR or Western-blot assay. The serum levels of ACE and Ang-Ⅱcontent were detected by enzyme-linked immunosorbent assay . The hepatic and renal function were analyed by automatic biochemical method.
4) At the end of the experiment, the whole heart (HW) and left ventricular weight (LW) were weighted to achieve heart / body weight, left ventricular / body weight ratio. And preparation of heart, kidney and aortic Organize frozen section to observe PEG- CS-EGFP-ACE-shRNA distribution under fluorescence microscope, inspect histology change under light microscopy and transmission electron microscopy.
Results:
1. Preparation and biological characteristics of PEG-chitosan nanoparticles containing ACEsh RNA
1.1 The DNA plasmid concentration was 0.5ug/ul, the purity was 1.88 detected by UV.
1.2 The impact of physical nature of various factors on chitosan nano-composite: (1) the particle size of chitosan nanoparticles increased with the solution pH value increasing. when the pH value is 5.5 , PEG chitosan nanoparticles average diameter is about 125.8±5.6nm with size uniformity, smallest multi-dispersity, more concentrated distribution and positive zeta potential which is benefited to combine of the negatively charged plasmid. (2) After PEG modified, zeta potential of particle size had no significant effect, but compared to the same conditions, the dispersity were significantly reduced, showing more suitable to prepare uniform nanoparticles and combine plasmids. (3) A smaller average chitosan particle size was obtained with 1:1 volume ratio (mass ratio) of chitosan nanoparticles to plasmid , with the character of the better permeability through the cell membrane, smaller dispersity , distributed more concentrate and positive zeta potential, which all benefit to combine the plasmid with negative charge.
1.3 The impact of plasmid-binding capacity of chitosan of various factors: (1) Chitosan nano-and PEG-chitosan nano-plasmid complexes have effective combination of plasmid , and the plasmid negative charge were decreased. When gel electrophoresis, the plasmid stayed in the holes and naked plasmid went out of the hole. (2) When PH <7 most of amino with positively charged of the chitosan molecules can effectively combine plasmid DNA plasmid to make it stay in the hole. (3) At volume ratio of 1:1,1:2,1:3, the chitosan nanoparticles can be effectively combine plasmid.
1.4 The impact of embedding rate of various factors on complex: (1) The DNA-embedded rate of chitosan nano-composite was highest (90.43±3.9)% at pH =5.5.. (2) Embedding rate increased significantly in each group after the surface modification by PEG. . (3) the embedding rate were higher at the volume ratio of 1:1,1:2 , respectively (88.87±13.2)% and (80.13±12.9)%, no significant difference between the two groups; And as the ratio increased embedding rate decreased.
1.5. the impact of vitro release of the compound with various factors : (1) The release of CS / DNA nanocomposites in PBS solution is basically same at pH = 3.5-7.5. There are the beginning stages of the release burst, the release of the preceding curve near a straight line, about after 4 days (96 hours), the stability release began, the release curve risen slowly, maintaining a smooth release of about eight days. And the nanoparticles quickly released in pH = 9.0 PBS solution, only lasted about 4 days. (2) The release curve of different gene complexes with different volume ratio all had the begining burst release when fixed at PH = 5.5 . The release curve rapid increased at the beginning, about after 4 days (96 hours)the stability release started. The duration of the release shortened with volume ratio increasing. At volume ratio 1:1, the duration of release was longest, maintained smoothly release about eight days; at volume ratio 1:5, the release time was shortest, only maintained about four days. (3) The beginging burst release characteristics of chitosan nanoparticles containing ACEshRNA were not enfluenced by PEG modified when fixed volume ratio 1:1 and pH = 5.5 . The release begun to stabilized after about 4 days (96 hours) , the release curve slowly raise, but the release duration was extended to 10 days.
1.6 DNaseⅠdigestion experiments: (1) PEG-CS-ACEshRNA gene complexes can be effective against DNA degradation at the volume ratio 1:1-1:3, and the plasmid was completely blocked into the hole. At the volume ratio of 1:4,1:5 bands can be showed indicating the nuclease protective effect decreased. (2) PEG-chitosan nanoparticles can effectively protect the plasmid from DnaseⅠdigestion. (3) The protection of plasmid DNA nuclease degradation can be observed at PH = 3, 5.5, 7. At pH = 11.0, the obvious bands can be seen, suggesting that the nuclease protective effect decreased.
2. The appropriate conditions of transfecting PEG-chitosan nano Containing ACEshRNA on vascular endothelial cells
2.1 Cultivate stable rat vascular endothelial cells were confirmed by immunofluorescence identification with cell survival rate of over 95%.
2.2 Determination of transfection efficiency: (1) Using fluorescence microscopy we can see there is the expression of green fluorescence, while the naked plasmid group and blank control group were no fluorescent expression. (2) Flow cytometry transfection efficiency: 1) Without PEG-modified chitosan-based nanoparticles on the endothelial cells of plasmid transfection rate is low, up to (26.0±3.9)%, After PEG chemical modification, the transfection efficiency has significantly increased,up to (59.4±5.2)%. 2) The transfection efficiency was highest at the volume of 100ul, that is, the plasmid DNA content of 25ug. At a certain range, transfection efficiency and the amount transfection complex volume was proportional . Instead, transfection efficiency rates begin to decline with the volume of transfection increase 3) At fixed volume of transfection, as time increased, the transfection efficiency gradually increased and reached the peak at 72h (57.1±7.1)%, (p <0.05); It began to decreased at 96 hours , but still was significantly higher than at 0h (p <0.05).
2.3 Determination of cytotoxicity: There was no significant difference compared with blank control group when transfection complexes volume was <100ul ,suggesting there were non-toxic to cells. The cell growth was significantly inhibited at the composite volume was 120 ul after transfected 24h, and with the time prolong inhibition gradually increasing (p <0.05).
3. The inhibit of ACE expression after PEG-chitosan nano containing ACEshRNA transfected on rat vascular endothelial cells
3.1 ACE gene mRNA expression changes before and after transfection in different groups: ACE mRNA expression levels decreased after 24 hours PEG chitosan nanocomposites plasmid transfection cells (14.7±5.9)%, up to (53.6±5.4)% after 48 hours, up to (60.1±2.1)% after 72-hour compared with the blank control group and the plasmid control group (all P <0.05).
3.2 ACE protein expression changes before and after transfection in different groups: protein expression had no significant change in PEG-CS/DNA group at 24 hours after transfection , significantly reduced at 48 hours compared to blank control group and plasmid control group (P <0.05), reduced lower at 72 hours (P <0.01). ACE protein expression was no significant change in the blank control group and the control plasmid group at different time points before and after transfected, according to the RT-PCR results.
3.3 The content of ACE and Ang-Ⅱin Vascular Endothelial Cell culture medium; ACE and Ang-Ⅱwere significantly reduced in culture medium in PEG-CS/DNA group after 72 hours transfection compared to the blank control group and the group of naked plasmid.(P <0.05),suggesting that PEG-CS nano-ACEshRNA transfection system can not only significantly reduced ACE gene at the molecular protein level but also on the function of a significant reduction in ACE content, reducing the synthesis of Ang-Ⅱ.
4. The RNAi effect mediated by PEG-CS-nano containing ACE-shRNA in spontaneously hypertensive rats
4.1 Tail arterial pressure and heart rate changes: The arterial pressure was no significant difference in each SHR group before the intervention (P> 0.05),. arterial pressure in each SHR group were significantly higher than that in normal blood pressure control group before intervention (P <0.01). In SHR gene therapy group at the first 3 days after injection, tail arterial pressure decreased significantly about (22±4) mmHg, there is significant difference compared with before treatment (P <0.05), after that the pressure declined slowly, sustained 8 days, and the biggest pressure drop-down was as 33mmHg. At about 11 days after injection, blood pressure began to rise; after second the injection, tail arterial pressure further significantly dropped about (24±5) mmHg, antihypertensive effect sustained 10 days, then blood pressure began rise, the biggest drop-down rate was about 25mmHg. The largest accumulated drop-down rate after second injection was up to 50mmHg. In Lotensin treatment group the blood pressure started to decrease after 3 days , continued to 13 days up to the maximum drop-down 39 mmHg, significantly lower than the maximum drop-down rate in gene therapy group (p<0.05). Followed by 7 days, no significant drop in blood pressure was observed in Lotensin treatment group. Tail arterial pressure continued to rise in SHR control group and the chitosan control group. In normal blood pressure control group no significant changes in arterial pressure were observed. There was no significant change in heart rate Rats in each group before and after treatment (P> 0.05).
4.2 PEG-CS-EGFP-ACE-shRNA distribution in tissues and organs: The expression of green fluorescent substantial can be found in heart, aorta, kidney tissue, these tissures were known as rich in ACE.
4. 3 The downward effect of ACE features of heart, kidney, aorta:after PEG - CS - EGFP - ACE - shRNA transfection 72h . 1) RT - PCR results: ACE mRNA expression of myocardium, aorta, renal was significantly lower in SHR gene therapy group than other SHR control groups, (P <. 05), but had no statistical difference compared with normal blood pressure control group (P>. 05)., suggesting with PEG - CS - EGFP - ACE - shRNA treatment can significantly reduce the expression of ACE mRNA. 2) Western blot results: ACE protein expression of myocardium, aorta, renal in SHR gene therapy group was significantly lower than those in other SHR control groups (P <. 05), no statistical difference compared to normal blood pressure control group. (P>. 05), according to RT - PCR results. 3) Serum ACE and Ang -Ⅱcontent: At 72 hours after transfection, in SHR ACE gene therapy group , ACE as well as Ang -Ⅱsignificantly reduced, which was lower than other SHR control groups. (P <. 05), had no statistical difference compared with normal blood pressure control group (P >. 05). In SHR drug treatment group, before and after treatment ACE had no significant change, but Ang -Ⅱsignificantly reduced, confirming ACEI drugs can inhibit Ang -Ⅱsynthesis.
4.4 Influence of left ventricular structure and function: 1) Detection of left ventricular hemodynamic function: left ventricular diastolic pressure (LVDP), left ventricular end diastolic pressure (LVDEP) and maximal diastolic velocity (- dp / dtmax) in the each SHR group were higher than normal blood pressure group. Those in the blank control group, chitosan control group were significantly higher than those in the drug treatment group and the gene therapy group (P <. 05), suggesting that in the early stage of hypertension diastolic dysfunction existed. Gene therapy group and drug treatment group can be significantly improved diastolic dysfunction, there is obvious difference between the two groups, the former more significantly reduced LVDP and (- dp / dtmax), suggesting that PEG - CS - ACEshRNA can more significantly improved diastolic dysfunction. 2) The heart / body weight, left ventricular / body weight: the heart / body / weight and left ventricular / body weight was significantly higher in SHR control group and the chitosan control group than those in normal blood pressure blood pressure group (P <. 05), suggesting there is a clear cardiac hypertrophy in untreat SHR groups. The above indexes in SHR Drug treatment group and the gene therapy group were significantly decreased (P <. 05), the latter reduced more obviously, but didn’t fell to normal levels in the control group blood pressure, suggesting compared with the ACEI drugs, PEG - CS - EGFP - ACE - shRNA more obviously reduce cardiac hypertrophy. 3) Histological observation: under light microscopy, myocardial cells hypertrophy were observed in SHR control group and SHR Chitosan control group than those in the normal control group. Myocardial hypertrophy were reduced significantly in SHR gene therapy group and drug treatment group. Observed under electron microscopy: myocardial cell membrane integrity, myofibrillar clear, with a more tidy, clear cross striations can be seen in SHR gene therapy group and the drug treatment group compared to those in SHR control group and chitosan control group. And mitochondria without swelling, a small amount of local increase in myocardial interstitial collagen fibers ,without obvious hyperplasia also can be found in both treatment groups,suggesting that both PEG-CS-EGFP-ACE-shRNA and ACEI drugs can improve the SHR myocardial ultrastructure changes.
4.5 The liver and kidney function: in each group were no significant difference (P> 0.05).It showed that PEG-CS-nanoparticles containing ACE-shRNA had no effect on the liver and kidney functions.
Conclusions
1. We have prepared the PEG -chitosan nanoparticles containing ACEshRNA with uniform size, narrower particle size distribution. Under the condition of PH = 5.5 and the volume ratio 1:1, PEG chitosan nanoparticles as a gene vector have good drug encapsulation rate, a prolong release reaction in vitro release characteristics, and can effectively inhibit the nuclease degradation of plasmid DNA,Which provided a foundation for the later in vitro transfection on cultured cells and in vivo transfection experiments.
2. After compared transfection efficiency rate under different conditions as well as surface modification with PEG we have gotten the appropriate transfection conditions: DNA25μg, DNA: CS nanoparticles volume ratio of 1:1, transfected for 72 hours.
3. The RNA interference mediated by ACEshRNA PEG-CS-nanoparticles played a successful downward biology effect on ACE gene expression ,ACE inhibition and Ang-Ⅱsynthetic in cultured rat vascular endothelial cells, proved ACEshRNA PEG-CS-nanoparticles transfection vector can effectively carry gene into target cell and produce gene downward effect.
4. ACE-shRNA-PEG-CS nanoparticles was injected into spontaneously hypertensive rats through the tail vein, which successfully played a role in gene silencing, effectively inhibited ACE mRNA and the corresponding functional protein expression. Compared to the traditional long-acting drugs ACEI , blood pressure was droped even more, one time drug delivery can maintained antihypertensive effect about 10 days, and significantly improved the myocardial remodeling and cardiac diastolic function. Therefore, the ACE-shRNA- PEG-CS-nanoparticles mediated RNA interference technology is a potential new strategy for the treatment of hypertension.
引文
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