超声/微气泡SonoVue介导体内和体外基因转导的研究

目的初步探讨超声联合微气泡超声造影剂SonoVue介导体外培养细胞和体内肌肉组织基因转导的可行性,并研究不同超声波参数和基因用量与转基因效率之间的关系.方法利用不同强度和不同剂量超声波和声学造影剂SonoVue,介导人肝癌细胞株HepG2和BALB/c小鼠右侧胫前肌内增强型绿色荧光蛋白报告质粒的转导,分别直接或组织冰冻切片后在荧光显微镜下观察,并计算转导效率.结果超声联合微气泡造影剂SonoVue可介导体外培养的HepG2细胞和小鼠胫前肌内pEGFP C1基因转导,HepG2细胞以1 MHz 1.5 W/cm2超声波基因转导效果最好;小鼠胫前肌以1 MHz 3 W/cm2的超声波转基因效果最好.结论超声联合微气泡造影剂SonoVue可介导体外培养细胞和体内肌肉组织基因转导,1 MHz超声波是转基因的理想频率.     

超声微泡造影剂介导LMP-1基因转染树突状细胞的实验研究

目的 探讨超声微泡造影剂介导LMP-1基因转染外周血来源的树突状细胞的有效性及最佳的超声辐照参数.方法 体外培养树突状细胞,在不同的超声波强度、机械指数、照射时间及不同的超声微泡造影剂浓度作用下,观察LMP-1基因与绿色荧光蛋白共表达的融合重组质粒pEGFP-C3-LMP1在树突状细胞的定向转染.在荧光显微镜下观察荧光蛋白质粒在树突状细胞中的表达,流式细胞仪评价重组质粒的转染效率,台盼蓝染色检测细胞活性.结果 超声辐照机械指数1.0、照射时间60 s、微泡造影剂浓度为20%,达到最佳的基因转染效率(14.37±2.12)%,且细胞生存率>90%.结论 微泡造影剂在超声辐射下可以有效地介导基因转染树突状细胞.     

体外培育牛黄诱导人肝癌HepG2细胞凋亡的实验研究

目的研究体外培育牛黄诱导人肝母细胞瘤HepG2细胞凋亡的作用及机制。方法用单层培养法培养HepG2细胞，用不同剂量的体外培育牛黄作用于HepG2细胞不同时间，用荧光显微镜、透射电镜观察细胞形态的改变及细胞凋亡，用流式细胞仪检测HepG2细胞的亚G1峰细胞（凋亡）率的变化。结果体外培育牛黄100～800ug／ml作用于HepG2细胞48h后，HepG2细胞表现为细胞皱缩、核质浓缩、核碎裂、细胞起泡以及凋亡小体形成等凋亡特征性形态学改变；流式细胞直方图上可见亚二倍体峰，不同浓度作用于HepG2细胞24、36、48、72h后的细胞凋亡率均显著高于空白对照组（P〈0．01），400ug／ml浓度组与阳性对照FT207（100ug／ml）组比较无明显差异。结论体外培育牛黄能诱导人肝母细胞瘤HepG2细胞凋亡。     

超声联合SonoVue介导荧光蛋白质粒转染人胚肾293T细胞的研究

目的 探讨超声协同微泡造影剂SonoVue介导荧光蛋白质粒转染人胚肾293T细胞的方法.方法 以不同的超声辐照时间、超声机械指数和不同浓度的微泡造影剂SonoVue的参数组合,将含有绿色荧光蛋白报告基因的真核表达质粒pEGFP-N1转染人胚肾293T细胞,接种于6孔板中,以荧光显微镜观察观察293T细胞中的绿色荧光蛋白表达情况并用流式细胞仪测算转染率.结果 当微泡造影剂SonoVue浓度为40%、超声机械指数为1.0、辐照时间5 min时,质粒的转染率最高(28.11±1.63)%.结论 超声联合微泡造影刺SonoVue能够作为载体介孚质粒转染细胞,但需优化各项超声辐照的条件以达到最佳效果.     

超声微泡造影剂增强重组腺相关病毒rAAV-sTRAIL感染肝癌细胞的实验观察

目的 探讨携带可溶性肿瘤坏死因子相关凋亡诱导配体(sTRAIL)基因的重组腺相关病毒载体rAAV-sTRAIL对肝癌细胞的增殖抑制及诱导凋亡作用,以及采用超声辐照联合微泡造影剂介导rAAV-sTRAIL转染肝癌细胞的有效性.方法 单纯重组腺相关病毒rAAV-sTRAIL感染肝癌细胞株HepG2,以及利用超声辐照联合微泡造影剂介导重组腺相关病毒rAAV-sTRAIL感染HepG2细胞,分别以RT-PCR方法和Westen印迹法检测细胞内sTRAIL基因mRNA及蛋白表达量,噻唑蓝法检测细胞的增殖抑制率,流式细胞术检测细胞凋亡率.结果 重组腺相关病毒rAAV-sTRAIL可有效转染HepG2细胞,sTRAIL mRNA表达水平在微泡造影剂组高于单纯病毒组,二者差异有统计学意义(P<0.05);rAAV-srrRAIL可抑制HepG2细胞增殖,其抑制率在微泡造影剂组高于单纯病毒组,二者差异有统计学意义(P<0.05);rAAV-sTRAIL可诱导HepG2细胞凋亡,其凋亡率在微泡造影剂组高于单纯病毒组,二者差异有统计学意义(P<0.05).结论 构建出的重组腺相关病毒rAAV-sTRAIL在肝癌细胞中能有效表达,sTRAIL基因对肝癌细胞有明显的抑制增殖和促凋亡作用;联合超声微泡造影剂及低强度超声,可有效提高腺相关病毒载体在肝癌细胞中的感染率,从而增强sTRAIL对肝癌细胞的增殖抑制及诱导凋亡作用.     

携目的基因的靶向超声微泡对肝癌HepG2细胞增殖活性的影响

目的 制备一种靶向肝癌HepG2细胞的载单纯疱疹病毒胸腺嘧啶核苷激酶(HSV-TK)基因超声微泡,并考察其体外寻靶能力及对HepG2细胞的增殖抑制作用.方法 用机械振荡法制备超声微泡,生物素-亲和素桥连方式构建靶向载HSV-TK超声微泡.检测其一般特性,并进行体外实验检测其对HepG2细胞增殖的影响.结果 靶向载HSV-TK超声微泡可较多地聚集在HepG2细胞表面,通过检测增殖细胞核抗原(PCNA)及噻唑蓝(MTT)法,发现载基因靶向微泡组的增殖能力明显下降,细胞凋亡明显增加,细胞侵袭实验表明载基因靶向微泡组(22.18±2.01)较对照组及载基因非靶向微泡组明显减少,对HepG2细胞增殖及侵袭能力有较好的抑制作用.结论 携目的基因靶向超声微泡对肝癌HepG2细胞在体外有较好抑制效果.     

超声微泡促腺病毒载体转染MDR1基因效率的体外实验研究

目的 探讨超声微泡造影剂在体外促进腺病毒载体介导的MDRl基因转染兔骨髓单个核细胞的效率,并初步探讨其安全性.方法 根据是否加入超声微泡造影剂和超声辐照将分选的兔骨髓单个核细胞分为常规培养组(A)、腺病毒MDRl基因转染组(B)、腺病毒MDRl基因转染+超声辐照组(C)、微泡造影剂+腺病毒MDRl基因转染组(D)、微泡造影剂+腺病毒MDRl基因转染+超声辐照组(E).收集各组细胞,应用RT-PCR法、流式细胞技术等分别从基因、功能及细胞生物学特性等方面检测外源性MDR1基因在骨髓单个核细胞内的功能性表达及安全性.结果 收获高滴度的Ad5-MDR1腺病毒载体并成功制备腺病毒微泡造影剂;RT-PCR结果 显示,除A组外其余4组在194 bp处均观察到特异性MDR1电泳条带,证实外源性MDR1基因通过腺病毒载体整合到细胞基因组中,且E组MDR1电泳条带的光密度比值明显高于B、C、D组(P<0.05);柔红霉素排出实验证实外源性MDR1基因表达产物P-gP有药物外排泵功能,且E组P-gp阳性率最高(P<0.05),而柔红霉素阳性率显著降低;一定强度的超声辐照及微泡造影剂对骨髓单个核细胞的细胞周期无明显影响.结论 低频超声波击碎微泡造影剂,能促进以腺病毒为载体介导的外源性MDR1基因转染兔骨髓单个核细胞的效率,且安全可行.     

稳定表达人细胞色素P450 1A2的HepG2细胞的建立及其代谢效应

目的：建立稳定表达人细胞的色素P450 1A2(CYP1A2)的HepG2细胞。方法：将所克隆的野生型CYP1A2 cDNA从重组质粒pGEM-CYP1A2中用Kpn Ⅰ／BamHⅠ双酶切，并亚克隆到哺乳动物细胞表达栽体pREP9中。再将重组质粒转化感受态大肠杆菌Top10，用氨苄青霉素抗性筛选和限制酶谱鉴定。改良的磷酸钙介导的细胞转染法将重组质粒pREP9-CYP1A2转染肝癌细胞HepG2，用RT-PCR技术对转基因细胞的CYP1A2mRNA表达作了分析，并用MTT法比较转基因细胞对黄曲霉素B1(AFB1)细胞毒敏感试验。结果：与HepG2细胞相比，HepG2-CYP1A2转基因细胞表达CYP1A2 mRNA，能增强AFB1的细胞毒作用。结论：建立了稳定表达CYP1A2的转基因细胞系，可用于由CYP1A2参与的毒理学与药物代谢研究。     

超声微泡造影剂对人肝癌 HepG2细胞活性的影响

目的：观察超声六氟化硫（SF6）微泡对人肝癌 HepG2细胞的细胞毒性作用，筛选最优微泡转染浓度。方法应用噻唑蓝（MTT）比色分析与台盼蓝染色两种方法检测 SF6对人肝癌 HepG2细胞活性的影响。结果当微泡造影剂浓度小于10％时，不同浓度的 SF6均不会引起明显的细胞死亡（细胞存活率＞80％）；随着微泡浓度增高，细胞活性下降，各组间比较差异有统计学意义（P ＜0.05）；当微泡浓度达20％时，其对细胞的杀伤力明显增强（细胞存活率＜70％）。结论MTT 和台盼蓝法检测显示，当微泡造影剂浓度小于10％时，SF6不会引起明显的细胞死亡，可作为最优细胞转染浓度进行后续研究。     

大蒜素诱导人肝癌HepG2细胞凋亡的影响及机制

目的 探讨大蒜素对体外培养的人肝癌HepG2细胞凋亡的促进作用及其分子机理.方法 采用TUNEL法检测大蒜素对HepG2细胞凋亡的影响,提取Bcl-2基因的mRNA,以RT-PCR方法研究大蒜素对Bcl-2基因表达的影响.结果 大蒜素使人HepG2细胞凋亡指数增加(P<0.01),且呈剂量依赖效应;Bc1-2基因mRNA水平随大蒜素浓度升高而降低.结论 大蒜素可促进人HepG2细胞的凋亡过程,并使Bcl-2基因mRNA水平降低,从而起到抗肿瘤的治疗作用.探讨大蒜素体内外诱导人肝癌细胞HepG2细胞凋亡的作用及机制.     

Gene Transfection Mediated by Ultrasound and Pluronic P85 in HepG2 Cells

In order to assess whether gene transfection could be mediated by ultrasound in associa- tion with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irra- diated by ultrasound at 1 MHz, 0.4-2.0 W/cm2 and 50% duty cycle with plasmid encoding enhanced green fluorescent protein (EGFP) as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal trans- fection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm2 for 30 s. The transfection efficacy in ulstrasound P85 group was three times higher than in single ultrasound group [(17.63±1.07)% vs (5.57±0.56)%, P<0.05]. The cell viability was about 81% and 62% in ultrasound group and ultrasound P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm2 for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound.     

超声介导自制白蛋白微泡和声诺维对体外报告基因转染效率的比较研究

目的:探讨超声介导微泡破裂法促进外源基因的安全转移的方法,为临床上肿瘤等疾病的基因治疗研究提供新的思路。方法:以绿色荧光蛋白基因为标记基因,以人大肠癌细胞株SW480为靶细胞,分别以自制白蛋白微泡和声诺维(SonoVue)微泡作为载体,通过超声介导微泡破裂法促进绿色荧光蛋白GFP基因在人大肠癌细胞株的定向转染,以激光共聚焦显微镜来定性和半定量观察GFP在靶细胞的表达情况,以椎虫蓝染色法检测超声作用于细胞的安全性。结果:当超声的强度为0.75W/cm2,作用时间为40s时,对细胞比较安全;自制白蛋白微泡和声诺维微泡的浓度为10%时,分别达到最佳的基因转染效率,两者无显著性差异,但后者的相对表达强度较高。结论:超声介导微泡破裂法促进外源基因的转移是一种比较安全而有效的基因转染方法。     

Gene transfection mediated by ultrasound and Pluronic P85 in HepG2 cells

Summary In order to assess whether gene transfection could be mediated by ultrasound in association with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irradiated by ultrasound at 1 MHz, 0.4–2.0 W/cm² and 50% duty cycle with plasmid encoding enhanced green fluorescent protein (EGFP) as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal transfection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm² for 30 s. The transfection efficacy in ulstrasound+P85 group was three times higher than in single ultrasound group [(17.63±1.07)% vs (5.57±0.56)%, P<0.05]. The cell viability was about 81% and 62% in ultrasound group and ultrasound+P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm² for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound. This project was supported by a grant from National Natural Sciences Foundation of China (No.30670620).     

Combined pluronic P85- and ultrasound contrast agents-mediated gene transfection to HepG2 cells

This study examined the effect of P85 (a pluronic block copolymer) and microbubble (MB)ultrasound contrast agents under ultrasound irradiation on gene transfection and expression.The pEGFP plasmids tha...     

RNAi cyclin E对肝癌细胞生长的影响

目的:探讨RNAi cyclin E对肝癌细胞生长的影响。方法:肝癌细胞系HepG2采用体外培养,选取siRNA合成双链发卡样DNA;将其重组入pGFP-V-RS得到重组子pGFP-V-RS-siCE951和pGFP-V-RSsiCE1122;两重组子分别进行DNA序列测定、同源性比较;将两重组子和pGFP-V-Con分别转入人肝癌HepG2细胞中,同时设空白对照组(不转染任何质粒,仅加转染试剂)。检测4组HepG2细胞cyclin E mRNA表达情况及细胞的增殖情况。结果:经过同源性检索和优选确定cyclin E基因的siRNA载体pGFP-V-RSsiCE951和pGFP-V-RS-siCE1122;干扰1组(转染pGFP-V-RS-siCE951)和干扰2组(转染pGFP-V-RSsiCE1122)细胞cyclin E mRNA的相对表达量显著低于空白对照组和无关siRNA对照组(P<0.05);干扰1组和干扰2组与空白对照组和无关siRNA对照组比较,在3、4、5 d细胞孔内的吸光度值显著减少,差异有统计学意义(P<0.05)。结论:siRNA载体pGFP-V-RS-siCE951和pGFP-V-RS-siCE1122转染人肝癌HepG2细胞能显著降低细胞cyclin E基因的表达,是理想的siRNA靶区段。抑制肝癌HepG2细胞cyclin E基因表达,可以降低肝癌HepG2细胞的生长速度。     

Ultrasound and Microbubbles： Their Functions in Gene Transfer In Vitro

To examine the role of ultrasound in gene delivery in vitro, three cells lines were exposed to the low-frequency ultrasound of varying intensities and for different durations to evaluate their effect on gene transfection and cell viability of the cells. Microbubble （MB）, Optison （10%）, was also used to observe the role of the microbubbles in gene transfection. The results demonstrated that as the ultrasound intensity and the exposure time increased, the gene transfer rate increased and the cell viability decreased, but at high energy intensities, the cell viability decreased dramatically, which caused the transfer rate to decrease. The most efficient ultrasound intensity for inducing gene transfer was 1 W/cm^2 with duration being 20 s. At the same energy intensity, higher ultrasound intensity could achieve maximal gene transfer rate earlier. Microbubbles could increase ultrasound-induced cell gene transfer rate by about 2 to 3 times mainly at lower energy intensities. Moreover, microbubbles could raise the maximum gene transfer rate mediated by ultrasound. It is concluded that the low-frequency ultrasound can induce cell gene transfer and the cell gene transfer rate and viability are correlated with not only the ultrasound energy intensity but also the ultrasound intensity, the higher ultrasound intensity achieves its maximal transfer rate more quickly and the ultrasound intensity that can induce optimal gene transfer is 1 W/cm^2 with duration being 20 s, and microbubbles can significantly increase the maximal gene transfer rate in vitro.     

超声靶向破坏微泡技术介导小鼠肝癌细胞株JNK1基因表达、细胞迁移和侵袭抑制

目的: 探讨应用超声靶向破坏微泡 (UTMD) 技术介导小鼠肝癌细胞株JNK1基因的表达、细胞迁移和侵袭抑制的作用,阐明其作用机制。方法: 构建并筛选RNA干扰效果最好的短发夹RNA(shRNA)。将小鼠肝癌细胞株Hca-F分为正常Hca-F细胞组、shRNA质粒组、脂质体组、超声微泡结合超声辐照组及脂质体结合超声微泡加超声辐照组。采用倒置荧光显微镜观察各组细胞转染率,荧光定量PCR和Western blotting 法检测JNK1基因mRNA和蛋白表达水平,CCK-8法检测各组细胞的细胞活性,应用Transwell 实验检测各组细胞的体外迁移能力。结果: 脂质体结合超声微泡加超声辐照组细胞转染率高于shRNA质粒组、脂质体组和超声微泡结合超声辐照组(均P<0.05),脂质体组和超声微泡结合超声辐照组比较差异无统计学意义(P>0.05)。脂质体结合超声微泡加超声辐照组JNK1 mRNA和蛋白表达水平低于其他各组(P<0.05);脂质体结合超声微泡加超声辐照组细胞活性和平均穿膜细胞数均低于其他各组(P<0.05)。结论: UTMD技术结合脂质体转染法可以提高小鼠肝癌细胞株JNK1 shRNA的转染效率,增强其对基因表达、细胞活力、迁移和侵袭能力的抑制。     

Efficient Gene Delivery to Myocardium with Ultrasound Targeted Microbubble Destruction and Polyethylenimine

The aim of present study was to evaluate the feasibility and efficiency of enhanced green fluorescent protein （EGFP） gene delivery to myocardium in vivo by ultrasound targeted microbubble destruction （UTMD） and polyethylenimine （PEI）. SonoVue/DNA and PEI/DNA/SonoVue complexes were prepared. Gel electrophoresis analysis was performed to determine the structural integrity of plasmid DNA or PEI/DNA after UTMD. Solutions of plasmid DNA, SonoVue/DNA, PEI/DNA complexes or PEI/DNA/SonoVue complexes were respectively transduced into BALB/c mice hearts by means of transthoracic ultrasound irradiation. Mice undergoing PBS injection, plasmid injection or PEI/DNA complexes injection without ultrasound irradiation served as controls. Gene expression in myocardium was detected 4 days after treatment. Cryosections and histological examinations were conducted. Electrophoresis gel assay showed no damage to DNA or PEI/DNA complexes after UTMD. When the heart was not exposed to ultrasound, the expression of EGFP was observed in the subendocardial myocardium obviously. The strongest expression was detected in the anterior wall of the left ventricle when the heart was exposed to ultrasound alone. Injection of PEI/DNA complexes and UTMD resulted in the highest transfection efficiency and the distributional difference of EGFP was not obvious. No tissue damage was seen histologically. In conclusion, a combination of UTMD and PEI was highly effective in transfecting mice hearts without causing any apparently adverse effect. It provides an alternative to current clinical gene therapy and opens a new concept of non-viral gene delivery for the treatment of cardiac disease.     

新型斑蝥素类似物LB1协同阿霉素杀伤肝癌细胞的实验研究

目的通过水溶性化学小分子蛋白磷酸酶2A抑制剂LB1与阿霉素(doxorubicin,DOX)联合应用,研究其在肝癌化疗过程中的协同作用。方法体外细胞活性分析检测不同剂量LB1对HepG2细胞活性影响及其与阿霉素(DOX)的协同杀伤效应;通过裸鼠体内荷瘤模型以及组织化学分析,进一步观察LB1对肿瘤生长影响以及在体与阿霉素的协同化疗作用。结果 LB1对HepG2细胞生长与作用剂量相关,低剂量时可促进其生长,高剂量时则促进其凋亡,而且在联合阿霉素后,可显著提高对HepG2肿瘤细胞的杀伤作用;体内实验也发现,单纯给予阿霉素抑瘤率为57%,而LB1联合应用后,抑瘤率可达77%,显著提高了阿霉素对肿瘤的杀伤作用(P<0.05),且无明显毒副作用。结论 LB1联合应用可显著提高阿霉素(DOX)对肝癌细胞的化学杀伤作用。     

超声靶向微泡破坏对支持细胞的生物学效应研究

目的:探讨超声靶向微泡破坏对体外培养大鼠支持细胞的影响.方法:分离与培养大鼠支持细胞,制作细胞悬液,添加0.01ml微泡造影剂后即行超声辐照,超声辐照时间为60s,输出声强为0.5W/cm~2,频率为1MHz,分别取照射后5min,2、6、12、24h作为分组时间点,在各时间点及进行波形蛋白免疫组化染色、测定细胞超氧化物歧化酶(Superoxide dismutase,SOD)、丙二醛(Malondialdehyde,MDA)以及细胞计数.结果:超声联合微泡可以降低支持细胞内波形蛋白的表达,引起细胞内早期MDA含量可逆性升高,SOD活性下降,后期(24h)MDA含量恢复正常,SOD活性升高,而支持细胞的存活不受影响.结论:超声靶向微泡破坏支持细胞的结构和功能引起可逆性损伤,通过降低支持细胞紧密连接蛋白的表达从而开放血睾丸屏障,继而影响生精细胞功能,为男性避孕的研究提供新的思路.