外源性PTEN基因体外转染C6胶质母细胞瘤株及稳定表达蛋白产物的鉴定

目的 探讨外源性PTEN抑癌基因转染胶质母细胞瘤株并表达活性蛋白产物的可行性.方法 对获赠的包含有人PTENcDNA全长序列的PRK-PTEN质粒进行扩增和酶切鉴定后,用脂质体介导PRK-PTEN质粒转染大鼠C6胶质母细胞瘤株,荧光染色检测转染细胞中PRK-PTEN质粒携带的绿色蛋白荧光报告基因,免疫组化染色鉴定稳定转染后C6细胞中PTEN功能蛋白.结果 转染后的C6胶质母细胞瘤株稳定表达绿色荧光蛋白和PTEN功能蛋白.结论 PRK-PTEN质粒载体在脂质体的介导下可以将PTEN基因整合到C6胶质母细胞瘤的基因序列中去,并且通过宿主基因序列的表达稳定地产生PTEN功能蛋白,为以PTEN基因为靶点的胶质瘤基因治疗提供了实验理论依据.     

多聚胺阳离子脂质体转染效率及细胞毒性的评价

目的评估四种多聚胺阳离子脂质体的转染效率及细胞毒性,筛选高效低毒的阳离子脂质体。方法多聚胺阳离子脂质TC-Chol、DC-Chol与中性磷脂DOPE分别以1：1、3：1摩尔比制备多聚胺阳离子脂质体,转染以增强型绿色荧光蛋白为报告基因的质粒PIRES2-EGFP入Hela细胞、Hep2细胞,倒置荧光显微镜下检测转染细胞的报告基因表达,筛选出高效的阳离子脂质体,通过MTT法检测转染细胞毒性,从而筛选相对高效低毒的阳离子脂质体。结果所制备的多聚胺阳离子脂质体在透射电镜下呈圆形、椭圆形囊泡样结构,少数呈管状、不规则状,粒径50-200nm,可将质粒PIRES2-EGFP有效转染入Hela细胞、Hep2细胞,其中多聚胺阳离子脂质TC-Chol与DOPE以3：1摩尔比制备的多聚胺阳离子脂质体是一种相对高效低毒的阳离子脂质体,转染效率39．5％～42．1％。结论多聚胺阳离子脂质TC-Chol、DC-Chol与中性磷脂DOPE以一定的摩尔比可制备高效低毒的多聚胺阳离子脂质体,其转染效率和细胞毒性与阳离子脂质的结构和中性磷脂DOPE的比例有关,在基因转染和基因治疗方面具有较广阔的应用前景。     

Chitosan-DNA介导关节基因转移的体外研究

目的探索体外Chitosan对关节软骨细胞、滑膜细胞的转染.方法分离培养兔正常滑膜细胞、软骨细胞,使用荧光质粒pEGFP-C3作为报道基因,制备Chitosan-DNA超微颗粒转染兔正常软骨细胞、滑膜细胞,荧光显微镜观察绿色荧光蛋白的表达.结果Chitosan能将DNA带入胞内,该DNA超微颗粒随时间的延长可缓慢进入胞内,其包被的DNA也可以被缓慢释放,且对软骨细胞的转染能力强.结论Chitosan 在体外可介导关节软骨细胞、滑膜细胞基因转移,为今后关节疾病非病毒基因转移的研究提供实验依据.     

经导管动脉注入脂质体介导的p53基因治疗肝癌的实验研究

目的 以兔VX2肝癌模型为对象,探讨经导管动脉注入脂质体介导的p53基因治疗肝癌的可行性及转染和表达情况.方法 将pCMV-myc-p53质粒、阳离子脂质体LipofectAMINE以及pCMV-myc-p53和LipofectAMINE的复合体分别注入兔VX2肝癌模型的肿瘤供血动脉,并提取肿瘤组织蛋白,采用蛋白印迹法及免疫组化检测基因转染及其表达.以不同量的pCMV-myc-p53与LipofectAMINE形成的复合体分别注入兔VX2肝癌模型的肿瘤供血动脉内,同法检测基因的转染及其表达.结果 脂质体介导的p53基因经动脉途径成功转染了兔VX2肝癌模型的肿瘤组织并进行表达,其转染效率明显高于单纯基因导入,基因的量与转染效率之间存在量效关系.结论 经动脉途径导入脂质体介导的p53基因治疗肝癌是可行、有效的,具有广阔的应用前景.     

经导管动脉输送转铁蛋白增强p53基因转染效率的研究

目的 研究转铁蛋白(Tf)对p53基因转染效率的影响,并探讨介入技术与Tf联合应用对基因治疗肝癌的双重靶向作用.方法 应用pCMV-myc-p53质粒与阳离子脂质体LipofectAMINE复合物,转染3种肝癌细胞株LM6、Hep3B、YY以及正常肝细胞株L02,以不同浓度Tf(0、10、25、50及100 μg)介导转染,蛋白印迹法检测各细胞株中p53表达情况,并对Tf影响p53转染效率的关系进行分析.再建立兔VX2肝癌动物模型,经介入导管输注Tf-质粒-脂质体复合物,提取肿瘤组织蛋白,蛋白印迹法检测P53表达.结果 4种细胞应用p53-脂质体复合物以及不同浓度Tf转染后48 h的蛋白印迹法检测,发现Tf在10～100 μg可明显增强p53-脂质体的转染效率,且转染效率随Tf浓度增加而增强.动物模型组织提取蛋白检测结果,显示Tf明显增强P53蛋白表达.结论 Tf能增强脂质体-基因转染,介入技术与Tf联合双重靶向对肝癌的基因治疗具有相当的应用前景.     

CB-GFP融合基因在COS-7细胞中的表达与定位

目的：构建以绿色荧光蛋白（green fluorescent protein，GFP）为报告基因的重组表达质粒pEGFP-N1-CB，转染体外培养的COS-7细胞，以观察CB重组蛋白在真核细胞中的表达及定位。方法：PCR方法扩增得到去除终止密码的cB融合基因序列，克隆入真核表达载体pEGFP-N1中，构建重组表达载体pEGFP-N1-CB。脂质体法转染体外培养的COST细胞后，以RT-PCR和Western印迹方法验证其mRNA及蛋白的表达，并在活细胞状态下用荧光显微镜、激光共聚焦显微成像技术直接观察CB-GFP融合蛋白在细胞中的分布和定位。结果：RT．PCR及Western印迹结果均证明CB—GFP融合基因表达载体pEGFP-N1-CB在COS．7细胞中获得了表达。荧光显微镜观察显示，在空载体pEGFP-N1转染组中，COST细胞内荧光呈弥散分布；重组质粒pEGFP-N1-CB转染组中，绿色荧光主要聚集在细胞浆中。结论：CB融合基因能在真核细胞COST中得到高效表达，且蛋白表达主要定位于细胞浆中，本试验为CB重组蛋白的提取及进一步功能研究奠定了基础。     

pEBFP/FP6共表达质粒转染神经干细胞的实验研究

目的：将α2巨球蛋白cDNA片段（FP6）-增强型绿色荧光蛋白（pEBFP）共表达质粒pEBFP／FP6转染至神经干细胞（NSC）中，观察其对NSC生长、分化、增殖的影响。方法：培养NSC，采用脂质体转染方法将pEBFP／FP6共表达质粒转染进NSC中，利用其携带的绿色荧光和RT—PCR观测其转染情况。结果：经鉴定所培养的细胞是NSC，采用脂质体转染方法成功的将pEBFP／FP6双表达质粒转染进NSC。结论：脂质体转染能将pEBFP／FP6共表达质粒转染进NSC中，pEBFP／FP6基因对神经干细胞生长、分化、增殖无明确影响。     

神经特异性转录因子DAT1绿荧光蛋白表达载体的构建及其在C8细胞中的表达定位

目的：构建神经特异性转录因子DAT1与绿荧光蛋白融合表达的表达载体，并检测其在C8星形胶质细胞中的表达。方法：采用基因重组技术将DAT1基因和绿色荧光蛋白基因融合，构建绿荧光蛋白表达载体pEGFP—C1-DAT1，测序验证序列无误后。用脂质体法将重组质粒转入C8细胞，荧光显微镜观察DAT1的表达及定位。结果：测序验证结果表明，重组质粒含有DAT1全长编码序列，转染实验表明EGFP—DAT1能在C8细胞中正确表达，且：DAT1蛋白主要定位于细胞核中。结论：DAT1全长cDNA基因绿色荧光蛋白表达载体构建成功，在C8细胞中可以正确表达，为进一步研究DAT1的功能奠定了基础。     

骨关节病滑膜细胞凋亡和癌基因表达

为了探索骨关节病在细胞和分子水平上的发病机理，观察了骨关节病的关节滑膜细胞的凋亡和癌基因表达。采用ＤＮＡ末端转移酶介导的凋亡细胞原位检测技术，检测正常滑膜和病理滑膜细胞程序化死亡，同时，采用免疫组化法观察ｐ５３，ｅｒｂＢ－２和ＰＣＮＡ在滑膜细胞中的表达。结果发现骨关节病中有，７７．８％出现滑膜细胞凋亡，与正常对照有明显差别。病理组中癌基因表达昨３阳性占７１．４％，ｅｒｂＢ－２阳性占７１．４％，ＰＣＮＡ在正常和病理滑膜细胞中均呈阴性表达。说明细胞凋亡和多种癌基因的产物有关，ｐ５３和ｅｒｂＢ－２对细胞凋亡有调节作用。     

一种用于上调内源性人SOD1表达多肽筛选的随机文库及细胞系的构建

目的以增强型绿色荧光蛋白(EGFP)为示踪物,建立一种简便、经济、有效的随机多肽文库,为进一步筛选出可上调内源性人SOD1表达多肽的药物奠定基础。方法采用点突变技术,在pET-14b-His-Tat-EGFP载体多克隆位点XhoI之后插入12个随机多肽的36个随机碱基序列。用阳离子脂质体LipofectamineTM2000包裹已构建的重组质粒pDsRed1-1-SOD1p后,转染NIH/3T3细胞,荧光显微镜观察SOD1启动子驱动的红色荧光蛋白的表达。用G418筛选稳定表达SOD1启动子驱动的红色荧光蛋白的细胞系,荧光显微镜检测红色荧光蛋白的表达及定位。结果在示踪载体的基础上构建的随机多肽文库理论上至少包含了107个独立克隆,且几乎所有克隆插入的随机片段都是36个碱基。稳定转染3个月后,重组质粒转染的细胞质及细胞膜上均有携带SOD1启动子的红色荧光蛋白的表达。结论成功建立随机多肽表达文库,获得了稳定表达SOD1启动子驱动的红色荧光蛋白的NIH3T3细胞系。     

The human norepinephrine transporter in combination with 11C-m-hydroxyephedrine as a reporter gene/reporter probe for PET of gene therapy.

Although the herpes simplex virus thymidine kinase gene has been frequently applied as a reporter gene for monitoring gene transfection in animals, it has some intrinsic limitations for use in humans. In our search for a reporter gene that lacks these limitations, we have evaluated the feasibility of the human norepinephrine transporter (hNET) as a reporter gene in combination with the reporter probe 11C-m-hydroxyephedrine (mHED) for PET. METHODS: An adenoviral vector (AdTrack-hNET) containing the hNET gene as reporter gene and the enhanced green fluorescent protein (EGFP) as a substitute for a therapeutic gene was constructed. After COS-7, A2780, and U373 cells were transiently transduced with AdTrack-hNET, hNET protein expression, EGFP fluorescence, and cellular uptake of 11C-mHED were determined. In rats, U373 tumor xenografts were grown and transiently transduced with either AdTrack-hNET or an AdTrack-Luc control adenovirus. Intratumoral accumulation of 11C-mHED was determined by PET and ex vivo biodistribution. The tumors were subsequently examined for EGFP fluorescence. RESULTS: 11C-mHED uptake was positively correlated with AdTrack-hNET viral titer and hNET protein expression. However, large differences in transfection efficiency between cell lines were observed. The highest 11C-mHED uptake was found in hNET transfected U373 cells, in which tracer uptake was >70-fold higher than that in control cells. 11C-mHED accumulation could be inhibited by desipramine, a potent inhibitor of hNET. In all cell lines, 11C-mHED uptake was positively correlated with EGFP fluorescence, implying that imaging of hNET with 11C-mHED would enable monitoring of a coexpressed therapeutic gene. In the animal model, gene transfection efficiencies were very low, as determined by EGFP fluorescence. Still, a significantly higher 11C-mHED uptake in hNET transduced tumors than that in control tumors was demonstrated by ex vivo biodistribution studies. PET with a clinical camera could visualize 1 of 3 hNET transduced tumors, indicating that the transfection efficiency was near the detection limit. CONCLUSION: These results indicate that monitoring of gene therapy using the hNET/11C-mHED reporter gene/probe is feasible, but further investigation with regard to the sensitivity of the technique is required.     

Development of echogenic, plasmid-incorporated, tissue-targeted cationic liposomes that can be used for directed gene delivery

RATIONALE AND OBJECTIVES: Echogenic antibody-conjugated anionic liposomes have been developed that allow directed tissue targeting and acoustic enhancement. These are not efficient for gene delivery. A cationic formulation that allows directed gene delivery while retaining acoustic properties may provide more efficient transfection. METHODS: Cationic liposomes were prepared and acoustic reflectivity was determined. Anti-fibrinogen-conjugated liposomes were laid on fibrin-coated slides and adherence was quantified using fluorescence techniques. Liposomes were combined with a reporter gene and plated on cell cultures. Human umbilical vein endothelial cells were stimulated to upregulate intercellular adhesion molecule-1 (ICAM-1) and were treated with anti-ICAM-1-conjugated liposomes, and gene expression was quantified. RESULTS: Cationic liposomes retained their acoustic reflectivity and demonstrated specific adherence to fibrin under flow conditions. Significant transfection of human umbilical vein endothelial cells was demonstrated, with higher gene expression seen with specific antibody-conjugated liposomes. CONCLUSIONS: Novel acoustic cationic liposomes have been developed that can be antibody conjugated for site-specific adherence and directed cell modification. This presents exciting potential for a vector that allows tissue enhancement and targeted gene delivery.     

定量超声基因导入与效应控制

目的研究低频超声裸基因导入新方法与基因导入效应的控制,为疾病的基因治疗提供安全可靠、无细胞毒性的导入新方法。方法用不同参数的35.1kHz超声进行裸基因导入细胞实验,用激光共聚焦显微镜、荧光显微镜、流式细胞仪和分光光度法分析细胞膜形态、细胞膜通透性与损伤阈值、细胞存活率和基因表达。结果实验所见90%细胞存活点的超声能量积分是细胞膜通透安全控制参数,80%细胞存活点的能量积分是细胞的损伤阈值。绿色荧光蛋白(GFP)在安全超声参数下成功导入了S180细胞,细胞转染率为35.83%±2.53%(n=6),荧光表达强度明显高于病毒转染法和控制组(P<0.001)。结论安全参数下低频超声可有效地将裸基因导入S180肿瘤细胞,其基因表达强度随超声能量积累而增强,并有细胞凋亡倾向。     

siRNA对体外培养大鼠海马神经细胞NgR基因表达的抑制

目的 研究siRNA对体外培养的大鼠海马神经细胞NgR基因表达抑制作用,为进一步研究中枢神经损伤修复提供实验基础.方法 使用阳离子脂质体转染试剂将体外化学合成的针对NgR基因的siRNA转染体外培养的海马神经细胞,GAP43对培养细胞进行鉴定,分别于转染后48小时、1周收集转染细胞,提取总蛋白并定量.结果 海马神经细胞成功培养,转染48小时后NgR蛋白的含量为阴性对照组的45.11%,差异显著P＜0.05;转染1周后NgR蛋白的含量为阴性对照组的99.18%,差异不明显.结论 化学合成的针对NgR基因的siRNA在转染48小时后能显著下调NgR基因表达,但在转染1周后对NgR基因表达影响不明显.     

A multimodality reporter gene for monitoring transplanted stem cells

IntroductionThe aim of this study is to explore the feasibility of a triple-fused reporter gene, termed TGF [herpes simplex virus type 1 thymidine kinase (HSV1-tk), enhanced green fluorescent protein (eGFP) and firefly luciferase (Fluc)], to monitor stem cells using multimodality molecular imaging.MethodsA recombinant adenovirus vector carrying the triple-fused reporter gene (Ad5-TGF) was constructed. Bone marrow mesenchymal stem cells (BMSCs) were transfected with different virus titers of Ad5-TGF [multiplicities of infection (MOIs) were 0, 50, 100, 150, 200 and 250]. The mRNA and protein expressions of HSV1-tk, eGFP and Fluc in the transfected BMSCs were evaluated using polymerase chain reaction and Western blot. After the transfection of the BMSCs with different virus titers of Ad5-TGF (MOIs were 25, 50, 75, 100 and 125), their uptake rates of ¹³¹I-FIAU were measured. Whole-body fluorescence, bioluminescence and micro-positron emission tomography (PET) images were acquired 1 day after the transfected BMSCs were injected into the left forelimb of rats.ResultsAfter the transfection with different titers of Ad5-TGF, the positive transfection rate reached a peak (70%) when the MOI was 100. HSV1-tk, eGFP and Fluc mRNA and protein were detected in the Ad5-TGF-transfected BMSCs, which implies their successful transfection and expression. The BMSCs uptake of ¹³¹I-FIAU increased with the adenovirus titer and incubation time and reached a plateau (approximately 5.3%) after 3 h. Strong signals were observed in the injected left forearms in the fluorescence, bioluminescence and micro-PET images.ConclusionsA triple-fused reporter gene, TGF, can be used as a multifunctional molecular probe for multimodality imaging.     

关节腔内注射裸露DNA治疗关节炎机制的实验研究

关节腔内注射裸露ＤＮＡ,是实现关节内基因转移的一种最简便的方法,但其机制尚不了解。本实验利用差速离心法,提取类风湿性关节炎患者关节滑膜细胞的非核膜蛋白,以大鼠肝细胞非核膜蛋白作为对照,应用32Ｐ标记的 ＤＮＡ,进行 ＤＮＡ膜蛋白结合实验。我们发现在滑膜细胞微粒体膜的抽提物中存在一种非核 ＤＮＡ结合蛋白质,分子量约为８３ＫＤａ,这种蛋白质能特异地与双链线状ＤＮＡ结合,可能在关节滑膜介导的基因转移中起一定作用。     

重组人血管能抑制素分泌型表达载体的构建及其生物学效应研究

目的　构建表达分泌型人血管能抑制素 (canstatin)蛋白的真核表达载体 ,观察其生物学效应。方法　以SOEPCR法将canstatin及癌胚抗原 (CEA)引导肽cDNA体外连接 ,并定向克隆到真核蛋白表达载体pCMV Script上 ,脂质体介导转染HUVEC及A5 4 9细胞 ,荧光定量PCR法检测转染细胞canstatin基因表达量 ,并采用多种方法检测该重组载体的生物学活性。结果　成功构建canstatin分泌型载体 ,在其转染的肿瘤细胞和人脐静脉内皮细胞中均检测到不同拷贝数的表达。转染后HUVEC生长增殖缓慢 ,出现G0 ～G1期阻滞并伴有大量凋亡 ,与空载体转染组相比差异有统计学意义 (P <0 0 5 ) ;而A5 4 9细胞转染后与空载体转染组在以上几个方面差异均无统计学意义 (P >0 0 5 )。重组载体转染A5 4 9细胞上清液干预的鸡胚绒毛尿囊膜血管生成明显减少 ,且颜色苍白、轮廓模糊 ,空载体转染组和未干预组血管生长旺盛、脉络清晰。结论　SOE法构建的含有CEA引导肽的canstatin分泌型表达载体能在转染的内皮细胞及肺癌细胞中稳定表达 ,可有效抑制内皮细胞生长增殖并诱导凋亡。重组载体转染的细胞可分泌有生物学活性的canstatin。