骨形成蛋白2重组腺病毒的构建与鉴定

目的　通过构建骨形成蛋白 (BMP)重组腺病毒 ,为骨缺损的基因治疗提供可能。方法　应用逆转录 聚合酶链反应 (RT PCR)法克隆出BMP2全长基因 ,构建重组腺病毒载体 ,DNA 磷酸钙共沉法将辅助质粒PJM 17转染 2 93细胞 ,同源重组构建出重组腺病毒。测滴度并用氯化铯梯度离心纯化备用。结果　逆转录克隆出BMP2全长基因 1.2kb ,测序后连接于重组腺病毒载体 ,酶切鉴定后转染构建活病毒 ,再次感染 2 93细胞扩增 ,测滴度为× 10E10 pfu/ml ,并应用PCR法测定目的序列。结论　BMP2重组腺病毒的构建为骨缺损等疾病的基因治疗提供可能     

抑瘤细胞运动和侵袭的鼠FRNK重组腺病毒的构建

目的应用AdEasy复制缺陷型腺病毒载体系统构建小鼠FRNK基因重组腺病毒，并在293HEK细胞中扩增制备重组病毒。方法把小鼠FRNK基因克隆入腺病毒穿梭载体pAdTrack—CMV中，构建腺病毒穿梭质粒pAdTrack—CMV-FRNK，经PmeⅠ内切酶酶切成线性化后，采用电穿孔转化将其转化到含有腺病毒骨架载体pAdEasy-1的感受态大肠杆菌BJ5183中，通过卡那霉素筛选获得阳性腺病毒重组质粒。再将获得的腺病毒重组质粒转染到293HEK细胞进行包装，获得FRNK重组腺病毒pAdFRNK。荧光显微镜下观察绿荧光的表达。结果经酶切和绿荧光表达证明了小鼠FRNK基因的重组腺病毒载体pAdFRNK构建成功，并制备出高滴度的重组病毒。结论成功构建携带小鼠FRNK基因的重组腺病毒pAdFRNK，为研究FRNK的基因治疗奠定了基础。     

腺病毒介导的人骨形成蛋白2基因治疗的免疫学研究

目的评价机体对腺病毒介导的人骨形成蛋白2(adenovirusmediatedhumanbonemorphogeneticprotein2,Ad-hBMP-2)基因治疗的免疫学反应。方法崇明山羊12只,手术截去右侧胫骨中段2.1cm,制备胫骨干缺损模型,随机分为2组。将Ad-hBMP-2转染的山羊骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs,转染组,n=7)及未转染的MSCs(未转染组,n=5)分别植入骨缺损内。于术后4、8、16及24周摄X线片检查骨缺损愈合情况,并对治疗前后机体对腺病毒的细胞和体液免疫反应进行检测。结果X线片示,转染组4～8周,骨缺损内有连续性骨痂形成;24周,有6侧骨缺损完全愈合,部分骨髓腔再通。未转染组4～8周,骨缺损内骨痂形成较少,与骨端界限清楚;24周,仅2侧骨缺损愈合。淋巴细胞与MSCs混合培养示淋巴细胞刺激指数(stimulationindex,SI)于植入后14d升高,转染组4.213±1.278,未转染组-0.310±0.147,且差异有统计学意义(P<0.05);28d后下降,转染组2.544±0.957,未转染组3.104±0.644,差异无统计学意义(P>0.05)。治疗后14、28、49和120d,转染组的血浆腺病毒中和抗体滴度分别为2.359±0.226、2.297±0.200、2.214±0.215和2.297±0.210,未转染组分别为-0.175±0.335、-0.419±0.171、0±0.171和0.874±0.524,两组各时间点差异均有统计学意义(P<0.05)。结论腺病毒介导的基因治疗可引起机体对腺病毒的细胞及体液免疫反应,从而逐渐消除腺病毒基因和相关蛋白的影响。     

腺病毒介导的LacZ基因在培养许旺细胞中的表达

目的 观察腺病毒介导的LacZ基因在培养许旺细胞中的表达。方法 用报告基因LacZ重组腺病毒感染原代培养的许旺细胞，X-gal组织化学染色检测LacZ基因的表达。结果 用LacZ重组腺病毒感染培养的许旺细胞时，有较好的量效关系和时效关系。当感染增殖率分别为200、100时，24、48h后接近100％的许旺细胞被感染。而且感染后许旺细胞的形态无明显异常，增殖能力也没有受到影响。结论 腺病毒介导的LacZ基因可转入体外培养的许旺细胞并高效表达，同时许旺细胞的生长特性并不受影响，为腺病毒介导神经营养因子等基因治疗促进外周周围神经损伤再生奠定了基础。     

腺病毒载体介导的双自杀基因对人胆管癌细胞QBC939的体外杀伤实验

目的观察腺病毒介导的胞嘧啶脱氨酶/单纯疱疹病毒-1-胸苷激酶(CD/TK)双自杀基因对人胆管癌细胞 QBC939的体外杀伤作用。方法 CD/TK 克隆入穿梭载体成为 pAdtrack-CMV-CD/TK,与骨架载体 pAdeasy-1在细菌内同源重组为 pAd-CD/TK,经 PacⅠ酶切,293细胞包装、扩增、纯化后,体外转染人胆管癌细胞 QBC939,并给予前药5-FC 或 GCV,观察其体外杀伤效果。结果含 CD/TK 基因的重组腺病毒鉴定正确,扩增纯化后,病毒滴度为1×10~(11)颗粒/ml。重组腺病毒对 QBC939细胞在感染倍数(m.o.i)为100时的转染效率为90%,在 m.o.i50感染时,0.1mmool/L的5-FC 及10 μmol/L 的 GCV 对 QBC939细胞的杀伤率为80%,明显高于单用5-FC 与 GCV 的效应。结论双自杀基因以腺病毒为载体对人胆管癌细胞转染效率高,体外杀伤效应明显。腺病毒介导的双自杀基因治疗有望成为治疗胆管癌的有效方法。     

重组腺病毒载体介导人OSM 基因对胰腺癌细胞增殖的抑制作用及对裸鼠致瘤性的影响

目的　构建了含人抑瘤素M (humanoncostatinM ,hosm)基因的复制缺陷型重组腺病毒载体 ,观察其对胰腺癌细胞株BxPC3 在体外生长抑制情况及在裸鼠致瘤性改变的作用 ,为胰腺癌的基因治疗提供参考。方法　用含hosm基因的缺陷型腺病毒载体转染人胰腺癌细胞株 ,用RT PCR法检测外源hosm基因在其中的表达 ;苔盘蓝染色、细胞计数法检测ad hosm对BxPC3 的体外增殖抑制情况 ;裸鼠皮下接种转染ad hosm的BxPC3 ,观察其对肿瘤形成率的影响。结果　hosm基因在转染细胞能有效的表达。体外试验示ad hosm能明显抑制BxPC3 的生长 ,ad hosm能抑制BxPC3 在裸鼠中的成瘤作用。结论　重组腺病毒介导hosm基因表达能有效抑制BxPC3 在体外、体内的增殖 ,提示重组腺病毒介导的hosm基因治疗可能成为胰腺癌治疗的侯选方案。     

骨形态发生蛋白-2基因转染兔骨髓基质干细胞及复合纳米羟基磷灰石体外构建组织工程骨的研究

目的 探讨腺病毒介导的人骨形态发生蛋白-2表达载体(Ad-BMP-2)转染兔骨髓基质干细胞(rBMSCs)后复合到纳米羟基磷灰石(Nano-HA)体外构建仿生人工骨的可行性. 方法 采用GatewayTM技术构建腺病毒载体,溶胶-絮凝法制备Nano-HA,用Ad-BMP-2转染体外培养的rBMSCs,免疫组化、RT-PCR和蛋白印迹方法检测转染后细胞的BMP-2表达.将转染48 h的细胞均匀接种到Nano-HA支架上,第3、5天取细胞载体复合物扫描电镜观察细胞贴附、生长状况,消化收集贴附支架的细胞,蛋白印迹检测贴附支架细胞BMP-2的表达.结果 转染后BMP-2基金在mRNA水平和蛋白水平均有高表达;扫描电镜见转染细胞在支架材料分布均匀,黏附良好,转染后及复合后Western Blot检测BMP-2表达较高.结论 利用转基因技术,用含目的基因的腺病毒载体转染靶细胞复合到Nano-HA,细胞载体复合物稳定长效地分泌生长因子,体外成功地构建了仿生人工骨.     

BMP—2基因转染的人骨髓基质干细胞复合PLA／PCL支架体外构建组织工程骨

目的 用人骨形态发生蛋白2腺病毒表达载体(Ad—BMP—2)转染的人骨髓基质干细胞(hBMSC)，复合PLA／PCL(聚乳酸／聚己内酯)生物降解支架体外构建组织工程骨。方法 用Ad—BMP—2转染体外培养的成人BMSC，免疫组化、原位杂交染色和蛋白印迹方法检测细胞BMP—2的表达，并通过流式细胞仪和ALP活性检测分析其对细胞增殖、分化的影响。然后将转染后细胞接种到PLA／PCL支架上，扫描电镜观察细胞贴附、生长状况。结果 转染后，hBMP—2基因在mRNA水平和蛋白水平均有表达；S期细胞比例和ALP活性明显增高。扫描电镜见转染细胞分布均匀，伸展良好。结论 Ad—BMP—2可高效转染hBMSC，且促进细胞增殖及成骨转化。转染后细胞在PLA／PCL上生长良好，BMP—2基因治疗的组织工程骨构建成功。     

腺病毒介导HGF基因转染对人脂肪干细胞生物学特性影响的研究

目的观察腺病毒介导的HGF基因转染对人脂肪干细胞(Human adipose-derived stem cells,hADSCs)生物学特性的影响。方法利用腺病毒介导HGF(MOI=200)转染hADSCs,检测转染组细胞目的基因HGF表达情况,并与未转染组细胞的形态、细胞生长曲线、细胞表面标志物、细胞周期和成脂、成骨诱导分化能力进行比较。结果 hADSCs转染24 h后,即可检测出HGF,并于48 h后达到高峰,1周后HGF表达逐渐降低。hADSCs转染后仍呈成纤维细胞样生长,其生长增殖能力与未转染hADSCs无明显差别;转染后的hADSCs仍表达CD44、CD90、CD49d、CD105,不表达CD45、CD34、CD31、CD106和STRO-1。转染组与未转染组细胞周期、成脂和成骨诱导分化能力无明显差别。结论腺病毒表达载体介导HGF基因转染hADSCs并不影响其生物学特性,是将hADSCs用于基因治疗的良好途径。     

表达人骨形成蛋白4的非复制型腺病毒的构建与鉴定

目的构建含有人骨形成蛋白4（human bone morphogenetic protein4,hBMP-4）的非复制型腺病毒表达载体pAdE/hBMP-4并检测其表达。方法酶切质粒pCS2（＋）/hBMP-4获得目的基因片段,克隆至真核表达载体pcDNA3.1（＋）,再亚克隆至pShuttle-CMV,电转化至含有pAdEasy-1骨架质粒的E.coliBJ5183/p中进行同源重组,重组质粒转染HEK293细胞包装成含有hBMP-4基因的非复制型腺病毒。病毒颗粒感染HEK293和HeLa细胞,提取总RNA和总蛋白,进行RT-PCR和Western-blot检测。结果获得了含有目的基因hBMP-4的非复制型腺病毒表达载体pAdE/hBMP-4,酶切鉴定提示构建正确,RT-PCR检测到hBMP-4基因的转录,Western-blot检测到hBMP-4蛋白表达。结论将目的基因hBMP-4克隆至非复制型腺病毒表达载体中,为进一步研究其在基因治疗骨缺损中的应用奠定基础。     

骨形成蛋白2基因修复兔桡骨缺损的实验研究

目的观察携带人骨形成蛋白2（bone morphogenetic protein 2，BMP-2）基因的腺病毒载体（adenovirus carrying BMP-2 gene，Ad—BMP2），通过纤维蛋白凝胶与牛松质骨支架（bovine cancellous bone，BCB）复合，修复骨缺损的效果。方法将60只新西兰大耳白兔随机分为4组，每组15只。制成双侧桡骨中段1．5cm骨缺损模型，采用4种材料植入修复。A组：Ad-BMP-2＋BCB；B组：重组BMP-2＋BCB；C组：携带D-半乳糖酐酶基因的腺病毒对照载体（adenovirus carrying β—galgene，Ad—Lacz）＋BCB；D组：单纯BCB支架。修复术后各组于4、8和12周各处死动物5只取材，行X线片、组织学、生物力学和免疫组织化学染色检查。结果A、B两组骨缺损均得到了修复，但术后各时间点，A组在成骨活跃程度、新生骨量、力学强度及BMP-2表达等方面均明显优于B组；C、D两组均无新骨形成。结论BMP-2直接基冈治疗，操作简便、骨诱导能力强，是修复节段性骨缺损的有效方法。     

A realistic chance for gene therapy in the near future

The expanding knowledge of the genetic and cellular mechanisms of human diseases in the post-genomic era coupled with the development of different vector systems to efficiently transfer genes to a variety of cell types and organs in vivo gave rise to the concept of gene therapy as a promising therapeutic option for genetic and acquired diseases. Gene therapy has been the focus of both enthusiasm and critique in the past years. Major progress has been achieved in evaluating gene therapy in clinical trials. However, a number of hurdles must still be overcome to make gene therapy safe and applicable for human diseases. Increased knowledge of the interaction of the gene therapy vehicles with the host has resulted in modifications of existing and the development of new vector systems, as well as adjustments of future clinical applications. Adeno-associated virus vectors, retrovirus- and lentivirus-based vectors show great promise for the correction of monogenic diseases. Correction of the genetic defect can be attempted by either in vivo administration to directly target a diseased organ or by administration of ex vivo genetically modified cells, e.g., bone marrow stem cells. The lack of persistent expression and the immune responses of the host have limited the use of adenovirus vectors for the permanent correction of monogenic diseases. However, the ease of production and the number of cell types and organs that can be efficiently infected make adenovirus-based vectors a promising tool for applications where permanent gene expression is not the therapeutic goal or where the induction of immune responses is the desired response, as for genetic vaccines. Overall, gene therapy remains promising for the correction of genetic as well as acquired disorders, where permanent or transient expression of a gene product will be therapeutic.     

Transcription factor decoy oligonucleotide-based therapeutic strategy for renal disease

Renal disease, including slight renal injuries, has come to be seen as one of the risk factors for cardiovascular events. At present, most conventional therapy is inefficient, and tends to treat the symptoms rather than the underlying causes of the disorder. Gene therapy based on oligonucleotides (ODN) offers a novel approach for the prevention and treatment of renal diseases. Gene transfer into somatic cells to interfere with the pathogenesis contributing to renal disease may provide such an approach, leading to the better prevention and treatment of renal disease. The major development of gene transfer methods has made an important contribution to an intense investigation of the potential of gene therapy in renal diseases. Amazing advances in molecular biology have provided the dramatic improvement in the technology that is necessary to transfer target genes into somatic cells. Gene transfer methods, especially when mediated by several viral vectors, have improved to a surprising extent. In fact, some (retroviral vectors, adenoviral vectors, or liposome-based vectors, etc.) have already been used in clinical trials. On the other hand, recent progress in molecular biology has provided new techniques to inhibit target gene expression. The transfer of cis-element double-stranded ODN (= decoy) has been reported to be a powerful novel tool in a new class of antigene strategies for gene therapy. The transfer of decoy ODN corresponding to the cis sequence will result in attenuation of the authentic cis-trans interaction, leading to the removal of trans-factors from the endogenous cis-elements with a subsequent modulation of gene expression.     

骨形成蛋白2基因修饰的老年大鼠骨髓间充质干细胞成骨分化能力的研究

目的观察年龄因素对骨髓间充质干细胞(marrow mesenchymal stem cells, MSCs)成骨分化能力的影响;了解基因治疗对老年大鼠MSCs成骨分化能力的影响. 方法 1月龄(幼年组)、9月龄(成年组)及24月龄(老年组)雄性Wistar大鼠各6只,取MSCs经体外分离、培养及携带骨形成蛋白2(bone morphogenetic protein 2,BMP-2)基因的腺病毒载体(Ad-BMP-2)转染后,定量检测BMP-2、碱性磷酸酶(alkaline phosphate,ALP)表达,以及成骨细胞标志性蛋白:Ⅰ型胶原、骨涎蛋白(bone sialoprotein,BSP)和骨桥素(osteopontin, OPN)的表达.将转染的各组MSCs分别与磷酸三钙(tricalcium phosphate, TCP)复合后植入裸鼠体内,3周后取材,比较各组诱导异位成骨能力. 结果 ELISA检测表明BMP-2基因修饰的MSCs可以有效表达BMP-2,且表达量在各年龄组间差异无统计学意义(P>0.05);各组ALP于诱导后第9天达高峰,但组间差异均无统计学意义(P＞0.05);诱导后第7天,RT-PCR半定量检测示各组均有成骨细胞特征性蛋白,即:Ⅰ型胶原、OPN及BSP的明显表达,表达量在各组间差异无统计学意义(P>0.05);BMP-2基因转染的MSCs与TCP复合后可诱导裸鼠体内异位成骨,各组成骨量差异无统计学意义(P>0.05). 结论 BMP-2基因修饰的老年大鼠MSCs可以恢复成骨分化能力,基因治疗可能为老年性骨骼疾病提供一种新的治疗途径.     

Gene therapy for meningioma: improved gene delivery with targeted adenoviruses

OBJECT: Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with current treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors investigated to determine whether adenoviruses could be used for gene transfer in meningioma cells. METHODS: The presence of the high-affinity Coxsackievirus and adenovirus receptor (CAR) for adenovirus type 5, as well as endothelial growth factor receptor (EGFR) and alpha, integrins (ITGAVs), were analyzed in primary tumors by using immunohistochemical studies and in primary meningioma cell cultures by using fluorescence-activated cell sorting. Targeting of adenoviruses to EGFR was achieved using bispecific antibodies, whereas targeting of adenoviruses to the ITGAVs was accomplished by insertion of an RGD (arginine-glycine-aspartic acid) motif in the adenovirus fiber H1 loop. Gene transfer efficiency of untargeted and targeted vectors was compared in primary cell cultures and in spheroids derived from patients' resected tumor material. The presence of CARs was observed in all tumors and in all but one of the derived primary meningioma cells. The higher expression of EGFRs and ITGAVs indicated that these receptors could be used as alternative targets to redirect the adenoviruses. Redirection of adenoviruses to the EGFRs or integrins enhanced gene transfer threefold (range two-sevenfold) for EGFRs in primary meningioma cells and ninefold (range three-23-fold) for integrins (p = 0.002, analysis of variance). The effect of adenovirus targeting was confirmed in spheroids composed of primary meningioma cells. CONCLUSIONS: Gene transfer with adenoviruses targeted to tumor-specific receptors is very effective in primary meningioma cells and spheroids. These vectors are promising agents for gene therapy of meningiomas.     

Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model

The objective of this study was to compare the efficacy of adenoviral and lentiviral regional gene therapy in a rat critical sized femoral defect model. The healing rates and quality of bone repair of femoral defects treated with syngeneic rat bone marrow cells (RBMCs) transduced with either lentiviral vector (Group I) or adenoviral vector (Group II) expressing bone morphogenetic protein-2 (BMP-2) gene were assessed. RBMCs transduced with the adenoviral vectors produced more than 3 times greater (p < 0.001) BMP-2 when compared to RBMCs transduced with lentiviral vectors in an in vitro evaluation. Serial bioluminescent imaging demonstrated short duration luciferase expression (less than 3 weeks) in defects treated with RBMCs co-transduced with two adenoviral vectors (Group IV; adenovirus expressing BMP-2 and luciferase [Ad-BMP-2 + Ad-Luc]). In contrast, the luciferase signal was present for 8 weeks in defects treated with RBMCs co-transduced with two lentiviral vectors (Group III; lentivirus expressing BMP-2 and luciferase gene [LV-BMP-2 + LV-Luc]). There were no significant differences with respect to the radiological healing rates (p = 0.12) in defects treated with lentiviral versus adenoviral mediated BMP-2 gene transfer. Biomechanical testing of healed Group I femoral specimens demonstrated significantly higher energy to failure (p < 0.05) when compared to Group II defects. Micro CT analysis revealed higher bone volume/tissue volume fraction (p = 0.04) in Group I defects when compared to Group II defects. In conclusion, prolonged BMP-2 expression associated with lentiviral mediated gene transfer demonstrated a trend towards superior quality of bone repair when compared to adenoviral mediated transfer of BMP-2. These results suggest that the bone repair associated with regional gene therapy is influenced not just by the amount of protein expression but also by duration of protein production. This observation needs validation in a more biologically challenging environment where differences in healing rates and quality of bone repair are more likely to be significantly different.     

压缩冲击对骨形成蛋白2基因修饰的组织工程骨的影响

目的观察直接压缩冲击对骨形成蛋白2(bone morphogenetic protein 2,BMP-2)基因修饰的组织工程骨细胞存活和成骨的影响,以探讨组织工程骨压缩植骨的可行性.方法体外培养扩增犬骨髓基质干细胞(marrow stromal stem cells,MSCs)转染腺病毒介导的人BMP-2(adenovirus mediated human BMP-2,Adv-hBMP-2)基因,与颗粒状的犬异体冻干松质骨复合.复合后4 d进行模拟压缩实验,分别于复合后24 h、4 d,压缩后即刻、1、4 d以扫描电镜观察细胞形态及数量.将单纯冻干骨、未经压缩的Adv-hBMP-2基因修饰的组织工程骨和压缩后4 d的Adv-hBMP 2基因修饰的组织工程骨植入裸鼠背部皮下,于术后6周行组织学观察新骨形成及冻干骨吸收情况.结果复合24 h冻干骨表面细胞展开,部分孔隙可见单层细胞生长;4 d后冻干骨表面细胞复层生长,胶原量多;压缩后即刻,冻干骨与冲击器接触的外表面基本无细胞存在,剖开面可见细胞片状掀起,碎片多;压缩后1 d,冻干骨表面细胞大部分脱落掀起,形成较多的细胞碎片;4 d后细胞数量明显减少,胶原分泌量增多.植入裸鼠背部皮下后,单纯冻干骨新骨形成极少,孔隙内主要为纤维组织,未经压缩的Adv-hBMP-2基因的组织工程骨组可见大量新骨形成,材料中心与周围新骨分布均匀,压缩后的Adv-hBMP-2基因的组织工程骨组新骨量明显减少,且主要在外周.结论体外模拟压缩植骨可明显减少基因修饰的组织工程骨中的细胞存活及体内成骨,但存活细胞的功能仍存在,可适用于压缩植骨重建假体周围骨缺损.     

携带人血管内皮细胞生长因子基因细菌内重组腺病毒转染兔骨髓基质干细胞的表达

目的应用高效细菌内同源重组系统pAd-Easy,制备含人血管内皮细胞生长因子121 (VEGF121)基因的重组腺病毒,感染兔骨髓基质干细胞(BMSCs),并检测外源基因的表达,为其进一步应用于血管化组织工程骨组织的构建打下基础。方法自pCDNA-VEGF121质粒中切取VEGF121基因,将VEGF基因克隆到穿梭质粒,在BJ5183细菌内重组,在293细胞中构建携带VEGF基因的重组腺病毒,将腺病毒感染BMSCs,利用ELISA、免疫组织化学的方法检测VEGF121在BMSCs中的表达。结果通过pAd-Easy系统成功构建高滴度的携带VEGF121基因重组腺病毒pAd-VEGF,ELISA检测显示经转染的BMSCs中VEGF121的表达均明显增强,随着感染病毒MOI值的增高,VEGF121的表达量相应增高,差异有统计学意义(P<0．01),免疫组化法显示经基因转染的BMSCs中有强阳性信号,未转染组出现阴性结果。结论Ad-VEGF转染BMSCs后能稳定提高VEGF蛋白的表达。