利用报告基因rNIS监测大鼠骨髓间充质干细胞移植大鼠心肌的可行性研究

Objective To investigate the feasibility of rat sodium/iodide symporter (rNIS) as a reporter gene monitoring rat bone marrow mesenchymal cells (rBMSC) transplanted to rat myocardium in vivo.Methods Recombinated adenovirus vector was constructed by rNIS/enhanced green fluorescence protein (EGFP) (Ad-rNIS/EGFP).rBMSC transfected by Ad-rNIS/EGFP were studied using fluorescence microscope.Fifteen rats were transplanted with rBMSC and randomly divided into three groups:rNIS group (with rNIS transfection), blocked group (with rNIS transfection) by oral intake of perchloric sodium before planar imaging(GE Millennium MPR SPECT), and control group (without rNIS transfection).All rats underwent 99Tcm-pertechnetate planar imaging.The biological distribution of 99Tcm-pertechnetate was studied.The expressions of rNIS gene and protein in myocardium were measured by real time polymerase chain reaction (PCR) and western blot, respectively.The expressions of CD29, CD44, CD90, CD11b, CD34 and CD45 were measured by immunohistochemistry.Results rBMSC transfected by Ad-rNIS/EGFP showed EGFP expression under fluorescence microscope.The transplanted rat myocardium could be visualized on 99Tcm-pertechnetate planar imaging in rNIS group.The relative uptake ratio( Rheart/Rhmb, RUR) was 6.7 ±0.4.RUR in control group (3.0 ±0.2) was lower than that in rNIS group (t =2.78, P=0.03).The percentage injection dose per gram of tissue (% ID/g) of the transplanted myocardium was 60.2 ± 20.8 in rNIS group,which was higher than that (2.5 ± 0.4) % ID/g of control group ( t = 7.13, P＜0.001 ).rNIS gene and protein were highly expressed in transplanted myocardium in rNIS group but less expressed in control group.The expressions of CD29, CD44 and CD90 were positive, CD45 and CD45 negative CD11b mildly positive in the myocardium transplanted with infective rBMSC.Conclusion rNIS can efficiently monitor rBMSC transplanted to rat myocardium.     

利用报告基因rNIS监测大鼠骨髓间充质干细胞移植大鼠心肌的可行性研究

Objective To investigate the feasibility of rat sodium/iodide symporter (rNIS) as a reporter gene monitoring rat bone marrow mesenchymal cells (rBMSC) transplanted to rat myocardium in vivo.Methods Recombinated adenovirus vector was constructed by rNIS/enhanced green fluorescence protein (EGFP) (Ad-rNIS/EGFP).rBMSC transfected by Ad-rNIS/EGFP were studied using fluorescence microscope.Fifteen rats were transplanted with rBMSC and randomly divided into three groups:rNIS group (with rNIS transfection), blocked group (with rNIS transfection) by oral intake of perchloric sodium before planar imaging(GE Millennium MPR SPECT), and control group (without rNIS transfection).All rats underwent 99Tcm-pertechnetate planar imaging.The biological distribution of 99Tcm-pertechnetate was studied.The expressions of rNIS gene and protein in myocardium were measured by real time polymerase chain reaction (PCR) and western blot, respectively.The expressions of CD29, CD44, CD90, CD11b, CD34 and CD45 were measured by immunohistochemistry.Results rBMSC transfected by Ad-rNIS/EGFP showed EGFP expression under fluorescence microscope.The transplanted rat myocardium could be visualized on 99Tcm-pertechnetate planar imaging in rNIS group.The relative uptake ratio( Rheart/Rhmb, RUR) was 6.7 ±0.4.RUR in control group (3.0 ±0.2) was lower than that in rNIS group (t =2.78, P=0.03).The percentage injection dose per gram of tissue (% ID/g) of the transplanted myocardium was 60.2 ± 20.8 in rNIS group,which was higher than that (2.5 ± 0.4) % ID/g of control group ( t = 7.13, P＜0.001 ).rNIS gene and protein were highly expressed in transplanted myocardium in rNIS group but less expressed in control group.The expressions of CD29, CD44 and CD90 were positive, CD45 and CD45 negative CD11b mildly positive in the myocardium transplanted with infective rBMSC.Conclusion rNIS can efficiently monitor rBMSC transplanted to rat myocardium.     

^13N—NH3 PET及冠状动脉造影评价CD151基因转染对小型猪心肌梗死后血流灌注的影响

目的用^13N—NH,PET及冠状动脉造影共同评价CD151基因转染促小型猪心肌梗死后血运重建。方法结扎20头小型猪冠状动脉左前降支（LAD）,建立心肌梗死模型。对梗死区及梗死周围心肌直接注射CD151及绿色荧光蛋白（GFP）重组腺相关病毒（rAAV）进行基因转染。8周后用免疫组织化学方法分析心肌组织CD151蛋白的表达和心肌组织微血管密度,用^13N—NH,PET显像评价心肌血流灌注,用LAD造影评价侧枝循环的建立。采用SPSS11．0软件行配对t检验或方差分析（ANOVA）。结果CD151基因转染促进局部心肌组织CD151高表达并增加缺血区心肌组织微血管密度。rAAV—CD151组心肌血流灌注明显增加,心肌缺血总分值为10．82±2．36,明显小于rAAV—GFP组（19．33±1．67,t=5．86,P=0．002）。冠状动脉造影显示rAAV—CD151组缺血心肌的侧枝循环建立明显较rAAV—GFP组增加。结论CD151基因转染可以明显促进心肌梗死后血运重建、增加血流灌注。^13N—NH,PET及冠状动脉造影能直观地评价心肌血运重建。     

^99Tc^m-亚锡葡庚糖酸钠结合GGC序列监测基因治疗的实验研究

目的 构建以金属结合肽（双甘氨酰半胱氨酸,即GGC序列）为核素报告基因、人VEGF165为治疗基因的重组腺病毒载体,以99Tcm-GH为报告探针,探讨该报告系统监测治疗基因表达的可行性.方法 pcDNA3-VEGF165质粒线性化后,将金属结合肽GGC序列连于VEGF165基因C端,通过内部核糖体切入位点（IRES）连接增强型绿色荧光蛋白（EGFP）,构建重组腺病毒载体Ad5-VEGF165GGCmotif-IRES-EGFP（Ad5-VIE）,同时构建腺病毒包装EGFP（Ad5-EGFP）为对照.以不同感染复数（MOI=0,10,25,50,100）Ad5-VIE感染大鼠骨髓间充质干细胞（MSC）后,用99Tcm-GH为报告探针,研究感染细胞摄取动力学（30,60,90和120 min）情况,以检测GGC序列在MSC中的表达,并与实时定量PCR、Western-blot蛋白印迹、免疫组织化学等方法鉴定的VEGF165表达进行对比分析.通过荧光显微镜及实时定量PCR检测EGFP在细胞中的表达.采用SPSS 13.0软件进行统计学处理,组间比较运用独立样本成组t检验、q检验和直线（Pearson）相关分析.结果 以Ad5-VIE感染MSC后,不同MOI摄取实验结果示细胞对99Tcm-GH的摄取率随病毒MOI的增加而逐渐增加（r2=0.86,P＜0.05）,并且在MOI=100时达到（7.94±0.75）%;不同时间摄取实验示随99Tcm-GH孵育时间延长,细胞摄取率逐步增高,至120 min时达到（7.72±0.22）%.Ad5-VIE感染组与Ad5-EGFP感染组摄取率在各个不同时间点差异均有统计学意义（t=15.10～54.92,P均＜0.05）.在mRNA水平上,VEGF165及EGFP表达均随病毒MOI增加而增加（r2=0.99,P＜0.05）.不同MOI下细胞对99Tcm-GH的摄取与VEGF165蛋白表达呈较好的相关性（r2=0.90,P＜0.05）.免疫组织化学检查结果表明人VEGF165目的 基因在MSC中成功表达.荧光显微镜下可以观测到被感染细胞中的EGFP蛋白.结论 成功构建的重组腺病毒系统Ad5-VIE感染MSC对99Tcm-GH的摄取与VEGF165表达呈正相关.以GGC多肽为报告基因可以监测治疗基因VEGF165的表达,为核素报告基因显像提供了理论依据.     

血红素加氧酶-1基因转染对大鼠心肌缺血再灌注损伤的保护作用

目的：观察腺病毒介导的血红素加氧酶-1基因（rHO-1）转染后,HO-1在大鼠心肌的表达及其对缺血再灌注损伤心肌的保护作用。方法：雄性SD大鼠随机分为4组：假手术对照组（SH）、生理盐水组（NS）、空载体组（Ad）和Ad-HO-1转染组（HO）,各组12只。后3组分别于心尖部分别注射1ml生理盐水、含空载体腺病毒（5．0×10^9PFU）和重组HO-1腺病毒50μl（7．5×10^9PFU）。在基因转染3d后,采用左冠状动脉前降支结扎开放建立心肌缺血再灌注模型。每组大鼠测定心功能后取心腔内血,做心肌酶检测,处死大鼠并取左心室标本,测定左心室心肌梗死面积,于荧光显微镜下观察心肌组织荧光蛋白的表达情况,RT—PCR、Westernblot检测心肌组织HO-1mRNA、蛋白的表达。结果：HO组中HO-1表达量明显高于Ad组和NS组,Ad和HO组左室心肌均见有荧光蛋白表达,其转染率为（55．5±3．5）％。各时相点HO组的SBP、DBP、MAP、＋dp／dtmax和-dp／dtmax等指标恢复率均显著高于Ad组和NS组（P〈0．01）。与SH组比较,NS组、Ad组血清LDH和CK—MB水平、梗死心肌重量、心肌梗死面积升高（P〈0．01）。与NS组或Ad组比较,HO组血清LDH和CK-MB水平、梗死心肌重量、心肌梗死面积减少（P〈0．01）。结论：腺病毒携带的HO-1基因能有效的转染心肌组织,并在体内稳定表达;HO-1基因转染对缺血再灌注的心肌有明显的保护作用。     

131I-FIAU／TK报告基因系统监测大鼠脑梗死模型中移植细胞的研究

目的检测经不同途径移植骨髓间充质干细胞后大鼠脑梗死模型中131I一氟一碘阿糖呋喃基尿嘧啶（FIAU）的生物分布及脑组织中胸苷激酶（TK）基因的表达,为核素报告基因显像无创性监测细胞移植治疗脑梗死提供实验依据。方法制备携带TK一内部核糖体连接位点一脑源性神经生长因子（IRES—BDNF）基因的腺病毒重组体Ad5一TK—IRES—BDNF一增强型绿色荧光蛋白（EGFP）;线栓法制作大鼠脑梗死模型;将转基因的干细胞分别通过脑实质、侧脑室、颈动脉和尾静脉注射人大鼠脑梗死模型中,以正常大鼠为对照组;Iodogen固相氧化法标记氟·阿糖呋喃基尿嘧啶（FAU）后进行生物分布研究,计算％ID／g;采用实时定量PCR和Westernblot定量分析TK基因的表达;数据分析采用独立样本t检验、单因素方差分析及Pearson相关分析。结果原位移植组梗死侧脑组织中的％ID／g为0．124±0．013,明显高于侧脑室移植组（0．052±0．004）、颈动脉移植组（0．061±0．002）、尾静脉移植组（0．059±0．005）和对照组（0．005±0．001）,差异有统计学意义（t=2．913～5．652,P均〈0．05）,其他移植细胞组组间差异无统计学意义（￡=0．694—1．448,P均〉0．05）。所有移植细胞组内两侧脑组织％ID／g的差异有统计学意义（f=9．004～15．734,P均〈0．05）,而对照组两侧间差异无统计学意义（t=1．511,P=0．182）。此外,原位移植组梗死侧脑组织中TK基因的表达高于其他各组,差异有统计学意义（t=7．482～12．371,P均〈0．05）;且脑组织TK基因表达的相对量与％ID／g呈正相关（r=0．971,P〈0．001）;脑组织内TK／[3一actin的比值与％ID／g呈正相关（r=0．899,P：0．002）。结论原位注射是治疗脑梗死的最佳细胞移植途径;选择适当示踪剂,可利用PET或SPECT进行无创性活体监测细胞移植治疗脑梗死的效果。     

新型核素报告基因系统hERL/核素标记雌二醇的实验研究

目的 在体外细胞摄取实验及在体显像基础上,评价新型核素报告基因系统人ER配体结合域( hERL)/核素标记雌二醇(E2)应用的可行性,为其用于基因治疗的监测提供依据.方法 以内部核糖体进入位点序列(IRES)方式构建携带hERL和治疗VEGF165的重组质粒pDC316-hERL-IRES-VEGF165(简写为EIV),将其用腺病毒包装构建重组腺病毒复合体(Ad-EIV).采用悬浮贴壁法从SD大鼠股骨和胫骨原代提取骨髓MSCs并培养.将Ad-EIV和脂质体2000包裹重组质粒(LipoEIV)分别转染MSCs,利用RT-PCR和Western blot,对hERL和VEGF165 mRNA和蛋白质水平的表达分别进行检测.测定Ad-EIV组、Lipo-EIV组和未转染组MSCs对125I-E2在不同时间(1、3、6、9、12和24h)的摄取率.将转染Ad-EIV的MSCs注射至大鼠左前肢、未转染MSCs注射至右前肢后1d,注射16α-18 F-17β-E2(18F-FES)进行micro PET/CT活体显像.2组间均数比较采用t检验,相关性研究采用Pearson相关分析.结果 Ad-EIV转染MSCs后RT-PCR检测示hERL和VEGF165 mRNA的表达随着感染滴度的增加而增加,且呈正相关(r2分别为0.953和0.966,P均＜0.05);感染复数(MOI)=25、50、75和100时,其mRNA表达高于Lipo-EIV组.Western blot检测蛋白质水平的表达也得到同样的结果.Ad-EIV组和Lipo-E1V组MSCs对125I-E2的摄取率均随时间延长逐渐增高,24h最高,分别达到( 10.94 ±0.30)％和(8.93±0.18)％;各个时间点2种载体转染组均明显高于未转染对照组(3.54％～5.52％;t值分别为15.489～26.560、10.523～24.204,P均＜0.05),而Ad-EIV组摄取率高于Lipo-EIV组(t=4.132～16.168,P均＜0.05).Micro PET/CT大鼠活体显像显示注射Ad-EIV转染的MSCs之左前肢放射性浓聚明显高于对侧.结论 报告基因hERL可以通过腺病毒转染、脂质体包裹等多种形式转染细胞并成功表达,应用核素标记E2可以对其进行探测和显像.应用腺病毒转染报告基因的方式转染率更高.     

腺病毒介导的AT2R基因转染对大鼠颈动脉新生内膜增生的抑制作用

目的构建AngⅡ2型受体(AT2R)重组腺病毒载体,并研究AT2R基因转染对大鼠颈动脉新生内膜增生的影响。方法用细菌内同源重组方法构建带有绿色荧光蛋白(GFP)报告基因的AT2R重组腺病毒载体pAdCMV/AT2R。大鼠颈动脉球囊损伤后,将pAdCMV/AT2R或空病毒pAdGFP局部导入,分别用RTPCR、免疫荧光染色、激光共聚焦技术检测目的基因AT2RmRNA及蛋白在血管中的表达,利用图像分析仪进行组织形态学分析。结果重组腺病毒载体pAdCMV/AT2R经PCR鉴定正确,其滴度为1.5×1012pfu/ml。pAdCMV/AT2R局部转染后14天,大鼠颈动脉新生内膜、外膜、中层有绿色荧光,阳性面积约40%;转染后7、14、21天大鼠颈动脉AT2RmRNA及蛋白表达显著高于pAdGFP转染组,21天时其内膜与中层的面积比显著低于pAdGFP组(0.78±0.06vs1.36±0.21,P<0.01)。结论pAdCMV/AT2R在体转染能上调血管中AT2R的表达,显著抑制大鼠颈动脉球囊损伤后新生内膜增生。     

神经突触结合蛋白Ⅰ-C2A区重组表达及其在心脏缺血-再灌注大鼠模型中的显像研究

目的 利用基因工程方法重组表达神经突触结合蛋白Ⅰ的C2A片段,探讨其在心肌细胞凋亡显像中的应用价值.方法 （1）将C2A基因连接到含有谷胱甘肽转移酶（GST）的重组原核表达载体pGEX-6P-1上,转化感受态细菌BL21,异内基硫代半乳糖苷（IPTG）诱导后纯化.（2）用异硫氰酸荧光素（FITC）标记纯化的蛋白,通过细胞结合实验鉴定蛋白质活性.（3）采用2-亚氨基噻吩方法,进行99TcmO-4标记C2A-GST融合蛋白,标记后的蛋白质用纸层析法测定其放化纯.（4）制备大鼠心肌缺血-再灌注模型,通过尾静脉注射99Tcm-C2A-GST,1 h后用SPECT仪进行显像 显像结束后,处死大鼠,取出心肌,用氯化三苯基四氮唑（TTC）染色,分离缺血心肌和存活心肌,测质量及其放射性计数,比较缺血心肌与正常心肌每克组织百分注射剂量率（%ID/g）值之间差别.采用SPSS12.0软件行统计分析,数据间比较采用t检验.结果 （1）成功表达的C2A-GST蛋白,相对分子质量约为3.8×104.（2）荧光显微镜下观察FITC-C2A-GST具有结合凋亡细胞的功能.（3）标记后的99Tcm-C2A-GST放化纯为（98.90±0.43）%.（4）大鼠显像示缺血损伤心肌显影清晰,体外测定缺血心肌摄取99Tcm-C2A-GST为（2.41±0.32）%ID/g,对照组99Tcm-C2A-GST-N-羟基琥珀酰亚胺（C2A-GST-NHS）的摄取为（0.82±0.24）%ID/g,两者之间差别具有统计学意义（t=10.6,P＜0.01）.结论 通过基因工程方法重组神经突触膜蛋白Ⅰ的C2A区域,重组后其具有监测缺血-再灌注大鼠模型中的心肌凋亡的作用.     

SD大鼠放射性肺纤维化模型的建立及评价

目的 建立SD大鼠放射性肺纤维化模型,探寻实验中纤维化蛋白、细胞因子等作为组织纤维化程度评价指标的可行性,为放射性肺纤维化的研究提供基础。 方法 选取体重为180~200 g的SD雄性大鼠37只,完全随机分为对照组（5只）和照射组（32只）。照射组采用X射线行右肺单次照射,照射剂量分别为13 Gy（10只）、15 Gy（10只）和17 Gy（12只）,分别于照后4个月和6个月,采用苏木精-伊红染色和马松三色染色评价大鼠肺组织纤维化程度;采用Western blot检测纤维黏连蛋白1（FN1）、基质金属蛋白酶2（MMP2）、α-平滑肌肌动蛋白（α-SMA）在肺组织中的表达;测定肺组织中羟脯氨酸含量以评价肺组织中胶原蛋白表达水平。酶联免疫吸附测定法测定支气管肺泡灌洗液中转化生长因子β（TGF-β）、白细胞介素6、肿瘤坏死因子α（TNF-α）、γ干扰素（IFN-γ）的变化。组间比较采用独立样本 t 检验。 结果 与对照组相比,照射组4~6个月后均出现肺纤维化,纤维化程度随照射剂量的增大和照射时间的增长而增加;照射组大鼠肺组织中FN1、α-SMA的蛋白表达水平较对照组升高,MMP2的蛋白表达水平较对照组降低;6个月照射组羟脯氨酸含量较对照组升高,分别从（514.19 ±282.20）μg/mg 增加至（886.13 ±145.01）、（1188.70 ±273.84）、（1700.70 ±590.95）μg/mg,且差异均有统计学意义（t=2.621、3.609、4.004,均P<0.05）;支气管肺泡灌洗液中TGF-β、白细胞介素6、TNF-α分泌量较对照组上调,差异有统计学意义（t=4.030~12.780,均P<0.05）,IFN-γ较对照组下调,差异有统计学意义（t=2.498~4.303,均P<0.05）。 结论 SD大鼠放射性肺纤维化模型构建成功。大鼠肺组织纤维蛋白含量未能反映肺组织纤维化程度,肺组织羟脯氨酸含量与支气管肺泡灌洗液中细胞因子在重度纤维化时可作为评价指标。     

99TcmO4-全身显像联合颈胸SPECT/CT在DTC患者术后肺转移灶显影中的应用

目的探讨99TcmO₄-全身显像联合颈胸SPECT/CT在分化型甲状腺癌（DTC）术后肺转移灶检出中的应用。方法回顾性分析2013年1月至2016年1月收治的34例DTC肺转移患者在术后131I治疗前的99TcmO₄-全身显像联合颈胸SPECT/CT中肺转移灶的检出情况,计算99TcmO₄-全身显像联合颈胸SPECT/CT检出肺转移灶的灵敏度、阳性预测值,并评估其对治疗决策的影响。结果34例肺转移患者中,2例表现为阳性（放射性浓聚+结构异常）,10例表现为可疑阳性（仅有典型肺转移CT表现）,22例表现为可疑阴性,若将可疑阳性与阳性均视为阳性发现,则99TcmO₄-全身显像联合颈胸SPECT/CT发现肺转移灶的灵敏度为35.3%（12/34）,阳性预测值为100%（12/12）,11例患者在后续的131I治疗中增加了剂量。结论99TcmO₄-全身显像联合颈胸SPECT/CT能发现部分无99TcmO₄-浓聚的肺转移灶,提高了肺转移灶的检出率;指导临床调整131I治疗剂量,改善预后。     

99锝m N-2-巯基吡啶-N-氧化物与99锝m-甲氧基异丁基异腈对犬急性心肌梗死模型的对照研究

目的 在正常和心肌梗死犬模型上,研究99TcmN-2-巯基吡啶-N-氧化物(99TcmN-MPO)的药物代谢动力学特征、生物学分布特征和对急性心肌梗死的诊断能力,并与传统示踪剂99锝m-甲氧基异丁基异腈(99Tcm-MIBI)进行对比研究.方法 正常杂种犬12只.静脉注射99TcmN-MPO,于不同时间点(30 s及1、2、3、4、5、10、20、30、40、60和90 min)静脉分别采血1 ml,经γ探测器测量其放射活性;注药后10、20、30、60、90及120 min行全身SPECT显像,并于不同器官上各取同样大小的ROI,测量其放射性计数进行定量研究.每只犬均注射相同剂量的99TcmMIBI,进行相同的实验作为对照.介入学方法构建犬急性心肌梗死模型,24 h后,静脉注入99TcmN-MPO(5只)和99Tcm-MIBI(5只),并于注药后30、60min进行心肌SPECT显像.结果 在静脉注射90 min内,99TcmN-MPO和99Tcm-MIBI均表现为快速的血液清除.两种示踪剂的初始注射剂量均为370 MBq.注射后1 min,每毫克血浆的放射活性小于初始注射剂量的50%[99TcmN-MPO为(35.77±6.31)%ID/mg;99Tcm-MIBI为(34.46±6.83)%ID/mg],30 min时＜5%[99TcmN-MPO:(3.11±1.44)%ID/mg;99Tcm-MIBI:(2.93±0.39)%ID/mg].99TcmN-MPO在心脏的浓聚明显,且存留时间很长,由于其在肝脏清除的速度较快,因此可获得良好的心/肝摄取比值,注射后10 min为0.54±0.06,30 min为1.02±0.06,60 min上升到1.38±0.06.相比之下,99Tcm-MIBI的心/肝摄取比值上升较为缓慢(注射后10 min为0.46±0.03,30 min为0.63±0.03,60 min为0.62±0.12).犬心肌梗死后SPECT心肌断层扫描显示,在注射99TcmN-MPO 30 min后,心脏与肝脏分界清楚,可清楚显示心肌缺血、梗死灌注缺损区域、范围和程度;而99Tcm-MIBI注射后60 min,心脏和肝脏的分界依然不清,尤其是肝脏的高放射性对心脏下壁和左心室壁的影响很大,对心脏左心室壁的心肌梗死灌注缺损区域的显示不佳.结论 99TcmN-MPO具有心肌摄取量较高,肝脏代谢快的特点,是一种具有广阔临床应用前景的SPECT心肌灌注成像显像剂.

Abstract：

Objective The purpose of the present study is to compare the pharmacokinetic and biodistribution properties of 99Tcm N-mercaptopyridine-N-oxide (99 Tcm N-MPO) with 99 Tcm-sestamibi (99 Tcm-MIBI) in normal dogs, and to investigate the potential of 99TcmN-MPO as a myocardial perfusion agent in canines with acute myocardial infarction. Methods Twelve healthy mongrel dogs were injected intravenously with 99TcmN-MPO (n = 6) or 99Tcm-MIBI (n = 6). Tracer kinetics in body fluids were determined by collecting blood of 1 ml via a femoral vein catheter at 30 s, 1,2,3,4,5, 10, 20, 30, 40, 60and 90 min post-injection (p. i.). The collected blood samples were weighed and counted for radioactivity in a γ-counter. Anterior and posterior planar γ-camera images were collected at 10, 20, 30, 60, 90, and 120 min after injection, with organ uptake quantified by region-of-interest (ROIs) analysis. For comparison, 99Tcm-MIBI was also evaluated in the same twelve dogs. Canine infarct models were set up by micro-invasive interventional embolization. SPECT images in the canine infarct model were collected 24 hours after myocardial infarction at 30 min and 60 min after the administration of 99Tcm N-MPO (n = 5) or 99Tcm-MIBI (n = 5). Results Both of 99Tcm N-MPO and 99Tcm-M1BI had a rapid blood clearance with less than 50% of initial radioactivity remaining at 1 min [99TcmN-MPO: (35. 77 ± 6. 31)% ID/mg ,99Tcm-MIBI (34. 46 ± 6. 83) % ID/mg] and less than 5% at 30 min p. i. [99Tcm N-MPO(3. 11 ± 1.44) % ID/mg,99Tcm-MIBI (2.93 ±0. 39)% ID/mg] . After injection, 99TcmN-MPO showed significant accumulation in the myocardium and prolonged retention. This rapid liver clearance of 99TcmN-MPO led to favorable heart-to-liver ratios, reaching values of 0. 54 ±0. 06 at 10 min, 1.02 ±0. 06 at 30 min, and 1.38 ±0. 06 at 60 min p. i.In contrast, the heart/liver ratio of 99Tcm-MIBI remained low at all time points (0. 46 ± 0. 03 at 10 min,0. 63 ±0. 03 at 30 min, and 0. 62 ± 0. 12 at 60 min p. i.). SPECT imaging studies in canines with acute myocardial infarction indicated that good visualization of the left ventricular wall and perfusion defects could be achieved at 30 min after administration of 99TcmN-MPO, but not 99Tcm-MIBI. Conclusion The combination of high heart uptake and rapid liver clearance makes 99TcmN-MPO a promising new radiotracer for myocardial perfusion imaging.     

Evaluation of the angiogenesis inhibitor KR-31831 in SKOV-3 tumor-bearing mice using (64)Cu-DOTA-VEGF(121) and microPET

KR-31831 ((2R,3R,4S)-6-amino-4-[N-(4-chloropheyl)-N-(1H-imidazol-2ylmethyl)amino]-3-hydroxyl-2-methyl-2-dimethoxymethyl-3,4-dihydro-2H-1-benzopyran), an angiogenesis inhibitor, was evaluated in tumor-bearing mice using molecular imaging technology. Pre-treatment microPET images were acquired on SKOV-3 cell-implanted nude mice after injection with (64)Cu-DOTA-VEGF(121). KR-31831 (50 mg/kg) was then injected intraperitoneally into the treatment group (n=3), while injection vehicle was injected into the control (n=4) and blocking (n=3) groups. After injections occurred daily for 28 days, all groups of mice underwent post-treatment microPET imaging after injection with (64)Cu-DOTA-VEGF(121). The post-treatment images showed high tumor uptake in the control group and reduced tumor uptake in both the blocking and treatment groups. ROI analysis of the tumor images revealed 6.25%±1.18% ID/g at 1 h, 6.55%±0.69% ID/g at 2 h, and 4.68%±0.63% ID/g at 16 h in the control group; 3.87%±0.45% ID/g at 1 h, 4.50%±0.44% ID/g at 2 h, and 3.63%±0.25% ID/g at 16 h in the blocking group; and 4.03%±0.74% ID/g at 1 h, 4.37%±0.67% ID/g at 2 h, and 3.83%±0.90% ID/g at 16 h in the treatment group. Biodistribution obtained after the post-treatment microPET imaging also demonstrated high tumor uptake (3.74%±0.27% ID/g) in the control group and reduced uptakes in both the blocking group (2.69%±0.73% ID/g, P<.05) and the treatment group (3.11%±0.25% ID/g, P<.05), which correlated well with microPET imaging data. Immunofluorescence analysis showed higher levels of VEGFR2 and CD31 expressions in tumor tissues of the control and blocking groups than in tumor tissues of the treatment group. These results suggest that the antiangiogenic activity of KR-31831 is mediated through VEGFR2 and microPET serves as a useful molecular imaging tool for evaluation of a newly developed angiogenesis inhibitor, KR-31831.     

99mTc-枸橼酸盐显像鉴别骨转移癌与良性退行性骨病变

目的 探讨99mTc-枸橼酸盐(99mTc-citrate)显像在骨转移癌与良性退行性骨病鉴别诊断中的价值.方法 对99mTc-亚甲基二膦酸盐(99mTc-MDP)骨显像阳性患者39例(92个病灶)在显像后的2～7 d内行99mTc-citrate显像,并分别进行定性及半定量分析,所有患者的临床诊断均经病理学、影像学、临床随访等证实.结果 定性分析:23例(48个病灶)骨转移癌患者的99mTc-citrate显像示72.92%病灶(35/48)呈异常浓聚;16例良性退行性骨病变者99mTc-citrate显像示88.64%病灶(39/44)无异常浓聚.半定量分析:99mTc-citrate显像示48个骨转移灶的病灶与健侧放射性摄取比(RUR)=1.47±0.42,44个良性退行性骨病灶RUR=1.09±0.38,两者相比差异有显著性(t=2.887,P＜0.01);而99mTc-MDP显像示48个骨转移灶RUR=1.96±0.25,44个良性退行性骨病灶RUR=1.87±0.21,差异无显著性(t=1.178,P＞0.20).结论 对99mTc-MDP骨显像阳性患者,99mTc-citrate显像在鉴别骨转移癌和良性退行性骨病中具有一定价值.     

用99Tcm标记突触结合蛋白I-C2A片段评价缺血预处理对心肌缺血再灌注损伤的保护作用

目的 应用99TcmO-4标记突触结合蛋白I-C2A片段(99Tcm-Syt I-C2A)评价缺血预处理对心肌缺血再灌注损伤的保护作用.方法 (1)采用2-亚氨基噻吩盐酸盐(2-IT)方法标记Syt I-C2A片段,纸层析法测定99Tcm-Syt I-C2A放化纯,用喜树碱处理的Jurket细胞测定标记蛋白质活性.(2)制备心肌缺血再灌注大鼠模型(A组)和心肌缺血预处理大鼠模型(B组)各6只,分别由尾静脉注射99Tcm-syt I-C2A 7.4 MBq,注射后1h处死动物,取出心脏,用生理盐水将心肌冲洗干净,并进行氯化三苯基四氮唑(TTC)染色,根据染色结果,分别取2组缺血损伤心肌和正常心肌,测量质量及放射性计数,比较2组缺血损伤心肌和正常心肌的每克组织百分注射剂量率(%ID/g).采用SPSS 12.0软件行统计分析,数据间比较用t检验.结果 (1)标记后的99Tcm-Syt I-C2A放化纯为(98.90±o.43)%,标记蛋白质与喜树碱处理组细胞结合测定的放射性计数是未处理组细胞的(10.99±O.55)倍.(2)A组缺血损伤心肌摄取99Tcm-Syt I-C2A(2.41±0.32)%ID/g,正常心肌为(O.16±O.02)%ID/g;而B组缺血损伤心肌和正常心肌的放射性摄取则分别为(0.46±0.05)和(0.20±0.05)%ID/g.B组缺血损伤心肌的99Tcm-Syt I.C2A摄取量明显低于A组(t=8.52,P相似文献   

99mTcO(BAT-NI), a novel nitroimidazole tracer: in vivo uptake studies in ischaemic myocardium

Myocardial perfusion single-photon emission tomography (SPET) performed with cationic technetium-99m complexes indicates ischaemic areas as cold lesions. By contrast, nitroimidazole derivatives labelled with fluorine-18 or (99m)Tc have recently shown promising results for hot spot imaging of ischaemic myocardium. This study evaluates (99m)TcO(BAT-NI), a new (99m)Tc complex comprising the nitroimidazole ligand, 2,10-dimercapto-2,10-dimethyl-4,8-diaza-6-[4-(2-nitroimidazolyl)butyl]undecane, in a low-flow in vivo model of myocardial ischaemia in thoracotomised rats. To elucidate the influence of the 2-nitroimidazole group on ischaemia-induced uptake, comparisons with ligand derivatives were performed where (a) the 2-nitro group was deleted [(99m)TcO(BAT-I)], (b) the 2-nitroimidazole functionality was replaced by a Br atom [(99m)TcO(BAT-Br)] and (c) the (99m)TcO(BAT) moiety was replaced by an iodine-125 iodophenoxybutyl ligand ((125)IP-NI). The radiolabelled compounds were i.v. injected 15 min after reducing resting myocardial blood flow by 50-60% and the uptake of radioactivity was assessed 90 min post injection. Autoradiography of left ventricular short-axis slices showed median uptake ratios of ischaemic/non-ischaemic myocardium (I/N) of 3.4, 4.5 and 3.4 for (99m)TcO(BAT-NI), (99m)TcO(BAT-I) and (99m)TcO(BAT-Br), respectively. In contrast, (125)IP-NI was not preferentially taken up by ischaemic myocardium. Accumulation of (99m)TcO(BAT-NI) in ischaemic heart regions was comparable to that in the liver. Biodistribution studies showed a median uptake of 0.65% ID/g of (99m)TcO(BAT-NI) in ischaemic tissue and an I/N of 3.3. On planar images of the thorax and upper abdomen the ischaemic hearts were visualised faintly; the median heart to lung count ratio for (99m)TcO(BAT-NI) was 1.7, and the median heart to liver count ratio was 1.0. We conclude that uptake of (99m)TcO(BAT-NI) in ischaemic myocardium does not depend on the nitroimidazole moiety but is intrinsic to the BAT complex. Clinical use of the (99m)TcO(BAT)-labelled tracers seems unlikely owing to their low uptake and their low ischaemic tissue contrast on planar images in vivo.     

稳定表达人VEGF165的NIH/3T3细胞株的筛选与鉴定

 目的 培养NIH/3T3细胞,并进行慢病毒载体介导的VEGF165基因转染,为建立转基因小鼠血管瘤模型奠定基础.方法 采取贴壁培养法分离培养NIH/3T3细胞,细胞随机分为3组,分别为Lenti-VEGF165-EGFP 重组慢病毒载体转染组、Lenti-EGFP慢病毒转染组和未转染组,转染后72 h荧光显微镜观察细胞绿色荧光蛋白的表达,流式细胞仪分选后用ELISA方法检测VEGF165外分泌.结果 ELISA 检测转染后小鼠NIH/3T3细胞,Lenti-VEGF165-EGFP 重组慢病毒载体感染组、Lenti-EGFP慢病毒转染组和未转染组培养上清中VEGF165浓度分别为(205±15)、0、0 ng/L(P<0.05) 结论 NIH/3T3细胞获得慢病毒载体介导的VEGF165表达基因修饰且稳定传代,为下一步血管瘤动物模型研究奠定了基础.     

99Tcm-MIBI评价缺血预适应心肌的实验研究

目的　研究     

99mTc-MIBI心肌灌注显像诊断小儿病毒性心肌炎的临床价值

目的探讨和评价99mTc-MIBI心肌灌注显像诊断小儿病毒性心肌炎的临床价值.材料与方法73例临床诊断为病毒性心肌炎和58例非病毒性心肌炎患儿空腹静息下静脉注射111～740MBq99mTe-MIBI,60min后行常规体位平面/断层显像.结果73例临床确诊病毒性心肌炎患儿中有50例在不同体位的平面影像(36例)、断层影像(2例)和平面+断层影像(12例)有1～3节段呈花斑样改变,其敏感性为68.5%.非病毒性心肌炎组58例,8例有花斑样改变,假阳性率为13.8%,特异性86.2%,诊断准确率为76.3%.结论99mTc-MIBI心肌灌注显像不失为一种诊断小儿病毒性心肌炎的简便、无创、经济而又有较高的辅助诊断价值的检查方法.     

用99^Tc^m标记突触结合蛋白Ⅰ-C2A片段评价缺血预处理对心肌缺血再灌注损伤的保护作用

目的应用99^Tc^mO4^-标记突触结合蛋白Ⅰ-C2A片段（99^Tc^m-SytⅠ-C2A）评价缺血预处理对心肌缺血再灌注损伤的保护作用。方法（1）采用2-亚氨基噻吩盐酸盐（2-IT）方法标记SytⅠ-C2A片段,纸层析法测定99^Tc^m-SytⅠ-C2A放化纯,用喜树碱处理的Jurket细胞测定标记蛋白质活性。（2）制备心肌缺血再灌注大鼠模型（A组）和心肌缺血预处理大鼠模型（B组）各6只,分别由尾静脉注射99^Tc^m-SytⅠ-C2A7．4MBq,注射后1h处死动物,取出心脏,用生理盐水将心肌冲洗干净,并进行氯化三苯基四氮唑（TTC）染色,根据染色结果,分别取2组缺血损伤心肌和正常心肌,测量质量及放射性计数,比较2组缺血损伤心肌和正常心肌的每克组织百分注射剂量率（％ID／g）。采用SPSS12．0软件行统计分析,数据间比较用t检验。结果（1）标记后的99^Tc^m-SytⅠ-C2A放化纯为（98．90±0．43）％,标记蛋白质与喜树碱处理组细胞结合测定的放射性计数是未处理组细胞的（10．99±0．55）倍。（2）A组缺血损伤心肌摄取99^Tc^m-SytⅠ-C2A（2．41±0．32）％ID／g,正常心肌为（0．16±0．02）％ID／g;而B组缺血损伤心肌和正常心肌的放射性摄取则分别为（0．46±0．05）和（0．20±0．05）％ID／g。B组缺血损伤心肌的99^Tc^m-SytⅠ-C2A摄取量明显低于A组（t=8．52,P〈0．01）。结论99^Tc^m-SytⅠ-C2A可用于定量评价缺血预处理抑制心肌细胞凋亡而产生的对缺血再灌注致心肌损伤的保护作用。