【作者】 王瑞华；

【导师】 曹国祥； 安锐；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2007， 硕士

【摘要】 目的制备¹²⁵I- FIAU,培养原代心肌细胞,构建含HSV1-tk的腺病毒重组体,通过一系列实验评价心肌细胞对该腺病毒重组体的易感性及感染后的活力,评估感染滴度及感染时间对心肌细胞体外摄取¹²⁵I-FIAU的影响,为进一步探讨用腺病毒作为载体、HSV1-tk作为标志性基因、放射性核素标记的FIAU作为标志性底物进行心脏报告基因显像的可行性打下基础。方法1. FAU的碘化标记及其在昆明小鼠体内的生物分布研究利用Iodogen固相氧化法碘化标记FAU,标记反应产物用Sep-Pak C18反相色谱柱进行纯化,用硅胶板薄层层析法测定¹²⁵I-FIAU的放射化学纯度和体外稳定性,并研究其在昆明小鼠体内的生物分布情况,着重分析在心肌及血液中的分布特性。2.原代SD大鼠乳鼠心肌细胞的培养及鉴定取1～3 d龄的SD大鼠乳鼠的心室组织,切成1mm3的小块采用含牛血清白蛋白（bovine serum albumin,BSA）的Ⅱ型胶原酶单次消化心肌,1 h后用差速贴壁法纯化心肌细胞后培养,全过程在25℃-37℃条件下进行。在生物倒置显微镜下动态观察培养的心肌细胞的形态,用免疫组织化学染色的方法进行纯度测定。3. Ad-CMV-HSV1-tk重组体的制备及鉴定用HSV1-tk作为报告基因,用巨细胞病毒作为启动子,腺病毒作为载体,构建腺病毒重组体,本部分与北京本元正阳公司合作。4.感染Ad5-tk的心肌细胞活性及感染滴度和感染时间对其体外摄取¹²⁵I-FIAU的影响。应用腺病毒重组体将HSV1-tk基因转染到心肌细胞中,用MTT的方法评价心肌细胞感染病毒重组体后的活力,分析细胞活力与感染滴度的关系;用RT-PCR的方法监测心肌细胞感染病毒重组体后有无HSV1-tk mRNA存在,并在更换含¹²⁵I-FIAU的培养基后分别于0.5h、1h、2h、3h、4h、5h时测定不同感染滴度下的心肌细胞对¹²⁵I-FIAU的摄取率,分析摄取率与感染滴度和感染时间的相关性。结果1. Iodogen固相氧化法能有效碘化标记FAU,标记反应产物经Sep-Pak C-18反相色谱柱纯化后,¹²⁵I-FIAU的放射化学纯度为98.60%±0.52%（n=5）;¹²⁵I-FIAU在正常人血清及PBS中37℃条件下稳定,在0.5h～24h的时段内放射化学纯度>95%。生物分布实验显示,¹²⁵I-FIAU从血液中清除迅速,肾脏是主要排泄器官,心肌24h后几乎无滞留。2.用含牛血清白蛋白（bovine serum albumin,BSA）的Ⅱ型胶原酶单次消化心肌,心肌组织在6 h时基本消化完全。5、10、15、20、25和30天时台盼兰染色法计算得到心肌细胞存活率均大于95%,30 d时仍为95.2%;生物倒置显微镜下观察,24 h左右心肌细胞之间形成交联,并有细胞搏动;培养48 h的细胞经免疫组织化学染色法测定心肌细胞纯度为（97.1±1.9）%。3.与本元正阳公司协作构建腺病毒重组体,pDC316-tk质粒经PCR、酶切鉴定和测序鉴定证明序列正确,得到Ad-CMV-HSV1-tk腺病毒重组体的物理滴度为1.1×1012,感染滴度达到1.6×1010。4. MTT实验结果显示,心肌细胞感染腺病毒重组体后的活力与感染滴度存在负相关的关系;RT-PCR结果显示,心肌细胞感染腺病毒重组体后有HSV1-tk mRNA存在。摄取率结果显示,腺病毒重组体感染后的心肌细胞体外对¹²⁵I-FIAU的摄取率与感染滴度和感染时间呈明显正相关关系。结论用Iodogen固相氧化法和Sep-Pak C-18反相色谱柱纯化可以快速制备符合实验要求的¹²⁵I-FIAU,并且所制备的¹²⁵I-FIAU具有较好的生物分布特性。应用改良的方法可以更简单的培养出符合大多数实验要求的心肌细胞。用腺病毒重组体做载体,可以将HSVI-tk基因成功转导入心肌细胞,并且对¹²⁵I-FIAU有较高的摄取率。因此,用腺病毒重组体作为载体,HSV1-tk作为标志性基因,¹²⁵I-FIAU作为标志性底物有望进行心脏报告基因显像。更多还原

【Abstract】 ObjectiveThe aim of this study was to evaluate the possibility of HSV1-tk which was transduced by an adenoviral vector, and radiolabled FIAU as a marker gene / marker substrate for imaging reporter gene expression in the heart. In this study, we radiolabeled ¹²⁵I-FIAU, cultured myocardial cells, constructed recombinant adenovirus carrying HSV1-tk gene. The susceptibility to the vector, ¹²⁵I-FIAU uptake ratios and the relations to MOIs and times were evaluated through a series of tests.Methods1. FAU was labeled with ¹²⁵I-NaI and the biodistribution of ¹²⁵I-FIAU in the normal mice was to evaluate. FAU was radiolabeled with ¹²⁵I-NaI by Iodogen iodization. The product was purified through Sep-Pak C-18 reverse phase chromatograph column. Radiochemical purity and the stability in vitro were measured by TLC-SG. We studied the biodistribution of ¹²⁵I-FIAU in the normal mice, and the distributions in cardiac muscle and blood should be emphasized.2. To culture normal SD neonatal rat myocardial cells. To culture purified neonatal rat myocardial cells by the simplified and modified methods. The neonatal rat heart tissue was digested once in collagenase typeⅡincluding BSA. The purified cadiocytes were obtained by differential adhesion after 1 hour. All of these steps were carried out in 25-37℃. We evaluated the survival rate through staining the cell with typsin on the day of 5th, 10th, 15th, 20th, 25th, 30th, and then observed cell form under the inverted biological microscop and detected the purity of the cadiocytes which were cultured for 48 hours by the method of immunohistochemistry.3. To construct the Ad-CMV-HSV1-tk recombinant. The compounds of Ad-CMV-HSV1-tk were recombinated by using HSV1-tk as a reporter gene, CMV as a promoter and adenoviral as a vector. This part of the research was collaborated with Vector Gene Company LTD.4. Study viability of the myocardical cells after Ad5-tk infection and the relations between ¹²⁵I-FIAU uptake and MOIs or time. HSV1-tk was transferred into myocardial cells after the fact that cells was infected by Ad -CMV-HSV1-tk. MTT test was used to evaluate the viability of the myocardial cells, and the relation between cell viability and the MOIs was analysised. RT-PCR test was used to identify HSV1-tk expression. Cultural medium of myocardial cells was changed to those including ¹²⁵I-FIAU at the time of 24 hours after being transferred. The uptake rates at the different times of 30min, 1h, 2h, 3h, 4h and 5h were observed. The relationship between uptake rates and different times or different MOIs was evaluated. Results1. FAU was effectively radiolabeled with ¹²⁵I-NaI by Iodogen iodization. The radiochemistry purity of product which was purified through Sep-Pak the C-18 reverse phase chromatograph column was 98.60%±0.52%. ¹²⁵I-FIAU was steady and the radiochemistry purity was exceeded 95% from 0.5 h to 24h in PBS and fresh normal human serum at the temperature of 37℃. The biological distribution experiment of ¹²⁵I-FIAU in the normal Kunming mice suggested that the distribution in blood was washed out quickly, the kidney was the main metabolism organ in which biodistribution of ¹²⁵I-FIAU was highest at the beginning. Little distribution of ¹²⁵I-FIAU was observed in cardiac muscle after 24h.2. The neonatal rat heart tissue was digested once in collagenase typeⅡincluding BSA. The heart tissue was digested almost completely at the 6th hour. The survival rate was more than 95% on the day of 5th, 10th, 15th, 20th, 25th and 30th. Under the inverted biological microscop, we observed the cells connecting each other and beating after 24 hours. The purity of the cadiocytes which were cultured for 48 hours was （97.1±1.9）% by the method of immunohistochemistry.3. Collaborated with Vector Gene Company LTD., the identification of pDC316-tk was correct and we obtained Ad–CMV–HSV1-tk recombinant of which v.p. was 1.1×1012 and TCID was 1.6×10¹⁰.4. MTT test suggested that relation between the MOIs and cell viability after Ad5-HSV1-tk infection was negative. RT-PCR showed that HSV1-tk gene expression in myocardial cells after transferring the HSV1-tk gene by adenovirus. RT-PCR demonstrated the susceptibility of myocardial cells to adenovirus. The statistic analysis showed that uptake ratio was positive related to MOI and time.Conclusions¹²⁵I-FIAU which had ideal biodistribution in normal mice could be radiolabeled by Iodogen iodization and be purified through Sep-Pak C-18 reverse phase chromatograph column. It was possible to get purer neonatal rat myocardial cells which survived long enough for many researches by the simplified and modified methods. Using an adenoviral vector, HSV1-tk can be successfully expressed in myocardial cell in vitro, yielding high uptake of radiolabled FIAU. Therefore, cardiac reporter gene imaging could be achievement by using adenovirus as vector, HSV1-tk as reporter gene and ¹²⁵I-FIAU as imaging agent.更多还原