CD40突变体靶向MR分子成像探针制备及体外卵巢癌成像的实验研究

Preparation of MR molecular probes targeting CD40 mutant and the preliminary study of imaging ovarian cancer in vitro

目的探讨超微超顺磁氧化铁（USPIO）粒子负载的，CD40突变体抗体分子探针的构建方法和其生物、理化性状，以及在体外对卵巢癌的靶向作用。方法采用化学交联法将单克隆抗体交联于二巯基丁二酸（DMSA）修饰的USPIO，形成具有免疫活性的分子探针，进行磁学性能鉴定。USPIO标记的抗人CD40突变体单克隆抗体5H6（5H6一USPIO）作为实验组，USPIO标记的抗人CD40单克隆抗体5C11（5C11-USPIO）及USPIO为对照组。通过流式细胞术、共聚焦显微镜及普鲁士蓝染色分析其体外生物学特性，采用3．0TMR对探针与高表达CD40突变体卵巢癌（H08910）进行体外细胞成像。信号变化数据组问比较采用单因素方差分析和LSD检验。采用CellCountingKit．8试剂盒检测探针对H08910细胞的增殖影响。结果携带USPIO的抗CD40突变体分子探针被成功构建并分离纯化。合成的探针同USPIO相比具有相似的磁学特性和良好的稳定性。流式细胞术、共聚焦显微镜及普鲁士蓝染色证实抗体分子探针能够特异性识别H08910细胞表面的CD40突变体，对细胞H08910的增殖无影响。体外MRI显示探针同H08910细胞结合后T1、T2。值明显缩短，T2图像较对照组明显变暗。5H6-USPIO组的T2、T2’弛豫时间分别为（40．05±1．62）、（3．08±0．11）ms，短于5C11-USPIO[分别为（85．38±4．74）和（11．82±1．00）ms]和USPIO组[分别为（91．62±3．35）和（13．60±1．92）ms]，差异均有统计学意义（F值分别为196．29、60．73，P值均〈0．01），而5C11-USPIO、USPIO两组T2、T2＊弛豫时间差异无统计学意义（P值均〉0．05）。结论化学交联法可制备出CD40突变体单克隆抗体超顺磁氧化铁粒子探针，该探针具有良好磁学特性及较高生物活性，能够特异性识别卵巢癌细胞H08910。

Objective To develop an uhrasmall superparamagnetic iron oxide （USPIO） based MR probe targeting CD40 mutant and investigate its biological and chemical properties and its targeting effect on ovarian cancer cells in vitro. Methods To prepare immunologically competent probe, the monoclonal antibody was conjugated with USPIO particles modified by DMSA based on chemical crosslinking method. The USPIO labeled anti-human CD40 mutant monoclonal antibody 5H6 （5H6-USPIO） was the experimental probe, and the USPIO labeled anti-human CIM0 monoclonal antibody 5Cll （5Cll-USPIO） and USPIO served as control agents. The flow cytometry, confocal microscopy and Prussian blue staining were employed to assess the magnetic performance and analyze its bioactivity of the probe. The probe＇s cell MR imaging in vitro was carried out using ovarian caner cells （HO8910）with high CD40 mutant expression. The analysis of signal data of different groups was conducted by using one-way ANOVA and LSD test. The probe＇s effect on ovarian caner cells＇ growth was measured by CCK-8 kit. Results The stable molecular probe carryingnanopartieles and CD40 mutant antibody was built and purified successfully. The probe had similar magnetic property compared with original USPIO. Immunofluorescence and Prussian blue staining confirmed that the molecular probe could recognize CD40 mutant on ovarian cancer cells （HO8910） with high specificity. The probe had no effect on the growth of H08910 cells. MR cell imaging in vitro showed that the value of T2 and T2 ＊ decreased significantly after the probe binding with HO8910 cells and T2WI became darker than control groups.