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Objective To prepare 99Tcm-succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH)-AC133 and evaluate its targeting ability on CD133 positive colon cancer by Gamma imaging,and to explore its feasibility as a molecular probe for cancer stem cells (CSCs).Methods CD133 expression was detected by immunofluorescence assay and flow cytometry on Lovo cell lines and tumor xenografts.CD133 specific monoclonal antibody (AC133) was conjugated with SHNH,and then labeled with 99Tcm to prepare 99Tcm-SHNH-AC133.The radiolabeled yield and radiochemical purity were investigated.Colon cancer xenografts were developed and Gamma imaging were performed.Region of interest (ROI) was drawn and the tumor/non-tumor (T/NT) ratio was calculated.For the blocking experiment,animals were pre-injected with excess unlabeled AC133.Two-sample t test was used to analyze the data.Results CD133 was expressed on the surface of Lovo cells.And the percentage of CD133 positive cells in Lovo tumor tissues was (29.5±3.4)％.The radiolabeled yield of 99Tcm-SHNH-AC133 was more than 85％ and radiochemistry purity was (97.7±2.4)％.Gamma imaging demonstrated that 99Tcm-SHNH-AC133 could specifically target to Lovo tumors which could be gradually visible after 12 h.The tumor uptake was obvious at 24 h and T/NT ratio was 8.16±0.45.In blocking study,the tumor uptake was significantly reduced by pre-injection of excess unlabeled AC133 (3.52±0.13;t=19.8,P＜0.05).Conclusions 99Tcm-SHNH-AC133 has high labeling yield and radiochemistry purity.The excellent targeting properties of 99Tcm-SHNH-AC133 on CD133 positive colon cancer demonstrate that it is a promising imaging agent for CSCs tracking in vivo.
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BACKGROUND:Currently, there is a lack of efficient, non-invasive way to transplant stem cels to the target organ or tissue. Exploring a way to guide targeting transplantation of stem cels and to improve the efficiency of stem cel homing is now one of focuses in the field of stem cels research. OBJECTIVE: To establish a simple and feasible method to chemicaly modify the cel surface using biotin-streptavidin reaction system, and to evaluate the efficiency of this method to label bone marrow mesenchymal stem cels (BMSCs) and its effects on cel biological functions. METHODS: Passage 3 BMSCs were obtained by whole bone marrow culture method and verified by flow cytometry. Biotin, streptavidin, sulfonated biotin-N-hydroxy-succinimide were used to equip the adhesion molecule ligand, sialyated LewisX (SLeX), to the BMSCs surface. The labeling rate of BMSCs was assessed using fluorescence microscope, the vitality of BMSCs was evaluated by trypan blue staining, and the proliferation of BMSCs was evaluated by cel counting kit-8 assay. Adipogenic and osteogenic inductions were used to evaluate the effect of the method on the multi-differentiation function of BMSCs. RESULTS AND CONCLUSION: After culture for 2 weeks, passage 3 BMSCs were obtained and confirmed by expressing CD90, CD29 and lack of CD34, CD45. Biotin, streptavidin, sulfonated biotin-N-hydroxy-succinimide were successfuly used to equip sialyated LewisX (SLeX) to the BMSCs surface and had minor effects on the vitality, proliferation, and differentiation of BMSCs. This method was simple for surface modification and had a high modification rate of 88%. The homing of BMSCs modified by this method to the target organ or tissue could be greatly enhanced. Therefore, this method potentialy could have extensive and important applications.
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Objective To evaluate the normalized correction effect of the ROX reference fluorescence on the real-time quantitative RT-PCR assessment for the Nrf2 mRNA.Methods The Taq-man probe was utilized in the real-time quantitative RT-PCR method to assess the Nrf2 mRNA; The reaction specificity of the Nrf2 PCR products obtained from the rat cDNA was assessed using gel electrophoresis and sequencing identification.The PCR calibrators with different concentrations varied at 10 gradients(from 2.0 × 109 to 2.0 × 100) were prepared for real-time quantitative RT-PCR assessment,and then the influence of the ROX reference fluorescence correction on the linear range of the PCR standard curve was investigated experimentally.The recombinant plasmids with different concentration levels(e.g,high,medium or low concentration) were assessed by the real-time RT-PCR and 20 replicate measurements were performed for all the samples in both intra- and inter-assays.The repeatability differences of both the inter- and intra-assays were analyzed systemically with/without the ROX reference fluorescence correction.Results The primers and probes required for Nrf2 mRNA real-time quantitative RT-PCR assessments were constructed,and the reaction system and conditions were also established.The length of the amplified fragment agreed well with the expected length(122 bp),and the sequencing analysis showed the amplified fragment was verified by the sequencing identification.After applying the ROX reference fluorescence correction,the linear range ofthe PCR standard curve was measured as 2.0 × 109 -2.0 × 100 copies/μl(R2 ＞ 0.99),which was 100times wider than that measured without ROX reference fluorescence correction(2.0 × 109 - 2.0 × 102copies/μl,R2 ＞ 0.99).Without the ROX reference fluorescence correction,the intra-assay CVs of the Ct value were measured as 4.1%,2.7% and 2.1% for the recombinant plasmids with high-,medium-,and low-level concentration,respectively.The inter-assay CVs of the Ct value were 4.3% 、3.0% 、2.4%.By contrast,with the ROX reference fluorescence correction,the intra-assay CVs of the Ct value became 0.7%,0.5%,0.4% and the inter-assay CVs of the Ct value turned to 1.0%,0.8% and 0.7%.The discrete degree of the Ct value with the correction was lower than that without the correction.The intra- and interassay Ct values of the high- and medium-level samples exhibited statistic significance for the measurements with/without the correction.In the high-level group,the t values of the intra- and inter-assays were 4.843 and 2.566,with P＜0.05.In the medium-level group,the t values of the intra- and inter-assays were 4.293 and 4.423,with P＜0.05.However,no statistic significance was observed the low-level group(the t values of the intra- and inter-assays were 0.753 and 1.279,with P ＞.0.05).Conclusions The utilization of the ROX reference fluorescence in the real-time quantitative PCR assessment for Nrf2 mRNA could widen the linear range of the standard PCR calibration curve and improve the repeatability of the assessment,which should be quite helpful for accurately assessing the expression level of the Nrf2 mRNA.
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Objective To develop and optimize a module for the automatic production of N-succinimidyl-4-[18F] fluorobenzoate (18F-SFB) that is used for further 18F labelling C2A domain of Synaptotagmin Ⅰ . The conjugated compound was applied for detecting the tumor apoptosis in rabbit model after chemotherapy. Methods GE TRACERlab and TRACERlab FXF-N modules were modified and programmed to automatically produce 18 F-SFB which was further analyzed by high performance liquid chromatography (HPLC).C2A-glutathione S transferase (GST) was conjugated with 18F-SFB (18F-FB-C2A-GST) and subsequently purified by HPLC. Two rabbits grafted with VX2 lung cancer were first treated with chemotherapy and then,37 MBq of 18F-FB-C2A-GST was administered via the auricular vein. Serial PET/CT imagings were performed at 0.5, 1 and 2 h post-injection respectively. Tumor apoptosis was examined by pathological study. Results The TRACERlab FXFoG and TRACERlab FXF-N modules were successfully adapted to synthesize18F-SFB, with the radiochenmical yield (76.41 ±4.00)% (n = 10), the corrected yield (45.43 ±5.90 ) % and the radiochemical purity about 95%. The whole procedure for labeling 18 F-SFB was about 87 min.From PET/CT imagings, significant uptake was found in the tumor after chemotherapy, but no obvious up-take was found in heart, lungs and liver. HE staining demonstrated large number of apoptotic bodies within the tumor tissues. Conclusions 18 F-SFB can be automatically synthesized. 18F-FB-C2A-GST might be useful for the detection of apoptosis in tumor after chemotherapy.