ABSTRACT
Objective To develop a specific trans-splicing intron ribozyme type Ⅰ-mediated dual reporter gene system (Rib53-Fluc-tk) for targeting CEA.Methods The novel CEA-targeting trans-splicing ribozyme with the downstream reporter system (Rib53-Fluc-tk) was constructed by genetic engineering technology.The trans-splicing reaction product was evaluated using the 131I-5-iodo-2'-fluro-l-beta-D-arabinofuranosy-luracil (FIAU) cellular uptake rates and the bioluminescence.Two-sample t test,the analysis of variance and the least significant difference (LSD) t test was performed for data analysis.Results The sequence of Rib53-Fluc-tk was proved by gene-sequencing test.Human MCF-7 breast cancer cells showed a high ratio of firefly luciferase/renilla luciferase (0.64±0.10,n =4).A 520 bp band of product existed,which matched with the predicted size using RNA from cells transfected with Rib53-Fluc-tk in MCF-7.Signals were detected by bioluminescence in human embryonic kidney 293T cells co-transfected with Rib53-Fluc-tk and pCDNA3.1-CEA.The labelling rate of 131I-FIAU was (64.02±4.79)％ (n =3).The radiochemical purity was (95.96± 1.07)％ (n=3),and the stability of the radiocompound remained high in human serum at least for 24 h.The uptake of 131I-FIAU in 293T cells transfected with Rib53-Fluc-tk was (0.31±0.01)％ (n=4),while it increased with the incubation time in 293T cells co-transfected with pCDNA3.1-CEA and Rib53-Fluc-tk and reached (1.40±0.06)％ at 4.5 h (F=1 007.29,t=136.34,both P＜0.01).Conelusions A novel and specific reporter gene in the cellular level was established.Taking advantage of trans-splicing reaction of the ribozyme,it could improve the specificity of the reporter gene imaging.
ABSTRACT
Objective To study the biodistribution of 131 I-2'-deoxy-1-β-D-arabinofuranosy1-5-iodouracil (FIAU) in the rat middle cerebral artery occlusion model and the expression of thymidine kinase (TK) gene in brain tissue after gene-modified stem cell transplantation,and thus evaluate the possibility of further noninvasive monitoring of stem cell transplantation therapy in cerebral infarction.Methods Adenovirus recombinant Ad5-TK-intemal ribosome entry site-brain derived heurotrophic factor-enhanced green florecent protein(IRES-BDNF-EGFP) carrying TK-IRES-BDNF gene was prepared.Cerebral infarction model was established in rats by intraluminal middle cerebral artery occlusion with nylon monofilament.Gene modified bone marrow mesenchymal stem cells were transplanted via intraparenchymal route,lateral ventricle,carotid artery and tail vein,respectively.The normal rats were used as controls.131 I- FAU was prepared to be the tracer for biodistribution study and the ％ ID/g was calculated based on measurement of the tissue radioactivity counts.The expression of TK gene was evaluated by quantitative real-time PCR (QR-PCR) and Western blot analysis.Data were analyzed with independent-samples t-test,one-way analysis of variance (ANOVA) test,and Pearson linear correlation test.Results The ％ ID/g of infarcted brain tissue in the intraparenchymal group was 0.124 ± 0.013,which was significantly higher than that in lateral ventricle group (0.052 ±0.004),carotid artery group (0.061 ±0.002),tail vein group (0.059 ±0.005) and control group (0.005 ±0.001) (t =2.913 - 5.652,all P＜0.05),while there were no statistically significant differences among the other route transplanted groups ( t =0.694 - 1.448,all P ＞ 0.05 ).The differences of ％ ID/g between the infarcted and contralateral sides of brain tissue in all transplanted groups were statistically significant (t =9.004 - 15.734,all P ＜ 0.05 ),while there was no statistically significant difference of this parameter between both sides of brain tissue in control group (t =1.511,P =0.182).The expression of TK gene in intraparenchymal group was significantly higher than other groups (t =7.482 -12.371,all P ＜0.05).The expression levels ofTK gene on QR-PCR showed a positive correlation with ％ID/g of the brain tissue ( r =0.971,P ＜ 0.001 ).Similarly,the ratio of TK/β-actin by the Western blot analysis correlated with the ％ ID/g ( r =0.899,P =0.002 ).Conclusion Intraparenchymal route may be the way of choice for cell transplantation therapy of cerebral infarction.If suitable radionuclide tracer is available,PET or SPECT may be potentially used for noninvasive monitoring of stem cell transplantation in cerebral infarction in vivo.