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.
ABSTRACT
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.