Bimodal Imaging Research On Ischemic Stroke In Endothelial Progenitor Cellstransplantation Treatment

Part One Construction and Identification of Lentiviral Vector Carrying Dual Reporter GenesObjective:To construct the lentiviral vector FUGW-Luc2-eGFP carrying reporter gene luciferase and green fluorescent protein. Infectious virions are packed and produced in HEK293T cells, and in-vivo detection is conducted for the infection ability of virions, so as to lay a solid foundation for future cell infection and living imaging.Methods:Through gene cloning, the sequence of luciferase gene (Luc2) in pGL4.17 vector is expanded. After purification, the Luc2 fragment is inserted into FUGW vector carrying eGFP sequence to obtain the dual reporter vector FUGW-Luc2-eGFP. After digestion and sequence detection, infectious virions are packed in HEK293T cells via lentivirus three-plasmid system.Results:Through enzyme digestion, PCR and sequencing experiment, lentiviral vector FUGW-Luc2-eGFP carrying dual reporter gene is successfully built. The titer of virions after packaging and concentration was 108 IU/mL. After injecting virus solution into the encephalic of mouse, the bioluminescence signal lasted until 28 days after injection. Comparing the Luc2-eGFP with the luciferase and fluorescent protein structure indicates that fusion protein does not change the protein structure of eGFP or the activity of luciferase.Conclusion:Lentiviral vector carrying dual reporter gene is successfully built and packed to produce the virions with infection abilityPart Two Labeling and Bimodal Imaging of Human Umbilical Cord Derived Endothelial Progenitor CellsObjective:To separate, cultivate and identify endothelial progenitor cells (EPCs)from umbilical cord. Double labeling is conducted for the hEPCs with FUGW-Luc2-eGFP carrying dual reporter gene and SPIONs, and in vitro imaging is realized against labeled cells with bioluminescent imaging (BLI) and magnetic resonance imaging (MRI).Methods :Density gradient centrifugation is adopted for cultivation of hEPCs from umbilical cord of newborns, and hEPCs are identified in terms of cellular morphology, immunofluorescent staining of marker cells, and cellular function test.Double labeling is conducted for hEPCs with FUGW-Luc2-eGFP and SPIONs to obtain D-hEPCs, and with the help of BLI and MRI, imaging detection of D-hEPCs is realized. Histological staining of D-hEPCs were carried on. Besides, the changes of cell cycle and apoptosis between hEPCs and D-hEPCs are compared.Results:hEPCs was in the shape of paving stones, and immunofluorescent staining CD31, CD34 and VEGFR2 were all positive. Phagocytic reaction of FITC-UEA-1 and Dil-ac-LDL was positive. Besides, the cells could form tubular structures on matrigel. Bimodal imaging test was conducted on D-hEPCs with BLI and MRI, which shows that the signal strength of different cell numbers varies.Prussian blue staining of D-hEPCs almost 100% could be seen in the cytoplasm Through flow cytometry detection and immunofluorescent staining, it could be seen that GFP expression in D-hEPCs reached 85%. As for cell cycle and apoptosis detection, there was no obvious difference between D-hEPCs and hEPCs (p >0,05).Conclusion:hEPCs are successfully separated, cultivated and identified from umbilical cord blood. The lentiviral virion of Luc-eGFP carrying reporter gene and USPIO can effectively and noninvasively label hEPCs, and realize imaging detection through BLI and MRI, providing feasible cell sources for future experimental cellular treatment and cellular in-vivo tracking.Part Three Research on Ischemic Stroke in hEPCs Transplantation TreatmentObjective: To set up mouse ischemic stroke model with photochemical method,so as to transplant hEPCs (V-hEPCs) only through lentiviral infection into mouse.Through BLI, the homing in mouse between caudal vein injection and left ventricular injection of V-hEPCs is compared. In stroked mice, left ventricular injection of D-hEPCs is chosen to realize BLI and MRI in-vivo bimodal tracking, and the treatment of exogenous EPCs for stroked mice is also discussed.Methods: hEPCs obtained through in-vitro cultivation is infected with lentiviral virions carrying dual reporter gene to get the singly labeled cells (V-hEPCs). 106 V-hEPCs are transplanted through left ventricular and caudal vein injection into mouse ischemic stroke model established through photochemical method. The homing after 1, 4 and 7 days of cellular transplantation through two different means.Lentiviral virions carrying dual reporter gene and USPIO are used to label hEPCs to obtain double labeled D-hEPCs. Through the injection of D-hEPCs into stroked mouse by left ventricular injection, BLI and MRI in-vivo tracking and monitoring are conducted after 1,4, 7 and 14 days of cellular transplantation. Neurological scoring 1,7 and 14 days before and after cellular transplantation is rated. MR T2WI is used for scanning 7 days after cellular transplantation to evaluate the size of ischemic tissue.Histological staining detection is conducted 14 days after cellular transplantation to observe angiogenesis, neurogenesis and apoptosis of brain tissues.Results: BLI showed that V-hEPCs through caudal vein and left ventricular injection could home to ischemia brain tissues of stroked mouse, but the homing ability of left ventricular injection was obviously stronger than that of caudal vein injection,hence displaying a statistical difference (p<0.05). Through BLI and MRI,the in-vivo tracking of prognosis could be realized for D-hEPCs through left ventricular into thrombosis mouse. Brain tissue pathological section staining displayed a positive staining of Prussian blue and GFP staining, and the positive cells were around infarction border region, demonstrating D-hEPCs homing to ischemia tissues. Neurological scoring and infarction size determination through T2WI scanning indicated that D-hEPCs transplantation can lower down the behavioral defects and the size of infarction in stroked mouse. The CD31 staining and DCX staining of D-hEPCs transplanted brain tissue pathological section were obviously higher than those in PBS control group, yet the TUNEL staining was lower, both displaying a statistical difference (p<0.05).Conclusions: Left ventricular transplantation of V-hEPCs can increase the number of cells homing to ischemia tissues. BLI and MRI in-vivo tracking and brain tissue pathological staining prove that D-hEPCs home to surrounding area of ischemia infarction. D-hEPCs transplantation can improve behavioral infects, decrease size of infarction, promote angiogenesis and neurogenesis in infarction, and lower down cell apoptosis in thrombosis mice.