Genetically Modified Bone Marrow Hematopoietic Stem Cell Targeting Inhibition Of Colorectal Cancer Angiogenesis In Experimental Research

Objective:Lenti-virus plasmid was reconstructed with the promotor and enhancer of Tie2 gene, together with Endostatin gene at downstream. Hematopoietic stem cell was transfected by this plasmid. Methods:The promotor and enhancer of Tie2 gene were extracted from the liver of mouse. After identification, they were cloned to the trans-plasmid pGL-3basic and then transfered into lenti-virus plasmid TG005. Endostatin gene, following the same procedure, was cloned to pGL-3basic-Tie2p/e and then transfered into TG005. Both of them were reconstructed in 293T cell to become TG005-Tie2p/e and TG005-Tie2p/e-ES. The sequence was evaluated with PCR. The hemotaopietic stem cells were extracted from the bone marrow of mouse. They are infected with the reconstucted lenti-virus. Those successfully infected cells were picked up. The ES product was tested. Result:Tie2 gene promoter and enhancer were successfully received from the liver of mice, were cloned into the lentivirus vector and identified by enzyme digestion. ES gene was also cloned into the lentiviral vector and identified. PCR confirmed the transformation of lentivirus. Hematopoietic stem cells extracted from bone marrow were screened, selected and transfected by the corresponding virus and expressed ES. Conclusion:Lentiviral vector is an efficient, simple and fast virus vector, can effectively infect cells, transfer and express the corresponding gene. Objective:To assess the bone marrow hematopoietic stem cells carrying the Tie2 promoter, enhancer and endostatin gene in the suppression of angiogenesis ans tumor proliferation in the animal models of colorectal cancer. Methods:Lovo colorectal cancer cell line was injected into nude mice to establish the model of colorectal cancer. 40 nude mice were divided into NS, HSC, lentivirus, Tie2, and ES 5 groups, n= 8. The appropriate treatment was given through the tail vein of each group. The tumor volumn was measured periodically during the treatment. After the treatment, the volumn and weight of tumor were measured.â…§factor and Tunel staining were used to evaluated the tumor blood vessels and the number of apoptotic cells. Vascular morphology was observed with electron microscope and formation of blood vessels of other organs was checked. Results:At the beginning of treatment, tumor volume was no significant difference among the various experimental groups(p= 0.5). In the termination, tumors in Tie2 group were significantly larger than those in ES group (P <0.01). Microvessel count showed that Tie2 group was significantly increased than ES group (P<0.01). Apoptotic index showed that Tie2 group was significantly decreased than ES group (P<0.01). The vascular damage was obvious in ES group under the electron microscopy. The vascular proliferation of other organswas not obvious in two group contained Tie2. Conclusion:The bone marrow hematopoietic stem cells carrying a Tie2 promoter, enhancer and endostatin gene have good therapeutic targeting. Endostatin was released in tumor angiogenesis district and the tumor growth was significantly inhibited in colorectal cancer animal models..