Effects Of Adenovirus-mediated Stem Cell Leukemia Gene Transfer And Administration Of Stem Cell Factor On Restoration Of Interstitial Cells Of Cajal In Vivo

BackgroundInterstitial cells of Cajal(ICC) within the gastrointestinal(GI), the pacemaker cells in GI muscles, generate and propagate electrical slow waves and mediate excitatory and inhibitory motor neurotransmission. Researches have indicated that ICC were decreased in the colon in patients with gastrointestinal motility dysfunction including slow transit constipation. Recent several studies have shown that cells with an ICC phenotype redifferentiate toward a smooth musclelike phenotype, rather than die, and further tests fail to reveal evidence of apoptosis, which could be the common feature that in many of the motility disorders.The stem cell leukaemia(SCL) gene encoding a basic helix-loop-helix transcription factor play a key role in both haematopoiesis and endothelial development. It acts as a positive regulator of upstream transcription of c-kit expression and up-regulates c-kit expression by regulating c-kit promoter.In our previous study we have demonstrated that gene transfection of SCL and administration of SCF can up-regulate c-kit expression, promote SCF/c-kit signaling pathway and restore redifferentiated ICC in vitro. So we want to investigate further whether gene transfection of SCL and administration of SCF can up-regulate c-kit expression and promote SCF/c-kit signaling pathway and restore redifferentiated ICC in vivo. The present study was primarily designed to construct and identificate the recombinant adenovirus vector containing human stem cell leukemia(hSCL) gene, to transfect of SCL gene, and to explore whether it can up-regulate c-kit expression and promote SCF/c-kit signaling pathway and restore redifferentiated ICC in vivo.Objective1. To investigate the rapid construction and identification of the green fluorescent protein(GFP) labeled recombinant adenovirus vector containing hSCL gene.2. To explore distribution and efficiency of GFP labeled recombinant adenovirus vector containing hSCL in mice with ICC loss and to observe its ability to infect ICC cultured in vitro and Hela cells.3. To detect expression of c-kit protein in mice with ICC loss after administration of recombinant adenovirus vector containing hSCL and to study the effects of gene transfection of SCL by recombinant adenovirus vector and administration of SCF on restoring its normal phenotype of interstitial cells of Cajal.Methods1 . Firstly, the hSCL gene fragment was subcloned into the shuttle plasmid pAdTrack-CMV. The recombinant plasmid pAdTrack-CMV/SCL was lined with Pme I and transfected into E.coli BJ5183 containing pAdeasy-1 plasmid. Then the screened recombinant plasmid was transfected into HEK293 cells with liposome and was identified by observing GFP and by PCR.2. Secondly, the culture media containing virus was transfected into Hela cells and ICC cultured in vitro, and the expression of SCL was proved by observing the GFP and SCL mRNA by RT-PCR. Then 1.6×10⁹ PFU of recombinant adenovirus was injected into Balb/c mice with ICC loss via the tail vein. The distribution and efficiency of recombinant adenovirus mediated hSCL in vivo was observed by GFP under the fluorescent microscope at 2d,7d,15d and 30d. The expression of SCL mRNA was measured by RT-PCR at the same time. The damages were also observed in different organs by HE staining.3. Thirdly, 1.6×10⁹ PFU of recombinant adenovirus containing hSCL gene was injected into Balb/c mice with ICC loss via the tail vein. Then the protein expression of c-kit were determined using Western blot analysis at 2d,7d,15d and 30d; After 1.6×10⁹ PFU of recombinant adenovirus containing hSCL gene via the tail vein and SCF 30μg/kg intraperitoneally for 5d continuously were injected into Balb/c mice with ICC loss simultaneously, the distribution and configuration of ICC were observed with immunohistochemistry and electronmicroscope. the area occupied by ICC were calculated with an image analysis system.Results1. Restriction endonuclease and PCR analyses confirmed that the recombinant adenovirus vector containing human SCL gene was successfully constructed by the method of homogenous recombination in bacteria. Then the screened recombinant plasmid was transfected into HEK293 cells with liposome and was identified by observing the green fluorescence protein (GFP) and by PCR. The titer of the recombinant adenovirus was 1.6×10¹¹pfu/ml. 2. The recombinant adenovirus had a high transfection efficiency on Hela cells and ICC cultured in vitro and it had no effect on growth of Hela cells by MTT cell proliferation assay. RT-PCR showed SCL mRNA expressed significantly in Hela cells and ICC cultured in vitro after infected with Ad-SCL.3. Under the fluorescent microscope, GFP was revealed highest at 2d and lowest at 30d in the heart, lung, liver, spleen, kidney, small intestine and large intestine of mice with ICC loss after infected with Ad-SCL. By RT-PCR, SCL mRNA expressed significantly for more than 30d after infected with Ad-SCL in such organs.4. No obvious damages were observed in different organs by HE staining after infected with Ad-SCL.5. By western blot analysis, expression of c-kit protein in colon of Balb/c mice with ICC loss was detected at 2d after infected with recombinant Ad-SCL, highest at 7d and return to normal levels at 30d.6. At 14d after infected with recombinant Ad-SCL and administration of SCF, the distribution and configuration of ICC observed with immunohistochemistry and electronmicroscope in Balb/c mice model with ICC loss. ICC-MP were mainly detected firstly,largely detected at 21d. At 28d ICC-MP,ICC-CM,ICC-LM and ICC-SMB were detected to restore its normal phenotype.7. The distribution and configuration had no difference between only infected recombinant Ad-SCL or administration of SCF in Balb/c mice and in Balb/c mice with ICC loss.Conclusions1. The recombinant adenovirus containing human SCL had a high transfection efficiency in Hela cells and ICC cultured in vitro and could enhance expression of SCL mRNA significantly and it could be used safely in vivo.2. The recombinant adenovirus up-regulated the expression of c-kit protein in colon of Balb/c mice model with ICC loss in vivo. The mechanism of it might be the increase of SCL protein, which enhanced the expression of c-kit protein.3. After infected with recombinant Ad-SCL and administrated of SCF, the distribution and configuration of ICC observed with immunohistochemistry and electronmicroscope in Balb/c mice model with ICC loss showed ICC-MP,ICC-CM,ICC-LM and ICC-SMB could restore its normal phenotype at 28d. Only infected with recombinant Ad-SCL or only administrated of SCF could not totally induce restoration its normal phenotype in Balb/c mice model with ICC loss.4. Both infected with recombinant Ad-SCL and administrated of SCF, can up-regulate c-kit expression and might promote SCF/c-kit signaling pathway and restore redifferentiated ICC in vivo.