Lentiviral Vector-mediated Runx3Overexpression Regulates T-help Cell Lineage Commitment In Patients With Chronic Hepatitis B

Part1The expression of Runx3mRNA in peripheral CD4~+T cellsfrom patients with chronic hepatitis BObjective To investigate the expression of Runx3mRNA in peripheral CD4~+T cellsfrom patients with chronic hepatitis B (CHB) and its significance.Methods37CHB patients and19healthy controls were enrolled. Peripheral CD4+Tcells derived from all subjects were separated by density gradient centrifugation and thenthe expression of Runx3mRNA was detected by quantitative real-time PCR.Results The expression of Runx3mRNA in the CHB group (0.48±0.09) wassignificantly lower than that in the healthy control group (0.79±0.17)(P<0.01).Conclusions Runx3gene may be involved in the pathogensis of CHB.Part2Construction and identification of the recombinant lentiviralvector containing human Runx3geneObjective To construct a recombinant lentiviral vector carrying human Runx3gene.Methods Human Runx3gene was amplified by PCR. Next, the Runx3genefragment and the lentiviral vector (pGC-FU) were ligated with In-Fusion technique togenerate the recombinant lentiviral plasmid (pGC-FU-Runx3). Then pGC-FU-Runx3was transformed into Escherichia coli DH5α competent cells. The positive clones werescreened by PCR and confirmed by sequencing and comparative analysis. The recombinant lentiviral plasmid (pGC-FU-Runx3) was mixed with the packaging plasmid(pHelper1.0) and the envelope plasmid (pHelper2.0) and then they were co-transfectedinto293T cells. The expression of Runx3-EGFP in293T cells was detected by bothfluorescence microscopy and Western blot. The viral titer was measured by quantitativereal-time PCR.Results The recombinant lentiviral plasmid (pGC-FU-Runx3) was confirmed thatthe target gene sequence was correct by sequencing and comparative analysis. Stronggreen fluorescence was observed in293T cells under fluorescent microscope followingco-transfection with three plasmids (pGC-FU-Runx3, pHelper1.0and pHelper2.0) intothe cells. Western blot identified the stable expression of Runx3-EGFP fusion protein inthe transfected293T cells. The virus in the supernatant reached a titer of2.0×10~8TU/ml.Conclusions The recombinant lentiviral vector (pGC-FU-Runx3) carrying humanRunx3gene was successfully constructed.Part3The effect of Runx3overexpression on Th cell differentiationin patients with chronic hepatitis BObjective To investigate the effect of Runx3overexpression on Th celldifferentiation in patients with CHB.Methods Peripheral CD4~+T cells derived from29CHB patients were transfectedwith the recombinant lentiviral vector (pGC-FU-Runx3) and the negative controllentiviral vector (pGC-FU) respectively. Then the cell culture supernatants were collectedat different times (days3,5and7) and the CD4~+T cells were collected on day5. Theexpression of Th1-type cytokines (IFN-γ, IL-2) and Th2-type cytokines (IL-4, IL-10) wasmeasured by ELISA and the experssion of T-bet and GATA3mRNA was assayed byquantitative real-time PCR.Results1. Compared with the pGC-FU transfected group, the expression of IFN-γin the pGC-FU-Runx3transfected group significantly increased on days3(P<0.05),5 (P<0.01) and7(P<0.01). Furthermore, the expression of IL-2in the pGC-FU-Runx3transfected group significantly increased on days5(P<0.05) and7(P<0.01). However,there was no difference in IL-2expression between the pGC-FU transfected group andthe pGC-FU-Runx3transfected group on day3(P>0.05).2. Compared with the pGC-FUtransfected group, the expression of IL-4in the pGC-FU-Runx3transfected groupsignificantly decreased on days5(P<0.01) and7(P<0.05). Nevertheless, there was nodifference in IL-4expression between the pGC-FU transfected group and thepGC-FU-Runx3transfected group on day3(P>0.05). Moreover, the expression of IL-10in the pGC-FU-Runx3transfected group significantly decreased on days5(P<0.05) and7(P<0.05). Nonetheless, there was no difference in IL-10expression between thepGC-FU transfected group and the pGC-FU-Runx3transfected group on day3(P>0.05).3. Compared with the pGC-FU transfected group, the ratio of IFN-γ/IL-4in thepGC-FU-Runx3transfected group significantly increased on days3(P<0.01),5(P<0.01)and7(P<0.01).4. There was no difference in T-bet and GATA3mRNA expressionbetween the pGC-FU transfected group and the pGC-FU-Runx3transfected group.However, compared with the pGC-FU transfected group, the ratio of T-bet/GATA3in thepGC-FU-Runx3transfected group significantly increased (P<0.01).Conclusions Runx3overexpression can promote the secretion of Th1-typecytokines and inhibit the secretion of Th2-type cytokines in CHB patients. It can alsoinduce Th cell differentiation into Th1cell lineage and improve the Th1/Th2imbalancein CHB patients.