【作者】 张毅；

【导师】 余永胜；

【作者基本信息】 苏州大学 ， 内科学， 2013， 硕士

【摘要】 第一部分慢性乙型肝炎患者外周血CD4~+T细胞Runx3的表达目的探讨慢性乙型肝炎患者外周血CD4~+T细胞Runx3mRNA的表达及意义。方法选取37例慢性乙型肝炎患者和19例健康对照者，应用密度梯度离心法分离外周血CD4~+T细胞，应用实时定量PCR检测CD4~+T细胞Runx3mRNA的表达水平。结果慢性乙型肝炎组Runx3mRNA的表达水平（0.48±0.09）较健康对照组（0.79±0.17）明显降低（P<0.01）。结论Runx3基因可能参与了慢性乙型肝炎的发病过程。第二部分人Runx3基因重组慢病毒载体的构建与鉴定目的构建人Runx3基因重组慢病毒载体。方法PCR扩增人Runx3基因，应用In-Fusion技术将Runx3基因PCR扩增产物交换进入线性化慢病毒载体pGC-FU以构建重组慢病毒质粒pGC-FU-Runx3，转化大肠杆菌DH5α感受态细胞。对长出的阳性克隆进行PCR鉴定，并进行测序和比对分析。将构建成功的重组慢病毒质粒pGC-FU-Runx3与包装质粒pHelper1.0、包膜质粒pHelper2.0共转染293T细胞进行病毒包装。荧光显微镜下观察重组慢病毒质粒pGC-FU-Runx3转染293T细胞后细胞内绿色荧光表达情况。Western blot检测Runx3与EGFP融合蛋白表达情况。实时定量PCR测定病毒滴度。结果重组慢病毒质粒pGC-FU-Runx3经测序和比对分析证实目的基因序列正确。三质粒共转染293T细胞后荧光显微镜下观察细胞内可见明显的绿色荧光。Western blot证实Runx3与EGFP融合蛋白在293T细胞内稳定表达。浓缩病毒后测定滴度为2.0×10~8TU/ml。结论成功构建携带人Runx3基因的重组慢病毒载体pGC-FU-Runx3。第三部分Runx3基因过表达对慢性乙型肝炎患者外周血Th细胞分化的影响目的探讨Runx3基因过表达对慢性乙型肝炎患者外周血Th细胞分化的影响。方法重组慢病毒载体pGC-FU-Runx3与阴性对照慢病毒载体pGC-FU分别转染29例慢性乙型肝炎患者外周血CD4~+T细胞，收集培养3d、5d和7d的细胞培养液上清，应用ELISA检测Th1型细胞因子IFN-γ、IL-2和Th2型细胞因子IL-4、IL-10的表达水平。收集培养5d的CD4~+T细胞，应用实时定量PCR检测转录因子T-bet、GATA3mRNA的表达水平。结果1.与pGC-FU转染组比较，pGC-FU-Runx3转染组Th1型细胞因子IFN-γ的表达水平在3d（P<0.05）、5d（P<0.01）和7d（P<0.01）时均明显升高；IL-2的表达水平在3d时无显著性差异（P>0.05），但在5d（P<0.05）和7d（P<0.01）时均明显升高。2.与pGC-FU转染组比较，pGC-FU-Runx3转染组Th2型细胞因子IL-4的表达水平在3d时无显著性差异（P>0.05），但在5d（P<0.01）和7d（P<0.05）时均明显降低；IL-10的表达水平在3d时无显著性差异（P>0.05），但在5d和7d时均明显降低（P均<0.05）。3.与pGC-FU转染组比较，pGC-FU-Runx3转染组IFN-γ/IL-4比值在3d、5d和7d时均明显增大（P均<0.01）。4.与pGC-FU转染组比较，pGC-FU-Runx3转染组T-bet、GATA3mRNA的表达水平无显著性差异（P>0.05），但pGC-FU-Runx3转染组T-bet/GATA3比值较pGC-FU转染组明显增大（P<0.01）。结论Runx3基因过表达可促进慢性乙型肝炎患者Th1型细胞因子的分泌，抑制Th2型细胞因子的分泌，促使慢性乙型肝炎患者外周血Th细胞向Th1细胞分化，使其Th1/Th2失平衡得到改善。更多还原

【Abstract】 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.更多还原