【作者】 江虹；

【导师】 黄茵；

【作者基本信息】 杭州师范大学 ， 人体解剖与组织胚胎学， 2012， 硕士

【摘要】 乙型肝炎病毒(Hepatitis B virus, HBV)持续感染的主要机理之一是特异性细胞免疫应答不足,而诱导有效细胞免疫的治疗性疫苗有望增强抗病毒免疫,可作为HBV感染综合治疗的有效手段之一。因此,有效性、持续性和安全性兼顾的治疗性疫苗是慢性乙肝免疫治疗研究的一个重要目标。目前市售的含铝佐剂乙肝表面抗原(hepatitis B surface antigen, HBsAg)重组疫苗是有效的预防用疫苗,但由于诱导的细胞免疫应答不足,因而一般不用于抗病毒治疗。CD137/CD137L (4-1BB/4-1BBL)是一对重要的协同刺激信号分子,对增强和维持免疫应答,尤其是特异性T细胞的扩增、存活和记忆性杀伤等均有极其重要的作用,在抗病毒免疫治疗中具有潜在的临床应用价值。本研究构建小鼠CD137L的真核表达质粒,观察该重组质粒和CD137L重组蛋白在小鼠体内对HBsAg重组蛋白和含铝佐剂乙肝疫苗诱导特异性免疫应答的免疫调节作用,探讨CD137L分子作为乙肝重组疫苗的佐剂以增强抗病毒免疫效应的可能性,为慢性乙型肝炎的治疗性疫苗提供新的优化思路。第一部分：小鼠CD137L真核表达质粒的构建及鉴定目的构建小鼠CD137L的真核表达质粒,并将该质粒体外转染NIH3T3细胞,检测其CD137L mRNA和蛋白的表达,为进一步用于动物实验做好前期准备。方法1.重组质粒pcD137L的构建分离BALB/c小鼠脾细胞,体外经ConA刺激培养后提取细胞总RNA,通过RT-PCR克隆小鼠CD137L全长cDNA, PCR产物经回收和纯化后克隆到载体pcDNA3.1(+)上。重组质粒分别经PCR、双酶切和测序鉴定。2.重组质粒pcD137L的表达及检测以脂质体法将重组质粒pcD137L转染到NIH3T3细胞中,并通过RT-PCR、流式细胞术和免疫荧光法分别检测转染细胞中CD137L mRNA和蛋白的表达。结果1.重组质粒pcD137L的PCR产物扩增出与目的片段相符的单一条带；双酶切鉴定得到与目的基因大小一致的片段。2.测序结果证实所插入片段的核苷酸序列与基因库中小鼠CD137L基因序列完全一致。3.经RT-PCR从pcD137L转染细胞中扩增出与目的基因相符的单一条带。4. pcD137L转染细胞经PE-抗小鼠CD137L单抗染色后,流式细胞仪检测到其表面CD137L分子的表达,并在荧光显微镜下观察到这些细胞的胞膜呈现荧光。结论1.成功构建了小鼠CD137L的真核表达载体(pcD137L).2.证实重组质粒pcD137L在真核细胞内能成功表达CD137L mRNA和目的蛋白。第二部分：CD137L重组质粒和CD137L重组蛋白对HBsAg重组疫苗的佐剂作用目的分别以CD137L重组质粒(pcD137L)和重组蛋白(rCD137L)与HBsAg重组疫苗共免疫小鼠,探讨CD137L分子作为重组乙肝表面抗原(rHBsAg)疫苗的佐剂以增强抗病毒免疫治疗作用的可能性。方法1.动物分组和免疫：BALB/c小鼠随机分8个实验组和5个对照组,每组5只。在0,3,6周各免疫1次,共3次,最后1次免疫后1周处死小鼠。实验组为：(1) rHBsAg组(S);(2) rHBsAg+pcDNA3.1(+)组(S+pcD);(3)rHBsAg+pcD137L组(S+pcD137L);(4)rHBsAg+铝佐剂组(S+Al);(5)rHBsAg+铝佐剂+pcDNA3.1(+)组(S+Al+pcD);(6) rHBsAg+铅佐剂+pcD137L组(S+Al+pcD137L);(7) rHBsAg+rCD137L组(S+rCD137L);(8) rHBsAg+铝佐剂+rCD137L组(S+Al+rCD137LX对照组为：(1)NS组；(2) pcDNA3.1(+)组(pcD)；(3)铝佐剂组(A1)；(4) pcD137L组；(5) rCD137L组。2. ELISA法检测免疫后小鼠血清IgG, IgGl及IgG2a水平。3.流式细胞术检测脾细胞中CD8+T细胞内IFN-y/IL-4表达水平。4.流式细胞术分析脾细胞中CD44+high釉CD127+CD44+highCD8+T记忆细胞百分比。5.流式细胞术分析特异性CTL细胞诱导靶细胞凋亡的活性。结果1.血清特异性抗体抗-HBs水平检测(DpcD137L能促进rHBsAg诱导抗体应答(总IgG、IgG2a和IgGl增高),对含铝佐剂乙肝疫苗抗体应答无调节作用。(2) rCD137L增强rHBsAg诱导Thl型抗体应答(总IgG和IgG2a增高),并可增强含铝佐剂乙肝疫苗诱导Thl型抗体而下调Th2型抗体(上调IgG2a,下调IgGl).(3)铝佐剂对rHBsAg诱导抗体有显著增强作用(总IgG、IgG2a和IgGl增高)。2. CD8+T细胞内IFN-y/IL-4检测(1) pcD137L可促进rHBsAg诱导特异性CD8+T细胞IFN-y的产生(Tcl),并可增强含铝佐剂乙肝疫苗诱导Tcl反应。(2) rCD137L对rHBsAg诱导Tcl细胞应答无明显作用,但对含铝佐剂乙肝疫苗诱导Tcl细胞免疫应答有显著促进作用。(3)铝佐剂可增强rHBsAg的免疫原性,提高Tcl细胞免疫应答水平。(4) pcD137L和rCD137L对rHBsAg或含铝佐剂rHBsAg刺激特异性CD8+T细胞分泌IL-4均无明显调节作用。3.脾细胞CD8+记忆T细胞分析(1) pcD137L促进rHBsAg产生CD44+high和CD127+CD44+highCD8+T记忆细胞,但对含铝佐剂乙肝疫苗诱导CD8+记忆T细胞无明显作用。(2) rCD137L可促进rHBsAg诱导CD8+记忆T细胞形成,并可增强含铝佐剂乙肝疫苗产生CD8+记忆T细胞。(3)铝佐剂促进rHBsAg疫苗诱导CD8+记忆T细胞数。4.特异性CTL诱导靶细胞凋亡的活性分析(1) rCD137L增强rHBsAg及含铝佐剂rHBsAg诱导特异性CTL应答。(2)铝佐剂可增强rHBsAg疫苗的CTL活性。以上差异均有统计学意义。结论1. CD137L重组质粒增强HBsAg重组疫苗在小鼠体内诱导的抗体应答、Tcl细胞应答及CD8+记忆T细胞形成,但对含铝佐剂乙肝疫苗无明显免疫增强作用。2.CD137L重组蛋白显著促进含铝佐剂HBsAg重组疫苗在小鼠体内产生IgG2a (Thl型)抗体反应和以分泌IFN-y为特征的Tc1(I型)细胞免疫,增强其特异性CTL活性及CD8+记忆T细胞形成,可能是市售乙肝疫苗由预防用制剂转为治疗性疫苗的有效佐剂。更多还原

【Abstract】 One of the main mechanisms of HBV persistent infection is that the specific cellular immune response is insufficient. But it is hoped that the therapeutic vaccine which induce effective cellular immunity will enhance the antiviral immune, and will be used as one of the effective means about comprehensive treatment of HBV infection. Therefore, to research of therapeutic vaccine, which is effective, sustainable and safety, is an important goal of chronic hepatitis B immunotherapy.The recombinant vaccine which combined with HBsAg and aluminum hydroxide adjuvant is effective preventive vaccine. But because of the insufficient of cellular immune response which be induced, it is generally not be used in antiviral treatment.CD137/CD137L (4-1BB/4-1BBL) is an important pair of costimulatory signal molecule, which is the important effect to enhance and keep immune responses, especially to proliferation, survival and mnemonic killer of specific T cell. It has potential clinical application of antiretroviral immune treatment.This study is to construct the eukaryotic expression plasmid of mouse CD137L at first. At second, the immunologic enhancement effect of CD137L recombinant plasmid and CD137L recombinant protein of the specific immune responses in mice inducted by the HBsAg recombinant protein and the hepatitis B vaccine combined with aluminum hydroxide adjuvant was observed. At last, the possibility of CD137L molecule as the adjuvant of recombinant hepatitis B vaccine to enhance the antiviral immune effect also was explored. Part1construction and identification of the eukaryotic expression plasmid of mouse CD137LObjectiveThis study will focus on the expression of mRNA and protein in NIH3T3cells by constructing the eukaryotic expression plasmid of CD137L, meanwhile, lay the experiment foundation for the further research of the animal study.Methods1. Construction of the recombinant plasmid of CD137LThe splenic cells of BALB/c mouse were isolated, and the total RNA from those cells which cultured in vitro and stimulated by ConA were extracted. Then the PCR products which had extracted and purified were recombinanted into pcDNA3.1(+).At last the recombinant plasmid was identified by PCR, enzyme digest and sequencing, respectively..2. Expression and examinations of the recombinant plasmid of CD137LEukaryotic expression vector containing the full length of mouse CD137L cDNA sequence (pcD137L) was transfected into NIH3T3cells using Lipofectamine2000, and then the expression of CD137L mRNA and protein in the transfected cells were detected by RT-PCR, flow cytometry and immunofluorescence method, respectively.Results1. A single band which amplified by PCR products of recombinant plasmid (pcD137L) is consistent with the target fragment. And the size of fragment which gained by enzyme digest is consistent with target fragment too.2. The result of sequencing proved that the nucleotide sequence of inserted fragment is consistent to mouse CD137L gene sequence in Genebank.3. A single band which amplified from pcD137L transfected cells by RT-PCR is consistent with target fragment.4. After pcD137L transfected cells stained by PE-anti mouse CD137L, the expression of CD137L molecular on the cell surface were detect by flow cytometry, and membranes of those cells showed fluorescence under fluorescence microscope. Conclusions1. An eukaryotic expression vector (pcD137L) containing mouse CD137L cDNA was successfully constructed.2. It was proved that the recombinant plasmid (pcD137L) can express CD137L mRNA and protein in eukaryotic cells successfully. Part2Adjuvant effect of CD137L recombinant plasmid and CD137L recombinant protein to recombinant HBsAg vaccineobjectiveMice were immunized with CD137L recombinant plasmid (pcD137L) or recombinant protein (rCD137L) combined with recombinant HBsAg, respectively. And the possibility of CD137L molecule as the adjuvant of recombinant HBsAg enhance the role of antiviral immune was explored.MethodsBALB/c mice were randomly divided into eight experimental groups and five control groups with5in each group. And mice were immuned three times at the week of0,3,6and killed at one week after the last immunity.Experimental groups:(1) rHBsAg (S);(2) rHBsAg+pcDNA3.1(+)(S+pcD);(3) rHBsAg+pcD137L (S+pcD137L);(4) rHBsAg+aluminum hydroxide adjuvant (S+Al);(5) rHBsAg+aluminum hydroxide adjuvant+pcDNA3.1(+)(S+Al+pcD);(6) rHBsAg+aluminum hydroxide adjuvant+pcD137L (S+Al+pcD137L);(7) rHBsAg+rCD137L (S+rCD137L);(8) rHBsAg+aluminum hydroxide adjuvant+rCD137L (S+Al+rCD137L).Control groups:(1) NS;(2) pcDNA3.1(+)(pcD);(3) aluminum hydroxide adjuvant (Al);(4) pcD137L;(5) rCD137L.2. Serum IgG, IgGl and IgG2a of immunization mice were detected by ELISA.3. The expression level of IFN-γ/IL-4in splenic CD8+T cells was detected by flow cytometry.4. The percentage of CD44+high and CD127+CD44+higbin splenic memory CD8+T cells was analied by flow cytometry.5. The activity of apoptotic target cells which induced by specific CTL was analied by flow cytometry.Results1. Detection of serum specific antibodies of anti-HBs(1)pcD137L promoted the antibody response inducted by rHBsAg(The titer of total IgG、IgG2a and IgGlwere rised), but can not play regulatory role of antibody response to the hepatitis B vaccine combined with aluminum hydroxide adjuvant.(2)pcD137L enhanced the Thl antibody response inducted by the rHBsAg(The titer of total IgG and IgG2a were rised), and increased the Thl antibodies but decreased Th2antibodies inducted by the hepatitis B vaccine combined with aluminum hydroxide adjuvant(The titer of IgG2a was rised but IgGl was decreased).(3) Aluminum hydroxide adjuvant substantially increased the level of antibodies inducted by the rHBsAg (The titer of total IgG、IgG2a and IgGlwere rised).2. Detection of the percentage of IFN-y/IL-4in CD8+T cells(1) pcD137L elicited higher level of IFN-y secreted by CD8+T cell inducted by the rHBsAg, and enhanced the Tcl response inducted by hepatitis B vaccine combined with aluminum hydroxide adjuvant.(2) rCD137L can not played regulatory role of the Tcl cellular immune response inducted by the rHBsAg, but obviously promoted the Tel cellular immune response inducted by the hepatitis B vaccine combined with aluminum hydroxide adjuvant.(3) Aluminum hydroxide adjuvant enhanced the immunogenicity of the rHBsAg and improved the level of cellular immune response of Tcl.(4) Both pcD137L and rCD137L can not played an obvious regulatory role of the IL-4secreted by specific CD8+T cells which stimulated by the rHBsAg or the rHBsAg combined with aluminum hydroxide adjuvant.3. Analysis of the percentage of memory CD8+T cells in splenic cells(1) pCD137L increased productions of CD44+high CD8+and CD127+CD44+high CD8+T cells induced by the rHBsAg but not induced by the hepatitis B vaccine combined with aluminum hydroxide adjuvant.(2) rCD137L elicited higher level of memory CD8+T cells both induced by the rHBsAg and hepatitis B vaccine combined with aluminum hydroxide adjuvant.(3) Aluminum hydroxide adjuvant increased the number of memory CD8+T cells inducted by the rHBsAg.4. Analysis of the activity of apoptotic target cells induced by CTL(1) rCD137L enhanced specific CTL response which induced by the rHBsAg and the rHBsAg combined with aluminum hydroxide adjuvant.(2) Aluminum hydroxide adjuvant enhanced the activity of CTL induced by the rHBsAg.The differences of all the above have statistical significance.Conclusions1.CD137L recombinant plasmid enhanced the antibody responses, Tcl cellular responses and the production of memory CD8+T cells inducted by the rHBsAg in mice but not to by the rHBsAg combined with aluminum hydroxide adjuvant.2.CD137L recombinant protein enhanced the IgG2a (Thl) antibody response and cellular response characterized by secrete IFN-y which inducted by the rHBsAg in mice, the activity of specific CTL and the production of memory CD8+T cells. It obesrvely enhanced the type1cellular immune responses and humoral immune responses to the hepatitis B vaccine combined with aluminum hydroxide adjuvant. It may be an effective adjuvant for translating prophylactic vaccines into therapeutic vaccine to HBV.更多还原