HA纳米载体介导转hGM-CSF基因的HepG2疫苗诱导的 抗瘤效应研究

目的:观察HA纳米颗粒载体介导转染hGM-CSF基因的HepG2细胞疫苗体外抗肿瘤效应,为hGM-CSF基因修饰的HepG2细胞疫苗的临床应用提供依据。方法:HA纳米颗粒载体介导hGM-CSF基因转染HepG2细胞制备转GMCSF基因的HepG2细胞疫苗。密度梯度离心法分离人PBMC,体外诱导人PBMC。WST-1法测定PBMC的增殖活性及对HepG2细胞的杀伤效应,流式细胞术分析CD4+和CD8+的阳性表达率,ELISA法测定INF-γ的分泌。结果:WST-1结果显示,转基因HepG2组疫苗能诱导PBMC增殖,其增殖率优于野生型疫苗(p<0.05);其诱导的PBMC对HepG2的杀伤率高于各野生型疫苗组和各空白对照组(p<0.05)。FCM结果显示,转基因HepG2疫苗组PBMC中CD4+和CD8+阳性表达率均高于各野生型疫苗组和各空白对照组(p<0.05)。ELISA结果显示,转基因组PBMC培养上清中IFN-γ含量为1989.76±254.21pg/ml,高于各野生型疫苗组和各空白对照组(p<0.05)。结论:HA纳米颗粒载体介导转染hGM-CSF基因能增加HepG2细胞疫苗的免疫原性,转hGM-CSF基因HepG2细胞疫苗可有效诱导PBMC增殖、分化,增加INF-γ的分泌,提高其对HepG2细胞的杀伤作用。     

HA纳米载体介导转hGM-CSF基因的HepG2疫苗诱导的抗瘤效应研究

目的：观察HA纳米颗粒载体介导转染hGM-CSF基因的HepG2细胞疫苗体外抗肿瘤效应,为hGM-CSF基因修饰的HepG2细胞疫苗的临床应用提供依据。方法：HA纳米颗粒载体介导hGM-CSF基因转染HepG2细胞制备转GMCSF基因的HepG2细胞疫苗。密度梯度离心法分离人PBMC,体外诱导人PBMC。WST-1法测定PBMC的增殖活性及对HepG2细胞的杀伤效应,流式细胞术分析CD4＋和CD8＋的阳性表达率,ELISA法测定INF-γ的分泌。结果：WST-1结果显示,转基因HepG2组疫苗能诱导PBMC增殖,其增殖率优于野生型疫苗（p〈0.05）;其诱导的PBMC对HepG2的杀伤率高于各野生型疫苗组和各空白对照组（p〈0.05）。FCM结果显示,转基因HepG2疫苗组PBMC中CD4＋和CD8＋阳性表达率均高于各野生型疫苗组和各空白对照组（p〈0.05）。ELISA结果显示,转基因组PBMC培养上清中IFN-γ含量为1989.76±254.21pg/ml,高于各野生型疫苗组和各空白对照组（p〈0.05）。结论：HA纳米颗粒载体介导转染hGM-CSF基因能增加HepG2细胞疫苗的免疫原性,转hGM-CSF基因HepG2细胞疫苗可有效诱导PBMC增殖、分化,增加INF-γ的分泌,提高其对HepG2细胞的杀伤作用。     

转hGM-CSF 基因HepG2 细胞疫苗侵袭性和生长活性初步检测

目的:探讨经60Co处理的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性变化。方法:体外培养三种肝癌细胞(①野生型HepG2肝癌细胞②转染hGM-CSF基因的HepG2肝癌细胞③60Co射线处理的转hGM-CSF基因的HepG2肝癌疫苗)采用MTT方法检测三种细胞在24h、48h、72h的OD值并绘出生长曲线;利用transwell小室进行体外侵袭实验来观察上述三种细胞侵袭性;用RT-PCR技术检测上述三种细胞基质金属蛋白酶2(MMP-2)在mRNA水平上表达的变化;结果:经60Co照射处理的转hGM-CSF基因的HepG2肝癌疫苗组OD值在相同培养时间点较其他两组明显变小且差异有显著性(P<0.05)。三种细胞(上述①②③种细胞)transwell侵袭试验显示:③组穿过人造基底膜的细胞数量明显少于前两组;PT-PCR示:③组细胞的MMP-2的mRNA的表达明显低于①②。结论:经过60Co处理过的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性均明显降低。     

转hGM-CSF基因HepG2细胞疫苗侵袭性和生长活性初步检测

目的：探讨经60Co处理的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性变化。方法：体外培养三种肝癌细胞（①野生型HepG2肝癌细胞②转染hGM-CSF基因的HepG2肝癌细胞③60Co射线处理的转hGM-CSF基因的HepG2肝癌疫苗）采用MTT方法检测三种细胞在24h、48h、72h的OD值并绘出生长曲线;利用transwell小室进行体外侵袭实验来观察上述三种细胞侵袭性;用RT-PCR技术检测上述三种细胞基质金属蛋白酶2（MMP-2）在mRNA水平上表达的变化;结果：经60Co照射处理的转hGM-CSF基因的HepG2肝癌疫苗组OD值在相同培养时间点较其他两组明显变小且差异有显著性（P〈0.05）。三种细胞（上述①②③种细胞）transwell侵袭试验显示：③组穿过人造基底膜的细胞数量明显少于前两组;PT-PCR示：③组细胞的MMP-2的mRNA的表达明显低于①②。结论：经过60Co处理过的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性均明显降低。     

GM-CSF与IL-21基因共转染人卵巢癌细胞在裸鼠体内的抗瘤效应

目的:研究GM-CSF与IL-21基因共转染人卵巢癌细胞系SKOV3后在裸鼠体内的抗肿瘤效应.方法:将体外培养的SKOV3、SKOV3/Neo、SKOV3/GM-CSF、SKOV3/IL21及SKOV3/GM-CSF-IL21细胞分别皮下接种BALB/c裸小鼠,监测荷瘤鼠肿瘤生长情况,观察GM-CSF与IL-21单独及联合诱导的抗肿瘤效应.ELISA法检测血清IFN-γ和TNF-α含量,MTT比色法测定裸鼠脾细胞NK活性,RT-PCR检测裸鼠脾细胞中NKG2D分子的表达.结果:与SKOV3及SKOV3/Neo相比,用SKOV3/GM-CSF、SKOV3/IL21和SKOV3/GM-CSF-IL21攻击的裸鼠,体内肿瘤形成受到明显抑制,其中SKOV3/GM-CSF-IL21组更明显,与其他各组比较均有显著差异;用SKOV3/GM-CSF-IL21攻击的裸鼠外周血,白细胞总数、中性粒细胞数、单核细胞数及IFN-γ和TNF-α含量均显著升高,脾细胞NK活性及NKG2D表达增强.结论:GM-CSF与IL-21基因共转染SKOV3细胞后,可在裸鼠体内诱导明显的抗肿瘤效应,其效果显著优于单-GM-CSF或IL-21基因转染的SKOV3细胞.     

结核杆菌Ag85B基因疫苗的免疫保护效果研究

为研究结核杆菌Ag85B基因疫苗的免疫原性和免疫保护效果,将雌性C57BL/6N小鼠32只,随机分为4组,即结核杆菌Ag85B基因疫苗组、BCG组、pcDNA3.1( )组和PBS组.取各免疫组小鼠脾细胞培养上清检测细胞因子水平;同时进行CTL杀伤活性检测;进一步用结核杆菌H37Rv国际标准强毒株静脉注射攻击小鼠,计数肺和脾组织中的结核杆菌菌落数,对小鼠部分肺和脾组织作病理切片,HE染色观察组织病变程度,Z-N染色检查抗酸杆菌,观察该疫苗对小鼠结核杆菌感染的免疫保护效果.结果表明,结核杆菌Ag85B基因疫苗对小鼠结核杆菌感染有一定的免疫保护效果,能够诱导较强的抗原特异性Th1型细胞免疫应答,细胞因子IFN-γ和IL-1分泌增加,IL-4分泌减少,CTL特异性活性增加;使小鼠肺和脾组织中的结核杆菌菌落数较空载体组显著减少,组织病变明显减轻.     

HIV—1CN嵌合基因与IL—2基因共表达产物诱导产生CTL的实验研究

经脂质体介导共表达中国流行株HIV-1gag-gp120基因与IL-2基因的核酸疫苗质粒pGPIL-2转染BHK-21细胞,以间接免疫荧光法鉴定其表达.取pGPIL-2免疫鼠脾细胞,检测pGPIL-2诱导的细胞毒性T细胞杀伤活性.杀伤实验结果证明,经基因免疫获得的CTL效应细胞,可杀伤HIV-1\{CN\}嵌合基因转染的靶细胞.提示pGPIL-2可有效诱导CTL的产生,该研究结果为进一步设计中国流行株HIV-1核酸疫苗提供了重要实验依据.     

肿瘤抗原致敏的GM-CSF基因转染巨噬细胞对实验性肺转移肿瘤的治疗作用

研究将对巨噬细胞双重功能均具有激活作用的细胞因子GM-CSF的基因转染小鼠腹腔巨噬细胞,再经肿瘤抗原致敏后通过静脉注射用于实验性CT26结肠癌肺转移小鼠的治疗.结果表明,腺病毒介导的GM-CSF基因转染小鼠腹腔巨噬细胞在转染后4h即可分泌较高水平的GM-CSF.转染后10d仍可有效表达;转染后16h左右小鼠腹腔巨噬细胞MHCⅡ类分子的表达明显增强,其抗原提呈能力也达最高水平;对肿瘤细胞的杀伤活性显著增高;荷瘤3d的实验性CT26结肠癌肺转移小鼠经肿瘤抗原致敏的GM-CSF基因转染巨噬细胞治疗后第16天肺部转移结节数明显减少;经治疗小鼠脾细胞经诱导的CTL杀伤活性也明显升高.以上结果提示,GM-CSF基因转染及表达能有效增强小鼠腹腔巨噬细胞的抗原提呈能力及效应功能;经肿瘤抗原刺激后其对转移性肿瘤也有明显的治疗作用.     

IL-18 DNA免疫对HIV-1核酸疫苗诱导的免疫应答的影响

为了研究白细胞介素-18(IL-18)基因对人免疫缺陷病毒(HIV-1)核酸疫苗诱导免疫应答的影响,将人IL-18基因插入到真核表达载体pVAX1中,构建了真核表达载体pVAX1-IL-18;将pCI-neoGAG联合pVAX1-IL-18或者pCI-neoGAG单独免疫Balb/c小鼠,检测免疫小鼠的特异性抗体和IFN-γ,同时观察免疫小鼠脾淋巴细胞增殖和小鼠特异性细胞毒性T淋巴细胞(CTL)反应.酶切及测序结果表明成功地构建了人IL-18基因真核表达载体;与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的抗HIV-1p24抗体滴度降低(P<0.01);而与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的IFN-γ升高(P<0.01);pCI-neoGAG联合pVAX1-IL-18免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pCI-neoGAG免疫组(P<0.01).IL-18基因联合HIV-1核酸疫苗免疫小鼠,可能增强特异性Th1细胞和CTL反应,白细胞介素-18基因对体液免疫有抑制作用.     

IL—18DNA免疫对HIV—1核酸疫苗诱导的免疫应答的影响

为了研究白细胞介素-18(IL-18)基因对人免疫缺陷病毒(HIV-1)核酸疫苗诱导免疫应答的影响,将人IL-18基因插入到真核表达载体pVAX1中,构建了真核表达载体pVAX1-IL-18;将pCI-neoGAG联合pVAX1-IL-18或者pCI-neoGAG单独免疫Balb/c小鼠,检测免疫小鼠的特异性抗体和IFN-γ,同时观察免疫小鼠脾淋巴细胞增殖和小鼠特异性细胞毒性T淋巴细胞(CTL)反应。酶切及测序结果表明成功地构建了人IL-18基因真核表达载体;与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的抗HIV-1p24抗体滴度降低(P<0.01);而与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的IFN-γ升高(P<0.01);pCI-neoGAG联合pVAX1-IL-18免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pCI-neoGAG免疫组(P<0.01)。IL-18基因联合HIV-1核酸疫苗免疫小鼠,可能增强特异性Th1细胞和CTL反应,白细胞介素-18基因对体液免疫有抑制作用。     

In Vivo Modulation of Vaccine-Induced Immune Responses toward a Th1 Phenotype Increases Potency and Vaccine Effectiveness in a Herpes Simplex Virus Type 2 Mouse Model

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4⁺-T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.     

IL-4 和IFN-γ mRNA 在子痫前期患者胎盘组织中的表达及意义

目的:检测胎盘组织中IFN-γ和IL-4的表达,探讨IFN-γ和IL-4在子痫前期的发病中的作用.方法:采用原位杂交法检测了20例正常妊娠孕妇和34例子痫前期组(包括16例轻度和18例重度)中的IFN-γ、IL-4 mRNA的表达水平,并通过图像分析系统对染色结果进行定量分析.结果:(1)IL-4 mRNA的表达在正常妊娠组、子痫前期轻度组和子痫前期重度组的表达无差异(P＞0.05).(2)与正常妊娠组、子痫前期轻度组相比,子痫前期重度组IFN-γ mRNA的表达有差异性(P＜0.05);子痫前期轻度组与正常妊娠组相比无差异(P＞0.05).(3)与正常妊娠组相比,子痫前期轻度组、重度组IFN-γ mRNA/IL-4 mRNA的比值均有差异性(P＜0.05),且随病情的加重比值增大.结论:Th1/Th2细胞因子的平衡偏离可能是导致子痫前期发病的病因之一.     

Superiority of the ear pinna over a subcutaneous tumour inoculation site for induction of a Th1-type cytokine response

 This study examines whether a correlation may be found between Th1- or Th2-type cytokine responses and resistance or susceptibility to tumour growth. Cytokine profiles were investigated in a well-defined mouse tumour model in which the injection site and the genetic background determine the phenotype of either tumour resistance or tumour susceptibility. DBA/2-derived ESb lymphoma variant cells with high metastatic capacity were inoculated into syngeneic mice either s.c., where they grow and metastasize, or into the ear pinna (i.e.), where they do not grow because of induction of protective immunity. Alternatively, the tumour cells were injected s.c. or i.e. into allogeneic B10.D2 mice, which are resistant to the tumour although they are identical at the MHC locus. Between 1 and 10 days after tumour cell injection the spleen-derived mRNA was tested for cytokine gene expression or the spleen cells were analysed by FACScan for T cell activation. The strongest cytokine response was observed in i.e. inoculated B10.D2 mice. This was characterized by an early (days 2–3) peak of interferon γ (INF-γ), interleukin-2 (IL-2), IL-2 receptor α (IL-2Rα) and IL-4. The cytokine mRNA response of i.e. inoculated DBA/2 mice was quite similar except that no IFN-γ could be detected. In s.c. inoculated B10.D2 mice, the IL-2, IL-2Rα and IFN-γ responses were weaker than after i.e. injection while the IL-4 response was comparable. The most striking difference between these cytokine profiles from tumour-resistant mice and those of s.c. inoculated tumour-susceptible DBA/2 mice was a delay in the latter in the IL-2, IL-2Rα and IFN-γ responses and the observation that the IL-4 response was not down-regulated. The persisting IL-4 response could down-regulate a Th1-type response and thereby explain tumour susceptibility as a consequence of host conditioning. Received: 4 September 1997 / Accepted: 2 October 1997     

Proinflammatory properties of IL-4 in the intestinal microenvironment

IL-4 is involved in type 2 T helper cell (Th)2-type immune responses and, in some cases, can promote Th1 responses. However, the proinflammatory potential of IL-4 alone is unclear. In this study, we examined the ability of IL-4 to induce colitis after its overexpression in the colon using an adenoviral vector (Ad5) and compared results with those obtained after overexpression of IL-12, a cytokine implicated in several models of colitis. Overexpression of IL-4 or IL-12 caused a fatal colitis within 24 h in 60% of animals and was dose and strain dependent. IL-12-induced colitis was accompanied by the local expression of IFN-gamma and TNF-alpha but not IL-4 mRNA and protein. Conversely, IL-4-induced colitis was accompanied by the local expression of IL-4 and TNF-alpha but not IFN-gamma mRNA and protein. The Ad5-IL4-induced colitis did not persist beyond 3 days and was present in recombinase activation gene-2 (RAG-2)-/- mice but not in STAT6-/- mice. Acute lethal colitis induced by Ad5IL12 was T cell mediated and IFN-gamma receptor (IFN-gamma R) dependent. Furthermore, TNF-alpha was found to be important in the pathogenesis of Ad5IL-4 and Ad5IL-12-induced colitis. Results of this study indicate that IL-4 alone can act as a proinflammatory cytokine in the gut of normal mice, inducing a rapid onset and short-lived colonic injury while maintaining a Th2-type cytokine profile that functions via a local T cell-independent mechanism involving TNF-alpha.     

Antitumor effects and immunoregulation mechanisms of IL-23 gene in mouse mammary cancer mediated by retrovirus

Background: Interleukin (IL)-23, composed of p19 and p40 subunits, has diverse functions in regulating immune systems, enhancing cell-mediated immunity. In the present study, we investigated whether forced expression of the p19-linked p40 gene in murine mammary cancer cells (MA891) produced antitumor effects in vivo. Tumor growth of MA-891 cells expressing IL-23 (IL-23/MA891) in mice was retarded compared with parental and vector DNA-transduced tumors and survival of the mice inoculated with IL-23/MA-891 cells was prolonged. Expressions of the CD4⁺ T cells and CD8⁺ T cells were up-regulated not only in IL-23/MA-891 tumor specimens but also in spleen cells of mice inoculated with IL-23/MA-891 as compared with those of mice inoculated with parental or vector DNA-transduced tumors. Cytotoxic CD8⁺ T lymphocyte (CTL) activity of spleen cells from mice inoculated with IL-23/MA-891 was also significantly higher than the other two groups. Th1-type cytokines such as interferon-γ, TNF-α and IL-12p70 secreted from spleen cells of mice bearing IL-23/MA-891 tumors were increased while Th2-type cytokine IL-4 was negative regulated. Moreover, we have identified that the quantity of DC in spleen cells of mice bearing IL-23/MA-891 tumors was increased as compared with those mice bearing parental or vector DNA-transfected tumors.