重组鼠IL-12腺病毒转染库普弗细胞对脾T淋巴细胞免疫功能调节的体外实验研究

发布时间：2018-05-06 19:13

本文选题：库普弗细胞 + 腺病毒载体　；参考：《苏州大学》2007年硕士论文

【摘要】： 目的:(1)分离纯化并鉴定SD大鼠库普弗细胞(Kupffer Cell,KC),得到较高纯度及活力的KC。(2)重组腺病毒Adv-IL-12体外转染KC,检测IL-12及MHC-Ⅰ类分子(H2-Kb)、MHC-Ⅱ类分子(I-Ab)的表达情况。(3)观察Adv-IL-12转染的KC对脾T细胞免疫功能的影响,为靶向治疗肿瘤肝内微转移的实验研究提供依据。方法: (1)采用Ⅳ型胶原酶、链霉蛋白酶E (PronaseE)灌注消化,Nycodenz密度梯度离心法得到SD大鼠肝脏非实质细胞(nonparenchymal cells,NPC),再以选择性贴壁法纯化KC。以台盼蓝拒染实验判断细胞产率和存活率;以细胞自发荧光现象、吞噬实验及ED-2单克隆抗体染色法对KC进行鉴定。(2)24孔细胞培养板中培养KC,细胞密度为1×106/ml,共设置3组,分别为KC细胞对照组(下称细胞对照组)、KC+Adv-EGFP病毒对照组(下称病毒对照组)和KC+Adv-IL-12试验组(下称实验组),每组设三个复孔。分别于转染后24h、48h收取3组细胞上清,以ELISA试剂盒检测细胞上清中IL-12的表达量;转染后48h收集各组细胞,以半定量RT-PCR法检测MHCⅠ类分子(H-2Kb)及MHCⅡ类分子(I-Ab)mRNA对应于内参照三磷酸甘油醛(GAPDH)的相对含量。(3)常规制备大鼠脾淋巴细胞(Splenic lymphocyte,SLC),调整密度为2×106/ml,加入ConA(终浓度为5μg/ml)刺激培养72小时后,与事先以重组IL-12腺病毒转染的KC混合培养,SLC与KC之比为10:1,分4组:SLC+Adv-IL-12-KC组(A组)、SLC+Adv-EGFP-KC组(B组)、SLC+KC组(C组)、单独SLC组(D组),每组设三个复孔。共同培养72h后,收集各孔培养上清及SLC,ELISA法测定培养上清中IFN-γ和IL-4蛋白的水平;以荧光标记的抗大鼠CD4、CD8单抗染色,流式细胞仪测定各组SLC中CD4T、CD8T细胞亚群变化。结果: (1)实验中每只SD大鼠可得到3.5-5.0×107个KC,细胞的存活率可达到93%以上;ED-2单抗染色鉴定,KC纯度达95%以上。(2)50 MOI剂量的重组腺病毒感染KC, ELISA检测:转染后24h,实验组上清IL-12浓度即明显升高,达对照组的10倍左右,48h测定IL-12浓度继续升高,实验组IL-12浓度可达1000pg/ml以上水平,与对照组比较差别具有显著统计学意义;而细胞对照组和病毒对照组KC也有一定的基础分泌,前者IL-12分泌量略低于后者,但差别不具有统计学意义。半定量RT-PCR法检测表明实验组H-2Kb /GAPDH和I-Ab /GAPDH比值分别为0.835,0.739,分别上调了1.5倍(H-2Kb)和2.0倍(I-Ab),与病毒对照组和细胞对照组比较差异有统计学意义,而病毒对照组和细胞对照组比较差异无统计学意义。(3)ELISA检测可见: A组、B组、C组和D组培养上清中IFN-γ含量依次为2026.29±103.37、988.55±94.39、1005.36±120.18、505.12±55.28(pg/mL),而IL-4含量依次为206.94±22.43、320.57±34.73、349.84±49.61、828.26±62.83(pg/mL)。FCM检测显示:A组SLC中CD4T细胞比例、T细胞总数(CD4T细胞和CD8T细胞之和)及CD4T/ CD8T比值均显著增大,与其他3组相比有显著差异,但CD8T细胞比例在各组间无明显差异;B组与C组、D组之间在各项指标上的差异不具有统计学意义;C组的CD4T细胞比例及T细胞总数高于D组,但两组CD8T细胞比例及CD4T/CD8T比值之间无明显差别。结论: (1)采用Ⅳ型胶原酶、PronaseE灌注消化,Nycodenz密度梯度离心及选择性贴壁法能够分离出较高纯度和活力的KC,能满足进一步的实验要求。(2)在体外腺病毒载体能有效的转染KC;IL-12基因修饰后,KC能高表达IL-12蛋白;其MHCⅠ和MHCⅡ分子基因转录增强,说明IL-12基因修饰能够上调KC的抗原提呈能力。(3)IL-12基因修饰的KC细胞与脾淋巴细胞混合培养,能显著增加混合淋巴细胞中CD4T细胞的比例,而对CD8T细胞无明显影响,结果导致CD4T/ CD8T比值增大;由IL-12诱导的CD4T细胞增殖主要分化为Th1细胞,培养上清中Th1型细胞因子IFN-γ的含量显著增加反映了这一变化,与此同时Th2方向的分化受到抑制,并表现为Th2型细胞因子IL-4分泌量的显著减少。
[Abstract]:Objective: (1) to isolate and purify and identify Kupffer Cell (KC) in SD rats, and to obtain KC. (2) recombinant adenovirus Adv-IL-12 with high purity and vigor to transfect KC in vitro, to detect the expression of IL-12 and MHC- class I molecules (H2-Kb), MHC- class II molecules (I-Ab). (3) observe the effect of transfection on the immune function of spleen cells. To provide evidence for the experimental study of tumor micrometastasis in liver.
Methods: (1) using type IV collagenase, streptomycin E (PronaseE) perfusion and digestion, Nycodenz density gradient centrifugation was used to obtain SD rat liver non parenchymal cells (nonparenchymal cells, NPC), and then purified KC. with trypan blue exclusion test to determine the cell yield and survival rate by selective adherence method, and the cell spontaneous fluorescence phenomenon, phagocytic experiment and ED-2. KC was identified by monoclonal antibody staining. (2) 24 hole cell culture plates were cultured for KC, and the cell density was 1 x 106/ml. A total of 3 groups were set up, respectively, the control group of KC cells (the lower cell control group), the KC+Adv-EGFP virus control group (the lower virus control group) and the KC+Adv-IL-12 test group (hereinafter referred to as the experimental group) with three compound holes in each group. 24 after transfection, respectively. H, 48h collected 3 groups of cell supernatants and detected the expression of IL-12 in the cell supernatant by ELISA kit. After transfection, 48h collected all groups of cells and detected the MHC class I molecules (H-2Kb) and MHC class II molecules (I-Ab) mRNA corresponding to the relative content of glyceraldehyde (GAPDH) of internal reference three phosphoric acid (GAPDH) by semi quantitative RT-PCR method. (3) normal rat spleen lymphocytes were prepared. Phocyte, SLC), the adjustment density was 2 x 106/ml, and the ConA (final concentration of 5 mu g/ml) was stimulated for 72 hours. The ratio of SLC to KC was 10:1, and the ratio of SLC to KC was 10:1, which was divided into 4 groups: SLC+Adv-IL-12-KC group (A group), individual group (Group), each group had three compound holes. Co culture After 72h, the level of IFN- gamma and IL-4 protein in culture supernatant was measured by the culture supernatant of each hole, and the level of IFN- gamma and IL-4 protein in the culture supernatant. The CD4T and CD8T cell subgroups of SLC in each group were measured by the fluorescent labeled anti CD4, CD8 mAb staining and the flow cytometry.
Results: (1) each SD rat could get 3.5-5.0 x 107 KC, the survival rate of the cell could reach more than 93%, and the purity of KC was above 95%. (2) the recombinant adenovirus infected with the 50 MOI dose KC, ELISA detection: 24h after transfection, the IL-12 concentration of the experimental group Rose obviously, reaching about 10 times of the control group and 48h determination IL-12 concentration. The concentration of IL-12 in the experimental group was up to 1000pg/ml, and the difference between the control group and the control group had significant statistical significance, while the cell control group and the virus control group had a certain basal secretion, the former IL-12 secretion was slightly lower than the latter, but the difference was not statistically significant. The semi quantitative RT-PCR test showed that the experimental group was H-2Kb /GAPD. The ratio of H and I-Ab /GAPDH was 0.835,0.739, respectively up 1.5 times (H-2Kb) and 2 times (I-Ab), compared with the virus control group and cell control group, but there was no significant difference between the virus control group and the cell control group. (3) ELISA detection could be seen in A group, B group, C group and D group. 2026.29 + 103.37988.55 + 94.391005.36 + 120.18505.12 + 55.28 (pg/mL), while the content of IL-4 was 206.94 + 22.43320.57 + 49.61828.26 + 62.83 (pg/mL).FCM. There was no significant difference in the proportion of CD8T cells in each group, but there was no statistical difference between group B and group C and group D in each index. The proportion of CD4T cells and the total number of T cells in group C were higher than that in D group, but there was no significant difference between the proportion of CD8T cells and the ratio of CD4T/CD8T in the two groups.
Conclusion: (1) the use of type IV collagenase, PronaseE perfusion and digestion, Nycodenz density gradient centrifugation and selective adherence can separate the higher purity and vitality of KC, and can meet further experimental requirements. (2) KC can be transfected effectively by adenovirus vector in vitro, and KC can express high expression of IL-12 protein after IL-12 gene modification; and its MHC I and MHC II molecular basis The IL-12 gene modification could increase the antigen presentation ability of KC. (3) the mixed culture of KC cells modified by IL-12 gene and spleen lymphocyte can significantly increase the proportion of CD4T cells in mixed lymphocytes, but have no obvious effect on CD8T cells, resulting in the increase of CD4T/ CD8T ratio, and the proliferation of CD4T cells induced by IL-12 is the main factor. A significant increase in the content of Th1 type cytokine IFN- gamma in the culture supernatant reflects this change, at the same time, the differentiation of Th2 direction is inhibited, and the secretion of Th2 type cytokine IL-4 is significantly reduced.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】