蛔虫感染小鼠前体树突状细胞免疫刺激活性的实验研究

发布时间：2018-05-21 08:35

本文选题：骨髓细胞 + 前体树突状细胞　；参考：《南昌大学》2007年硕士论文

【摘要】： 目的:通过研究蛔虫感染小鼠骨髓前体树突状细胞(precursor dendritic cells,pDCs)刺激淋巴细胞增殖的作用及其细胞毒T淋巴细胞的毒性作用,探讨蛔虫感染小鼠pDCs的免疫刺激活性。方法: 1.动物模型的建立:用灌胃方法建立人蛔虫-C57BL/6小鼠感染模型,按1000个/只剂量。 2.实验分组:A—感染组DCs与感染组脾淋巴细胞混合培养;B—感染组DCs与未感染组脾淋巴细胞混合培养;C—未感染组DCs与感染组脾淋巴细胞混合培养; E—用ABF冲击未感染组的DCs后与未感染组脾淋巴细胞混合培养;F—用B16肿瘤抗原冲击未感染组的DCs后与未感染组脾淋巴细胞混合培养;并设立一对照组D—未感染组DCs与未感染组脾淋巴细胞混合培养。通过MTT法检测脾淋巴细胞增殖情况。共分为感染后1、11、34d三个时间段。 3.DC的诱导培养及鉴定:无菌条件下制备小鼠骨髓细胞(详见全文)。培养体系中加入诱导分化因子培养其成为DCs,倒置显微镜下观察DCs的生长情况。用流式细胞术检测DCs表达CD80的情况,电镜观察DCs细胞形态。 4.脾细胞的分离培养培养:常规无菌状态(详细见全文)制备小鼠脾细胞,调整细胞密度为1×106/ml,置37℃,5%CO2培养箱中培养。 5.CTL细胞因子的表达:CTL用ELISA方法检测培养72h后收集的CTL上清中IL-2,TNF-α,IFN-γ, IL-4,IL-10,IL-5的表达量。 6.绘制B16细胞生长曲线:取对数生长期的B16细胞常规消化计数,准确将细胞分别接种于24孔培养板中,每日取3孔计数,连续观察7d。掌握靶细胞基本生长规律。 7.CTL对B16细胞的影响:将CTL作为效应细胞,B16细胞作为靶细胞,通过MTT法检测CTL对小鼠黑色素瘤细胞株(B16)的影响。结果: 1. DCs培养不同阶段细胞分化程度和形态各异,培养7d后,细胞表面可见明显的树突状突起。透射电镜可见典型的DCs的形态特征。同时流式细胞术检测CD80处于高表达状态。其中感染小鼠CD80的表达为龴74.53%±6.42%㖞,未感染小鼠CD80的表达为(50.12%±3.25%)。 2.细胞增值情况在感染后1d, B组OD值高于D组;在感染后11d,B组OD值稍低于D组;在感染后34d,B组OD值稍高于D组。在三个时间段的任一时间段B、D组间OD值无显著性差异,在不同的时间段各组OD值具有显著性差异。 3.细胞因子分泌情况,在感染后1d、11d B组CTL分泌的IL-4高于D组;在感染后34d,B组CTL分泌的IL-4低于D组。在感染后1d ,B组CTL分泌的IFN-γ高于D组;感染后11d、34d D组CTL分泌的IFN-γ高于B组。在不同时间段以及在同一时间段各组CTL分泌的IFN-γ和IL-4有显著性的差异。但在各时间段未检测出CTL有IL-2,IL-5,IL-10,TNF-α细胞因子的分泌。 4. B16细胞从第2d开始进入对数生长期,在第5d生长最为旺盛,之后增殖减慢逐渐进入平台期。 5.蛔虫感染34天时各组CTL对B16细胞的生长无抑制作用。结论: ①受感染小鼠pDCs具有免疫刺激的作用。 ②受感染小鼠pDCs能够刺激脾淋巴细胞增殖,随感染条件不同增殖率不同。 ③受感染小鼠pDCs诱导的CTL分泌的细胞因子IFN-γ和IL-4,随感染条件不同表达量不同。 ④本实验中,蛔虫感染小鼠34dpDCs诱导的CTL对靶细胞B16细胞株的生长无抑制作用。
[Abstract]:Objective: To investigate the effect of precursor dendritic cells (pDCs) on lymphocyte proliferation and the toxicity of cytotoxic T lymphocytes in mice infected with Ascaris infection, and to explore the immune activity of pDCs in mice infected with Ascaris.
Method:
1. establishment of animal model: -C57BL/6 mouse infection model of Ascaris Ascaris was established by intragastric administration, according to 1000 doses per dose.
2. experimental groups: A - infection group DCs and infected group splenic lymphocyte mixed culture; B - infection group DCs and uninfected group splenic lymphocyte mixed culture; C - uninfected group DCs and infection group splenic lymphocyte mixed culture; E - E with ABF impact uninfected group and uninfected group splenic lymphocyte mixed culture; F - B16 tumor antigen impact The splenic lymphocytes in the uninfected group were mixed with the uninfected group, and a control group of D uninfected group DCs was mixed with the uninfected group of spleen lymphocytes. The splenic lymphocyte proliferation was detected by MTT method. The 1,11,34d three period after infection was divided into three periods after infection.
3.DC induced culture and identification: the mouse bone marrow cells were prepared under aseptic conditions (detailed in full text). In the culture system, the differentiation factor was induced to become DCs. The growth of DCs was observed under the inverted microscope. The expression of CD80 in DCs was detected by flow cytometry and the morphology of DCs cells was observed by electron microscope.
4. the isolation and culture of spleen cells: the mice splenocytes were prepared in the normal aseptic state (in detail), the cell density was 1 x 106/ml, and the cells were incubate in the 5%CO2 culture box at 37.
5.CTL cytokine expression: CTL was used to detect IL-2, TNF- alpha, IFN-, IL-4, IL-10, and IL-5 expression in CTL supernatants collected after culture 72h by ELISA.
6. plot B16 cell growth curve: take the regular digestibility count of B16 cells in logarithmic growth period, inoculate the cells in 24 hole culture plate accurately, take 3 holes per day and observe the basic growth rule of target cells continuously by 7d..
Effects of 7.CTL on B16 cells: CTL was used as the effector cell, B16 cells as target cells, and the effect of CTL on mouse melanoma cell line (B16) was detected by MTT.
Result:
1. DCs culture at different stages of cell differentiation and morphology, after culture 7d, the cell surface visible dendritic protuberance. Transmission electron microscope can see the typical morphological characteristics of DCs. Meanwhile, flow cytometry detected the high expression of CD80. The expression of CD80 in infected mice was 74.53% + 6.42%? And the expression of CD80 in the uninfected mice was (50.12) % + 3.25%).
The value of 2. cell increment was higher than that of group D after infection, and the OD value of group B was higher than that of group D after infection. After infection 11d, the OD value of B group was slightly lower than that of D group; after infection 34d, the OD value of B group was slightly higher than that of D group. There was no significant difference between B and D groups at any time period of three periods, and there was significant difference in each group of OD values at different time periods.
The secretion of 3. cell factor was higher than that of D group in 1D, 11d B group after infection, and the IL-4 of CTL secreted in 34d, B group was lower than that in the D group after infection. After infection, the secretory gamma secreted by the group was higher than that in the group. Significant differences were observed. However, CTL did not show IL-2, IL-5, IL-10 and TNF- alpha cytokine secretion at any time.
4. B16 cells entered the logarithmic growth phase from 2D, the most vigorous growth at 5D, and then gradually slowed down to platform stage.
5. CTL on the 34 day of Ascaris infection did not inhibit the growth of B16 cells.
(1) the infected mice pDCs has the effect of immune stimulation.
(2) pDCs of infected mice can stimulate the proliferation of splenic lymphocytes.
(3) cytokine IFN- gamma and IL-4 secreted by CTL induced by pDCs in infected mice were different depending on the infection conditions.
(4) in this experiment, 34dpDCs induced CTL in Ascaris infected mice had no inhibitory effect on the growth of B16 cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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