结核分枝杆菌重组Bb-ESAT-6-MPT64疫苗保护力及其免疫机制研究
发布时间：2018-06-08 17:56
本文选题：结核分枝杆菌 + rBb-ESAT-6-MPT64疫苗 ； 参考：《重庆医科大学》2008年硕士论文
【摘要】： 目的 用结核分枝杆菌重组Bb-ESAT-6-MPT64疫苗免疫BALB/c小鼠,比较各种途径接种疫苗,结核减毒株攻击后产生的免疫保护力及鼠脾细胞培养上清液IFN-γ、IL-12、TNF-α和IL-10的变化;动态观察rBb-ESAT-6-MPT64疫苗皮下注射接种和鼻腔粘膜接种BALB/c小鼠后,血清IgE、IgG及其亚类变化脾T淋巴细胞CD4+和CD8+亚群变化、脾细胞培养上清液IFN-γ、IL-12、TNF-α和IL-10的变化脾T淋巴细胞增殖产生的免疫应答。从而为TB的防治提供一种有价值的疫苗。 方法 为了研究rBb-ESAT-6-MPT64疫苗免疫BALB/c小鼠后,抗MTB攻击产生的免疫保护力,将65只雌性BALB/c小鼠随机分为5组,每组13只。A组: 5×106CFU rBb-ESAT-6-MPT64疫苗悬浮于100μl PBS于小鼠后背部皮下单次注射;B组:5×106CFU rBb-ESAT-6-MPT64疫苗悬浮于100μl PBS于小鼠后腿股四头肌肌肉注射;C组:5×105CFUrBb-ESAT-6-MPT64疫苗悬浮于10μl PBS于小鼠鼻腔粘膜单次接种;D组:5×106CFU Bb悬浮于100μl PBS于小鼠后背部皮下单次注射;E组:PBS皮下注射对照。5组小鼠免疫8w后用MTB H37Ra减毒株鼻腔粘膜攻击感染。在MTB攻击感染后6w,杀鼠取肝、肺作细菌负荷量测定;ELISA方法检测各组鼠血清特异性IgG、IgG1、IgG2a、IgG2b、IgG3和IgE抗体水平和各组鼠脾细胞体外培养原液及在受到MTBAg和ConA或LPS刺激后分别产生的IFN-γ、IL-12、TNF-α和IL-10的水平。 为了研究rBb-ESAT-6-MPT64疫苗皮下注射和鼻腔粘膜接种免疫BALB/c小鼠诱导的免疫应答类型及动态变化,将60只雌性BALB/c小鼠随机分为2组,每组30只。Ⅰ组:5×106CFU rBb-ESAT-6-MPT64疫苗悬浮于100μl PBS于小鼠后背部皮下单次注射;Ⅱ组:5×105CFU rBb-ESAT-6-MPT64疫苗悬浮于10μl PBS于小鼠鼻腔粘膜单次接种。于免疫前和免疫后2、4、6、8和10w分别剖杀4只小鼠,收集血清,ELISA方法检测各组鼠在免疫后不同时间血清特异性IgG、IgG1、IgG2a、IgG2b、IgG3和IgE抗体水平以及脾细胞体外培养原液及在受到MTBAg和ConA或LPS刺激后分别产生的IFN-γ、IL-12、TNF-α和IL-10的变化;用FCM检测各组鼠在免疫前和免疫后不同时间(免疫后2、4、6、8和10w)脾细胞CD4+和CD8+亚群的变化;用MTT比色法检测脾细胞增殖水平。 结果 rBb-ESAT-6-MPT64疫苗免疫BALB/c小鼠8w后用5×105CFU MTBH37Ra减毒株进行鼻腔粘膜内攻击感染,在H37Ra攻击感染后6w杀鼠取肝、肺作细菌负荷量测定。其结果:鼻腔粘膜接种组(IN)和皮下注射组(SC)明显低于PBS对照组(P0.01),鼻腔粘膜接种组(IN)也明显低于皮下注射组(SC),皮下注射组(SC)的肝、肺组织荷菌量低于肌肉注射组(IM)。 ELISA法检测抗体结果:A～C组中,小鼠IgG、IgG1、IgG2a和IgG2b水平均较对照组D、E组显著增高,IgG3和IgE水平均较对照组D、E组无显著差异;3组之间的相互比较:C组IgG、IgG1、IgG2a和IgG2b水平较A、B组显著增高,C组IgG3和IgE水平均较A、B组无显著差异,A、B组之间各抗体水平无显著差异。 ELISA法检测细胞因子变化结果:A～C组中,小鼠脾细胞原液组、MTBAg刺激组和ConA刺激组或LPS刺激组IFN-γ、IL-12和TNF-α水平均显著高于PBS对照组,A～C组中,小鼠脾细胞原液组、MTBAg刺激组和ConA刺激组IL-10水平均显著低于PBS对照组。rBb-ESAT-6-MPT64疫苗免疫BALB/c小鼠后ELISA法检测抗体结果: 皮下接种组IgG从免疫后2w开始升高,免疫后8w达最高水平,IgG1变化甚微,仅在免疫后8w时有所降低,IgG2a从免疫后2w开始升高,免疫后6w达最高水平,IgG2b从免疫后2w开始升高并且达到最高水平,IgG3免疫后2w开始降低,免疫后8w和10w最低,IgE免疫后2w开始降低,免疫后8w和10w最低;鼻腔粘膜接种组IgG从免疫后2w开始升高,免疫后8w达最高水平,IgG1变化甚微,仅在免疫后8w、10w时有所降低,IgG2a从免疫后2w开始升高,免疫后6w达最高水平,IgG2b从免疫后2w开始升高并且达到最高水平,IgG3免疫后2w开始降低,免疫后8w和10w最低,IgE免疫后2w开始降低,免疫后8w和10w最低。 用ELISA法检测细胞因子变化结果:IFN-γ水平皮下注射组从免疫后2w开始升高,在免疫后6w达到高峰,鼻腔粘膜接种组从免疫后2w开始升高,在免疫后6w和8w达到高峰;IL-12水平皮下注射组从免疫后2w开始升高,在免疫后4w达到高峰,鼻腔粘膜接种组从免疫后2w开始升高,在免疫后4w、6w和8w达到高峰;TNF-α水平皮下注射组从免疫后2w开始升高,在免疫后6w达到高峰,鼻腔粘膜接种组从免疫后2w开始升高,在免疫后6w和8w达到高峰;IL-10水平皮下注射组从免疫后2w开始升高,在免疫后6w和8w达到高峰,鼻腔粘膜接种组从免疫后2w开始升高,在免疫后8w达到高峰。 用FCM检测CD4+和CD8+亚群水平结果:皮下注射组和鼻腔粘膜接种组分别在免疫后2w和4、6w CD4+亚群水平分别较0w有统计学意义增加;皮下注射组和鼻腔粘膜接种组免疫后4w时CD8+亚群水平较0w增加,但无显著差异;两组之间在同一时间点两两比较,CD4+亚群水平中,皮下注射组较鼻腔粘膜接种组有所增加;CD8+亚群水平中,两组间同一时间无统计学意义。 用MTT法检测的小鼠脾淋巴细胞增殖结果:皮下注射组从免疫后2w开始升高,MTB Ag刺激组和ConA刺激培养组淋巴细胞增殖在2w达到高峰;鼻腔粘膜接种组从免疫后2w开始升高,MTB Ag刺激组淋巴细胞增殖在2w达到高峰,ConA刺激培养组淋巴细胞增殖在4w达到高峰。 结论 rBb-ESAT-6-MPT64疫苗免疫,H37Ra攻击感染后,小鼠肝、肺细菌负荷量明显降低;小鼠IgG、IgG1、IgG2a和IgG2b水平明显升高,IgG3和IgE水平变化不大,其中以鼻腔粘膜接种组的保护力最好。 rBb-ESAT-6-MPT64疫苗免疫加攻击组产生高水平的IFN-γ、IL-12和TNF-α,以及低水平的IL-10,提示产生较强的Th1细胞免疫应答。 rBb-ESAT-6-MPT64疫苗免疫BALB/c小鼠后产生高水平的IgG、IgG2a和IgG2b抗体,低水平的IgG1、IgG3和IgE,提示IgG、IgG2a和IgG2b可能参与体液免疫应答机制。 rBb-ESAT-6-MPT64疫苗能诱导BALB/c小鼠产生较强的特异性CD4+Th1型细胞免疫、体液免疫及微弱的CD8+CTL反应。 rBb-ESAT-6-MPT64疫苗鼻腔粘膜效果优于皮下注射和肌肉注射。
[Abstract]:objective
The recombinant Bb-ESAT-6-MPT64 vaccine of Mycobacterium tuberculosis was used to immunization BALB/c mice, to compare the immunization of various routes, the immune protection force and the changes of IFN- gamma, IL-12, TNF- alpha and IL-10 in the culture supernatant of the rat spleen cells, and the dynamic observation of rBb-ESAT-6-MPT64 vaccination and nasal mucosa inoculation of BALB/c mice. After that, changes in serum IgE, IgG and its subclasses? Changes in the CD4+ and CD8+ subgroups of splenic T lymphocytes, changes in IFN- gamma, IL-12, TNF- A and IL-10 in the supernatant of spleen cells, and the immune response to the proliferation of spleen T lymphocytes, thus providing a valuable vaccine for TB control.
Method
In order to study the immune protective effect of anti MTB attack on BALB/c mice immunized with rBb-ESAT-6-MPT64 vaccine, 65 female BALB/c mice were randomly divided into 5 groups with 13.A groups in each group: 5 x 106CFU rBb-ESAT-6-MPT64 vaccine was suspended in 100 mu L PBS in the dorsal subcutaneous subcutaneous injection of mice; B group: 5 x 106CFU rBb-ESAT-6-MPT64 vaccine was suspended in 100 micron. Intramuscular injection of four muscles of the hind leg femoris in mice; group C: 5 x 105CFUrBb-ESAT-6-MPT64 vaccine was suspended in 10 mu L PBS for single inoculation of nasal mucosa of mice; group D: 5 x 106CFU Bb suspended in 100 mu L PBS in the dorsal subcutaneous subcutaneous injection of mice; E group: PBS subcutaneous injection of the nasal mucosa of the mice. TB was used to attack 6W after infection, the rat liver was killed and the lung was measured, and the serum specific IgG, IgG1, IgG2a, IgG2b, IgG3 and IgE antibody levels were detected by ELISA method and the culture liquid of spleen cells in vitro and the levels of MTBAg and ConA or LPS stimulated respectively.
In order to study the immune response types and dynamic changes induced by immunization of rBb-ESAT-6-MPT64 vaccinated and nasal mucosal immunization BALB/c mice, 60 female BALB/c mice were randomly divided into 2 groups, 30 rats in each group. Group I: 5 x 106CFU rBb-ESAT-6-MPT64 vaccine was suspended in a single subcutaneous injection of 100 mu L PBS in the back of the rat back, and group II: 5 x 105CFU rBb- The ESAT-6-MPT64 vaccine was suspended in 10 mu L PBS for single inoculation in the nasal mucosa of mice. 4 mice were killed before and after immunization with 2,4,6,8 and 10W, and serum was collected respectively. The serum specific IgG, IgG1, IgG2a, IgG2b, IgG3 and IgE anti body levels, and the culture of spleen cells in vitro at different time after immunization were detected by ELISA method. The changes of IFN- gamma, IL-12, TNF- alpha and IL-10 were produced after Ag and ConA or LPS stimulation, and the changes in CD4+ and CD8+ subgroups of spleen cells before and after immunization were detected by FCM, and the proliferation of splenocytes was detected by colorimetric assay.
Result
RBb-ESAT-6-MPT64 Vaccine Immunized BALB/c mice 8W with 5 x 105CFU MTBH37Ra attenuated strain to attack the nasal mucous infection. After H37Ra attack, the liver was taken to take the liver and the bacterial load was measured in the lungs. The results showed that the nasal mucosa inoculation group (IN) and the subcutaneous injection group (SC) were significantly lower than the PBS control group (P0.01), and the nasal mucosa inoculation group (IN) was also obvious. Compared with subcutaneous injection group (SC), the amount of bacteria in lung tissue of subcutaneous injection group (SC) was lower than that in muscle injection group (IM).
In group A to C, the levels of IgG, IgG1, IgG2a and IgG2b in the group of mice were all higher than the control group D, and the E group was significantly higher than that of the control group. There was no significant difference between the IgG3 and IgE levels. The comparison between the 3 groups was significantly higher than that between the 3 groups. There was no significant difference in the level of each antibody.
In the group of A to C, the level of IFN- gamma, IL-12 and TNF- alpha in the group of splenocytes, MTBAg and LPS stimulation group were significantly higher than that of the PBS control group in the group of the spleen cells of the splenocytes, the ConA stimulation group and the LPS stimulation group, and the level of the spleen cell fluid group, the stimulation group and the stimulation group were significantly lower than those of the control group in the group of the spleen cells of the splenocytes, the ConA stimulation group and the ConA stimulation group. After immunization with BALB/c mice, the antibody was detected by ELISA method.
The subcutaneous inoculation group IgG began to increase from 2W after immunization, and the 8W reached the highest level after immunization, and the IgG1 changed slightly, only 8W decreased after immunization. IgG2a began to rise from 2W after immunization. 6W reached the highest level after immunization. IgG2b began to rise and reached the highest level after immune 2W. 2W began to decrease after IgG3 immunization. 2W began to decrease and 8W and 10W were lowest after immunization, and IgG increased from 2W after immunization. 8W reached the highest level after immunization. IgG1 changed slightly, only 8W, 10W decreased after immunization. IgG2a began to rise after immunization, and 6W reached the highest level after immunization. After 2W began to decrease, 8W and 10W were the lowest after immunization, 2W began to decrease after IgE immunization, and 8W and 10W were lowest after immunization.
The results of cytokine changes were detected by ELISA: IFN- gamma level in the subcutaneous injection group began to rise from 2W after immunization, and reached the peak of 6W after immunization. The nasal mucosa inoculation group began to rise from 2W after immunization, and reached the peak of 6W and 8W after immunization; IL-12 level subcutaneous injection group began to rise from immune 2W, and reached the peak in 4W after immunization, and the nasal mucosa was connected to the nasal mucosa. The group began to rise from 2W after immunization, and reached the peak of 4W, 6W and 8W after immunization. The TNF- alpha subcutaneous injection group began to rise from 2W after immunization, and reached the peak in 6W after immunization. The nasal mucosa inoculated group began to rise from the immune 2W, and the 6W and 8W reached the peak after immunization; IL-10 level subcutaneous injection group began to increase from immunization 2W, and after immunization. 8W reached its peak. The nasal mucosa inoculation group began to rise from 2W after immunization and reached its peak after 8W.
The results of CD4+ and CD8+ subgroup levels were detected by FCM: the levels of 2W and 4,6w CD4+ subgroups in the subcutaneous injection group and the nasal mucosa inoculation group were significantly higher than those of the 0W, respectively. The level of CD8+ subgroup in the subcutaneous injection group and the nasal mucosa inoculated group was higher than that of 0W, but there was no significant difference between the two groups at the same time point 22. Compared with the CD4+ subgroup level, the subcutaneous injection group increased compared with the nasal mucosa inoculation group, and there was no significant difference in the CD8+ subgroup level between the two groups at the same time.
The proliferation of spleen lymphocyte in mice was detected by MTT method: the subcutaneous injection group began to increase from 2W after immunization. The lymphocyte proliferation in the MTB Ag stimulation group and the ConA stimulation group reached the peak of 2W, and the nasal mucosa inoculation group began to increase from the 2W after immunization, and the lymphocyte proliferation in MTB Ag stimulation group reached the peak in 2W and ConA stimulated the lymphocyte culture group. The proliferation of 4W reaches the peak.
conclusion
After immunization with rBb-ESAT-6-MPT64 vaccine, after H37Ra attack, the load of liver and lung bacteria in mice decreased obviously; the level of IgG, IgG1, IgG2a and IgG2b in mice increased obviously, and the level of IgG3 and IgE changed little, and the protective ability of the nasal mucosa inoculation group was the best.
The rBb-ESAT-6-MPT64 vaccine plus immunization group produced high levels of IFN- gamma, IL-12 and TNF- alpha, and low levels of IL-10, suggesting a strong Th1 cell immune response.
RBb-ESAT-6-MPT64 vaccines immunized BALB/c mice to produce high levels of IgG, IgG2a and IgG2b antibodies, low levels of IgG1, IgG3 and IgE, suggesting that IgG, IgG2a, and IgG2b may be involved in the humoral immune response mechanism.
RBb-ESAT-6-MPT64 vaccine can induce specific CD4+Th1 cell immunity, humoral immunity and weak CD8+CTL reaction in BALB/c mice.
The efficacy of rBb-ESAT-6-MPT64 vaccine in nasal mucosa is superior to subcutaneous injection and intramuscular injection.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
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