本文選題：結(jié)核分枝桿菌 + rBb-ESAT-6-MPT64疫苗��； 參考：《重慶醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的 用結(jié)核分枝桿菌重組Bb-ESAT-6-MPT64疫苗免疫BALB/c小鼠,比較各種途徑接種疫苗,結(jié)核減毒株攻擊后產(chǎn)生的免疫保護(hù)力及鼠脾細(xì)胞培養(yǎng)上清液IFN-γ、IL-12、TNF-α和IL-10的變化;動(dòng)態(tài)觀察rBb-ESAT-6-MPT64疫苗皮下注射接種和鼻腔粘膜接種BALB/c小鼠后,血清IgE、IgG及其亞類變化脾T淋巴細(xì)胞CD4+和CD8+亞群變化、脾細(xì)胞培養(yǎng)上清液IFN-γ、IL-12、TNF-α和IL-10的變化脾T淋巴細(xì)胞增殖產(chǎn)生的免疫應(yīng)答。從而為TB的防治提供一種有價(jià)值的疫苗。 方法 為了研究rBb-ESAT-6-MPT64疫苗免疫BALB/c小鼠后,抗MTB攻擊產(chǎn)生的免疫保護(hù)力,將65只雌性BALB/c小鼠隨機(jī)分為5組,每組13只。A組: 5×106CFU rBb-ESAT-6-MPT64疫苗懸浮于100μl PBS于小鼠后背部皮下單次注射;B組:5×106CFU rBb-ESAT-6-MPT64疫苗懸浮于100μl PBS于小鼠后腿股四頭肌肌肉注射;C組:5×105CFUrBb-ESAT-6-MPT64疫苗懸浮于10μl PBS于小鼠鼻腔粘膜單次接種;D組:5×106CFU Bb懸浮于100μl PBS于小鼠后背部皮下單次注射;E組:PBS皮下注射對(duì)照。5組小鼠免疫8w后用MTB H37Ra減毒株鼻腔粘膜攻擊感染。在MTB攻擊感染后6w,殺鼠取肝、肺作細(xì)菌負(fù)荷量測定;ELISA方法檢測各組鼠血清特異性IgG、IgG1、IgG2a、IgG2b、IgG3和IgE抗體水平和各組鼠脾細(xì)胞體外培養(yǎng)原液及在受到MTBAg和ConA或LPS刺激后分別產(chǎn)生的IFN-γ、IL-12、TNF-α和IL-10的水平。 為了研究rBb-ESAT-6-MPT64疫苗皮下注射和鼻腔粘膜接種免疫BALB/c小鼠誘導(dǎo)的免疫應(yīng)答類型及動(dòng)態(tài)變化,將60只雌性BALB/c小鼠隨機(jī)分為2組,每組30只。Ⅰ組:5×106CFU rBb-ESAT-6-MPT64疫苗懸浮于100μl PBS于小鼠后背部皮下單次注射;Ⅱ組:5×105CFU rBb-ESAT-6-MPT64疫苗懸浮于10μl PBS于小鼠鼻腔粘膜單次接種。于免疫前和免疫后2、4、6、8和10w分別剖殺4只小鼠,收集血清,ELISA方法檢測各組鼠在免疫后不同時(shí)間血清特異性IgG、IgG1、IgG2a、IgG2b、IgG3和IgE抗體水平以及脾細(xì)胞體外培養(yǎng)原液及在受到MTBAg和ConA或LPS刺激后分別產(chǎn)生的IFN-γ、IL-12、TNF-α和IL-10的變化;用FCM檢測各組鼠在免疫前和免疫后不同時(shí)間(免疫后2、4、6、8和10w)脾細(xì)胞CD4+和CD8+亞群的變化;用MTT比色法檢測脾細(xì)胞增殖水平。 結(jié)果 rBb-ESAT-6-MPT64疫苗免疫BALB/c小鼠8w后用5×105CFU MTBH37Ra減毒株進(jìn)行鼻腔粘膜內(nèi)攻擊感染,在H37Ra攻擊感染后6w殺鼠取肝、肺作細(xì)菌負(fù)荷量測定。其結(jié)果:鼻腔粘膜接種組(IN)和皮下注射組(SC)明顯低于PBS對(duì)照組(P0.01),鼻腔粘膜接種組(IN)也明顯低于皮下注射組(SC),皮下注射組(SC)的肝、肺組織荷菌量低于肌肉注射組(IM)。 ELISA法檢測抗體結(jié)果:A～C組中,小鼠IgG、IgG1、IgG2a和IgG2b水平均較對(duì)照組D、E組顯著增高,IgG3和IgE水平均較對(duì)照組D、E組無顯著差異;3組之間的相互比較:C組IgG、IgG1、IgG2a和IgG2b水平較A、B組顯著增高,C組IgG3和IgE水平均較A、B組無顯著差異,A、B組之間各抗體水平無顯著差異。 ELISA法檢測細(xì)胞因子變化結(jié)果:A～C組中,小鼠脾細(xì)胞原液組、MTBAg刺激組和ConA刺激組或LPS刺激組IFN-γ、IL-12和TNF-α水平均顯著高于PBS對(duì)照組,A～C組中,小鼠脾細(xì)胞原液組、MTBAg刺激組和ConA刺激組IL-10水平均顯著低于PBS對(duì)照組。rBb-ESAT-6-MPT64疫苗免疫BALB/c小鼠后ELISA法檢測抗體結(jié)果: 皮下接種組IgG從免疫后2w開始升高,免疫后8w達(dá)最高水平,IgG1變化甚微,僅在免疫后8w時(shí)有所降低,IgG2a從免疫后2w開始升高,免疫后6w達(dá)最高水平,IgG2b從免疫后2w開始升高并且達(dá)到最高水平,IgG3免疫后2w開始降低,免疫后8w和10w最低,IgE免疫后2w開始降低,免疫后8w和10w最低;鼻腔粘膜接種組IgG從免疫后2w開始升高,免疫后8w達(dá)最高水平,IgG1變化甚微,僅在免疫后8w、10w時(shí)有所降低,IgG2a從免疫后2w開始升高,免疫后6w達(dá)最高水平,IgG2b從免疫后2w開始升高并且達(dá)到最高水平,IgG3免疫后2w開始降低,免疫后8w和10w最低,IgE免疫后2w開始降低,免疫后8w和10w最低。 用ELISA法檢測細(xì)胞因子變化結(jié)果:IFN-γ水平皮下注射組從免疫后2w開始升高,在免疫后6w達(dá)到高峰,鼻腔粘膜接種組從免疫后2w開始升高,在免疫后6w和8w達(dá)到高峰;IL-12水平皮下注射組從免疫后2w開始升高,在免疫后4w達(dá)到高峰,鼻腔粘膜接種組從免疫后2w開始升高,在免疫后4w、6w和8w達(dá)到高峰;TNF-α水平皮下注射組從免疫后2w開始升高,在免疫后6w達(dá)到高峰,鼻腔粘膜接種組從免疫后2w開始升高,在免疫后6w和8w達(dá)到高峰;IL-10水平皮下注射組從免疫后2w開始升高,在免疫后6w和8w達(dá)到高峰,鼻腔粘膜接種組從免疫后2w開始升高,在免疫后8w達(dá)到高峰。 用FCM檢測CD4+和CD8+亞群水平結(jié)果:皮下注射組和鼻腔粘膜接種組分別在免疫后2w和4、6w CD4+亞群水平分別較0w有統(tǒng)計(jì)學(xué)意義增加;皮下注射組和鼻腔粘膜接種組免疫后4w時(shí)CD8+亞群水平較0w增加,但無顯著差異;兩組之間在同一時(shí)間點(diǎn)兩兩比較,CD4+亞群水平中,皮下注射組較鼻腔粘膜接種組有所增加;CD8+亞群水平中,兩組間同一時(shí)間無統(tǒng)計(jì)學(xué)意義。 用MTT法檢測的小鼠脾淋巴細(xì)胞增殖結(jié)果:皮下注射組從免疫后2w開始升高,MTB Ag刺激組和ConA刺激培養(yǎng)組淋巴細(xì)胞增殖在2w達(dá)到高峰;鼻腔粘膜接種組從免疫后2w開始升高,MTB Ag刺激組淋巴細(xì)胞增殖在2w達(dá)到高峰,ConA刺激培養(yǎng)組淋巴細(xì)胞增殖在4w達(dá)到高峰。 結(jié)論 rBb-ESAT-6-MPT64疫苗免疫,H37Ra攻擊感染后,小鼠肝、肺細(xì)菌負(fù)荷量明顯降低;小鼠IgG、IgG1、IgG2a和IgG2b水平明顯升高,IgG3和IgE水平變化不大,其中以鼻腔粘膜接種組的保護(hù)力最好。 rBb-ESAT-6-MPT64疫苗免疫加攻擊組產(chǎn)生高水平的IFN-γ、IL-12和TNF-α,以及低水平的IL-10,提示產(chǎn)生較強(qiáng)的Th1細(xì)胞免疫應(yīng)答。 rBb-ESAT-6-MPT64疫苗免疫BALB/c小鼠后產(chǎn)生高水平的IgG、IgG2a和IgG2b抗體,低水平的IgG1、IgG3和IgE,提示IgG、IgG2a和IgG2b可能參與體液免疫應(yīng)答機(jī)制。 rBb-ESAT-6-MPT64疫苗能誘導(dǎo)BALB/c小鼠產(chǎn)生較強(qiáng)的特異性CD4+Th1型細(xì)胞免疫、體液免疫及微弱的CD8+CTL反應(yīng)。 rBb-ESAT-6-MPT64疫苗鼻腔粘膜效果優(yōu)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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