本文選題：結(jié)核分枝桿菌　切入點(diǎn)：H37Ra　出處：《重慶醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:1.探討結(jié)核分枝桿菌H37Ra與有機(jī)硒聯(lián)合免疫小鼠后,細(xì)菌在小鼠體內(nèi)定植的情況,以及誘導(dǎo)機(jī)體產(chǎn)生的特異性細(xì)胞免疫功能。2.構(gòu)建可表達(dá)結(jié)核分枝桿菌分泌蛋白Ag85B的靶細(xì)胞,并研究結(jié)核分枝桿菌H37Ra與有機(jī)硒聯(lián)合免疫小鼠后的特異性細(xì)胞毒作用及其免疫機(jī)制。 方法:1.將C57BL/6小鼠分為6組(每組12只),即⑴H37Ra+Se組、⑵BCG+Se組、⑶H37Ra組、⑷BCG組、⑸Se組和⑹PBS對(duì)照組。小鼠連續(xù)灌服有機(jī)硒1周后,采用皮內(nèi)注射方式接種H37Ra或BCG,然后繼續(xù)喂硒4周。免疫4周和8周后,檢測(cè)小鼠臟器指數(shù)及結(jié)核桿菌在臟器內(nèi)的定植情況。2.分離各組小鼠脾淋巴細(xì)胞,體外用PPD刺激,MTT法檢測(cè)脾淋巴細(xì)胞的刺激指數(shù)(Stimulation Index,SI);ELISA法檢測(cè)培養(yǎng)上清液中IFN-γ、IL-2的產(chǎn)量。3.構(gòu)建可表達(dá)結(jié)核分枝桿菌分泌蛋白Ag85B的靶細(xì)胞,MTT法檢測(cè)免疫小鼠脾淋巴細(xì)胞的殺傷活性。4.免疫8周后,RT-PCR法檢測(cè)脾淋巴細(xì)胞中穿孔素、顆粒酶B mRNA的表達(dá)。 結(jié)果:1.免疫4周或8周后,H37Ra+Se組的脾指數(shù)、胸腺指數(shù)和脾肺荷菌量略高于BCG+Se組、H37Ra組,但差異無顯著性(P0.05)。2. H37Ra+Se組的SI、IL-2和IFN-γ均顯著高于H37Ra組(P0.05),但與BCG+Se組相比,IFN-γ有顯著性差異,其他指標(biāo)無顯著性差異(P0.05);隨著時(shí)間的延長(zhǎng),各項(xiàng)指標(biāo)均下降。3.轉(zhuǎn)染陽(yáng)性細(xì)胞中有結(jié)核分枝桿菌分泌蛋白Ag85B的表達(dá),H37Ra+Se組脾淋巴細(xì)胞的殺傷活性顯著高于H37Ra組(P0.05),略高于BCG+Se組(P0.05)。4. H37Ra+Se組小鼠穿孔素及顆粒酶B mRNA的表達(dá)量均顯著高于PBS組(P0.05),略高于BCG+Se組(P0.05);H37Ra+Se組與H37Ra組比較,穿孔素mRNA的表達(dá)量有顯著性差異(P0.05),而顆粒酶B mRNA的表達(dá)量,前者略多于后者,但無統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論:1. H37Ra和有機(jī)硒聯(lián)合免疫小鼠后結(jié)核菌能在小鼠脾臟、肺臟定植至少60天。2. H37Ra和有機(jī)硒聯(lián)合免疫小鼠后誘導(dǎo)機(jī)體產(chǎn)生的細(xì)胞免疫效果顯著優(yōu)于H37Ra或BCG單獨(dú)免疫,略優(yōu)于BCG和有機(jī)硒聯(lián)合免疫。3.構(gòu)建表達(dá)結(jié)核分枝桿菌分泌蛋白Ag85B的靶細(xì)胞及MTT法檢測(cè)特異性殺傷活性,為研究結(jié)核疫苗免疫的細(xì)胞毒作用提供了較好的研究手段。4. H37Ra和有機(jī)硒聯(lián)合免疫小鼠后可以誘導(dǎo)脾淋巴細(xì)胞的殺傷活性,其殺傷機(jī)制可能與細(xì)胞中穿孔素、顆粒酶B mRNA表達(dá)增加有關(guān)。
[Abstract]:Objective 1. To investigate the colonization of mycobacterium tuberculosis (H37Ra) combined with organic selenium in mice and the specific cellular immune function induced by mycobacterium tuberculosis. 2. To construct the target cells expressing the secreting protein Ag85B of Mycobacterium tuberculosis. The specific cytotoxic effect and immune mechanism of mice immunized with mycobacterium tuberculosis H37Ra and organic selenium were studied. Methods: 1. C57BL/6 mice were divided into 6 groups (12 rats in each group, I. e., 1H37Ra se group, 2 BCG se group, 3 BCG group, 4 BCG group, 5 se group and 6PBS control group). After one week of continuous administration of organic selenium, the mice were given organic selenium for 1 week. The mice were inoculated with H37Ra or BCG by intradermal injection, and then given selenium for 4 weeks. After immunization for 4 weeks and 8 weeks, the viscera index and the colonization of Mycobacterium tuberculosis in the organs were detected. The stimulation index of splenic lymphocytes was detected by PPD stimulation assay in vitro. Elisa was used to detect the production of IL-2 in culture supernatant. The target cells expressing Ag85B secreted by Mycobacterium tuberculosis were constructed to detect the spleen lymphocytes of immunized mice. After 8 weeks of immunization, the perforin in splenic lymphocytes was detected by RT-PCR. Expression of granzyme B mRNA. Results 1. The spleen index, thymus index and the amount of splenic and pulmonary bacilli in H37Ra se group were slightly higher than those in BCG se group, but there was no significant difference between H37Ra se group and H37Ra group, but there was significant difference between H37Ra se group and BCG se group, but there was a significant difference between H37Ra se group and H37Ra group, but there was a significant difference between H37Ra se group and BCG se group, but there was no significant difference between H37Ra se group and BCG se group, but there was no significant difference between H37Ra se group and H37Ra group (P 0.05), but there was no significant difference between H37Ra se group and BCG se group, but there was no significant difference between H37Ra se group and H37Ra group. There was no significant difference in other indexes (P 0.05). The activity of splenic lymphocytes in H37Ra se group was significantly higher than that in H37Ra group, and slightly higher than that in BCG se group. 4. Perforin and granzyme B in H37Ra se group. The expression of mRNA was significantly higher than that of PBS group (P 0.05) and slightly higher than that of BCG se group (P 0.05) and H37Ra se group compared with H37Ra group. The expression of perforin mRNA was significantly different (P 0.05), while the expression of granzyme B mRNA was slightly higher in the former than in the latter, but there was no significant difference between the two groups. Conclusion H37Ra combined with organic selenium can immunize mice with tuberculous bacillus at least 60 days after inoculation of mice spleen and lung. The cellular immunity of mice immunized with H37Ra and organic selenium is better than that of H37Ra or BCG alone. The target cells expressing the secreting protein Ag85B of Mycobacterium tuberculosis were constructed and the specific killing activity was detected by MTT method. 4. H37Ra and organoselenium combined immunized mice can induce the killing activity of spleen lymphocytes, which may be related to the cytotoxicity of perforin in cells. The increased expression of granzyme B mRNA was related to the expression of granzyme
【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】：2007
【分類號(hào)】：R392

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