本文選題：弓形蟲 + 排泄-分泌抗原��； 參考：《山西醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的本研究首先篩選體外制備弓形蟲排泄-分泌抗原(excreted/secreted antigen,ESA)的最適條件,再觀察兩種不同來源的弓形蟲ESA(即Vero細(xì)胞體外培養(yǎng)弓形蟲獲取的ESA和感染鼠腹水中分離的ESA)以及可溶性速殖子抗原(soluble tachyzoite antigen,STAg)鼻內(nèi)免疫小鼠誘導(dǎo)的黏膜及系統(tǒng)免疫應(yīng)答及其抗弓形蟲感染的能力。探討三種抗原鼻內(nèi)免疫誘導(dǎo)的免疫應(yīng)答及抗感染機(jī)制,為弓形蟲鼻內(nèi)復(fù)合疫苗的研制奠定理論基礎(chǔ)。 方法本研究分三部分:第一部分觀察弓形蟲與Vero細(xì)胞不同共培養(yǎng)條件對ESA含量的影響。首先觀察不同血清濃度及含血清培養(yǎng)時(shí)間對ESA蛋白濃度的影響。按血清濃度0.5%、1%、2%、4%分4組,組內(nèi)又按含血清培養(yǎng)時(shí)間(3h、6h、12h、24h)分4個(gè)時(shí)間點(diǎn)。弓形蟲速殖子與Vero細(xì)胞分別在含不同濃度血清的培養(yǎng)基中共培養(yǎng),并分別于3h、6h、12h或24h后改為無血清培養(yǎng)基繼續(xù)培養(yǎng),第7d收集培養(yǎng)上清液并檢測蛋白含量。其次觀察共培養(yǎng)時(shí)間及含血清培養(yǎng)時(shí)間對ESA蛋白濃度的影響。按共培養(yǎng)時(shí)間(7d、9d、11d、13d)分為4組,組內(nèi)又按不同的含血清培養(yǎng)時(shí)間(12h、24h、48h、72h)分4個(gè)時(shí)間點(diǎn)。弓形蟲速殖子與Vero細(xì)胞在含1%血清的培養(yǎng)基中共培養(yǎng),各組分別于培養(yǎng)12h、24h、48h、72h后改為無血清培養(yǎng)基繼續(xù)培養(yǎng),并分別于第7d、9d、11d、13d收集培養(yǎng)上清液,檢測蛋白含量。 第二部分觀察兩種不同來源的ESA及STAg鼻內(nèi)免疫小鼠誘導(dǎo)的黏膜及系統(tǒng)免疫應(yīng)答。BALB/c小鼠隨機(jī)分為4組,分別用PBS 20μl/只、體外ESA、腹腔ESA或STAg各20μg/只鼻內(nèi)免疫2次,間隔14d。分別于末次免疫后14d和44d處死,計(jì)數(shù)腸上皮內(nèi)淋巴細(xì)胞(intestinal intraepithelial lymphocytes,IEL)和脾淋巴細(xì)胞,檢測血清IgG和腸液sIgA。 第三部分觀察兩種不同來源的ESA及STAg鼻內(nèi)免疫小鼠誘導(dǎo)的抗弓形蟲作用。BALB/c小鼠隨機(jī)分為4組,分別用PBS 20μl/只、體外ESA、腹腔ESA和STAg各20μg/只鼻內(nèi)免疫2次,間隔14d。末次免疫后14d用1×10~4個(gè)速殖子/只灌胃攻擊,觀察小鼠健康狀況及體重變化,攻擊后30d處死小鼠,計(jì)數(shù)脾、腦內(nèi)速殖子。 結(jié)果弓形蟲速殖子與Vero細(xì)胞在不同培養(yǎng)條件中共培養(yǎng),培養(yǎng)基中血清濃度為1%、共培養(yǎng)12h或24h改為無血清培養(yǎng)基所提取的ESA蛋白含量明顯高于其它培養(yǎng)條件(P＜0.05)。12h或24h改為無血清培養(yǎng)基并在共培養(yǎng)第13d收集培養(yǎng)上清液獲取的ESA蛋白含量顯著高于其它時(shí)間點(diǎn)(P＜0.001)。 體外ESA、腹腔ESA和STAg鼻內(nèi)免疫小鼠后,腹腔ESA組小鼠狀態(tài)欠佳,其它各組小鼠健康狀況良好。末次免疫后14d,各抗原組脾淋巴細(xì)胞(P＜0.05)及IEL(P＜0.01)均增殖活躍,至免疫后44d,兩種ESA組脾淋巴細(xì)胞數(shù)及IEL仍高于PBS組(P＜0.05)。與PBS組比較,各抗原組血清IgG水平在免疫后14d(P＜0.001)和44d(P＜0.05)均明顯增高。腸液sIgA水平,體外ESA(P＜0.05)、腹腔ESA(P＜0.001)和STAg(P＜0.001)組在免疫后14d明顯高于PBS組,兩種ESA組在免疫后44d仍與PBS組有差異(P＜0.05)。 體外ESA、腹腔ESA和STAg鼻內(nèi)免疫小鼠后,腹腔ESA組小鼠出現(xiàn)輕微豎毛、倦怠等異常表現(xiàn),其它各組小鼠健康狀況良好。攻蟲后,PBS組小鼠體重逐漸降低,15d后逐漸趨于平緩,而體外ESA組和STAg組(P＜0.05)小鼠體重仍呈增高趨勢,腹腔ESA組未見明顯升高。各抗原組脾、腦組織內(nèi)蟲荷顯著低于PBS組(P＜0.01)。 結(jié)論弓形蟲速殖子與Vero細(xì)胞在含1%血清的培養(yǎng)基中共培養(yǎng)12h或24h后,換無血清培養(yǎng)基繼續(xù)培養(yǎng),第13d收集培養(yǎng)上清可獲得較高含量的弓形蟲ESA。體外ESA、腹腔ESA和STAg鼻內(nèi)免疫均可誘導(dǎo)黏膜及系統(tǒng)的細(xì)胞和體液免疫應(yīng)答,產(chǎn)生抗弓形蟲感染的部分保護(hù)。體外ESA和腹腔ESA誘導(dǎo)的免疫應(yīng)答較STAg持久,但腹腔ESA可能對機(jī)體有毒副作用,不適宜直接鼻內(nèi)免疫,體外ESA和STAg符合疫苗設(shè)計(jì)的安全性及有效性原則,可用于弓形蟲黏膜疫苗的研制。
[Abstract]:Objective this study first screened the optimum conditions for the preparation of Toxoplasma excretory antigen (excreted/secreted antigen, ESA) in vitro, and then observed two different sources of Toxoplasma ESA (ESA obtained by Vero cells in vitro culture of Toxoplasma gondii and ESA isolated from infected rat ascites), and soluble tachyonus antigen (soluble tachyzoite antigen, STAg). The immune response and the ability to resist Toxoplasma infection induced by intranasal immunization in mice were studied. The immune response and anti infection mechanism of three antigens in nasal immunization were discussed, which lay a theoretical foundation for the development of the compound vaccine of Toxoplasma gondii.
Methods this study was divided into three parts: the first part observed the effect of different co culture conditions of Toxoplasma and Vero cells on the content of ESA. First, the effects of different serum concentration and serum culture time on the concentration of ESA protein were observed. According to the serum concentration of 0.5%, 1%, 2%, 4% points, 4 groups were divided into 4 time points according to the serum incubation time (3H, 6h, 12h, 24h). The tachytachus and Vero cells were cultured in the medium containing different concentrations of serum, respectively, after 3h, 6h, 12h or 24h, to continue to be cultured in serum-free medium. 7d was used to collect and culture supernatant and to detect protein content. Secondly, the co culture time and the influence of serum culture time on the concentration of ESA protein were observed. Co culture time (7d,) 9D, 11d, 13D) were divided into 4 groups, and the group was divided into 4 time points according to the different serum culture time (12h, 24h, 48h, 72h). The tachygonite and Vero cells were cultured in the medium containing 1% sera. Each group was cultured 12h, 24h, 48h, after 72h, and continued to be cultured in serum-free medium. The content of protein was measured.
In the second part, the.BALB/c mice induced by two different sources of ESA and STAg intranasal immunization were randomly divided into 4 groups, with PBS 20 mu l/, ESA in vitro, 20 micron 20 micron in the abdominal cavity, and 20 micron in the nasal cavity, and the interval 14D. was killed after the last immunization, and the lymphocytes were counted. Nal intraepithelial lymphocytes, IEL) and splenic lymphocytes were used to detect serum IgG and intestinal fluid sIgA..
The third part observed the.BALB/c mice induced by two different sources of ESA and STAg intranasal immunization mice. The mice were randomly divided into 4 groups, with PBS 20 mu l/, ESA in vitro, 20 micron g/ in the abdominal cavity of ESA and STAg for 2 times, and 1 * 10~4 tachyonus / gavage after the last immunization at the end of 14D.. The health status and body of the mice were observed. After the attack, 30d was killed and the spleen and intracerebral tachycardia were counted.
Results the serum concentration of Toxoplasma gondii and Vero cells in different culture conditions was 1%. The content of ESA protein extracted from co culture 12h or 24h to serum-free medium was significantly higher than that of other culture conditions (P < 0.05).12h or 24h was changed into serum-free medium and collected in co culture 13D for culture supernatant. Protein content was significantly higher than other time points (P < 0.001).
In vitro ESA, intraperitoneal ESA and STAg intranasal immunization mice, the state of the abdominal cavity ESA mice was not good, the other mice were in good health condition. After the last immunization, the spleen lymphocyte (P < 0.05) and IEL (P < 0.01) proliferated active after the last immunization, and the number and IEL of the splenic lymphocyte in the two ESA groups were still higher than those in the PBS group (0.05). The levels of serum IgG in each antigen group were significantly higher in 14d (P < 0.001) and 44d (P < 0.05) after immunization. The level of sIgA in the intestinal fluid, ESA (P < 0.05) in vitro, ESA (P < 0.001) in the abdominal cavity and STAg (P < 0.001) groups were significantly higher than those in the group after immunization. The two groups were still different from those of the group after immunization (0.05).
After ESA, intraperitoneal ESA and STAg intranasally immunized mice, the mice in the ESA group of the abdominal cavity showed slight erection and burnout, and the other mice were in good health. After the attack, the body weight of the PBS group gradually decreased and the 15d gradually tended to slow, while the body weight of the ESA group and the STAg group (P < 0.05) in the in vitro ESA group was still higher, and the ESA group in the abdominal cavity was not clear. The weight of spleen and brain in each antigen group was significantly lower than that in group PBS (P < 0.01).
Conclusion the Toxoplasma gondii and Vero cells were cultured for 12h or 24h in the culture medium containing 1% sera. The culture medium without serum was continued to be cultured. The high content of Toxoplasma ESA. in vitro ESA was obtained from the culture supernatant of 13D. The immune response of the mucous and systemic cells and body fluids could be induced in the intraperitoneal and intraperitoneal ESA and STAg, and the anti Toxoplasma gondii could be produced. Partial protection of infection. The immune response induced by ESA and ESA in vitro is longer than that of STAg, but ESA in abdominal cavity may have toxic side effects to the body. It is not suitable for direct intranasal immunity. In vitro ESA and STAg conform to the safety and effectiveness principles of vaccine design. It can be used for the development of Toxoplasma mucous membrane vaccine.

【學(xué)位授予單位】：山西醫(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

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 董玉蘭,王樹迎;動(dòng)物黏膜免疫細(xì)胞研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2003年01期

2 賈雪梅;弓形蟲致密顆粒蛋白的分子生物學(xué)研究進(jìn)展[J];國外醫(yī)學(xué)(寄生蟲病分冊);2000年03期

3 薄梅花;寄生蟲排泄分泌抗原的保護(hù)性及在疫苗研制中的作用[J];國外醫(yī)學(xué)(寄生蟲病分冊);2001年01期

4 李華文,陳觀今;弓形蟲致密顆粒蛋白結(jié)構(gòu)功能及免疫[J];國外醫(yī)學(xué)(寄生蟲病分冊);2004年02期

5 吳春風(fēng),李淑紅;弓形蟲病免疫學(xué)研究現(xiàn)狀[J];國外醫(yī)學(xué)(寄生蟲病分冊);2004年02期

6 黃復(fù)深,易新元;寄生蟲的粘膜免疫研究進(jìn)展[J];國外醫(yī)學(xué)(寄生蟲病分冊);2004年03期

7 弓鴻飛;殷國榮;;腸上皮內(nèi)淋巴細(xì)胞抗弓形蟲感染的免疫功能研究進(jìn)展[J];國際醫(yī)學(xué)寄生蟲病雜志;2006年04期

8 祁贊梅;細(xì)胞因子與腸上皮內(nèi)淋巴細(xì)胞的發(fā)育分化和生物學(xué)功能[J];國外醫(yī)學(xué)(免疫學(xué)分冊);2000年05期

9 孫怡,何深一,古欽民,李瑛,叢華,周懷愉,趙群力;弓形蟲P30-P22復(fù)合DNA疫苗免疫小鼠誘導(dǎo)的體液免疫應(yīng)答[J];臨沂醫(yī)學(xué)專科學(xué)校學(xué)報(bào);2004年06期

10 劉潤芳,王新彩,趙榮坡;國內(nèi)不同患病人群弓形蟲感染情況研究[J];河南科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年02期

相關(guān)碩士學(xué)位論文 前2條

1 孟曉麗;STAg和霍亂毒素滴鼻免疫小鼠誘導(dǎo)的弓形蟲特異性黏膜及系統(tǒng)免疫應(yīng)答[D];山西醫(yī)科大學(xué);2005年

2 劉娟娟;弓形蟲排泄—分泌抗原（ESA）鼻內(nèi)免疫小鼠誘導(dǎo)的免疫應(yīng)答的觀察[D];山西醫(yī)科大學(xué);2006年

，

本文編號(hào)：1829126