本文選題：IFIT1 + 多克隆抗體��； 參考：《重慶醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 第一部分:小鼠干擾素誘導(dǎo)蛋白IFIT1多克隆抗體的制備 目的:獲取小鼠IFIT1 ( Interferon-induced protein with tetratricopetide repeats 1)特異性多克隆抗體,為深入研究IFIT1的結(jié)構(gòu)和功能奠定基礎(chǔ)。 方法:擴(kuò)增培養(yǎng)高效表達(dá)IFIT1的重組子pMAL-C2X-IFIT1,超聲破碎后的細(xì)胞上清過(guò)Amylose Pre-packed column親和層析柱,將所得純化的融合蛋白MBP-IFIT1作為抗原,添加福氏佐機(jī)劑后免疫兔,收集抗血清,Western blot和ELISA方法測(cè)定抗血清特異性反應(yīng)和效價(jià)。 結(jié)果:純化的MBP-IFIT1可誘導(dǎo)兔產(chǎn)生特異性免疫應(yīng)答,所得抗體能夠特異性識(shí)別融合蛋白中的IFIT1,ELISA結(jié)果顯示其效價(jià)為1:12800。 結(jié)論:MBP-IFIT1具有良好的抗原性,以該融合蛋白為抗原免疫兔成功制備出IFIT1特異性多克隆抗體。 第二部分:小鼠多器官及細(xì)胞株干擾素誘導(dǎo)蛋白IFIT1的表達(dá)情況目的:觀察小鼠多器官和多細(xì)胞株干擾素誘導(dǎo)蛋白IFIT1 RNA水平的表達(dá)情況。 方法:將10只雄性正常小鼠C57/129小鼠斷頸處死,取心、肝、脾、肺、腎、小腸、淋巴細(xì)胞和骨骼肌提RNA。用5μg/ml的內(nèi)毒素(LPS)刺激Raw264.7細(xì)胞株、3T3細(xì)胞株和10T1/2細(xì)胞株5h后,終止刺激,收集細(xì)胞提RNA,同時(shí)設(shè)定正常對(duì)照組。將提純的RNA分別進(jìn)行RT-PCR,觀察IFIT1的表達(dá)情況。 結(jié)果:小鼠心、肝、脾、肺、腎、小腸、淋巴細(xì)胞和骨骼肌中均表達(dá)IFIT1,且存在表達(dá)量上的差別,心表達(dá)量最高,肝與小腸比較P=0.307,其它各組織間比較P0.05。Raw264.7細(xì)胞株、3T3細(xì)胞株和10T1/2細(xì)胞株LPS刺激5h后,均能誘導(dǎo)IFIT1表達(dá),正常對(duì)照組表達(dá)為陰性。 結(jié)論:IFIT1在小鼠體內(nèi)為廣泛表達(dá),LPS刺激可多種細(xì)胞株表達(dá)IFIT1。 第三部分:放射性損傷對(duì)小鼠干擾素誘導(dǎo)蛋白IFIT1及JAB1表達(dá)的影響 目的:觀察放射性損傷后早期小鼠肝組織IFIT1、JAB1各時(shí)相點(diǎn)變化特點(diǎn),初步探討創(chuàng)傷應(yīng)激早期糖皮質(zhì)激素抵抗的分子機(jī)制。 方法:將20只C57小鼠,不拘雌雄,隨機(jī)分為正常對(duì)照組和致傷組,致傷組采用5Gry 60Co一次性全身照射,分別于傷后1h、4h、和12h脫頸處死取肝組織。用western blot測(cè)定肝組織IFIT1、JAB1表達(dá)變化。 結(jié)果:放射損傷后1h IFIT1開(kāi)始增高,12h達(dá)最高,呈上升趨勢(shì),差異顯著P0.01;同時(shí),JAB1在1h開(kāi)始下降,12h降到最低,呈下降趨勢(shì),差異顯著P0.01。 結(jié)論:放射損傷早期不僅引起小鼠肝組織IFIT1的顯著增高,同時(shí)引起JAB1的顯著下降,成相反趨勢(shì),揭示創(chuàng)傷應(yīng)激早期糖皮質(zhì)激素抵抗可能與IFIT1對(duì)其的阻遏作用和JAB1對(duì)其的正性調(diào)控有關(guān)。 第四部分:頸交感神經(jīng)阻滯對(duì)燒傷早期小鼠肝干擾素誘導(dǎo)蛋白IFIT1表達(dá)的影響 目的:探討頸交感神經(jīng)阻滯(cervical sympathetic block,SB)對(duì)燒傷后早期小鼠肝臟干擾素誘導(dǎo)蛋白IFIT1表達(dá)的影響。方法:雄性C57/129小鼠50只,隨機(jī)分為對(duì)照組和SB治療組,TBSA 15-20%Ⅲ度小鼠燒傷模型,分別于燒傷前、燒傷后1h、6h、12h和24h取肝組織,采用免疫印跡法(Western blot)觀察IFIT1的蛋白表達(dá)情況。 結(jié)果:對(duì)照組燒傷后肝IFIT1表達(dá)顯著增高(P0.01),燒傷后治療組IFIT1表達(dá)量較對(duì)照組相應(yīng)時(shí)相點(diǎn)顯著降低(P0.01)。結(jié)論:燒傷后早期GR功能抑制可能與IFIT1負(fù)性調(diào)控有關(guān),SB對(duì)燒傷的治療作用可能是通過(guò)抑制IFIT1表達(dá)來(lái)實(shí)現(xiàn)的。
[Abstract]:Part one: preparation of mouse polyclonal antibody against interferon inducible protein IFIT1
Objective: to obtain the specific polyclonal antibody of mouse IFIT1 (Interferon-induced protein with tetratricopetide repeats 1), and to lay the foundation for the in-depth study of the structure and function of IFIT1.
Methods: the recombinant IFIT1 recombinant pMAL-C2X-IFIT1 was amplified and cultured, and the cells after ultrasonic fragmentation were cleared by Amylose Pre-packed column affinity chromatography column. The purified fusion protein MBP-IFIT1 was used as antigen, and the rabbit was immunized with FFL, and antiserum was collected, Western blot and ELISA methods were used to determine the specific reaction of antiserum and the antiserum. Titer.
Results: the purified MBP-IFIT1 can induce a specific immune response in rabbits. The antibody can specifically identify the IFIT1 in the fusion protein, and the ELISA results show that its titer is 1:12800..
Conclusion: MBP-IFIT1 has good antigenicity. IFIT1 specific polyclonal antibody was successfully prepared by immunizing rabbits with the fusion protein as antigen.
The second part: the expression of interferon induced protein IFIT1 in multiple organs and cell lines in mice: To observe the expression of interferon induced protein IFIT1 RNA in multiple organs and multicell lines in mice.
Methods: 10 male normal mice were killed in C57/129 mice, the heart, the liver, the spleen, the spleen, the lung, the kidney, the small intestine, the lymphocyte and the skeletal muscle were used to stimulate the Raw264.7 cell line, the 3T3 cell line and the 10T1/2 cell line 5h, and the 3T3 cell line and the 10T1/2 cell line 5h, to collect the cell extract RNA and set the normal control group. The purified RNA was carried out RT-PC, respectively. R, observe the expression of IFIT1.
Results: IFIT1 was expressed in the heart, liver, spleen, spleen, lung, kidney, kidney, kidney, small intestine, lymphocyte and skeletal muscle, and there was a difference in expression, the expression of heart was the highest, the liver and small intestine were compared with P=0.307, the other tissues were compared with P0.05.Raw264.7 cell lines, 3T3 cell lines and 10T1/2 fine cell LPS stimulated 5h, and the expression of the normal control group was expressed as the normal control group. Negative.
Conclusion: IFIT1 is widely expressed in mice, and LPS can stimulate IFIT1. expression in many cell lines.
The third part: the effect of radiation injury on the expression of interferon inducible protein IFIT1 and JAB1 in mice.
Objective: To observe the changes of the phase points of IFIT1 and JAB1 in early mice liver tissue after radiation injury, and to investigate the molecular mechanism of glucocorticoid resistance at the early stage of trauma stress.
Methods: 20 C57 mice were randomly divided into normal control group and injury group, which were randomly divided into normal control group and injury group. The injured group was irradiated by 5Gry 60Co in one time. The liver tissues were killed after 1h, 4h, and 12h removed respectively. The expression of IFIT1 and JAB1 in liver tissue was measured with Western blot.
Results: after radiation injury, 1H IFIT1 began to increase, and 12h reached the highest, showing an upward trend, and the difference was significant P0.01. At the same time, JAB1 began to decline in 1H, and the 12h decreased to the lowest level, and the difference was significant P0.01..
Conclusion: early radiation injury not only caused a significant increase in the IFIT1 of liver tissue in mice, but also caused a significant decrease in JAB1. It was suggested that the early glucocorticoid resistance of traumatic stress may be related to the repression of IFIT1 and the positive regulation of JAB1 on it.
The fourth part: the effect of cervical sympathetic blockade on the expression of interferon inducible protein IFIT1 in the early stage of burn injury in mice.
Objective: To investigate the effect of cervical sympathetic block (SB) on the expression of interferon induced protein IFIT1 in the liver of mice after burn. Methods: 50 male C57/129 mice were randomly divided into control group and SB treatment group, TBSA 15-20% III degree mice burn model, before burn, 1H, 6h, 12h, and liver tissue after burn, respectively. Western blotting (Western blot) was used to observe the protein expression of IFIT1.
Results: the expression of IFIT1 in the control group was significantly increased (P0.01), and the expression of IFIT1 in the treatment group was significantly lower than that of the control group (P0.01). Conclusion: the inhibition of GR function in the early stage of burn may be related to the negative regulation of IFIT1. The therapeutic effect of SB on burn may be achieved by inhibiting the expression of IFIT1.
【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

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 鐘河江,楊天德,粟永萍;JAB1、LPS對(duì)糖皮質(zhì)激素受體轉(zhuǎn)錄激活活性的影響[J];重慶醫(yī)學(xué);2004年11期

2 鄧正陽(yáng),李淑蓉,王蒙,王軍平,廖蘭,徐建明,蔣建新,黃躍生,粟永萍;人P56蛋白及其各種缺失突變體酵母表達(dá)質(zhì)粒的構(gòu)建及鑒定[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2004年07期

3 李淑蓉,粟永萍,劉曉宏,樓淑芬,程天民;干擾素誘導(dǎo)蛋白P56與糖皮質(zhì)激素受體的相互作用及對(duì)其轉(zhuǎn)錄活性的影響[J];生物化學(xué)與生物物理進(jìn)展;2004年09期

4 林筱潔,羅建紅,宋英,朱麗君,余應(yīng)年;人細(xì)胞色素P450 1A1與谷胱甘肽S-轉(zhuǎn)移酶的融合蛋白及其抗體的制備[J];中國(guó)藥理學(xué)與毒理學(xué)雜志;2000年06期

5 王軍平,趙景宏,粟永萍;糖皮質(zhì)激素受體調(diào)控與創(chuàng)傷應(yīng)激紊亂關(guān)系的研究進(jìn)展[J];中華創(chuàng)傷雜志;2001年08期

6 劉都戶,粟永萍,張渭,樓淑芬;α-MSH和合成肽KPV對(duì)病理性應(yīng)激大鼠血漿皮質(zhì)酮的調(diào)節(jié)作用[J];中國(guó)急救醫(yī)學(xué);2001年10期

，

本文編號(hào)：1975727