重组BPI蛋白对感染绵羊支原体肺炎的盘羊杂交羊的免疫调节及治疗效果研究

发布时间：2018-06-13 22:55

本文选题：杀菌通透性增加蛋白 + 绵羊肺炎支原体　；参考：《石河子大学》2015年硕士论文

【摘要】：目的:本试验以感染绵羊肺炎支原体(MO)的绵羊为疾病模型,使用重组BPI蛋白(r BPI)治疗感染MO的盘羊杂交羊,然后用real-time QPCR与ELISA的方法,测定BPI和相关细胞因子的含量,用血常规方法检测血液各项生理指标,以研究r BPI对感染MO的盘羊杂交羊的免疫调节效果。方法:(1)绵羊肺炎支原体的人工感染:将冻存的MO菌种进行复壮培养,测定菌液浓度并稀释到备用浓度106CCU/m L。然后将6只巴什拜羊为A组,12只盘羊杂交羊随机分为B、C组,全部气管内注射绵羊肺炎支原体,每天观察临床症状并测量记录体温。两周后每组随机取3只试验羊的鼻腔分泌物,进行MO的培养与生化鉴定,检测人工感染情况。(2)r BPI对感染MO的盘羊杂交羊的治疗作用:在试验的第4 d,C组杂交羊使用纯化的r BPI对其治疗,150mg/只/天,而A组和B组每天注射生理盐水,继续观察临床症状并记录体温。实验结束后将所有羊解剖观察肺脏病理变化,并制作病理切片进行病理评分,对比各组羊的差别。(3)r BPI对感染MO的盘羊杂交羊的内源BPI m RNA水平的影响:分别在感染前(0 d)、感染后第二天(2 d)、感染后五天(5 d)、一周(7 d)、两周(14 d)、三周(21 d),采血分离嗜中性粒细胞,用real time-QPCR方法检测试验羊BPI m RNA的相对表达水平。(4)r BPI对感染MO的盘羊杂交羊的免疫调节作用:分别在第0 d、1 d、5 d、7 d、14 d、21 d,采血分离血清,用ELISA方法检测试验羊IL-1β、IL-4、IL-6、IL-8、IL-10、TNF-α及IFN-γ的含量,比较各组羊细胞因子的表达变化差异。(5)r BPI对感染MO的盘羊杂交羊的血液部分生理指标测定:分别在第0 d、7 d、14 d,采集各组羊的外周血进行血常规检测。结果与结论:(1)鼻腔分泌物用支原体专用培养基培养5d培养液变黄,葡萄糖发酵试验显示培养液由红变黄呈阳性;水解精氨酸试验显示培养液未变色呈阴性;洋地黄皂苷试验显示抑制带0.3cm为阳性;氯化四氮唑还原实验显示琼脂培养基呈红色;红细胞吸附试验显示菌落表面布满红细胞,溶血试验为β溶血。说明鼻腔分泌物中含有MO,试验羊人工感染成功。(2)A组巴什拜羊在感染后2-3d均表现为食欲减退、体温一过性升高(40-41℃)、呼吸Up音,无死亡病例,剖检后肺表面有轻微出血点;B组杂交羊体温升高(40-41℃)持续较久,有咳嗽、流浆液性鼻涕,甚至严重呼吸Up音及腹泻症状,其中2只死亡,剖检肺表面有不同程度的粉色肉变和肝变,有出血点和出血斑。C组杂交羊起初体温升高、食欲减退、咳嗽症状,剖检肺表面有出血点和出血斑。组织病理学评分结果:A组平均分为9.4,B组平均分19.9,C组平均分为12.8,B组评分显著高于A、C组(P0.05;P0.05)。说明杂交羊在治疗后临床症状及肺部炎症减轻。(3)在第7 d,C组BPI基因的相对表达水平显著低于A、B组(P0.05),在第14-21d C组极显著高于B组(P0.01),而在第7-14 d A组极显著高于B组(P0.01)。说明了注射r BPI对体内BPI的表达水平有影响。(4)从感染后的第7-21d,C组的IL-1β含量分别极显著低于A、B(P0.01)。在感染后的第7-21 d,C组IL-4含量分别极显著高于对照组A、B(P0.01);在第14-21d,A组IL-4显著高于B组(P0.05)。第14-21 d,C组的IL-6极显著低于A、B组(P0.01),A组也显著低于B组(P0.05)。在感染后第7 d,C组IL-8浓度显著低于A组(P0.05);在第14-21d,B组的极显著高于A组(P0.01;P0.01)。在第5 d,C组IL-10显著高于A组(P0.05),极显著低于B组(P0.01);在第14 d,C组极显著低于A组(P0.01),显著高于B组(P0.05);在第21 d,C组显著低于A组(P0.05),极显著高于B组(P0.01)。在第14 d,C组IFN-γ含量显著高于A组(P0.05);在第21 d,C组显著低于B组(P0.05),但极显著高于A组(P0.01)。在感染第14 d,C组TNF-α显著低于B组(P0.05),显著高于A组(P0.05);在第21 d,C组的TNF-α显著低于B组(P0.05),但极显著高于A组(P0.01)。(5)在感染前,杂交羊WBC、LYM、LYM%、RBC显著高于巴什拜羊,而MID、MID%、MCHC低于巴什拜羊。注射r BPI后,在第14 d,C组WBC、GRAN(109/L)及GRAN%显著低于B组(P0.05),而LYM%、MCHC显著高于B组(P0.05)。说明注射BPI对杂交羊的血液生理指标有一定影响。
[Abstract]:Objective: in this experiment, the sheep of the sheep infected with Mycoplasma pneumoniae (MO) were used as the disease model and the recombinant BPI protein (R BPI) was used to treat the sheep of the sheep infected with MO. Then the content of BPI and related cytokines were measured by real-time QPCR and ELISA, and the physiological indexes of the blood were detected by blood routine method to study the disk of R BPI on the infection MO. Methods: (1) artificial infection of Mycoplasma pneumoniae: Rejuvenate the frozen MO strains, determine the concentration of the bacteria and dilute the concentration of the bacteria to the standby concentration 106CCU/m L., and then divide the 6 bash sheep into group A, 12 sheep cross sheep randomly divided into B, C group, all trachea injection of Mycoplasma pneumoniae in the trachea, every day view Observe the clinical symptoms and measure the body temperature. Two weeks after two weeks, each group of 3 sheep's nasal secretions were randomly selected to carry out the culture and biochemical identification of the sheep and detect the artificial infection. (2) the therapeutic effect of R BPI on the cross sheep of MO infected sheep: in the experiment fourth D, the C group was treated with the purified R BPI, 150mg/ only / day, and A and B groups. Daily injection of saline, continued to observe the clinical symptoms and record the body temperature. After the experiment, all the sheep were dissected to observe the pathological changes of the lungs, and the pathological sections were made to compare the pathological scores of the sheep. (3) the effect of R BPI on the endogenous BPI m RNA level of the sheep infected with MO: before infection (0 d), and second after infection, respectively. Day (2 D), five days after infection (5 d), one week (7 d), two weeks (14 d), three weeks (21 d), isolated neutrophils, and real time-QPCR method was used to detect the relative expression level of sheep BPI m RNA. (4) r BPI on the immune modulation of the sheep of the sheep of zeroth, 1, 5, 7, 7, 7, 14 Test the content of IL-1 beta, IL-4, IL-6, IL-8, IL-10, TNF- A and IFN- gamma in sheep, and compare the difference of the expression of sheep cell factors in each group. (5) the physiological indexes of R BPI on the blood of sheep with MO in the sheep were measured in zeroth D, 7 D, 14. The results and conclusions: (1) nasal secretion use The 5D culture medium was cultured in the special medium of mycoplasma, and the glucose fermentation test showed that the culture medium was positive from red to yellow, and the hydrolyzed arginine test showed that the culture solution was negative; digitalis saponins test showed that the inhibitory zone 0.3cm was positive; the experiment of chlorinated tetrazolium reduction experiment showed that the agar medium was red; the red cell adsorption test showed that the chlorinated tetrazoloside experiment showed that the culture medium was positive. The surface of the colony was full of red blood cells and hemolysis test was beta hemolysis. It indicated that MO in the nasal secretions was contained and the artificial infection of sheep was successful. (2) in group A, after infection, 2-3D showed anorexia, hyperthermia (40-41 degrees C), respiratory Up sound, no death cases, slight bleeding point on the lung surface after caesarean section, and B group hybrid sheep body temperature rise (40- 41 C) lasted for a long time, with cough, runny nose, and even severe respiratory symptoms of Up and diarrhea. 2 of them died, and the surface of the lung had different degrees of pink flesh change and liver change. There were hemorrhagic spots and hemorrhagic spot.C group at first, the body temperature, anorexia, cough symptoms, and histopathology of the lung surface. The average score was 9.4 in group A, 19.9 in group B and 12.8 in group C. The score of group B was significantly higher than that in A and C group (P0.05; P0.05). (3) the relative expression level of the BPI gene in the C group was significantly lower than that of A, but in the group of C, the relative expression level was significantly higher than that in the group. The 7-14 D A group was significantly higher than the B group (P0.01). It shows that the R BPI injection has an effect on the expression level of BPI in the body. (4) from the infected 7-21d, the IL-1 beta content of the C group is significantly lower than that of A, and the 7-21 after infection is significantly higher than that of the control group. In group 14-21 D, the IL-6 of group C was significantly lower than that of A, B group (P0.01), and A group was also significantly lower than that of B group (P0.05). P0.01) was significantly higher than group B (P0.05), and in twenty-first D, group C was significantly lower than group A (P0.05), significantly higher than that of group B (P0.01). In the fourteenth D, C group was significantly higher than that of the group, but in twenty-first, the group was significantly lower than that of the group. 1 D, C group TNF- alpha was significantly lower than group B (P0.05), but significantly higher than group A (P0.01). (5) before infection, the hybrid sheep WBC, LYM, LYM%, RBC were significantly higher than the basbai sheep, and MID was significantly lower than that of basbai sheep. I has a certain influence on the blood physiological indexes of the hybrid sheep.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.26

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