本文選題：殺菌通透性增加蛋白 + 綿羊肺炎支原體　； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：目的:本試驗以感染綿羊肺炎支原體(MO)的綿羊為疾病模型,使用重組BPI蛋白(r BPI)治療感染MO的盤羊雜交羊,然后用real-time QPCR與ELISA的方法,測定BPI和相關(guān)細(xì)胞因子的含量,用血常規(guī)方法檢測血液各項生理指標(biāo),以研究r BPI對感染MO的盤羊雜交羊的免疫調(diào)節(jié)效果。方法:(1)綿羊肺炎支原體的人工感染:將凍存的MO菌種進(jìn)行復(fù)壯培養(yǎng),測定菌液濃度并稀釋到備用濃度106CCU/m L。然后將6只巴什拜羊為A組,12只盤羊雜交羊隨機分為B、C組,全部氣管內(nèi)注射綿羊肺炎支原體,每天觀察臨床癥狀并測量記錄體溫。兩周后每組隨機取3只試驗羊的鼻腔分泌物,進(jìn)行MO的培養(yǎng)與生化鑒定,檢測人工感染情況。(2)r BPI對感染MO的盤羊雜交羊的治療作用:在試驗的第4 d,C組雜交羊使用純化的r BPI對其治療,150mg/只/天,而A組和B組每天注射生理鹽水,繼續(xù)觀察臨床癥狀并記錄體溫。實驗結(jié)束后將所有羊解剖觀察肺臟病理變化,并制作病理切片進(jìn)行病理評分,對比各組羊的差別。(3)r BPI對感染MO的盤羊雜交羊的內(nèi)源BPI m RNA水平的影響:分別在感染前(0 d)、感染后第二天(2 d)、感染后五天(5 d)、一周(7 d)、兩周(14 d)、三周(21 d),采血分離嗜中性粒細(xì)胞,用real time-QPCR方法檢測試驗羊BPI m RNA的相對表達(dá)水平。(4)r BPI對感染MO的盤羊雜交羊的免疫調(diào)節(jié)作用:分別在第0 d、1 d、5 d、7 d、14 d、21 d,采血分離血清,用ELISA方法檢測試驗羊IL-1β、IL-4、IL-6、IL-8、IL-10、TNF-α及IFN-γ的含量,比較各組羊細(xì)胞因子的表達(dá)變化差異。(5)r BPI對感染MO的盤羊雜交羊的血液部分生理指標(biāo)測定:分別在第0 d、7 d、14 d,采集各組羊的外周血進(jìn)行血常規(guī)檢測。結(jié)果與結(jié)論:(1)鼻腔分泌物用支原體專用培養(yǎng)基培養(yǎng)5d培養(yǎng)液變黃,葡萄糖發(fā)酵試驗顯示培養(yǎng)液由紅變黃呈陽性;水解精氨酸試驗顯示培養(yǎng)液未變色呈陰性;洋地黃皂苷試驗顯示抑制帶0.3cm為陽性;氯化四氮唑還原實驗顯示瓊脂培養(yǎng)基呈紅色;紅細(xì)胞吸附試驗顯示菌落表面布滿紅細(xì)胞,溶血試驗為β溶血。說明鼻腔分泌物中含有MO,試驗羊人工感染成功。(2)A組巴什拜羊在感染后2-3d均表現(xiàn)為食欲減退、體溫一過性升高(40-41℃)、呼吸Up音,無死亡病例,剖檢后肺表面有輕微出血點;B組雜交羊體溫升高(40-41℃)持續(xù)較久,有咳嗽、流漿液性鼻涕,甚至嚴(yán)重呼吸Up音及腹瀉癥狀,其中2只死亡,剖檢肺表面有不同程度的粉色肉變和肝變,有出血點和出血斑。C組雜交羊起初體溫升高、食欲減退、咳嗽癥狀,剖檢肺表面有出血點和出血斑。組織病理學(xué)評分結(jié)果:A組平均分為9.4,B組平均分19.9,C組平均分為12.8,B組評分顯著高于A、C組(P0.05;P0.05)。說明雜交羊在治療后臨床癥狀及肺部炎癥減輕。(3)在第7 d,C組BPI基因的相對表達(dá)水平顯著低于A、B組(P0.05),在第14-21d C組極顯著高于B組(P0.01),而在第7-14 d A組極顯著高于B組(P0.01)。說明了注射r BPI對體內(nèi)BPI的表達(dá)水平有影響。(4)從感染后的第7-21d,C組的IL-1β含量分別極顯著低于A、B(P0.01)。在感染后的第7-21 d,C組IL-4含量分別極顯著高于對照組A、B(P0.01);在第14-21d,A組IL-4顯著高于B組(P0.05)。第14-21 d,C組的IL-6極顯著低于A、B組(P0.01),A組也顯著低于B組(P0.05)。在感染后第7 d,C組IL-8濃度顯著低于A組(P0.05);在第14-21d,B組的極顯著高于A組(P0.01;P0.01)。在第5 d,C組IL-10顯著高于A組(P0.05),極顯著低于B組(P0.01);在第14 d,C組極顯著低于A組(P0.01),顯著高于B組(P0.05);在第21 d,C組顯著低于A組(P0.05),極顯著高于B組(P0.01)。在第14 d,C組IFN-γ含量顯著高于A組(P0.05);在第21 d,C組顯著低于B組(P0.05),但極顯著高于A組(P0.01)。在感染第14 d,C組TNF-α顯著低于B組(P0.05),顯著高于A組(P0.05);在第21 d,C組的TNF-α顯著低于B組(P0.05),但極顯著高于A組(P0.01)。(5)在感染前,雜交羊WBC、LYM、LYM%、RBC顯著高于巴什拜羊,而MID、MID%、MCHC低于巴什拜羊。注射r BPI后,在第14 d,C組WBC、GRAN(109/L)及GRAN%顯著低于B組(P0.05),而LYM%、MCHC顯著高于B組(P0.05)。說明注射BPI對雜交羊的血液生理指標(biāo)有一定影響。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.26

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