豚鼠结膜炎衣原体噬菌体phiCPG1衣壳蛋白Vp1的生物学作用

发布时间：2018-08-13 10:40

【摘要】：人类许多的疾病与沙眼衣原体(Chlamydia trachomatis,Ct)地感染存在着密切的关系,Ct感染眼结膜可以引起包涵体结膜炎、沙眼和感染婴幼儿肺脏引起婴幼儿的肺炎,感染泌尿生殖道能引起非淋菌性尿道炎、非淋菌性阴道炎、宫颈炎、性病淋巴肉芽肿等疾病。目前许多国家和地区性传播疾病(sexually transimitted disease,STD)都是以Ct为病原体的,Ct在世界范围内已经成为STD的主要病原体之一了。在欧美的许多国家和地区,由Ct感染所致STD已成为最常见的STD,其发病率已经超过了淋病。沙眼衣原体感染泌尿生殖道(genital Chlamydia trachomatis infections,GCI)的患病人数在我国也在逐年增加。沙眼衣原体在女性患者经过性传播可以导致女性阴道炎、子宫内膜炎、宫颈炎、输卵管炎、非淋菌性尿道炎等,还可以造成盆腔炎、异位妊娠、自然流产、胎膜早破、早产和不孕等多种不良后果,通过垂直传播会使分娩的感染患者传染新生儿使其患上包涵体性的结膜炎[1]。目前因为不当应用抗生素的治疗Ct感染的问题日趋严重,引起Ct抗生素耐药问题日渐突出,使可选择用来治疗Ct感染的敏感性抗生素越来越少,甚至无药可用,大大增加了Ct感染治疗难度。因此,全世界的专家学者均期望通过对沙眼衣原体感染的进一步的深入研究,来发现新的预防或者治疗Ct感染的方法,来解决抗生素耐药的问题。许多研究学者们发现衣原体噬菌体感染宿主衣原体的途径是与衣原体的原体结合而得以实现的,它能使衣原体停留在网状体阶段形成异常增大的网状体,从而阻止衣原体发育为成熟原体,造成原体数量减少,使衣原体的感染性大大降低,甚至杀灭衣原体。被γ-干扰素抑制发育的衣原体也能产生异常增大的网状体,这些网状体的形态与感染噬菌体后衣原体形成的网状体的形态相似,这从一个侧面可以说明噬菌体具有在临床上能够治疗Ct感染的可能。衣原体噬菌体衣壳蛋白Vp1是噬菌体主要的结构蛋白,该蛋白对于黏附与植入衣原体起到重要的作用,其相对的分子质量是61805,并且Vp1蛋白的保守性和特异性强。[目的]表达、鉴定、纯化豚鼠结膜炎衣原体噬菌体phi CPG1的衣壳蛋白Vp1;在细胞培养E血清型和K血清型沙眼衣原体过程中用其进行干预,观察其对Ct的抑制作用;将Vp1蛋白作用于生殖道感染E型Ct的小鼠模型,观察其对模型小鼠阴道内衣原体的作用;将Vp1蛋白作用于生殖道感染K型Ct的小鼠模型,观察其对沙眼衣原体在模型小鼠生殖道内上行感染的作用。[方法]表达、鉴定、纯化豚鼠结膜炎衣原体噬菌体phi CPG1衣壳蛋白Vp1,分别将沙眼衣原体的标准株E、K分别和浓度为50μg/ml和100μg/ml Vp1于室温下孵育3小时,在单层致密Mc Coy细胞中接种孵育好的沙眼衣原体的标准株E、K,在整个沙眼衣原体的培养过程中均加入Vp1蛋白并保持其浓度为50μg/ml和100μg/ml,最后通过碘染色计数包涵体数量,观察Vp1蛋白对Mc Coy细胞培养的沙眼衣原体增殖的抑制作用。构建生殖道感染E型Ct的BALB/c雌性小鼠模型,经阴道和肌肉注射给予Vp1蛋白干预,于不同时间取小鼠阴道分泌物进行细胞培养和实时定量PCR检测,并观察小鼠阴道口红肿情况,观察Vp1蛋白对小鼠阴道内沙眼衣原体增殖的作用。构建生殖道感染K型Ct的BALB/c雌性小鼠模型,经阴道给予Vp1蛋白干预,于不同时间取小鼠子宫角、输卵管和卵巢进行病理切片、组织匀浆细胞培养检测,观察Vp1蛋白对小鼠沙眼衣原体阴道感染引起的生殖系统炎症的作用。[结果]本实验表达纯化的噬菌体phi CPG1衣壳蛋白Vp1在沙眼衣原体细胞培养过程中能够抑制其生长,E型和K型标准株的包涵体数量均有较明显的减少。计数每个视野下2种标准株包涵体的数量,得到平均值,计算出100 ug/ml、50ug/ml浓度Vp1作用的抑制率,E型91%、79%;K型94%、70%。成功构建了生殖道感染E型标准株Ct的BALB/c雌性小鼠模型,通过肌肉注射和阴道给予不同浓度的Vp1蛋白干预,于不同时间取小鼠阴道分泌物行细胞培养和实时定量PCR进行定量检测,肌注组与感染组、对照组差别无统计学意义,阴道干预高浓度组较低浓度组衣原体浓度低,低浓度组较感染组、对照组衣原体浓度低,差异有统计学意义。成功构建了生殖道感染K型标准株Ct的BALB/c雌性小鼠模型,经阴道给予Vp1蛋白干预,于接种后35天取小鼠生殖系统组织,进行HE染色病理观察,评价子宫角、输卵管、卵巢炎症情况,干预组较感染组炎症轻,差异有统计学意义。第7天感染组和干预组小鼠组织匀浆细胞培养结果:感染组和干预组小鼠的子宫角组织匀浆细胞培养结果均为阳性,输卵管、卵巢组织匀浆细胞培养结果均为阴性。第35天感染组和干预组剩余小鼠子的宫角、输卵管、卵巢组织匀浆细胞培养结果均为阴性,[结论]豚鼠结膜炎衣原体噬菌体phi CPG1衣壳蛋白Vp1在体外细胞培养中对沙眼衣原体增殖的抑制作用明显。Vp1蛋白对感染小鼠阴道内沙眼衣原体的增殖具有抑制作用,缩短了小鼠阴道内沙眼衣原体的自净时间。Vp1蛋白具有减轻沙眼衣原体感染小鼠引起的上生殖道炎症的作用。为进一步研究临床上沙眼衣原体感染的治疗提供新的思路。
[Abstract]:Many human diseases are closely related to Chlamydia trachomatis (Ct) infection. Ct infection of conjunctiva can cause inclusion body conjunctivitis, trachoma and lung infection of infants and young children can cause pneumonia in infants and young children. Infection of urogenital tract can cause non-gonococcal urethritis, non-gonococcal vaginitis, cervicitis, venereal lymphadenitis. At present, many countries and regions sexually transmitted diseases (STD) are caused by CT. CT has become one of the main pathogens of STD in the world. STD caused by CT infection has become the most common STD in many countries and regions of Europe and the United States, and its incidence has exceeded that of gonorrhea. The number of genital Chlamydia trachomatis infections (GCI) is increasing year by year in China. Sexual transmission of Chlamydia trachomatis in female patients can lead to female vaginitis, endometritis, cervicitis, salpingitis, non-gonococcal urethritis, pelvic inflammation and ectopic pregnancy. Natural abortion, premature rupture of membranes, premature delivery, infertility and other adverse consequences, through vertical transmission will make the delivery of infected neonates infected with neonatal inclusion conjunctivitis [1].At present, due to the improper use of antibiotics in the treatment of CT infection is becoming increasingly serious, causing the problem of CT antibiotic resistance is becoming increasingly prominent, making it optional for treatment. So, experts and scholars all over the world are expecting to find new methods to prevent or treat Chlamydia trachomatis infection and to solve the problem of antibiotic resistance through further in-depth study of Chlamydia trachomatis infection. The researchers found that Chlamydia phages infect the host Chlamydia by binding to Chlamydia, which causes Chlamydia to stay in the reticulum stage and form abnormally enlarged reticulum, thus preventing the development of Chlamydia into mature protoplasts, reducing the number of protoplasts, greatly reducing the infectivity of Chlamydia, and even killing it. Chlamydia. Chlamydia, which is inhibited by interferon-gamma, also produces abnormally enlarged reticulae similar to those formed by Chlamydia after infection with phages. This suggests that phages may be able to treat CT infection clinically. Chlamydia phage capsid protein Vp1 is [Objective] To express, identify and purify the capsid protein Vp1 of Chlamydia conjunctivitis phage phi CPG1 from guinea pigs, and to culture E serotype and K serotype sand in cells. Chlamydia ophthalmica was used to intervene in the process of infection and observe its inhibitory effect on CT; Vp1 protein was used to treat E-type CT in the reproductive tract of mice, and its effect on the vaginal endothelium of the model mice was observed; Vp1 protein was used to treat K-type CT in the reproductive tract of mice, and its effect on Chlamydia trachomatis in the reproductive tract of the model mice was observed. [Methods] Chlamydia conjunctivitis phage phi CPG1 capsid protein Vp1 was expressed, identified and purified. The standard strains E, K and 100 ug/ml Vp1 of Chlamydia trachomatis were incubated at room temperature for 3 hours, respectively. The standard strains E and K of Chlamydia trachomatis were inoculated in monolayer dense Mc Coy cells and incubated in the whole sand. Vp1 protein was added to Chlamydia trachomatis culture and kept at 50 and 100 ug/ml. The number of inclusion bodies was counted by iodine staining to observe the inhibitory effect of Vp1 protein on the proliferation of Chlamydia trachomatis cultured in Mc Coy cells. The effect of Vp1 protein on the proliferation of Chlamydia trachomatis in vagina of mice was observed. A female BALB/c mouse model of K-type CT infection was established. Vp1 protein was given to the vagina at different intervals. The effects of Vp1 protein on the reproductive system inflammation induced by vaginal infection of Chlamydia trachomatis in mice were observed by pathological section of uterine horn, oviduct and ovary, and homogenate cell culture. The number of inclusion bodies of E-type and K-type standard strains was significantly reduced. The average number of inclusion bodies of the two standard strains was counted under each field of vision, and the inhibition rates of 100 ug/ml, 50 ug/ml Vp1, E-type 91%, 79%; K-type 94%, 70%. A female BALB/c mouse model of E-type standard strain CT was successfully constructed by intramuscular injection. Different concentrations of Vp1 protein were injected into the vagina and the vagina of mice at different time for cell culture and real-time quantitative PCR. There was no significant difference between the intramuscular injection group and the infection group. The concentration of Chlamydia in the high concentration group was lower than that in the low concentration group, and the concentration of Chlamydia in the low concentration group was lower than that in the infection group. The BALB/c female mice model of genital tract infection with standard strain CT of K type was successfully constructed. The reproductive system tissues of the mice were taken out 35 days after inoculation by Vp1 protein. The pathological observation of HE staining was carried out to evaluate the inflammation of uterine horn, fallopian tube and ovary. On the 7th day, the results of homogenate cell culture in the infected and intervention groups were all positive, but the results of oviduct and ovary were negative. On the 35th day, the corners of uterus, oviduct and ovary of the remaining mice in the infection group and intervention group were homogeneous. The results of plasma cell culture were negative. [Conclusion] The capsid protein Vp1 of Chlamydia conjunctivitis phi CPG1 can inhibit the proliferation of Chlamydia trachomatis in vitro. Vp1 can inhibit the proliferation of Chlamydia trachomatis in the vagina of infected mice and shorten the self-purification time of Chlamydia trachomatis in the vagina of mice. Vp1 protein can alleviate the inflammation of upper genital tract caused by Chlamydia trachomatis infection in mice.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R374.1

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