發(fā)布時(shí)間：2018-08-13 10:40

【摘要】：人類許多的疾病與沙眼衣原體(Chlamydia trachomatis,Ct)地感染存在著密切的關(guān)系,Ct感染眼結(jié)膜可以引起包涵體結(jié)膜炎、沙眼和感染嬰幼兒肺臟引起嬰幼兒的肺炎,感染泌尿生殖道能引起非淋菌性尿道炎、非淋菌性陰道炎、宮頸炎、性病淋巴肉芽腫等疾病。目前許多國(guó)家和地區(qū)性傳播疾病(sexually transimitted disease,STD)都是以Ct為病原體的,Ct在世界范圍內(nèi)已經(jīng)成為STD的主要病原體之一了。在歐美的許多國(guó)家和地區(qū),由Ct感染所致STD已成為最常見的STD,其發(fā)病率已經(jīng)超過了淋病。沙眼衣原體感染泌尿生殖道(genital Chlamydia trachomatis infections,GCI)的患病人數(shù)在我國(guó)也在逐年增加。沙眼衣原體在女性患者經(jīng)過性傳播可以導(dǎo)致女性陰道炎、子宮內(nèi)膜炎、宮頸炎、輸卵管炎、非淋菌性尿道炎等,還可以造成盆腔炎、異位妊娠、自然流產(chǎn)、胎膜早破、早產(chǎn)和不孕等多種不良后果,通過垂直傳播會(huì)使分娩的感染患者傳染新生兒使其患上包涵體性的結(jié)膜炎[1]。目前因?yàn)椴划?dāng)應(yīng)用抗生素的治療Ct感染的問題日趨嚴(yán)重,引起Ct抗生素耐藥問題日漸突出,使可選擇用來治療Ct感染的敏感性抗生素越來越少,甚至無藥可用,大大增加了Ct感染治療難度。因此,全世界的專家學(xué)者均期望通過對(duì)沙眼衣原體感染的進(jìn)一步的深入研究,來發(fā)現(xiàn)新的預(yù)防或者治療Ct感染的方法,來解決抗生素耐藥的問題。許多研究學(xué)者們發(fā)現(xiàn)衣原體噬菌體感染宿主衣原體的途徑是與衣原體的原體結(jié)合而得以實(shí)現(xiàn)的,它能使衣原體停留在網(wǎng)狀體階段形成異常增大的網(wǎng)狀體,從而阻止衣原體發(fā)育為成熟原體,造成原體數(shù)量減少,使衣原體的感染性大大降低,甚至殺滅衣原體。被γ-干擾素抑制發(fā)育的衣原體也能產(chǎn)生異常增大的網(wǎng)狀體,這些網(wǎng)狀體的形態(tài)與感染噬菌體后衣原體形成的網(wǎng)狀體的形態(tài)相似,這從一個(gè)側(cè)面可以說明噬菌體具有在臨床上能夠治療Ct感染的可能。衣原體噬菌體衣殼蛋白Vp1是噬菌體主要的結(jié)構(gòu)蛋白,該蛋白對(duì)于黏附與植入衣原體起到重要的作用,其相對(duì)的分子質(zhì)量是61805,并且Vp1蛋白的保守性和特異性強(qiáng)。[目的]表達(dá)、鑒定、純化豚鼠結(jié)膜炎衣原體噬菌體phi CPG1的衣殼蛋白Vp1;在細(xì)胞培養(yǎng)E血清型和K血清型沙眼衣原體過程中用其進(jìn)行干預(yù),觀察其對(duì)Ct的抑制作用;將Vp1蛋白作用于生殖道感染E型Ct的小鼠模型,觀察其對(duì)模型小鼠陰道內(nèi)衣原體的作用;將Vp1蛋白作用于生殖道感染K型Ct的小鼠模型,觀察其對(duì)沙眼衣原體在模型小鼠生殖道內(nèi)上行感染的作用。[方法]表達(dá)、鑒定、純化豚鼠結(jié)膜炎衣原體噬菌體phi CPG1衣殼蛋白Vp1,分別將沙眼衣原體的標(biāo)準(zhǔn)株E、K分別和濃度為50μg/ml和100μg/ml Vp1于室溫下孵育3小時(shí),在單層致密Mc Coy細(xì)胞中接種孵育好的沙眼衣原體的標(biāo)準(zhǔn)株E、K,在整個(gè)沙眼衣原體的培養(yǎng)過程中均加入Vp1蛋白并保持其濃度為50μg/ml和100μg/ml,最后通過碘染色計(jì)數(shù)包涵體數(shù)量,觀察Vp1蛋白對(duì)Mc Coy細(xì)胞培養(yǎng)的沙眼衣原體增殖的抑制作用。構(gòu)建生殖道感染E型Ct的BALB/c雌性小鼠模型,經(jīng)陰道和肌肉注射給予Vp1蛋白干預(yù),于不同時(shí)間取小鼠陰道分泌物進(jìn)行細(xì)胞培養(yǎng)和實(shí)時(shí)定量PCR檢測(cè),并觀察小鼠陰道口紅腫情況,觀察Vp1蛋白對(duì)小鼠陰道內(nèi)沙眼衣原體增殖的作用。構(gòu)建生殖道感染K型Ct的BALB/c雌性小鼠模型,經(jīng)陰道給予Vp1蛋白干預(yù),于不同時(shí)間取小鼠子宮角、輸卵管和卵巢進(jìn)行病理切片、組織勻漿細(xì)胞培養(yǎng)檢測(cè),觀察Vp1蛋白對(duì)小鼠沙眼衣原體陰道感染引起的生殖系統(tǒng)炎癥的作用。[結(jié)果]本實(shí)驗(yàn)表達(dá)純化的噬菌體phi CPG1衣殼蛋白Vp1在沙眼衣原體細(xì)胞培養(yǎng)過程中能夠抑制其生長(zhǎng),E型和K型標(biāo)準(zhǔn)株的包涵體數(shù)量均有較明顯的減少。計(jì)數(shù)每個(gè)視野下2種標(biāo)準(zhǔn)株包涵體的數(shù)量,得到平均值,計(jì)算出100 ug/ml、50ug/ml濃度Vp1作用的抑制率,E型91%、79%;K型94%、70%。成功構(gòu)建了生殖道感染E型標(biāo)準(zhǔn)株Ct的BALB/c雌性小鼠模型,通過肌肉注射和陰道給予不同濃度的Vp1蛋白干預(yù),于不同時(shí)間取小鼠陰道分泌物行細(xì)胞培養(yǎng)和實(shí)時(shí)定量PCR進(jìn)行定量檢測(cè),肌注組與感染組、對(duì)照組差別無統(tǒng)計(jì)學(xué)意義,陰道干預(yù)高濃度組較低濃度組衣原體濃度低,低濃度組較感染組、對(duì)照組衣原體濃度低,差異有統(tǒng)計(jì)學(xué)意義。成功構(gòu)建了生殖道感染K型標(biāo)準(zhǔn)株Ct的BALB/c雌性小鼠模型,經(jīng)陰道給予Vp1蛋白干預(yù),于接種后35天取小鼠生殖系統(tǒng)組織,進(jìn)行HE染色病理觀察,評(píng)價(jià)子宮角、輸卵管、卵巢炎癥情況,干預(yù)組較感染組炎癥輕,差異有統(tǒng)計(jì)學(xué)意義。第7天感染組和干預(yù)組小鼠組織勻漿細(xì)胞培養(yǎng)結(jié)果:感染組和干預(yù)組小鼠的子宮角組織勻漿細(xì)胞培養(yǎng)結(jié)果均為陽性,輸卵管、卵巢組織勻漿細(xì)胞培養(yǎng)結(jié)果均為陰性。第35天感染組和干預(yù)組剩余小鼠子的宮角、輸卵管、卵巢組織勻漿細(xì)胞培養(yǎng)結(jié)果均為陰性,[結(jié)論]豚鼠結(jié)膜炎衣原體噬菌體phi CPG1衣殼蛋白Vp1在體外細(xì)胞培養(yǎng)中對(duì)沙眼衣原體增殖的抑制作用明顯。Vp1蛋白對(duì)感染小鼠陰道內(nèi)沙眼衣原體的增殖具有抑制作用,縮短了小鼠陰道內(nèi)沙眼衣原體的自凈時(shí)間。Vp1蛋白具有減輕沙眼衣原體感染小鼠引起的上生殖道炎癥的作用。為進(jìn)一步研究臨床上沙眼衣原體感染的治療提供新的思路。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R374.1

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7 凹凸;沙眼衣原體可致不孕[N];大眾衛(wèi)生報(bào);2001年

8 江錦琦;女性感染沙眼衣原體可致不孕[N];中國(guó)中醫(yī)藥報(bào);2001年

9 林國(guó)瑞;女性濫交易染衣原體[N];醫(yī)藥養(yǎng)生保健報(bào);2007年

10 于泓珍;淋球菌與沙眼衣原體引起的感染[N];農(nóng)村醫(yī)藥報(bào)（漢）;2007年

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