本文選題：結核分枝桿菌 + 復蘇促進因子��； 參考：《安徽醫(yī)科大學》2014年博士論文

【摘要】：結核病(Tuberculosis, TB)是由結核分枝桿菌(Mycobacterium tuberculosis, Mtb)感染引起的慢性傳染病,目前仍然嚴重威脅著人類健康。目前對于結核病的控制情況還不盡如人意,主要原因在于人群中(尤其是發(fā)展中國家)大量潛伏感染者(Latently TB infected individuals,LTBI)的存在,其中約10%感染者在一定時間內可發(fā)展成為活動性結核病,而大多數則始終保持LTBI的狀態(tài)。人們對疾病最終轉歸結果的影響因素進行了很多的研究,是否發(fā)生感染主要與結核暴露的強度、頻率以及持續(xù)時間有關,而發(fā)生感染后是否進展為活動性的結核病則主要取決于結核暴露者的自身身體條件以及結核暴露的方式。與TB病人的直接接觸,其風險明顯要大于一般人群。接觸了空氣中含有Mtb的飛沫后,一些人將會被感染并發(fā)生TB。疾病有可能在6個星期之內發(fā)生,也有可能在數年之后。如果我們能夠在大量的LTBI感染者中篩選出可能發(fā)展為TB的高危人群,那么將有益于對TB的控制,因為對LTBI進行持續(xù)治療是有效的。 TB的傳播主要是通過TB病人釋放出的含有傳染性病菌的飛沫。有效和及時的治療可以使這些病人盡快的失去傳染性,因此對控制TB也非常關鍵。治療和控制則主要依賴及時準確的診斷。然而,由于Mtb具有生長緩慢的特性,因此常規(guī)的培養(yǎng)需要耗費4-8周的時間。 復蘇促進因子(Resuscitation-promoting factor, Rpf)是細菌分泌的具有生長因子特性的蛋白。Mtb的Rpf是由生長旺盛期的細菌分泌的,其主要特點是能夠通過其溶菌酶和肽聚糖水解酶的特性在體內和體外復蘇非復制型的Mtb。Rpf有5個同源蛋白質,包括RpfA (Rv0867c), RpfB (Rv1009),RpfC (Rv1884c),RpfD(Rv2389c)和RpfE (Rv2450c)。 已有的結果表明,通過密切接觸而導致的LTBI個體相對于TB病人和健康人群,即使結核暴露發(fā)生在數年以前,Rpf刺激后仍然能夠產生較強的T細胞反應。然而,對于那些由于低強度的社區(qū)暴露而引起的LTBI個體來說,對Rpf的反應就不得而知。對于不同暴露水平而導致的LTBI個體,其針對Rpf的免疫反應是否存在差異是很值得研究的。如果存在差異,那么針對Rpf的免疫反應就具有預測疾病轉歸的潛力,將有利于TB的控制。 由于在TB病人的痰液中發(fā)現了依賴于Rpf才能被培養(yǎng)出來的Mtb存在,所以Rpf被認為具有提升臨床Mtb培養(yǎng)實驗效果的潛力。但是這種潛力是否具有真正的臨床應用價值,始終沒有得到確認。因此,其能否有效地縮短標本培養(yǎng)時間,提高培養(yǎng)陽性率,滿足臨床的需要,也是很值得研究的。本課題主要分為兩個部分：一、Rpf在結核病接觸者篩查中的應用。二、Rpf對結核桿菌培養(yǎng)敏感性的影響。 一、復蘇促進因子在結核病接觸者篩查中的應用 我們選取了因不同暴露程度而導致的潛伏感染患者,實驗共分為4組：肺結核患者組(Pulmonary tuberculosis patients,PTB),社區(qū)暴露組(Community exposure,CE),密切接觸組(Household contacts,HHC),健康對照組(Healthy control, HC)。提取外周血單個核細胞后分別用RpfA或RpfD刺激,取刺激后的細胞培養(yǎng)上清采用酶聯免疫吸附實驗(Enzyme-linked immunosorbent assay,ELISA)測定γ-干擾素(Interferon-γ, IFN-γ)、腫瘤壞死因子-α (Tumor necrosis factor, TNF-α)、超氧陰離子(Superoxide anion, O2-)。采用Griess法檢測一氧化氮(Nitric oxide, NO).收取細胞后采用實時熒光定量PCR (Real time quantity polymerase chain reaction, RT-qPCR)實驗檢測IFN-γ及TNF-α的轉錄水平；流式細胞術檢測CD4+IFN-γ+細胞數比例。 通過IFN-γ釋放試驗(Interferon-y release assays, IGRAs)和Mtb-IgG檢測的雙陽性結果明確了CE和HHC中的潛伏感染者,分別命名為LTBI-CE和LTBI-HHC。結果顯示,LTBI-HHC和LTBI-CE組中,TB-IGRA實驗所釋放的IFN-y量沒有明顯的差別(P0.05)。各組PBMC經RpfA或RpfD刺激后,IFN-y的釋放量存在明顯差異。對于RpfA, LTBI-HHC組高于其他組(P0.01), LTBI-CE組高于PTB組(P0.05)；對于RpfD, LTBI-HHC組高于其他組(P0.01), LTBI-CE組與PTB組無差異(P0.05)。各組間TNF-α,O2-和NO釋放量無明顯差異(P0.05)。提取了Rpf刺激后的PBMC總RNA,經逆轉錄后進行RT-qPCR實驗。各組PBMC經RpfA或RpfD刺激后,IFN-γ的轉錄存在明顯差異。對于RpfA, LTBI-HHC組高于其他組(P0.01)；對于RpfD, LTBI-HHC組高于LTBI-CE和HC組(P0.01),PTB組高于HC組(P0.05)。各組間TNF-α的轉錄水平無明顯差異(P0.05)。利用流式細胞術檢測產生IFN-γ的CD4+細胞比例。對于RpfA, LTBI-HHC組高于LTBI-CE和HC組(P0.05)；對于RpfD, LTBI-HHC組高于LTBI-CE、PTB組(P0.05)和HC組(P0.01)。各組間CD4+TNF-a+的細胞頻率無明顯差異(P0.05)。另外,我們比較了PTB, HHC和CE組中TB-IGRA陽性個體經Rpf誘導后IFN-γ釋放量及CD4+IFN-y+比例。結果顯示RpfA或RpfD刺激HHC-IGRA(+)后可釋放較多的IFN-γ,但CD4+IFN-γ+細胞的比例沒有明顯的變化(P0.05)。 總之,在中國這樣的TB高發(fā)國家,潛在的TB暴露可能隨時隨地發(fā)生。我們的結果顯示低強度的CE暴露可能不足以誘發(fā)針對RpfA和RpfD的免疫反應。而在LTBI-HHC中,高強度的暴露水平足以使機體誘發(fā)此反應。但是對于ESAT-6和CFP-10這些具有強免疫原性的蛋白來說,LTBI-CE的暴露水平也可以誘發(fā)機體的免疫反應。所以,針對RpfA和RpfD的免疫反應可以區(qū)別不同的暴露水平,而TB-IGRA則不能。而不同的暴露水平與日后的疾病轉歸密切相關,因此,針對RpfA和RpfD的IFN-γ反應具有在接觸者篩查研究中用以評判暴露水平和疾病進展風險的應用價值。 二、復蘇促進因子對結核桿菌培養(yǎng)敏感性的影響 首先,我們成功獲得了重組的可溶性的RpfB和RpfE蛋白。為了鑒定Rpf對Mtb培養(yǎng)敏感性的影響,我們在臨床上用于培養(yǎng)Mtb的培養(yǎng)基內加入重組RpfB或RpfE蛋白,與標準培養(yǎng)體系的結果進行對比。另外,我們利用熱處理的方法對樣本進行處理,并利用金胺O尼羅紅復合染色判定熱處理對樣本中非復制型(Non-replicating persistence, NRP)并含有脂滴的Mtb (Lipid body-positive Mtb, LBP-Mtb)的作用。我們同時收集了處于生長旺盛期的臨床Mtb菌種,使用含有RpfB或RpfE的培養(yǎng)基對其進行培養(yǎng),用以判定Rpf對復制型Mtb的影響。 經過對臨床23例Mtb痰涂片陽性樣本的培養(yǎng),我們首先發(fā)現培養(yǎng)時間與Ziehl-Neelsen抗酸染色結果之間無明顯相關性(R2=0.35)。在培養(yǎng)基中加入RpfB和RpfE后,23份標本的總體培養(yǎng)時間并沒有得到明顯的縮短。但在培養(yǎng)時間大于20天樣本中,相對于標準的培養(yǎng)體系,RpfB和RpfE均可以明顯的縮短培養(yǎng)時間(P0.05)。分別使用60℃,63℃和70℃熱處理臨床標本10min,使用金胺O尼羅紅復合染色鑒定LBP-Mtb。發(fā)現經63℃和70℃熱處理10min后,金胺O尼羅紅復合染色未能觀察到細菌存在。而經60℃處理10min后,樣本中LBP-Mtb的比例明顯增高(P0.01),且樣本中剩余Mtb的抗酸性也普遍偏低。另外,經60℃作用10mmin后,標準培養(yǎng)體系均無法檢測到Mtb。培養(yǎng)基中添加RpfB或RpfE后,其中分別有4例和3例熱處理后的樣本也可以被培養(yǎng)出來,而它們正是那些先前能夠被Rpf影響的樣本。我們對處于生長旺盛期的Mtb菌種按照104CFU/ml,106CFU/ml和108CFU/ml的濃度分別接種至含RpfB或RpfE終濃度為2nM,20nM和200nM的培養(yǎng)系統中,發(fā)現RpfB和RpfE對臨床標本中復制菌的培養(yǎng)時間沒有影響。 所以,我們發(fā)現60℃熱處理10min可以有效殺滅樣本中的復制菌,但不影響LBP-Mtb。那些在標準培養(yǎng)體系下培養(yǎng)時間較長的樣本(大于20天),RpfB和RpfE可以縮短其培養(yǎng)時間；并且在熱處理后,可以使這些樣本能夠重新被檢測出來,而這很有可能是因為其成功復蘇了經熱處理后仍然存活在樣本中的LBP-Mtb。因此,我們發(fā)現Rpf可以提高那些在正常培養(yǎng)條件下培養(yǎng)時間較長樣本的檢測敏感性,說明其具有在臨床Mtb檢測中的應用潛力。但是仍需要更大樣本量的研究來進一步驗證。
[Abstract]:Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) infection. It is still a serious threat to human health. At present, the control of tuberculosis is not satisfactory. The main reason is the large number of latent infection (Latently TB in) in the population (especially in developing countries). The presence of fected individuals, LTBI), of which about 10% of the infected people can develop into active TB for a certain period of time, and most of them remain in the state of LTBI. Many studies have been conducted on the factors affecting the outcome of the final outcome of the disease, whether the infection is mainly associated with the intensity, frequency, and duration of tuberculosis exposure. The risk of progressing to active tuberculosis after infection depends mainly on the physical condition of the person who is exposed to tuberculosis and the way of tuberculosis exposure. Direct contact with TB patients is significantly greater than that of the general population. After exposure to Mtb droplets in the air, some people will be infected and TB. is likely to be in 6 It will happen within a week and may be a few years later. If we can screen a high risk population that may develop into TB among a large number of LTBI infected people, it will be beneficial to the control of TB, since continuous treatment of LTBI is effective.
The spread of TB is mainly through droplets of infectious pathogens released by TB patients. Effective and timely treatment can make these patients lose contagion as soon as possible. Therefore, it is also critical to control TB. Treatment and control are mainly dependent on timely and accurate diagnosis. However, because of the slow growth of Mtb, conventional cultivation It takes 4-8 weeks.
Resuscitation-promoting factor (Rpf) is the growth factor protein.Mtb secreted by bacteria, which is secreted by the growing bacteria, which is characterized by the ability to recover 5 homologous proteins from the non replicating Mtb.Rpf in vivo and in vitro through the characteristics of its lysozyme and peptidoglycan hydrolase. It includes RpfA (Rv0867c), RpfB (Rv1009), RpfC (Rv1884c), RpfD (Rv2389c) and RpfE (Rv2450c).
The results have shown that LTBI individuals, resulting from close contact with TB patients and healthy people, can still produce stronger T cell responses after Rpf stimulation years ago, even if the TB exposure occurred several years ago. However, the response to Rpf is unknown to those of LTBI individuals caused by low intensity community exposure. For LTBI individuals with different exposure levels, it is worth studying whether there is a difference in the immune response to Rpf. If there is a difference, the immune response to Rpf will have the potential to predict the outcome of the disease and will be beneficial to the control of the TB.
Because of the presence of Mtb in the sputum of patients with TB, which is dependent on Rpf to be cultured, Rpf is considered to have the potential to improve the effect of clinical Mtb culture. But whether this potential has real clinical application value has never been confirmed. Therefore, it can effectively shorten the time of culture and raise the culture Yang. The sex rate, which meets the needs of the clinic, is also worthy of study. This topic is divided into two parts: first, the application of Rpf in the screening of TB contacts. Two, the effect of Rpf on the sensitivity of TB culture.
1. Application of recovery promoting factor in screening of TB contacts.
We selected the patients with latent infection caused by different exposure levels. The experiment was divided into 4 groups: Pulmonary tuberculosis patients (PTB), community exposure group (Community exposure, CE), close contact group (Household contacts, HHC), healthy control group (Healthy control,). Don't use RpfA or RpfD to stimulate the cell culture supernatant after the stimulation with the enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) for the determination of interferon (Interferon- gamma, IFN- gamma), tumor necrosis factor - alpha (Tumor necrosis factor, TNF- alpha), and superoxide anion. Tric oxide, NO). After collecting cells, real time fluorescence quantitative PCR (Real time quantity polymerase chain reaction, RT-qPCR) test was used to detect the transcriptional level of IFN- gamma and alpha, and flow cytometry was used to detect the number of gamma + cells.
The double positive results of the IFN- gamma release test (Interferon-y release assays, IGRAs) and Mtb-IgG detected the latent infection in CE and HHC, which were named as LTBI-CE and LTBI-HHC. respectively. The release amount of IFN-y was significantly different. For RpfA, group LTBI-HHC was higher than that of other groups (P0.01), LTBI-CE group was higher than PTB group (P0.05); for RpfD, LTBI-HHC group was higher than other groups (P0.01), LTBI-CE group was not different from PTB group. After RpfA or RpfD stimulation, the transcription of IFN- gamma was significantly different. For RpfA, the LTBI-HHC group was higher than the other groups (P0.01), and for RpfD, the LTBI-HHC group was higher than LTBI-CE and HC groups. There was no significant difference in the transcriptional level of the IFN- group. The ratio of CD4+ cells to IFN- gamma was measured. For RpfA, group LTBI-HHC was higher than that of LTBI-CE and HC group (P0.05), and for RpfD, the LTBI-HHC group was higher than LTBI-CE, PTB group (P0.05) and group. The results showed that RpfA or RpfD could release more IFN- gamma after stimulation of HHC-IGRA (+), but the proportion of CD4+IFN- gamma + cells did not change significantly (P0.05). CD4+IFN-y+ and IFN- were also detected.
In TB countries like China, potential TB exposure may occur anytime and anywhere. Our results show that low intensity CE exposure may not be sufficient to induce immune responses to RpfA and RpfD. In LTBI-HHC, high intensity exposure levels are sufficient to induce the body to induce this reaction. But there is a strong immunity to ESAT-6 and CFP-10. The exposure level of LTBI-CE can also induce the immune response of the body. Therefore, the immune response to RpfA and RpfD can distinguish different levels of exposure, while TB-IGRA is not. The different levels of exposure are closely related to the future outcome of the disease. Therefore, the IFN- gamma response to RpfA and RpfD is screened by the contact screen. It is used to assess the exposure level and the risk of disease progression.
Two, the effect of resuscitation promoting factor on the sensitivity of Mycobacterium tuberculosis culture.
First, we successfully obtained the recombinant soluble RpfB and RpfE protein. In order to identify the effect of Rpf on the sensitivity of Mtb culture, we use the recombinant RpfB or RpfE protein in the culture base of the culture of Mtb to compare the results of the standard culture system. In addition, we use the heat treatment method to deal with the samples and profit. The effect of heat treatment on non replicating (Non-replicating persistence, NRP) and Mtb (Lipid body-positive Mtb, LBP-Mtb) containing lipid droplets in the sample was determined by the compound coloring of gold amine O Nile. We also collected the clinical Mtb strains at the peak growth period, and used the culture medium containing RpfB or RpfE. The effect of F on the replicative Mtb.
After training 23 cases of Mtb sputum smear positive samples, we first found that there was no significant correlation between the culture time and the results of Ziehl-Neelsen anti acid staining (R2=0.35). After adding RpfB and RpfE to the medium, the total culture time of the 23 specimens was not significantly shortened, but the culture time was greater than 20 days. The standard culture system, RpfB and RpfE could significantly shorten the culture time (P0.05). The clinical specimens were treated with 60 C, 63 and 70 C, respectively, to heat treatment of clinical specimens, 10min. Using gold amine O Nile Red compound staining, LBP-Mtb. found that after 63 and 70 centigrade heat treatment 10min, the presence of gold amine O Nile Red complex staining failed to observe the existence of bacteria. And the treatment of 10m at 60 centigrade After in, the proportion of LBP-Mtb in the sample increased significantly (P0.01), and the acid resistance of the remaining Mtb in the sample was generally low. In addition, after 10mmin at 60 C, the standard culture system could not detect the addition of RpfB or RpfE in the Mtb. medium, of which 4 and 3 samples of heat treated, respectively, could be cultivated, and they were those Samples that were previously affected by Rpf were inoculated to the growth period of Mtb strains at the concentration of 104CFU/ml, 106CFU/ml and 108CFU/ml to 2nM, 20nM and 200nM in 2nM, 20nM and 200nM containing RpfB or RpfE concentrations, and found that RpfB and RpfE had no effect on the incubation time of the replicating bacteria in the clinical specimens.
Therefore, we found that the 60 C heat treatment 10min can effectively kill the replicating bacteria in the sample, but does not affect the LBP-Mtb. and the longer samples (more than 20 days) under the standard culture system (more than 20 days), RpfB and RpfE can shorten the incubation time; and after heat treatment, these samples can be redetected, and this is very possible. Because of its successful recovery of LBP-Mtb., which is still alive in the sample after heat treatment, we have found that Rpf can improve the detection sensitivity of those samples with longer incubation time under normal culture conditions, indicating its potential for clinical Mtb detection. However, a larger sample size is still needed to be further verified.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R52

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