复苏促进因子在结核病接触者筛查及临床诊断中的应用

发布时间：2018-05-21 18:26

本文选题：结核分枝杆菌 + 复苏促进因子　；参考：《安徽医科大学》2014年博士论文

【摘要】：结核病(Tuberculosis, TB)是由结核分枝杆菌(Mycobacterium tuberculosis, Mtb)感染引起的慢性传染病,目前仍然严重威胁着人类健康。目前对于结核病的控制情况还不尽如人意,主要原因在于人群中(尤其是发展中国家)大量潜伏感染者(Latently TB infected individuals,LTBI)的存在,其中约10%感染者在一定时间内可发展成为活动性结核病,而大多数则始终保持LTBI的状态。人们对疾病最终转归结果的影响因素进行了很多的研究,是否发生感染主要与结核暴露的强度、频率以及持续时间有关,而发生感染后是否进展为活动性的结核病则主要取决于结核暴露者的自身身体条件以及结核暴露的方式。与TB病人的直接接触,其风险明显要大于一般人群。接触了空气中含有Mtb的飞沫后,一些人将会被感染并发生TB。疾病有可能在6个星期之内发生,也有可能在数年之后。如果我们能够在大量的LTBI感染者中筛选出可能发展为TB的高危人群,那么将有益于对TB的控制,因为对LTBI进行持续治疗是有效的。 TB的传播主要是通过TB病人释放出的含有传染性病菌的飞沫。有效和及时的治疗可以使这些病人尽快的失去传染性,因此对控制TB也非常关键。治疗和控制则主要依赖及时准确的诊断。然而,由于Mtb具有生长缓慢的特性,因此常规的培养需要耗费4-8周的时间。复苏促进因子(Resuscitation-promoting factor, Rpf)是细菌分泌的具有生长因子特性的蛋白。Mtb的Rpf是由生长旺盛期的细菌分泌的,其主要特点是能够通过其溶菌酶和肽聚糖水解酶的特性在体内和体外复苏非复制型的Mtb。Rpf有5个同源蛋白质,包括RpfA (Rv0867c), RpfB (Rv1009),RpfC (Rv1884c),RpfD(Rv2389c)和RpfE (Rv2450c)。已有的结果表明,通过密切接触而导致的LTBI个体相对于TB病人和健康人群,即使结核暴露发生在数年以前,Rpf刺激后仍然能够产生较强的T细胞反应。然而,对于那些由于低强度的社区暴露而引起的LTBI个体来说,对Rpf的反应就不得而知。对于不同暴露水平而导致的LTBI个体,其针对Rpf的免疫反应是否存在差异是很值得研究的。如果存在差异,那么针对Rpf的免疫反应就具有预测疾病转归的潜力,将有利于TB的控制。由于在TB病人的痰液中发现了依赖于Rpf才能被培养出来的Mtb存在,所以Rpf被认为具有提升临床Mtb培养实验效果的潜力。但是这种潜力是否具有真正的临床应用价值,始终没有得到确认。因此,其能否有效地缩短标本培养时间,提高培养阳性率,满足临床的需要,也是很值得研究的。本课题主要分为两个部分：一、Rpf在结核病接触者筛查中的应用。二、Rpf对结核杆菌培养敏感性的影响。一、复苏促进因子在结核病接触者筛查中的应用我们选取了因不同暴露程度而导致的潜伏感染患者,实验共分为4组：肺结核患者组(Pulmonary tuberculosis patients,PTB),社区暴露组(Community exposure,CE),密切接触组(Household contacts,HHC),健康对照组(Healthy control, HC)。提取外周血单个核细胞后分别用RpfA或RpfD刺激,取刺激后的细胞培养上清采用酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)测定γ-干扰素(Interferon-γ, IFN-γ)、肿瘤坏死因子-α (Tumor necrosis factor, TNF-α)、超氧阴离子(Superoxide anion, O2-)。采用Griess法检测一氧化氮(Nitric oxide, NO).收取细胞后采用实时荧光定量PCR (Real time quantity polymerase chain reaction, RT-qPCR)实验检测IFN-γ及TNF-α的转录水平；流式细胞术检测CD4+IFN-γ+细胞数比例。通过IFN-γ释放试验(Interferon-y release assays, IGRAs)和Mtb-IgG检测的双阳性结果明确了CE和HHC中的潜伏感染者,分别命名为LTBI-CE和LTBI-HHC。结果显示,LTBI-HHC和LTBI-CE组中,TB-IGRA实验所释放的IFN-y量没有明显的差别(P0.05)。各组PBMC经RpfA或RpfD刺激后,IFN-y的释放量存在明显差异。对于RpfA, LTBI-HHC组高于其他组(P0.01), LTBI-CE组高于PTB组(P0.05)；对于RpfD, LTBI-HHC组高于其他组(P0.01), LTBI-CE组与PTB组无差异(P0.05)。各组间TNF-α,O2-和NO释放量无明显差异(P0.05)。提取了Rpf刺激后的PBMC总RNA,经逆转录后进行RT-qPCR实验。各组PBMC经RpfA或RpfD刺激后,IFN-γ的转录存在明显差异。对于RpfA, LTBI-HHC组高于其他组(P0.01)；对于RpfD, LTBI-HHC组高于LTBI-CE和HC组(P0.01),PTB组高于HC组(P0.05)。各组间TNF-α的转录水平无明显差异(P0.05)。利用流式细胞术检测产生IFN-γ的CD4+细胞比例。对于RpfA, LTBI-HHC组高于LTBI-CE和HC组(P0.05)；对于RpfD, LTBI-HHC组高于LTBI-CE、PTB组(P0.05)和HC组(P0.01)。各组间CD4+TNF-a+的细胞频率无明显差异(P0.05)。另外,我们比较了PTB, HHC和CE组中TB-IGRA阳性个体经Rpf诱导后IFN-γ释放量及CD4+IFN-y+比例。结果显示RpfA或RpfD刺激HHC-IGRA(+)后可释放较多的IFN-γ,但CD4+IFN-γ+细胞的比例没有明显的变化(P0.05)。总之,在中国这样的TB高发国家,潜在的TB暴露可能随时随地发生。我们的结果显示低强度的CE暴露可能不足以诱发针对RpfA和RpfD的免疫反应。而在LTBI-HHC中,高强度的暴露水平足以使机体诱发此反应。但是对于ESAT-6和CFP-10这些具有强免疫原性的蛋白来说,LTBI-CE的暴露水平也可以诱发机体的免疫反应。所以,针对RpfA和RpfD的免疫反应可以区别不同的暴露水平,而TB-IGRA则不能。而不同的暴露水平与日后的疾病转归密切相关,因此,针对RpfA和RpfD的IFN-γ反应具有在接触者筛查研究中用以评判暴露水平和疾病进展风险的应用价值。二、复苏促进因子对结核杆菌培养敏感性的影响首先,我们成功获得了重组的可溶性的RpfB和RpfE蛋白。为了鉴定Rpf对Mtb培养敏感性的影响,我们在临床上用于培养Mtb的培养基内加入重组RpfB或RpfE蛋白,与标准培养体系的结果进行对比。另外,我们利用热处理的方法对样本进行处理,并利用金胺O尼罗红复合染色判定热处理对样本中非复制型(Non-replicating persistence, NRP)并含有脂滴的Mtb (Lipid body-positive Mtb, LBP-Mtb)的作用。我们同时收集了处于生长旺盛期的临床Mtb菌种,使用含有RpfB或RpfE的培养基对其进行培养,用以判定Rpf对复制型Mtb的影响。经过对临床23例Mtb痰涂片阳性样本的培养,我们首先发现培养时间与Ziehl-Neelsen抗酸染色结果之间无明显相关性(R2=0.35)。在培养基中加入RpfB和RpfE后,23份标本的总体培养时间并没有得到明显的缩短。但在培养时间大于20天样本中,相对于标准的培养体系,RpfB和RpfE均可以明显的缩短培养时间(P0.05)。分别使用60℃,63℃和70℃热处理临床标本10min,使用金胺O尼罗红复合染色鉴定LBP-Mtb。发现经63℃和70℃热处理10min后,金胺O尼罗红复合染色未能观察到细菌存在。而经60℃处理10min后,样本中LBP-Mtb的比例明显增高(P0.01),且样本中剩余Mtb的抗酸性也普遍偏低。另外,经60℃作用10mmin后,标准培养体系均无法检测到Mtb。培养基中添加RpfB或RpfE后,其中分别有4例和3例热处理后的样本也可以被培养出来,而它们正是那些先前能够被Rpf影响的样本。我们对处于生长旺盛期的Mtb菌种按照104CFU/ml,106CFU/ml和108CFU/ml的浓度分别接种至含RpfB或RpfE终浓度为2nM,20nM和200nM的培养系统中,发现RpfB和RpfE对临床标本中复制菌的培养时间没有影响。所以,我们发现60℃热处理10min可以有效杀灭样本中的复制菌,但不影响LBP-Mtb。那些在标准培养体系下培养时间较长的样本(大于20天),RpfB和RpfE可以缩短其培养时间；并且在热处理后,可以使这些样本能够重新被检测出来,而这很有可能是因为其成功复苏了经热处理后仍然存活在样本中的LBP-Mtb。因此,我们发现Rpf可以提高那些在正常培养条件下培养时间较长样本的检测敏感性,说明其具有在临床Mtb检测中的应用潜力。但是仍需要更大样本量的研究来进一步验证。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R52

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