肠道菌群及其代谢产物短链脂肪酸和肠黏膜损伤标记物在HIV感染组和健康组的对比研究

发布时间：2018-06-15 01:54

本文选题：肠道菌群 + 短链脂肪酸　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:艾滋病是一种常见的性传播疾病,当HIV病毒感染后会引起严重的细胞免疫缺陷并导致各种机会性感染,严重危害人类健康。临床上绝大多数发展至AIDS期的患者会出现慢性腹泻、腹痛等消化道症状,研究证明可能与肠道黏膜损伤、肠道菌群易位、肠道微生态失衡等病理机制有关,但具体机制尚不明确。正常状况下,机体与微生物群间存在互利共惠的共生关系,肠道微生物群及代谢产物在促进营养物质消化吸收、维持肠道正常生理功能,调节机体免疫等众多生命活动起重要作用。本实验的研究对象将选择尚未发展至AIDS期的HIV感染者,观察其肠道微生态及肠道黏膜损伤情况,以及探究其肠道微生态与肠黏膜损伤、细胞免疫状态是否具有相关性。本实验将从以下几个方面进行研究:1.对比HIV感染者和健康人群肠道菌群分布及其重要代谢产物短链脂肪酸的差异;2.对比HIV感染者与健康人群肠粘膜损伤标记物的差异;3.分析肠道菌群分布及不同短链脂肪酸与肠粘膜损伤标记物有无相关性;4.分析肠道菌群分布及不同短链脂肪酸与HIV感染者的CD4+、CD8+T细胞数和CD4/CD8比值有无相关性;从而为进一步完善HIV感染引起肠道损伤的机制以及肠道微生态相关的治疗方法用于艾滋病的预防和治疗打下理论基础。方法:收集来自于传染病医院的符合入选标准的HIV感染者15例以及符合入选标准的健康者10例,所有入选对象均在实验前签订知情同意书,并获四川省人民医院伦理道德委员会批准。采集两组人群粪便标本后-80℃低温保存,用高通量测序法检测两组人群肠道菌群的分布情况及其差异;用气相色谱-质谱法(gc-ms)联合分析两组人群粪便中各短链脂肪酸的含量;采集血液标本,离心后用酶联免疫吸附法(elisa)测定两组人群血清中能够反映肠粘膜损伤情况的循环标记肠型脂肪酸结合蛋白(i-fabp)和d-乳酸的含量;用流式细胞术(fum)检测反映hiv感染者细胞免疫状态的cd4+、cd8+t细胞数和cd4/cd8比值。最后使用spss19.0统计软件,采用mann-whitney检验,t检验进行分析细菌种属或者otus组间统计学差异分析,采用person法进行相关性分析,p0.05提示差异有统计学差义。结果:1.肠道菌群分布:和健康对照组相比,在门水平上,hiv感染组肠道的拟杆菌门、厚壁菌门、变形菌门、梭菌门、放线菌门不存在显著差异;在纲水平上,只有negativicutes菌显著增多;在科水平上,理研菌rikenellaceae和分支杆菌microbacteriaceae菌显著减少,莫拉菌moraxellaceae显著增多;在属水平上,罗氏菌roseburia、毛螺旋菌lachnospiraceae、alistipes菌、瘤胃球菌ruminococcaceae明显减少;嗜冷杆菌psychrobacter明显增多。2.各短链脂肪酸定量:和健康对照组相比,hiv感染组肠道丁酸和戊酸含量明显减少,其他短链脂肪酸含量不存在差异。3.血清肠道黏膜损伤标记物:hiv感染组和健康对照组相比,血清肠道黏膜损伤标记物i-fabp和d-乳酸值不存在差异。4.细胞免疫:hiv感染者的cd4+t细胞数目200/μl,cd8+t细胞升高,cd4/cd8比值下降,符合尚未发展至aids期hiv感染者的细胞免疫状态。5.肠道菌群分布与丁酸和戊酸的相关性分析:在科水平上,hiv感染者肠道丁酸含量的降低主要与理研菌科rikenellaceae、瘤胃球菌科ruminococcaceae呈正相关。戊酸含量降低主要与理研菌科rikenellaceae、消化球菌科peptococcaceae、瘤胃球菌科ruminococcaceae呈正相关。在属水平上,丁酸含量降低主要与理研菌科的alistipes属;毛螺旋菌科的roseburia属、blautia属、lachnospiraceaeucg-005属、lachnospiraceaefcs020group属;瘤胃球菌科的ruminococcaceaeucg-003属、ruminococcus1属呈正相关。戊酸含量降低主要与理研菌科的alistipes属、瘤胃球菌科的eubacteriumcoprostanoligenesgroup属、ruminococcaceaeucg-002属、ruminococcaceaeucg-003属、ruminococcaceaenk4a214group属、ruminococcaceae1属;毛螺旋菌科的eubacteriumruminantiumgroup属;消化球菌科的peptococcus属呈正相关。6.肠道菌群与cd4+、cd8+t细胞数及cd4/cd8比值的相关性:我们未发现与cd4+t细胞呈相关性的菌群。与cd8+t细胞呈正相关的主要菌群有:变形菌门下的oxalobacer、undibacterium、burkholderia、achromobacter、ramlibacter属;拟杆菌门下sphingobacteriia科。7.短链脂肪酸与CD4+、CD8+T细胞数及CD4/CD8比值的相关性:CD4+T细胞、CD4/CD8比值与乙酸呈负相关,CD8+T细胞与戊酸呈正相关。结论:1.HIV感染者在尚未进入艾滋病期,发生消化道症状前,肠道微生态环境可能已经与健康者存在差异;2.HIV感染者的重要肠道粘膜保护因素较健康者降低,如罗氏菌属Roseburia、毛螺旋菌属Lachnospiraceae、Alistipes菌属、瘤胃球菌属Ruminococcaceae、理研菌科Rikenellaceae等的菌群负荷降低,相关细菌的代谢产物丁酸和戊酸水平下降;3.HIV感染者肠道粘膜的破坏性因素增加,如变形菌门中的莫拉菌科Moraxellaceae和冷杆菌属Psychrobacter丰度在肠腔中显著升高,推测其可能与菌群易位密切相关,从而诱导持续的炎症反应,加重HIV病程的发展;4.尚未发展至AIDS期的HIV感染者的细胞免疫水平与肠道菌群分布不一定具有相关性;5.尚未发展至AIDS期的HIV感染者的细胞免疫水平与肠道各短链脂肪酸SCFAs水平相关性不明确。
[Abstract]:Objective: AIDS is a common type of sexually transmitted disease. When HIV virus is infected, it can cause serious cellular immune deficiency and cause various opportunistic infections, which seriously harm human health. Most of the patients who develop to AIDS stage may have chronic diarrhea, abdominal pain and other digestive tract symptoms, and the study has proved that it may be associated with intestinal mucosal damage, intestinal mucosa damage, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa injury, intestinal mucosa damage, intestinal mucosa damage, intestinal mucosa damage, intestinal mucosa damage, intestinal mucosa damage, intestinal mucosa damage, intestinal mucosa damage, intestinal mucosa damage, intestinal mucosa damage, intestinal mucosa damage The pathomechanism of the translocation of the flora and the imbalance of the intestinal microflora is not clear. Under normal conditions, there is a symbiotic relationship between the organism and the microorganism. The intestinal microbes and the metabolites can promote the digestion and absorption of nutrients, maintain the normal physiological function of the intestinal tract, and regulate the many life activities of the body. The subjects of this study will choose HIV infected people who have not developed to AIDS stage, observe the intestinal microflora and intestinal mucosal damage, and explore whether the intestinal microflora and intestinal mucosa damage, and whether the cell immune state is related. This experiment will be studied from the following aspects: 1. comparison of HIV infected persons and The distribution of intestinal microflora and the difference between the important metabolites of short chain fatty acids in the healthy population; 2. compared the difference of intestinal mucosal damage markers between HIV infected people and healthy people; 3. analysis of the distribution of intestinal flora and the correlation between different short chain fatty acids and intestinal mucosal damage markers; 4. analysis of the distribution of intestinal microflora and different short chain fatty acids and HIV There is no correlation between the number of CD4+, CD8+T cells and the ratio of CD4/CD8 in the infected people, which provides a theoretical basis for further improving the mechanism of intestinal damage caused by HIV infection and the treatment of the intestinal microecology related to the prevention and treatment of AIDS. Methods: 15 cases of HIV infected with the criteria of admission from infectious diseases hospitals were collected. And 10 healthy persons who met the standard of admission, all the selected subjects signed informed consent before the experiment, and approved by the ethics committee of Sichuan Provincial People's Hospital. Two groups of fecal specimens were collected at -80 C and preserved at low temperature. The distribution and difference of intestinal microflora of two groups of people were detected by high flux sequencing method, and gas chromatography-quality was used. The content of the short chain fatty acids in the feces of two groups of people was analyzed by GC-MS. Blood samples were collected, and after centrifugation, the contents of circulating labeled intestinal fatty acid binding protein (I-FABP) and d- lactic acid in the serum of two groups were measured by enzyme linked immunosorbent assay (ELISA); the flow cytometry (fum) was used to detect the content of the intestinal mucosa injury. Cd4+, cd8+t cell number and cd4/cd8 ratio in the cell immune state of HIV infected people. Finally, using the spss19.0 statistics software, using Mann-Whitney test, t test to analyze the statistical difference between the bacteria species and the OTUs group, and using person method to analyze the correlation. The P0.05 suggests the difference is statistically difference. Results: 1. intestinal flora distribution: Compared with the healthy control group, there was no significant difference in the intestinal bacteriobacteria, the thicker door, the deformable bacteria gate, the Clostridium, and the actinomycetes in the HIV infection group. At the level of the class, only the negativicutes bacteria increased significantly; at the level of the family, the rikenellaceae and the Mycobacterium microbacteriaceae were significantly reduced, Molarand moraxell Aceae was significantly increased; at the level of the genus Roche Roseburia, spirulae lachnospiraceae, alistipes, and rumen cocci ruminococcaceae decreased significantly; Psychrobacter significantly increased.2. short chain fatty acids in.2.: compared with the healthy control group, the content of butyric acid and valeric acid in the intestinal tract of HIV infection group decreased significantly, and the other short chain fatty acids were found in the HIV infection group. There was no difference in.3. serum intestinal mucosa damage markers: the value of serum intestinal mucosal damage markers I-FABP and d- lactate in HIV infection group and healthy control group did not differ from.4. cell immunity: the number of cd4+t cells in HIV infected persons was 200/ U L, cd8+t cells increased and cd4/cd8 ratio decreased, which was in line with those who had not developed to AIDS stage. The relationship between the distribution of.5. intestinal flora and the relationship between butyric acid and valerate: at the level of the family, the decrease of butyric acid content in the intestinal tract of HIV infected people is mainly related to the rikenellaceae of the family of the family of LAD and the ruminococcaceae of the family of rumen, and the decrease of the content of valerate is mainly with the rikenellaceae in the family of science and medicine, the peptococcaceae of the family of digestive bacteria, and the tumor. A positive correlation is found in the ruminococcaceae of the family of stomach cocci. At the level of the genus, the decrease of butyric acid content is mainly with the alistipes genus of the family of the family, Roseburia, blautia, lachnospiraceaeucg-005, lachnospiraceaefcs020group, ruminococcaceaeucg-003, ruminococcus1, and valeric acid in the family of the family of the family of the family rumen. The genus alistipes, eubacteriumcoprostanoligenesgroup, ruminococcaceaeucg-002, ruminococcaceaeucg-003, ruminococcaceaenk4a214group, ruminococcaceae1, eubacteriumruminantiumgroup, and eubacteriumruminantiumgroup genera of the family helioris; peptococcus genera in the family of digestistate families are positively related to.6. intestines. The correlation between the number of cd4+, cd8+t cells and the ratio of cd4/cd8: we did not find the bacteria associated with the cd4+t cells. The main groups that were positively related to the cd8+t cells were: oxalobacer, undibacterium, Burkholderia, Achromobacter, ramlibacter genera under the Proteus, sphingobacteriia family under the bacteriobacteria and the.7. short chain fatty acids. The correlation between CD8+T cell number and CD4/CD8 ratio: CD4+T cells, CD4/CD8 ratio is negatively correlated with acetic acid, CD8+T cells are positively correlated with valerate. Conclusion: 1.HIV infected people may have different intestinal microecological environment before the onset of AIDS, and the intestinal microecological environment may have been different from those of healthy people; the important intestinal mucosa protection of 2.HIV infected people The factors such as Roseburia of Roche, Lachnospiraceae, Alistipes, Ruminococcaceae of rumen, and Rikenellaceae in the family of Rikenellaceae and so on were reduced, and the levels of butyric acid and valeric acid in the metabolites of the related bacteria decreased, and the destructive factors of intestinal mucosa in the 3.HIV infected people were increased, such as the deformable bacteria. The abundance of Moraxellaceae and Psychrobacter in the genus Mora and colibacilli was significantly increased in the intestinal cavity. It is suggested that it may be closely related to the bacterial translocation, thus inducing sustained inflammatory response and aggravating the development of the HIV disease course; 4. the cellular immune level of the HIV infected persons who have not yet developed to the AIDS stage is not necessarily related to the distribution of intestinal flora; 5 The correlation between the level of cellular immunity and the short chain fatty acid SCFAs level of intestinal tract in HIV infected persons who have not yet developed to AIDS stage is not clear.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91

【参考文献】