【作者】 李青；

【导师】 于红刚；

【作者基本信息】 武汉大学 ， 内科学， 2022， 博士

【摘要】 研究背景与目的随着时间推移及研究的不断深入,学者们逐渐意识到微生物与宿主之间维持着微妙的平衡,他们之间互利共生。然而当机体不能维持这种平衡时,宿主体内可能会发生一系列病理生理改变而导致疾病的发生。目前已有部分研究表明,宿主免疫系统的成熟与肠道微生物群的存在有不可或缺的联系,肠道微生物群的失调与肠道肿瘤的发生发展有关。与之相似,在机体的胃脏内,除人们熟知的幽门螺杆菌外,亦有大量非幽门螺杆菌的菌群定植。其中,幽门螺杆菌的存在被认为是诱导胃癌发病的主要原因,其在人群中胃脏的定植率超过50%。但研究发现,在被幽门螺杆菌感染的人群中,胃癌的发病率只有约1-3%,并且经幽门螺杆菌根除治疗的患者仍有罹患胃癌的几率,这便给人们以疑问,胃癌的发病是否也受到其他菌群的诱导?近期,部分学者对胃黏膜中的菌群进行分析,发现与健康人群相比,胃癌组织中的菌群结构发生了明显的改变。由此,我们猜想胃内非幽门螺杆菌的菌群或许也参与了胃癌的发生和发展。本研究旨在探索这些紊乱菌群在胃癌发生和进展过程中所发挥的作用及其相关机制,研究结果可望为胃癌的防治提供新的诊治思路,并有望为胃癌的研究提供新的探索方向。材料与方法1.于胃肠外科收集了17对手术切下标本中的胃癌及癌旁正常组织,同时收集患者的临床病理学特征信息。采用16S rRNA基因测序,分析胃癌组织及癌旁正常组织中的微生物菌群。比较胃癌和癌旁正常组织中菌群丰富度、多样性等,进一步分析胃癌和癌旁正常组织中的差异菌群(分析得到的差异细菌为痤疮丙酸杆菌,Propionibacterium acnes,P.acnes)。2.收集福尔马林固定后石蜡包埋的组织:13例癌旁正常组织,58例胃癌组织(包括I期21例,II期21例,III期16例),利用针对P.acnes的特异性探针采用荧光原位杂交检测这些组织切片中P.acnes的丰度。进一步验证P.acnes在胃癌组织及癌旁正常组织中的分布,并分析其丰度与胃癌进展的相关性。采用免疫荧光分析这些组织中M2型巨噬细胞(CD206+)的丰度,分析P.acnes细菌丰度与M2巨噬细胞丰度的相关性。3.体外培养胃癌细胞系BGC-823及SGC-7901。购买P.acnes标准菌株ATCC6919,对照菌株E.coli DH5α。在不同MOI值下予以P.acnes及E.coli DH5α刺激胃癌细胞,通过CCK-8,划痕,Trans-well等实验方法检测P.acnes对胃癌细胞增殖、迁移的影响。接着培养人单核细胞THP-1,并使用PMA诱导其分化为巨噬细胞。在不同MOI值下予以P.acnes及E.coli DH5α刺激THP-1分化的巨噬细胞后得到条件培养基,使用条件培养基处理胃癌细胞BGC-823及SGC-7901,再通过CCK-8,划痕,Trans-well等实验方法检测条件培养基对胃癌细胞增殖、迁移的影响。采用细胞因子芯片分析条件培养基中的细胞因子,以了解何种细胞因子可能促进胃癌细胞的迁移。4.用P.acnes及E.coli DH5α分别刺激THP-1分化的巨噬细胞,随后采用免疫荧光分析巨噬细胞向M1(CD86+)及M2(CD206+)极化的情况,并采用RT-PCR分析M1及M2代表性细胞因子表达情况。5.采用RT-PCR分析经P.acnes刺激的THP-1分化的巨噬细胞中TLR1,TLR2,TLR4,TLR5的表达。并采用TLR4抑制剂TAK-242阻滞TLR4信号后,采用免疫荧光检测对M1及M2极化的影响,采用RT-PCR分析相应细胞因子的变化。最后通过免疫荧光分别分析高丰度及低丰度P.acnes胃癌组织中TLR4与M2(CD206+)巨噬细胞共定位情况。6.使用PI3K抑制剂LY2940022阻滞PI3K信号后,采用免疫荧光检测对M1及M2极化的影响,采用RT-PCR分析相应细胞因子的变化。并采用蛋白免疫印迹检测TLR4抑制剂TAK-242阻滞TLR4信号后PI3K、Akt、p-Akt的变化。结果1.胃癌组织中P.acnes丰度增加,并与胃癌进展有关采用16S rRNA基因测序分析胃癌组织及癌旁正常组织中菌群组成,我们发现胃癌组织中发生了菌群紊乱:在胃癌及癌旁正常组织中,细菌在门水平组成有所不同。通过进一步分析,我们发现在种水平,胃癌组织中P.acnes丰度明显增加。在分析了幽门螺杆菌阴性及幽门螺杆菌阳性的组织后,我们发现P.acnes丰度在幽门螺杆菌阴性组织中差异更为明显。通过进一步扩大样本量,我们采用荧光原位杂交分析P.acnes在胃癌组织及癌旁正常组织中的分布,以及不同TNM分期胃癌组织中的分布,结果显示:P.acnes丰度在胃癌组织中较癌旁正常组织多,并且随TNM分期进展,其丰度逐渐增加。2.P.acnes可能通过促进巨噬细胞M2极化促进胃癌细胞迁移CCK-8分析显示P.acnes对BGC-823及SGC-7901细胞的增殖无影响;划痕及Trans-well结果分析P.acnes的直接刺激对胃癌细胞的迁移无影响。由P.acnes刺激THP-1分化的巨噬细胞所得到的条件培养基,对胃癌细胞的增殖无影响,但促进了胃癌细胞的迁移能力。采用细胞因子芯片分析条件培养基中细胞因子,发现IL-6和MIP-1β较对照组分别升高了584倍和508倍。接下来,通过免疫荧光分析胃癌组织中M2巨噬细胞的数量,并将其与荧光原位杂交结果行相关分析,发现胃癌组织中P.acnes丰度与M2巨噬细胞的数量成正相关。免疫荧光分析P.acnes处理对THP-1分化的巨噬细胞极化的影响,发现P.acnes可促进巨噬细胞向M2型极化。RT-PCR分析M1及M2巨噬细胞产生的代表性细胞因子,发现P.acnes促进巨噬细胞分泌更多的CCR-2和IL-10。3.P.acnes通过TLR4/PI3K/Akt通路促进巨噬细胞M2极化RT-PCR结果分析发现,THP-1分化的巨噬细胞经P.acnes刺激后,细胞中中TLR4表达明显升高。通过免疫荧光分析发现,TAK-242(TLR4抑制剂)减少了P.acnes所诱导的M2巨噬细胞的数量。通过RT-PCR分析发现,较未使用TAK-242比较,使用TAK-242后P.acnes诱导的M2型巨噬细胞相关细胞因子CCR-2、IL-10的水平明显降低。研究同时发现,在高丰度P.acnes的胃癌组织中,TLR4与M2巨噬细胞共表达细胞数目较低丰度的胃癌组织明显增多。经PI3K抑制剂LY2940022阻滞PI3K信号后,通过免疫荧光检测发现PI3K抑制剂减弱了P.acnes对巨噬细胞M2极化的促进作用。同时,通过RT-PCR分析后发现,使用PI3K抑制剂后,P.acnes诱导的M2型巨噬细胞相关细胞因子CCR-2、IL-10的水平明显降低。通过蛋白免疫印迹分析发现,P.acnes促进了TLR4、PI3K、p-Akt的表达,但对Akt的表达无影响。而使用TAK-242阻滞TLR4能够抑制PI3K、p-Akt的表达,对Akt的表达无影响。结论本研究首次发现P.acnes在胃癌组织中丰度明显上升,尤其是在幽门螺杆菌阴性的组织中;P.acnes可能通过诱导巨噬细胞M2极化而促进胃癌细胞迁移从而促进胃癌进展;P.acnes促进巨噬细胞M2极化可能是经由TLR4/PI3K/Akt信号通路实现的。更多还原

【Abstract】 Background and objectiveIn the last few decades,we have gradually realized that there was a close relationship between microorganisms and their hosts and found that they maintained a delicate balance and symbiosis with each other.However,a series of pathophysiological changes may occur in the host when this balance was broken.At present,many studies have shown that the maturity of the host immune system is indispensable to the intestinal microbiota,and the dysregulation of the intestinal microbiota is related to the occurrence and development of intestinal tumors.Similarly,in addition to the wellknown Helicobacter pylori,there are also a large number of non-Helicobacter pylori flora colonization in the stomach of the body.Among these bacteria,the presence of Helicobacter pylori is considered to be the main risk factor for inducing gastric cancer,and its colonization rate in the stomach exceeds 50% in the population.However,studies have found that among people infected with Helicobacter pylori,only 1-3% of Helicobacter pylori-infected individuals will eventually develope gastric cancer,and patients who have undergone Helicobacter pylori eradication therapy still have the risk of developing gastric cancer.Those results raise questions that whether the incidence of gastric cancer could be induced by other flora.Recently,some scholars have analyzed the flora in gastric mucosa and found that the flora structure in gastric cancer tissue changed significantly when compared with healthy people.On this basis,we speculate that non-Helicobacter pylori flora in the stomach may also be involved in the occurrence and development of gastric cancer.The purpose of this study was to explore the roles and related mechanisms of these disordered flora in the occurrence and progression of gastric cancer.The results of the study are expected to provide new ideas for diagnosis and treatment of gastric cancer,and are expected to provide new exploration directions for gastric cancer research.Materials and methods1.Seventeen pairs of gastric cancer and adjacent normal tissues were collected from gastric cancer patients who underwent gastrectomy surgery at Renmin Hospital of Wuhan University.At the same time,clinicopathological characteristics of these patients were obtained.16 S rRNA gene sequencing was performed to analyze the microbiota in gastric cancer tissues and adjacent normal tissues.The abundance and diversity of the bacterial flora in gastric cancer and adjacent normal tissues were compared.We found that Propionibacterium acnes(P.acnes)was highly increased in gastric cancer tissues.2.A total of 13 adjacent normal tissues and 58 cases of gastric cancer tissues(including 21 cases of stage I,21 cases of stage II,and 16 cases of stage III)were collected,which were fixed by formalin and embedded with paraffin.Specifical primers targeted 16 s rRNA of P.acnes was designed,and fluorescence in situ hybridization(FISH)was carried out to detect the abundance of P.acnes in these tissues.The abundance of M2 macrophages(CD206+)in these tissues was analyzed by immunofluorescence.Correlation analysis was performed between the abundance of P.acnes and the abundance of M2 macrophages.3.P.acnes and E.coli DH5α were given to stimulate gastric cancer cells under different MOI values,and the effects of P.acnes on the proliferation and migration of gastric cancer cells were detected by CCK-8,wound healing assay,and trans-well assays.Human monocytes THP-1 were cultured and differentiate into macrophages by using PMA.P.acnes and E.coli DH5α were given to stimulate THP-1-derived macrophages under different MOI values to obtain conditioned medium.The conditioned medium was used to treat gastric cancer cell lines(BGC-823 and SGC-7901),and then CCK-8,wound healing assay,and trans-well assays were performed to analyze proliferation and migration of gastric cancer cells treated with conditioned medium.The Luminex liquid suspension chip was employed to measure the cytokines in the conditioned medium.4.Immunofluorescence was used to analyze the polarization of THP-1-derived macrophages treated with P.acnes and E.coli DH5α,respectively.RT-PCR was used to analyze the m RNA levels of representative cytokines secreted by M1 and M2 macrophages.5.The expressions of TLR1,TLR2,TLR4,and TLR5 in THP-1 derived macrophages stimulated with P.acnes were measured using RT-PCR.After the TAK-242(TLR4 inhibitor)was used to block the TLR4 signal,immunofluorescence was used to detect the polarization of M1 and M2,and the changes of corresponding cytokines were analyzed by RT-PCR.Finally,immunofluorescence was used to analyze the co-localization of TLR4 and M2(CD206+)macrophages in gastric cancers tissues with high-abundance and low-abundance of P.acnes,respectively.6.After the LY2940022(PI3K inhibitor)was used to block the PI3 K signal,immunofluorescence was used to detect the influence on the polarization of macrophages,and the changes of the corresponding cytokines were analyzed by RTPCR.Western blot was used to detect the changes of PI3 K,Akt and p-Akt after TAK242 blocked.Results1.The abundance of P.acnes was increased in gastric cancer tissue,and correlated with the progression of gastric cancer.We found that dysbiosis of microbiota occurred in gastric cancer tissues and the bacterial diversity was decreased by using 16 S rRNA gene sequencing.The composition of bacteria at the phylum level was different in gastric cancer and adjacent normal tissues.After analyzing the micorbiota at the species level,we found that the abundance of P.acnes significantly increased in gastric cancer tissues.After further analyzing of Helicobacter pylori-negative and Helicobacter pylori-positive tissues separately,we found that P.acnes abundance was more significantly different in Helicobacter pylori-negative tissues.To further expand the sample size,fluorescence in situ hybridization was used to analyze the distribution of P.acnes in gastric cancer tissues and adjacent normal tissues,as well as gastric cancer tissues of different TNM stages.We found that the abundance of P.acnes in gastric cancer tissues was higher than that in adjacent normal tissues,and its abundance gradually increased with the progression of TNM staging.2.P.acnes may favor gastric cancer cell migration by promoting M2 polarization of macrophagesAfter perfoming CCK-8 analysis,wound healing assay and trans-well assays,we found that P.acnes stimulation alone had no effect on the proliferation and migration of BGC-823 and SGC-7901.Next,the conditioned medium obtained by THP-1differentiated macrophages treated with P.acnes also did not promote the proliferation of gastric cancer cells.However,the conditioned medium could promote the migration of gastric cancer cells.The Luminex liquid suspension chip assay was used to measure the cytokines in the conditioned medium,it was found that IL-6 and MIP-1β were 584 times and 508 times higher than those in the control group,respectively.A positive correlation was found between M2 macrophages and abundance of P.acnes in gastric cancer tissues.Immunofluorescence analysis showed that P.acnes promoted M2 polarization of macrophages.Representative cytokines produced by M1 and M2 macrophages was measured by RT-PCR,and we finally confirmed that THP-1derived macrophages stimulated with P.acnes produced higher levels of CCR-2 and IL-10.3.P.acnes promote M2 polarization of macrophages through the TLR4/PI3K/AKT pathwayRT-PCR analysis showed that P.acnes promoted the expression of TLR4 in THP-1-derived macrophages.Immunofluorescence analysis showed that M2 polarization of macrophages was attenuated by using TAK-242(TLR4 inhibitor).Moreover,CCR-2and IL-10 produced by M2 macrophages was decreased in macrophages pretreated with TAK-242.In addition,the number of cells co-expressing TLR4 and CD206 was significantly higher in gastric cancer tissues with high abundance of P.acnes.After LY2940022 blocked the PI3 K signal,immunofluorescence detection found that LY2940022 inhibited the effect of P.acnes in promoting the M2 polarization of macrophages.The IL-10 and CCR-2 were also reduced,which was proved by RT-PCR analysis.Western blot analysis showed that P.acnes promoted the expression of TLR4,PI3 K,and p-Akt,but had no effect on the expression of Akt.After TLR4 blockade,the expression of PI3 K and p-Akt were down-regulated,but also had no effect on the expression of Akt.ConclusionThis study found that the abundance of P.acnes in gastric cancer tissues significantly increased for the first time,especially in Helicobacter pylori-negative tissues.P.acnes may promote the progression of gastric cancer by promoting the M2 polarization of macrophages.P.acnes may promote M2 polarization of macrophages through TLR4/ PI3K/Akt signaling pathway.更多还原