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Article(id=1194613944959606947, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1194613942065533315, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.0129.2024.0711, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1706457600000, receivedDateStr=2024-01-29, revisedDate=null, revisedDateStr=null, acceptedDate=1716393600000, acceptedDateStr=2024-05-23, onlineDate=1762747759327, onlineDateStr=2025-11-10, pubDate=1743091200000, pubDateStr=2025-03-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1762747759327, onlineIssueDateStr=2025-11-10, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1762747759327, creator=13701087609, updateTime=1762747759327, updator=13701087609, issue=Issue{id=1194613942065533315, tenantId=1146029695717560320, journalId=1189873630562394117, year='2025', volume='50', issue='3', pageStart='245', pageEnd='365', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1762747758641, creator=13701087609, updateTime=1762749141462, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1194619742100103439, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1194613942065533315, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1194619742100103440, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1194613942065533315, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=261, endPage=268, ext={EN=ArticleExt(id=1194613945278374056, articleId=1194613944959606947, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Improvement effects of fecal microbiota transplantation on chemotherapy-induced diarrhea in mice, columnId=1194613943957164424, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Special Issue on Role of Gut Microbiota in Disease Occurrence and Treatment, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the improvement effects of homogeneous fecal microbiota transplantation (FMT) on chemotherapy-induced diarrhea (CID) in mice. Methods Fifteen C57BL/6N mice were divided into control group, CID model group and CID+FMT group according to the random number distribution and remainder grouping method, with 5 mice per group. Control group received no intervention, and their feces were used to prepare fecal bacteria suspension. CID model group was injected intraperitoneally with fluorouracil (65 mg/kg) for 5 consecutive days to construct the CID mouse model, followed by gavage with 0.1 ml of saline on alternate days. CID+FMT group was given 0.1 ml fecal bacteria suspension gavage on alternate days for one week, followed by intraperitoneal injection of fluorouracil (65 mg/kg) for 5 consecutive days to construct the CID mouse model, with the experiment ending on the 14th day. During the experiment, the mice's food intake and body weight were recorded. At the end of the experiment, the mice were euthanized with deep carbon dioxide anesthesia, and the mice colonic specimens from cecum to anus were collected for hematoxylin and eosin (HE) staining and histopathological examination. Fecal samples were collected for 16S rRNA gene sequencing. Shannon index, Simpson index and Chao1 algorithm were used to analyze the α-diversity species of the intestinal flora in each group of mice. Similarity analysis (Anosim) was used to perform non-parametric on the inter-group differences of intestinal flora among the mice. Linear discriminate analysis size effect (LEfSe) and nonmetric multidimensional scaling (NMDS) were employed to analyze the intestinal dominant flora and the similarity classification relationships in each group of mice. Results The colonic specimen's length from cecum to anus in CID model group was significantly shorter than that in control group (P<0.05), while there was no significant difference between CID+FMT group and CID model group (P>0.05). The weight of mice in CID model group decreased by 42.04%, while control group mice gained 10.24%, with a significant difference between the two groups (P<0.05). The weight of mice in CID+FMT group decreased by 8.12%, which was significantly improved compared to CID model group (P<0.05). HE staining results revealed the intestinal mucosal structure in CID model group was severely damaged, with atrophy and deformation, accompanied by inflammatory cell infiltration, and the pathological score was higher than that of control group (P<0.05). Compared with CID model group, the intestinal mucosal integrity and crypt cells in the CID+FMT group were improved, with less damage, and the pathological score was lower than that of CID model group, but the difference was not statistically significant (P>0.05). The α-diversity analysis showed that there were significant differences in the Shannon, Simpson and Chao1 indices among the three groups (P<0.05). ANOSIM and NMDS analysis revealed that the intestinal flora in CID+FMT group was closer to the normal intestinal flora compared to CID model group. LEfSe analysis showed that the intestinal flora in CID model group was enriched in famliy_Bacteroidaceae, and the intestinal flora in CID+FMT group was similar to that of control group, with an enrichenment of familiy_Enterobacteriaceae. Conclusion Homogeneous FMT can improve the abundance of intestinal flora in CID mice, making it more similar to normal intestinal flora, thereby protecting intestinal mucosa, reducing damage and alleviating the severity of CID.

, correspAuthors=Ze-Xian Fu, authorNote=null, correspAuthorsNote=
E-mail:
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目的 探讨同种菌群移植(FMT)对小鼠化疗相关性腹泻(CID)的改善作用。方法 15只C57BL/6N小鼠按照随机数分配、余数归组法分为空白对照组、CID模型组与CID+FMT组，每组5只。空白对照组不做任何干预，取其粪便制作粪菌悬液；CID模型组采用氟尿嘧啶(65 mg/kg)连续5 d腹腔注射构建CID小鼠模型，然后给予0.1 ml生理盐水隔日灌胃；CID+FMT组给予0.1 ml粪菌悬液隔日灌胃，干预7 d后采用氟尿嘧啶(65 mg/kg)连续5 d腹腔注射构建CID小鼠模型，至第14天结束实验。实验期间记录小鼠进食量和体重，实验结束时采用二氧化碳深度麻醉处死小鼠，采集肛门至盲肠肠管标本进行HE染色和组织学观察，采集粪便样本进行16S rRNA基因测序，采用Shannon指数、Simpson指数、Chao1算法对各组小鼠肠道菌群进行α多样性物种分析，采用相似性分析(Anosim)对各组小鼠肠道菌群的组间差异进行非参数检验，采用线性判别分析效应大小(LEfSe)和非度量多维尺度分析(NMDS)法分析各组小鼠肠道的优势菌群及相似性归类关系。结果 CID模型组小鼠肛门至盲肠肠管长度较空白对照组明显缩短(P<0.05)，CID+FMT组与CID模型组小鼠肛门至盲肠肠管长度比较差异无统计学意义(P>0.05)。CID模型组小鼠体重降低42.04%，空白对照组小鼠增重10.24%，两组间差异有统计学意义(P<0.05)；CID+FMT组小鼠体重降低8.12%，与CID模型组小鼠比较体重下降明显缓解，差异有统计学意义(P<0.05)。HE染色结果显示，与空白对照组相比，CID模型组肠黏膜结构损伤较重，出现萎缩和变形，伴有炎性细胞浸润，病理学评分升高(P<0.05)；与CID模型组相比，CID+FMT组肠黏膜完整性和隐窝细胞有所改善，损伤较轻，病理学评分差异无统计学意义(P>0.05)。菌群α多样性分析结果显示，三组Shannon、Simpson及Chao1指数差异有统计学意义(P<0.05)；Anosim相似性分析和NMDS分析结果显示，与CID模型组相比，CID+FMT组肠道菌群更接近正常的肠道菌群；LEfSe分析结果显示，CID模型组小鼠肠道f_Bacteroidaceae(拟杆菌科)菌群富集，CID+FMT组小鼠肠道菌群种类与空白对照组相似，且f_Enterobacteriaceae(肠杆菌科)菌群富集。结论 同种FMT可恢复CID小鼠肠道菌群丰度，使之更接近正常肠道菌群，从而保护肠道黏膜，减轻损伤，减轻CID的严重程度。

, correspAuthors=付泽娴, authorNote=null, correspAuthorsNote=
付泽娴，E-mail：
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杨秋雨，临床医学硕士，主要从事肠道菌群及胃肠肿瘤治疗方面的研究

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杨秋雨，临床医学硕士，主要从事肠道菌群及胃肠肿瘤治疗方面的研究

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杨秋雨，临床医学硕士，主要从事肠道菌群及胃肠肿瘤治疗方面的研究

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CID. 化疗性腹泻；FMT. 菌群移植；与空白对照组比较，(1)P<0.05

, figureFileSmall=bm3kR2s3Y6Hfu7kFKDLKTg==, figureFileBig=1rvwqGjTtAREpz1tL57i9A==, tableContent=null), ArticleFig(id=1194634232824832730, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194613944959606947, language=EN, label=Fig.2, caption=Changes in body weight of mice in each group, figureFileSmall=bQD8AwG39kg8ZrFSyvKPyg==, figureFileBig=T9VWO/ribPkxZ/djFFGOog==, tableContent=null), ArticleFig(id=1194634232908718811, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194613944959606947, language=CN, label=图2, caption=各组小鼠体重变化情况

CID. 化疗性腹泻；FMT. 菌群移植；CID模型组小鼠建模后出现明显体重下降，在实验第8天出现死亡。CID+FMT组小鼠在FMT干预下，CID症状减轻，体重也出现下降但下降幅度明显低于CID模型组。与空白对照组比较，(1)P<0.05；与CID模型组比较，(2)P<0.05

, figureFileSmall=bQD8AwG39kg8ZrFSyvKPyg==, figureFileBig=T9VWO/ribPkxZ/djFFGOog==, tableContent=null), ArticleFig(id=1194634232980021980, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194613944959606947, language=EN, label=Fig.3, caption=Pathological changes of colorectal tissue of mice in each group, figureFileSmall=KrVa7KcD6QOOokipIQxRYQ==, figureFileBig=6Pta6JC3lSm2FUP8culMjw==, tableContent=null), ArticleFig(id=1194634233051325149, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194613944959606947, language=CN, label=图3, caption=各组小鼠结直肠组织病理学变化

CID. 化疗性腹泻；FMT. 菌群移植；A. 各组小鼠结直肠组织HE染色；B. 各组小鼠结直肠组织病理学评分；与空白对照组比较，(1)P<0.01

, figureFileSmall=KrVa7KcD6QOOokipIQxRYQ==, figureFileBig=6Pta6JC3lSm2FUP8culMjw==, tableContent=null), ArticleFig(id=1194634233122628318, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194613944959606947, language=EN, label=Fig.4, caption=Effect of FMT on the α-diversity of intestinal flora in each group of mice, figureFileSmall=rU7DFvRlYQv72VJchUuU/A==, figureFileBig=EW7DglGrVbTYOZ1hsZqmsA==, tableContent=null), ArticleFig(id=1194634233181348575, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194613944959606947, language=CN, label=图4, caption=FMT对各组小鼠肠道菌群α多样性的影响

CID. 化疗性腹泻；FMT. 菌群移植；A. 各组小鼠肠道优势菌群韦恩图；B. 各组小鼠肠道菌群丰度Shannon、Simpson和Chao1指数分析；C. 各组小鼠肠道菌群ANOSIM相似性分析；D. 各组小鼠肠道菌群NMDS分析；与空白对照组比较，(1)P<0.05，(2)P<0.01；与CID模型组比较，(3)P<0.01

, figureFileSmall=rU7DFvRlYQv72VJchUuU/A==, figureFileBig=EW7DglGrVbTYOZ1hsZqmsA==, tableContent=null), ArticleFig(id=1194634233248457440, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194613944959606947, language=EN, label=Fig.5, caption=Effect of FMT on intestinal flora species in CID mice, figureFileSmall=Be9Oe5yTfXKyQLvvlEipOg==, figureFileBig=hLkb28je69PVCNlmWMFWPQ==, tableContent=null), ArticleFig(id=1194634233311372001, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1194613944959606947, language=CN, label=图5, caption=FMT对CID小鼠肠道菌群物种的影响

CID. 化疗性腹泻；FMT. 菌群移植；A. 各组小鼠肠道菌群物种标志物进化树图；B. 各组小鼠肠道菌群物种标志物LDA评分

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菌群移植对小鼠化疗相关性腹泻的改善作用
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杨秋雨 1 , 檀梦天 1 , 白净 2 , 任行 1 , 张浚琪 1 , 杨勇 3 , 孙宇航 4 , 李磊 3 , 付泽娴 1, *
解放军医学杂志 | 肠道菌群在疾病发生及治疗中的作用专题 2025,50(3): 261-268
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解放军医学杂志 | 肠道菌群在疾病发生及治疗中的作用专题 2025, 50(3): 261-268
菌群移植对小鼠化疗相关性腹泻的改善作用
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杨秋雨1, 檀梦天1, 白净2, 任行1, 张浚琪1, 杨勇3, 孙宇航4, 李磊3, 付泽娴1, *
作者信息
  • 1河北工程大学临床医学院，河北邯郸 056038
  • 2河北工程大学附属医院老年病科，河北邯郸 056038
  • 3河北工程大学附属医院普外中心，河北邯郸 056038
  • 4河北工程大学附属医院医学整形美容中心，河北邯郸 056038

    • 杨秋雨，临床医学硕士，主要从事肠道菌群及胃肠肿瘤治疗方面的研究

通讯作者:

付泽娴，E-mail：
Improvement effects of fecal microbiota transplantation on chemotherapy-induced diarrhea in mice
Qiu-Yu Yang1, Meng-Tian Tan1, Jing Bai2, Xing Ren1, Jun-Qi Zhang1, Yong Yang3, Yu-Hang Sun4, Lei Li3, Ze-Xian Fu1, *
Affiliations
  • 1School of Clinical Medicine, Hebei University of Engineering, Handan, Hebei 056038, China
  • 2Department of Geriatric, Affiliated Hospital of Hebei Engineering University, Handan, Hebei 056038, China
  • 3Department of General Surgery, Affiliated Hospital of Hebei Engineering University, Handan, Hebei 056038, China
  • 4Medical Cosmetic and Plastic Surgery Center, Affiliated Hospital of Hebei Engineering University, Handan, Hebei 056038, China
出版时间: 2025-03-28 doi: 10.11855/j.issn.0577-7402.0129.2024.0711
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摘要
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目的 探讨同种菌群移植(FMT)对小鼠化疗相关性腹泻(CID)的改善作用。方法 15只C57BL/6N小鼠按照随机数分配、余数归组法分为空白对照组、CID模型组与CID+FMT组，每组5只。空白对照组不做任何干预，取其粪便制作粪菌悬液；CID模型组采用氟尿嘧啶(65 mg/kg)连续5 d腹腔注射构建CID小鼠模型，然后给予0.1 ml生理盐水隔日灌胃；CID+FMT组给予0.1 ml粪菌悬液隔日灌胃，干预7 d后采用氟尿嘧啶(65 mg/kg)连续5 d腹腔注射构建CID小鼠模型，至第14天结束实验。实验期间记录小鼠进食量和体重，实验结束时采用二氧化碳深度麻醉处死小鼠，采集肛门至盲肠肠管标本进行HE染色和组织学观察，采集粪便样本进行16S rRNA基因测序，采用Shannon指数、Simpson指数、Chao1算法对各组小鼠肠道菌群进行α多样性物种分析，采用相似性分析(Anosim)对各组小鼠肠道菌群的组间差异进行非参数检验，采用线性判别分析效应大小(LEfSe)和非度量多维尺度分析(NMDS)法分析各组小鼠肠道的优势菌群及相似性归类关系。结果 CID模型组小鼠肛门至盲肠肠管长度较空白对照组明显缩短(P<0.05)，CID+FMT组与CID模型组小鼠肛门至盲肠肠管长度比较差异无统计学意义(P>0.05)。CID模型组小鼠体重降低42.04%，空白对照组小鼠增重10.24%，两组间差异有统计学意义(P<0.05)；CID+FMT组小鼠体重降低8.12%，与CID模型组小鼠比较体重下降明显缓解，差异有统计学意义(P<0.05)。HE染色结果显示，与空白对照组相比，CID模型组肠黏膜结构损伤较重，出现萎缩和变形，伴有炎性细胞浸润，病理学评分升高(P<0.05)；与CID模型组相比，CID+FMT组肠黏膜完整性和隐窝细胞有所改善，损伤较轻，病理学评分差异无统计学意义(P>0.05)。菌群α多样性分析结果显示，三组Shannon、Simpson及Chao1指数差异有统计学意义(P<0.05)；Anosim相似性分析和NMDS分析结果显示，与CID模型组相比，CID+FMT组肠道菌群更接近正常的肠道菌群；LEfSe分析结果显示，CID模型组小鼠肠道f_Bacteroidaceae(拟杆菌科)菌群富集，CID+FMT组小鼠肠道菌群种类与空白对照组相似，且f_Enterobacteriaceae(肠杆菌科)菌群富集。结论 同种FMT可恢复CID小鼠肠道菌群丰度，使之更接近正常肠道菌群，从而保护肠道黏膜，减轻损伤，减轻CID的严重程度。

菌群移植  /  化疗性腹泻  /  肠道菌群  /  高通量测序

Objective To investigate the improvement effects of homogeneous fecal microbiota transplantation (FMT) on chemotherapy-induced diarrhea (CID) in mice. Methods Fifteen C57BL/6N mice were divided into control group, CID model group and CID+FMT group according to the random number distribution and remainder grouping method, with 5 mice per group. Control group received no intervention, and their feces were used to prepare fecal bacteria suspension. CID model group was injected intraperitoneally with fluorouracil (65 mg/kg) for 5 consecutive days to construct the CID mouse model, followed by gavage with 0.1 ml of saline on alternate days. CID+FMT group was given 0.1 ml fecal bacteria suspension gavage on alternate days for one week, followed by intraperitoneal injection of fluorouracil (65 mg/kg) for 5 consecutive days to construct the CID mouse model, with the experiment ending on the 14th day. During the experiment, the mice's food intake and body weight were recorded. At the end of the experiment, the mice were euthanized with deep carbon dioxide anesthesia, and the mice colonic specimens from cecum to anus were collected for hematoxylin and eosin (HE) staining and histopathological examination. Fecal samples were collected for 16S rRNA gene sequencing. Shannon index, Simpson index and Chao1 algorithm were used to analyze the α-diversity species of the intestinal flora in each group of mice. Similarity analysis (Anosim) was used to perform non-parametric on the inter-group differences of intestinal flora among the mice. Linear discriminate analysis size effect (LEfSe) and nonmetric multidimensional scaling (NMDS) were employed to analyze the intestinal dominant flora and the similarity classification relationships in each group of mice. Results The colonic specimen's length from cecum to anus in CID model group was significantly shorter than that in control group (P<0.05), while there was no significant difference between CID+FMT group and CID model group (P>0.05). The weight of mice in CID model group decreased by 42.04%, while control group mice gained 10.24%, with a significant difference between the two groups (P<0.05). The weight of mice in CID+FMT group decreased by 8.12%, which was significantly improved compared to CID model group (P<0.05). HE staining results revealed the intestinal mucosal structure in CID model group was severely damaged, with atrophy and deformation, accompanied by inflammatory cell infiltration, and the pathological score was higher than that of control group (P<0.05). Compared with CID model group, the intestinal mucosal integrity and crypt cells in the CID+FMT group were improved, with less damage, and the pathological score was lower than that of CID model group, but the difference was not statistically significant (P>0.05). The α-diversity analysis showed that there were significant differences in the Shannon, Simpson and Chao1 indices among the three groups (P<0.05). ANOSIM and NMDS analysis revealed that the intestinal flora in CID+FMT group was closer to the normal intestinal flora compared to CID model group. LEfSe analysis showed that the intestinal flora in CID model group was enriched in famliy_Bacteroidaceae, and the intestinal flora in CID+FMT group was similar to that of control group, with an enrichenment of familiy_Enterobacteriaceae. Conclusion Homogeneous FMT can improve the abundance of intestinal flora in CID mice, making it more similar to normal intestinal flora, thereby protecting intestinal mucosa, reducing damage and alleviating the severity of CID.

fecal microbiota transplantation  /  chemotherapy-induced diarrhea  /  intestinal flora  /  high-throughput sequencing
杨秋雨, 檀梦天, 白净, 任行, 张浚琪, 杨勇, 孙宇航, 李磊, 付泽娴. 菌群移植对小鼠化疗相关性腹泻的改善作用. 解放军医学杂志, 2025 , 50 (3) : 261 -268 . DOI: 10.11855/j.issn.0577-7402.0129.2024.0711
Qiu-Yu Yang, Meng-Tian Tan, Jing Bai, Xing Ren, Jun-Qi Zhang, Yong Yang, Yu-Hang Sun, Lei Li, Ze-Xian Fu. Improvement effects of fecal microbiota transplantation on chemotherapy-induced diarrhea in mice[J]. Medical Journal of Chinese People’s Liberation Army, 2025 , 50 (3) : 261 -268 . DOI: 10.11855/j.issn.0577-7402.0129.2024.0711
正文
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近年来，结直肠癌发病率逐渐升高[1-2]，采用手术、辅助放化疗等综合治疗方案可明显提高结直肠癌Ⅲ期患者的术后生存率[3]。临床上使用5-氟尿嘧啶(5-fluorouracil，5-FU)、卡培他滨和伊立替康[4]等化疗药物治疗时，患者往往因为出现严重的化疗性腹泻(chemotherapy-induced diarrhea，CID)而不得不提前终止化疗[5]。CID是一种较为复杂的胃肠道并发症，主要临床表现为腹泻、恶心等，严重者可出现多器官衰竭，降低了抗癌效果，进而影响患者的生活质量[6]。化疗药物治疗可导致肠道通透性增加，引起肠道菌群紊乱，从而诱发CID[7]。菌群移植(fecal microbiota transplantation，FMT)是从健康人体的粪便中提取出功能菌群进行移植，以重建患者肠道菌群，实现肠道菌群的稳态[8]。将健康人群的粪便混悬液移入炎症性肠病患者肠道中，其腹泻症状可得到明显缓解[9]。但FMT的临床应用仍存在一些问题。有研究表明，采用FMT持续治疗克罗恩病时，部分患者治疗效果欠佳，分析原因可能与多供体提供菌群有关[10]。此外，临床采用FMT治疗需要大量数据进行安全性评估[11]。基于此，本研究通过同种FMT干预CID小鼠模型，旨在恢复其肠道菌群的健康状态，减轻化疗药物对机体的损伤，增强免疫力，为临床开展同种FMT治疗CID提供理论依据。
1 材料与方法
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1.1 主要试剂及仪器
氟尿嘧啶注射液(2202131，天津金耀药业有限公司)；核酸提取试剂(GHFDE100，杭州谷禾信息技术有限公司)；Phusion High-Fidelity PCR Master Mix(M0531，美国NEB公司)；Agencourt AMPure XP 60 ml Kit (A63881，美国Beckman Coulter公司)；QIAquick Gel Extraction Kit (250)(28706，德国Qiagen公司)；Qubit dsDNA HS Assay Kit(Q32851，美国Life Tech公司)；Library Quant Kit illumina GA revised primer-SYBR Fast Universal(KK4824，美国KAPA公司)。
1.2 实验方法
1.2.1 实验动物及分组
SPF级雌性C57BL/6N小鼠15只，6~7周龄，体重16~19 g，购自北京维通利华实验动物技术有限公司[实验动物生产许可证号：SCXK(京)2021-0006]。小鼠自由进食、饮水，动物房温度(20±5) ℃，相对湿度55%±5%，12 h光暗循环。适应性饲养1周后，根据小鼠体重，按照随机数分配、余数归组法分为空白对照组、CID模型组与CID+FMT组，每组5只。本研究获河北工程大学临床医学院生物医学伦理委员会审批(BER-YXY-2022038)。
1.2.2 菌液制备及干预
适应性饲养期间，每3 d取各组小鼠粪便1次，部分送检，同时取空白对照组粪便0.4 g加入常温无菌生理盐水20 ml进行溶解，组织研磨器混匀后经细胞筛反复过滤，去除大颗粒物质后获得粪菌悬液备用[12]。
1.2.3 小鼠CID模型构建
CID模型组小鼠给予氟尿嘧啶65 mg/kg腹腔注射(连续5 d)构建CID模型后给予生理盐水0.1 ml灌胃，隔日1次。CID+FMT组给予粪菌悬液0.1 ml灌胃，隔日1次连续灌胃1周后，给予氟尿嘧啶65 mg/kg腹腔注射(连续5 d)构建CID模型。空白对照组不做任何处理。实验期间，每日记录小鼠进食量、体重、活动情况及腹泻状态，按照动物实验3R原则，在第14天结束实验，采用二氧化碳深度麻醉处死小鼠，解剖并自肛门逆行分离小鼠结直肠至回盲部，取距肛门端1.0 cm肠管置于包埋盒中并加入组织液固定。
1.2.4 HE染色
结肠组织于4%多聚甲醛溶液中固定24 h后，常规包埋，0.4 μm连续切片，HE染色后于显微镜下观察结肠黏膜损伤情况。高倍镜下随机选取10个视野，观察肠道黏膜病理变化并进行评分。评分标准：(1)黏膜变薄或杯状细胞数量有所减少，甚至出现隐窝与腺体远离基底膜，计1分；(2)隐窝消失且伴有以中性粒细胞为主的炎性细胞浸润，计2分；(3)上皮出现破坏且基底浆细胞与淋巴细胞聚集量明显增多，计3分[13]。
1.2.5 粪便DNA提取及测序
适应性饲养时采集小鼠粪便作为检测基准，二氧化碳深度麻醉处死小鼠前采集粪便并置于无菌采样管进行肠道菌群检测。16S rRNA基因测序于杭州谷禾信息技术有限公司Illumina Novaseq 6000平台进行。使用515F-806R通用引物扩增基因组DNA中16S rRNA的V4区，每个样本扩增产物通过PCR加入barcode，混合后2×150 bp测序。测序序列使用Vsearch进行双端最小5 bp重叠的拼接，并去除嵌合体。使用QIIME2(v2022.2)进行Amplicon序列型变体(amplicon sequence variant，ASV)的聚类和丰度表分析。使用QIIME2的SILVA 138版本进行后续注释分析。
1.2.6 肠道菌群分析
(1)采用Shannon指数评估各组小鼠肠道菌群群落的丰富度和均匀度，侧重群落的丰富度和群落中的稀有运算分类单元(operational taxonomic unit，OTU)。(2)采用Simpson指数评估各组小鼠肠道菌群的多样性，侧重均匀度和群落中的优势OTU。(3)采用Chao1算法估算粪便样本中OTU的数量以估计物种总数。(4)采用相似性分析(analysis of similarities，Anosim)对各组小鼠肠道菌群的组间差异进行非参数检验。横轴Between表示组间距离，其他表示对应分组组内距离，R值表示组间与组内的差异程度。(5)采用线性判别分析效应大小(linear discriminate analysis size effect，LEfSe)分析各组小鼠肠道中丰度差异显著的优势菌群，用成组的Wilcoxon秩和检验进行组间差异分析，再用线性判别分析(linear discriminate analysis，LDA)对数据进行降维后评估差异显著的影响力(即LDA评分)。(6)采用非度量多维尺度分析(nonmetric multidimensional scaling，NMDS)对各组小鼠肠道菌群进行非约束排序，以矩阵中各组菌群的距离进行相似性归类分析。
1.3 统计学处理
采用GraphPad Prism 6软件进行统计分析。所有实验数据均以$\bar{x}±s$表示，多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验；采用R软件对肠道菌群测序结果进行非参数检验。P<0.05为差异有统计学意义。
2 结果
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2.1 各组小鼠一般情况
空白对照组小鼠生活习性较好，进食量持续增加，小鼠处死后检查肠腔可见粪便颗粒，数目正常，肠壁未见出血点。CID模型组小鼠注射氟尿嘧啶后进食量明显减少，活动度降低，肠壁可见出血点。CID+FMT组小鼠较CID模型组进食量增加，活动度略好，肠壁出血点略少。与空白对照组比较，CID模型组肛门至盲肠肠管长度明显缩短，差异有统计学意义(P<0.05)，CID+FMT组肛门至盲肠肠管长度稍缩短，但差异无统计学意义(P>0.05)；CID+FMT组与CID模型组肛门至盲肠肠管长度比较差异无统计学意义(P>0.05，图1)。
2.2 FMT对CID小鼠体重的影响
空白对照组、CID模型组、CID+FMT组小鼠初始体重依次为(18.06±0.46) g、(17.84±0.39) g、(17.73±0.65) g；实验结束时，各组小鼠体重依次为(19.91±0.25) g、(12.56±0.49) g、(16.29±0.58) g。空白对照组小鼠增重10.24%，CID模型组体重降低42.04%，CID+FMT组体重降低8.12%。与空白对照组比较，CID模型组体重明显下降，差异有统计学意义(P<0.05)；CID+FMT组小鼠体重与空白对照组相比，差异无统计学意义(P=0.06)。与CID模型组比较，CID+FMT组小鼠体重下降明显减缓(P<0.05)。各组小鼠体重变化情况如图2所示。
2.3 FMT对CID小鼠结肠黏膜屏障的影响
HE染色结果显示，空白对照组小鼠结肠黏膜结构较完整，组织排列整齐且紧密，未见明显异常；CID模型组小鼠肠黏膜结构损伤较重，出现萎缩和变形，隐窝细胞远离基底膜甚至消失，杯状细胞明显减少，黏膜层炎性细胞增多；与CID模型组相比，CID+FMT组小鼠肠黏膜结构完整性和隐窝细胞有所改善，炎性细胞浸润减少(图3A)。空白对照组、CID模型组、CID+FMT组结直肠组织病理学评分依次为(0.2±0.2)分、(2.0±0.4)分、(1.8±0.3)分。与空白对照组相比，CID模型组和CID+FMT组结直肠组织病理学评分均增高，差异有统计学意义(P<0.01)；与CID模型组相比，CID+FMT组结直肠组织病理学评分差异无统计学意义(P>0.05) (图3B)。
2.4 FMT对CID小鼠肠道菌群多样性的影响
三组共有1093个OTU，空白对照组与CID+FMT组共有1255个OTU，空白对照组与CID模型组共有1166个OTU，CID+FMT组与CID模型组共有1267个OTU，空白对照组、CID模型组和CID+FMT组独有的OTU分别为105、51和49个(图4A)。
菌群α多样性分析结果显示，三组Shannon、Simpson及Chao1指数差异有统计学意义(P<0.05)，表明三组间菌群α多样性差异显著。与空白对照组比较，CID模型组、CID+FMT组Shannon指数明显降低(P<0.01)，CID模型组Simpson、Chao1指数明显降低(P<0.05)；与CID模型组比较，CID+FMT组Shannon指数明显降低(P<0.01)，但两组Simpson、Chao1指数差异无统计学意义(P>0.05，图4B)。
ANOSIM相似性分析结果显示，三组肠道菌群组间差异明显(R=0.822，P=0.001，图4C)。NMDS分析结果显示，空白对照组与CID模型组样本未出现重叠，表明两组小鼠肠道菌群存在明显差异；CID+FMT组的样本距离更接近空白对照组，表明FMT可以调节小鼠肠道菌群，使之更接近正常的肠道菌群(图4D)。
2.5 FMT对CID小鼠菌群物种的影响
LEfSe结果分析显示，空白对照组中o_Bacteroidales(拟杆菌目)、p_Bacteroidota(拟杆菌门)、c_Bacteroidia(拟杆菌纲)、f_Muribaculaceae(拟杆菌科)、g_Muribaculaceae(拟杆菌属)丰度较高；CID模型组中f_Bacteroidaceae(拟杆菌科)、g_Bacteroides(拟杆菌属)丰度较高；CID+FMT组中o_Enterobacterales(肠杆菌目)、c_Gammaproteobacteria(γ‐变形菌纲)、p_Proteobacteria(变形菌门)、f_Enterobac-teriaceae(肠杆菌科)、g_Escherichia_Shigella(埃希菌属)丰度较高(图5)。
3 讨论
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FMT可重塑肠道微生物的组成[14]，也可改善机体的代谢[15-17]，应用较广泛。但由于供体与受体之间存在疾病传播的风险，限制了其临床应用[8]。文献报道FMT的治疗效果存在明显差异，其原因之一是供体不同[18]。有研究发现，同种FMT可调节特定菌群并改善微生物代谢途径，控制血糖的效果较好[19]。基于此，本研究主要探讨同种FMT干预对氟尿嘧啶所致CID的改善作用。
有研究发现，氟尿嘧啶注射液可引起大鼠体重下降及腹泻，对肠黏膜造成损伤[20]；而FMT可调节机体的肠道菌群，减轻肠道损伤，缓解肠道炎症[21]。本研究结果显示，连续腹腔注射氟尿嘧啶后，CID模型组和CID+FMT组小鼠均出现进食量和体重下降，虽未观察到明显腹泻，但实验结束时CID模型组小鼠肛门至盲肠肠管长度缩短；与CID模型组相比，CID+FMT组肠壁，出血点减少。该结果表明同种FMT可通过调节肠道菌群，有效缓解化疗导致的肠黏膜炎症，与既往研究一致[21]。
分泌蛋白具有润滑肠道、保护肠道免受外来物质侵害的作用，氟尿嘧啶给药后会严重损伤杯状细胞，使杯状细胞和分泌蛋白减少，进而破坏肠道屏障[22]。有研究表明，FMT可有效改善肠道菌群，减轻炎症性肠病的腹泻程度[23]。本研究HE染色结果显示，空白对照组肠黏膜结构完整，杯状细胞排列紧密，隐窝细胞数量正常；CID模型组肠黏膜出现萎缩和变形，隐窝细胞远离基底膜甚至消失，杯状细胞明显减少，黏膜层炎性细胞增多；CID+FMT组肠黏膜隐窝细胞有所改善，炎性细胞浸润较少。该结果表明同种FMT可通过调节肠道菌群，减轻肠组织上皮缺失、黏膜溃疡、隐窝细胞及杯状细胞丢失等病理学变化，有利于维持肠道完整性，修复肠道屏障。
有研究表明，产肠毒素脆弱拟杆菌(enterotoxigenic Bacteroides fragilis，ETBF)可通过信号转导和转录激活因子3(signal transducer and activator of transcription 3，STAT3)介导IL-8 mRNA及蛋白的表达，促进结直肠癌的发生[24]。生物被膜由ETBF产生，定植在肠道，可促进脆弱拟杆菌毒素的产生，引起机体发生一系列炎症，导致肠道黏膜组织损伤，在结直肠癌的发生发展中起重要作用[25]。以上结果提示，ETBF的增多激发了机体的炎症反应，与结直肠癌的发生发展密切相关。本研究发现，CID模型组小鼠出现了较为明显的体重减轻现象；通过对菌群进行分析，发现f_Bacteroidaceae(拟杆菌科)和g_Bacteroides(拟杆菌属)丰度较高，表明体重减轻可能与ETBF增多有关。大肠埃希菌(Escherichia coli)是常见的革兰阴性菌，属于机体正常的肠道菌群，具有维持肠道菌群稳定的功能[26]。有研究通过对比分析APC(结肠腺瘤样息肉病基因)缺陷小鼠与氧化偶氮甲烷/葡萄糖硫酸钠(AOM/DSS)肠道腺瘤-癌模型小鼠的实验数据，发现大肠埃希菌在正常小鼠肠道菌群中具有较高的丰度[27]。本研究中，CID+FMT组小鼠经干预后o_Enterobacterales(肠杆菌目)相对丰度明显升高，这可能是同种FMT干预引导肠道菌群朝向健康状态转变的一个特征。本研究从肠道菌群的角度展开分析，结果显示，CID模型组小鼠肠道中f_Bacteroidaceae(拟杆菌科)菌群较为丰富，这种现象与体重减轻及黏膜损伤严重密切相关。在经过同种FMT处理后，CID+FMT组小鼠的肠道菌群状况接近空白对照组，且f_Enterobacteriaceae(肠杆菌科)菌群富集较多，发挥了保护性作用，从而减轻了肠道损伤的程度。
综上所述，本研究结果表明，同种FMT可通过调节拟杆菌科和肠杆菌科菌群的丰度，优化肠道菌群结构，缓解肠道炎症及黏膜损伤，未来有望应用于化疗引发的肠道菌群紊乱。本研究还揭示了同种FMT的潜在价值，即通过收集患者自身粪便进行提纯，实现个性化移植，使患者肠道菌群恢复至健康状态，进而减轻以氟尿嘧啶为主的化疗药物引发的不良反应，提高结直肠癌患者的生活质量，这有望成为临床新的个性化预防策略。但本研究仍存在不足之处，如样本量较小，后期需进一步增加样本量完善实验。此外，虽然FMT可减轻机体的炎症反应，缓解CID，但肠道菌群组成复杂，后续仍需进一步鉴别并分析纯化在抗腹泻中发挥重要作用的菌群。
基金
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  • 邯郸市科技局基金(21422083309)
文献
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doi: 10.11855/j.issn.0577-7402.0129.2024.0711
  • 接收时间：2024-01-29
  • 首发时间：2025-11-10
  • 出版时间：2025-03-28
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出版历史
  • 收稿日期：2024-01-29
  • 录用日期：2024-05-23
基金
Handan Science and Technology Bureau Fund(21422083309)
邯郸市科技局基金(21422083309)
作者信息
    1河北工程大学临床医学院，河北邯郸 056038
    2河北工程大学附属医院老年病科，河北邯郸 056038
    3河北工程大学附属医院普外中心，河北邯郸 056038
    4河北工程大学附属医院医学整形美容中心，河北邯郸 056038

通讯作者:

付泽娴，E-mail：
参考文献
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https://castjournals.cast.org.cn/joweb/jfjyxzz/CN/10.11855/j.issn.0577-7402.0129.2024.0711
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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