【作者】 张凯；

【导师】 孙思予；

【作者基本信息】 中国医科大学 ， 内科学， 2019， 博士

【摘要】 内镜超声引导下消化道与胆囊暂时性支架吻合的动物实验目的:内镜超声引导下消化道与胆囊暂时性支架吻合对内镜医师来说是一项具有挑战性的技术,需要高水平的技术。内镜超声引导下消化道与胆囊暂时性支架吻合具有挑战性的一个非常重要的原因是胆囊很容易塌陷。为了解决这个问题,我们为内镜超声引导下消化道与胆囊暂时性支架吻合开发一种可回收的穿刺锚牵拉(RPAT)方法。我们使用猪模型评估并比较了内镜超声引导下消化道与胆囊暂时性支架吻合的RPAT方法与现有的内镜超声引导下消化道与胆囊暂时性支架吻合方法的成功率,安全性和结果;消化道-胆囊瘘管的形成是EUS引导下保胆取石术中内镜可以进入胆囊的关键。消化道-胆囊瘘管的闭合是EUS引导下保胆取石术结束的关键。消化道-胆囊瘘管的闭合之后的长期情况是本术式关注的重点。如果术后胆囊与消化道粘连很重,甚至持续与周围粘连严重,这一术式的意义和必要性就需要重新考量。反之,若术后胆囊与消化道以及周围组织粘连很轻甚至没有粘连,胆汁中即使出现小的结晶或沉淀,由于胆囊的的收缩功能良好,可能会把这些结晶或沉淀排出,避免形成结石;对于结石复发的患者,不论是再次行消化道与胆囊支架吻合术还是选择外科手术切除都不会产生很大影响,那么这个术式也将有着广阔的临床应用前景。因此,我们将进行内镜超声引导下消化道与胆囊暂时性支架吻合后胆囊-消化道瘘管形成及闭合的初步研究;胆囊炎及胆囊结石是目前临床上较为常见的疾病之一,在我国发病率逐年增加,影响人们的健康。本课题组进一步研究并报道了EUS引导下胆囊引流术及结石取出的手术方式,初步显示了此技术的可行性与安全性。EUS引导下胆囊与消化道暂时性支架吻合在建立胆囊和消化道之间的通道过程中,胆囊和消化道形成通路。消化道内部不是无菌环镜,细菌经通路进入胆囊,对胆囊内细菌微生态可能造成影响,而这些均可引发或加重胆囊炎和胆囊结石,因此有必要对行术后胆囊菌群评价及培养进行深入研究。方法:将16只巴马小型猪(15-20kg)随机分为实验组(n=8)和对照组(n=8)。可回收的穿刺锚牵拉方法在实验组中进行,并且在对照组中进行没有可回收穿刺锚的内镜超声引导下消化道与胆囊暂时性支架吻合。在实验组和对照组之间比较和分析手术不良事件,伤口愈合和内镜超声引导下消化道与胆囊暂时性支架吻合成功率;将巴马小型猪(15-20kg)采用可回收的穿刺锚牵拉方法进行消化道与胆囊暂时性支架吻合,观察消化道胆囊瘘管的形成情况。拔掉支架后,模拟胆囊取石,观察瘘管消化道侧的闭合情况,并进行解剖观察瘘管长期闭合后的情况;将未手术的巴马小型猪直接解剖的胆囊粘膜及胆汁为对照组。每只巴马小型猪支架置入术前采集的采集的胆汁及胆囊粘膜标本作为术前组,支架取出后不同时间采集的胆汁及胆囊粘膜标本作为术后组。选择胆囊功能良好的实验动物在EUS引导下穿刺并抽取胆囊胆汁,进行消化道与胆囊支架吻合,术后用内镜模拟胆囊取石,清理胆囊,并打入造影剂造影见胆囊管通畅后,然后将动物解剖取胆汁。将收集来的标本分组,进行如下实验:1、从胆囊粘膜中提取16SrDNA进行高通量测序,比较各组菌群的变化。2、从胆汁中提取16SrDNA进行高通量测序,比较各组菌群的变化。结果:所有内镜超声引导下消化道与胆囊暂时性支架吻合在实验组的8只动物中成功进行(8/8;100%)。然而,在对照组中,内镜超声引导下消化道与胆囊暂时性支架吻合仅在四头猪上成功进行(4/8;50%);其余四头猪因胆囊塌陷导致支架释放不成功,进而导致内镜超声引导下消化道与胆囊暂时性支架吻合失败而死亡;在瘘管形成情况方面,形成瘘管比较坚实的时间为2-4周,并且初步探索瘘管的胆囊连接处为相对薄弱位置。在瘘管闭合方面,课题组初步通过术后的胃镜检查发现,在支架拔除术后24-48小时左右瘘管口就会闭合。术后的动物解剖发现,在支架拔除术后1个月实验动物解剖时发现胆囊与消化道形成片状粘连瘢痕,但我们在支架拔除术后4个月动物解剖发现胆囊-消化道之间行成极细的瘢痕带,支架拔除术后6个月的实验动物解剖室发现实验动物胆囊-消化道吻合处光滑无粘连;胆囊粘膜菌群基于Ion S5TMXL测序平台,利用单端测序(Single-End)的方法,构建小片段文库进行单端测序。通过对Reads剪切过滤,平均每样品测得78,102条reads,经过质控平均得到73,517条有效数据,质控有效率达94.24%。以97%的一致性(Identity)将序列聚类成为OTUs(Operational Taxonomic Units),共得到7,057个OTUs,然后对OTUs序列与Silva132数据库进行物种注释。注释结果中,共有2,578(36.53%)个OTU注释到属水平。胆汁菌群通过对Reads剪切过滤,平均每样品测得77,458条reads,经过质控平均得到73,312条有效数据,质控有效率达94.66%。以97%的一致性(Identity)将序列聚类成为OTUs(Operational Taxonomic Units),共得到5,542个OTUs,然后对OTUs序列与Silva132数据库进行物种注释。注释结果中,共有2,415(43.58%)个OTU注释到属水平。术前术后胆囊粘膜的菌群在门水平上,我们发现占据主导地位的主要包括变形杆菌(Proteobacteria)、黄粉杆菌(Tenericutes)、硬杆菌(Firmicutes);在纲水平上主导的为γ-变形杆菌(Gammaproteobacteria)、Mollicutes、梭状芽孢杆菌(Clostridia);在目水平上的优势物种为黄单胞菌属(Xanthomonadales)、支原体(Mycoplasmatales)、梭状芽孢杆菌(Clostridiales);在科水平上的优势物种为黄单胞菌科(Xanthomonadaceae)、支原体科(Mycoplasmataceae)、脱硫弧菌科(Desulfovibrionaceae);在属水平上的优势物种为寡养单胞菌(Stenotrophomonas)、支原体(Mycoplasma)、嗜胆菌属(Bilophila);优势种为副嗜血杆菌(Haemophilus_parasuis)、莫拉菌(Moraxella catarrhalis)。而胆汁的菌群在门水平上,我们发现占据主导地位的主要包括变形杆菌(Proteobacteria)、拟杆菌(Bacteroidetes)、硬杆菌(Firmicutes);在纲水平上主导的为γ-变形杆菌(Gammaproteobacteria)、Bacteroidia、杆菌(Bacilli);在目水平上的优势物种为肠杆菌类(Enterobacteriales)、几丁质噬菌体(Chitinophagales)、乳酸杆菌类(Lactobacillales);在科水平上的优势物种为肠杆菌科(Enterobacteriaceae)、几丁质噬菌体科(Chitinophagaceae)、脱硫弧菌科(Desulfovibrionaceae);在属水平上的优势物种为肠杆菌科(Enterobacteriaceae)、定会杆菌(Dinghuibacter)、嗜双胞菌(Bilophila);优势种为大肠杆菌(Escherichia_coli)、戊糖球菌(Pediococcus_pentosaceus)、假单胞菌(Pseudomonas_hibiscicola)。结论:可回收的穿刺锚牵拉方法有助于提高内镜超声引导下消化道与胆囊暂时性支架吻合的成功率,并可减少胆囊塌陷。因此,我们相信这种方法可以在不久的将来应用于临床的内镜超声引导下消化道与胆囊暂时性支架吻合。此外,可回收的穿刺锚牵拉方法将提高内镜超声引导下消化道与胆囊暂时性支架吻合的安全性;内镜超声引导下消化道与胆囊暂时性支架吻合及关闭吻合口中,消化道-胆囊瘘管牢固形成时间为2-4周,闭合时间为24-48小时,瘘管闭合后长期可变细甚至消失;实验动物在内镜超声引导下消化道与胆囊支架吻合及拔除支架后,手术前后胆囊粘膜及胆汁菌群组成发生改变,而且随着时间的增加胆囊内菌群也在发生不断地变化,但是术前术后菌群相关的导致结石活性物质无增高趋势,因此该手术对胆囊结石复发的影响可能比较小。可回收穿刺锚牵拉法应用于内镜超声引导下消化道与胆囊暂时性支架吻合的临床研究目的:评价可回收穿刺锚牵拉法应用于内镜超声引导下消化道与胆囊暂时性支架吻合对胆囊息肉、结石等胆囊疾病的治疗效果,并评价分析该方法的安全有效性。通过观察不同方法的内镜超声引导下消化道与胆囊吻合术的各项临床指标,分析该方法的安全有效性。研究方法:将2014年6月至2019年1月于我院行内镜超声引导下消化道与胆囊暂时性支架吻合术的患者纳入研究。观察指标为:超声内镜引导下两种方法的吻合术操作时间的长短,吻合位置,吻合手术的成功率,术后的腹痛评分(采用视觉模拟评分法,VAS)、手术后是否有发热,有无其他术后相关的并发症发生。使用SPSS Statistics 23.0(SPSS,Inc,Chicago,Ill,USA)进行统计学分析。结果:本研究共纳入2014年6月至2019年1月期间在我院因胆囊结石、胆囊息肉等胆囊疾病行超声内镜引导下消化道与胆囊暂时性支架吻合术的治疗45例患者。男性16例,女性29例。其中进行可回收穿刺锚牵拉(RPAT)法手术的14例患者作为实验组,其中男性4例,女性10例,未使用可回收穿刺锚牵拉(RPAT)法手术的31例患者作为对照组,其中男性12例,女性19例。实验组的技术成功率100%(14/14),临床成功率100%(14/14),而在对照组技术成功率87.1%(27/31),临床成功率83.9%(26/31)。实验组发生术后发热7例,一过性白细胞升高9例,对照组发生术后发热13例,一过性白细胞升高18例。对照组术后出现并发症7例,其中腹腔感染1例,气腹2例,而实验组内均未出现。实验组术后使用抗生素天数及术后禁食时间均短于对照组(3.14d vs 4.43d,2.36d vs2.93d)。结论:综上所述,可回收穿刺锚牵拉法EUS引导下消化道与胆囊吻合术是一项安全有效、操作快捷简便,成功率高且不良事件发生率低。这种方法将来可以应用于临床的内镜超声引导下胆囊的引流。更多还原

【Abstract】 Animal experiment of endoscopic ultrasound guided gastrointestinal tract and temporary stent anastomosis Objective: Endoscopic ultrasound-guided temporary gastrointestinal tract anastomosis with the gallbladder is a challenging technique for endoscopists and requires a high level of skill.A very important reason for the endoscopic ultrasound-guided anastomosis of the digestive tract and the temporary stent of the gallbladder is that the gallbladder is easily collapsed.In order to solve this problem,we developed a retrievable puncture anchor traction(RPAT)method for endoscopic ultrasound-guided temporary anatomy of the digestive tract and gallbladder.We used a pig model to evaluate and compare the success rate,safety,and outcome of the RPAT method of endoscopic ultrasound-guided temporary stent anastomosis between the digestive tract and the gallbladder and the existing endoscopic ultrasound-guided method of temporary stent anastomosis of the digestive tract and gallbladder.The formation of the digestive tract-capsule fistula is the key to endoscopic access to the gallbladder during EUS-guided gallstone removal.The closure of the digestive tract-the gallbladder fistula is the key to the end of the EUS-guided gallstone removal procedure.The long-term condition after the closure of the digestive tract-the gallbladder fistula is the focus of this procedure.If the postoperative gallbladder and the digestive tract are very heavy,and even persist in adhesion to the surrounding area,the significance and necessity of this procedure need to be reconsidered.Conversely,if the postoperative gallbladder adheres to the digestive tract and surrounding tissues very lightly or even without adhesion,even if small crystals or precipitates appear in the bile,due to the good contraction function of the gallbladder,these crystals or precipitates may be discharged to avoid the formation of stones;For patients with recurrence of stones,whether it is re-analysis of the digestive tract and gallbladder stent or surgical resection will not have a great impact,then this procedure will also have broad clinical application prospects.Therefore,we will conduct a preliminary study of the formation and closure of the gallbladder-digestive fistula after endoscopic ultrasound-guided anastomosis of the digestive tract and the gallbladder;cholecystitis and gallstones are one of the more common clinical diseases in China.The rate is increasing year by year,affecting people’s health.The research team further studied and reported the surgical procedure of gallbladder drainage and stone removal guided by EUS,which showed the feasibility and safety of this technique.The EUS guides the gallbladder to the temporary stent of the digestive tract.During the process of establishing the passage between the gallbladder and the digestive tract,the gallbladder and the digestive tract form a pathway.The inside of the digestive tract is not a sterile loop mirror.The bacteria enter the gallbladder through the pathway,which may affect the bacterial micro-ecology in the gallbladder.These can cause or aggravate cholecystitis and gallstones.Therefore,it is necessary to evaluate the postoperative gallbladder flora.Cultivate for in-depth research.Methods: Sixteen Bama miniature pigs(15-20 kg)were randomly divided into an experimental group(n = 8)and a control group(n = 8).The retrievable puncture anchor traction method was performed in the experimental group,and endoscopic ultrasound guided endoscopic ultrasound guided anastomosis of the gallbladder without a retrievable puncture anchor in the control group.Comparison and analysis of operative adverse events,wound healing and endoscopic ultrasound-guided succession of temporary stent anastomosis between the experimental group and the control group;Recoverable puncture anchors were used for Bama miniature pigs(15-20 kg)The traction method was performed to make the digestive tract and the temporary stent of the gallbladder anastomosis,and the formation of the gallbladder fistula of the digestive tract was observed.After the stent was removed,the gallbladder was taken for stone removal,and the closure of the digestive tract side of the fistula was observed.The anatomical observation of the long-term closure of the fistula was performed.The gallbladder mucosa and bile directly dissected from the untreated Bama mini-pig were used as the control group.Bima and gallbladder mucosa specimens collected before surgery were taken as preoperative group,and bile and gallbladder mucosa specimens collected at different times after stent removal were used as postoperative group.The experimental animals with good gallbladder function were selected under the guidance of EUS and the gallbladder bile was taken out.The digestive tract was anastomosed with the gallbladder stent.After the operation,the gallbladder was used to simulate the gallbladder,the gallbladder was removed,and the contrast agent was used to see the cystic duct.The animals were dissected for bile.Group the collected specimens and perform the following experiment: 1.16SrDNA was extracted from the gallbladder mucosa for high-throughput sequencing,and the changes of each group were compared.2.16SrDNA was extracted from bile for high-throughput sequencing,and the changes of each group were compared.Results: All endoscopic ultrasound-guided anastomoses of the digestive tract and the gallbladder were successfully performed in 8 animals in the experimental group(8/8;100%).However,in the control group,endoscopic ultrasound-guided temporary gastrointestinal tract anastomosis with the gallbladder was successfully performed on only four pigs(4/8;50%);the remaining four pigs were unsuccessful in stent release due to gallbladder collapse.Furthermore,endoscopic ultrasound guided death of the digestive tract and the temporary stent of the gallbladder failed;in the formation of the fistula,the formation of the fistula was relatively solid for 2-4 weeks,and the junction of the gallbladder was initially explored as a relatively weak position.In terms of fistula closure,the team initially found that the fistula was closed 24 to 48 hours after the stent was removed by postoperative gastroscopy.Postoperative animal anatomy found that the anatomy of the experimental animal at 1 month after stent extraction revealed a flaky adhesion scar between the gallbladder and the digestive tract,but we found that the gallbladder-digestive tract was found 4 months after stent extraction.A very thin scar band,6 months after stent removal,the experimental animal anatomy chamber found that the gallbladder-digestive tract anastomosis was smooth and non-adhesive;the gallbladder mucosal flora was based on the IonS5 TM XL sequencing platform,using single-end sequencing(Single-End)The method of constructing a small fragment library for single-ended sequencing.By cutting and filtering the Reads,an average of 78,102 reads per sample was obtained,and 73,517 valid data were obtained by quality control,and the quality control efficiency was 94.24%.The sequences were clustered into OTUs(Operational Taxonomic Units)with 97% identity(Identity),and a total of 7,057 OTUs were obtained,and then the OTUs sequence and the Silva132 database were species annotated.In the annotated results,there are 2,578(36.53%)OTU annotations to the genus level.The bile flora was cut and filtered by Reads,and an average of 77,458 reads per sample was measured.After quality control,73,312 valid data were obtained,and the quality control efficiency was 94.66%.The sequences were clustered into OTUs(Operational Taxonomic Units)with 97% identity(Identity),a total of 5,542 OTUs were obtained,and the OTUs sequence was then annotated with the Silva132 database.In the annotated results,there are 2,415(43.58%)OTU annotations to the genus level.Preoperatively,the flora of the gallbladder mucosa at the door level,we found that the dominant ones include Proteobacteria,Tenericumes,and Firmicutes;γ-deformation dominated at the level Gammaproteobacteria,Mollicutes,Clostridia;dominant species at the target level are Xanthomonadales,Mycoplasmatales,Clostridiales;at the level of the family The dominant species are Xanthomonadaceae,Mycoplasmataceae,Desulfovibrionaceae;the dominant species at the genus level are Stenotrophomonas,Mycoplasma,and biliary Bilophila;the dominant species are Haemophilus_parasuis and Moraxella catarrhalis.While the bile flora is at the level of the door,we found that the dominant ones include Proteobacteria,Bacteroidetes,and Firmicutes;the gamma-proteobacteria(Gammaproteobacteria)is dominant at the level.,Bacteroidia,Bacilli;the dominant species at the target level are Enterobacteriales,Chitinophagales,Lactobacillales;the dominant species at the family level is Enterobacteriaceae(Enterobacteriaceae),Chitinophagaceae,Desulfovibrionaceae;dominant species at the genus level are Enterobacteriaceae,Dinghuibacter,Bilophila;The dominant species are Escherichia_coli,Pediococcus_pentosaceus,Pseudomonas_hibiscicola.Conclusion: The retrievable puncture anchor traction method helps to improve the success rate of endoscopic ultrasound guided digestive tract and temporary cholecyst stent anastomosis,and can reduce gallbladder collapse.Therefore,we believe that this method can be applied to the endoscopic ultrasound-guided temporary stent anastomosis of the gallbladder in the near future.In addition,the retrievable puncture anchor traction method will improve the safety of endoscopic ultrasound guided digestive tract and temporary stent anastomosis;endoscopic ultrasound guided digestive tract and gallbladder temporary stent anastomosis and closure of the anastomosis,digestive tract-The gallbladder fistula is firmly formed for 2-4 weeks,the closure time is 24-48 hours,and the long-term variable or even disappears after the fistula is closed.The endoscopic ultrasound guided digestive tract and gallbladder stent anastomosis and removal of the stent,before and after surgery The composition of the gallbladder mucosa and bile flora changed,and the flora in the gallbladder also changed continuously with time.However,the preoperative postoperative flora-related causes no increase in the activity of the stone,so the operation on the gallstone The effects of recurrence may be small.Clinical study of retractable puncture anchor traction method for endoscopic ultrasound guided gastrointestinal tract and temporary stent anastomosis Objective: To evaluate the therapeutic effect of retrievable puncture anchor traction method on endoscopic ultrasound-guided gastrointestinal tract and temporary stent anastomosis for gallbladder polyps,stones and other gallbladder diseases,and evaluate the safety and efficacy of the method.The safety and effectiveness of the method were analyzed by observing the clinical parameters of different methods of endoscopic ultrasound guided digestive tract and gallbladder anastomosis.Methods: Patients who underwent endoscopic ultrasonography guided laparoscopic and gallbladder temporary stent anastomosis from June 2014 to January 2019 were included in the study.The observation indicators were: length of anastomosis operation time,anastomotic position,anastomosis success rate,postoperative abdominal pain score(using visual analogue scale,VAS),and whether there was fever after surgery,postoperative complications.Statistical analysis was performed using SPSS Statistics 23.0(SPSS,Inc,Chicago,Ill,USA).Results: A total of 45 patients with gallbladder disease,such as gallbladder stones and gallbladder polyps,underwent endoscopic laparoscopic and cystic stent anastomosis in our hospital from June 2014 to January 2019.There were 16 males and 29 females.Among them,14 patients underwent retractable puncture anchor traction(RPAT)surgery as the experimental group,including 4 males and 10 females,and 31 patients who did not use the retractable puncture anchor(RPAT)operation as a control group.There are 12 males and 19 females.The technical success rate of the experimental group was 100%(14/14),and the clinical success rate was 100%(14/).14),while in the control group,the technical success rate was 87.1%(27/31),and the clinical success rate was 83.9%(26/31).In the experimental group,there were 7 cases of postoperative fever,9 cases of transient leukopenia,13 cases of postoperative fever and 18 cases of transient leukocytes in the control group.There were 7 cases of complications in the control group,including 1 case of abdominal infection and 2 cases of pneumoperitoneum,but none of them in the experimental group.The days of antibiotic use and postoperative fasting time in the experimental group were shorter than those in the control group(3.14 d vs 4.43 d,2.36 d vs 2.93d).Conclusion: In summary,the retrievable puncture anchor traction method EUS guided gastrointestinal and gallbladder anastomosis is a safe and effective,quick and easy operation,high success rate and low incidence of adverse events.This method can be applied to clinical endoscopic ultrasound-guided drainage of the gallbladder.更多还原