PLGA-PEG-PLGA热致水凝胶溶液作为粘膜下注射液在消化内镜手术中的应用

发布时间：2018-05-31 20:12

本文选题：内镜粘膜下剥离术 + 粘膜下注射　；参考：《第二军医大学》2014年博士论文

【摘要】：第一部分目的：内镜粘膜下剥离术（endoscopic submucosal dissection，ESD）是消化道早期肿瘤病变的首选治疗方法，它普及的重要障碍在于手术的操作难度较大和穿孔的发生率较高。通过消化道粘膜下注射形成一个粘膜下液体垫层（submucosal fluid cushion,SFC）是避免穿孔的最好方法，因此合适的注射材料成为改进这种微创内镜技术的重要环节。在本研究中，我们将一种可注射的热致水凝胶的生物材料作为新型的粘膜下注射材料尝试用于ESD术中，以观察可行性、安全性、持久性和组织相容性。方法：这种水凝胶成分是聚乳酸羟基乙酸-聚乙二醇-聚乳酸羟基乙酸三嵌段共聚物Poly(lactic acid-co-glycolic acid)-poly(ethylene glycol)-poly(lactic acid-co-glycolicacid)(PLGA-PEG-PLGA)，这种高分子水凝胶溶液在室温下是一种粘性液体，因此可以注射，在注射到体内之后接触体温，变成一种不能流动的凝胶。我们在活体外和活体内分别将这种热致水凝胶进行粘膜下注射，来评估它的生物安全性、注射可行性、粘膜隆起的高度和时间以及对ESD手术的帮助。具体来说，合成该水凝胶溶液之后，将该溶液经皮下注射到小型猪的后腿内侧，观察皮下隆起灶形成情况；购买新鲜的离体猪胃，分别抽取水凝胶溶液和甘油果糖、透明质酸注射到胃的粘膜下层，在不同时间点评价不同的注射剂形成的粘膜隆起，相机拍照记录；将活体小型猪麻醉后，通过内镜腔道，采用内镜注射针，将三种注射剂注射（水凝胶溶液、甘油果糖和透明质酸）到粘膜下层，形成的粘膜隆起在0，15，30min和1周时通过内镜观察，留取相应内镜图片，1周后处死取材做HE染色评价，光学显微镜观察；将活体小型猪麻醉后，使用该水凝胶溶液进行粘膜下注射并进行ESD手术，3只立即处死以检查组织急性损伤，另2只1周后处死以观察迟发性组织损害。粘膜下注射和ESD手术部位的胃黏膜组织用福尔马林固定，石蜡包埋后HE染色，光镜观察。结果：在将热致水凝胶溶液皮下注射到猪的大腿内侧之后，注射部位因为生理体温所致的快速凝胶化（约30s）在注射后形成明显的椭圆形隆起，它的形状保持时间超过了1月，高度和大小因为胶体的活体内降解逐渐减小，没有观察到红肿热痛、坏死等副作用；在离体猪胃的粘膜下注射实验中，虽然3种注射液注射之后都产生了明显的粘膜隆起，但水凝胶形成的SFC明显更为持久，观察60分钟之后没有见到水凝胶的粘膜隆起灶的大小、外形和硬度有明显变化，相反，其他两者的隆起在15min之后逐渐塌陷；在活体猪的粘膜下注射实验中，三种溶液都造成了明显的粘膜隆起灶，在刚注射完毕时，三者形成的粘膜隆起外形明显，高度上没有明显的差异，但透明质酸和甘油果糖注射完毕15min之后粘膜隆起逐渐塌陷，到了30min时候，内镜下已经观察不到粘膜隆起灶，相反，水凝胶形成的粘膜隆起的外形和清晰边界在30min时都没有改变，1周之后水凝胶形成的粘膜隆起仍然清楚存在且边界清晰，局部没有缺血征象或者溃疡，病理显示没有明显的上皮损伤，也没有观察到炎症细胞；在活体猪的ESD实验中，所有的ESD手术都成功完成，当用电刀切开粘膜隆起灶时，隆起灶的环周切除能够很方便地完成，接下来，粘膜下形成的胶体很容易地就通过内镜吸取孔道被吸走，粘膜下形成一个干净的空腔，最终病变用圈套器被完整的整块切除下来，整个过程中无需任何重复注射，没有发生大量出血和穿孔等严重并发症，病理没有观察到除手术切缘以外其他部位粘膜和固有肌层的损伤，炎症仅停留在浅层。结论：在该凝胶的协助下，ESD手术能够精确地施行病变的整块切除，并且显著缩短了手术时间。同时，没有发生大出血、穿孔和组织损伤等并发症。该凝胶的使用不仅使得ESD程序变得简便，也增加了ESD的有效性和安全性。PLGA-PEG-PLGA热致水凝胶适合作为一个理想的粘膜下注射材料来形成更高的粘膜抬举和维持更长抬举时间，从而减少了并发症。它的独特的粘膜下剥离功能简化了ESD的操作步骤，避免了传统方法中繁琐冗长的剥离过程，因此有利于ESD手术的普及和应用。第二部分目的：隧道内镜是一种创新的内镜技术，在包括经口内镜食管括约肌切开术(peroral endoscopic myotomy, POEM)在内的多种内镜手术中起到核心作用。但目前的隧道内镜技术仍然复杂而费时，而且在某些解剖部位和病理情况下无法成功建立粘膜下隧道(submucosal tunnel, SMT)，这大大限制了它的临床应用，，亟待开发能够解决这些局限性的新方法。PLGA-PEG-PLGA热致水凝胶溶液在我们的前期研究中已经证实了在ESD手术中，它是一种很好的粘膜下注射材料，因此本研究进一步评价它在消化道粘膜下隧道建立过程中的作用。方法：小型猪禁食麻醉后，经猪的肛门将1个三腔二囊管插入结肠，生理盐水冲洗以建立清洁的肠段。在3头小型猪的食管、胃、结肠依次采用PLGA-PEG-PLGA水凝胶溶液作为粘膜下注射液来建立粘膜下隧道，将内镜通过口部或肛门插入消化道并到达手术部位，选择合适的位置通过一个注射针进行水凝胶溶液的粘膜下注射，然后在垫层的一侧用内镜电刀做一个1.5cm左右的横行切口，然后内镜进入粘膜下空间并通过吸引孔道吸引凝胶逐渐前进，视频记录整个操作过程，评价可行性和并发症。手术完成之后，处死所有的猪并进行病理解剖，同时，在进行粘膜下注射之后尽快取消化道粘膜组织，10%中性福尔马林固定，石蜡包埋，HE染色，光镜观察。结果：所有3只猪的食管、胃和结肠均成功建立隧道，没有并发症发生。食管隧道的位置位于距门齿约20-25cm处，胃隧道的位置位于胃体，结肠隧道的位置位于距肛门15-20cm左右的肠段。在食管、胃、结肠建立每个隧道所需要的注射胶的体积约为20ml，每次粘膜下注射都形成了一个厚的粘膜下垫层，在切开粘膜隆起灶之后，带帽的内镜顺利的进入粘膜下空间并吸取胶体，凝胶基本吸取干净后隧道自然形成，无需使用电刀剥离或者钝性分离。在三个部位建立隧道的中位操作时间分别是13.6,11.5和9.4分钟。形成的隧道长度约5cm。解剖没有发现明显的粘膜层和固有肌层的撕裂伤，也没有邻近器官的损伤。结论：我们的初步实验证实了PLGA-PEG-PLGA热致水凝胶溶液作为一种新的粘膜下注射来建立胃肠道SMT是安全可行的，未来需要进行一系列的对照研究来比较它与目前临床上常用的粘膜下注射物质对手术疗效的影响。第三部分目的：经自然腔道内镜手术(natural orifice transluminal endoscopic surgery，NOTES)是近年兴起的一类新型手术类型，它使微创手术从腹壁无可见瘢痕变为真正的无瘢痕，彻底消除了腹壁切口以及相关的切口感染、疼痛、切口疝等问题，患者术后恢复快，心理应激程度低，费用也低。但是内镜在进行NOTES手术时有一些固有的缺陷：内镜镜身柔软，轴向力不足，在进行腹腔内分离时操作起来很困难，分离过程成为内镜进行NOTES手术中最耗时和风险最大的步骤，开发能够帮助内镜在腹腔内分离过程的方法，有利于NOTES的发展和应用。我们的前期研究用PLGA-PEG-PLGA热致水凝胶溶液作为粘膜下注射液，可以极大地方便ESD手术和消化道隧道的建立，受此启发，我们提出设想：如果在NOTES手术进入腹腔之前，用EUS的定位优势将此类凝胶直接注射到手术的目标区域附近，溶液固化后因其本身的占位效应，形成一个直达手术目标的胶体通路，然后NOTES进入腹腔后通过吸走固化的凝胶，从而形成一个直达手术目标的“隧道”，从而省去了费时和复杂繁琐的腹腔内剥离过程，简化了手术过程。因此，我们利用热致水凝胶溶液尝试在动物身上进行这种基于腹腔内生物材料注射的NOTES-腹腔神经丛松解术(celiac plexus neurolysis, CPN)，研究其可行性和安全性。方法：活体小型猪禁食麻醉后，将超声内镜通过猪口插入，在EUS实时监测下将穿刺针刺入，注射胶约30ml。接着沿食道下方用常规隧道技术打一隧道，切开后进入腹腔，发现凝胶位置后在蓝色凝胶块的一侧用内镜电刀切开，内镜镜头进入胶块内空间，通过吸引孔道吸走凝胶，边吸引边前进，直到看到腹腔干和腹主动脉汇合处，前进过程中避免暴力分离，以防出血影响视野，少量出血采用电活检钳进行电凝处理。腹腔干和腹主动脉汇合处附近即为腹腔神经节的位置。采用电活检钳电凝周围组织。视频记录整个操作过程，评价可行性和并发症。手术完成之后，处死所有的猪并进行病理解剖，仔细检查手术部位和附近的器官有无损伤。结果：所有三头猪均成功完成这种方式的NOTES-CPN手术，没有明显的并发症（穿孔或者大出血）。注射所用的水凝胶溶液平均体积为36ml (range25-28ml)。每次粘膜下注射都形成了一个半球状的蓝色凝胶团块。带帽的内镜进入团块内空间之后，凝胶很容易就被吸走，从而形成一个内镜前进的隧道。无需电刀剥离或者其他形式的钝性分离即可到达手术部位。在病理解剖时没有看到粘膜层或者固有肌层的撕裂伤，邻近器官组织也没有任何损伤。操作的平均时间是35mins (range28-41mins)。结论：本研究中我们成功地将一种对温度敏感可逆的PLGA-PEG-PLGA水凝胶溶液作为一种腹腔内注射材料来引导NOTES手术中内镜快速到达目标区域。因此，PLGA-PEG-PLGA热致水凝胶除了可作为内镜粘膜下注射液之外，它的胶化特性和生物相容性使得它可以作为体内手术的标志物和引导物，在NOTES等新兴手术中可能也有更大的应用范围。
[Abstract]:Part one
Objective: endoscopic submucous dissection (endoscopic submucosal dissection, ESD) is the first choice for the early digestive tract lesions of the digestive tract. Its important obstacle is the difficulty of operation and the high incidence of perforation. A submucosal liquid cushion (submucosal fluid cushion, SF) is formed by Submucous injection of the digestive tract. C) is the best way to avoid perforation, so appropriate injection material is an important part of improving this minimally invasive endoscopic technique. In this study, we tried to use an injectable thermo hydrogel biomaterial as a new submucosal injection material in ESD to observe the feasibility, safety, persistence and histocompatibility.
Method: this hydrogel is a three block copolymer Poly (lactic acid-co-glycolic acid) -poly (ethylene glycol) -poly (lactic acid-co-glycolicacid) (lactic acid-co-glycolicacid) (lactic acid-co-glycolicacid) (PLGA-PEG-PLGA). The polymer hydrogel solution is a viscous liquid at room temperature, so it can be injected. After being injected into the body, the body is exposed to body temperature and becomes an inflow gel. We have submucous injection of this thermohydrogel in both living and living bodies to assess its biological safety, the feasibility of the injection, the height and time of the protruding of the mucous membrane, and the help of the ESD operation. Specifically, after the synthesis of the hydrogel solution, The solution was injected subcutaneously into the inside of the hind leg of a small pig to observe the formation of the subcutaneous eminence, and the fresh isolated pig stomach was purchased and the hydrogel and glycerol fructose were extracted respectively. Hyaluronic acid was injected into the submucous layer of the stomach, and the mucosal protruding of different injections was evaluated at different time points and the camera was photographed; the living body was recorded and the living body was small. After anaesthesia, the three kinds of injections (hydrogel, fructose and hyaluronic acid) were injected into the submucosa by endoscopic injection, and the mucosal bulge formed by endoscopy was observed by endoscopy at 0,15,30min and 1 weeks. After 1 weeks, HE staining was performed and the optical microscope observation was used. After anaesthesia, submucous injection of the hydrogel solution and ESD operation were performed with the hydrogel solution. 3 rats were killed immediately to examine the acute tissue injury. The other 2 were executed 1 weeks later to observe the tardive tissue damage. The submucous injection and the ESD surgical site of the gastric mucosa were fixed with Faure Marin, paraffin embedded HE staining, and light microscopy.
Results: after subcutaneous injection of the thermohydrogel solution into the inner thigh of the pig, the rapid gelation (about 30s) caused by the physiological body temperature of the injection site formed an obvious elliptical uplift after the injection. Its shape retention time exceeded January. The height and size of the injection site decreased gradually and the swelling of the body was gradually reduced and the swelling was not observed. Heat pain, necrosis and other side effects; in the submucosal injection experiment of the isolated pig stomach, although 3 kinds of injection produced obvious mucosal protruding after injection, but the SFC formed by hydrogel was obviously more lasting. After 60 minutes, the size of the mucosa of the mucous membrane of the hydrogel was not seen, the shape and hardness of the gel were obviously changed, on the contrary, the other two. The protuberances were gradually collapsed after 15min; in the submucosal injection experiment of living pigs, the three solutions all caused obvious mucosal protuberances. At the end of the injection, the mucosal protuberances formed in the three groups were obvious, and there was no obvious difference in height. However, after the hyaluronic acid and glycerin fructose were injected, the mucosal bulge gradually collapsed after the injection of hyaluronic acid and glycerol. By the time of 30min, the mucosal protrusion was not observed under endoscopy. On the contrary, the morphology and clear boundary of the mucous membrane formed by the hydrogel did not change at 30min. After 1 weeks, the mucous protruding of the hydrogel was still clear and the boundary was clear. There were no ischemic signs or ulcers in the local area. Pathology showed no obvious epithelial injury. No inflammatory cells were observed. In the ESD experiment of living pigs, all the ESD operations were successfully completed. The circumferential resection of the bulge can be easily completed when an electric knife is cut into the mucosal bulge. Then the colloids formed under the mucous membrane are easily sucked away through the endoscope and form a clean space under the mucous membrane. In the cavity, the final lesion was removed with a complete block. No repeated injections were needed during the whole process. No massive bleeding and perforation occurred. The pathology did not observe the damage of the mucosa and the inherent muscularis in other parts except the cutting edge of the operation. The inflammation only stayed in the shallow layer.
Conclusion: with the help of the gel, the ESD operation can accurately perform an integral resection of the lesion and significantly shorten the operation time. At the same time, there are no complications such as massive hemorrhage, perforation and tissue damage. The use of the gel not only makes the ESD program easier, but also increases the effectiveness and safety of ESD heat induced water. The gel is suitable as an ideal submucosal injection material to form higher mucosal lifts and maintain longer lifts, thus reducing complications. Its unique submucosal dissection simplifies the procedure of ESD operation and avoids the tedious and lengthy stripping process in traditional methods, which is conducive to the popularization and application of ESD surgery.
The second part
Objective: tunnel endoscopy is an innovative endoscopic technique that plays a core role in a variety of endoscopic surgery including peroral endoscopic myotomy (POEM), but the current tunnel endoscopy is still complex and time-consuming and can not be successfully established under some anatomical and pathological conditions. Submucosal tunnel (SMT), which greatly restricts its clinical application, is urgent to develop a new method to solve these limitations,.PLGA-PEG-PLGA thermo hydrogel solution. In our previous study, it has been proved that it is a very good sub mucous injection material in ESD operation. Therefore, this study further evaluated it in this study. The role of the submucosal tunnel of the digestive tract during the establishment of the tunnel.
Methods: after a small pig's anaesthesia, 1 three cavities and two cystic tubes were inserted into the colon through the pig's anus, and the normal saline was washed to establish a clean intestinal segment. The esophagus, stomach and colon of 3 miniature pigs were used as submucous submucous tunnel by PLGA-PEG-PLGA hydrogel solution in order to insert the endoscope through the mouth or anus into the digestive tract. At the site of the operation, an injection needle is selected for the submucous injection of the hydrogel solution, and an endoscopic scalpel is used to make a 1.5cm transverse incision on one side of the cushion, then the endoscope enters the submucosal space and draws the gel by attracting the channel to advance gradually. The video records the entire operation process, and the evaluation can be made. After the operation was completed, all pigs were killed and pathologically dissected. At the same time, the mucosal tissue was removed as soon as submucous injection. 10% neutral formalin fixation, paraffin embedding, HE staining, and light microscopy were observed.
Results: the esophagus, stomach and colon were successfully established in all 3 pigs, without complications. The location of the tunnel was located about 20-25cm from the teeth, the position of the stomach tunnel was located in the body of the stomach and the location of the tunnel in the colon was located around the 15-20cm of the anus. In the esophagus, stomach and colon, the volume of injection needed for each tunnel was established. For 20ml, each submucosal injection formed a thick submucous cushion. After opening the mucosal bulge, the endoscopy with the cap entered the submucosal space and absorbed the colloid. The tunnel was naturally formed after the gel was basically clean. The middle operation time of the tunnel was established in three parts without the need of electric knife stripping or blunt separation. It was 13.6,11.5 and 9.4 minutes respectively. The length of the tunnel was about 5cm., and there was no obvious tear in the mucous layer and the inherent muscularis, and there was no injury to the adjacent organs.
Conclusion: our preliminary experiments confirm that the PLGA-PEG-PLGA thermal hydrogel solution is safe and feasible as a new submucous injection to establish the gastrointestinal tract SMT. In the future, a series of controlled studies are needed to compare the effect of the submucosal injections that are commonly used in clinic on the surgical effect.
The third part
Objective: natural orifice transluminal endoscopic surgery (NOTES) is a new type of surgery in recent years. It makes minimally invasive surgery from the invisible scar of the abdominal wall to a real no scar. It completely eliminates the abdominal wall incision and related incision infection, pain, incisional hernia, and so on. It is fast, low psychological stress and low cost. But endoscopy has some inherent defects in NOTES operation: the endoscopy is soft, the axial force is insufficient and it is difficult to operate in the abdominal cavity. The separation process becomes the most time-consuming and most risky step in the endoscopy for NOTES operation, and the development can help the endoscope in abdominal cavity. The method of internal separation is beneficial to the development and application of NOTES. Our previous study using PLGA-PEG-PLGA thermal hydrogel solution as submucosal injection can greatly facilitate the establishment of ESD surgery and the tunnel of the digestive tract. Inspired by this, we suggest that if the NOTES operation enters the abdominal cavity, the EUS positioning advantage will be used. The gel is injected directly near the target area of the operation. After the solution is solidified, a colloid pathway is formed because of its own occupying effect. Then NOTES enters the abdominal cavity and absorbs the solidified gel through the intraperitoneal cavity to form a "tunnel" that reaches the objective of the operation, thereby saving the time-consuming and complicated intricate intraperitoneal stripping. The procedure simplifies the procedure. Therefore, we try to use a thermohydrogel solution to try the NOTES- plexus neurolysis (CPN) based on intraperitoneal biological materials (celiac neurolysis, CPN) to study the feasibility and safety of the intraperitoneal injection of the intraperitoneal (CPN).
Methods: after the living miniature pig was fasting anaesthesia, the ultrasound endoscopy was inserted through the porcine mouth. The puncture was punctured under the EUS real-time monitoring. The injection glue was inserted into the tunnel under the conventional tunnel technique under the esophagus, and then entered the abdominal cavity after the incision. After the gel position was found on one side of the blue gel block, the endoscopic scalpel was cut and the endoscope lens entered the glue. The intraperitoneal space takes away the gel by attracting the passage and draws the edge to attract the edge, until the abdominal trunk and abdominal aorta converge to avoid violent separation in order to prevent the hemorrhage to affect the field of vision. A small amount of bleeding is electrocoagulated by electrobiopsy forceps. The abdominal trunk and abdominal aorta are the location of the celiac ganglion near the confluence of the abdominal and abdominal aorta. The whole operation was recorded by video. The whole operation was recorded to evaluate the feasibility and complications. After the operation was completed, all pigs were executed and the pathological anatomy was carried out. The surgical site and the adjacent organs were carefully examined.
Results: all three pigs successfully completed this NOTES-CPN operation without obvious complications (perforation or massive bleeding). The average volume of the hydrogel solution used for injection was 36ml (range25-28ml). Each submucosal injection formed a hemispherical blue gelation group. The glue is easily sucked away to form an endoscope tunnel. The surgical site is reached without an electric knife or other form of blunt separation. There is no tear in the mucous layer or the inherent muscularis in the pathological anatomy, and there is no damage to the adjacent organs. The average time of operation is 35mins (range28-41mins).
Conclusion: in this study, we successfully used a temperature sensitive and reversible PLGA-PEG-PLGA hydrogel solution as an intraperitoneal injection material to guide the rapid arrival of endoscopy in the target area in the NOTES operation. Therefore, the PLGA-PEG-PLGA thermo induced hydrogel, in addition to the injection of the endoscopic mucosal injection, its gelation properties and biocompatibility. Sex enables it to be used as a biomarker and guide in vivo. It may also have a wider application in new operations such as NOTES.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R735

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