【作者】 邓晶晶；

【导师】 靳瑞；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2010， 博士

【摘要】 目的本研究采用靳三针组穴中的“足三针”远道取穴防治腹部术后胃肠运动功能紊乱,深入探讨针刺对腹部术后胃肠运动功能紊乱的调整作用和机制。临床研究部分采用传统手法针刺与电针治疗进行对照,旨在丰富针刺调整腹部术后胃肠运动功能紊乱的治疗方式。临床研究严格按照随机对照原则进行,可为针刺调整腹部术后胃肠运动功能紊乱提供有力的循证医学支持。机理研究部分以胃肠起搏细胞ICC为主要切入点,从ICC的数量,超微结构,ENS-ICC-SMC网络结构,Kit-SCF系统的SCF基因表达,血液胃肠激素,胃肠组织的炎症反应等不同层面对针刺调整腹部术后胃肠功能紊乱的机制做了较为全面的探讨,旨在为其临床的推广应用提供科学的理论基础。方法文献研究回顾了近20年来术后胃肠运动功能紊乱的临床研究和实验研究文献,对现代医学关于术后胃肠运动功能紊乱的认识、机理研究和治疗进展,以及中医对其病因病机的认识和治法进行了归纳总结,并采用系统评价的方法对所查及的文献进行分析。临床研究严格按照诊断标准和纳入、排除标准收集广州中医药大学第一附属医院胃肠外科和肝胆外科患者105例,按随机数字表法随机分为手法针刺组、电针组、空白组,每组35人。手法针刺组与电针组取穴相同,均取靳三针组穴里的“足三针”(足三里、三阴交、太冲)进行治疗。手法针刺组的针刺操作为垂直刺入腧穴后持针候气,拇、食、中三指微用力握持针柄,将辨证与辨针下气结合,补虚泻实,施行徐疾提插补泻。若正气不实不虚则采用导气同精手法徐入徐出导之,并配合呼吸调气,随患者一呼一吸将针插入相应深度,再随患者一呼一吸将针提出浅层,待感觉针下正气充实平和,患者自觉症状改善。每穴每次行针约2分钟,治疗30分钟后出针。电针组的针刺操作为足三里和太冲针刺后接一对电极,足三里负极,太冲正极,双三阴交只针刺不加电针。连续疏波,脉冲频率2Hz,电流1～2mA,以患者耐受为度,治疗30分钟后出针。两组均每日2次,直至患者出现排气,最长治疗5天,5天仍未排气者终止治疗。空白组不予针刺。观察并记录术后第一次肛门排气时间,第一次肛门排便时间,恢复流质饮食时间,肠鸣音出现时间及恢复正常时间,腹胀痛分级,胃肠反应分级,对观察结果进行统计分析。实验研究制作大鼠结肠肠肠吻合模型。设针刺组、模型组、假手术组(开腹操作)、空白组进行比较。术后麻醉清醒即予针刺。针刺组、假手术组取穴均为足三针(足三里、三阴交、太冲),用直径0.18mm,长10mm的针直刺入穴位,每5分钟缓慢提插捻转2～3次。模型组、空白组每天同一时间放于自制固定器中15min。每日针刺1次,每次15分钟,连续治疗3天。观察并记录大鼠术后首次排便时间,术后1-3天每天排便粒数、排便重量。术后第四天采用双探头可变角度单光子发射型计算机断层显像仪(SPECT)对大鼠进行核素扫描胃液相排空实验检测,观察大鼠胃半排时间和60min胃排空率的改变。处死采血进行血浆胃动素和血清胃泌素的含量检测。取胃体中部距贲门1／3的胃运动起搏区、盲肠下2cm处结肠(相当吻合口处)组织,分为四部分：一部分制作病理切片观察组织炎症反应情况；一部分制作冰冻切片后,用C-Kit和VAChT免疫荧光双重标记,用激光扫描共聚焦显微镜观测ENS-ICC-SMC网络结构的改变；一部分用于制作电镜标本以观察ICC超微结构的改变；最后一部分用RT-PCR法检测SCF、CALM1的基因表达。结果临床研究1.术后第一次肛门排气情况：手法组与空白组的差异具有显著性(P<0.05),而手法组与电针组、电针组与空白组的差异均无显著性(P>0.05)2.术后第一次肛门排便情况：术后排便时间手法组与空白组、手法组与电针组的差异均具有显著性(P<0.05)。就首次排便质量(是否成形)看,手法组与电针组比较P<0.0125。3.恢复流质饮食时间：手法组与空白组、电针组与空白组均有显著差异,手法组较电针组的差异更大(P手法<0.01,P电针＜0.05)。4.肠鸣音情况：①肠鸣音出现与恢复情况：肠鸣音从出现到恢复所需的时间,手法组与电针组、电针组与空白组间有显著性差异(P<0.05),手法组与空白组无明显差异(P>0.05)。②肠鸣音评分：电针组与空白组评分差值两组差异显著(P<0.01),手法组与空白组评分差值两组差异显著(P<0.01)。5.腹胀痛评分：电针组的治疗前后评分差值与空白组相比有显著差异(P<0.05)。6.胃肠反应评分：电针组治疗前后胃肠反应评分差值与空白组比较,差异有显著性(P<0.05)。7.疗效评定①症状积分评价：手法组治疗前后评分差值与空白组比较有显著性(P<0.05),电针组治疗前后症状评分差值与空白组比较具显著性差异(P<0.01)。②疗效指数分析：手法组与空白组、电针组与空白组均具有显著性差异(P<0.01)。③疗效分级：手法组与空白组在等级程度和总有效率方面的差异具有显著性(P<0.01)。8.术后首次排气时间与针刺介入时间的相关分析：以排气时间为y,针刺介入时间为x,采用曲线拟合,求得曲线方程为y=x1.4(5<x≤39),决定系数R2=0.97,P<0.001。实验研究1.排便观察①首次排便时间：针刺组术后首次排便时间与模型组比较有显著性差异(P<0.05)。②术后第一天大鼠排便例数：术后第一天大鼠排便例数模型组与空白组(P<0.01)、针刺组与空白组(P<0.01)、假手术组与空白组(P<0.05)均有显著性差异。③术后大鼠排便粒数变化：术后第一至三天排便粒数模型组与空白组、针刺组与空白组、假手术组与空白组均有显著性差异(P<0.01)。针刺组与模型组比较,术后第一天无明显差异,术后第二第三天出现显著性差异(P<0.05)④术后大鼠排便重量变化：术后第一至三天排便重量模型组与空白组、针刺组与空白组、假手术组与空白组均有显著性差异(P<0.01)。针刺组与模型组比较,术后第一天排便重量无明显差异,术后第二第三天出现显著性差异(P<0.05)。2.核素扫描胃液相排空实验①半排时间(GET1,2)：模型组与空白组存在显著性差异(P<0.05)。针刺组与模型组比较差异显著(P<0.01)。针刺组与空白组比较无显著性差异。②60分钟排空率：模型组与空白组存在显著性差异(P<0.05)。针刺组与模型组比较差异显著(P<0.01)。针刺组与空白组比较无显著性差异。3.血浆胃动素和血清胃泌素的改变：模型组与空白组比较,差异显著(P<0.01)。针刺组与模型组比较差异显著(P<0.01)。针刺组与空白组比较无显著性差异。4.胃肠组织光镜下病理形态变化：模型组炎症水肿最重,可见急慢性炎症细胞浸润,肉芽肿组织。针刺组炎症水肿程度较模型组轻。模型组和针刺组均可见手术线头和异物反应,存在粘膜溃疡。5.胃肠ENS-ICC-SMC网络结构的变化：模型组ICC数量明显减少,荧光强度减弱,与空白组比较P<0.05。ICC细胞突起不明显,完整的网络样结构消失,网络出现大片空缺,ICC之间以及与其平滑肌和神经纤维之间的紧密样连接缺损。胆碱能神经网状结构严重残缺,呈片状分布,神经纤维间的连接大大减少,VAChT阳性神经纤维明显减少,荧光强度减弱,与空白组比较P＜0.05。ICC与胆碱能神经纤维分布不均匀,胆碱能神经-ICC-平滑肌网络结构紊乱。针刺组ICC分布较为连续,保持网络状结构,ICC细胞突起可见,细胞突触以及与平滑肌和神经纤维之间连接较紧密,无明显的间隙,细胞的数量以及荧光强度比模型组有所增强(P均<0.05),较空白组无显著性差异(P>0.05)。VAChT阳性神经纤维较模型组明显多,维持神经网络样结构,胆碱能神经节之间的连接较为紧密,荧光强度有所增强(P均<0.05),较空白组无显著性差异(P>0.05)。ICC与胆碱能神经纤维间的长突起较模型组增多,相互间的连接结构较为完整,维持网络样结构。6.胃肠ICC电镜超微结构的改变：模型组ICC细胞核皱缩,异染色质趋边,呈斑块状；突起明显减少或消失,许多末梢突起破裂,失去胞浆内容物；胞浆空泡形成,胞膜泡状化；胞浆内细胞器数量明显减少,结构出现异常：线粒体数量减少,出现肿胀、嵴断裂、溶解、形成空泡、甚至破裂；内质网扩张,粗面内质网脱颗粒；许多中间丝排空；出现大的脂滴和空的膜结合泡；次级溶酶体增多,与融合性脂滴和成簇的糖原颗粒密切相关；基底膜缺乏或不完整。部分细胞胞浆内微细结构辨认不清。针刺组ICC细胞核保持正常形态,细胞突起损伤不明显,异染色质部分趋边；存在大量线粒体、核糖体、内质网和高尔基体；胞浆内细胞器形态结构较为清楚,少量线粒体肿胀、内质网扩张；基底膜维持完整。7.胃肠组织SCF基因表达的改变：模型组与空白组存在显著性差异(P<0.01)。针刺组与模型组比较差异存在显著性(P<0.05)。针刺组与空白组无显著性差异。8.胃动素与胃肠排空的关系：胃动素与胃半排时间的方程为y=165.179-0.732x(0<x≤162),决定系数R2=0.309,P<0.001。胃动素与胃排空率的方程为y=0.008+0.003x(0<x≤162),决定系数R2=0.234,P<0.001。胃动素与排便粒数的曲线方程为y=-0.793x+0.017x2-7.734×10-5x3(0<x≤162),决定系数R2=0.65,P<0.001。结论1.针刺能缩短腹部术后首次排气时间,改善术后排便,促使患者提前恢复流质饮食,减轻腹胀痛、恶心呕吐等胃肠反应。2.手法针刺和电针各有优势。手法针刺在整体调节首次排气排便时间、改善排便质量、促使患者提前恢复流质饮食方面疗效较好,而电针在改善和促进肠鸣音恢复、减轻腹胀和胃肠反应方面疗效较好。不同的优势特点可能与其作用机理有关。手法针刺善于整体调节,而电针则长于改善胃肠电节律,提高阈值。3.术后首次排气时间与针刺介入时间呈幂相关。4.针刺对腹部术后胃肠运动功能紊乱的调整作用主要在于促进胃肠蠕动和排空功能的恢复,此功效可能是通过调节血浆胃动素的含量,减轻术后炎症反应,改善胃肠起搏细胞ICC的数量、结构和功能等多方面机制共同作用的结果。4.针刺能促进腹部术后ICC细胞的再生,ENS-ICC-SMC网络结构的恢复,改善ICC超微结构,这可能与针刺具有调整Kit-SCF系统中SCF基因表达的作用有关。更多还原

【Abstract】 ObjectiveTo further discuss the regulative effects and its mechanism of acupuncture on abdominal postoperative gastrointestinal dysmotility, this research used the 3-Leg point in Jin’3-needle technique to prevent abdominal postoperative gastrointestinal dysmotility. In the clinical research part comparison of traditional acupuncture and electroaucpuncture was made in order to enrich acupuncture treatment modality on abdominal postoperative gastrointestinal dysmotility. And this also benefits to inheritance of traditional culture in TCM. The controlled and randomized clinical research part could offer a strong surpport of evidence-based medicine to acupuncture treatment on abdominal postoperative gastrointestinal dysmotility. The experimental research part made gastric movement’s pacemaking cell ICC (interstitial cell of Cajal) as the incision, comprehensively studied the mechanism of acupuncture on abdominal postoperative gastrointestinal dysmotility from different aspects, for example, the number and ultrastructure of ICC, the network structure of ENS-ICC-SMC, the gene expression of SCF mRNA in Kit-SCF system, blood gastrointestinal hormone, inflammatory reponse of gastrointestinal tissue, etc. This could offer a scientific theoretical basis to further clinical application of acupuncture treatment on abdominal postoperative gastrointestinal dysmotility.MethodLiterature researchReviewed literatures of clinical and experimental research on postoperative gastrointestinal dysmotility in the past 20 years, summarized the understanding of domestic and foreign scholars on postoperative gastrointestinal dysmotility, the study methods of gastrointestinal movement, the machanism study and the treatment progress in this field. Analysed the literature by using the method of systematic review.Clinical research105 patients was selected out strictly according to the diagnostic criteria and inclusive and exclusive criteria in gastrointestinal surgery and hepatobiliary surgery department of the No.l Affiliated Hospital of Guangzhou University of TCM. They were divided randomly into traditional acupuncture group, electroacupuncture group and blank group by means of random number table, each group 35 persons. The 3-Leg point in Jin’3-needle technique(Zusanli, Sanyinjiao, Taichong) was used in both acupuncture group and electroacupuncture group.The acupuncture operation of traditional acupuncture group was to insert the needle perpendicularly into the point, hold the needle lightly by using thumb, forefinger and midfinger waiting for Qi. When Qi arrived, made discrimination and compared it with the differentiation of symptoms and signs, then did aucupuncture manipulation, reinforced deficiency and reduced excess by lifting and thrusting the needle. If there was no deficiency or excess, the manipulation of guiding Qi was used coordinately with regulating patient’s breathing. Inserted the needle to certain depth while the patient breathed once, then lifted it to the surficial layer while patient breathed again. Manipulation was done 2 min per time each point. The total treatment lasted 30minutes. In the electroacupuncture group Zusanli and Taichong were given electrol stimulation. Zusanli was connected to cathode while Taichong was connected to anode. Sanyinjiao was not given electrol stimulation. Used rarefaction wave, impulse frequency was 2Hz, current was 1-2mA, according to patient’s tolerence. The total treatment lasted 30minutes. Both traditional acupuncture group and electroacupuncture group were treated twice a day till the patient exhausted. The longest treatment lasted 5 days. Stop treating when 5 day came though the patient still didn’t exhaust. Blank group had no acupuncture treatment.Obsevred and recorded the time of first exhaust, first defecation after operation, the time began to take liquid diet, the time bowelsound appeared and recover, classification of abdominal distension and pain, classification of gastrointestinal reaction, etc. Analyzed and did statistics.Experimental research40 rats were devieded randomly into acupuncture group, model group, sham-operation group and blank group, each group 10 rat. Rats in acupuncture group and model group were given colon transection and anastomosis. Rats in sham-operation group were given open abdominal operation. Both acupuncture group and sham-operation group were given acupuncture treatment when came round after operation. The 3-Leg point in Jin’3-needle technique (Zusanli, Sanyinjiao, Taichong) was used. Inserted the needle and do manipulation like lifting and thrusting, twirling and rotating 2-3 times every 5 minutes. The treatment lasted 15 minutes and was given once a day for 3 days. The model group and blank group were trapped into the same self-made fixator for 15min each day.Observed and recorded the time of first defecation after operation, everyday’s granule and weight of excrement in the first 3 days after operation. Radionuclide scanning on gastric emptying was taken on the forth day after operation to observe the gastric emptying. Rats were killed and the blood was taken to detect plasma MLT and seru GAS. Tissue in pacemaking eara of gas, which was in the middle part of gas 1/3 from cardia and tissue of colon which was 2cm under caecum (anastomosis part) were taken and divided into 4 parts: one was used to make pathology section to observe the inflammatory reponse; one was used to make frozen section, do immunofluorescence double labeling combined with confocal microscopic to observe the network structure of ENS-ICC-SMC; one was used to make electron microscope specimen to observe ultrastructure of ICC; the last one was used to detect gene expression of SCF mRNA and CALM1 mRNA by RT-PCR.ResultClinical research1. The time of first exhaust after operation:The time in traditional acupuncture group was significantly shorter than that in blank group(P<0.05). While the difference between traditional acupuncture group and electroacupuncture group, the difference between electroacupuncture group and blank group, had no significant meaning(P>0.05).2. The time of first defecation after operation:The time in traditional acupuncture group was significantly shorter than that in blank group and electroacupuncture group(P<0.05). The quality of first defecation in traditional acupuncture group was also significantly better than that in electroacupuncture group(P<0.0125).3. The time began to take liquid diet:Both the traditional acupuncture group (P<0.05) and the electroacupuncture group(P<0.01) had signifucantly difference with blank group. And the difference between traditional acupuncture group and blank group was more significant.4. Bowelsound:The time bowelsound from appeared to recovered had significant difference between traditional acupuncture group and blank group, electroacupuncture group and blank group(P<0.05). While there was no significant difference between traditional acupuncture group and electroacupuncture group(P>0.05). The grade change from before and after treatment in both traditional acupuncture group and electroacupuncture group was significantly greater than that in blank group(P<0.01).5. Grade of abodaomimal distension and pain:The grade change from before and after treatment in electroacupuncture group was significantly greater than that in blank group(P<0.05).6. Grade of gastrointestinal reaction:The grade change from before and after treatment in electroacupuncture group was significantly greater than that in blank group(P<0.05).7. Effecacy evaluation:The symptoms score change from before and after treatment in traditional acupuncture group(P<0.01) and electroacupuncture group(P<0.05) was significantly greater than that in blank group. The therapeutic index of traditional acupuncture group and electroacupuncture group was significantly greater than that in blank group(P<0.01). The traditional acupuncture group had significant difference with blank group in therapeutic grade constitution and total effective rate(P<0.01).8. Correlation analyze of the first exhaust time and acupuncture inervention time:The first exhaust time was seen as y, the acupuncture inervention time was seen as x, according to curve fitting, such equation was get:y=x1.4 (5<x≤39), R2=0.97, P<0.001.Experimental research1. Defecation:The first defecation time after operation in acupuncture group was significantly shorter than that in blank group(P<0.05). The number of rats which defecated on the first day after operation had significant difference with blank group(P＜0.05). The decrease of granule and weight of excrement was significant different in acupuncture group, model group and sham-operation group from that in blank group(P<0.01). Compared the acupuncture group with model group, there was no significant difference on the first day after operation, but from the second day on the difference became significant(P<0.05).2. Radionuclide scanning on gastric emptying:GET1/2 was significantly longger in model group than that in blank group(P<0.05) and acupuncture group(P<0.01). The 60min emptying rate was significantly smaller in model group than that in blank group(P<0.05) and acupuncture group(P<0.01). There was no significant difference between acupuncture group and blank group(P>0.05) in both GET1/2 and 60min emptying rate.3. Blood gastrointestinal hormone change:The plasma MLT was significantly less in model group than that in blank group and acupuncture group(P<0.01). There was no significant difference between acupuncture group and blank group(P>0.05). There was no significant difference of seru GAS among the four groups(P>0.05).4. The pathological morphology change of gastrointestinal tissue observed under microscope:The inflammation and hydroncus was most serious in model group. Acute and chronic inflammatory cells soaked. Granuloma was seen. The inflammation and hydroncus in acupuncture group was not so serious. Thread ends and foreign body reaction was seen in both model group and acupuncture group. Mucosa ulcer existed.5. The gastrointestinal ENS-ICC-SMC network structure change:The number of ICC and the fluorescence intensity was greatly decreased in model group compared to blank group(P<0.05). The synapsis were not obvious. The integrated network structure disappear, huge vacancy was found. The tightness connects between ICCs, ICC and SMC, ICC and nerve fibre were deficient. Cholinerqic nerve network structure was seriously imcomplete, present schistose distribution. Connect between nerve fibre were greatly decreased. VAChT positive nerve fibre was obviously reduced, the fluorescence intensity was significantly weaken compared to blank group. The distribution of ICC and cholinerqic nerve was nonuniform. The ENS-ICC-SMC structure was confused. The ICC distribution in acupuncture group was kept network structure. The synapsis were seen. The connections of ICCs, ICC and SMC, ICC and and nerve fibre were tight and there were no obvious gaps. The number of ICC and the fluorescence intensity were increased compared to the model group(P<0.05) and there were no difference with blank group. VAChT positive nerve fibre was obviously increased compared to model group. The network structure was kept and connects between cholinerqic nerves were tight. The fluorescence intensity was significantly stronger than model group(P<0.05) and there were no difference with blank group. Long tubers between ICC and cholinerqic nerve increased. Connect structure was integrated. Network structure existed.6. The gastrointestinal ICC ultrastructure change under electron microscope:The nucleus of ICC in model group contracted, heterochromatin appeared near the nucleus membrance shaped like spot. Tubers were greatly decreased and the tip fractured, cytoplasm content were lost. Vacuole was formed in cytoplasm. The number of organelle was greatly reduced, the structure of organelle was abnormal:the number of mitochondria decreased, mitochondria swelled and dissolved, ridge broke, vacuole formed; endoplasmic reticulum expanded, rough surfaced endoplasmic reticulum dropped granule; lots of intermediate fiber evacuation; large lipid droplet and empty membrane concrement vacuole was seen; secondary lysosome increased, closely related with fused lipid droplet and cluster glycogen grain; basement membrane was incompleted. Some cytoplasm ultrastructure was hard to identified. The nucleus of ICC in acupuncture group kept normal shape. Damage of tubers was not obvious. Heterochromatin partly appeared near the nucleus membrance. Lots of mitochondrion, ribosome, endoplasmic reticulum and Gorky existed. The shape and structure of organelle was clear. A few mitochondrion swelled. some endoplasmic reticulum expanded. basement membrane was completed7. The gene expression of SCF mRNA change:The gene expression of SCF mRNA was significantly less in model group than that in blank group(P<0.01) and acupuncture group(P<0.05). There was no significant difference between acupuncture group and blank group(P>0.05).8. Correlation analyze of plasma MLT and gastrointestinal emptying:MLT was seen as x, the GET1/2, gas emptying rate and granule of excrement were seen as y, according to curve fitting, such equations were get: y=165.179-0.732x(0<x≤162), R2=0.309, P＜0.001; y=0.008+0.003x(0<x≤162), R2=0.234, P＜0.001; y=-0.793x+0.017x2-7.734×10-5x3(0<x≤162), R2=0.65, P＜.001。Conclusion1. Acupuncture can shorten the time of first exhaust after abdominal operation, improve the quality of defecation, help patient take in liquid diet earily, reduce abdominal distension and pain, reduce gastrointestinal reaction like nausea, vomit, etc.2. Both traditional acupuncture and electroacupuncture have its own superiority. Traditional acupuncture is good at shortening the time of first exhaust after abdominal operation, improving the quality of defecation, helping patient take in liquid diet earily. While electroacupuncture is good at recovering bowlsound, reduce abdominal distension and pain, reduce gastrointestinal reaction. Different superiority is likely related to the different mechanism of these two therapy. Traditional acupuncture emphasizes on entire accommodation while electroacupuncture emphasizes on regulate gastrointestinal electrical rhythm and improve threshold.3. The first exhaust time and acupuncture inervention time have a power relation.4. The regulative effect of acupuncture on abdominal postoperative gastrointestinal dysmotility is mainly on helping gastrointestinal movement and emptying function to recover. This effect is achieved perhaps through the combined action of improving plasma MLT, reducing inflammatory reaction, increasing ICC’s number, bettering ICC’s structure and function, etc.5. Acupuncture can increase ICC’s number, better ICC’s ultrastructure and function, recover the gastrointestinal ENS-ICC-SMC network structure. And this is perhaps related to the effect of regulate the gene expression of SCF mRNA in Kit-SCF system.更多还原