| Objective:1. Cholecystolithiasis is one of the most common gastrointestinal diseases. Gallbladder hypomotility may be a key factor in the pathogenesis of cholelithiasis. Nevertheless previous researches more focus on the decreased density of cholecystokinin (CCK), CCK-receptor dysfunction, gallbladder smooth muscle impairment and extrahepatic biliary system regulating function disorder, etc. With further studies on interstitial cell of Cajal (ICC) in gastrointestinal tract, several digestive dynamic disorders have been associated with the loss of ICC. The latest research shows that ICC exist in the animals and human gallbladder, its role in the regulation of gallbladder motility and relationship with pathogenesis of cholecystolithiasis has become the research hotspot. This study aims to explore some new pathogenesis, precaustions in cholelithiasis. On the base of rabbit cholesterol gallstone models, the changes and relative mechanisms of gallbladder motility and ICC were explored by a series of experiments in vivo and in vitro.2. Cholecystectomy is considered the "golden standard" in treatment of gallstone, but various complications and postoperative side effects exist. For these reasons, some experts suggested a novel endoscopic minimal invasive cholecystolithotomy (EMIC) to substitute cholecystectomy, which is beneficial for patients who can selectively preserve the gallbladder. With the years of clinical practice, some details of indications and surgical techniques in EMIC deserve to be improved. Therefore, we provide some theoretical and clinical research for amelioration of EMIC. According to the distribution of ICC in gallbladder, some explorations in surgical methodology of EMIC have been conducted, and preliminary results have been achieved.Methods:(1) 52 New Zealand rabbit were divided into control group (20) and gallstone group (32). Gallstone models were established with high cholesterol diet, and the gallstone formation rate was assessed ultrasonographically. The gallbladder contractile function was estimated by SPECT. The basal electrical rhythm (BER) of gallbladder was recorder in vivo, and gallbladder muscle strip contractile responses to CCK in vitro were conducted alone. Changes of ICC quantities were analyzed by whole mount immunofluorescence. Ultrastructure changes of ICC in gallbladder tisssues were studied using electron microscope. The protein expression of c-kit in gallbladder was detected by Western blot analysis. (2) Using automatic biochemical analyzer detected bile cholesterol, phospholipids and bile acid concentrations and calculated cholesterol saturation index (CSI). The parameters of oxidative stress reaction (SOD, MDA, GSH-PX) in gallbladder tissues were analyzed between two groups. The ICC apoptosis of gallbladder tissue in two groups was assessed by TUNEL ASSAY KIT. After successful isolation and purification of ICC from the rabbit gallbladder in vitro, ICCs were cultured in increased cholesterol concentration medium (0,25,50, 100mg/L). Then the increased concentration cholesterol scavenger, methyl-?-cyclodextrin (0, 2,4,6mM), was added into the ICC culture medium, which contained cholesterol 100mg/L. The parameters of oxidative stress reaction, pacemaker potential and apoptotic rate of ICC were detected by patch clamp and flow cytometer in respective concentration group. (3) A total 65 patients with cholecystolithiasis were underwent EMIC. According to the distribution of ICC in gallbladder, the incision positions of gallbladder were selected differently, which located in gallbladder fundus (35 patients) and gallbladder corpus (30 patients). We also ameliorated the surgical techniques and supplemented surgical indications of EMIC. Follow-up including gallbladder contractile function between two groups, clinical assessment and recurrent of stone was conducted.Results:(1) In experiment group, the rabbit gallstone-forming rate was 74.2%(23/31). Compared with the control group, SPECT scan shows the gallbladder contractile function was obviously decreased in the experiment group (P<0.05). Furthermore, the frequency and amplitude of BER in the experiment group in vivo was reduced significantly. The tension value of gallbladder muscle strip to CCK was lowered in the in the lithogenic group, which suggested the contractile function of gallbladder was impaired. The whole mount immunofluorescence shows that c-kit positive ICCs were existed in the gallbladder tissues, however, the decreased number of ICCs were present obviously in the gallstone group. Moreover, the ultrastructure of ICC has changed in the gallstone group, including mitochondrial swelling, autophagosome formation, nucleus invagination and chromatin margination, etc. The protein expression of c-kit was also attenuated in the gallstone group. (2) The CSI was higher in the lithogenic bile. In the experiment group, the marks of antioxidant stress (SOD, GSH-PX) were damped, but the mark of oxidative stress (MDA) was ascended. Moreover, there were seldom TUNEL/c-kit double positive apoptotic ICCs in the normal gallbladder tissues, while increased quantities in the gallstone group. ICCs were successfully derived from gallbladder tissue of New Zealand White Rabbit by enzymatic dissociation of collagenase type ?, and purified by gradient centrifugation and microdissection technique, which the purification rate was about 85%. With the increased cholesterol concentration medium (0,25,50,100mg/L), the pacemaker potential of ICC was damped, the oxidative stress reaction of ICC was stronger and the ICC apoptosis rate was increased, which present dose dependency. However, with the increased concentration cholesterol scavenger, methyl-?-cyclodextrin (0,2,4,6mM), was used in the high concentration cholesterol (100mg/L) ICC culture medium, the parameters of pacemaker potential, marks of oxidative stress and apoptotic rate of ICC was gradually improved, which also present dose dependency. (3) In 61 cases of EMIC, the gallbladder was preserved perfectly with no complications. The other 4 cases were switched to laparoscopic cholecystectomy because of the diffuse cholesterolosis or submucosal stones and jujube paste bile filled in gallbladder lumen. The success rate was 93.8%. Of the 61 patients who were followed up at a mean of 18months (range 6-24), gallstone recurrence was detected in 3 cases. The overall stone recurrence rate was 4.92%. The patients with preoperative symptoms were symptom-free. Compared with the recovery of postoperative gallbladder contractibility between different incisions in gallbladder, the gallbladder ejection fraction (GEF) was bottom in the 1st month after operation and without significance between two groups. However, the GEF which the incision choosed on gallbladder fundus was markedly superior to the GEF which the incision choosed on the gallbladder corpus at the 3 rd and the 6th months after operation.Conclusions:(1) During the process of diet-induced cholesterol gallstone formation in rabbits, the number of ICC decreased significantly and the motility of gallbladder was impaired. (2) An increased CSI in lithogenic bile contributes to strong oxidative stress reaction, which increases apoptosis of ICC. (3) The pacemaker potential of ICC is weakened by high concentration cholesterol, which results in the attenuated BER of gallbladder and motility disorder. (4) The optimal position of incision is choosed on the gallbladder fundus, which is benefit for postoperative recovery of gallbladder contraction function. (5)EMIC has strict operative indications and contraindications. The long-term outcomes of EMIC should be evaluated by prospective, large sample, multicenter randomized controlled study... |
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