台灣地區膽結石疾病成因及相關之研究

The pathogenesis of and related studies on gallstone diseases in Taiwan region

黃興睦(Shing-Moo Huang)

指導教授 ： 潘惠錦 ； 賴德仁

中山醫學大學/醫學院/醫學研究所/博士(2010年)

https://doi.org/10.6834/CSMU.2010.00029

摘要

本論文主要為膽源性結石疾病的相關研究，從臨床膽結石的手術治療改進及膽囊體積變化與膽囊結石患者的臨床病理生理機制關聯性，到碳酸鈣膽囊結石的晶體學研究以及膽固醇結石患者遺傳分析。本論文共包括五個獨立研究，第一項研究是要評估比較腹腔鏡縫合膽總管結合經皮肝穿刺引流術和腹腔鏡T 管置入術治療膽總管結石的有效性和安全性。我們收集50 例膽總管直徑大於或等於1 釐米的膽總管結石病人接受腹腔鏡探索，其中研究組包括 10 例接受腹腔鏡縫合Ⅵ膽總管結合經皮肝穿刺引流術病人，對照組包括 40例接受腹腔鏡T 管置入術的病人。對各參數進行了統計分析，研究組表現出較高的女性/男性比例 (6/4 vs. 8/32, P = 0.02)、 較少結石數(1.90 ± 0.88 vs 3.40 ± 1.65, P = 0.0078)，更短的手術時間 (138 ± 37 分鐘 vs. 191 ± 75 分鐘，P = 0.014)，和縮短術後停留時間 (7 ± 3 天 vs. 10 ± 3 天，P = 0.0013）。此結果顯示腹腔鏡縫合膽總管結合經皮肝穿刺引流術比起腹腔鏡T 管置入術治療膽總管結石可以縮短手術時間和術後停留時間。
第二項研究是要比較底端往下和常規腹腔鏡膽囊切除術治療收縮性膽囊併結石的療效和併發症。64 例收縮性膽囊結石被列入研究。主要的結果評估包括轉換率、 併發症發生率、 膽道損傷率、 手術時間和術後住院時間。底端往下術的平均術後住院時間是 5 ± 3 天，常規技術則為7 ± 3 天（P = 0.003）。轉換率和併發症發生率在底端往下術分別為0%(0/33)和3%(1/33)，在常規技術則為32.3%(10/31)和22.6%(7/31)。在次群分析中，底端往下腹腔鏡膽囊切除術比常規技術似可降低膽道損傷率從2/31(6.5%)到0/33(0%)。因此，底端往下腹腔鏡膽囊切除術與較低的轉換和併發症率及較短術後住院時間有相關聯。
第三項研究是探討膽囊結石患者的膽囊體積和射出比例(ejection fraction)改變的病理生理意義。急性膽囊炎膽囊結石患者(n = 99)，慢性膽囊炎膽囊結石患者(n = 85)和非膽結石患者(n = 240)的空腹膽囊體積在術前先以電腦攝影測量，在膽囊切除術後再以直接鹽水注射測量膽囊體積。另外我們以腹部超音波測量了 65 例膽囊結石與慢性膽囊炎和 53例健康人的空腹和餐後膽囊體積。空腹膽囊體積在後期比在早期/第二階段急性膽囊炎為大(84.66 ± 26.32 cm3、 n = 12，與 53.19 ± 33.80 cm3 、n = 87, = 0.002）。空腹的膽囊體積及射出比例，在慢性膽囊炎患者比正常人大及低（28.77 ± 15.00 cm3 vs. 6.77 ± 15.75 cm3, P < 0.0001)/(34.6 ± 10.6%, n = 65, vs. 53.3 ± 24.9%, n = 53, P < 0.0001)。急性膽囊炎進展至壞疽和/或蓄膿時膽囊體積會增加。膽結石的形成與較差的膽囊收縮力和較大的膽囊體積有相關性。
第四項研究的目的是調查碳酸鈣膽結石的化學成分和晶體結構，以及石灰膽汁可能的病因學意義。我們以原子吸收分光光度法和結晶學x 射線繞射法、 紅外吸收光譜和偏光顯微鏡法分析膽囊結石患者的結石粉末，從401例膽囊結石患者中找出58例為碳酸鈣膽囊結石。碳酸鈣是石灰膽汁的主要成分。它在膽囊結石中所佔的重量範圍從36.0%到90.7%, 0平均 60.5%.在結晶學中，碳酸鈣有三種不同的多態結晶形式：方解石、 文石結晶和霰石球。每個異構體有自己特定的紅外光吸收光譜帶，在偏光顯微鏡底下有特定的晶體結構，在 x 射線繞射有特定的d間距模式。在58 個病人中只有 13人與石灰膽汁有關聯。在人體中可以找到碳酸鈣沉澱。它是人類膽囊結石中第三個最常見的組成。在我們的案例中，文石結晶最普遍存在，發生率為 92.3%,方解石發生率為 73.1%,霰石球53.8%。此外， 58例中.23例包含所有三種形式。
第五項研究是調查GPBAR1 基因是否在人類膽固醇膽囊結石的形成中扮演角色。我們收集41例膽囊結石患者(包括22例膽固醇膽囊結石、18例膽色素膽囊結石及1例碳酸鈣結石患者)，110例正常人和 28例癌症患者，萃取全血 DNA以聚合酶鏈鎖反應和定序分析其GPBAR1基因。我們在一位肝癌和一位膽固醇膽囊患者檢體各發現一個 GPBAR1 基因點突變，分別是G721A和C329T，導致胺基酸改變為V241M及R110H。由於這二個胺基酸在各物種間的GPBAR1蛋白質有高度保留性，暗示其突變可能造成蛋白質功能改變。此外我們確認了二個臺灣地區GPBAR1 基因單核苷酸多形性， G336T 和 G903A.由於這些單核苷酸多形性尚未在其他族群發現，很可能它們是台灣地區特有的變化。我們建立的資料除了提供將來研究參考，這些 GPBAR1基因序列的改變是否與功能有關值得進一步研究。

關鍵字

膽結石疾病成因 ； 腹腔鏡縫合膽總管結合經皮肝穿刺引流術 ； 收縮性膽囊併結石 ； 底端往下腹腔鏡膽囊切除術 ； 膽囊體積改變的病理生理意義 ； 碳酸鈣膽結石 ； GPBAR1 基因點突變

並列摘要

Objectives, Methods and Materials:
The aims of this study are to investigate the whole aspect of gallstone diseases from the micro world of the genetic profiles of cholesterol gallstone patients and the crystallographic study of calcium carbonate gallbladder stones to the macro world of clinical pathophysiological mechanistic gallbladder volume changes of gallstone patients and therapeutic aspects of gallstone diseases. The study comprises 5 separate parts.
Results, Conclusion and Suggestion:
The aim of the first study was to compare the efficacy and safety of laparoscopic primary closure of the common bile duct (CBD) combined with percutaneous transhepatic cholangiographic drainage (PTCD) and laparoscopic choledocholithotomy with T-tube placement for the treatment of CBD stones. Between January 1991 and July 2002, 50 patients with choledocholithiasis and a CBD diameter larger than or equal to 1 cm underwent laparoscopic CBD explorations. The study group consisted of 10 patients undergoing laparoscopic primary closure of the CBD combined with PTCD. The control group consisted of 40 patients undergoing laparoscopic choledocholithotomy with T-tube placement. Parameters were compared statistically. The study group showed higher female/male ratio (6/4 Ï'S 8/32, P = 0.02), less stone numbers (1.90 ± 0.88 vs 3.40 ± 1.65, P = 0.0078), shorter operation time (138 ± 37 minutes vs 191 ± 75 minutes, P = 0.014), and shorter postoperative stay (7 ± 3 days vs 10 ± 3 days, P = 0.0013).It seems that laparoscopic primary closure of the CBD combined with PTCD can shorten the operation time and postoperative stays as compared with laparoscopic choledocholithotomy with T-tube placement for the treatment of CBD stones.
The aim of the second study is to compare efficacy and complications between fundus-down and conventional laparoscopic cholecystectomy (LC) in treating contracted gallbladders with gallstones. Between January 1999 and May 2008, 64 patients with contracted gallbladders and gallstones were included in the study. Main outcome measures included conversion rate, complication rate, bile duct injury rate, operation time, and postoperative stay. The average postoperative hospital stay for fundus-down technique was 5 ± 3 days, and 7 ± 3 days for conventional technique (P = 0.003). The conversion rate and complication rate were 0% (0/33) and 3.00% (1/33) for fundus-down technique, and 32.3% (10/31) and 22.6% (7/31) for conventional technique (P = 0.0009 and 0.02, respectively). In subgroup analysis, fundus-down LC seemed to lower the bile duct injury rate from 2/31 (6.5%) to 0/33 (0%) compared with 6/1,468 (0.4%) (P = 0.01 vs P = 1.00). It appears that fundus-down laparoscopic cholecystectomy is associated with lower conversion and complication rates and shorter postoperative hospital stay as compared with conventional laparoscopic cholecystectomy when used to treat patients with contracted gallbladders and gallstones.
The aims of the third study are to study the pathophysiological significance of gallbladder volume (GBV) and ejection fraction changes in gallstone patients.The fasting GBV of gallstone patients with acute cholecystitis (n = 99), chronic cholecystitis (n = 85) and non-gallstone disease (n = 240) were measured by preoperative computed tomography. Direct saline injection measurements of GBV after cholecystectomy were also performed. The fasting and postprandial GBV of 65 patients with gallstones and chronic cholecystitis and 53 healthy subjects who received health examinations were measured by abdominal ultrasonography. Proper adjustments were made after the correction factors were calculated by comparing the preoperative and postoperative measurements. Pathological correlations between gallbladder changes in patients with acute calculous cholecystitis and the stages defined by the Tokyo International Consensus Meeting in 2007 were made. Unpaired Student’s t tests were used. P < 0.05 was deemed statistically significant. The fasting GBV was larger in late stage than in early/second stage acute cholecystitis gallbladders (84.66 ± 26.32 cm3, n = 12, vs. 53.19 ± 33.80 cm3, n = 87, P = 0.002). The fasting volume/ejection fraction of gallbladders in chronic cholecystitis were larger/lower than those of normal subjects (28.77 ± 15.00 cm3 vs. 6.77 ± 15.75 cm3, P < 0.0001)/(34.6 ± 10.6%, n = 65, vs. 53.3 ± 24.9%, n = 53, P < 0.0001). GBV increases as acute cholecystitis progresses to gangrene and/or empyema. Gallstone formation is associated with poorer contractility and larger volume in gallbladders that contain stones.
The aim of the fourth study is to investigate the chemical contents and crystallographic structures of calcium carbonate gallstones as well as the possible etiologic significance of limy bile. Between August 2001 and July 2007, calcium carbonate gallstones from 58 cases out of 401 gallstone patients were chemically analyzed by atomic absorption spectrophotometry and crystallographically studied by the powder X-ray diffraction method, infrared absorption spectroscopy and polarizing microscopy. Chemically, calcium carbonate is the major constituent of limy bile. Its weight in gallstones ranges from 36.0 % to 90.7 % and averaged 60.5 %. Crystallographically, calcium carbonate has three different polymorphic crystalline forms: calcite, aragonite and vaterite. Each isoform has its own characteristic absorption bands in infrared absorption spectroscopy, crystal structures in polarizing microscopy and d-spacings in x-ray diffraction patterns. Only 13 out of 58 patients were associated with limy bile in their gallbladders. Calcium carbonate precipitation can be found in human body. It comprises the third most frequent compositions in human gallstones. In our series, aragonite was most commonly found, with an occurrence rate of 92.3%, while that of calcite was 73.1%, vaterite 53.8%. Moreover, 23 out of 58 cases contained all three forms.
The aim of the fifth study is to investigate whether GPBAR1 gene plays a role in the formation of cholesterol gallbladder stones in humans. We collected 41 gallstones patients (including 22 CGS, 18 PGS, and 1 calcium carbonate patient) , 110 normal subjects and 28 cancer patients for genetic study of GPBAR1 gene. The whole blood genomic DNA was subjected to PCR-sequencing analysis for the GPBAR1 gene coding region. We identified 2 point mutations, G721A in a hepatocellular carcinoma patient and C329T in a cholesterol gallstone patient, respectively. The two mutations resulted in V241M and R110H amino acid substitution, respectively, which were not found in normal subjects. Both V241 and R110 amino acids are highly conserved in other species, implicating that these two mutations may cause functional alteration of GPBAR1 protein. In addition, we found two novel SNPs, G336T and G903A in normal subjects. Because these GPBAR1 gene sequence changes have not been reported in other populations, they could have evolved in Taiwanese population. Future functional study on these mutant GPBAR1 genes identified in this study may provide further insight into the mechanism of gallbladder stone formation.

並列關鍵字

laparoscopic primary closure of the common bile duct combined with percutaneous transhepatic cholangiographic drainage ； contracted gallbladders with gallstones ； fundus-down laparoscopic cholecystectomy ； pathophysiological significance of gallbladder volume ； calcium carbonate gallstones ； GPBAR1 genes mutations

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