高三酸甘油酯胰臟炎之基因易感性及致病機轉研究

Hypertriglyceridemic Pancreatitis: Studies of Genetic Susceptibility and Pathogenesis

張毓廷(Yu-Ting Chang)

指導教授 ： 翁昭旼

共同指導教授 ： 高嘉宏

國立臺灣大學/醫學院/臨床醫學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.02184

摘要

高三酸甘油酯血症是導致急性胰臟炎的原因之一，約有3∼38%的急性胰臟炎是由高三酸甘油酯血症所引起。在台灣，高三酸甘油酯血症是造成急性胰臟炎第三常見的原因。至於高三酸甘油酯血症如何引發急性胰臟炎，目前致病機轉並不清楚。過去對lipoprotein lipase (LPL)及apolipoprotein基因的研究，發現這類基因的突變與高三酸甘油酯血症有關，但並不能解釋為何有些高三酸甘油酯血症病人會發生急性胰臟炎，而有些病人卻從不發生急性胰臟炎。而與胰臟發炎有關的基因如：cystic fibrosis transmembrane conductance regulator (CFTR)、cationic trypsinogen gene (PRSS1)、serine protease inhibitor Kazal type 1 (SPINK1)及致發炎基因像是腫瘤壞死因子(Tumor necrosis factor, TNF)-α等，這些基因在高三酸甘油酯血症合併急性胰臟炎的病人，從未被研究過。之前動物實驗研究顯示，當cftr 基因被完全毁壞的老鼠，因其易發炎的特徵而容易導致嚴重的急性胰臟炎。過去高三酸甘油酯血症急性胰臟炎的宿主易發病傾向並未被深入研究。本研究的第一部分是高三酸甘油酯胰臟炎的相關基因研究，假說之一是高三酸甘油酯血症的病人其CFTR、SPINK1、PRSS1及發炎基因像是TNF-α的突變或變異，使宿主易發生急性胰臟炎。為了研究此假說及高三酸甘油酯血症的致病機轉，本研究分析高三酸甘油酯血症病人合併急性胰臟炎，與高三酸甘油酯血症無急性胰臟炎病人，其CFTR、SPINK1、PRSS1及TNF-α之基因變化。我們對126位高三酸甘油酯血症的病患進行基因分析，其中包含46位曾有過高三酸甘油酯血症急性胰臟炎和另外80位從未發生過急性胰臟炎高三酸甘油酯血症病患。這126位高三酸甘油酯血症病患的PRSS1、SPINK 1及CFTR三個基因的突變與變異以及腫瘤壞死因子驅動子的多型性，全部都以變性高效能液相色層分析（Denaturing High Performance Liquid Chromatography, DHPLC）或以直接核苷酸定序來確定基因的變異。吾人發現在126位高三酸甘油酯血症的病患中，13位(10.3%)有CFTR基因的突變，而全部的病患都沒有PRSS1或 SPINK1的基因突變發生。CFTR基因突變的比例在高三酸甘油酯血症合併急性胰臟炎病患及高三酸甘油酯血症無合併急性胰臟炎病患各為46%(12/26)及1.3%(1/80)，兩者在統計學上有顯著的差異(p<0.0001) 。而CFTR基因的突變都是Ile 556 Val。多變項分析發現血中三酸甘油酯的濃度, CFTR470val及腫瘤壞死因子α驅動子-863A基因多型性各為高三酸甘油酯血症產生急性胰臟炎的獨立危險因子。而高三酸甘油酯胰臟炎的基因研究中我們也分析與脂質代謝有關的基因包括脂蛋白脂肪酶(lipoprotein lipase; LPL)及Apolipoprotein CⅡ (APO CII) 的基因突變是否與高三酸甘油酯血症產生急性胰臟炎的發作或導致慢性胰臟炎有相關。我們共分析了134位高三酸甘油酯血症的病患，其中53位曾發生過急性胰臟炎，而另外81位則從未曾發生急性胰臟炎。這134位高三酸甘油酯血症病患的LPL及APO CII的基因變異，都用變性高效能液相色層分析或高解析熔點分析(High Resolution Melting ; HRM)來篩檢，而篩檢出來的變異均用核苷酸定序來確認。我們發現在134位高三酸甘油酯血症病患中有9.7%(134位中有13位)有LPL基因的突變。LPL在高三酸甘油酯血症發生急性胰臟炎的病患有基因突變的機率是17%(53位中有9位)，遠比在高三酸甘油酯血症沒有急性胰臟炎病患的4.7%(81位中有4位)來的高，兩者在統計學上有顯著的差異(p<0.0001) 。最常見的LPL基因突變位置是L252V及S447X，而在高三酸甘油酯血症合併急性胰臟炎且有LPL基因突變的病患中77.8%(9位中有7位)是S447X的變異。多變項分析發現LPL基因的突變與急性胰臟炎發作的次數是產生胰臟鈣化及脂便等慢性胰臟炎特徵的獨立危險因子。本研究第二部分我們提出假說認為三酸甘油酯的成份也就是飽和脂肪酸及不飽和脂肪酸的比例會影響高三酸甘油酯血症的病患是否會發生急性胰臟炎有關。為了檢驗這個假說我們建立老鼠胰臟腺細胞的胰臟炎研究模式，探討各種不同濃度，不同飽合及不飽和脂肪酸對胰臟腺細胞細胞質中鈣離子濃度的影響。我們發現高濃度的不飽和脂肪酸如亞麻油酸(linoleic acid)、油酸(oleic acid)及棕梠油酸(palmitoleic acid)等會引發胰臟腺細胞細胞質中鈣離子濃度的上升，而低濃度的不飽和脂肪酸如亞麻油酸、油酸及棕梠油酸等，以及高濃度與低濃度的飽和脂肪酸如棕梠酸(palmitic acid) 及硬脂酸(stearic acid)均不能引發胰臟腺細胞細胞質中鈣離子濃度的上升。而高濃度的三酸甘油酯也無法立即引發腺細胞細胞質中鈣離子濃度的上升，顯示高濃度的三酸甘油酯本身並不能引發急性胰臟炎的發作，而必須在三酸甘油酯水解成脂肪酸之後且不飽和脂肪酸/飽和脂肪酸的比例偏高時，胰臟腺細胞才會受損而引發急性胰臟炎。這結果提供臨床上為什麼只有部份高三酸甘油酯血症病患會產生急性胰臟炎的一個可能解釋。綜合本研究結果顯示CFTR基因的不同變異與各種不同成因的胰臟炎的表現型有相關。而高三酸甘油酯血症的病患如果同時有CFTR基因的變異產生急性胰臟炎的風險比較高，而若同時再有腫瘤壞死因子α驅動子基因多型性-863A的特徵，胰臟炎的發生機率會更高。另外LPL基因變異與高三酸甘油酯急性胰臟炎的發作也與高三酸甘油酯血症形成慢性胰臟炎有關。高三酸甘油酯血症產生急性胰臟炎或高三酸甘油酯血症產生慢性胰臟炎是多因子，多基因共同作用的結果並與高三酸甘油酯的組成成份有關。

關鍵字

高三酸甘油酯血症 ； 高三酸甘油酯急性胰臟炎 ； cystic fibrosis transmembrane conductance regulator (CFTR) ； 腫瘤壞死因子TNF-α ； lipoprotein lipase ； Apolipoprotein CⅡ ； 高效能液相色層分析（Denaturing High Performance Liquid Chromatography, DHPLC） ； 高解析熔點分析(High Resolution Melting , HRM) ； 鈣離子 ； 不飽和脂肪酸 ； 飽和脂肪酸

並列摘要

Hypertriglyceridemia (HTG) is a well known association of acute pancreatitis and found in 3-38% of patients with acute pancreatitis. HTG is the third frequent etiology of acute pancreatitis in Taiwan, account for about 10-15% of our patients suffering acute pancreatitis. The mechanism that how hypertriglyceridemia leads to pancreatitis attack and whether HTG could cause chronic pancreatitis are not clear. Genetic factors such as lipoprotein lipase and apolipoprotein mutations are reported to be associated with occurrence of HTG but could not explain why some patients experienced acute pancreatitis and why some patients with HTG seldom develop pancreatitis, even with marked elevation of triglyceride level. Whether the genes involved in pancreatic ductal or acinar cell injury including cationic trypsinogen gene (PRSS1), pancreatic secretory trypsin inhibitor gene (SPINK1), cystic fibrosis transmembrance conductance regulator (CFTR) and inflammation genes (tumor necrosis factor, TNF-alpha) are associated with hyperlipidemic pancreatitis (HLP) in patients with hypertriglyceridemia (HTG) has never been studied. In an animal study with cftr(-/-) mice, a baseline proinflammatory state and an antiapoptotic phenotype were observed and the cftr(-/-) mice was susceptible to severe acute pancreatitis. We hypothesized those genetic variations, including CFTR, PRSSI, SPINK1 genes, and proinflammatory cytokine gene: such as TNF-α, etc., make patients with HTG susceptible to developing acute pancreatitis. To investigate the hypothesis, we analyzed the genetic variations of CFTR and TNF-α in patients with HTG and acute pancreatitis, and in patients with HTG but without acute pancreatitis. we performed genetic analysis of 126 HTG patients in Taiwan (46 with HLP and 80 without HLP). The entire coding and intronic regions of the PRSS1, SPINK1, and CFTR genes were identified by heteroduplex analysis techniques and were confirmed by sequencing analysis. The presence of 125G/C, 1001 + 11C>T, 1540A>G (Met470Val), 2694T>G, and 4521G>A in CFTR, the presence of 272C>T in SPINK1, and TNF promoter polymorphisms (nucleotide positions -1031, -863, -857, -308, and -238) were measured by direct sequencing. Of the 126 HTG patients, 13 (10.3%) carried a CFTR mutation. No PRSS1 or SPINK1 mutations were detected in our patients or in HTG controls. The CFTR gene mutation rates in HTG with and without HLP were 26.1% (12 of 46) and 1.3% (1 of 80), respectively (P <0.0001). The CFTR gene mutations were all Ile556Val. A multivariate analysis of HTG patients indicated that triglycerides, CFTR 470Val, and TNF promoter -863A were independent risk markers for HLP. In addition, we sought to determine whether the genes involved lipid metabolism including lipoprotein lipase (LPL) and apolipoprotein CII (APO CII) are associated with HLP and chronic pancreatitis in patients with HTG. We performed genetic analysis of 134 patients in Taiwan with HTG (53 with HLP and 81 without HLP). The entire coding and intronic regions of the LPL and APO CII genes were identified with heteroduplex analytical techniques or high resolution melting analysis. All mutations were confirmed by sequencing analysis. Correlation of phenotype and genotype also analyzed. The frequency of LPL gene mutation rates in HLP patients17.0 %( 9 of 53) is significantly higher than that without HLP attack 4.9% (4 of 81) (P<0.0001). A total of 9.7% (13 of 134) of our HTG patients carried LPL mutation. The most two common LPL gene mutations were L252V and S447X. Our results also firstly demonstrate a high prevalence (77.8%) of HLP attack in HTG patients carrying S447X mutation. Multivariate analysis in HLP patients indicated that the presence of LPL mutation and episode of acute attack were independent risk markers for pancreatic calcification occurrence and steatorrhea. This genetic study is the first one to address the association of HLP with the CFTR mutation/variant/haplotype and TNF promoter polymorphism in a Chinese HTG population. This is also the first complete genetic study to address the association of HLP with the LPL mutation in a Chinese HTG population. LPL gene mutation is associated with pancreatic calcification in HLP patient. The results enhance that the occurrence of HLP is multifactorial and polygenic. In the second part of the studies, we hypothesized that the components of triglyceride, that is: saturated and unsaturated fatty acids, may influence the susceptibility to develop acute pancreatitis in HTG patients. To investigate the hypothesis, we set up primary pancreatic acinar cell culture to measure the cytosolic Ca2+ signal change in acinar cells after treated with different saturated and unsaturated fatty acids. We found that the unsaturated fatty acids, including Oleic acid, Linoleic acid, and Palmitoleic acid, in high concentration induced the persistent rise of cytosolic Ca2+ concentration in acinar cell similar to the condition with supra-maximal concentrations of CCK-induced elevation of baseline Ca2+ in isolated pancreatic acinar cells. However, unsaturated fatty acids, including Oleic acid, Linoleic acid, and Palmitoleic acid, in low concentration and saturated fatty acids, including Palmitic acid and Stearic acid, in low and high concentrations could not induce the rise of Ca2+ concentration in acinar cell. Triglyceride in high concentration itself initially did not cause the rise of cytosolic Ca2+ concentration in acinar cell. The results indicted that the HTG itself could not induce the attack of acute pancreatitis. Only under circumstance of the hydrolysis of triglyceride by lipase into free fatty acids, and the ratio of unsaturated/saturated fatty acid high enough, the acinar cells become injured then pancreatitis developed. The results provide a potential explanation that why clinically only a portion of HTG patients develop HLP. The unsaturated fatty acids may play a crucial role in the pathogenesis of HLP. In conclusion, CFTR mutations are associated with a broad spectrum of pancreatic phenotypes. Identification of the association of CFTR and other genes with HLP has provided evidence that HTG patients with CFTR mutations/variants are more susceptible to developing HLP. This susceptibility increased after CFTR mutation/variant interaction with proinflammatory cytokines such as TNF. In addition, LPL gene mutations are associated with attack of HLP in HTG populations. Identification of distinct genes associated with HTG has provided evidence that HTG patients with LPL mutations are more susceptible to HLP attack and development of chronic pancreatitis. The development of HLP in HTG patients and chronic pancreatitis related to HTG appear to be a multifactorial and polygenic event and related to the components of triglyceride.

並列關鍵字

hyperlipidemic pancreatitis(HLP) ； hypertriglyceridemia (HTG) ； cystic fibrosis transmembrane conductance regulator (CFTR) ； Tumor necrosis factor-alpha(TNF-α) ； lipoprotein lipase(LPL) ； Apolipoprotein CII(APO CII) ； Denaturing High Performance Liquid Chromatography(DHPLC) ； High Resolution Melting(HRM) ； Ca2+ ； saturated fatty acid ； unsaturated fatty acid

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