肝癌营养支持的临床与基础研究
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摘要
研究背景
原发性肝癌是我国常见的恶性肿瘤之一,近年来其发病率有增高趋势。据1995年卫生部统计,我国肝癌死亡率占肿瘤死亡率的第二位,严重威胁我国居民的健康。肝切除术仍然是目前治疗肝癌首选和最有效的治疗方法,但肝切除术系创伤较大的手术,对机体的代谢和内环境影响较为严重,加之应激反应使机体处于高分解代谢状态,可导致患者术后营养不良,恢复延迟,并抑制免疫系统功能。
营养支持可以减轻患者术后的负氮平衡,维持重要脏器的功能,减少外科手术并发症,已成为共识。对于原发性肝癌患者术后营养支持,肠内和肠外两个途径那一个更安全尚未定论。一些动物实验研究表明,静脉营养支持有促进恶性肿瘤细胞生长的表现。目前不主张对恶性肿瘤患者进行长期的术前营养支持,以防止营养支持促进肿瘤的增殖和转移,尤其是静脉营养支持。与静脉营养相比,肠内营养具有以下优点:①营养底物从肠道吸收,经门静脉进入肝脏符合生理,有利于内脏蛋白的合成与代谢;②为黏膜细胞提供营养物质,可维持肠黏膜屏障,防止细菌异位;③经肠道吸收的脂肪酸不能为肿瘤细胞利用;④可减少肝胆并发症等。而且,与胃肠道手术相比,肝切除术患者胃肠道完整,肠内营养途径较为安全。近年来随着对肠道功能的再认识,早期肠内营养(EN)受到重视。早期营养支持是指术后6～24h内开始给予的营养支持。一些研究显示,术后早期营养支持可以改善患者的营养状态。手术后早期进行EN支持由于受到“肛门排气方可进食”观念的影响,加之实施麻烦,患者有时出现腹胀、腹泻等不适症状,临床应用一直受到限制。有研究表明,小肠功能正常在术后6～12 h即可恢复;手术后小肠吸收功能虽然受到一定抑制,但是保留的功能足以吸收满足机体需要的营养素。
随着免疫营养概念的提出,一些对恶性肿瘤生长具有抑制作用的特殊营养物质被发现。ω-3多不饱和脂肪酸(polyunsaturated fatty acid,PUFA)是近年来发现的免疫营养支持的营养底物之一,主要存在于深海鱼油等食物中,具有抗血栓、降血脂、降血压、抗动脉硬化等作用,并对过度的炎症反应有抑制作用。PUFA还可以改变恶性肿瘤的细胞膜脂质成分,进而使其流动性变化,而增强恶性肿瘤对各种治疗的敏感性。流行病学调查发现ω-3PUFA对于前列腺癌、乳腺癌、结直肠癌等的发生、发展具有抑制作用。体外细胞培养实验和动物实验也证实ω-3 PUFA对多种肿瘤和肿瘤细胞具有抑制或杀伤作用。
ω-3 PUFA对肿瘤细胞的杀伤作用机制尚不清楚,复习国内外的相关文献研究,共提出了如下几种假说:
(1)改变生物膜的结构和功能,增加脂质过氧化物的产生。PUFA是构成生物膜的重要结构脂肪酸,补充PUFA可以改变生物膜的构成,导致细胞膜、线粒体膜的正常活性和功能发生改变。Stoll等研究发现,肿瘤细胞膜ω-3/ω-6 PUFA升高,可以造成细胞膜流动性和通透性增加,细胞毒药物容易透过细胞膜,增强肿瘤对化疗的敏感性。Chapkin等发现,肿瘤线粒体膜ω-3/ω-6升高,导致线粒体膜电位下降,细胞呼吸链和电子传递受损,线粒体膜PT(permeabilitytransition)孔开放,膜通透性增加,使细胞色素C和凋亡诱导因子的释放、半胱氨酸蛋白酶(caspase)酶解级联反应得以进行,导致细胞凋亡。
(2)导致细胞代谢障碍,促进细胞凋亡或死亡。肿瘤细胞的共同特征是代谢旺盛,细胞周期调控的破坏及细胞凋亡的减少和中止。细胞凋亡在肿瘤的发生和生长过程中发挥重要作用。有证据表明,PUFA抗肿瘤机制也体现在对促进凋亡的作用上。文献报道大多数被PUFA抑制的肿瘤细胞系,如前列腺癌、乳腺癌、大肠癌等,尽管抑制程度不同及所报道的机制不同,但均可见细胞凋亡的发生。
(3)脂质过氧化代谢产物的细胞毒作用
脂质过氧化产物和自由基对细胞具有杀伤作用。正常细胞有完备的抗氧化反应系统,可免受过氧化反应的损伤。而肿瘤细胞内由于抗氧化酶含量低及相应的细胞抗氧化屏障缺陷,抗脂质过氧化反应能力降低,随着脂质过氧化产物和自由基积聚,容易遭受过氧化损伤。
ω-3脂肪酸是细胞内脂质过氧化的主要底物,所含不饱和双键越多越易氧化,产生的过氧化产物和自由基越多。这些物质能损伤肿瘤细胞膜,改变细胞成分或细胞骨架的组装,调节膜转运系统或酶,或抑制聚合酶反应和/或聚胺合成。导致细胞死亡或凋亡,信号转导通路改变,细胞生长的抑制。
(4)抑制花生四烯酸来源的类花生酸类物质的生物合成。花生四烯酸(AA)是细胞膜中的主要多不饱和脂肪酸,因此,大多数的类花生酸类物质是其来源的2系列前列腺素(前列腺素和血栓素)和4系列白细胞三烯,这些物质具有强促炎症和促细胞增殖活性。Leahy KM和Cianchi F等发现PGE2具有阻止肿瘤细胞凋亡、刺激肿瘤细胞增殖和促进肿瘤血管新生的作用,Pidgeon GP等发现,12-Lox对AA的作用产物12-HETE参与调控细胞周期、肿瘤血管生成及抗凋亡和粘附转移相关基因的表达,Calder PC等发现白三烯B4(LTB4)与肿瘤的黏附有关并能增加活性氧的产生,攻击DNA导致癌的发生。Chen JK等发现细胞色素P450单氧合酶对AA的作用产物14,15-环氧二十碳四烯酸可以抑制细胞凋亡。
(5)肿瘤相关基因表达的改变。近期研究发现,ω-3多不饱和脂肪酸(PUFA)可以调控多种凋亡相关基因,发挥对肿瘤细胞的细胞毒作用,并可以诱导肿瘤细胞的凋亡。关于多不饱和脂肪酸(PUFA)影响肿瘤相关基因表达的文献数量越来越多,Clarke,Deckelbaum等对此做过总结,包括与转录相关的基因,癌基因、抑癌基因、与增殖分裂有关的基因、与侵袭转移相关的基因、与凋亡相关的基因等,还有不断增加的趋势。其中有关ω-3多不饱和脂肪酸(PUFA)与bcl-2家族的关系研究文章较多。有研究显示,经EPA诱导HL-60细胞和人乳腺癌细胞可以发生凋亡,并观察到具有促进凋亡作用的bax、bcl-xs的表达显著上调,而具有抗凋亡作用的bcl-2、bcl-xl基因表达均明显受抑,表明EPA能通过调节bcl-2家族的表达活性来诱导肿瘤细胞的凋亡。此外,Bax可以在凋亡信号的刺激下从细胞质移位到线粒体外膜,转变为膜内蛋白,形成转运通道,促进细胞色素C的释放,加速诱导细胞凋亡的发生。
近年来随着对肠道功能的再认识,早期肠内营养(EN)受到重视,但对于肝癌肝切除术后早期肠内营养对患者术后恢复情况影响的临床价值尚无统一意见。而且有关ω-3PUFA对肝癌细胞的作用报道较少。因此,我们从两个方面研究肝癌的营养支持作用。
1.早期肠内营养对肝切除患者术后恢复情况的影响;
2.ω-3多不饱和脂肪酸对肝癌细胞生长抑制的作用及机制。
第一部分:早期肠内营养对肝切除患者术后恢复情况的影响
目的
探讨早期肠内营养与肠外营养对肝切除患者术后恢复情况的影响
方法
(1)选择44例肝癌肝切除术患者随机分为PN组和EN组;
(2)手术后分别接受EN和PN一周;
(3)观察两种营养支持方法对患者基础营养状态、肝功能、肠道功能、术后并发症和淋巴细胞数、白细胞介素-2(IL-2)等营养和免疫功能方面的影响。
结果
(1)经统计学分析,两组患者术前肝功能Child-Pugh分级(x~2=0.121,P=0.50)和营养状况均无明显差别(P＞0.05);两组手术范围也无统计学差异(x~2=1.126,P=0.771),具有可比性。
(2) EN和PN组患者手术后日均热量摄入分别为(7412±368)kJ和(7149±421)kJ,日均氮摄入量分别为(9±7)g和(10±13)g,差异无统计学意义(P＞0.05)。
(3) EN与PN组手术后肛门排气时间分别为(59±14)h和(73±17)h,差异有统计学意义(t=-2.960,P=0.005)。
(4) EN和PN两组病例手术后体重和上臂肌围均下降,与术前比较差异有统计学意义。
(5)两组患者的血清总蛋白、白蛋白和前白蛋白手术后均明显下降,前白蛋白于营养支持一周后均能恢复到术前水平,但总蛋白和白蛋白不能恢复到术前水平。
(6)两组患者血清甘油三酯术后均明显下降,静脉营养组患者一周后能恢复到术前水平,而肠内营养组患者不能恢复到术前水平。
(7) EN组外周血淋巴细胞数术后1d明显下降,至术后8d恢复至术前水平。PN组外周血淋巴细胞数术后1d明显下降,至术后8d仍不能恢复至术前水平。
(8)手术后营养支持1周,PN组血清IL-2无明显变化,EN组血清IL-2明显升高(P＜0.05)。
结论
临床研究结果提示,早期营养支持对减轻肝切除术患者蛋白的分解,改善其术后营养状况,对促进患者的术后恢复是有帮助的。早期EN还能明显促进患者的肠道功能恢复,明显促进肝切除患者术后淋巴细胞数量的恢复,并能提高血清IL-2水平,在改善肝切除术患者的免疫功能方面优于PN。因此,早期EN是肝切除术后可靠、有益的营养支持方式。
第二部分:ω-3多不饱和脂肪酸对肝癌细胞生长抑制的作用及机制
目的
探讨ω-3多不饱和脂肪酸对肝癌细胞生长增殖及细胞凋亡的作用,以评估ω-3多不饱和脂肪酸对肝癌的作用,并初步探讨其作用机制。
方法
(1)用MTT法、生长曲线选择对数生长期细胞的合适浓度;
(2)应用MTT法分别测定DHA和EPA对肝癌细胞HepG2生长增殖的影响;
(3)应用免疫组织化学法观察DHA和EPA分别作用后PCNA在肝癌细胞HepG2中的表达;
(4)用荧光染色法观察DHA和EPA分别作用后肝癌细胞HepG2形态的改变;
(5)应用透射电镜观察DHA和EPA分别作用后肝癌细胞HepG2超微形态的改变;
(6)应用流式细胞仪检测DHA和EPA分别作用后肝癌细胞HepG2的细胞凋亡情况。
结果
(1)根据各浓度细胞生长情况,1×10~5/mL浓度细胞处于对数生长期,选定为药物干预试验的细胞浓度。
(2) MTT比色法显示DHA对细胞HepG2的生长有明显的抑制作用;随着剂量的增加和作用时间的延长,细胞生长抑制率显著上升,各组间两两比较差异均有统计学意义(P＜0.05)。DHA对HepG2细胞的抑制作用呈时间、剂量依赖关系。
(3) MTT比色法显示EPA对HepG2细胞的生长有明显的抑制作用,随着剂量和作用时间的增加,细胞生长抑制率显著提高,各组间两两比较差异有统计学意义(P＜0.05)。EPA对HepG2细胞的抑制作用亦呈时间、剂量依赖关系。
(4)经两种浓度(45μg/mL和60μg/mL)DHA/EPA作用24h后,PCNA的表达随药物浓度增高而减弱。
(5) HepG2细胞经DHA/EPA(45μg/mL)单独作用48h后,在相差显微镜下观察,结果显示:经两种脂肪酸作用后的HepG2细胞数量明显减少,细胞皱缩,出现凋亡小体或核碎片。
(6)与对照组细胞相比,经PUFA作用后的HepG2细胞出现线粒体空泡化增加,粗面内质网扩张,染色质浓聚,边聚等改变,胞浆内可见大量大小不等的电子致密物,可见到凋亡小体。
(7)不同浓度的药物(45μg/mL和60μg/mL DHA/EPA)干预HepG2细胞后,可明显观察到肿瘤细胞的凋亡。与对照组比较,DHA/EPA能明显促进HepG2细胞凋亡(P＜0.05)。相对于低浓度(45μg/mL),高浓度(60μg/mL)DHA/EPA促细胞凋亡的效果更明显(P＜0.05)。
结论
细胞培养研究结果提示,DHA、EPA对HepG2细胞的生长有明显的抑制作用,呈时间、剂量依赖关系。两种脂肪酸均可导致肝癌HepG2细胞早期凋亡和死亡,对HepG2细胞起杀伤作用。因此,ω-3多不饱和脂肪酸能显著抑制肝癌细胞生长,并诱导其凋亡。
Background
Primary hepatic cancer(PHC) is one of the most common malignant tumors in our country.The morbidity of PHC has increased in recent years. According to the statistic of the Ministry of Health in 1995,the mortality of PHC is the second place in all the malignant tumors,and it threatened the health of residents in our country heavily.Nowadays,partial hepatoectomy is still the first choice and the most effective treatment in the patients with PHC.While because of trauma,influence on the metabolism and the internal environment,and the stress reaction,the patients received partial hepatoectomy are always found with malnutrition, delayed recovery,immune suppression.
It was agreed that the Nutritional support can relieve the negative nitrogen balance,maintain the function of important organs,and reduce the complication of partial hepatectomy.It was disagreement that enteral or parenteral nutritional support postoperative was safe for patients with primary liver cancer.Some animal experiments studies showed that parenteral nutrition support could promote malignant cell growth.It was not in favor of malignant tumors with long-term preoperative nutritional support in patients in order to prevent promoting tumor proliferation and metastasis,especially in parenteral nutritional support at present. Compared with parenteral nutrition,enteral nutrition has the following advantages:①Nutrition substrates are absorbed from the intestinal,and go into the liver by portal vein,which is consistent with the physical and in favor of visceral protein synthesis and metabolism.②Enteral nutrition provides nutrients for the mucosal cells in order to maintain intestinal mucosal barrier and prevent bacteria ectopic.③Fatty acids absorbed in the intestinal can not be used by tumor cells.④Enteral nutrition can reduce complications of liver and gallbladder,et al. Moreover,compared with gastrointestinal,enteral nutritional support is a safer way for patients with partial hepatectomy because patients have gastrointestinal integrity.With re-recognition with the intestinal function,early enteral nutrition(EN) was paid much attention on in recent years.Early postoperative nutritional support means nutritional support is given in 6-24 hours postoperative.Some studies have shown that early postoperative nutritional support could improve the patient's nutritional status.Due to the concept of "anal exhaust before eating", trouble of implementation,and patients with abdominal distension, diarrhea and other symptoms during enteral nutritional support, application of early post-operative enteral nutritional supports in clinical has been restricted.Studies showed that small bowel function was retuned to normal in 6～12 h after surgery.Although it could not be fully restored,they could absorb enough nutrients of the body needs. Meanwhile,with the concept of immune nutrition,some nutrients with the tumor-growth inhibition were found recently.ω-3 Polyunsaturated Fatty Acid(PUFA) was one of the immune nutritional substrate found in recent years.It was mainly in the Deep Sea Fish Oil food,with anti-thrombosis, reducing blood fat,blood pressure,anti-arteriosclerosis,and inhibiting excessive inflammatory response.PUFA also could change the lipid composition on malignant cell membrane,thus change its fluidity, and enhance the sensitivity of all kinds of treatments.Epidemiological survey found thatω-3PUFA could inhibit the development and the occurrence of prostate cancer,breast cancer,and colorectal cancer.The inhibited effect ofω-3 PUFA on a variety of tumors and tumor cells was confirmed by cell culture experiments in vitro and animal experiments. Mechanisms of the effect of PUFA on tumor cells are not clear.There are the following hypotheses provided by relationship articles.
(1) PUFAchanges the structure and function of biofilm and increases the generation of lipid peroxide.
PUFA is important structural fatty acids of biofilm.Adding PUFA can change the composition of biofilm,and lead to changing function and the normal activity of cell membrane and mitochondrial membrane.Stoll,etc. found thatω-3/ω-6 PUFA increasing in tumor cell membrane could lead to cell membrane fluidity and permeability,cytotoxic drugs go easily through the cell membrane,and enhance the sensitivity of the tumor to chemotherapy.Chapkin,etc.found thatω-3/ω-6PUFA increasing in tumor mitochondrial membrane could lead mitochondrial membrane to decline, cellular respiration and electron transport chain damage,mitochondrial membrane PT(permeability transition) hole to open,membrane permeability, cytochrome C and apoptosis-inducing factor to release,cysteine protease (caspase) enzyme cascade can be carried out,at last lead to apoptosis.
(2) PUFA causes cell dysbolism and promotes cell apoptosis or death The common features of tumor cells are strong metabolism,cell cycle regulation destruction and apoptosis reduction and suspension.Apoptosis plays an important role in tumor incidence and growth.There was evidence that promotion of apoptosis was also anti-tumor mechanism of PUFA.Most literatures have been reported that the inhibition mechanism of PUFA inhibition of tumor cell lines was different,such as prostate cancer, breast cancer,colorectal cancer,est.,but the common feature was apoptosis.
(3) PUFA causes Lipid peroxidation metabolites and cytotoxicity Products of lipid peroxidation and free radicals have a killing effect on cells.Normal cells have a complete response to anti-oxidation system and should be exempted from peroxidation damage.Because of low content of antioxidant enzymes and the corresponding cell antioxidant barrier defects,tumor cells have low anti-lipid peroxidation ability.As accumulating products of lipid peroxidation and free radicals,tumor cells are vulnerable to peroxidation.
ω-3 fatty acids are the major lipid peroxidation substrate in cells,who contains more unsaturated double bonds more be easily oxidized,resulting in excessive free radical oxidation products.These substances can damage tumor cell membrane and change the cell composition or assembly of the cytoskeleton and regulating membrane transport systems or enzymes,or PCR inhibition and or polyamine synthesis,and lead to cell death or apoptosis, signal transudation pathway change,cell growth inhibition.
(4) PUFAinhibits biosynthesis of arachidonic acid source material type peanut
Arachidonic acid(hA) is the major cell membrane polyunsaturated fatty acids.Therefore,most of the categories of peanut acids are the source material 2 series prostaglandins(prostaglandins and thromboxane) and interleukin 4 series of leukotrienes,these substances have a strong pro-inflammatory and promote cell proliferation.Leahy KM and Cianchi F, et al.found that PGE2 could prevent tumor cell apoptosis and stimulate tumor cell proliferation and promote the role of tumor angiogenesis. Pidgeon GP,etc.found that product of 12-Lox effect on AA 12-HETE participated in cell cycle regulation,tumor angiogenesis and anti-apoptotic and adhesion expression of metastasis-related genes. Calder PC,etc.found that leukotriene B4(LTB4) had a related with tumor adhesion and increased the generation of reactive oxygen species and attacked DNA leading to cancer.Chen JK,etc.found that 14,15-epoxyeicosatrienoic acid which was c product of ytochrome P450 single-oxygenase effect on AA could inhibit cell apoptosis.
(5) Changes of cancer-related gene expression
Recent studies found thatω-3 polyunsaturated fatty acids(PUFA) could control a variety of apoptosis-related genes,exert cytotoxicity to tumor cells and induce tumor cell apoptosis.There were more and more literatures about polyunsaturated fatty acids(PUFA) effect on tumor-associated gene expression.Clarke and Deckelbaum,etc.have summed up the related literatures,including transcription-related genes, oncogenes,tumor suppressor gene,with the proliferation of split-related genes,with the invasion and metastasis-related genes,and apoptosis-related genes,as well as the increasing trend.Articles of relationship betweenω-3 polyunsaturated fatty acids(PUFA) and bcl-2 family were majority.Some studies have shown that HL-60 cells and human breast cancer cell induced by EPA could occur apoptosis,and observed that bax,bcl-xs expression with the promotion of apoptosis significantly increased,bcl-2,bcl-x1 gene expression with the role of anti-apoptotic were significantly inhibited,indicating that EPA could regulate bcl-2 family expression of activity-induced tumor cell apoptosis.In addition, Bax could translocate from the cytoplasm to the mitochondrial outer membrane,under stimulated in the apoptotic signal,into a membrane protein,the formation of transit access,and promote the release of cytochrome C to accelerate the occurrence of induced apoptosis However,there were few reports on the effect ofω-3PUFA on liver cancer cells.Therefore,we will study the role of nutritional support on patients liver cancer in two ways.
With the recognition of the function of GI tract,the early enteral nutrition was emphasized.However,there were no unified views about clinical value of the effect of early enteral nutrition on postoperative recovery in patients with partial hepatectomy.Moreover,there are few reports on the effect ofω-3PUFA on liver cancer cells.Therefore,we will study the role of nutritional support on patients liver cancer in two ways.
1.The effect of early enteral nutrition on postoperative recovery in patients with partial hepatectomy.
2.Mechanism and inhabited effects ofω-3 polyunsaturated fatty acid on the proliferation of hepatocellular carcinoma cells.
PartⅠThe effect of early enteral nutrition on postoperative recovery in patients with partial hepatectomy
Objectives
To explore the effect of early enteral nutrition on postoperative recovery in patients with liver resection.
Methods
(1) 44 cases of liver resection were randomly divided into two groups to receive enteral or parenteral nutrition support for a week respectively.
(2) The effect of two nutrition supports on nutrition and immune function, such as the basis of nutritional status,liver function,intestinal function,post-operative complications,lymphocytes and interleukin-2 (IL-2),were observed.
Results
(1) There was no significant difference between preoperative liver function with Child-Pugh classification(see Table 1,x~2=0.121,P=0.50) and the nutritional status of two groups of patients(P＞0.05).There was no significant difference between the scopes of liver resection of two groups of patients(x~2=1.126,P=0.771),which was comparability.
(2) There was no significant difference between the heat(EN:7412±368Kj; PN:7149±421Kj) and nitrogen(EN:9±7g;PN:10±13g) which was given in two groups of patients everyday.
(3) Intestinal function recovery time in enteral nutrition group of patients(59±14) h was shorter than that in parenteral nutrition group of patients(73±17) h(t=-2.960,P=0.005).
(4) The body weight and the upper arm circumference decreased significantly after operation.Serum total protein,albumin and prealbumin decreased significantly after operation.
(5) After a week of nutritional support,prealbumin could return to preoperative levels,but the total protein,albumin could not return to pre-operative level.
(6)Serum triglycerides significant decreased after operation.After a week of nutritional support,serum triglycerides could return to preoperative levels in intravenous nutrition group,but it could not return to pre-operative level in enteral nutrition group.
(7) Blood lymphocytes decreased significantly after operation in two groups.After a week of nutritional support,blood lymphocytes could restore to pre-operative level in EN group,but it could not restore to pre-operative level in PN group.
(8) After a week of nutritional support,the level of serum IL-2 increased significantly(P＜0.05) in EN group,however,there was not significant change in the PN group.
Conclusions
Early nutrition support could reduce protein decomposition in patients with hepatectomy,and improve their nutritional status.So,it was helpful for recovery of patients received partial hepatectomy.Early EN also could significantly promote intestinal functional recovery in patients, compared with PN.EN could significantly promote the number of lymphocyte recovery and enhance the level of serum IL-2 after hepatectomy,which indicated that EN was better than PN in improving the patient's immune function.Therefore,early EN is a reliable,useful way of nutritional support after hepatectomy.
PARTⅡInhibited ofω-3 polyunsaturated fatty acid on the proliferation of hepatocellular carcinoma cells
Objectives
To explore inhibited ofω-3 polyunsaturated fatty acid on the proliferation and apoptosis of hepatocellular carcinoma cells.
Methods
(1) Appropriate concentration of logarithmic phase cells was selected by using MTT method and growth curve.
(2) The effect of DHA and EPA on the growth of hepatocellular carcinoma HepG2 cells proliferation was measured by using MTT method.
(3) The express of PCNA in hepatocellular carcinoma HepG2 cells were observed by Immunohistochemical method after incubated with DHAand EPA, respectively.
(4) Cell shape changes of hepatocellular carcinoma HepG2 cells were observed by fluorescence staining after incubated with DHA and EPA, respectively.
(5) Cell ultra structure changes of hepatocellular carcinoma HepG2 cells were observed by transmission electron microscopy after incubated with DHA and EPA,respectively.
(6) Cell apoptosis of hepatocellular carcinoma HepG2 cells were detected by flow cytometry after incubated with DHA and EPA,respectively.
Results
(1) According to the curve of cells growth,a cell in concentration of 1×105 cells/mL was in logarithmic phase,which was selected for drug intervention trials.
(2) MTT colorimetric showed that DHA inhibited the growth of HepG2 cells. With the increase in dosage and time,IR of cells growth increased significantly.There was statistically significant difference between each group(P＜0.05).The proliferation of HepG2 was inhabited significantly by DHA with a dosage-dependent and time-dependent manner.
(3) MTT colorimetric showed that EPA inhibited the growth of HepG2 cells. With the increase in dosage and time,IR of cells growth increased significantly.There was statistically significant difference between each group(P＜O.05).The proliferation of HepG2 was also inhabited significantly by EPA with a dosage-dependent and time-dependent manner.
(4) After incubated with DHA or EPA at two concentrations(45μg/mL and 60μg/mL) for 24 h,expression of PCNA in HepG2 cells decreased with the drug concentration increasing.
(5) After incubated with DHA/EPA at concentration of 45μg/mL for 48 h respectively,fluorescence staining showed that the amount of HepG2 cells apparently reduced,cells shrinked,apoptotic bodies or nuclear debris appeared.
(6) Compared with the control group cells,changes of vacuolization of mitochondria increasing,rough endoplasmic reticulum expansion and concentration of chromatin marginating appeared in HepG2 cells after incubated with PUFA.A large number of intracytoplasmic size electron-dense materials and apoptotic bodies could be seen.
(7) After incubated with DHA/EPA at concentrations of 45μg/mL and 60μg/mL,flow cytometry showed apoptosis appeared in HepG2 cells. Compared with the control group,DHAand EPA could significantly promote HepG2 cells apoptosis(P＜0.05).Compared with the low concentration (45μg/mL),DHA and EPA at high concentration(60μg/mL) could promote HepG2 cells apoptosis(P＜0.05) obviously.
Conclusions
The proliferation of HepG2 was inhabited significantly by DHA/EPA with a dosage-dependent and time-dependent manner.DHA/EPA could lead HepG2 cell early apoptosis and death which were detected by using flow cytometry. HepG2 cells were destructed at last.Therefore,ω-3 polyunsaturated fatty acid could suppress the proliferation of nepG2 cells and induce apoptosis.
原发性肝癌是我国常见的恶性肿瘤之一,近年来其发病率有增高趋势。据1995年卫生部统计,我国肝癌死亡率占肿瘤死亡率的第二位,严重威胁我国居民的健康。肝切除术仍然是目前治疗肝癌首选和最有效的治疗方法,但肝切除术系创伤较大的手术,对机体的代谢和内环境影响较为严重,加之应激反应使机体处于高分解代谢状态,可导致患者术后营养不良,恢复延迟,并抑制免疫系统功能。
营养支持可以减轻患者术后的负氮平衡,维持重要脏器的功能,减少外科手术并发症,已成为共识。对于原发性肝癌患者术后营养支持,肠内和肠外两个途径那一个更安全尚未定论。一些动物实验研究表明,静脉营养支持有促进恶性肿瘤细胞生长的表现。目前不主张对恶性肿瘤患者进行长期的术前营养支持,以防止营养支持促进肿瘤的增殖和转移,尤其是静脉营养支持。与静脉营养相比,肠内营养具有以下优点:①营养底物从肠道吸收,经门静脉进入肝脏符合生理,有利于内脏蛋白的合成与代谢;②为黏膜细胞提供营养物质,可维持肠黏膜屏障,防止细菌异位;③经肠道吸收的脂肪酸不能为肿瘤细胞利用;④可减少肝胆并发症等。而且,与胃肠道手术相比,肝切除术患者胃肠道完整,肠内营养途径较为安全。近年来随着对肠道功能的再认识,早期肠内营养(EN)受到重视。早期营养支持是指术后6～24h内开始给予的营养支持。一些研究显示,术后早期营养支持可以改善患者的营养状态。手术后早期进行EN支持由于受到“肛门排气方可进食”观念的影响,加之实施麻烦,患者有时出现腹胀、腹泻等不适症状,临床应用一直受到限制。有研究表明,小肠功能正常在术后6～12 h即可恢复;手术后小肠吸收功能虽然受到一定抑制,但是保留的功能足以吸收满足机体需要的营养素。
随着免疫营养概念的提出,一些对恶性肿瘤生长具有抑制作用的特殊营养物质被发现。ω-3多不饱和脂肪酸(polyunsaturated fatty acid,PUFA)是近年来发现的免疫营养支持的营养底物之一,主要存在于深海鱼油等食物中,具有抗血栓、降血脂、降血压、抗动脉硬化等作用,并对过度的炎症反应有抑制作用。PUFA还可以改变恶性肿瘤的细胞膜脂质成分,进而使其流动性变化,而增强恶性肿瘤对各种治疗的敏感性。流行病学调查发现ω-3PUFA对于前列腺癌、乳腺癌、结直肠癌等的发生、发展具有抑制作用。体外细胞培养实验和动物实验也证实ω-3 PUFA对多种肿瘤和肿瘤细胞具有抑制或杀伤作用。
ω-3 PUFA对肿瘤细胞的杀伤作用机制尚不清楚,复习国内外的相关文献研究,共提出了如下几种假说:
(1)改变生物膜的结构和功能,增加脂质过氧化物的产生。PUFA是构成生物膜的重要结构脂肪酸,补充PUFA可以改变生物膜的构成,导致细胞膜、线粒体膜的正常活性和功能发生改变。Stoll等研究发现,肿瘤细胞膜ω-3/ω-6 PUFA升高,可以造成细胞膜流动性和通透性增加,细胞毒药物容易透过细胞膜,增强肿瘤对化疗的敏感性。Chapkin等发现,肿瘤线粒体膜ω-3/ω-6升高,导致线粒体膜电位下降,细胞呼吸链和电子传递受损,线粒体膜PT(permeabilitytransition)孔开放,膜通透性增加,使细胞色素C和凋亡诱导因子的释放、半胱氨酸蛋白酶(caspase)酶解级联反应得以进行,导致细胞凋亡。
(2)导致细胞代谢障碍,促进细胞凋亡或死亡。肿瘤细胞的共同特征是代谢旺盛,细胞周期调控的破坏及细胞凋亡的减少和中止。细胞凋亡在肿瘤的发生和生长过程中发挥重要作用。有证据表明,PUFA抗肿瘤机制也体现在对促进凋亡的作用上。文献报道大多数被PUFA抑制的肿瘤细胞系,如前列腺癌、乳腺癌、大肠癌等,尽管抑制程度不同及所报道的机制不同,但均可见细胞凋亡的发生。
(3)脂质过氧化代谢产物的细胞毒作用
脂质过氧化产物和自由基对细胞具有杀伤作用。正常细胞有完备的抗氧化反应系统,可免受过氧化反应的损伤。而肿瘤细胞内由于抗氧化酶含量低及相应的细胞抗氧化屏障缺陷,抗脂质过氧化反应能力降低,随着脂质过氧化产物和自由基积聚,容易遭受过氧化损伤。
ω-3脂肪酸是细胞内脂质过氧化的主要底物,所含不饱和双键越多越易氧化,产生的过氧化产物和自由基越多。这些物质能损伤肿瘤细胞膜,改变细胞成分或细胞骨架的组装,调节膜转运系统或酶,或抑制聚合酶反应和/或聚胺合成。导致细胞死亡或凋亡,信号转导通路改变,细胞生长的抑制。
(4)抑制花生四烯酸来源的类花生酸类物质的生物合成。花生四烯酸(AA)是细胞膜中的主要多不饱和脂肪酸,因此,大多数的类花生酸类物质是其来源的2系列前列腺素(前列腺素和血栓素)和4系列白细胞三烯,这些物质具有强促炎症和促细胞增殖活性。Leahy KM和Cianchi F等发现PGE2具有阻止肿瘤细胞凋亡、刺激肿瘤细胞增殖和促进肿瘤血管新生的作用,Pidgeon GP等发现,12-Lox对AA的作用产物12-HETE参与调控细胞周期、肿瘤血管生成及抗凋亡和粘附转移相关基因的表达,Calder PC等发现白三烯B4(LTB4)与肿瘤的黏附有关并能增加活性氧的产生,攻击DNA导致癌的发生。Chen JK等发现细胞色素P450单氧合酶对AA的作用产物14,15-环氧二十碳四烯酸可以抑制细胞凋亡。
(5)肿瘤相关基因表达的改变。近期研究发现,ω-3多不饱和脂肪酸(PUFA)可以调控多种凋亡相关基因,发挥对肿瘤细胞的细胞毒作用,并可以诱导肿瘤细胞的凋亡。关于多不饱和脂肪酸(PUFA)影响肿瘤相关基因表达的文献数量越来越多,Clarke,Deckelbaum等对此做过总结,包括与转录相关的基因,癌基因、抑癌基因、与增殖分裂有关的基因、与侵袭转移相关的基因、与凋亡相关的基因等,还有不断增加的趋势。其中有关ω-3多不饱和脂肪酸(PUFA)与bcl-2家族的关系研究文章较多。有研究显示,经EPA诱导HL-60细胞和人乳腺癌细胞可以发生凋亡,并观察到具有促进凋亡作用的bax、bcl-xs的表达显著上调,而具有抗凋亡作用的bcl-2、bcl-xl基因表达均明显受抑,表明EPA能通过调节bcl-2家族的表达活性来诱导肿瘤细胞的凋亡。此外,Bax可以在凋亡信号的刺激下从细胞质移位到线粒体外膜,转变为膜内蛋白,形成转运通道,促进细胞色素C的释放,加速诱导细胞凋亡的发生。
近年来随着对肠道功能的再认识,早期肠内营养(EN)受到重视,但对于肝癌肝切除术后早期肠内营养对患者术后恢复情况影响的临床价值尚无统一意见。而且有关ω-3PUFA对肝癌细胞的作用报道较少。因此,我们从两个方面研究肝癌的营养支持作用。
1.早期肠内营养对肝切除患者术后恢复情况的影响;
2.ω-3多不饱和脂肪酸对肝癌细胞生长抑制的作用及机制。
第一部分:早期肠内营养对肝切除患者术后恢复情况的影响
目的
探讨早期肠内营养与肠外营养对肝切除患者术后恢复情况的影响
方法
(1)选择44例肝癌肝切除术患者随机分为PN组和EN组;
(2)手术后分别接受EN和PN一周;
(3)观察两种营养支持方法对患者基础营养状态、肝功能、肠道功能、术后并发症和淋巴细胞数、白细胞介素-2(IL-2)等营养和免疫功能方面的影响。
结果
(1)经统计学分析,两组患者术前肝功能Child-Pugh分级(x~2=0.121,P=0.50)和营养状况均无明显差别(P＞0.05);两组手术范围也无统计学差异(x~2=1.126,P=0.771),具有可比性。
(2) EN和PN组患者手术后日均热量摄入分别为(7412±368)kJ和(7149±421)kJ,日均氮摄入量分别为(9±7)g和(10±13)g,差异无统计学意义(P＞0.05)。
(3) EN与PN组手术后肛门排气时间分别为(59±14)h和(73±17)h,差异有统计学意义(t=-2.960,P=0.005)。
(4) EN和PN两组病例手术后体重和上臂肌围均下降,与术前比较差异有统计学意义。
(5)两组患者的血清总蛋白、白蛋白和前白蛋白手术后均明显下降,前白蛋白于营养支持一周后均能恢复到术前水平,但总蛋白和白蛋白不能恢复到术前水平。
(6)两组患者血清甘油三酯术后均明显下降,静脉营养组患者一周后能恢复到术前水平,而肠内营养组患者不能恢复到术前水平。
(7) EN组外周血淋巴细胞数术后1d明显下降,至术后8d恢复至术前水平。PN组外周血淋巴细胞数术后1d明显下降,至术后8d仍不能恢复至术前水平。
(8)手术后营养支持1周,PN组血清IL-2无明显变化,EN组血清IL-2明显升高(P＜0.05)。
结论
临床研究结果提示,早期营养支持对减轻肝切除术患者蛋白的分解,改善其术后营养状况,对促进患者的术后恢复是有帮助的。早期EN还能明显促进患者的肠道功能恢复,明显促进肝切除患者术后淋巴细胞数量的恢复,并能提高血清IL-2水平,在改善肝切除术患者的免疫功能方面优于PN。因此,早期EN是肝切除术后可靠、有益的营养支持方式。
第二部分:ω-3多不饱和脂肪酸对肝癌细胞生长抑制的作用及机制
目的
探讨ω-3多不饱和脂肪酸对肝癌细胞生长增殖及细胞凋亡的作用,以评估ω-3多不饱和脂肪酸对肝癌的作用,并初步探讨其作用机制。
方法
(1)用MTT法、生长曲线选择对数生长期细胞的合适浓度;
(2)应用MTT法分别测定DHA和EPA对肝癌细胞HepG2生长增殖的影响;
(3)应用免疫组织化学法观察DHA和EPA分别作用后PCNA在肝癌细胞HepG2中的表达;
(4)用荧光染色法观察DHA和EPA分别作用后肝癌细胞HepG2形态的改变;
(5)应用透射电镜观察DHA和EPA分别作用后肝癌细胞HepG2超微形态的改变;
(6)应用流式细胞仪检测DHA和EPA分别作用后肝癌细胞HepG2的细胞凋亡情况。
结果
(1)根据各浓度细胞生长情况,1×10~5/mL浓度细胞处于对数生长期,选定为药物干预试验的细胞浓度。
(2) MTT比色法显示DHA对细胞HepG2的生长有明显的抑制作用;随着剂量的增加和作用时间的延长,细胞生长抑制率显著上升,各组间两两比较差异均有统计学意义(P＜0.05)。DHA对HepG2细胞的抑制作用呈时间、剂量依赖关系。
(3) MTT比色法显示EPA对HepG2细胞的生长有明显的抑制作用,随着剂量和作用时间的增加,细胞生长抑制率显著提高,各组间两两比较差异有统计学意义(P＜0.05)。EPA对HepG2细胞的抑制作用亦呈时间、剂量依赖关系。
(4)经两种浓度(45μg/mL和60μg/mL)DHA/EPA作用24h后,PCNA的表达随药物浓度增高而减弱。
(5) HepG2细胞经DHA/EPA(45μg/mL)单独作用48h后,在相差显微镜下观察,结果显示:经两种脂肪酸作用后的HepG2细胞数量明显减少,细胞皱缩,出现凋亡小体或核碎片。
(6)与对照组细胞相比,经PUFA作用后的HepG2细胞出现线粒体空泡化增加,粗面内质网扩张,染色质浓聚,边聚等改变,胞浆内可见大量大小不等的电子致密物,可见到凋亡小体。
(7)不同浓度的药物(45μg/mL和60μg/mL DHA/EPA)干预HepG2细胞后,可明显观察到肿瘤细胞的凋亡。与对照组比较,DHA/EPA能明显促进HepG2细胞凋亡(P＜0.05)。相对于低浓度(45μg/mL),高浓度(60μg/mL)DHA/EPA促细胞凋亡的效果更明显(P＜0.05)。
结论
细胞培养研究结果提示,DHA、EPA对HepG2细胞的生长有明显的抑制作用,呈时间、剂量依赖关系。两种脂肪酸均可导致肝癌HepG2细胞早期凋亡和死亡,对HepG2细胞起杀伤作用。因此,ω-3多不饱和脂肪酸能显著抑制肝癌细胞生长,并诱导其凋亡。
Background
Primary hepatic cancer(PHC) is one of the most common malignant tumors in our country.The morbidity of PHC has increased in recent years. According to the statistic of the Ministry of Health in 1995,the mortality of PHC is the second place in all the malignant tumors,and it threatened the health of residents in our country heavily.Nowadays,partial hepatoectomy is still the first choice and the most effective treatment in the patients with PHC.While because of trauma,influence on the metabolism and the internal environment,and the stress reaction,the patients received partial hepatoectomy are always found with malnutrition, delayed recovery,immune suppression.
It was agreed that the Nutritional support can relieve the negative nitrogen balance,maintain the function of important organs,and reduce the complication of partial hepatectomy.It was disagreement that enteral or parenteral nutritional support postoperative was safe for patients with primary liver cancer.Some animal experiments studies showed that parenteral nutrition support could promote malignant cell growth.It was not in favor of malignant tumors with long-term preoperative nutritional support in patients in order to prevent promoting tumor proliferation and metastasis,especially in parenteral nutritional support at present. Compared with parenteral nutrition,enteral nutrition has the following advantages:①Nutrition substrates are absorbed from the intestinal,and go into the liver by portal vein,which is consistent with the physical and in favor of visceral protein synthesis and metabolism.②Enteral nutrition provides nutrients for the mucosal cells in order to maintain intestinal mucosal barrier and prevent bacteria ectopic.③Fatty acids absorbed in the intestinal can not be used by tumor cells.④Enteral nutrition can reduce complications of liver and gallbladder,et al. Moreover,compared with gastrointestinal,enteral nutritional support is a safer way for patients with partial hepatectomy because patients have gastrointestinal integrity.With re-recognition with the intestinal function,early enteral nutrition(EN) was paid much attention on in recent years.Early postoperative nutritional support means nutritional support is given in 6-24 hours postoperative.Some studies have shown that early postoperative nutritional support could improve the patient's nutritional status.Due to the concept of "anal exhaust before eating", trouble of implementation,and patients with abdominal distension, diarrhea and other symptoms during enteral nutritional support, application of early post-operative enteral nutritional supports in clinical has been restricted.Studies showed that small bowel function was retuned to normal in 6～12 h after surgery.Although it could not be fully restored,they could absorb enough nutrients of the body needs. Meanwhile,with the concept of immune nutrition,some nutrients with the tumor-growth inhibition were found recently.ω-3 Polyunsaturated Fatty Acid(PUFA) was one of the immune nutritional substrate found in recent years.It was mainly in the Deep Sea Fish Oil food,with anti-thrombosis, reducing blood fat,blood pressure,anti-arteriosclerosis,and inhibiting excessive inflammatory response.PUFA also could change the lipid composition on malignant cell membrane,thus change its fluidity, and enhance the sensitivity of all kinds of treatments.Epidemiological survey found thatω-3PUFA could inhibit the development and the occurrence of prostate cancer,breast cancer,and colorectal cancer.The inhibited effect ofω-3 PUFA on a variety of tumors and tumor cells was confirmed by cell culture experiments in vitro and animal experiments. Mechanisms of the effect of PUFA on tumor cells are not clear.There are the following hypotheses provided by relationship articles.
(1) PUFAchanges the structure and function of biofilm and increases the generation of lipid peroxide.
PUFA is important structural fatty acids of biofilm.Adding PUFA can change the composition of biofilm,and lead to changing function and the normal activity of cell membrane and mitochondrial membrane.Stoll,etc. found thatω-3/ω-6 PUFA increasing in tumor cell membrane could lead to cell membrane fluidity and permeability,cytotoxic drugs go easily through the cell membrane,and enhance the sensitivity of the tumor to chemotherapy.Chapkin,etc.found thatω-3/ω-6PUFA increasing in tumor mitochondrial membrane could lead mitochondrial membrane to decline, cellular respiration and electron transport chain damage,mitochondrial membrane PT(permeability transition) hole to open,membrane permeability, cytochrome C and apoptosis-inducing factor to release,cysteine protease (caspase) enzyme cascade can be carried out,at last lead to apoptosis.
(2) PUFA causes cell dysbolism and promotes cell apoptosis or death The common features of tumor cells are strong metabolism,cell cycle regulation destruction and apoptosis reduction and suspension.Apoptosis plays an important role in tumor incidence and growth.There was evidence that promotion of apoptosis was also anti-tumor mechanism of PUFA.Most literatures have been reported that the inhibition mechanism of PUFA inhibition of tumor cell lines was different,such as prostate cancer, breast cancer,colorectal cancer,est.,but the common feature was apoptosis.
(3) PUFA causes Lipid peroxidation metabolites and cytotoxicity Products of lipid peroxidation and free radicals have a killing effect on cells.Normal cells have a complete response to anti-oxidation system and should be exempted from peroxidation damage.Because of low content of antioxidant enzymes and the corresponding cell antioxidant barrier defects,tumor cells have low anti-lipid peroxidation ability.As accumulating products of lipid peroxidation and free radicals,tumor cells are vulnerable to peroxidation.
ω-3 fatty acids are the major lipid peroxidation substrate in cells,who contains more unsaturated double bonds more be easily oxidized,resulting in excessive free radical oxidation products.These substances can damage tumor cell membrane and change the cell composition or assembly of the cytoskeleton and regulating membrane transport systems or enzymes,or PCR inhibition and or polyamine synthesis,and lead to cell death or apoptosis, signal transudation pathway change,cell growth inhibition.
(4) PUFAinhibits biosynthesis of arachidonic acid source material type peanut
Arachidonic acid(hA) is the major cell membrane polyunsaturated fatty acids.Therefore,most of the categories of peanut acids are the source material 2 series prostaglandins(prostaglandins and thromboxane) and interleukin 4 series of leukotrienes,these substances have a strong pro-inflammatory and promote cell proliferation.Leahy KM and Cianchi F, et al.found that PGE2 could prevent tumor cell apoptosis and stimulate tumor cell proliferation and promote the role of tumor angiogenesis. Pidgeon GP,etc.found that product of 12-Lox effect on AA 12-HETE participated in cell cycle regulation,tumor angiogenesis and anti-apoptotic and adhesion expression of metastasis-related genes. Calder PC,etc.found that leukotriene B4(LTB4) had a related with tumor adhesion and increased the generation of reactive oxygen species and attacked DNA leading to cancer.Chen JK,etc.found that 14,15-epoxyeicosatrienoic acid which was c product of ytochrome P450 single-oxygenase effect on AA could inhibit cell apoptosis.
(5) Changes of cancer-related gene expression
Recent studies found thatω-3 polyunsaturated fatty acids(PUFA) could control a variety of apoptosis-related genes,exert cytotoxicity to tumor cells and induce tumor cell apoptosis.There were more and more literatures about polyunsaturated fatty acids(PUFA) effect on tumor-associated gene expression.Clarke and Deckelbaum,etc.have summed up the related literatures,including transcription-related genes, oncogenes,tumor suppressor gene,with the proliferation of split-related genes,with the invasion and metastasis-related genes,and apoptosis-related genes,as well as the increasing trend.Articles of relationship betweenω-3 polyunsaturated fatty acids(PUFA) and bcl-2 family were majority.Some studies have shown that HL-60 cells and human breast cancer cell induced by EPA could occur apoptosis,and observed that bax,bcl-xs expression with the promotion of apoptosis significantly increased,bcl-2,bcl-x1 gene expression with the role of anti-apoptotic were significantly inhibited,indicating that EPA could regulate bcl-2 family expression of activity-induced tumor cell apoptosis.In addition, Bax could translocate from the cytoplasm to the mitochondrial outer membrane,under stimulated in the apoptotic signal,into a membrane protein,the formation of transit access,and promote the release of cytochrome C to accelerate the occurrence of induced apoptosis However,there were few reports on the effect ofω-3PUFA on liver cancer cells.Therefore,we will study the role of nutritional support on patients liver cancer in two ways.
With the recognition of the function of GI tract,the early enteral nutrition was emphasized.However,there were no unified views about clinical value of the effect of early enteral nutrition on postoperative recovery in patients with partial hepatectomy.Moreover,there are few reports on the effect ofω-3PUFA on liver cancer cells.Therefore,we will study the role of nutritional support on patients liver cancer in two ways.
1.The effect of early enteral nutrition on postoperative recovery in patients with partial hepatectomy.
2.Mechanism and inhabited effects ofω-3 polyunsaturated fatty acid on the proliferation of hepatocellular carcinoma cells.
PartⅠThe effect of early enteral nutrition on postoperative recovery in patients with partial hepatectomy
Objectives
To explore the effect of early enteral nutrition on postoperative recovery in patients with liver resection.
Methods
(1) 44 cases of liver resection were randomly divided into two groups to receive enteral or parenteral nutrition support for a week respectively.
(2) The effect of two nutrition supports on nutrition and immune function, such as the basis of nutritional status,liver function,intestinal function,post-operative complications,lymphocytes and interleukin-2 (IL-2),were observed.
Results
(1) There was no significant difference between preoperative liver function with Child-Pugh classification(see Table 1,x~2=0.121,P=0.50) and the nutritional status of two groups of patients(P＞0.05).There was no significant difference between the scopes of liver resection of two groups of patients(x~2=1.126,P=0.771),which was comparability.
(2) There was no significant difference between the heat(EN:7412±368Kj; PN:7149±421Kj) and nitrogen(EN:9±7g;PN:10±13g) which was given in two groups of patients everyday.
(3) Intestinal function recovery time in enteral nutrition group of patients(59±14) h was shorter than that in parenteral nutrition group of patients(73±17) h(t=-2.960,P=0.005).
(4) The body weight and the upper arm circumference decreased significantly after operation.Serum total protein,albumin and prealbumin decreased significantly after operation.
(5) After a week of nutritional support,prealbumin could return to preoperative levels,but the total protein,albumin could not return to pre-operative level.
(6)Serum triglycerides significant decreased after operation.After a week of nutritional support,serum triglycerides could return to preoperative levels in intravenous nutrition group,but it could not return to pre-operative level in enteral nutrition group.
(7) Blood lymphocytes decreased significantly after operation in two groups.After a week of nutritional support,blood lymphocytes could restore to pre-operative level in EN group,but it could not restore to pre-operative level in PN group.
(8) After a week of nutritional support,the level of serum IL-2 increased significantly(P＜0.05) in EN group,however,there was not significant change in the PN group.
Conclusions
Early nutrition support could reduce protein decomposition in patients with hepatectomy,and improve their nutritional status.So,it was helpful for recovery of patients received partial hepatectomy.Early EN also could significantly promote intestinal functional recovery in patients, compared with PN.EN could significantly promote the number of lymphocyte recovery and enhance the level of serum IL-2 after hepatectomy,which indicated that EN was better than PN in improving the patient's immune function.Therefore,early EN is a reliable,useful way of nutritional support after hepatectomy.
PARTⅡInhibited ofω-3 polyunsaturated fatty acid on the proliferation of hepatocellular carcinoma cells
Objectives
To explore inhibited ofω-3 polyunsaturated fatty acid on the proliferation and apoptosis of hepatocellular carcinoma cells.
Methods
(1) Appropriate concentration of logarithmic phase cells was selected by using MTT method and growth curve.
(2) The effect of DHA and EPA on the growth of hepatocellular carcinoma HepG2 cells proliferation was measured by using MTT method.
(3) The express of PCNA in hepatocellular carcinoma HepG2 cells were observed by Immunohistochemical method after incubated with DHAand EPA, respectively.
(4) Cell shape changes of hepatocellular carcinoma HepG2 cells were observed by fluorescence staining after incubated with DHA and EPA, respectively.
(5) Cell ultra structure changes of hepatocellular carcinoma HepG2 cells were observed by transmission electron microscopy after incubated with DHA and EPA,respectively.
(6) Cell apoptosis of hepatocellular carcinoma HepG2 cells were detected by flow cytometry after incubated with DHA and EPA,respectively.
Results
(1) According to the curve of cells growth,a cell in concentration of 1×105 cells/mL was in logarithmic phase,which was selected for drug intervention trials.
(2) MTT colorimetric showed that DHA inhibited the growth of HepG2 cells. With the increase in dosage and time,IR of cells growth increased significantly.There was statistically significant difference between each group(P＜0.05).The proliferation of HepG2 was inhabited significantly by DHA with a dosage-dependent and time-dependent manner.
(3) MTT colorimetric showed that EPA inhibited the growth of HepG2 cells. With the increase in dosage and time,IR of cells growth increased significantly.There was statistically significant difference between each group(P＜O.05).The proliferation of HepG2 was also inhabited significantly by EPA with a dosage-dependent and time-dependent manner.
(4) After incubated with DHA or EPA at two concentrations(45μg/mL and 60μg/mL) for 24 h,expression of PCNA in HepG2 cells decreased with the drug concentration increasing.
(5) After incubated with DHA/EPA at concentration of 45μg/mL for 48 h respectively,fluorescence staining showed that the amount of HepG2 cells apparently reduced,cells shrinked,apoptotic bodies or nuclear debris appeared.
(6) Compared with the control group cells,changes of vacuolization of mitochondria increasing,rough endoplasmic reticulum expansion and concentration of chromatin marginating appeared in HepG2 cells after incubated with PUFA.A large number of intracytoplasmic size electron-dense materials and apoptotic bodies could be seen.
(7) After incubated with DHA/EPA at concentrations of 45μg/mL and 60μg/mL,flow cytometry showed apoptosis appeared in HepG2 cells. Compared with the control group,DHAand EPA could significantly promote HepG2 cells apoptosis(P＜0.05).Compared with the low concentration (45μg/mL),DHA and EPA at high concentration(60μg/mL) could promote HepG2 cells apoptosis(P＜0.05) obviously.
Conclusions
The proliferation of HepG2 was inhabited significantly by DHA/EPA with a dosage-dependent and time-dependent manner.DHA/EPA could lead HepG2 cell early apoptosis and death which were detected by using flow cytometry. HepG2 cells were destructed at last.Therefore,ω-3 polyunsaturated fatty acid could suppress the proliferation of nepG2 cells and induce apoptosis.
引文
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3.江志伟,李宁,黎介寿.恶性肿瘤病人营养支持的常见困惑.外科理论与实践.2004:9(5):357-358
4.Bisgaard T,Kehlet H.Early oral feeding after elective abdominal surgery-what are the issues? Nutrition,2002,18(11-12):944-948
5.童强,王国斌,卢晓明.早期肠内营养支持对胃肠肿瘤术后患者免疫功能及肠黏膜通透性的影响.中国康复.2005;10(5):292-294
6.Osland E,Yunus R,Khan S,ect.Early Enteral Nutrition Within 24 h of Intestinal Surgery Versus Later Commencement of Feeding:A Systematic Review and Meta-analysis.J Gastrointest Surg,2009;3(6):136-154
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8.黄洁夫.《腹部外科学》.人民卫生出版社,2001,6:1145
9.Daly JM,Redmon HP,Gallapher H.Preoperative nutrition in cancer patients.JPEN.1992,16(6s):100s-105s
10.Ward N.Nutrition support to patients undergoing gastrointestinal surgery[J],Nutr J,2003,2(18):1-5.
11.陈思增.刘新.林永堃等.术后早期肠内营养对结直肠癌病人营养状态、免疫功能及应激反应的影响.福建医科大学学报,2007,41(3):264-267
12.沈洪亮,柏津怀,戴腾昌.肝炎后肝硬化病人前白蛋白合成速率的测定及临床评价.临床肝胆病杂志.2004,20(1):48-49
13.朱建平,张同琳,袁炯,等.肝移植术后的营养支持策略[J].中国普通外科杂志,2007,8(16):741-743.
14.江志伟,李宁,黎介寿.恶性肿瘤病人营养支持的常见困惑.外科理论与实 践.2004;9(5):357-358
15.SandsromR,Drott C,Hyhander A,et al.The effect of postoperative intravenous feeding(TPN) on outcome following major surgery evaluated in a randomized study.Ann Surg.1993;217:185-192
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17.Bisgaard T,Kehlet H.Early oral feeding after elective abdominal surgery-what are the issues? Nutrition,2002,18(11-12):944-948
18.Duerksen DR,Bector S,Yaffe C,et al.Does je juna feeding with a polymeric immune-enhancing formula increase pancreatic exocrine output as compared with TPN? A case report.Nutrition,2000,16(1):47-49
19.杨孝清,朱良纲,车嘉铭等.术后早期肠内营养在食管和贲门癌病人中的应用价值.中国肿瘤临床与康复,2007,14(1):79-81
20.余彦,吕恩,唐朝辉.肠内营养支持对ICU病人细胞免疫的影响.中国危重病急救医学,2002,12(2):113-115
21.MeGowan I,Chalmers A,Smith GR,et al.Advanced in mucosal immunology[J].Gastroenteral Clin N Am,1997,26(2):145-173.
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2.Todorova NZ,Troic T.Effect of surgical trauma on patient nutritional status[J].Med Arh,2003,57(4sl):29-31.
3.江志伟,李宁,黎介寿.恶性肿瘤病人营养支持的常见困惑.外科理论与实践.2004:9(5):357-358
4.Bisgaard T,Kehlet H.Early oral feeding after elective abdominal surgery-what are the issues? Nutrition,2002,18(11-12):944-948
5.童强,王国斌,卢晓明.早期肠内营养支持对胃肠肿瘤术后患者免疫功能及肠黏膜通透性的影响.中国康复.2005;10(5):292-294
6.Osland E,Yunus R,Khan S,ect.Early Enteral Nutrition Within 24 h of Intestinal Surgery Versus Later Commencement of Feeding:A Systematic Review and Meta-analysis.J Gastrointest Surg,2009;3(6):136-154
7.梁力建,林建华.《外科学》第五版.人民卫生出版社,2005,5:590
8.黄洁夫.《腹部外科学》.人民卫生出版社,2001,6:1145
9.Daly JM,Redmon HP,Gallapher H.Preoperative nutrition in cancer patients.JPEN.1992,16(6s):100s-105s
10.Ward N.Nutrition support to patients undergoing gastrointestinal surgery[J],Nutr J,2003,2(18):1-5.
11.陈思增.刘新.林永堃等.术后早期肠内营养对结直肠癌病人营养状态、免疫功能及应激反应的影响.福建医科大学学报,2007,41(3):264-267
12.沈洪亮,柏津怀,戴腾昌.肝炎后肝硬化病人前白蛋白合成速率的测定及临床评价.临床肝胆病杂志.2004,20(1):48-49
13.朱建平,张同琳,袁炯,等.肝移植术后的营养支持策略[J].中国普通外科杂志,2007,8(16):741-743.
14.江志伟,李宁,黎介寿.恶性肿瘤病人营养支持的常见困惑.外科理论与实 践.2004;9(5):357-358
15.SandsromR,Drott C,Hyhander A,et al.The effect of postoperative intravenous feeding(TPN) on outcome following major surgery evaluated in a randomized study.Ann Surg.1993;217:185-192
16.Baskin WN.Advance in enteral nutrition techniques[J].Am J Gastroenterol,1992,87(11):1547-1553.
17.Bisgaard T,Kehlet H.Early oral feeding after elective abdominal surgery-what are the issues? Nutrition,2002,18(11-12):944-948
18.Duerksen DR,Bector S,Yaffe C,et al.Does je juna feeding with a polymeric immune-enhancing formula increase pancreatic exocrine output as compared with TPN? A case report.Nutrition,2000,16(1):47-49
19.杨孝清,朱良纲,车嘉铭等.术后早期肠内营养在食管和贲门癌病人中的应用价值.中国肿瘤临床与康复,2007,14(1):79-81
20.余彦,吕恩,唐朝辉.肠内营养支持对ICU病人细胞免疫的影响.中国危重病急救医学,2002,12(2):113-115
21.MeGowan I,Chalmers A,Smith GR,et al.Advanced in mucosal immunology[J].Gastroenteral Clin N Am,1997,26(2):145-173.
22.Sedman PC,Ramsden CW,Bernnan TG,et al.Pharmacological concentrations of lipid emulsion inhibit interleukin-2 dependent lymphocyte responses in vitro[J].JPEN J Parenter Enteral Nutr.,1990,14(1):12-17.
1. Supector AA. Polyunsaturated fatty acids in the treatment of cancer: a new twist [J]. Nutrition. 1990; 6(6):493～494.
2. Terry PD, Rohan TE, Wolk A. Intakes of fish and marine fatty acids and the risks of cancers of the breast and prostate and of other hormone-related cancers: a review of the epidemiologic evidence [J]. Am J Clin Nutr, 2003, 77(3): 532-543.
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