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Analysis of non-obstructive dysphagia with esophageal high-resolution manometry

Chinese Journal of Digestion.2013;33(10):664-668. doi:10.3760/cma.j.issn.0254-1432.2013.10.007

Objective To analyze the distribution and esophageal motility characteristics of patients with non obstructive dysphagia (NOD).Methods From June 2010 to June 2012,ninety seven patients with a sense of obstruction when swallowing were selected and patients with esophageal organic stenosis was excluded through endoscopic examination and upper gastrointestinal radiography.While nine healthy volunteers were recruited as healthy control.High-resolution esophageal manometry was performed with high-resolution multi-channel Netherlands CTD Synectics gastrointestinal function monitoring system (Pcpolygraf polysomnography recording system) and MMS digestive power detection system.The observation parameter included upper esophageal sphincter pressure (UESP),upper esophageal sphincter relaxation rate (UESRR),the length of the lower esophageal sphincter (LESL),lower esophageal sphincter pressure (LESP),intergrated relaxation pressure (IRP),lower esophageal sphincter relaxation rate (LESRR),esophageal subordinate segments pressure and esophageal effective peristalsis ratio.The rank sum test was performed for comparison between groups.Results Among 97 patients with NOD,the percentage of achalasia,nonspecific esophageal motor disorder andgastroesophageal reflux disease (GERD) was 41.2％ (40/97),39.2％ (38/97)and 19.6％(19/97),respectively.Among patients with nonspecific esophageal motor disorder,the percentage of abnormal peristalsis,absent peristalsis,normal pressure and distal esophegesl spasm was 39.5％(15/38),36.8％(14/38),15.8％(6/38) and 7.9％(3/38),respectively.The differences in lESL,LESP,LESRR,esophageal subordinate segments pressure and esophageal effective peristalsis ratio among achalasia,GERD,nonspecific esophageal motor disorder and healthy control were statistically significant (F 6.143,57.490,50.559,10.155 and 22.046,all P＜0.05).LESP oF patients with achalasia was higher than that of healthy control,however LESRR,esophageal subordinate segments pressure and esophageal effective peristalsis ratio were all lower than those of healthy control and the differences were statistically significant (F 2.276,11.113,-8.036 and -14.663,all P＜0.05).LESL and LESP of achalasia were both higher than those of GERD group,LESRR,esophageal subordinate segments pressure and esophageal effective peristalsis ratio were all lower than those of GERD group and the differences were statistically significant (F=4.325,15.983,-19.235,-3.410 and-4.351,all P＜0.05).LESL and LESP of achalasia group both were higher than those of nonspecific esophageal motor disorder group,LESRR and esophageal effective peristalsis ratio were both lower than those of GERD group and the differences were statistically significant (F=2.376,7.668,2.873 and-3.873,all P＜0.05).LESRR of GERDgroup was higher than that of healthy control group,LESL,LESP,esophageal subordinate segments pressure and esophageal effective peristalsis ratio were all lower than those of healthy control group and the differences were statistically significant (F=5.931,-2.483,-14.618,-3.071 and-4.516,all P＜0.05).LESL and LESP of GERD group were both lower than those of nonspecific esophageal motor disorder group,LESRR was higher than that of nonspecific esophageal motor disorder group and the differences were statistically significant (F =--2.113,6.578 and 10.979,all P ＜ 0.05).LESP,LESRR,esophageal subordinate segments pressure and esophageal effective peristalsis ratio of nonspecific esophageal motor disorder group were all lower than those of healthy control group and the differences were statistically significant (F=-6.313,-3.580,-3.511 and-8.150,all P＜0.05).IRP of 40 patients with achalasia were all beyond the normal range.Conclusions NOD mainly included achalasia,nonspecific esophageal motor disorder and GERD.The reduction of effective esophageal peristalsis may be an important pathophysiological mechanism of NOD.

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Relationship between microRNA-200c expression, epithelial-mesenchymal transition and the sensitivity to gefitinib in colon cancer cells

Ying XU ; Shubei WANG ; Haili WANG ; Yunwei SUN

Chinese Journal of Digestion.2013;33(10):690-695. doi:10.3760/cma.j.issn.0254-1432.2013.10.012

Objective To explore the relationship between microRNA (miRNA)-200c expression,epithelial-mesenchymal transition (EMT) and the sensitivity to gefitinib in colon cancer cells.Methods The inhibitory effects of gefitinib on four types of colon cancer cell lines (HT29,SW620,HCT1116,SW480) were examined by cell counting kit-8 (CCK-8) assay.The expressions of miRNA-200c,epithelial marker (E-cadherin) and mesenchymal markers (vimentin and zinc finger Ebox binding homeobox 1 ZEB 1) at mRNA level in four types of colon cancer cell lines were detected by fluorescence quantitative polymerase chain reaction.The expressions of E-cadherin,vimentin and ZEB 1 at protein level were determined by Western blot.After up-or down-regulated the expression of miRNA-200c,the changes of the expression of EMT related genes and the sensitivity to gefitinib were observed.Results HT29 cells were most sensitive to gefitinib (IC50 =(7.70 ± 0.31) μmol/L),in which the expressions of both miRNA-200c and E-cadherin were the highest,and the expressions of vimentin and ZEB1 were extremely low.HCT116 and SW480 were moderately sensitive to gefitinib (IC50=(11.88±0.97) and (16.63±0.45) μmol/L),and the expressions of miRNA-200c and Ecadherin were moderate.SW620 cell line was most insensitive to gefitinib (IC50 =(26.43 ± 3.68)μmol/L),the expressions of miRNA-200c and E-cadherin were the lowest.The expressions of vimentin and ZEB 1 in SW620 were higher than that of the other three types of cell lines.After upregulated the expression of miRNA 200c,the expression of E-cadherin in SW620 cells increased,the expression of ZEB 1 and vimentin decreased,and the sensitivity to gefitinib increased.After downregulated the expression of miRNA-200c,the expression of E-cadherin in HT29 cells decreased,the expression of ZEB 1 and vimentin increased,and the sensitivity to gefitinib decreased.Conclusion miRNA-200c may up-regulate the expression of E-cadherin through EMT regulation,and then influence the sensitivity to gefitinib in colon cancer cells.

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Clinicopathological characteristic analysis of three cases of colonic muco-submucosal elongated polyp

Jiajia XU ; Lihua ZHANG

Chinese Journal of Digestion.2013;33(10):696-699. doi:10.3760/cma.j.issn.0254-1432.2013.10.013

Objective To explore the clinicopathological features,diagnosis and differential diagnosis of colonic muco-submucosal elongated polyp (CMSEP).Methods The clinical manifestations,endoscopic and histological features of three cases of CMSEP were analyzed.The literature was reviewed with regard to the diagnostic criteria and differential diagnosis.Results Three patients were male,and aged 25,45 and 75 years old.Two polyps were at transverse colon and one at rectum.Under colonoscope,all three cases showed the long pedunculated polyps.The surfaces of two polyps were smooth and the colors were similar to colonic mucosa while one polyp appeared mucosal erosion and congestion on the top.Under optical microscope,three cases had similar lesions.The axis of the lesions was expanded submucosa and the surface was the layer of colonic mucosa.The expanded and thick walled blood vessels were seen paralleled to the long axis in the submucosa of the axis of lesions.One polyp with mucosal erosion on the top presented with adenocarcinomal changes.Conclusions CMSEP is a kind of specific polypoid lesion,with characteristic colonoscopic and pathological features.Most are benign changes,however malignant changes occur in a few cases.In histology,CMSEP should be identified with mucosal prolapse syndrome associated inflammatory polyp.

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Effects of deoxyribonucleic acid methylation on the expression of hepatocyte nuclear factor-4α

Jingxian HU ; Xin ZHANG ; Weifen XIE

Chinese Journal of Digestion.2013;33(9):621-625. doi:10.3760/cma.j.issn.0254-1432.2013.09.010

Objective To investigate the effect of deoxyribonucleic acid (DNA) methylation on the expression of hepatocyte nuclear factor-4α (HNF4c) and its role in the expression of HNF4α regulated by transforming growth factor-β1 (TGF-β1).Methods The expression of HNF4αP1 mRNA in six human hepatoma cell lines (HepG2,Huh-7,Hep3B,SMMC-7721,BEL-7405 and FOCUS),20 hepatic carcinoma specimens and corresponding adjacent tissues was detected by real-time reverse transcription polymerse chain reaction (real-time RT-PCR).The methylation status of the promoter region of HNF4αP1 in six human hepatoma cell lines was examined by bisulfite sequencing PCR (BSP).FOCUS cells were treated with 5-aza-2'-deoxycytidine (5-AZA-CdR) and then the methylation status of the promoter region of HNF4αP1 was examined by BSP.The expression of HNF4αP1 mRNA was detected by real-time RT-PCR.The six human hepatoma cell lines were treated with TGFβ1 and the expression of HNF4αP1 mRNA was detected by real-time RT-PCR.FOCUS cells were cotreated with 5-AZA-CdR,TGF-β1 and 5-AZA-CdR.The expression of HNF4αP1 mRNA was detected by real-time RT-PCR,and t test was performed for statistical analysis.Results Among 20 human hepatic carcinoma specimens and corresponding adjacent tissues,the expression of HNF4αP1 mRNA of 13 human hepatic carcinoma specimens was lower than that of corresponding adjacent tissues (t=2.350,P＜0.05).The relative quantity of the expression of HNF4αP1 mRNA was higher in Hep3B,HepG2 and Huh-7 cells,whereas that in SMMC-7721,BEL-7405 and FOCUS cells was lower.The methylation of the promoter region of HNF4αP1 in HepG2,Huh-7 and Hep3B was lower,but that in SMMC-7721,BEL-7405 and FOCUS was higher.Along with the increasing of the concentration of 5-AZA-CdR (0,0.1,1.0 and 2.5 μmol/L),the degree of the methylation of the promoter region of HNF4αP1 in FOCUS cells gradually decreased (61％,46％,32％ and 27％),and however the relative quantity of the expression of HNF4αP1 mRNA gradually increased ((9.661 ± 0.336)×10-7,(2.001±0.432)×10-6,(3.689±0.714)×10-6and (4.732±2.451)×10-6).After stimulated with TGF-β1,the relative quantity of the expression of HNF4αP1 mRNA was downregulated in HepG2,Huh-7 and Hep3B cells in which the methylation of the promoter region was low (t=12.994,8.441,and 9.032,all P＜0.01).There was no significant difference in the relative quantity of the expression of HNF4αP1 mRNA in SMMC-7721,BEL-7405 and FOCUS cells in which the methylation of the promoter region was high (all P ＞ 0.05).The relative quantity of the expression of HNF4αP1 mRNA in 5-AZA-CdR treated FOCUS cells ((4.972±0.035) × 10-6) was higher than that of control group ((1.411 ± 0.104) × 10-6) and the difference was statistically significant (t=13.212,P＜0.01).The relative quantity of the expression of HNF4αP1 mRNA in FOCUS cells co-treated with 5-AZA-CdR and TGF-β1 was lower than that in cells treated with 5-AZA-CdR alone and the difference was statistically significant ((1.181 ± 0.132) × 10-6 vs (4.972 ± 0.035) × 10-6,t=13.873,P＜0.01).Conclusions The expression of HNF4αP1 is down-regulated in hepatic carcinoma tissues.DNA methylation may regulate the expression of HNF4αP1 in hepatoma cells.The methylation of HNF4αP1 promoter region inhibits the regulating function of TGF-β1 in the expression of HNF4αP1.

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Consistency of gene amplification and its expression at protein level of human epidermal growth factor receptor 2 in gastric carcinoma

Xinyan ZHANG ; Juanjuan FU ; Wenjing PU ; Zhenzhu SUN

Chinese Journal of Digestion.2013;33(10):680-683. doi:10.3760/cma.j.issn.0254-1432.2013.10.010

Objective To investigate the consistency of gene amplification and its expression at protein level of human epidermal growth factor receptor 2 (HER2) in gastric carcinoma.Methods From 2010 to 2012,120 gastric cancer specimens of patients with gastric cancer were collected,of which 100 were surgical specimens and 20 were specimens from biopsy under gastroscope.The protein expression of HER2 in 120 specimens was detected by immunohistochemistry (IHC).According to the results of IHC,the positive parts of HER2 expression of IHC slices were developed into tissue microarrays for fluorescence in situ hybridization (FISH) to test the gene amplification of HER2.The different parts with different color intensity of focal (+ + +) (≤ 10％ tumor cell strongly staining) specimens detected by IHC detection were compared with the results of FISH.Kappa test was performed for statistical analysis.Results Among 120 gastric cancer specimens,the results of IHC indicated that 77 specimens were positive with different staining intensity including 16 strong positive (+++),six focal positive (+++),37 moderate positive (++) and 18 weak positive (+).The positive rate of HER2 protein expression detected by IHC was 18.3％ (22/120).The results of FISH showed 41 specimens were positive and the rate of gene amplification was 34.2％.Among which,21 were moderate positive (++) detected by IHC,15 were strong positive (+ + +) and five were focal positive (+ ++).The positive rate of HER2 was 35.0％ (42/120) with IHC and FISH combined detection.The consistent rate of IHC and FISH was 91.6 ％ (76/83).Kappa coefficient was 0.960 (P＜0.01).In five positive specimens detected by FISH and which were focal positive (+ + +) by IHC,the different parts with different color intensity were compared with the results of FISH and gene amplification was found in all specimens.Conclusion Tissue microarray technology is consistent with IHC in HER2 detection in gastric cancer specimens and could help to improve the detection rate.

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Quantitative detection and the significance of K-ras12 and K-ras13 mutations in peripheral blood of the population with high risk of pancreatic cancer

Yanping ZHU ; Lihua WANG ; Jun GAO ; Quanjiang LI ; Jing JIN ; Zhaoshen LI

Chinese Journal of Digestion.2013;33(11):746-749. doi:10.3760/cma.j.issn.0254-1432.2013.011.010

Objective To explore the clinical significance of the detection of K-ras12 and K-ras13 mutations in the peripheral blood of population with high risk group of pancreatic cancer.Methods From May 2010 to November 2011,a total of 160 patients with clear diagnosis and high risk of pancreatic cancer were collected.Among those patients,36 cases were finally diagnosed as pancreatic cancer,36 cases were chronic pancreatitis (CP),six cases were autoimmune pancreatitis (AIP),16 cases were other malignant tumor,13 cases were pancreatic benign tumor,13 cases were bile duct stones and 40 cases were other benign diseases.Fasting peripheral venous blood was collected in all selected candidates.The condition of K-ras12 and K-rasl3 mutation was detected by peptide nucleic acid-mediated real time fluorescen quantitative polymerase chain reaction.Mann-Whitney U test was performed to compare the mutation between the two groups.Chi-square test was used to compare the rates.According to the receiver operator characteristic (ROC) curve,the mutation positive decision value and the mutation positive rate of K-ras12 and K-ras13 were calculated.Results There were significant differences in K-ras12 mutation between pancreatic cancer group (1154(2207)) and other benign diseases (476(973)),CP group (446(808)),bile duet stones group (357(568)) (U=502,446 and 117,all P＜0.05).There were no significant differences in K-ras13 mutation between pancreatic cancer group and other benign diseases,CP group,bile duct stones group (all P＞0.05).When the mutation positive decision value of K-ras12 was set as mutation copy over 1000,the positive mutation rate of pancreatic cancer and non-pancreatic cancer was 55.6％ (20/36) and 25.0％ (31/124),respectively.When the area under the ROC curve of K-ras12 was 0.664,there was differential diagnostic value in identifying pancreatic cancer and non-pancreatic cancer (P=0.003).When the area under the ROC curve of K-ras13 was 0.522,there was no differential diagnostic value in identifying pancreatic cancer and non-pancreatic cancer (P=0.695).There was significant difference in K-ras13mutation between smokers and non-smokers (943 (1510) and 571 (964)).Conclusion K-ras12mutation in peripheral venous blood may be used for pancreatic cancer screening.

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Expression and significance of plasma circulation microRNA155 in ulcerative cilitis

Yulan YE ; Zhi PANG ; Chunli ZHOU ; Jiaju ZHENG

Chinese Journal of Digestion.2013;33(11):761-764. doi:10.3760/cma.j.issn.0254-1432.2013.011.013

Objective To explore the diagnostic value of the concentration of plasma circulation microRNA155 (miRNA155) in ulcerative cilitis (UC) and its correlation with clinical characteristic of UC.Methods From October 2010 to August 2012,a total of 136 patients diagnosed as UC were enrolled,and at same time,170 healthy individuals were set as healthy control.The blood samples of all participants were obtained and plasma was isolated.The adsorption column was used for RNA extraction according to miRNeasy kit instruction.RNA was reverse-transcribed into cDNA with miScript reverse transcription kit.cDNA was a template and miRNA155 real-time quantitative polymerase chain reaction (PCR) was performed with miScript SYBR Green PCR kit.The relative quantity of miRNA155 expression was calculated with 2-△△Ct method.Analysis of variance were performed for comparison between groups.Receiver operating characteristic curve analysis was used for the diagnostic value of miRNA155 concentration in UC.Multiple linear regression analysis was used for the correlation between miRNA155 concentration and clinical characteristics of UC.Results The concentration of plasma circulation miRNA155 of patients with UC ((1357.43±326.15) fmol/L)was higher than that of healthy controls ((1140.70 ± 312.47) fmol/L) and the differences were statistically significant (F=35.56,P＜0.01).The area under receiver operating characteristic curve of the concentration of plasma circulation miRNA155 of patients with UC was 0.847,and the 95 ％CI was 0.806 to 0.888 (P＜0.01).When the concentration of plasma circulation miRNA155 was 1404.51 fmol/L,its specificity in the diagnosis of UC was 94.7％,and sensitivity was 40.4％.There was correlation between the concentration of plasma circulation miRNA155 and the disease activity in patients with UC (F=12.91,P＜0.05).However there was no correlation with the severity and location of the disease (both P＞0.05).Conclusion Plasma circulation miRNA155 highly expressed in patients with UC,and its concentration is correlated with the disease activity.

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Prognosis of liver retransplantation in patients with viral hepatitis recurrence

Haiming ZHANG ; Liying SUN ; Hong ZHENG ; Cheng PAN ; Yonglin DENG ; Zhijun ZHU

Chinese Journal of Digestion.2013;33(9):606-610. doi:10.3760/cma.j.issn.0254-1432.2013.09.006

Objective To investigate the prognosis of liver retransplantation in patients with transplanted liver function failure caused by viral hepatitis recurrence.Methods From January 20th 2003 to November 20th 2012,the clinical data of 215 patients with liver retransplantation were retrospectively analyzed.The survival of transplanted liver of 18 cases with liver retransplantation because of hepatitis recurrence (eight cases of hepatitis C and 10 cases of hepatitis B) was compared with that of 115 cases with liver retransplantation for biliary complications.The dysfunction of transplanted liver after first transplantation and the survival after second liver retransplantation of patients with hepatitis C recurrence were compared with those of patients with hepatitis B recurrence.The prognosis analysis was compared by survival curves made by Kaplan-Meier method.Results Biliary complications were the most common reason in 215 patients with second liver retransplantation and which accounted for 115 cases (53.5 ％).Eighteen cases were hepatitis recurrence (8.4 ％).There was no significant difference in survival rate of the second transplanted liver between patients with hepatitis recurrence and biliary complication (P =0.543).The dysfunction of transplanted liver occurred at early stage (in three months) after first liver transplantation in part of patients with hepatitis C recurrence.The dysfunction of transplanted liver almost all occurred two years after first liver transplantation in patients with hepatitis B recurrence.Among eight patients with hepatitis C recurrence,the second transplanted liver of five cases survived more than one year.All the second transplanted liver of 10 patients with hepatitis B recurrence survived more than one year.There was no significant difference between them (P =0.060).Conclusions The prognosis of liver retransplantation in patients with hepatitis recurrence is similar with that of patients with biliary complications.The prognosis of liver retransplantation in patients with hepatitis B recurrence is good.

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Expression of hepatic stellate cells inhibited by receptor for advance glycoxidation end products specific small interfering RNA

Yajing LIU ; Jinrong XIA

Chinese Journal of Digestion.2013;33(9):611-615. doi:10.3760/cma.j.issn.0254-1432.2013.09.008

Objective To investigate the effect of specific small interfering RNA (siRNA) targeting receptor of advanced glycation end products (RAGE) on the production of fibrosis markers (laminin,hyaluronic acid (HA) and N-terminal procollagen Ⅲ propeptide (PⅢ NP) in hepatic fibrosis (HF).Methods The expression vectors of specific siRNA targeting RAGE were constructed.Primary rat hepatic stellar cells (HSC) were isolated and cultured.The primary rat HSC were transfected with the recombinant vector.The blank control group and unspecific siRNA vector pAKD-NC-transfected group were as controls.The expressions of RAGE,laminin,HA and PⅢ NP at mRNA and protein levels were detected by real-time polymerase chain reaction and Western blot,respectively.Least-significant difference (LSD) and Student-Newman-Keuls (SNK) were performed to analyze standard normal distribution or homogeneous variance.Non-normal distribution and heterogeneity of variance data were analyzed by non-parametric Wilcoxon test.Results The expressions of RAGE at mRNA and protein levels in pAKD-GR126-transfected primary HSC were (42.32 ± 6.16)％,(43.24±7.50)％,(51.06±13.79)％ and (47.94±5.36)％ in blank control group and pAKDNC group (F=7.791 and 36.513,all P＜0.05).The expressions of laminin at mRNA and protein levels were (41.07±3.13)％,(40.59±5.87)％,(53.89±2.25)％ and (52.46±4.68)％ in blank control group andpAKD-NC group (F=225.111 and 88.039,all P＜0.05).The expressions of HA at mRNA and protein levels were (45.69 ± 0.87) ％,(46.08 ± 2.36) ％,(54.20 ± 0.56) ％ and (52.30±3.42)％ in blank control group and pAKD-NC group (F=178.317 and 180.646,all P＜ 0.05).The expressions of PⅢ NP mRNA at mRNA and protein levels were (56.10±4.18)％,(55.15±2.39)％,(54.40±2.79)％ and (53.58±6.18)％ in blank control group and pAKD-NC group (F=141.633 and 49.670,all P＜0.05).Conclusion RAGE specific siRNA could inhibit the expression of RAGE in primary rat HSC and could significantly lower the expression of fibrosis markers laminin,HA and PⅢ NP at mRNA and protein level.

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Changes of caveolin-1 in the livers of mice with nonalcoholic fatty liver disease caused by high-fat diet

Yan QIU ; Hongtan CHEN ; Guoqiang XU ; Chaohui YU ; Xiaodong TENG ; Hongtian YAO

Chinese Journal of Digestion.2013;33(9):616-620. doi:10.3760/cma.j.issn.0254-1432.2013.09.009

Objective To explore the role of caveolin-1 in nonalcoholic fatty liver disease (NAFLD) caused by high-fat diet.Methods A total of 12 ten-week-old male C57BL/6 mice were fed with high-fat and high-cholesterol diet for 14 weeks to establish the NAFLD animal model.And six syngeneic mice fed with normal diet at the same time were taken as control.All the mice were sacrificed by the end of 14th week,body weight,liver weight and the changes of serum lipids of the two groups were compared.The changes of caveolin-1 at mRNA and protein levels in the liver of mice with NAFLD were detected by quantitative polymerase chain reaction (qPCR) and Western blot.The liver steatosis of the mice was observed under light microscopy after stained by hematoxylin and eosin.The changes of distribution of caveolin-1 in liver were examined by immunohistochemistry.The differences of caveolin-1 at mRNA and protein level in livers between the two groups were compared by t test.The differences of immunohistochemical scores of caveolin-1 expression in the livers of mice with different degree of fatty liver were analyzed by ordinal variables of two independent samples ranksum test analysis.Results After 14 weeks high-fat and high-cholesterol diet,all the mice of experiment group developed NAFLD.Nine of which were severe and three were moderate.Compared with the control group,serum total cholesterol,high density lipoprotein cholesterol and low density lipoprotein cholesterol of experiment group significantly increased ((1.940 ± 0.300) mmol/L vs (3.771±0.800) mmol/L,(1.120±0.066) mmol/L vs (2.224±0.420) mmol/L,(0.510±0.191) mmol/L vs (1.241±0.660) mmol/L,t=-3.760,-5.474,-3.332,all P＜0.01),however there was no significant difference in triglyceride (P＞0.05).The caveolin-1 of experiment group significantly increased at mRNA (1.536 ±0.226 vs 0.980± 0.272,t=3.371,P＜0.05) and protein levels (0.643±0.240 vs 0.100±0.130,t=4.847,P＜0.01).The immunohistochemical results indicated that the increased caveolin-1 expression mainly distributed in the membrane of hepatocytes,cytoplasm and membrane of lipid droplets.Conclusion The up-regulated caveolin-1 expression in the livers of NAFLD mice induced by high-fat and high-cholesterol may be involved in the mechanism of NAFLD.

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