Genome-wide analysis of long noncoding RNA expression profiles in patients with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder poising burgeoning health problem to humans. Recent studies have shown that long non-coding RNA (lncRNA) plays critical roles in a myriad of biological processes and human diseases. Since the roles of lncRNA in NAFLD remain unknown, they were investigated in the study. Microarray expression profiling of mRNAs and lncRNAs was conducted using RNA extracted from patients with and without NAFLD. One thousand seven hundred thirty-five lncRNAs and 1485 mRNA were found differentially expressed in NAFLD samples compared with those in control samples. Among them 535 and 1,200 lncRNAs were upregulated and downregulated in NAFLD, respectively; 760 and 725 mRNAs were upregulated and downregulated in NAFLD, respectively. Moreover, seven lncRNAs and seven mRNAs that were highly up- or downregulated in NAFLD samples were validated by quantitative real-time polymer chain reactions. Kyoto Encyclopedia of Genes and Genomes pathway analysis and Gene Ontology analysis for the differentially expressed mRNAs showed that these RNAs are involved in various metabolic processes, cellular components, and molecular functions. Our findings indicate that the expression profiles of lncRNAs have changed in NAFLD as compared with normal liver, and the identified regulated RNAs may provide novel insight into the molecular mechanisms underlying the disease and potential novel diagnostic or therapeutic targets for NAFLD.

A study on new method of noninvasive esophageal venous pressure measurement based on the airflow and laser detection technology.

Using optics combined with automatic control and computer real-time image detection technology, a novel noninvasive method of noncontact pressure manometry was developed based on the airflow and laser detection technology in this study. The new esophageal venous pressure measurement system was tested in-vitro experiments. A stable and adjustable pulse stream was produced from a self-developed pump and a laser emitting apparatus could generate optical signals which can be captured by image acquisition and analysis system program. A synchronization system simultaneous measured the changes of air pressure and the deformation of the vein wall to capture the vascular deformation while simultaneously record the current pressure value. The results of this study indicated that the pressure values tested by the new method have good correlation with the actual pressure value in animal experiments. The new method of noninvasive pressure measurement based on the airflow and laser detection technology is accurate, feasible, repeatable and has a good application prospects.

miR-21 regulates triglyceride and cholesterol metabolism in non-alcoholic fatty liver disease by targeting HMGCR.

Non-alcoholic fatty liver disease (NAFLD) has emerged as a public health issue with a prevalence of 15-30% in Western populations and 6-25% in Asian populations. Certain studies have revealed the alteration of microRNA (miRNA or miR) profiles in NAFLD and it has been suggested that miR-21 is associated with NAFLD. In the present study, we measured the serum levels of miR-21 in patients with NAFLD and also performed in vitro experiments using a cellular model of NAFLD to further investigate the effects of miR-21 on triglyceride and cholesterol metabolism. Furthermore, a novel target through which miR-21 exerts its effects on NAFLD was identified. The results revealed that the serum levels of miR-21 were lower in patients with NAFLD compared with the healthy controls. In addition, 3-hydroxy-3-methylglutaryl-co-enzyme A reductase (HMGCR) expression was increased in the serum of patients with NAFLD both at the mRNA and protein level. To mimic the NAFLD condition in vitro, HepG2 cells were treated with palmitic acid (PA) and oleic acid (OA). Consistent with the results obtained in the in vivo experiments, the expression levels of miR-21 were decreased and those of HMGCR were increased in the in vitro model of NAFLD. Luciferase reporter assay revealed that HMGCR was a direct target of miR-21 and that miR-21 exerted an effect on both HMGCR transcript degradation and protein translation. Furthermore, the results from the in vitro experiments revealed that miR-21 decreased the levels of triglycerides (TG), free cholesterol (FC) and total cholesterol (TC) in the PA/OA-treated HepG2 cells and that this effect was attenuated by HMGCR overexpression. Taken together, to the best of our knowledge, the present study is the first to report that miR-21 regulates triglyceride and cholesterol metabolism in an in vitro model of NAFLD, and that this effect is achieved by the inhibition of HMGCR expression. We speculate that miR-21 may be a useful biomarker for the diagnosis and treatment of NAFLD.

[A new system for noninvasive esophageal varices pressure measurement based on airflow and laser technology].

OBJECTIVE: Combined the optical principle with automatic control technology and computer real-time image detection technology to develop a non-contact system for noninvasive esophageal varices pressure measurement. METHODS: The system included the adjustable air pump, laser device, image collection and analysis program. The feasibility and accuracy of the system were verified by in vitro experiments. RESULTS: The bionic vascular pressure measured by this system had good correlation and repeatability with the actual pressure. CONCLUSIONS: This system is accurate, feasible and has good application prospects.

Association between reversal of multidrug resistance by methyl jasmonate and P-glycoprotein ATPase activity in hepatocellular carcinoma.

OBJECTIVE: To study the effects of methyl jasmonate on multidrug resistance in a mouse model of hepatocellular carcinoma. METHODS: Multidrug resistant H22 (H22/FAP) hepatocellular carcinoma cells were produced in vitro by continuous exposure to increasing doses of doxorubicin, cisplatin and 5-fluorouracil (FAP regimen). Cell toxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay. Survival time was calculated for BALB/c mice that received intraperitoneal injections of H22/FAP cells followed by treatment with methyl jasmonate or verapamil in combination with FAP for 7 days. Adenosine triphosphate (ATP) hydrolysis was used to measure the activity of permeability-glycoprotein (P-gp) ATPase activity in plasma membranes. RESULTS: The MTT assay showed that methyl jasmonate significantly enhanced the cytotoxicity of the FAP regimen in multidrug resistant H22/FAP cells. Methyl jasmonate (10 mg/kg and 5 mg/kg) combined with FAP significantly increased survival time in BALB/c mice by 44.25% and 48.01%, respectively, compared with FAP. Methyl jasmonate increased P-gp ATPase activity. CONCLUSION: The combined use of methyl jasmonate and the FAP regimen might be a novel strategy for overcoming the multidrug resistance often observed in hepatocellular carcinoma.

Amelioration of carbon tetrachloride-induced cirrhosis and portal hypertension in rat using adenoviral gene transfer of Akt.

AIM: To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride (CCl4). METHODS: Using cre-loxp technique, we created an Ad-myr-HA-Akt virus, in which Akt is labeled by a HA tag and its expression is driven by myr promoter. Further, through measuring enzyme levels and histological structure, we determined the efficacy of this Ad-myr-HA-Akt virus in inhibiting the development of cirrhosis induced by CCl4 in rats. Lastly, using western blotting, we examined the expression levels and/or phosphorylation status of Akt, apoptotic mediators, endothelial nitric oxide synthase (eNOS), and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus. RESULTS: The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment, which was consistent with the sequence reported in the GenBank. The concentrations of Ad-myr-HA-Akt and adenoviral enhanced green fluorescent protein (Ad-EGFP) virus used in the current study were 5.5 × 10(11) vp/mL. The portal vein diameter, peak velocity of blood flow, portal blood flow and congestion index were significantly increased in untreated, saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection. In contrast, these parameters in the Akt cirrhosis group were comparable to normal control group. Compared to the normal control, the liver function (Alanine aminotransferase, Aspartate aminotransferase and Albumin) was significantly impaired in the untreated, saline and Ad-EGFP cirrhosis groups. The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated, saline and Ad-EGFP cirrhosis groups. The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups. The results of HE and Van Gieson staining indicated that Akt group has better preservation of histological structure and less fibrosis than other cirrhosis groups. The percentage of apoptotic cell was greatly less in Akt cirrhosis group than in other cirrhosis groups. Akt group showed positive HA tag and an increased level of phosphorylated Akt as well as decreased levels of Fas. In contrast, Caspase-3 and Caspase-9 levels in Akt group were significantly lower than other cirrhosis groups. Noticeable decrease of DR5 and α-SMA and increase of phosphorylated eNOS were observed in the Akt group when compared to other cirrhosis groups. The NO level in liver was significantly higher in Akt group than other cirrhosis groups, which was consistent with the level of phosphorylated eNOS in these groups. CONCLUSION: This study suggest that Ad-myr-HA-Akt virus is a useful tool to prevent CCl4-induced cirrhosis in rat model and Akt pathway may be a therapeutic target for human cirrhosis.

[Comparison of endoscopic band ligation and propranolol for the primary prophylaxis of variceal bleeding in cirrhosis].

OBJECTIVE: To compare endoscopic variceal ligation (EVL) with propranolol for prophylaxis of first variceal bleeding. METHODS: We chose 168 patients with cirrhosis and esophageal varices in our hospital and allocated them to EVL and propranolol groups. Treatment effectiveness and safety in the 2 groups were observed. RESULTS: he parameters of two groups were similar before therapy. Follow-up period was 8-36 months. Variceal bleeding occurred in 24 (28.6%) of the EVL group and in 20 (23.9%) of the propranolol group (P>0.05). Overall mortality and death related to bleeding were similar (21.4% vs 17.9%; 7.1% vs 6.0%, P>0.05). Adverse events related to EVL were 43 (3 of them life-threatening) compared to 16 in the propranolol group (51.19% vs 19.05%, P<0.05). CONCLUSION: Propranolol may be the better choice in prophylaxis of variceal bleeding with similar effects and lower adverse events than with EVL.

High level of PTEN expression is associated with low-grade liver metastasis and satisfactory patient survival in pancreatic cancer.

BACKGROUND AND AIMS: We investigated PTEN expression in primary pancreatic cancer and pancreatic cancer liver metastasis in order to evaluate the interrelationship between PTEN expression and clinicopathological characteristics of pancreatic cancer patients with and without liver metastasis. METHODS: Eighty five primary pancreatic cancer specimens without liver metastasis were analyzed as controls. Eighty seven pancreatic cancer specimens and homologous liver metastasis specimens were investigated immunohistochemically, and the correlation between immunohistochemical findings and clinicopathological factors was investigated. RESULTS: A strong PTEN expression was observed in 52 (61.2%) specimens from patients without liver metastasis. In contrast, only 26 (29.9%) specimens were observed in patients with liver metastasis. A strong PTEN expression was apparently associated with low-grade lymph node metastasis (p <0.05) and TNM stage (p <0.05). PTEN expression in patients without liver metastasis was apparently stronger than that with liver metastasis. In addition, among patients with liver metastasis, the 5-year survival rate was markedly higher in patients with strong PTEN expression compared to those with weak PTEN expression. CONCLUSIONS: Our results suggest that a high level of PTEN expression is associated with low-grade liver metastasis and satisfactory patient survival in pancreatic cancer. The diagnostic evaluation of PTEN expression may provide valuable prognostic information to aid treatment strategies for pancreatic cancer patients.

[Laparoscope and endoscope for portal hypertension].

OBJECTIVE: To determine the therapeutic effect of laparoscopic splenectomy, perisoph-agogastric devascularization, and endoscopic variceal ligation (EVL) on patients with portal hypertension. METHODS: We randomly divided 105 patients into 3 groups: 40 had endoscopic band ligation (the ligation group), 35 had splenectomy and perisoph-agogastric devascularization (the laparotomy group), and the other 30 had laparoscopic splenectomy, perisoph-agogastric devascularization and endoscopic variceal ligation (the combination group). Blood samples were analyzed preoperatively and postoperatively on day 1,3,and 7,including alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),and directed bilirubin(DBIL). The length of stay, blood loss, operation time, anal exhaust time, azygos vein diameter, blood flow velocity and blood flow, recurrence of esophageal varices and rehaemorrhagia were compared. RESULTS: Between the combination group and the laparotomy group, the serum levels of TbIL and Dbil had difference on 1st postoperative day(P<0.05). AST had difference on 7th postoperative day(P<0.05). The length of stay, blood loss, operation time, and anal exhaust time had significant difference(P<0.05). Among the combination group, the laparotomy group and the ligation group, the azygos vein blood flow before and after the treatment, recurrence of esophageal varices and rehaemorrhagia had no difference(P<0.05). CONCLUSION: Laparoscopic splenectomy, perisoph-agogastric devascularization and endoscopic variceal ligation have less trauma, lower recurrence rate, fewer complications and rapid recovery, and may reduce the azygous vein blood flow. It can be used safely for portal hypertension.

Transversal inducing differentiation of human amniotic epithelial cells into hepatocyte-like cells.

OBJECTIVE: To evaluate the in vitro differentiation of human amniotic epithelial cells (hAECs ) into hepatocyte-like cells. METHODS: Combined approach of dexamethasone, HGF, IGF and other cytokines were used to induce the differentiation of hAECs into hepatocyte-like cells. The induction lasted 2 weeks. During the induction, the expression of albumin ALB, CYP1A1, CYP1A2, IGFR, c-met and key functional genes related to liver cells as well as transcription factors HNF3, HNF4 and C/EBPa were monitored by RT-PCR. Time dependent changes of the surface marker colony ALB, AFP and CK18 were analyzed by cell flow cytometry. RESULTS: After the 2 week induction, the expressions of liver hepatocyte-like cell functional genes such as albumin, CYP1A1, CYP1A2, c-met, and transcription factors such as HNF3, HNF4, C/EBPa and HNF1 were observed. Six days after the induction, hAECs mainly were stained AFP+, and the positive rate was (15.1 ± 2.1)%. While 10 days after the induction, part of the hAECs showed AFP+/ALB+ (6.5 ± 1.4)%; and on 14th day, hAECs only showed ALB+, and the rate was (13.9 ± 2.3)%. ALB+ cell increase indicated a gradual functional maturation from the hAECs to hepatocyte-like cells. Similaritly, the number of CK18+ cells in the whole population was also increased: On 10th day, the rate was (16.1 ± 1.2)%; on 14th day, that was (21.3 ± 4.6)%, which proved the above hypothesis of the trandifferentiation. By extending the induction time, the expression of functional genes increased gradually, and a maturing process of hAECs was detected by cell surface markers. CONCLUSION: The differentiation of hAECs induced in vitro has the characteristics of hepatocyte-like cells.

[Risk factors for anastomotic leakage after anterior resection for rectal cancer].

OBJECTIVE: To identify the risk factors associated with anastomotic leakage following an anterior resection for rectal cancer. METHODS: Between June, 1999 and June, 2009, 628 patients underwent anterior resection for rectal cancer. A retrospective study of the cases was performed to identify the risk factors for anastomotic leakage following the resection. RESULTS: The overall incidence rate of anatomic leak was 8.6% (54/628) in these patients. A low albumin level (less than 35 g/L), diabetes, absence of a protective stoma, a distance less than 7 cm from the tumor to the anal edge, and a tumor diameter over 5 cm were identified as the risk factors for anastomotic leakage after anterior resection. CONCLUSION: For patients at a high risk for anastomotic leakage, a protective stoma can significantly decrease the rate of clinical leaks and subsequent reoperation after low anterior resection for rectal cancer.

[Preliminary study on transdifferentiation of human amniotic epithelial cells and its intrasplenic transplantation].

OBJECTIVE: The human amniotic epithelial cells (hAECs) are a recently identified new type of stem cells. It has previously been shown that hAECs express hepatocyte-related gene and possess intracellular features and functional properties of hepatocytes. The hAECs may be a candidate seed cell for liver regeneration. To research the survival and migration in vivo of hAECs via adeno-associated virus-mediated the green fluorescent protein gene (AAV-GFP) transfection, and to explore the expression of hepatocyte-like function. METHODS: Thirty nude mice (aging 6-8 weeks, half males and females, and weighing 20-22 g) were randomly divided into 3 groups (groups A, B, and C, n=10). The mice of groups A and C were made the 2/3 partial hepatectomy model, and the mice of group B underwent open abdominal operation without hepatectomy. The hAECs transfected by AAV-GFP were transplanted into the inferior end of the spleen in groups A and B with a cell density of 5 x 10(6)/mL and a volume of 0.2 mL; the same volume of normal saline was injected in group C. At 4 hours, the nude mice were sacrificed and the samples of liver, spleen, heart, lung, brain, and kidney were harvested and the general observation, histological observation, and immunofluorescence detection were performed for the hAECs survival, migration, and the functional properties of hepatocytes. RESULTS: No tumor tissue was found in liver and spleen of 3 groups, and HE staining showed no tumor cells. There were a lot of roundlike and deeply-stained cells with less cytoplasm and large nucleus in the spleen and the liver of group A; no abnormal cells were found in liver and spleen of groups B and C and in kidney, heart, bung, and brain of groups A, B, and C. The GFP+ cells were detected in the spleen and liver of group A with expressing human albumin, but no GFP+ cells was found in liver and spleen of groups B and C and in heart, kidney, lung, and brain of groups A, B, and C. CONCLUSION: AAV-GFPA infected hAECs transplanted into SCID nude mice with hepatectomy can keep the hepatocyte-like function. It will be beneficial to further identify their biological characteristics.

[Therapeutic effect of subtotal gastrectomy and Roux-en-Y gastrojejunostomy on type 2 diabetes mellitus in non-obese patients].

OBJECTIVE: To evaluate the therapeutic effect of Roux-en-Y anastomosis following subtotal gastrectomy on type 2 diabetes mellitus (T2DM) in non-obese patients. METHODS: We performed a retrospective analysis of 16 non-obese patients with T2DM undergoing Roux-en-Y anastomosis following subtotal gastrectomy for stomach cancer and upper gastrointestinal tract ulcer. RESULTS: All the patients were followed up for 6 months after the surgery. Roux-en-Y gastrojejunostomy significantly lowered the levels of fasting plasma glucose (FPG), 2 h postprandial plasma glucose (2hPG), and glycated hemoglobin (HbA1c)(P<0.05). Of these patients, 8 (50%) achieved adequate glycemic control without antidiabetic medication and 5 (31.25%) showed obvious improvement. The total effectiveness rate of the surgery was 81.25%. CONCLUSION: Roux-en-Y gastrectomy can effectively ameliorate the diabetic symptoms and might serve as a new treatment option for T2DM in non-obese patients.

Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma.

Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23) of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.

[Treatment for chronic venous ulceration of the leg by subfascial endoscopic perforator vein surgery].

OBJECTIVE: To observe the effect of subfascial endoscopic perforator vein surgery (SEPS) in the treatment of chronic venous ulceration of the legs. METHODS: Chronic venous ulceration for 91 patients with 102 limbs was treated by SEPS from January 2005 to July 2008. The effect of SEPS on chronic venous ulceration of the leg, the symptoms during and after the operation, and the durations of hospital treatment were analyzed. RESULTS: The symptoms of the 102 legs conducted by SEPS operation, except the pigmentation, were obviously improved (P<0.01). Its cicatrisation rate, the recrudesce rate, and the cut infection rate were 93.1%, 1.96%, and 1.0%, respectively. The time of the operation was short and the hemorrhage was small during the surgery. The durations of hospitalization and the cicatrisation time of ulceration were (6.5+/-3.4) d and (12.2+/-13.7) d, respectively. CONCLUSION: SEPS is simple and effective in treating chronic venous ulceration of the leg, and particularly effective for patients classified into C5 and C6 in clinical-etiological-anatomical-pathophysiology (CEAP).

[Akt gene therapy for cirrhotic rats with portal hypertension].

OBJECTIVE: To determine whether there is an impaired Akt and eNOS activation in cirrhotic livers, and to investigate the feasibility of transferring adenovirus-mediated Akt gene to the liver for portal hypertension. METHODS: Recombinant adenovirus Ad-myr-HA-Akt and Ad-EGFP were produced by homologoas recombination in 293 cells . The Methods of compound factor, carbon tetrachloride (CCl4), corn flour, and cholesterol plus alcohol were used to construct the hepatic cirrhosis rat models. Ten normal rats were served as a normal control group, and 40 cirrhotic rats were divided into 4 groups randomly: an untreated group, an Ad-myr-HA-Akt treated group, an Ad-EGFP group, and a saline group. Ad-myr-HA-Akt, Ad-EGFP, and saline were transduced into the Ad-myr-HA-Akt treated group, Ad-EGFP group, and saline group via the tail vein respectively. Portal vein pressure, mean arterial pressure, and heart rate were measured in all rats. Protein abundance and phosphorylation status of Akt and eNOS were examined by Western blot. Spectrophotometry was used to measure the NO level. Frozen sections of the liver, heart, lung, kidney, brain, spleen, and testis were made to examine the expression of enhanced green fluorescent protein (EGFP) by fluorescence microscopy on Day 3 in the Ad-EGFP group. RESULTS: The concentration of recombinant adenovirus Ad-myr-HA-Akt after the purification was 5.5 x 10(11)vp/mL and that of Ad-EGFP was 6.0 x 10(11)vp/mL. Akt and eNOS phosphorylations in the liver of cirrhotic rats were obviously impaired. Adenoviral delivery of myr-Akt restored eNOS phosphorylation, increased the NO level and decreased the portal pressure after 3 days of adenoviral infection. In contrast, the livers infected with Ad-EGFP and saline were not changed. The EGFP expression was mainly found under the fluorescence microscopy on the frozen section of liver. Very little fluorescence was detected in the lung and kidney; and there was no detectable EGFP in other organs. CONCLUSION: There is an impaired Akt and eNOS activation in the cirrhotic livers; myr-Akt gene therapy can restore the Akt activation and NO production in the cirrhotic liver, suggesting that this therapy may be helpful in treating portal hypertension.

[Comparison of treatment response in primary hepatocellular carcinomas with contrast-enhanced ultrasonography and contrast-enhanced helical CT].

OBJECTIVE: To investigate the value of contrast-enhanced ultra sonography for non-surgical treatment response in hepatocellular carcinomas. METHODS: Non-surgical therapies were performed on 56 patients (64 liver neoplasms) who were diagnosed by ultrasonography-guided biopsy before the therapy. Contrast-enhanced ultrasonography(CEUS) and contrast-enhanced helical CT were performed to assess the treatment response. RESULTS: Forty-six of the 64 lesions were not enhanced with CEUS.Partial enhancement was demonstrated in the other 18 lesions. Forty-eight of the 64 lesions were not enhanced with contrast-enhanced helical CT. Partial enhancement were demonstrated in the other 16 lesions.The sensitivity, specificity, and accuracy were 94.4%, 97.8%, and 96.9% for CEUS and 83.3%, 97.8%, and 93.8% for contrast-enhanced helical CT (P>0.05). CONCLUSION: CEUS is a good method in assessing the non-surgical treatment response in hepatocellular carcinomas and is more sensitive and useful than contrast-enhanced helical CT in assessing the treatment response of transcatheter hepatic arterial chemoembolization.

Endoscopic variceal ligation combined with Hassab's procedure in preventing the recurrence of esophageal varices.

OBJECTIVE: To assess the efficacy of endoscopic variceal ligation (EVL) combined with Hassab's procedure in the prevention of variceal recurrence. METHODS: One hundred and thirty-five patients with esophageal varices were randomized to receive EVL alone, Hassab's procedure alone or a combination of EVL and Hassab's procedure for variceal eradication. Ultrasonographic venous network images were recorded by an esophageal microprobe before and after the EVL or Hassab's procedure. The clinical outcome and vascular network images of the 3 groups were analyzed. RESULTS: Esophageal varices were obliterated immediately after EVL alone, while both perforating veins and periesophageal collaterals remained patent, and 83% had recurrence of esophageal varices during an initial 3-year follow-up. Esophageal varices were reduced in size, periesophageal collaterals were obliterated after Hassab's procedure alone, and 30% experienced rebleeding and 95% with variceal recurrence. EVL combined with Hassab's procedure obliterated all esophageal varices, perforating veins and periesophageal collaterals, and only 3 patients (8%) recurred. CONCLUSION: The existence of patent perforating veins and periesophageal collaterals is the reason of esophageal variceal recurrence after EVL alone. EVL combined with Hassab's procedure can effectively prevent the recurrence, even if the cirrhojtic portal hypertension persisted.