Labeling and tracking human amniotic epithelial cells with green fluorescent protein in an adeno-associated virus vector.

Human amniotic epithelial cells (hAECs) are a recently identified type of stem cell. Thanks to their ready availability and the lower risk of teratoma formation, hAECs have been studied and tested for a variety of human disease treatments and tissue reconstruction efforts. This aim of this study was to establish a stable tracking system to further monitor hAECs in vivo after transplantation. hAECs were isolated from the placentas of patients who visited the Hunan Province Maternity and Child Care Hospitals between Jan 2008 and Jan 2009. Using the classic transfection/infection technique, we successfully introduced green fluorescent protein (GFP) into cultured hAECs with an adeno-associated virus (AAV) vector. The initial preparation of the AAV-GFP virus stock was titrated using HT1081 cells, and further used for the infection of hAECs. GFP(+) hAECs preserve the capacity of differentiation into hepatocyte-like cells with the expression of cytokeratin-18 (CK18) and albumin (ALB). AAV-GFP virus-infected hAECs were transplanted through the spleen into severe combined immune deficiency (SCID) mice via hepatectomy. Four weeks later, the GFP and human albumin expressions were examined in multiple organs through immunofluorescence staining. In culture, over 50% of the hAECs were GFP-positive 3 days after infection. Following transplantation, AAV-GFP-infected hAECs survived and continued to express GFP in the host for up to 4 weeks. These cells were primarily found in the spleen and liver, expressing human albumin. This study provides a feasible and stable system to track hAECs. It may prove useful to further identify their biological characteristics after transplantation and to elucidate their beneficial roles for therapeutic purposes.

[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.

[Effect of losartan on portal hypertensive gastropathy in rats].

OBJECTIVE: To determine the effect of angiotensin II receptor 1 antagonist losartan on portal hypertensive gastropathy (PHG) in rats and its mechanism. METHODS: Forty-eight Sprague-Dawley rats were randomly divided into 4 groups: a sham-operated group, a model group, a treatment group, and a prevention group. The partial portal vein and left suprarenal vein of rats were ligated to develop PHG. Portal vein pressure (PVP), the level of angiotensin IIin blood, gastric injury index(GI), and pathological diagnosis integral(PI) were measured. In situ hybridization was used to determine the expression and immunolocalization of angiotensin II receptor 1 in rat stomach wall. RESULTS: PVP, GI, and PI of the treatment group and the prevention group were evidently reduced (P<0.01), and the level of angiotensin IIin blood increased obviously. The expression of angiotensin II receptor 1 was negative in the control group, increased significantly in the model group, and decreased significantly in the treatment group and the prevention group. CONCLUSION: The expression of angiotensin II receptor 1 elevates in portal hypertensive gastropathy. Losartan can reduce PVP, inhibit the activation of angiotensin II receptor 1 in gastric submucous layer, and has therapeutic effect on PHG.

[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.

Glutathione-s-transferase A 4 (GSTA4) suppresses tumor growth and metastasis of human hepatocellular carcinoma by targeting AKT pathway.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies of cancers and its prognosis remains dismal due to the paucity of effective therapeutic targets. Up-regulation of glutathione-s-transferase A 4 (GSTA4) is associated with poor prognosis of HCC, but its functional mechanism in HCC remains unclear. In this study, we investigated the roles of GSTA4 in tumor growth and metastasis of HCC and found that GSTA4 was frequently up-regulated in HCC tissues. Through gain- and loss-of-function studies, GSTA4 was demonstrated to significantly regulate cell proliferation, migration, and invasion in vitro. Furthermore, GSTA4 overexpressing significantly promoted the tumorigenicity and metastasis of HCC cells in nude mice models bearing human HCC, whereas silencing endogenous GSTA4 caused an opposite outcome. Moreover, we demonstrated that GSTA4 enhanced HCC aggressiveness by activating protein kinase B (AKT) signaling. In multivariate analysis, our results GSTA4 overexpression promotes the progression of hepatocellular carcinoma and might represent a novel therapeutic target for its treatment.

[Effects of different treatment complex on esophageal vascular structures in patients with portal hypertension].

OBJECTIVE: To assess the effects of different treatment complex on esophageal vascular structures in patients with portal hypertension. METHODS: Patients (142 cases) with esophageal varices received either endoscopic variceal ligation (EVL) alone (54 cases), pericardial devascularization procedure (PDP) alone (23 cases), a combination of EVL and partial splenic embolization (PSE) (34 cases), or a combination of EVL and PDP (31 cases) for variceal eradication. Esophageal vascular structures were examined with miniature ultrasonic probe. The recurrence and rebleeding of esophageal varices were investigated. RESULTS: Esophageal submucous varices were obliterated and collateral veins remained unchanged in patients treated by EVL or EVL combined with PSE; esophageal submucous varices were diminished in size and collateral veins were obliterated by PDP, and both esophageal submucous varices and collateral veins were obliterated by the combination of EVL and PDP. CONCLUSIONS: The combination of EVL and Hassab's procedure can effectively shut off the portoazygous shunt, prevent esophageal varices from bleeding and recurrence. It's a simply and less cost procedure.

Expression of TNF-alpha and VEGF in the esophagus of portal hypertensive rats.

AIM: To investigate the expression of tumor necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor (VEGF) in the development of esophageal varices in portal hypertensive rats. METHODS: Thirty male Sprague-Dawley (SD) rats in the model group in which a two-stage ligation of portal vein plus ligation of the left adrenal vein was performed, were divided into three subgroups (M(7), M(14), and M(21)) in which the rats were kiued on the seventh day, the 14(th) d and the 21 d after the complete portal ligation. Thirty male SD rats, which underwent the sham operation in the control group, were also separated into three subgroups (C(7), C(14) and C(21)) corresponding to the models. The expression of TNF-alpha and VEGF in the esophagus of all the six subgroups of rats were measured with immunohistochemical SP technique. RESULTS: The portal pressure in the three model subgroups was significantly higher than that in the corresponding control subgroups (23.82+/-1.83 vs 11.61+/-0.86 cmH(2)O, 20.90+/-3.27 vs 11.43+/-1.55 cmH(2)O and 20.68+/-2.27 vs 11.87+/-0.79 cmH(2)O respectively, P<0.01), as well as the number (9.3+/-1.6 vs 5.1+/-0.8, 11.1+/-0.8 vs 5.4+/-1.3 and 11.7+/-1.5 vs 5.2+/-1.1 respectively, P<0.01) and the total vascular area (78 972.6+/-3 527.8 vs 12 993.5+/-4 994.8 mum(2), 107 207.5+/-4 6461.4 vs 11 862.6+/-5 423.2 mum(2) and 110 241.4+/-49 262.2 vs 11 973.7+/-3 968.5 mum(2) respectively, P<0.01) of submucosal veins in esophagus. Compared to the corresponding controls, the expression of TNF-alpha and VEGF in M(21) was significantly higher (2.23+/-0.30 vs 1.13+/-0.28 and 1.65+/-0.38 vs 0.56+/-0.30 for TNF-alpha and VEGF respectively, P<0.01), whereas there was no difference in M(7) (1.14+/-0.38 vs 1.06+/-0.27 and 0.67+/-0.35 vs 0.50+/-0.24 for TNF-alpha and VEGF respectively, P>0.05) and M(14) (1.20+/-0.25 vs 1.04+/-0.26 and 0.65+/-0.18 vs 0.53+/-0.25 for TNF-alpha and VEGF respectively, P>0.05). And the expression of TNF-alpha and VEGF in M(21) was significantly higher than that in M(7) (2.23+/-0.30 vs 1.14+/-0.38 and 1.65+/-0.38 vs 0.67+/-0.35 for TNF-alpha and VEGF respectively, P<0.01) and M(14) (2.23+/-0.30 vs 1.20+/-0.25 and 1.65+/-0.38 vs 0.65+/-0.18 for TNF-alpha and VEGF respectively, P<0.01), but there was no difference between M(7) and M(14) (1.14+/-0.38 vs 1.20+/-0.25 and 0.67+/-0.35 vs 0.65+/-0.18 for TNF-alpha and VEGF respectively, P>0.05). CONCLUSION: In the development of esophageal varices in portal hypertensive rats, increased TNF-alpha and VEGF may be not an early event, and probably play a role in weakening the esophageal wall and the rupture of esophageal varices.

[Dynamic variation of endothelin in portal hypertensive patients with hemorrhage and its clinical significance].

OBJECTIVE: To investigate variations of plasma endothelin (ET) and its clinical significance in portal hypertensive patients with esophageal variceal hemorrhage. METHODS: Sixty-six patients with portal hypertension were randomly divided into 2 groups. Group I (32 patients) received general therapy and Group II (34 patients) received general therapy and UTI after hemorrhage. The plasma ET concentration and liver function were determined at 1, 2, 4, 7, 10, and 14 d after the hemorrhage. Another 20 patients without the hemorrhage were elected as the control group. RESULTS: At 7 and 14 d after the hemorrhage, the levels of TBIL, ALT and AST were elevated at first and then decreased in Groups I and II. The decrease of TBIL, ALT and AST levels was significantly faster in Group II than in Group I (P < 0.05, P < 0.01, P < 0.05, respectively) on 14 d after the hemorrhage. At 1 d after the hemorrhage the ET concentration was markedly increased in Group I and II as compared with the control group (P < 0.01). Then it was gradually decreased on 10 d after the hemorrhage. The ET concentration in Group II was decreased more rapidly than that in Group I on 2, 4 and 7 d after the hemorrhage (P < 0.05; P < 0.01; P < 0.05, respectively). The ET concentration was positively correlated to TBIL levels in groups I and II (r = 0.734, P < 0.01). And the decreased index of ET concentration was negatively correlated to the increased index of TBIL (r = -0.486, P < 0.05). CONCLUSION: The increased plasma ET in portal hypertensive patients with hemorrhage may contribute to liver injury. UTI can protect the liver function by inhibiting ALT, AST, TBIL and ET level.

[Treatment of portal hypertension by endoscopic esophageal ligation of varicose veins combined with partial splenic embolization].

OBJECTIVE: To evaluate the feasibility and efficacy of a new method of endoscopic esophageal variceal ligation combined with partial splenic embolization (EVL-PSE) for the patients with portal hypertension. METHODS: From May 1999 to February 2003, sixty-eight patients with portal hypertension underwent EVL-PSE, and hemodynamics of the portal trunk (PT), the left gastric vein and azygos vein, including maximum velocity, flow volume, vein diameter, were assessed using color ultrasound Doppler. RESULTS: The esophageal varices and hypersplenism were greatly ameliorated after operation in patients who had undergone EVL-PSE. Postoperative portal trunk flow volume and velocity were significantly reduced (P < 0.05), and flow volume of the left gastric vein as well as the azygos vein were also reduced after operation. During 2 - 24-month follow-up, no recurrent bleeding was found. CONCLUSIONS: EVL-PSE is less traumatic with less complications, and results in marked eradication of esophageal varices, it can be carried out safely in the clinical treatment for patients with portal hypertension.

[Laser inducing mucosal fibrosis for preventing recurrence of esophageal varices].

OBJECTIVE: To investigate the prevention of esophageal varices recurrence by laser inducing esophageal mucosal fibrosis. METHODS: Our study included 42 patients after esophageal varices eradicated by endoscopic varices ligation, and they were divided into 2 groups randomly, each group included 21 patients. One group was assigned to received laser treatment, and indocyanine green solution (1 mg/ml) was injected submucosally, a diode laser (power 10 watts) was applied to the surface from the esophagogastric junction to 5 cm above it. Another group was controlling without any treatments. All patient were followed up by endoscopy every 3 months until 12 months. RESULTS: Laser irradiation was performed safely without any major complications. And lower esophageal mucosa produced fibrosis widely after laser irradiated 1 month. After 12 months follow up, the cumulative recurrence rate was significantly lower than the control group, 14% (3/21) vs 43% (9/21) (chi(2) = 4.20, P < 0.05). CONCLUSIONS: Our study indicates that laser inducing mucous fibrosis is safely and can prevent recurrence of esophageal varices.

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.

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.

[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.

[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.

[The expression of HIF-1alpha in scars and its relationship to apoptosis].

OBJECTIVE: To explore the mechanism of hypoxic environment lessening in the course of scar maturation. METHODS: The expression of hypoxia inducible factor 1alpha (HIF-1alpha) and p53 in granulation of burn wound, burn scars of different stages, and normal skin was detected with immunohistochemical staining. The expressions were quantified by the weight method. The relationship between HIF-1alpha and p53 was investigated. RESULTS: With the scar maturation, the expression of HIF-1alpha gradually decreased while the expression of p53 gradually increased within one year. There was a negative correlation between HIF-1alpha and p53 (P < 0.01). CONCLUSION: p53 plays an important role in scar maturation. Associated with p53, HIF-1alpha may induce apoptosis, which decreases oxygen consumption and lightens hypoxia in the scar maturation.