[Laparoscopic-assisted modified Soave procedures for adult Hirschsprung disease].

OBJECTIVE: To evaluate the clinical value of laparoscopy-assisted modified Soave procedure for Hirschsprung disease in adults. METHODS: Twenty-eight patients with a preoperative diagnosis of Hirschsprung disease underwent laparoscopy-assisted modified Soave procedure between March 2005 and December 2009. Clinical data were retrospectively analyzed. RESULTS: There were no conversions to open surgery. The mean operative time was (165±12) minutes (range: 135-185 minutes). Estimated blood loss ranged from 50 to 250 ml, and no patients required intraoperative blood transfusion. Postoperative pathologic examination showed Hirschsprung diseases in 19 patients and Hirschsprung allied diseases in 9. Only two patients developed rectal cuff infection and three mild seepage. Other patients had no postoperative complications. The mean hospital stay was (17.5±1.0) days. No fecal incontinence or recurrent constipation occurred during follow-up. CONCLUSION: Laparoscopy- assisted modified Soave procedure is safe and effective for Hirschsprung disease.

[Effects of magnetic gemcitabine stealth nano-liposomes on the characteristics of breast cancer cell line MCF-7].

The magnetic responsibility and antitumor effect of magnetic gemcitabine stealth nano-liposomes (MGSL) on breast cancer cell line MCF-7 in vitro and in vivo was evaluated. The magnetic response and targeting effect of MGSL in vivo were investigated. Morphological feature and ultrastructure changes of apoptosis of MCF-7 cells were observed. The effect of MGSL on proliferation inhibitory rate of MCF-7 cells was measured with MTT method. The FCM analysis was carried out to examine the cell cycle distribution and cell apoptotic rate. The antitumor effect on human breast cancer xenografts in nude mice was also studied. MGSL was able to converge at the targeting tissue under tridimensional magnetic field and the gemcitabine concentration around it increased, while the amount of gemcitabine in other organs decreased, such as in kidneys and heart. MCF-7 cell line was sensitive to MGSL and the cytotoxity was correlated with the loaded drug dose. The effect of MGSL on apoptosis of MCF-7 was obvious and the rate of apoptosis was 51.62%. The growth speed of tumor in the group of MGSL (+) significantly slowed down than that of other groups. MGSL prepared by reverse-phase evaporation method met with the demand of targeted delivery system, and it might be an effective antitumor agent.

[Efficacy and safety of adjuvant post-surgical therapy with imatinib in gastrointestinal stromal tumor patients with high risk of recurrence: interim analysis from a multicenter prospective clinical trial].

OBJECTIVE: To evaluate the efficacy and safety of postoperative adjuvant chemotherapy with imatinib in gastrointestinal stromal tumor(GIST) patients who had high risk of recurrence. METHODS: A prospective, open-label, multi-center trial conducted in sixteen teaching hospitals in China was carried out. The criteria of the enrolled patients included age more than 18 years old, CD117 positive GIST, tumor size more than 5 cm, pathological mitosis counts more than 5/50 HPF, and treatment beginning within 4 weeks after complete resection and with imatinib (400 mg, once a day) for at least 12 months. The 1, 3 year recurrence rates, disease free survival, overall survival rate and quality of life were evaluated. RESULTS: From Aug. 16th 2004 to Sep. 13th 2005, there were totally 74 patients screened and 57 patients (34 men, 23 women) enrolled in the imatinib treatment group. The primary tumors were located in the stomach in 50.9%, the small intestine in 38.6% and the colorectum in 10.5% of the cases. All the patients received radical resection. Until the cut-off date of interim analysis, there was no evidence of tumor relapse or metastasis in all patients and no death was reported either. Among the 57 enrolled patients with intention to treat(ITT), twelve patients finished the protocol (per protocol, PP). The disease free survival was (268.3 +/-120.2) d in ITT analysis, and (396.7+/-38.2) d in the PP analysis. The incidence of adverse effect was 44.4% . The score in quality of life showed no statistically significant difference between the baseline visit and the follow-up visits. CONCLUSION: Imatinib is a promising postoperative adjuvant chemotherapy in GISTs patients with high risk of recurrence, and the adverse effects are receivable.

[Double sites short hairpin RNAs targeting epidermal growth factor receptor to promote colon cancer cells apoptosis and enhance 5-fluorouracil chemotherapy effect].

OBJECTIVE: To investigate the differences about RNA interference (RNAi) technique which focuses on single or multiple sites to suppress colon cancer LoVo cell line's epidermal growth factor receptor (EGFR) mRNA and protein expression, induce cell apoptosis and enhance 5-fluorouracil (5-FU) sensitivity. METHODS: The human colon cancer LoVo cells were transfected by liposome with pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 expressive vectors which were established by p Genesil-1 plasmid and EGFR short hairpin RNA (shRNA) synthesized in vitro, then were selected for 4 weeks by using G418. Five groups were selected for the study: Group 1: the normal cultured LoVo cells; Group 2: the negative control plasmid HK; Group 3: pU6-EGFR-shRNA-1 plasmid vector; Group 4: pU6-EGFR-shRNA-2 plasmid vector; Group 5: pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2, half for each. The mRNA and protein expression were assessed using Real Time PCR and Western blot, the cell apoptosis was determined via flow cytometry, and the suppressive rate and IC(50) to LoVo cells by 5-FU of different concentrations and time points were carried out by using Cell Counting Kit-8 (CCK-8). RESULTS: Expression plasmids encoding shRNA were successfully established and transfected into the LoVo cells. In group 3, 4 and 5, the mRNA expression was decreased by (80.2 +/- 3.4)%, (81.3 +/- 2.8)% and (90.6 +/- 2.8)%, respectively, and protein expression was decreased by (74.1 +/- 4.0)%, (73.4 +/- 2.3)% and (90.4 +/- 3.3)%, respectively; meanwhile, cell apoptosis increased by (10.4 +/- 0.5)%, (10.1 +/- 0.4)% and (14.2 +/- 0.5)%, respectively. The IC(50) of 5-FU and cell suppressive rate analysis demonstrated that there were significant differences among group 5, groups 3 and 4, and groups 1 and 2, but there were no significant difference between group 1 and group 2, as well as group 3 and group 4. CONCLUSIONS: Both pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 were capable of suppressing EGFR expression of LoVo cells, and therefore promoting apoptosis and increasing the cell toxicity of 5-FU. The targeting double combined sites RNAi technique was significantly better than single site interference. The new therapeutic modalities in the treatment of human colon cancer are suggested by this study.

[Antisense RNA targeting survivin enhances the chemosensitivity of LOVO/Adr cells to taxotere].

OBJECTIVE: To investigate the ability of antisense RNA eukaryotic expression plasmid pcDNA3.0/survivin targeting survivin gene to inhibit survivin expression and enhance the sensitivity to taxotere in multidrug resistant colon carcinoma cell line LOVO/Adr. METHODS: The antisense RNA eukaryotic plasmid pcDNA3.0/survivin was transfected into LOVO/Adr cells by lipofectamine. The expression of survivin mRNA was measured using RT-PCR. After treated with taxotere, MTT assay and flow cytometry were used to evaluate the proliferation inhibition and apoptosis of LOVO/Adr cells. RESULTS: The expression of survivin mRNA in LOVO/Adr cells transfected with pcDNA3.0/survivin was down-regulated in a time- dependent manner. The inhibitory rate of taxotere (0.5 micromol/L) was (37.3 +/- 2.9)% in pcDNA3.0/survivin transfected cells, significantly higher than (21.9 +/- 2.3)% and (21.1 +/- 1.9)% in pcDNA3.0 transfected and untransfected control cells respectively (P< 0.01). The apoptosis rate of taxotere was (28.7 +/- 1.7)% in pcDNA3.0/survivin transfected cells,significantly higher than (13.4 +/- 1.6)% and (14.3 +/- 1.8)% in pcDNA3.0 transfected and untransfected cells respectively. CONCLUSION: The antisense RNA eukaryotic expression plasmid pcDNA3.0/survivin could down-regulate the expression of survivin gene and enhance the chemosensitivity of LOVO/Adr cells to taxotere, which may provide a novel therapy for colon carcinoma.

[Surgical treatment of massive rebleeding after gastrectomy for bleeding gastroduodenal ulcer].

OBJECTIVE: To summarize the reoperation experiences in treatment of massive rebleeding after subtotal gastrectomy for bleeding gastroduodenal ulcer. METHODS: From 1980 to 2002, clinical data of 26 cases with massive rebleeding after subtotal gastrectomy for bleeding gastrorenal ulcer were analyzed retrospectively. RESULTS: Preoperative gastroscopy was performed in 6 cases, intraoperative gastroscopy in 11, and preoperative superselective angiography in 2 cases. Eleven cases with left ulcer or post- bulb ulcer bleeding underwent resection of the left ulcer or longitudinal incision of the duodenal descending part and direct hemostasis. Thirteen cases with anastomotic stoma bleeding underwent local suture hemostasis or resection of the stoma plus Billroth II or Roux- en- Y gastrojejunostomy. Two cases with gastric bleeding received reexcision of the stomach remnant. Twenty- four cases (92.3% ) were cured and 2 cases (7.7% ) died of gastric bleeding. CONCLUSION: Preoperative superselective angiography and intraoperative gastroscopy are beneficial to clarify the bleeding position and causes for massive rebleeding after gastrectomy. It is very important to select proper operative method to prevent postoperative rebleeding.

Nitrite-derived nitric oxide by xanthine oxidoreductase protects the liver against ischemia-reperfusion injury.

BACKGROUND: It was demonstrated that xanthine oxidoreductase (XOR), during ischemia, catalyzes the formation of nitric oxide (NO) from nitrite (NO2-) and this NO2- -derived NO protects the isolated perfused rat heart against the damaging effects of ischemia-reperfusion (I/R) when conventional nitric oxide synthase (NOS)-dependent NO production is impaired. Liver is one of the organs with the highest XOR concentration. This study was designed to determine whether NO2- -derived NO by XOR protects liver against I/R injury in vivo. For its minute amounts and active reactivity, NO can not be detected directly in real time in vivo by this time. We have to prove the above hypothesis indirectly. METHODS: Wistar rats were pretreated with saline, NOS inhibitor L-NAME (10 mg/kg intravenously), XOR inhibitor allopurinol (1.5 mg/kg orally), L-NAME +allopurinol and NO scavenger carboxy-PTIO (0.6 mg/kg intravenously) respectively (12 animals per group). And then, they were subjected to total liver ischemia for 40 minutes followed by reperfusion. Blood samples and liver tissues were obtained for analysis after 3 hours of reperfusion. Survival was also investigated. RESULTS: Allopurinol-treated animals exhibited further increased serum alanine aminotransferase(ALT) levels and liver myeloperoxidase(MPO) activities, but further decreased liver adenosine triphosphate(ATP) stores after I/R compared to saline-treated counterparts (830.5+/-108.3 U/L, 56.5+/-11.0 U/mg protein and 1.93+/-0.47 mumol/g vs. 505.8+/-184.2 U/L, 41.5+/-10.2 U/mg protein and 3.05+/-0.55 micromol/g respectively, P < 0.01, P < 0.05 and P < 0.01 respectively). The hepatocyte injury was further exacerbated and the overall survival rate was significantly decreased after I/R in animals given by allopurinol compared to those pretreated by saline (P < 0.05). L-NAME and allopurinol co-treated animals exhibited more severe liver injury (P < 0.05 and P<0.01)and a further decreased overall survival rate (P < 0.05)compared to L-NAME or allopurinol alone-treated counterparts, but they were not different from carboxy-PTIO treated animals (P > 0.05). CONCLUSION: NO2- -derived NO by XOR in the hypoxic and acidic environment induced by hepatic I/R protects the liver against I/R injury in vivo.

Gender differences in hepatic ischemic reperfusion injury in rats are associated with endothelial cell nitric oxide synthase-derived nitric oxide.

AIM: This study was designed to examine the hypothesis that gender differences in I/R injury are associated with endothelial cell nitric oxide synthase (eNOS)-derived nitric oxide (NO). METHODS: Wistar rats were randomized into seven experimental groups (12 animals per group). Except for the sham operated groups, all rats were subjected to total liver ischemia for 40 min followed by reperfusion. All experimental groups received different treatments 45 min before the laparotomy. For each group, half of the animals (six) were used to investigate the survival; blood samples and liver tissues were obtained in the remaining six animals after 3 h of reperfusion to assess serum NO, alanine aminotransferase (ALT) and TNF-alpha levels, liver tissue malondialdehyde (MDA) content, and severity of hepatic I/R injury. RESULTS: Basal serum NO levels in female sham operated (FS) group were nearly 1.5-fold of male sham operated (MS) group (66.7+/-11.0 micromol/L vs 45.3+/-10.1 micromol/L, P<0.01). Although serum NO levels decreased significantly after hepatic I/R (P<0.01, vs sham operated groups), they were still significantly higher in female rat (F) group than in male rat (M) group (47.8+/-8.6 micromol/L vs 23.8+/-4.7 micromol/L, P<0.01). Serum ALT and TNF-alpha levels, and liver tissue MDA content were significantly lower in F group than in M group (370.5+/-46.4 U/L, 0.99+/-0.11 microg/L and 0.57+/-0.10 micromol/g vs 668.7+/-78.7 U/L, 1.71+/-0.18 microg/L and 0.86+/-0.11 micromol/g, respectively, P<0.01). I/R induced significant injury to the liver both in M and F groups (P<0.01 vs sham operated groups). But the degree of hepatocyte injury was significantly milder in F group than in M group (P<0.05 and P<0.01). The median survival time was six days in F group and one day in M group. The overall survival rate was significantly higher in F group than in M group (P<0.05). When compared with male rats pretreated with saline (M group), pretreatment of male rats with 17-beta-estradiol (E2) (M+E2 group) significantly increased serum NO levels and significantly decreased serum ALT and TNF-alpha levels, and liver tissue MDA content after I/R (P<0.01). The degree of hepatocyte injury was significantly decreased and the overall survival rate was significantly improved in M+E2 group than in M group (P<0.01 and P<0.05). The NOS inhibitor N(w) -nitro-L-arginine methyl ester (L-NAME) treatment could completely abolish the protective effects of estrogen in both male and female rats. CONCLUSION: The protective effects afforded to female rats subjected to hepatic I/R are associated with eNOS-derived NO.

[Construction of PTEN eukaryotic expression plasmid and its effects on breast carcinoma cell line MDA468].

OBJECTIVE: To investigate the effects of exogenous wild PTEN gene stably transfection on growth of breast cancer cells in vitro. METHODS: At first, a recombinant eukaryotic expression plasmid pcDNA3.1-PTEN was constructed. Human breast cancer cell line MDA468 was transfected with pcDNA3.1-PTEN or mock transfected plasmid pcDNA3.1(-) with lipofectamine. RT-PCR, immunohistochemical staining and Western blot were used to determine target gene expression. Cell viability was tested by MTT assay. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. RESULTS: The PTEN stably transfected cells demonstrated the integration of the exogenous target gene and corresponding mRNA and protein over-expression. There was a significant decline in cell viability of pcDNA3.1-PTEN transfected MDA468 cells in comparison with the mock-transfected ones (P < 0.01). The PTEN-trasfected MDA468 cells also showed an increase in the rate of apoptosis, compared with parental and mock-trasfected cells (P < 0.001). CONCLUSION: Stable expression of exogenous PTEN can suppress the malignant phenotypes of the human breast cancer cell line MDA468.

[Exogenous PTEN gene induces apoptosis in breast cancer cells].

OBJECTIVE: To investigate the effects of exogenous PTEN gene on apoptosis of breast cancer cells. METHODS: Human breast cancer cells of the line MDA468 were cultured. Recombinant plasmid pcDNA3.1-PTEN was constructed and transfected into the breast cancer cells with the lipofectAMINE 2000 transfection technique. Parental MDA468 cells and parental MDA468 cells transfected with blank vector pcDNA3.1(-) were used as control groups. RT-PCR was used to detect the expression of the PTEN mRNA and Western blotting was used to determine the expression of PTEN protein. Epithelial growth factor (EGF) was added into the cultures of MDA468 cells transfected with pcDNA3.1-PTEN or blank vector. Then Western blotting was used to detect the expression of phosphospecific protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) protein stimulated by EGF. The aapoptosis of the MDA468 cells was determined by flow cytometry with the double-staining method using FITC-conjugated annexin V and PI. RESULTS: Expressions of PTEN mRNA and protein were shown in the MDA468 cells transfected with pcDNA3.1-PTEN by RT-PCR and Western blotting. Both the expression of p-AKT and that of p-FAK were down-regulated in the MDA468 cells transfected with pcDNA3.1-PTEN in comparison with those in the control cells. The apoptotic rate of the MDA468 cells transfected with PCDNA3.1-PTEN was 21.68%, significantly higher than those of the blank control cells (1.17%) and pcDNA3.1(-)-transfected cells (3.55%, both P < 0.01). CONCLUSION: Exogenous PTEN suppress the growth of human breast cancer cell and induces apoptosis by phosphatase activity, which provides a new clue to gene therapy for breast cancer.

Tumor type M2 pyruvate kinase expression in gastric cancer, colorectal cancer and controls.

AIM: Tumor formation is generally linked to an expansion of glycolytic phosphometabolite pools and aerobic glycolytic flux rates. To achieve this, tumor cells generally overexpress a special glycolytic isoenzyme, termed pyruvate kinase type M(2). The present study was designed to evaluate the use of a new tumor marker, tumor M(2)-PK, in discriminating gastrointestinal cancer patients from healthy controls, and to compare with the reference tumor markers CEA and CA72-4. METHODS: The concentration of tumor M(2)-PK in body fluids could be quantitatively determined by a commercially available enzyme-linked immunosorbent assay (ELISA)-kit (ScheBo Tech, Giessen, Germany). By using this kit, the tumor M(2)-PK concentration was measured in EDTA-plasma of 108 patients. For the healthy blood donors a cut-off value of 15 U/mL was evaluated, which corresponded to 90% specificity. Overall 108 patients were included in this study, 54 patients had a histological confirmed gastric cancer, 54 patients colorectal cancer, and 20 healthy volunteers served as controls. RESULTS: The cut-off value to discriminate patients from controls was established at 15 U/mL for tumor M(2)-PK. The mean tumor M(2)-PK concentration of gastric cancer was 26.937 U/mL. According to the TNM stage system, the mean tumor M(2)-PK concentration of stage I was 16.324 U/mL, of stage II 15.290 U/mL, of stage III 30.289 U/mL, of stage IV 127.31 U/mL, of non-metastasis 12.854 U/mL and of metastasis 35.711 U/mL. The mean Tumor M(2)-PK concentration of colorectal cancer was 30.588 U/mL. According to the Dukes stage system, the mean tumor M(2)-PK concentration of Dukes A was 16.638 U/mL, of Dukes B 22.070 U/mL, and of Dukes C 48.024 U/ml, of non-metastasis 19.501 U/mL, of metastasis 49.437 U/mL. The mean tumor M(2)-PK concentration allowed a significant discrimination of colorectal cancers (30.588 U/mL) from controls (10.965 U/mL) (P<0.01), and gastric cancer (26.937 U/mL) from controls (10.965 U/mL) (P<0.05). The overall sensitivity of tumor M(2)-PK for colorectal cancer was 68.52%, while that of CEA was 43.12%. In gastric cancer, tumor M(2)-PK showed a high sensitivity of 50.47%, while CA72-4 showed a sensitivity of 35.37%. CONCLUSION: Tumor M(2)-PK has a higher sensitivity than markers CEA and CA72-4, and is a valuable tumor marker for the detection of gastrointestinal cancer.

[Relationship between estradiol and the mitogenic activated protein kinase signal transduction pathway].

OBJECTIVE: To discuss the relationship between estradiol and the mitogenic activated protein kinase signal transduction pathway and the expression of the MAPK in the MCF-7 breast cancer cell-line. METHODS: Epithelial growth factor (EGF) and different concentration of estradiol to induce the expression of phosphospecific ERK1/2 (pERK1/2) in MCF-7 cell line was used and the expression of pERK1/2 with western-blotting was detected. Then antiestrogen ICI 182780 and MAPK inhibitor PD98059 to inhibit the expression of pERK1/2 was used. The cell cycle of MCF-7 was detected by FACS. RESULTS: EGF could significantly induce the expression of pERK1/2. Estradiol could also induce the expression of pERK1/2, but the intensity was less than the induction of EGF. The percentage of cells in the G(2)/M cell cycle after estradiol induction increased (18.38%) compared to the control group (10.52%) (P < 0.05). CONCLUSIONS: MAPK is an important regulatory signal in breast cancer. Its measurement in breast cancer tissues provides information about the degree of activation of various growth factor pathways. This molecule may also provide a molecular target for compounds designed to block cell proliferation.

[Early superselective angiography and transarterial embolization for massive bleeding after gastrectomy].

OBJECTIVE: To evaluate the efficacy of early superselective angiography and embolization in the diagnosis and treatment of massive bleeding after gastrectomy. METHODS: The clinical data of 28 patients with massive bleeding after surgery from 1980 to 2001 were retrospectively analysed. All patients underwent emergency angiography and 27 of them were treated by transcatheter embolization. RESULTS: Bleeding was controlled in 26 of the 28 patients (93%), recurrent bleeding occurred in 1, an recognized bleeding in 1, and abdominal pain in 1. There was no death. CONCLUSIONS: Transarterial embolization for massive bleeding after gastrectomy is safe and effective. It is suggested that early emergency angiography should be considered in all patients with massive gastrointestinal bleeding after gastrectomy.