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This study was designed to investigate the in vitro and in vivo transfection efficiency of chitosan nanoparticles used as vectors for gene therapy. Three types of chitosan nanoparticles [quaternized chitosan -60% trimethylated chitosan oligomer (TMCO-60%), C(43-45 KDa, 87%), and C(230 KDa, 90%)] were used to encapsulate plasmid DNA (pDNA) encoding green fluorescent protein (GFP) using the complex coacervation technique. The morphology, optimal chitosan-pDNA binding ratio and conditions for maximal in vitro transfection were studied. The in vivo transfection was conducted by feeding the chitosan/pDNA nanoparticles to 12 BALB/C-nu/nu nude mice. Both conventional and TMCO-60% could form stable nanoparticles with pDNA. The in vitro study showed the transfection efficiency to be in the following descending order: TMCO-60%>C(43-45 KDa, 87%)>C(230 KDa, 90%). TMCO-60% proved to be the most efficient and the optimal chitosan/pDNA ratio being 3.2:1. In vivo study showed most prominent GPF expression in the gastric and upper intestinal mucosa. GFP expression in the mucosa of the stomach and duodenum, jejunum, ileum, and large intestine were found, respectively, in 100%, 88.9%, 77.8% and 66.7% of the nude mice examined. TMCO-60%/pDNA nanoparticles had better in vitro and in vivo transfection activity than the other two, and with minimal toxicity, which made it a desirable non-viral vector for gene therapy via oral administration.

Aim: To investigate the relationship between the polymorphism of class II transactivator (CIITA) gene promoters and chronic hepatitis B (CHB)., Methods: Genomic DNA was prepared from peripheral blood leukocytes. Promoters I, III and IV of gene were analyzed respectively with polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) in 65 patients with CHB, 26 patients with acute hepatitis B (AHB) and 85 normal controls., Results: No abnormal migration was found in PCR-SSCP analysis of the three promoters in the three groups. Also, no sequential difference was observed at the three promoters among the CHB patients, AHB patients and normal controls., Conclusion: No polymorphism in promoters I, III and IV of CIITA gene exists in CHB patients, ABH patients and normal controls, suggesting that the promoter of CIITA gene might be a conserved domain.

Purpose: To investigate the effect of curcumin on the proliferation of human fetal retinal pigment epithelium (hfRPE) cells in vitro., Methods: Subcultured hfRPE cells were treated with different concentrations of curcumin (2.5, 5, 10, 20 microg/ml) for 24 or 48 hours. MTT assay and flow cytometry were performed to evaluate the effect of curcumin on the proliferation and cell cycle of cultured hfRPE cells., Results: Curcumin was effective in inhibiting the proliferation of subcultured hfRPE, the IC50 value at 24 and 48 hours were found to be 14.27 microg/ml, 12.7 microg/ml, respectively. Cell cycle analysis indicated that CUR blocked cells in G2/M phase., Conclusion: Curcumin can inhibit the proliferation of hfRPE cells by cells cycle arrest.

Aim: To investigate p27 expression in hepatocellular carcinoma (HCC), adjacent nontumoral and normal liver tissues, and to verify whether the subcellular localization of p27 was altered in HCC., Methods: The level of p27 in tumoral, nontumoral, and normal liver tissues were assessed by immunohistochemical (IHC) analysis. Parallel immunostaining was done for proliferating cell nuclear antigen (PCNA) to evaluate cell proliferation., Results: The labeling index (LI) of p27 in tumoral lesions was significantly lower than that in adjacent nontumoral lesions (t=2.444, P=0.017) and normal controls (t=2.268, P=0.029). The LI of p27 significantly decreased in patients with massive type (t=2.227, P=0.037) and infiltration (t=2.197, P=0.036). The prognosis of patients with higher p27 LI was longer than that of patients with lower p27 LI (P=0.0247, log-rank test). The LI of PCNA was significantly higher in HCC than that in adjacent nontumoral lesions (t=2.092, P=0.041) and normal controls (t=3.533, P=0.002). There was no significant correlation between p27 expression and cell proliferation in tumor samples. The level of p27 in the cytoplasmic fraction was higher in tumoral and nontumoral liver tissues, and was associated with clinical stage (t=2.520, P=0.029) and the degree of invasion (t=2.640, P=0.019). Survival analysis showed that p27 was an independent prognosis marker for HCC patients., Conclusion: These results suggest that p27 underexpressing in patients with HCC is closely associated with infiltration, metastasis, and prognosis. Alterations in the subcellular localization of p27 protein may occur early during hepatocarcinogenesis.

Aim: To observe the effects of low molecular weight heparin (LMWH) on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis., Methods: Colitis was induced in female Sprague-Dawley rats by colonic administration of 2, 4, 6-TNBS. LMWH, a dalteparin (150 U/kg, 300 U/kg), was subcutaneously administrated one hour before induction of colitis and went on once a day for 6 days. Then a half dose was given for the next 7 days. Control animals received the same volume of normal saline once a day for 14 days after treated by TNBS. Animals were sacrificed at 24 h, days 7 and 14 after induction of colitis. The colon was excised for the evaluation of macroscopic and histological findings and TNF-alpha immunohistochemical assay. Platelet surface P-selectin expression was determined by radioimmunoassay and serum IL-8 production was assayed by ELISA method., Results: LMWH treatment in a dose of 300 U/kg for 14 days significantly improved colonic inflammation by histological examination. Serum IL-8 production in the 300 U/kg treatment group was more significantly decreased at day 14 than that at 24 h (P<0.05). However, platelet surface P-selectin expression and TNF-alpha staining in colonic tissue were not significantly different among the three groups., Conclusion: LMWH has an anti-inflammatory effect on TNBS induced colitis in rats. The effect is possibly related to inhibition of proinflammatory cytokine IL-8, but not involved platelet surface P-selectin expression.

Aim: To examine the expression of nuclear factor kappaB (NF-kappaB) and its target genes in intestinal metaplasia (IM), dysplasia (DYS) and gastric carcinoma (GC) infected with Helicobacter pylori (H pylori) and to investigate the mechanism underlying H pylori cytotoxin associated gene A (cag A) infection leading to gastric adenocarcinoma., Methods: Expressions of NF-kappaB/p65 and its target genes: c-myc, cyclinD1 and bcl-xl were immunohistochemically examined in 289 cases of gastric biopsy and resection specimens from patients with IM, DYS and GC infected with H pylori. H pylori in the above mentioned tissues was detected by Warthin-Starry stain and rapid urease tests. IgG antibody to cagA in sera of the patients was measured by ELISA., Results: The positive rates of NF-kappaB/p65 were significantly higher in groups with cagA of IMI-II(28/33), IM III(48/52), DYSI(27/31), DYS II-III(28/32), GC(35/40) than in groups without cagA of IMI-II(4/17), IMIII(3/20), DYSI(3/20), DYSII-III(6/21), GC(10/23). The expressions of c-myc, cyclinD1, and bcl-xl were significantly higher in groups with cagA of IM III(47/52, 49/52, 46/52), DYSII-III(29/32, 26/32, 25/32) than in groups without cagA of IM III(8/20, 7/20, 5/20), DYSII-III(10/21, 8/21, 3/21), which were in conformity with the expression of NF-kappaB in IM III, and DYSII-III. A significantly higher expression level of NF-kappaB/p65, c-myc, cyclinD1 and bcl-xl was detected in intestinal type GC(27/28, 18/28, 22/28, 24/28) than in diffuse type GC(8/12, 3/12, 3/12, 6/12), respectively., Conclusion: There may be two different molecular mechanisms in the occurrence of intestinal and diffuse type gastric carcinomas. Intestinal type gastric carcinoma is strongly associated with high expression of c-myc, cyclinD1 and bcl-xl through NF-kappaB/p65 activated by H pylori cagA. Inhibiting the activity of NF-kappaB is an effective and promising way to prevent intestinal type gastric carcinoma.

Purpose: To evaluate the management for eyes with retinal detachment due to giant retinal breaks., Methods: Retrospectively, we reviewed 33 eyes of 32 cases treated either by scleral buckling (2 eyes) or by pars plana vitrectomy (PPV) combined with 20% C3F8(3 eyes) or silicone oil (28 eyes) tamponade. Of the 31 eyes undergone PPV, lensectomy was also performed on 20 eyes and perfluorocarbon was used to unroll and flatten the flap of the giant retinal breaks (GRBs) as well as to help peeling membranes in 27 eyes. Cryopexy (9 eyes) or endophotocoagulation (24 eyes) was applied to close the GRBs., Results: After follow-up for 6-42 months (mean 16.97 +/- 9.88), total retinal reattachment was achieved in 26 eyes, macular reattachment in 4 eyes, and 3 eyes maintain detached. Of the reattached 30 eyes, the final acuity were less than 0.02 in 4 eyes, 0.02-0.04 in 3 eyes, 0.05-0.08 in 2 eyes, 0.1-0.3 in 14 eyes, 0.4-0.7 in 7 eyes., Conclusion: A majority of GRBs eyes may avoid blindness by appropriate surgical methods and intraocular tamponade according to the complexion of the GRBs. Early diagnosis and operation of GRBs are the main prerequisites of a better prognosis and results. Proliferative vitreo retinopathy (PVR) was the main cause of surgical failure.

Objective: To introduce a simplified method to remove silicone oil via limbus of corneal after previous lensectomy-vitrectomy., Methods: Fifty-one eyes received the modified procedures to eliminate silicone oil via one pars plana or limbus incision after previous lensectomy-vitrectomy. Local anesthesia was performed with 25G syringe needle through pars plana or corneal limbus infusion and super limbus incisions to eliminate silicone oil., Results: All operations were primarily successful in eliminating silicone oil. The complications occurred in all the patients with the early low intraocular pressure, in 4 (4/51, 7.8%) eyes with retinal detachment, and in 2 (2/51, 3.9%) eyes with less bleeding at the point of puncture. The visual acuity was significantly improved after the procedure (P < 0.05) in patients with stable retinal reattachment., Conclusion: The procedure via one pars plana and limbus or limbus incision after previous lensectomy-vitrectomy is useful in removing the silicone oil tamponade in aphakic eyes because of its simplicity, good curative effect, and fewer complications.

Aim: To investigate the relationship between Helicobacter pylori (H.pylori) infection and the expressions of the p53, Rb, c-myc, bcl-2 and hTERT mRNA in a series of diseases from chronic gastritis (CG), intestinal metaplasia type I or II(IMI-II), intestinal metaplasia type III (IMIII), mild or modest dysplasia (DysI-II), severe dysplasia (DysIII) to gastric cancer(GC) and to elucidate the mechanism of gastric carcinogenesis relating to H.pylori infection., Methods: 272 cases between 1998 and 2001 were available for the study including 42 cases of CG, 46 cases of IMI-II, 25 cases of IMIII, 48 cases of DysI-II, 27 cases of DysIII, 84 cases of GC. H.pylori infection and the expressions of p53, Rb, c-myc, bcl-2 were detected by means of streptavidin-peroxidase (SP) immunohistochemical method. HTERT mRNA was detected by in situ hybridization (ISH)., Results: The expressions of p53, Rb, c-myc, hTERT mRNA and bcl-2 were higher in the GC than in CG, IM, Dys. The expression of c-myc was higher in IMIII with H.pylori infection (10/16) than that without infection (1/9) and the positive rate in DysI-II and DysIII with H.pylori infection was 18/30 and 13/17, respectively, higher than that without infection (4/18 and 3/10, respectively). In our experiment mutated p53 had no association with H.pylori infection, the expression of Rb was associated with H.pylori infection in GC, but the p53-Rb tumor-suppressor system abnormal in DysI-II cases, DysIII and GC cases with H.pylori infection was 21/30, 15/17 and 48/48 respectively, higher than non-infection groups (4/18, 3/10, 28/36). Furthermore the level of hTERT mRNA in GC with H.pylori infection (47/48) was higher than that without infection (30/36), however the relationship between bcl-2 and H.pylori was only in IMIII. C-myc had a close association with hTERT mRNA in DysIII and GC (P=0.0 253,0.0 305 respectively)., Conclusion: In the gastric carcinogenesis, H.pylori might cause the severe imbalance of proliferation and apoptosis in the precancerous lesions (IMIII and GysIII) first, leading to p53-Rb tumor-suppressor system mutation and telomerase reactivation, and finally causes gastric cancer.

Aim: To investigate the role of TGFbeta1 in invasion and metastasis in colorectal cancer by analysing TGFbeta1 correlated with depth of tumor invasion, stage and metastasis., Methods: Serum TGFbeta1 levels were determined in 50 patients with colorectal cancer and 30 healthy volunteers using a TGFbeta1 enzyme-linked immunosorbent assay. TGFbeta1 expression in primary and lymph node metastatic lesions were detected in 98 cases of colorectal cancer by immunohistochemical staining and in situ hybridization., Results: Serum levels of TGFbeta1 in patients with colorectal cancer(40+/-18 microg.L(-1)) were significantly higher than those in the healthy control group(19+/-8 microg.L(-1)), P<0.05. Elevated levels of serum TGFbeta1 were found in 60 % of patients with colorectal cancer when the mean +2 s was used as the upper limit of the normal range (35.1 microg.L(-1)). Increases in serum TGFbeta1 levels were significantly associated with Duke's stage (P<0.05), but there was no significant difference between Duke's stage B patients and Duke's stage C patients. In the cytoplasm of cancer cells, TGFbeta1 was immunostained in 37.8 % (37/98) of colorectal cancer, and this expression was confirmed by in situ hybridization. Among 35 cases of colorectal cancer with lymph node metastatic lesions, TGFbeta1 positive staining was found in 18 (51.4 %) cases of primary tumor, and 25 (71.4 %) cases with lymph node metastatic lesions, respectively. Of 17 cases w ith no staining in the primary lesion, 7 (41.2%) casesshowed TGFbeta1 staining in the metastatic lesion. Serum TGFbeta1 levels and TGFbeta1 expression in colorectal cancer tissues were correlated significantly with depth of tumor invasion, stage and metastasis. Patients in stage C-D,T3-T4 and with metastasis had significantly higher TGFbeta1 levels than patients in stage A-B,T1-T2 and without metastasis (P<0.05)., Conclusion: These results suggest that transforming growth factor-beta1 is closely related to the invasion and metastasis of colorectal cancer. It increased the invasive and metastatic potential of tumor by altering a tumor microenvironment. TGFbeta1 may be used as a possible biomarker.

Aim: Transforming growth factor TGF beta1 is involved in a variety of important cellular functions,including cell growth and differentiation, angiogenesis, immune function and extracellular matrix formation. However, the role of TGF beta(1) as an angiogenic factor in colorectal cancer is still unclear. We investigate the relationship between transforming growth factor beta(1) and angiogenesis by analyzing the expression of transforming growth factor TGF beta(1) in colorectal cancer, as well as its association with VEGF and MVD., Methods: The expression of TGF beta(1),VEGF, as well as MVD were detected in 98 colorectal cancer by immunohistochemical staining. The relationship between the TGF beta(1) expression and VEGF expression,MVD was evaluated. To evaluate the effect of TGF beta(1) on the angiogenesis of colorectal cancers., Results: Among 98 cases of colorectal cancer,37 were positive for TGF beta(1) 37.8% 36 for VEGF 36.7% respectively. The microvessel counts ranged from 19 to 139.8, with a mean of 48.7(standard deviation,21.8). The expression of TGF beta(1) was correlated significantly with the depth of invasion, stage of disease, lymph node metastasis, VEGF expression and MVD. Patients in T3-T4, stage III-IV and with lymph node metastasis had much higher expression of TGF beta(1) than patients in T1-T2, stage I-II and without lymph node metastasis (P<0.05). The positive expression rate of VEGF(58.3%) in the TGF-beta(1) positive group is higher than that in the TGF-beta(1) negative group(41.7%, P<0.05). Also, the microvessel count (54+/-18) in TGF-beta(1) positive group is significantly higher than that in TGF-beta(1) negative group(46+/-15, P<0.05). The microvessel count in tumors with both TGF-beta(1) and VEGF positive were the highest (58+/-20 36-140, P<0.05). Whereas that in tumors with both TGF-beta(1) and VEGF negative were the lowest (38+/-16, 19-60, P<0.05)., Conclusion: TGF beta(1) might be associated with tumor progression by modulating the angiogenesis in colorectal cancer and TGF beta(1) may be used as a possible biomarker.