Nutritional Risk Screening 2002 as a Predictor of Postoperative Outcomes in Patients Undergoing Abdominal Surgery: A Systematic Review and Meta-Analysis of Prospective Cohort Studies.

BACKGROUND: The nutritional risk screening (NRS 2002) has been applied increasingly in patients who underwent abdominal surgery for nutritional risk assessment. However, the usefulness of the NRS 2002 for predicting is controversial. This meta-analysis was to examine whether a preoperative evaluation of nutritional risk by NRS 2002 provided prediction of postoperative outcomes in patients undergoing abdominal surgery. METHODS: A systematic literature search for published papers was conducted using the following online databases: MEDLINE, EMBASE, the Cochrane library, EBSCO, CRD databases, Cinahl, PsycInfo and BIOSIS previews. The pooled odds ratio (OR) or weight mean difference (WMD) was calculated using a random-effect model or a fix-effect model. RESULTS: Eleven studies with a total of 3527 patients included in this study. Postoperative overall complications were more frequent in nutritional risk patients versus patients without nutritional risk (the pooled OR 3.13 [2.51, 3.90] p<0.00001). The pooled OR of mortality for the nutritional risk group and non-nutritional risk group was 3.61 [1.38, 9.47] (p = 0.009). Furthermore, the postoperative hospital stay was significant longer in the preoperative nutritional risk group than in the nutritional normal group (WMD 5.58 [4.21, 6.95] p<0.00001). CONCLUSIONS: The present study has demonstrated that patients at preoperative nutritional risk have increased complication rates, high mortality and prolonged hospital stay after surgery. However, NRS 2002 needs to be validated in larger samples of patients undergoing abdominal surgery by better reference method.

Expression of gastrointestinal nesfatin-1 and gastric emptying in ventromedial hypothalamic nucleus- and ventrolateral hypothalamic nucleus-lesioned rats.

AIM: To determine the expression levels of gastrointestinal nesfatin-1 in ventromedial hypothalamic nucleus (VMH)-lesioned (obese) and ventrolateral hypothalamic nucleus (VLH)-lesioned (lean) rats that exhibit an imbalance in their energy metabolism and gastric mobility. METHODS: Male Wistar rats were randomly divided into a VMH-lesioned group, a VLH-lesioned group, and their respective sham-operated groups. The animals had free access to food and water, and their diets and weights were monitored after surgery. Reverse transcription-polymerase chain reaction and immunostaining were used to analyse the levels of NUCB2 mRNA and nesfatin-1 immunoreactive (IR) cells in the stomach, duodenum, small intestine, and colon, respectively. Gastric emptying was also assessed using a modified phenol red-methylcellulose recovery method. RESULTS: The VMH-lesioned rats fed normal chow exhibited markedly greater food intake and body weight gain, whereas the VLH-lesioned rats exhibited markedly lower food intake and body weight gain. NUCB2/nesfatin-1 IR cells were localised in the lower third and middle portion of the gastric mucosal gland and in the submucous layer of the enteric tract. Compared with their respective controls, gastric emptying was enhanced in the VMH-lesioned rats (85.94% ± 2.27%), whereas the VLH lesions exhibited inhibitory effects on gastric emptying (29.12% ± 1.62%). In the VMH-lesioned rats, the levels of NUCB2 mRNA and nesfatin-1 protein were significantly increased in the stomach and duodenum and reduced in the small intestine. In addition, the levels of NUCB2 mRNA and nesfatin-1 protein in the VLH-lesioned rats were decreased in the stomach, duodenum, and small intestine. CONCLUSION: Our study demonstrated that nesfatin-1 level in the stomach and duodenum is positively correlated with body mass. Additionally, there is a positive relationship between gastric emptying and body mass. The results of this study indicate that gastrointestinal nesfatin-1 may play a significant role in gastric mobility and energy homeostasis.

[Association of gastric emptying with ghrelin, obestatin and receptor (GHSR, GPR-39) in hypothalamus of diabetic rats].

OBJECTIVE: To investigate the association of gastric emptying with ghrelin, obestatin and GHSR, GPR-39 in hypothalamus of diabetic rats. METHODS: Sixty Wistar rats were randomly divided into three groups: a normal control group (NC, n = 20), a diabetes mellitus group (DM, n = 20) induced by intraperitoneal injection of streptozotocin (STZ) and an insulin treated group (INS, n = 20). After two and six weeks of STZ injection, gastric emptying was measured by intragastric administration of phenol red, ghrelin and obestatin in hypothalamus measured by ELISA (enzyme-linked immunosorbent assay) and GHSR and GPR-39 by RT-PCR (reverse transcription-polymerase chain reaction). RESULTS: After two weeks of STZ injection, gastric emptying (%) (74 +/- 8, 40 +/- 5), ghrelin level(ng/g) (52 +/- 9, 51 +/- 7) and ratio of ghrelin/obestatin (3.8 +/- 1.0, 2.8 +/- 1.0) increased significantly in DM and INS groups compared to those in NC group [32% +/- 7%, (39 +/- 11) ng/g, 2.1 +/- 0.8, all P < 0.05]. Obestatin level(ng/g) (14.2 +/- 2.0) of hypothesis decreased significantly in DM group as compared to those in NC group (21.7 +/- 4.7) while GHSR/beta-actin increased significantly (1.26 +/- 0.46 vs 0.77 +/- 0.21, P < 0.05). Gastric emptying was positively correlated with ghrelin, ghrelin/obestatin and GHSR/beta-actin of hypothalamus (r = 0.49; r = 0.63; r = 0.73; P < 0.01). But there was a negative correlation with obestatin of hypothalamus (r = -0.74, P < 0.01). After six weeks of STZ injection, gastric emptying (78.97% +/- 8.13% vs 44.06% +/- 5.06%) increased significantly in DM and INS groups as compared to those in NC group (35.06% +/- 3.91%, P < 0.01). Gastric emptying was positively correlated with ghrelin/obestatin of hypothalamus (r = 0.40, P < 0.05). There was no detection of GPR-39 in hypothalamus. CONCLUSION: The rapid gastric emptying may be due to the rising levels of ghrelin and GHSR in hypothalamus during early hyperglycemia. And the duration of hyperglycemia is affected by the rising ratio of ghrelin/obestatin.

[Inhibition of mutant-type p53 by a chimeric U6 maxizyme in hepatocellular carcinoma cell lines].

OBJECTIVE: To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines. METHODS: Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot. RESULTS: In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis. CONCLUSION: Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.

Effect of ribozyme against transforming growth factorbeta1 on biological character of activated HSCs.

Transforming growth factorbeta1 (TGFbeta1) is considered to be the principal contributor to liver fibrosis. So in this study the ribozymes against TGFbeta1 were designed. The in vitro cleavage activities of the ribozymes were assayed through incubation of (32)p-labeled target RNAs and (32)p-labeled ribozymes in different conditions. HSC-T6 cells were transfected with the eukaryotic constructs encoding ribozyme and disable ribozyme, then the stable cell clones were used to evaluate its antifibrotic characteristic through the effect of ribozyme on biological character of activated hepatic stellate cells (HSCs). The results demonstrated that two ribozymes (Rz803 and Rz1395) could cleave target RNAs into expected products effectively, Rz803 possessed better cleavage activity in vitro. Stable transfection of Rz803 into activated HSCs reduced TGFbeta1 expression in mRNA and protein level efficiently. The further studies demonstrated that Rz803 reduced deposition of collagen I, suppressed HSC proliferation, but had no effect on HSC activation in transfected HSC-T6 cells. Therefore, it indicated that Rz803 could reverse the character of activated HSCs by down-regulating TGFbeta1 expression efficiently and diminishing TGFbeta1 signaling underlying activation of hepatic stellate cells. As the consequence, it would provide a potential therapeutic approach for liver fibrosis.

Ribozymes against TGFbeta1 reverse character of activated hepatic stellate cells in vitro and inhibit liver fibrosis in rats.

BACKGROUND/AIMS: Transforming growth factor beta (TGFbeta1) is considered the key mediator in the process of liver fibrosis. The purpose of this investigation was to evaluate the activity of ribozymes against TGFbeta1 in a cell-free system and activated hepatic stellate cells (HSCs), and antifibrotic effect in activated HSCs in vitro and in rats. METHODS: Three ribozymes targeting against TGFbeta1 mRNA were designed, and then cloned into the U1 snRNA expression cassette. The chimeric ribozymes were selected for the analysis of their performances in activated HSCs through the detection of their cleavage activities in a cell-free system. After ribozyme-encoding plasmids had been transfected into HSC-T6 cells, the effects of ribozymes on activated HSCs were evaluated through the analysis of proliferation, activation and collagen deposition of HSC-T6. The adenoviral vector expressing the ribozymes was constructed, and then delivered into rat models of hepatic fibrosis induced by carbon tetrachloride. RESULTS: TGFbeta1 expression was efficiently down-regulated in activated HSCs by U1 snRNA chimeric ribozymes which possessed perfect cleavage activity in a cell-free system. Further studies demonstrated that U1 snRNA chimeric ribozymes inhibited the synthesis of collagen I, reduced deposition of collagen I, suppressed BrdU incorporation, but had no effect on desmin and alpha-SMA expression in transfected HSC-T6 cells. Histological analysis demonstrated that the adenoviral vector expressing ribozyme (Rz803) could alleviate fibrotic pathology in rats treated with carbon tetrachloride. CONCLUSIONS: The anti-TGFbeta1 ribozymes could reverse the character of activated HSCs in vitro and improve fibrotic pathology in vivo. It indicated that TGFbeta1 could be considered as a novel candidate for a therapeutic agent against hepatic fibrosis.

[Hammerhead ribozyme against human telomerase catalytic subunit (hTERT) induced apoptosis of liver cancer cells].

OBJECTIVE: To construct vector pEGFP-C1-hTERT-ribozyme (pGTRz-U6) and its mutant (pGTmRz-U6) against hTERT containing U6 promoter, then transfect them into human liver cancer cell line SMMC7721 to observe the action of the human telomerase catalytic subunit (hTERT) hammerhead ribozyme on proliferation and apoptosis of human liver cancer cell SMMC7721. METHODS: Eukaryotic expressing vector pGTRz-U6 and mutant pGTmRz-U6 were constructed and transfected into SMMC7721 using Lipofectamine2000 Reagent, with pEGFP-C1 as the control group. After strict screening by G418, positive clones were cultured; the amount of expression of ribozyme and hTERT was detected by RT-PCR; cell proliferation by MTT; telomerase activity by TRAP and silver staining assay; cell apoptosis by FCM. RESULTS: We found that the two ribozymes were expressed persistently in SMMC7721; different expression levels (P < 0.01) of hTERT among SMMC7721-Rz, SMMC7721-mRz and SMMC7721-pEGFP-C1 was exhibited by the analysis of variance with SPSS software. The difference between SMMC7721-Rz and the others is significant in t-test (P < 0.01), while there was no difference between SMMC7721-mRz and SMMC7721-pEGFP-C1 (P > 0.05). With the advance of cell division, telomerase activities of the cells treated by SMMC7721-Rz and SMMC7721-mRz decreased gradually, and the percentage of apoptosis of the cells transfected with Rz and mRz increased gradually. The apoptosis percentage of 7PDS SMMC7721-Rz was 29.86%, while those of SMMC7721-mRz and SMMC7721-pEGFP-C1 were 9.87% and 3.36%, respectively. CONCLUSION: The apoptosis level of SMMC7721 induced by hTERT ribozyme increases as cells divide, and this ribozyme maybe a potential approach for liver cancer gene therapy.

[The inhibitory effects on hepatitis B virus replication by stable expression of DN mutants of hepatitis B virus X gene pRev X-GFP].

OBJECTIVE: Persistent replication of hepatitis B virus (HBV) is one of the major obstacles in HBV infection treatment. Reduction or clearance of HBV propagation would be one of the aims of HBV therapy. The drugs approved in clinical used such as nucleotide analogs or interferon, were limited effects on HBV replication. The newly developing gene therapy method, dominant negative mutants, were be used as new promising HBV therapy strategy, and a dominant negative mutant of HBVX gene pRev X-GFP which we have reported in our previous study has some effects both on HBV replication and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG2 2.2.15 cells without transfection pRev X-GFP in the experiment. To make sure the effects of dominant negative mutant of pRev X-GFP, we established a HBX DN stable express cell clone, and evaluated the effects of HBX dominant negative mutant on HBV replication. METHODS: The X gene mutant, in which a specific point mutation of 3'-end ATG to AAG and fused with human green fluorescence protein (GFP) were cloned into pRev TRE vector, assigned to pRev HBX-GFP dominant mutant (pRev X-GFP). And the plasmid contains the wild type X gene or GFP gene was cloned into the same vector to construct pRev Xwt, pRev GFP constructs. All the constructs then transfected into HepG2 2.2.15 cells by liposome. After 7 days resistance selection of hygromycin (300 microg/ml), and cell clones which stable expression HBX-GFP, HBXwt, GFP were obtained. After reseeding of 106 cells of each clones in 12 wells with a 12 well cell plate and another 12 wells 2.2.15 cell were serve as blank control. The cells and media were harvested after cultured in DMEM with 10% FBS for 3 days. HBV-related DNA was assayed by dot blot and Southern blot. RESULTS: The 100% expression of pRev HBX-GFP, GFP and wild type X constructs were obtained. The stable expressed HBX-GFP can significantly reduce HBV DNA level both in cell media and cells by dot blot and Southern blot analysis, but not for pRev Xwt and pRev GFP. CONCLUSION: The dominant negative mutant pRev HBX-GFP can significantly inhibit the HBV gene expression. It also suggested that X gene might be one of promising target for HBV gene therapy.

[Inhibition of mutant p53 in hepatocellular carcinoma cells by hammerhead ribozyme].

OBJECTIVES: To investigate the inhibition of mutant type p53 in hepatocellular carcinoma by hammerhead ribozyme in both cell-free system and MHCC97 cells. METHODS: Hammerhead ribozyme genes (RZ) and control ribozyme (asRZ) directed against mutant p53 (249 codons, AGG --> AGT) were designed by computer. The in vitro transcription plasmid and eukaryotic expression plasmid were constructed into the vector pBSKU6 and pEGFPC1. Human mutant and wild type p53 gene fragment were cloned into the pGEM-T vector under T7 promoter control. In vitro cleavage reaction was carried out by mixing the RZ and target mRNAs which were labeled with [alpha-32P] dUTP. RZ was introduced into MHCC97 cells by LipofectAMINEAM2000 and mtp53 expression was analyzed by RT-PCR. RESULTS: In cell-free systems, RZ showed a specific cleavage activity against mtp53 with cleavage efficiency of 42%, while the wild type p53 was not cleaved. The mRNA level of mtp53 in MHCC97 cells after transfection was reduced by RT-PCR analysis. CONCLUSION: These findings suggest that the hammerhead ribozyme against the mtp53 is a new promosing gene therapeutic agent against hepatocellular carcinoma.

Maxizyme-mediated specific inhibition on mutant-type p53 in vitro.

AIM: To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG-AGT) in vitro. METHODS: Two different monomers of anti-mtp53 maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G(5)-A(5)) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR, pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme). Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEM-T vector under the T7 promoter control. The (32)p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with (32)p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis. RESULTS: In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 degrees and 25 mM MgCL(2). The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either. CONCLUSION: pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good foundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.

[Clinical study of oligonucleotide microarray on monitoring the lamivudine-resistance mutations in hepatitis B virus].

OBJECTIVE: To evaluate the mutations of lamivudine-resistance using oligonucleotide microarray in hepatitis B virus (HBV) infected patients. METHODS: A randomized clinical trial was conducted on 20 lamivudine-treated patients for 18 months and 10 patients as controls. The serum HBV DNA was amplified by PCR and the lamivudine-resistance mutations in YMDD region were assayed by 4 sites microarray developed before. RESULTS: This microarray could clearly differentiate the wide-type from mutated-type HBV with lamivudine-resistance mutations. The rate of mutations in YMDD region increased with the time of lamivudine treatment (chi2=6.69, P<0.01). The most common mutated type was M539V+L515M and next M539I. Continuous administration of lamivudine was no benefit for inhibiting the replication of HBV with YMDD mutation but helpful for wide-type HBV. CONCLUSION: The routine serum HBV DNA assay by PCR may introduce prejudice in monitoring HBV inhibitory effect by lamivudine, while the microarray technique can avoid this and is one of the best ways to monitor the lamivudine-resistance mutations in HBV. There is no effect of lamivudine on HBV with YMDD mutation in clinical practice.

Preparation and identification of anti-transforming growth factor beta1 U1 small nuclear RNA chimeric ribozyme in vitro.

AIM: To study the preparation and cleavage activity of anti-transforming growth factor (TGF)beta1 U1 small nuclear (sn) RNA chimeric hammerhead ribozymes in vitro. METHODS: TGFbeta1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. (32)p-labeled TGFbeta1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFbeta1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator. (32)p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE. RESULTS: Active U1snRNA chimeric ribozyme (U1Rz803) had the best cleavage activity at 50 degrees; at 37 degrees, it was active, K(m)=34.48 nmol/L, K(cat)=0.14 min(-1); while the point mutant ribozyme U1Rz803(m) had no cleavage activity, so these indicated the design of U1Rz803 was correct. CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFbeta1 in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.

Anti-HBV hairpin ribozyme-mediated cleavage of target RNA in vitro.

AIM: To study the preparation and cleavage activity of HpRz directed against the transcript of HBV core gene in vitro. METHODS: HpRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32p-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme in vitro. 32p-labeled pKC transcript containing HBV core region as target-RNA was transcribed using T7 RNA polymerase and purified by denaturing PAGE. Cold HpRz transcript was incubated with 32p-labeled target-RNAs under different conditions and radio autographed after denaturing polyacrylamide gel electrophoresis. RESULTS: HpRz has the specific ability of cleavage of target RNA at 37 degrees and 12 mM MgCl2. Km=26.31 nmol/L, Kcat=0.18/min. These results revealed that the design of HpRz was correct. CONCLUSION: HpRz prepared in this study possesses specific catalytic activity from the identification of cleavage activity. These results indicate that hairpin ribozyme may intracellularly inhibit the replication of HBV, therefore it may become a novel potent weapon for the treatment of hepatitis B.