Objective：The aim of this study was to investigate the relationship among the telomerase reverse transcriptase (TRT), the heparanase gene expressions, and the metastasis and prognosis of colorectal cancer. Method： The expression of TRT in 33 cases of formalin fixed, paraffin embedded colorectal adenocarcinoma tissues was examined using antibody of human TRT and Streptavidin HRP(SP) immunohistochemical method, meanwhile metastasis and 5 year survival rate of the patients were analyzed. The expression of heparanase gene in tweenty fresh colorectal carcinoma tissues were also analyzed by reverse transcriptase polymerase chain reaction (RT PCR). Results： The expression of TRT was found positive in all 33 patients with colorectal cancer, of which 14 cases were strongly positive and 19 weakly positive. TRT was expressed diffusely in the cytoplasm and partly in the nuclei of the cancer cells. Whereas only minimally or weakly positive signals for TRT located mainly in the nuclei with few cytoplasm around, were found in normal or in proliferative colorectal mucosa. The metastatic rate and the 5 year survival rate in tumors with strong expressing TRT were 50.0％ (7/14) and 21.4％ (3/14), respectively, whereas in those with weak expressing TRT were 15.8％ (3/19) and 84.2％ (16/19), respectively. There were significant differences between two groups in the metastatic rate (P< 0.05) and the 5 year survival rate (P < 0.001).The expression of heparanase gene was found positive in 20/20 cases of colorectal carcinoma when RT PCR using 0.5 μ g total RNA but positive was only in 11/20 cases of the cancer when using 0.1 μ g total RNA. The positive rate for metastatic tumors was 90.0％ (9/10), whereas for non metastatic tumors it was only 20.0％ (2/10) when using 0.1 μ g templet of total RNA. Five cases of normal colorectal mucosa were negative for heparanase. Conclusions： The expression of TRT may be corresponding to metastatic rate and 5 year survival rate. The expression of heparanase gene may be related to the malignant degree and metastasis of colorectal cancer. Both TRT and heparanase may be important new markers for predicting the prognosis of the patients with colorectal cancer.

Objective To evaluate the clinical efficacy and safty of le-vocarnitine combined with alprostadil in the treatment of diabetic ne-phropathy.Methods A total of 68 patients with diabetic nephropathy were randomly divided into control group ( n=34 ) and treatment group (n=34).Control group was given alprostadil 1 mL, qd by intravenous drip.Treatment group was received levocarnitine 1 g, qd by intravenous drip on the basis of control group .The course of two groups was 28 d. The clinical efficacy , biochemical indexes before and after treatment and the incidence of adverse drug reactions were compared between two groups.Results After treatment , the total effective rate of control group was significantly lower than that of treatment group ( 67.65% vs 94.12%, P<0.05).After treatment, the levels of serum 24 h urinary protein , urinary microglobulin , urea nitrogen , cystatin C , homocysteine , glycosylated hemoglobin , transforming growth factor -β1 ,Ⅳcollagen in two groups were significantly decreased ( P<0.05 ) , and those indexes of treatment group after treatment were significantly lower than those of control group (P<0.05).After treatment, the levels of serum of serum creatinine , 25 -hydroxy vitamin D 3 in two groups were decreased with significant difference ( P <0.05 ) , and the treatment group was more obvious ( P<0.05 ).The incidence of adverse drug reactions in control group was significantly higher than that in treat -ment group (23.53%vs 8.82%,P<0.05 ) .Conclusion Levocarnitine combined with alprostadil have a definitive clinical efficacy and safety for the treatment of diabetic nephropathy .

Animals ; Artemisinins ; pharmacology ; Cricetinae ; Leishmania donovani ; drug effects ; Male ; Mesocricetus ; Sesquiterpenes ; pharmacology

Objective To establish and optimize a sensitive and specific quantitative realtime polymerase chain reaction(PCR)method for detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA)in liver tissue. Methods Specific primers and probes were designed to detect HBV DNA(tDNA)and cccDNA. A series of plasmids(3.44 × 100-3.44 × 109 copies/μl)containing a full double-stranded copies of HBV genome(genotype C)were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma(HCC), 13 Chronic hepatitis B patients(CHB)and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase(PSAD)for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount(copies/cell)of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. Results The standard curves of real-time PCR with a linear range of 3.44 × 100 to 3.44 × 109 copies/μl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 100 copies/μl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 102 times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficience of variations on cycle threshold (Ct)were between 0.224%-0.609%. Conclusion A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.

Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-β1(TGF-β1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.
Humans ; Rats ; Animals ; Diabetic Nephropathies/drug therapy* ; Abelmoschus ; Flavones/pharmacology* ; Podocytes ; Tumor Necrosis Factor-alpha/metabolism* ; Necroptosis ; Receptor-Interacting Protein Serine-Threonine Kinases/metabolism* ; Fibrosis ; Threonine/pharmacology* ; Collagen/metabolism* ; Serine/pharmacology* ; Diabetes Mellitus/drug therapy*