Protective effects of mesenchymal stromal cells on adriamycin-induced minimal change nephrotic syndrome in rats and possible mechanisms.

BACKGROUND AIMS: Minimal change nephrotic syndrome is the most frequent cause of nephrotic syndrome in childhood. Current treatment regimes, which include glucocorticoid hormones and immunosuppressive therapy, are effective and have fast response. However, because of the side effects, long treatment course, poor patient compliance and relapse, novel approaches for the disease are highly desired. METHODS: The adriamycin-induced nephrotic rat model was established. Rats were allocated to a model group, a prednisone group or mesenchymal stromal cell (MSC) group. Clinical parameters in each treatment group were determined at 2 weeks, 4 weeks and 8 weeks. The messenger RNA (mRNA) levels of synaptopodin, p21 and monocyte chemoattractant protein-1 were determined through the use of quantitative real-time-polymerase chain reaction. Protein levels were determined by means of Western blot or enzyme-linked immunosorbent assay. Podocytes were isolated and apoptotic rate after adriamycin with or without MSC treatment was analyzed by means of flow cytometry. RESULTS: MSC intervention improved renal function as assessed by urinary protein, blood creatinine and triglyceride levels. MSC intervention reduced adriamycin-induced renal tissue damage visualized by immunohistochemistry and light and electron microscopic analysis and reduced adriamycin-induced podocyte apoptosis. After MSC intervention, mRNA and protein levels of synaptopodin and p21 in renal cortex were significantly increased. MSCs also restored synaptopodin mRNA and protein expression in isolated podocytes. In addition, monocyte chemoattractant protein-1 mRNA in renal cortex and protein level in serum of the MSC treatment group were significantly decreased compared with that in the adriamycin-induced nephropathy model group. CONCLUSIONS: Our data indicate that MSCs could protect rats from adriamycin-induced minimal change nephrotic syndrome, and the protective effects of MSCs are mediated through multiple actions.

Effect of the ACY-1 gene on HER2 and TRAIL expression in rectal carcinoma.

The incidence of rectal carcinoma (RC) is increasing and the age at onset of the disease is reducing. Therefore, elucidating the pathogenesis of RC is beneficial for early diagnosis and improving the prognosis. Aminoacylase-1 (ACY-1) is abnormally expressed in various malignant tumor tissues. Furthermore, the human epidermal growth factor receptor-2 (HER2) gene is involved in tumor metastasis and invasion, while tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumor cell apoptosis. The aim of the present study was to investigate the effect of the ACY-1 gene on the expression of HER2 and TRAIL in RC. Cancerous and adjacent tissues from RC patients were collected. ACY-1 expression was analyzed by immunohistochemistry. The rectal cancer cell lines HT29 and SW620, and normal colorectal mucosal epithelial fetal human cells were cultured in vitro. ACY-1 gene and protein expression levels were tested by reverse transcription-quantitative PCR and western blotting. ACY-1 small interfering RNA (siRNA) was transfected into HT29 and SW620 cells. Cell proliferation was detected by thiazolyl blue MTT assay. Caspase-3 activity was assessed using a commercial kit. HER2 and TRAIL expression levels were determined by western blotting. ACY-1 expression was significantly increased in cancer tissue compared with adjacent tissue (P<0.05). ACY-1 expression was elevated in HT29 and SW620 cells compared with normal colorectal mucosal epithelial cells (P<0.05). ACY-1 siRNA transfected into HT29 cells downregulated its expression, inhibited cell proliferation, enhanced caspase-3 activity, reduced HER2 expression and upregulated TRAIL expression (P<0.05). ACY-1 expression was found to be increased in rectal cancer tissue. Therefore, targeting the ACY-1 gene may regulate HER2 and TRAIL expression levels, and may reduce the occurrence and inhibit the development of rectal cancer.

[Expressing of N gene encoding nucleocapsid protein of vesicular stomatitis virus and elementary application in ELISA].

The gene encoding the nucleocapsid (N) protein of vesicular stomatitis virus (VSV-NJ) was subcloned from pMD-VN5, and inserted into pBAD/Thio TOPO vector. The recombinant plasmid was identified by restriction analysis and PCR. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted N gene. The results of SDS-PAGE and Western immunoblotting revealed that the N protein was expressed in Escherichia coli LGM194 in a high level and the recombinant fusion protein, which contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. It had a molecular mass of approximately 63.5 kD and immunologically reactive activity. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of vesicular stomatitis using 186 serum samples from experimentally infected goats and guinea-pigs with VSV-NJ and VSV-IN, and from field origin and reference serum samples. The sensitivity and specificity of the ELISA were compared with those of the standard microtiter serum neutralization (MTSN) tests. The ELISA and MTSN test results were highly correlated for detection of VSV antibodies. The ELISA was as sensitive as the SN assay in detecting positive serum to VSV. The correlation between SN titers and ELISA titers was statistically significant. These data suggest that the recombinant fusion N protein of VSV could be used as a recombinant test antigen for the serodiagnosis of vesicular stomatitis. The ELISA based on the reconmbinant nucleocapsid protein may offer the best combination of rapidity, sensitivity, simplicity, economy, and laboratory biosafety of any of the methods yet developed for VSV serodiagnosis. This study lay on foundation for the development of the diagnosis methods in serology for VSV.