[Thrombolytic effects of recombinant staphylokinase on coronary thrombosis in miniature pigs].

OBJECTIVE: To evaluate the thrombolytic effects of recombinant staphylokinase and compare it with those of recombinant streptokinase. METHODS: Thirty Chinese experimental miniature pigs were divided into five groups, namely, solvent control group, positive drug control group and three recombinant staphylokinase groups, six in each group. The thrombus of coronary artery was formed by surgical thoracotomy and direct current stimulation in anesthetized animals. Intravenous administration was started after the thrombus of coronary artery was formed for 30 minutes, and the method of first injection and then constant speed infusion by peristaltic pump was used. The solvent control group was injected intravenously with solvent, the positive drug control group was given recombinant streptokinase 4 mg·kg-1 intravenously, and the three recombinant staphylokinase groups were given recombinant staphylokinase at the doses of 4, 2 and 1 mg.kg-1 intravenously. The volume of intravenous injection was 5 ml, which was completed within 1 min, the speed of infusion was 0.5 ml·min-1, which was completed within 60 min, and the animals were sacrificed 120 minutes later. Before and 30, 60 and 120 min after administration, the venous blood samples were collected. At the end of the experiment, the coronary artery segments of the thrombosis site were taken, and the euglobulin dissolution time (ELT), blood fibrinogen content (FBG), fibrinogen degradation product (FDP) and wound bleeding volume were measured respectively. The coronary thrombolysis rate, myocardial ischemia degree and ischemia range were measured. RESULTS: Compared with the solvent control group, ELT in the experimental group was significantly shortened (P＜0.05 or P＜0.01), FBG degradation in a few experimental animals was more than 20%, FDP was significantly increased (P＜0.05 or P＜0.01), and there was no significant effect on blood pressure and heart rate of small pigs. Compared with the control group, the maximum thrombus area was decreased by 34.3% and 15.4% (P＜0.05) in the high and middle dose groups of the experimental group. Compared with the same dose of recombinant streptokinase, recombinant staphylokinase had stronger thrombolytic effect (P＜0.05 or P＜0.01) on the coronary thrombus caused by electrical stimulation, less bleeding side effects and the same effect on the degree and range of myocardial infarction as recombinant streptokinase. CONCLUSION: Compared with recombinant streptokinase, recombinant staphylokinase has faster thrombolysis speed, higher fibrin specificity and less bleeding side effects. In general, 2 mg.kg-1 recombinant staphylokinase has better efficacy and safety.

Bufalin and 5-fluorouracil synergistically induce apoptosis in colorectal cancer cells.

5-fluorouracil (5-FU) has been used in the treatment of colorectal cancer for >50 years. However, drug resistance remains an obstacle in the application of 5-FU-based chemotherapy. Bufalin, a type of steroid with anti-tumor activity, may be purified from the skin and parotid venom glands of toads. In order to improve the anti-tumor effect of 5-FU, the present study examined the combined effects of bufalin with 5-FU on human colorectal cancer HCT116 cells. Following treatment, cell proliferation was quantified using MTT assay and apoptotic cell percentage was assessed by flow cytometry. The apoptosis-associated protein expression was evaluated by western blotting. It was observed that bufalin enhanced the cytotoxicity of 5-FU in HCT116 cells via the induction of the mitochondrial apoptotic pathway. Additionally, bufalin combined with 5-FU reduced the expression levels of anti-apoptotic proteins, such as Mcl-1, XIAP and Bcl-2 and upregulated the levels of the pro-apoptotic proteins, Bax and Bad. To verify the role of Bax, RNA interference was used to knock-down Bax. It was determined that the synergistic effect between 5-FU and bufalin was diminished following the silencing of Bax. In summary, bufalin in combination with 5-FU may induce a higher level of apoptosis compared with monotherapy, and the combination mat be a potential therapeutic strategy for the treatment of colorectal cancer.

Suppression of scinderin modulates epithelial­mesenchymal transition markers in highly metastatic gastric cancer cell line SGC­7901.

Scinderin is a Ca2+­dependent filamentous actin (F­actin) severing and capping protein, which has a key role in regulated secretion. However, little is known regarding the function and mechanism of scinderin in human carcinoma development and progression. In the present study, the biological function of scinderin was investigated using a cell proliferation assay, flow cytometric analysis and a Transwell assay in highly tumorigenic and the metastatic human gastric cancer cell line SGC­7901 transfected with scinderin­small hairpin RNA lentivirus. The changes in the expression of epithelial­mesenchymal transition (EMT) markers were also investigated. The results indicated that scinderin knockdown effectively suppressed proliferation, reduced migration and arrested the cell cycle of the SGC­7901 cells at G2/M phase. Furthermore, scinderin knockdown altered the expression of EMT markers; the expression of E­cadherin was significantly upregulated, along with an evident decrease in N­cadherin and ß­catenin expression. In conclusion, the present study suggested that suppression of scinderin impaired proliferation and migration of gastric cancer SGC­7901 cells and attenuates its EMT process. Scinderin may therefore be a potential target for tumor EMT and therapy against gastric cancer.

[Estimation of the flux of inorganic nitrogen flowing into the East China Sea].

The flux of inorganic nitrogen flowing into the East China Sea was estimated based on the systematic analysis of all the pollution sources from 1980-2005. The result showed that the flux of inorganic nitrogen had been increasing from the early 1980s to the early 21st century. In detail, the flux was about 8.8 x 10(5) t x a(-1) in the early 1980s, and increased to about 2.6 x 10(6) t x a(-1) in the early of 21st century. The annual increasing rate was about 4.3%, and the mean flux was 1.8 x 10(6) t x a(-1). The flux of inorganic nitrogen of Yangtze River had also been increasing from early 1980s to the early 21st century. In detail, the flux was 4.0 x 10(5) t x a(-1) in the early 1980s, and increased to about 6.2 x 10(5) t x a(-1) in the middle 1980s, and was then kept at this value to the end of 1980s. After that, the flux value increased quickly from the early 1990s to 1.8 x 10(6) t x a(-1) in the early 21st century. Of all the sources, the proportion of land-source inorganic nitrogen was the largest, which was about 79%, among which, the river-source, the sewage-source and the non-point source accounted for 73%, 4% and 2%, respectively. Besides the land-source, the air-source and the mariculture-source accounted for 18% and 3%. The proportion of flux of Yangtze River in the river source was 76.5%.

[Expression of transforming growth factor-ß1 and its receptors in peripheral blood of patients with immune thrombocytopenic purpura].

This study was purposed to detect the expression of transforming growth factor ß1 (TGF-ß1) and its receptors (TGF-ßR) and to investigate their roles in pathogenesis of immune thrombocytopenic purpura (ITP). The expressions of TGF-ß1 and their receptors TGF-ßRI, TGF-ßRII and TGF-ßRIII in the peripheral blood of patients with ITP and healthy persons were detected by the real-time PCR, and differences of their expression levels were analysed. The results showed that the expression of TGF-ß1 and TGF-ßRII mRNA in ITP patients was significantly higher than that in the healthy controls, while the TGF-ßRI mRNA expression in ITP patient was significantly lower than that in the controls. The expression of TGF-ßRIII was not statistically different between the two groups. It is concluded that TGF-ß1 and its receptors including TGF-ßRI and TGF-ßRII express abnormally in the peripheral blood of ITP patients, which suggests that the TGF-ß signaling pathway probably play a vital role in the pathogenesis of the ITP.

A gold nanoparticle-based immunochromatographic assay: the influence of nanoparticulate size.

Four different sized gold nanoparticles (14 nm, 16 nm, 35 nm and 38 nm) were prepared to conjugate an antibody for a gold nanoparticle-based immunochromatographic assay which has many applications in both basic research and clinical diagnosis. This study focuses on the conjugation efficiency of the antibody with different sized gold nanoparticles. The effect of factors such as pH value and concentration of antibody has been quantificationally discussed using spectra methods after adding 1 wt% NaCl which induced gold nanoparticle aggregation. It was found that different sized gold nanoparticles had different conjugation efficiencies under different pH values and concentrations of antibody. Among the four sized gold nanoparticles, the 16 nm gold nanoparticles have the minimum requirement for antibody concentrations to avoid aggregation comparing to other sized gold nanoparticles but are less sensitive for detecting the real sample compared to the 38 nm gold nanoparticles. Consequently, different sized gold nanoparticles should be labeled with antibody under optimal pH value and optimal concentrations of antibody. It will be helpful for the application of antibody-labeled gold nanoparticles in the fields of clinic diagnosis, environmental analysis and so on in future.

[Ecological effect of No.0 diesel water accommodated fraction on marine algae].

With batch culture experiments in field and laboratory, the ecological effect of No. 0 diesel water accommodated fraction on marine algae was studied. A growth model of marine algae under grazing pressure and a model of growth effect on marine algae with different doses No.0 diesel water accommodated fraction were proposed. Based on the model and experiments, the growth effect of No.0 diesel water accommodated fraction on marine algae was studied. The results show that, the growth model of marine algae under grazing pressure is more suited for the marine ecological system than Logistic model. And the final biomass (B(f)) of marine algae with different doses No.0 diesel water accommodated fraction was calculated by the model with none-linear fitting software. The results also show that, under the field and laboratory conditions, lower doses No.0 diesel water accommodated fraction promotes the growth of marine algae, and the most promoting ratio are 180% and 120% respectively, however, higher doses hardly promotes but bates the growth of marine algae.

[Establishment of liver metastasis model of human gallbladder cancer and isolation of the subpopulation with high metastatic potential].

OBJECTIVE: To establish an experimental liver metastatic model of gallbladder cancer and isolate the subpopulation with high metastatic potential from the model, which may serve as a reliable tool in research on liver metastasis of gallbladder cancer in vivo and in vitro. METHODS: Human gallbladder cancer cells of the line GBC-SD were cultured. Ten nude athymic BALB/c mice, aged 4 approximately 5 weeks, underwent inoculation of suspension of GBC-SD cells into the spleens and then their spleens were resected. Three weeks later laparotomy was performed to observe if liver metastasis occurred. Once liver metastasis was discovered, the mice were killed and the livers were taken out to undergo microscopy, tumor cells were isolated from the metastatic foci and cultured in vitro, and then inoculated into other 10 mice in the same way as their parents cells for the second round of selection. The similar steps were repeated, altogether for three rounds of selection and a total 30 mice were inoculated in 3 rounds so as to find subpopulation with high metastatic potential. Another 10 mice underwent subcutaneous inoculation, 90 days later the mice were killed to observe if pulmonary metastasis occurred. PCR was used to detect the 3 microsatellite sequences D14S68, D18S69, and D20S199 in the DNA samples of the gallbladder cancer cells of the parent cell line GBC-SD, the isolated cells of the subpopulation with high metastatic potential, and the liver of experimental animal. RESULTS: 90% of the mice inoculated subcutaneously with the GBC-SD cells developed subcutaneous tumors, however, no mouse in this group died and no pulmonary metastasis was found. The liver metastatic rate was 65% in the 10 mice undergoing intrasplenic inoculation. Thus a metastatic model of human gallbladder cancer in nude mice was established. The liver metastatic tumors were uniformly distributed throughout the liver parenchyma with predominance to the periphery. Despite multiple sites of involvement, the left lobes were most commonly affected in all experimental animals. Histological examination of the metastatic lesion demonstrated adenocarcinoma. Gross hepatic metastasis was detected 10, 7, and 5 weeks after the inoculation respectively in the first, second, and third round selection respectively with an incidence rate of metastases of 60%, 70%, and 90% respectively. From the third round metastatic model a subpopulation with high metastatic potential was isolated and designated as GBC-SD/M, which exhibited the similar histological characteristics as its parent cell line GBC-SD under inverted light microscopy. The three amplified bands at the sites 14S68, D18S69, and D20S199, amplified with the three pairs of specific primers for the three homo-specific microsatellites, were detected in the GBC-SD cells and GBC-SD/M cells, but not in the liver of tumor-bearing animal. CONCLUSION: A liver metastasis model of human gallbladder cancer has been established, a reliable and efficient tool for study on the metastasis of gallbladder cancer to liver in vivo. Isolated from hepatic metastasis, the line of GBC-SD/M is a subpopulation with high metastatic potential, retaining the histological properties and identification of genetic background of its parent cell line GBC-SD.

Anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection.

BACKGROUND: This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection. METHODS: ANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and HepG2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of HepG2-ANGPTL4 cells in vivo and in vitro, respectively. RESULTS: The cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4 x 10(6) infective viral grains/ml, and the rate of HepG2-ANGPTL4 cells expressing GFP was 68.45%, with an average intensity of fluorescence 31.67 times greater in HepG2-ANGPTL4 cells than in HepG2 cells. The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was higher than in HepG2-pMSCV cells (154% higher) or HepG2 cells (161% higher). The proliferation rate of HepG2-ANGPTL4 cells in vitro was obviously lower than those of HepG2-pMSCV cells and HepG2 cells (P <0.01). The mean volume and weight of tumors seeded from HepG2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from HepG2 cells and HepG2-pMSCV cells (P <0.01). CONCLUSION: A stable ANGPTL4-transfected human liver cancer cell line HepG2-ANGPTL4 has been created. The transfer of the human ANGPTL4 gene mediated by a retroviral vector is a possibly effective approach for liver cancer therapy.