Objective To explore the objectivity and scientificity of fecal sampling , and to provide reference for investigating the relationship between intestinal microbes and diseases . Methods Terminal restriction fragment length polymorphism, degeneration gradient gel electrophoresis and real time fluorescent quantitative PCR techniques were applied to differentially analyze the bacterial community composition and abundance of intestinal contents and feces taken from dif -ferent sites of BALB/c mouse intestine .Results The predominant T-RFLP fragments ( T-RFs) in feces in the rectum and colon were 244 bp, 255 bp and 449 bp, however , those in feces of the small intestine including duodenum , jejunum and il-eum were 60 bp, 73 bp, 261 bp, 268 bp and 272 bp, and with a larger variation of the bacterial community composition in various parts of the small intestine .The bacterial abundance in the contents of duodenum and jejunum were 6.9 log ( cop-ies)/g and 8.3 log (copies)/g, fewer than in the other parts of the intestine , while the bacterial abundance in the feces was as high as 11.8 log (copies)/g, being about 2 times higher than that in the duodenum and jejunum (P<0.05), and similar to that in the ileum and colon content (P>0.05).Conclusions The inter-mouse variations of bacterial communi-ty composition in the large intestine contents are small .The bacterial composition and abundance are similar suggest that studies on the relationship between large intestine especially colorectal microbiota and diseases may be conducted via fecal sampling.

This study is to investigate the inhibitory effect of lidamycin (LDM) and its combination with methotrexate (MTX) on lung metastasis of fibrosarcoma by bioluminescence imaging in athymic mice. A stable luciferase transfected HT-1080 cell line was constructed and the capability to establish experimental lung metastasis in athymic mice was confirmed. The optical imaging system was applied to evaluate the formation of lung metastasis in vivo. In addition, metastatic nodules were counted for the evaluation of inhibition rates. As shown, the fluorescent intensity of luciferase-transfected HT-1080 cells was colinear with the cell population and the minimal detected cell population was 100 cells/well. Optical imaging showed that the fluorescent intensity of treated group was apparently lower than that of the control. The inhibition rates of lung metastasis by LDM alone at 0.025 mg x kg(-1) and 0.05 mg x kg(-1) were 53.9% and 75.9%, respectively, while that of MTX alone at 0.5 mg x kg(-1) was 70.2%. The combination of LDM at 0.025 mg x kg(-1) and MTX at 0.5 mg x kg(-1) showed an inhibition rate of 88.7%. The coefficient of drug interaction (CDI) was 0.82. The results herein demonstrated that LDM alone had strong anti-metastasis effect on human fibrosarcoma HT-1080 and the inhibition efficacy is strengthened when combined with MTX.

Objective To explore the impacts of over-expression of microRNA-7 (miRNA-7) on the sensitivity of cis-platin in esophageal carcinoma cell line TE-1, and the possible mechanism thereof. Methods Lipofectmin 2000 method was used to transient transfect with miRNA-7 mimic into esophageal cancer cell line TE-1, which was taken as transfection group, mimic negative control was taken as transfection conrtol group. The expressions of miRNA-7 and epidermal growth factor receptor (EGFR) mRNA were detected by RT-PCR in the above two groups and normal control group. The total EGFR and EGFR in cytoplasmic and nucleus were detected with Western blot assay in transfection group and transfection control group. CCK-8 was used to detect IC50 of cisplatin in transfection group and transfection control group. The expression of EGFR was observed with immunofluorescence confocal microscope in two groups. Results The miRNA-7 expression was signifi-cantly increased in transfection group than that of transfection conrtol group and control group. The expression of EGFR mRNA was significantly reduced in transfection group (P<0.001). The total EGFR was significantly decreased in transfec-tion group than that of transfection conrtol group. The level of nuclear EGFR was significantly increased ( P<0.01),and cyto-plasm EGFR expression was significantly decreased in transfection group than that of transfection control group ( P<0.05). CCK-8 results showed that after the over expression of miRNA-7 in TE-1, the IC50 of cisplatin (48 h) increased in transfec-tion group than that of control group (P<0.01). Immunofluorescence results showed that EGFG in nuclear was higher in transfection group than that of transfection control group but its expressions reduced in cell membrane and cytoplasm. Con-clusion The over-expressed miRNA-7 in esophageal cancer cells TE-1 can reduce cisplatin sensitivity by the increased EGFR in nuclear translocation.

Objective To investigate the antitumor effects of IL-18 gene transfected T cells on pancreatic cancer cell SW-1990.Methods Construction of IL-18 contained recombinant lentivirus using PCR method and packaged at HEK293T cell, then transfected the human T cells and evaluated the antitumor effects when cocultured with SW-1990.Results The IL-18 gene contained recombinant lentivirus was successfully constructed and packaged, and transfected the T cells, the LDH secretion and IL-2 and IFN-γ content all increased significantly (P<0.01) when cocultured with SW-1990 cell.Conclusions T cells transfected with IL-18 possessed more potent antitumor effects to pancreatic cancer cell SW-1990 as compared to the regular T cells.

Objective To investigate the difference of effect between interventional treatments and intravenous therapy of lidamycin on VX2 rabbit liver cancer.Methods VX2 Carcinoma cells were surgically implanted into the left liver lobe of 12 New Zealand white rabbits to establish the VX2 rabbit liver tumor model.Tumor size was detected by type-B ultrasonic diagnostic instrument.The rabbits were randomly divided into two groups of six,respectively treated with the hepatic inter-ventional administration of lidamycin (LDM)(1 ml,0.05 mg/kg)under the guidance of digital subtraction angiography (DSA)(group A)and with the auricular intravenous administration of LDMat the same dose (group B).All the rabbits were sacrificed and anatomized on day 10 after treatment,whose liver tumor was fixed with 4% paraformaldehyde solution and embedded in paraffin.Proliferating cell nuclear antigen (PCNA)and CD34 expression in the sample sections of tumor tissue were assessed through immunohistochemical staining.The levels of alanine transaminase (ALT)and aspartate trans-aminase(AST)were detected by Cobas 8000.Finally,the inhibition of VX2 tumor was evaluated.Results The VX2 tumor volumes were all increased at 10 day after LDMtreatment.However,the tumors in group A were smaller than those of group B (P <0.05).The results of immunohistochemistry showed that the intervention therapy of LDM could further lower the expression of CD34 and PCNA compared to group B.Conclusion Hepatic interventional administration of LDM under the guidance of DSA produces a better effect on attenuating the tumor growth than the intravenous administration of LDM.

To improve thrombolytic effect, a fusion protein SFH composed of staphylokinase (SAK) and hirudin (HV) with blood coagulation factor Xa (FXa) recognition peptide as a linker, was designed. SFH showed improved thrombolytic effect and low bleeding in vivo. Two thrombus-targeting mechanisms might account for the above features of SFH. This study was designed to study the two thrombus-targeting mechanisms of SFH. ELISA and immunohistochemistry assay were used to study the improved thrombus selectivity of SFH and the results showed that SFH, compared with SAK, displayed higher affinity for thrombin and thrombin-rich thrombus. To verify the thrombus-targeting release of anticoagulant activity of SFH, FH-a derivative of HV with only FXa recognition sequence at N terminus of HV was designed and used in animal tests. In inferior vena cava thrombosis model, FH showed equal antithrombotic effect as HV, indicating that HV could be successfully released from FH by FXa cleavage in vivo. More importantly, no prolongation of plasma TT, APTT and PT were found in FH group, but significant prolongations were discovered in HV group. This revealed that the anticoagulant activity of FH was released in thrombus-targeting way and limited in the vicinity of the thrombus, and this could be extrapolated to SFH. In conclusion, the high thrombus affinity and thrombus-targeting release of anticoagulant activity of SFH assigned low bleeding risk to SFH.
Animals ; Anticoagulants ; pharmacology ; Factor X ; pharmacology ; Hirudins ; biosynthesis ; genetics ; Metalloendopeptidases ; biosynthesis ; genetics ; Mice ; Rats ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Thrombolytic Therapy ; methods ; Thrombosis ; drug therapy ; Vena Cava, Inferior

[Abstract] Objective: To investigate the effects of interleukin-18 over-expression on the in vitro and in vivo proliferation of human colorectal cancer (CRC) HCT-116 cells. Methods: A recombinant lentivirus vector containing human IL-18 gene fragment was constructed. Then theCRC HCT-116 cell line stably expressing human IL-18 (HCT-116/IL-18) was obtained by recombinant lentivirus transfection. In vitro proliferation of HCT-116/IL-18 cells and wild-type HCT-116 cells was determined by CCK-8 method. The expressions of IL-18, Cyclin D1, proliferating cell nuclear antigen (PCNA) and DNA damage repair enzyme (PARP) were detected by Western blotting. HCT-116 and HCT-116/IL-18 cells were inoculated into left and right axillas of Balb/c nude mice, respectively. Then the tumorigenicity and the growth of transplanted tumor were observed. The expressions of IL-18 and PCNAin xenograft tissues were detected by immunohistochemistry analysis. Results: IL-18 gene over-expression in HCT-116 cells could delay the proliferation of HCT-116 cells (P<0.05 or P<0.01). PARP expression was increased significantly and PCNA, Cyclin D1 expression were decreased in HCT-116/ IL-18 cells as compared to that of HCT-116 cells (P<0.01).The tumorigenicity of HCT-116/IL-18 cell was significantly decreased in nude mice with a tumor-formation rate of 43%; Compared with control group, HCT-116/IL-18cell line had a longer tumorigensis time, slower growth and smaller tumor volume; moreover, PCNAprotein expression was down-regulated in HCT-116/IL-18 xenograft tissuesas shown by immunohistochemistry analysis (P<0.01). Conclusion: IL-18 over-expression inhibited the growth and proliferation of HCT-116 cells both in vitro and in vivo, and the mechanism might berelated with IL-18 regulating cell cycle and promoting DNA damage.

Objective:To explore the optimal time point for combining chemotherapy and immunotherapy and provide an experimen-tal basis for immunotherapy intervention in clinical. Methods:Twenty-three lung cancer patients who completed five chemotherapy cycles between November 2015 and December 2016 in the First Affiliated Hospital of Xinxiang Medical University were enrolled in this study. Numbers of T lymphocyte subsets, B lymphocytes, and NK lymphocytes in peripheral blood were counted. Expression levels of T lymphocyte co-suppression molecule and cytokines in the peripheral blood mononuclear cell were detected using flow cytometry to analyze the dynamic changes of such indicators from one cycle to five cycles of chemotherapy. Results:Significant decreases in the lev-els of CD8+T lymphocytes, CD19+B lymphocytes, and CD16+CD56+NK cells and an increase in CD4+T lymphocytes were observed in the course of multi-cycle chemotherapy for patients with lung cancer. Differences were statistically significant (P<0.05). The co-suppres-sion molecular expression of PD-1, CTLA-4, and CCR-4 with T lymphocytes was downregulated, and the differences were significant (P<0.05). Conclusion:Profiling the dynamic changes of lymphocyte subsets and the expression of T lymphocyte co-suppression molecule are significant in multiple chemotherapy cycles for patients with lung cancer. In the later stage, the combined application of PD-1, PD-L1, CTLA-4, or CCR-4 antibody may exert good therapeutic effects for patients with a high expression level of related immune check-points.

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