Effect of serine protease inhibitor Kazal-type 1 on the proliferation of hepatocellular carcinoma cell and its mechanism / 解剖学报

[Abstract] Objective To explore the effect of serine protease inhibitor Kazal-type 1(SPINK1) on the proliferation of hepatocellular carcinoma cells RH-35 and its underling molecular mechanism. Methods Spink1 gene expression in liver cancer and rat liver cancer models were analyzed by Gene Expression Omnibus (GEO) data, RH-35 cells were treated with rrSPINK1 protein, the effect of rrSPINK1 on the proliferation and apoptosis of RH-35 cells was explored by MTT, 2’-deoxy-5-ethynyluridine(EdU) and flow cytometry, the molecular mechanism of SPINK1 regulating liver cancer were detected by Real-time PCR and Western blotting. Results The results showed that Spink1 gene was over-expressed significantly in liver cancer and rat liver cancer models, rrSPINK1-treated RH-35 cells showed increased viability, EdUpositive cell rate, and the proportion of cells in S phase and G

Antitumor effects of mutant endostatin are enhanced by Bcl-2 antisense oligonucleotides in UM-UC-3 bladder cancer cell line / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endostatin is a potent inhibitor of tumor angiogenesis. In the preliminary studies, we developed a mutant endostatin containing Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) sequences. In this study, we compared the antitumor effects of mutant endostatin and Bcl-2 antisense oligonucleotides both in combination and individually.</p><p><b>METHODS</b>The artificially synthesized Bcl-2 ASODN (antisense oligonucleotides) included a translation-initiation site and was transfected into the bladder cancer cells by Lipofectamine. Cell growth was investigated by the tumor cell growth chart, MTT assay, caspase-3 activity detection assay, AO/EB fluorescein stain, and the annexin V-FITC apoptosis detection assay. In the in vivo study, UM-UC-3 bladder cancer cells were subcutaneously implanted into nude mice and the growth of tumor was examined. The ultrastructure of the tumor tissues in the treated and control groups were observed.</p><p><b>RESULTS</b>The cell growth chart showed that the cell population of the treated combination group decreased by 52.04% compared to the control group. The inhibition rate of the treated combination group was (79.66 ± 6.79)%, whereas those of the individual ASODN and ES groups were (53.39 ± 3.22)% and (50.22 ± 5.46)% respectively. In the caspase-3 activity detection using AO/EB fluorescein stain and annexin V-FITC apoptosis detection assay, the co-inhibitory effect was higher than the individual inhibitory effects (P < 0.05). There were significant differences in the inhibition of the solid tumor growth in the in vivo study.</p><p><b>CONCLUSIONS</b>Our findings indicated that Bcl-2 antisense oligonucleotides enhance the antitumor effects of mutant endostatin both in vitro and in vivo. We noted the synergistic effects of Bcl-2 antisense oligonucleotides combined with mutant endostatin.</p>

The genetic stability of recombinant adenovirus expressing human rotavirus VP6 gene which used Ad41 as vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the genetic stability of non-replicating recombinant adenovirus which used Ad41 as vector and could express VP6 gene of group A rotavirus during continous passage, in order to develop the vaccine of rotavirus.</p><p><b>METHODS</b>The recombinant adenovirus rvAd41-VP6 (o) was prepared by our laboratory early, it then was continuously propagated on 293TE7 cells for 14 passages. After that samples of the infected cells were collected at every 2 passages for the detection of the integration of the VP6 gene by PCR, and the expression of the target protein was detected by Western Blot analysis.</p><p><b>RESULTS</b>Analysis by PCR revealed that, there was stable integration of specific VP6 gene in the rvAd41-VP6 (o), Western Blot analysis confirmed that rvAd41-VP6 (o) could stably expressed the group-specific antigen structural protein VP6 (o), and it had preferable genetic stability.</p><p><b>CONCLUSION</b>The recombinant adenovirus rvAd41-VP6 (o) which could stably express the VP6 (o) gene had favorable biological property in vitro, and it has provided a basis for further research of animal immunization.</p>

Expression of nucleostemin in prostate cancer and its effect on the proliferation of PC-3 cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Nucleostemin is essential for the proliferation and survival of stem and cancer cells, but it is unknown whether this newly identified molecule is involved in prostate cancer pathogenesis.</p><p><b>METHODS</b>Total RNA and protein were extracted from prostate cancer tissues and PC-3, LNCap and DU145 cell lines. The nucleostemin mRNA and protein expression were measured by RT-PCR and Western blot. Immunohistochemistry was also used to detect the nucleostemin protein expression in prostate cancer tissues and PC-3 cells. A nucleostemin specific, short hairpin RNA, expression plasmid was used to transfect PC-3 cells. The changes of nucleostemin gene were detected and the proliferative capacity of the cells was determined.</p><p><b>RESULTS</b>Nucleostemin was highly expressed in prostate cancer tissues and cell lines. Nucleostemin expression level in the silencer group PC-3 cells remarkably reduced. The proliferation rate of silencer group PC-3 cells decreased and the percentage of G1 stage cells increased. The neoplasm forming capacity in nude mice of the silencer group PC-3 cells decreased significantly.</p><p><b>CONCLUSIONS</b>Nucleostemin is highly expressed in prostate cancer tissues and cell lines. The proliferative capacity of PC-3 cells is remarkably reduced after silencing nucleostemin gene expression.</p>

Inhibitory effect of silencing STAT3 gene with short hairpin RNA mediated by polyamidoamine dendrimers on growth of prostate cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the possibility of using generation 5 polyamidoamine dendrimers (G5-PAMAM-D) as gene vector for eukaryotic expression plasmid of siRNA in prostate carcinoma in vitro and vivo.</p><p><b>METHODS</b>Firstly, eukaryotic expression vector of siRNA pSilencing 4.1-EGFP-shRNA, specific for enhanced green fluorescent protein (EGFP), pSilencing 4.1-STAT3-shRNA for signal transducers and activators of transcription 3 (STAT3) was constructed. pEGFP-C1 and pSilencing 4.1-EGFP-shRNA were cotransfected into prostate cancer cells PC-3 and 22Rv1 with G5-PAPAM-D as vector, and to observe silencing of EGFP. Next, pSilencing 4.1-STAT3-shRNA was transfected into PC-3 and 22Rv1 cells by G5-PAPAM-D, Western blotting and apoptosis staining was used to detect silencing of STAT3 and growth inhibition. Thirdly, BALB/C mice subcutaneous tumor model was made with PC-3 cells. Polyplex of G5-PAMAM-D and pSilencing 4.1-STAT3-shRNA was injected intratumorally. The tumor volume was measured and recorded.</p><p><b>RESULTS</b>Fluorescence detection and Western blotting analysis demonstrated that G5-PAMAM-D was able to deliver Silencing 4.1-EGFP-shRNA and pSilencing 4.1-STAT3-shRNA into the two prostate cancer cell lines, and shRNA was expressed to induce silence of EGFP and STAT3. MTT results showed that proliferation of prostate cancer cells was suppressed by G5-PAMAM-D/pSilencing 4.1-STAT3-shRNA and induced apoptosis of PC-3 cells in vitro. Human prostate cancer in mice was successfully formed by inoculation of PC-3 cells into male BABL/C mice. In G5-PAMAM-D/pSilencing 4.1-STAT3-shRNA treated group, the tumor volume was shrank remarkably at 9 days after treatment and tumor growth was retarded compared with control groups.</p><p><b>CONCLUSION</b>GS-PAMAM-D nanoparticles can be used to deliver plasmid vector expressing shRNA into prostate cancer cells effectively in vitro and vivo. It appears to be a promising gene vector for RNA interference therapy in prostate cancer.</p>

Dynamics of in vitro amyloid fiber formation of yeast prion protein Sup35NM / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the dynamics of amyloid fiber formation of yeast (Saccharomyces cerevisiae) prion protein Sup35NM under the native condition to provide materials and clues for the elucidation of amyloid fiber formation.</p><p><b>METHODS</b>The Sup35NM gene was cloned and expressed in E. coli. The recombinant Sup35NM protein was purified under denaturing conditions through Nickel-Sepharose chromatography. Aliquots were removed at designated time points for transmission electron microscopy (TEM), circular dichroism (CD) spectra, protease K resistance assay, as well as thioflavin T (ThT) binding assay.</p><p><b>RESULTS</b>The Sup35NM expressed and purified under denaturing conditions. The morphological alteration of the Sup35NM in PBS (pH7.4) during the protein aggregation and amyloid fiber formation was visualized by TEM. The CD assay showed that the course of amyloid fiber formation underwent a conformational shift from alpha-helix to beta-sheet. The fibers had higher capacity of resistance to protease K digestion compared to the monomers. ThT fluorescence assay displayed a rapid growth phase before reaching a final equilibrium phase during the fiber formation, and the higher concentration of Sup35NM could greatly accelerate the fiber formation in vitro.</p><p><b>CONCLUSION</b>Yeast prion protein Sup35NM forms amyloid readily under native conditions in vitro. The dynamics of Sup35NM amyloid formation may provide supporting evidences for the nucleating polymerization models of amyloid fiber formation.</p>

Receptor-binding ability of fragments 260-600 and 397-796 of SARS-associated coronavirus spike protein / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the interaction between the host cell and the truncated S fragments to identify the receptor-binding domain of the spike (S) protein of SARS-associated coronavirus (SARS-CoV).</p><p><b>METHODS</b>Two different fragments S260-600 and S397-796 of the SARS-CoV S protein were expressed in Escherichia coli (E.coli) using a pET expression vector, respectively. The two recombinant proteins were separately verified by Western blot, purified by nickel-affinity chromatography, and incubated with Vero cells, a susceptible cell line of SARS-CoV infection, for cell binding assay. After the sequential probing with sera from convalescent SARS-patients and FITC-labeled anti-human IgG, the cells were analyzed by flow cytometry. The NIH 3T3 cell, a non-permissive cell line of SARS-CoV infection, was used as controls.</p><p><b>RESULTS</b>The recombinant proteins S260-600 and S397-796 were efficiently expressed in an insoluble form in E.coli. The appropriate expression of the proteins was confirmed by Western blotting using both SARS patients' sera and anti-6 x histidine antibody. The flow cytometry results showed that the both proteins were able to bind Vero cells, but the binding ability of S260-600 was somewhat stronger than that of S397-796. In contrast, the S260-600 protein did not bind NIH3T3 cells.</p><p><b>CONCLUSION</b>Both S260-600 and S397-796 exhibited different receptor binding activity. The S260-600 fragment probably contains the important receptor binding domain and could be a potential candidate for the development of SARS vaccine and anti-SARS therapeutics.</p>

Study of antileukemic effect of CpG-oligodeoxynucleotides treated cord blood / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the antileukemic effect of lymphocytes from cord blood treated by CpG-oligodeoxynucleotides (CpG-ODN).</p><p><b>METHODS</b>Lymphocytes from cord blood were exposed to different oligodeoxynucleotides containing a panel of CpG-ODN and were cultured with K562 cells. The cytotoxic effects were detected by MTT method. Immunological markers of cord blood treated by CpG-ODN(3) which showed highest activity were measured with flow cytometry.</p><p><b>RESULTS</b>Different CpG-motifs have different immunostimulatory activity and CpG-ODN(3) has the highest one. After treated by CpG-ODN(3), NK killing activity to K562 cells increased in a dose-dependent manner, and CD(3), CD(4), CD(19) and CD(56) increased to (60.6 +/- 7.9)%, (40.2 +/- 3.5)%, (22.4 +/- 1.9)% and (15.5 +/- 3.1)%, respectively.</p><p><b>CONCLUSION</b>CpG-ODN could reinforces the immunological competence of cord blood lymphocytes and their effects on K562 cells. This provides a new approach to reinforce the antitumor effects of cord blood.</p>

The construction of SCID-Hu IC mice model and application in rAd5HPV16L1-E7 vaccine / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct human-SCID chimeric mice through implantation of mononuclear cells from human cord blood and study the immunoreaction of SCID-Hu IC mice immunized with rAd5HPV16L1-E7 vaccine.</p><p><b>METHODS</b>(1) Experiment groups were injected with the suspension of mononuclear cells from human cord blood through a tail vein; the control ones were injected with non serum RPMI 1640 medium. Eight weeks after implantation, blood was collected and human serum IgG level in the mice were tested, and human CD45, CD3 and CD19 were determined. (2) SCID-Hu IC mice were divided into two groups: in group A the mice were immunized intraperitoneally with rAd5HPV16L1-E7 virus and in group B the mice were immunized through nasal drip with rAd5HPV16L1-E7 virus. At the end of fourth week, the serum specific IgG antibody to rAd5HPV16L1-E7 virus, IFN-gamma in culture medium of spleen lymphocyte and T-lymphocyte propagation were tested.</p><p><b>RESULTS</b>(1) In the experiment groups, the number of mice positive for human IgG was 10/15, the average values of CD45, CD3 and CD19 were (9.39+/-4.21), (3.25+/-3.99) and (1.69+/-0.75), respectively. In the control ones, the human IgG, CD45, CD3 and CD19 were negative. (2) The results in the experiment groups showed that the IFN-gamma and T-lymphocyte stimulated by HPV16 protein were higher than those in the non-stimulated group (P less than 0.05).</p><p><b>CONCLUSION</b>(1) The results indicated that the construction of human-SCID chimaera through the implantation of mononuclear cells from human cord blood into SCID mice was successful. They also indicated that the reconstructed SCID-Hu IC mice has the ability to produce immune response against rAd5HPV16L1-E7 recombinant virus.</p>