Comparison on DNA barcodes of nuclear and chloroplast gene fragments in Rehmannia Libosch. ex Fisch. et Mey. (Rehmanniaceae) / 中草药

Objective: To compare the usage of single-copy nuclear genes (SCNGs), chloroplast gene fragments and internal transcribed spacer (ITS) as DNA barcodes in plants of Rehmannia Libosch. ex Fisch. et Mey. and elucidate the interspecific relationships in the genus. Methods: Distance and tree-based methods were performed to analyze 12 SCNGs, six cpDNA regions, and ITS fragment in 13 populations of Rehmannia Libosch. ex Fisch. et Mey. Neighbor-joining tree was constructed to investigate the relationships among the species. Results: CpDNA regions (matK and rbcL) and SCNGs had lower identification rates than others, especially ITS. Multiple DNA barcodes combination would be helpful to improve the species identification rate. The NJ tree indicated that R. glutinosa and R. solanifolia were clustered together, other species in the genus were monophyletic, and R. piasezkii was the basal group. Conclusion: ITS should be used as the core barcode in the genus while psbA-trnH, trnL-F, trnM-V, and trnS-G would be considered as the candidates, and nuclear genes R255 and R257 might be utilized as unique barcode for the identification of R. glutinosa. The origin of tetraploid species (R. glutinosa and R. solanifolia) still remains unknown and further works should be done to solve the question.

Screening and identification of genes associated with multi-drug resistance in colonic cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To identify novel multi-drug resistance-related genes, and to explore the mechanisms of multi-drug resistance.</p><p><b>METHODS</b>Multi-drug resistant cell line Lovo/5-FU was established by incubation with increasing dose of 5-FU. The sensitivity to 5-FU and cis-diaminodichloroplatinum (CDDP) was measured by MTT assay. Two dimensional electrophoresis plus mass spectrum(2-DE/MS) was used to identify the differentially expressed protein between Lovo and Lovo/5-FU. The identified protein was then verified by Western blot analysis.</p><p><b>RESULTS</b>The IC50 concentrations of Lovo/5-FU to 5-FU and CDDP were increased by 31 and 3 times, compared with Lovo (both P<0.01). 2DE-MS showed that CAP-G and RhoGDI2 were up-regulated, whereas 6-PGL, DCI, Prdx-6 and Maspin were down-regulated in Lovo/5-FU. Western blot analysis confirmed that the expression levels of RhoGDI2 and CAP-G in Lovo/5-FU were increased by 6.14 and 2.98 fold respectively (both P<0.01), whereas Maspin was decreased to 5.2% of Lovo(P<0.01).</p><p><b>CONCLUSIONS</b>Multi-gene and multi-pathway are involved in the development of multi-drug resistance of colorectal cancer cells. CAP-G, RhoGDI2 and Maspin are potential multi-drug resistant genes.</p>

Silencing invariant chains of dendritic cells enhances anti-tumor immunity using small-interfering RNA / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Genetic modification of dendritic cells (DCs) has been used as an effective approach to enhance anti-tumor immunity. RNA interference (RNAi), which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA (siRNA) specific for the Ii gene was transfected into DCs, and the anti-tumor immunity of Ii-silenced DCs was assessed.</p><p><b>METHODS</b>The silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96(®) non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used.</p><p><b>RESULTS</b>The Ii expression of DCs was significantly reduced after Ii siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with Ii siRNA plus endogenous tumor antigen (P < 0.05). Furthermore, tumor growth was greatly inhibited when mice were immunized with DCs transfected with Ii siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4(+) and CD8(+) T cells were significantly activated in the Ii siRNA group (P < 0.05).</p><p><b>CONCLUSION</b>Silencing of the Ii gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.</p>

Isoflavone glycosides from the bark of Maackia amurensis / 药学学报

To study the chemical constituents of the Maackia amurensis, the constituents were isolated by various chromatographies and the structures were elucidated on the basis of chemical and spectroscopic data (ESI-MS, 1D and 2D NMR). Thirteen isoflavone glycosides were isolated from the n-BuOH-soluble fraction of the 70% ethanol extract and identified as 7-hydroxy-4',6-dimethoxyisoflavone-7-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (1), dalsympathetin (2), ononin (3), glycitin (4), genestin (5), saikoisoflavonoside A (6), afrormosin-7-O-beta-D-glucopyranoside (7), gehuain (8), kushenol O (9), 7-hydroxy-4',6-dimethoxyisoflavone-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (10), tectoridin (11), biochanin A 7-O-beta-D-gentiobioside (12) and 7-hydroxy-4'-methoxyisoflavone-7-O-beta-D-apiofuranosy l-(1-->6)-beta-D-glucopyranoside (13). Compound 1 is a new isoflavone glycoside, named as maackiaisoflavonoside, compounds 2, 8, 9, 10, 12 and 13 were isolated from this genus for the first time.

Analysis of human gastric cancer by transcriptome and proteome profiling / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To screen differential expression genes and proteins at transcriptome and proteome levels between human gastric cancer tissue and corresponding normal mucosa.</p><p><b>METHODS</b>Fresh-frozen gastric cancers were collected from patients treated at Ruijin Hospital. A total of 22 pairs of gastric cancer tissues and the corresponding noncancerous mucosa were analyzed. Commercially available cDNA microarray with 14 592 genes/ESTs was used. Genes were considered to be up-or down-regulated when the intensity ratio Cy3/Cy5 was > or = 2 or < or = 0.5 in over 50% samples (P<0.05). Immobilized pH gradient(IPG)-based 2-DE was applied to separate the total proteins of gastric cancer tissue and paired normal tissue. After staining and analysis by software,the differential expression proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS.</p><p><b>RESULTS</b>As compared with corresponding noncancerous tissue, there were totally 149 up-regulating genes/ESTs and 238 down-regulating genes/ESTs in gastric cancer, including 29 genes with 3-fold over-expression ratio and 21 genes with 5-fold under-expression. Fifteen protein spots were identified successfully, among whom there were ten over-expressed and five under-expressed proteins in gastric cancer tissue compared with normal tissue. Most of over-expressed genes and proteins were related to cell motility, cell proliferation, signal transduction, while those under-expressed genes and proteins were related to defense response, toxoid metabolism.</p><p><b>CONCLUSION</b>Studying gastric cancer at transcriptome and proteome levels can help demonstrate tumorigenesis and biological characteristics of gastric cancer comprehensively and provide powerful tools to find new biomarkers associated with gastric cancer and therapy targets.</p>

Galectin-9 isoforms influence the adhesion between colon carcinoma LoVo cells and human umbilical vein endothelial cells in vitro by regulating the expression of E-selectin in LoVo cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the regulatory effect of galectin-9 isoforms on some molecules involved in cell adhesion/invasion, and the influence of this regulation action on the adhesion between colon carcinoma LoVo cells and human umbilical vein endothelial cells (HUVECs) in vitro.</p><p><b>METHODS</b>Various expression vectors of galectin-9 isoforms were transfected into LoVo cells. 24 h after transfection, the expression of integrin-beta1, E-cadherin, E-selectin, ICAM-1, CD44 and MMP-9 was detected by RT-PCR and Western blot analysis. LoVo cell-HUVEC adhesion assay was performed under conditions of galectin-9 transfection, galectin-9 transfection + galectin-9 antibody, galectin-9 transfection + E-selectin antibody and galectin-9 transfection + beta-lactose, respectively.</p><p><b>RESULTS</b>Galectin-9L down-regulates the mRNA and protein levels of E-selectin while galectin-9M and galectin-9S up-regulate the expression of E-selectin. In LoVo cell-HUVEC adhesion assay, the average fluorescence intensity of vector transfection group, galectin-9L transfection group, galectin-9M transfection group and galectin-9S transfection group was 0.90 +/- 0.20, 0.94 +/- 0.24, 1.60 +/- 0.11 and 1.45 +/- 0.13, respectively, indicating that galectin-9M and galectin-9S facilitated the adherence of LoVo cells to HUVECs (P < 0.05). E-selectin antibody, galectin-9 antibody or beta-lactose inhibited that effect.</p><p><b>CONCLUSION</b>Galectin-9 isoforms regulate the E-selectin expression in LoVo cells differently and thus influence the adhesion between LoVo cells and HUVECs in vitro in different modes. The mechanisms through which galectin-9 isoforms participate in the metastasis process of colon cancer may not be the same.</p>

Hedgehog signaling pathway activates in gastric carcinoma and promotes the proliferation through GLI1 in MKN28 cell / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of Hedgehog (HH) pathway on proliferation and in vitro tumorigenicity of gastric cancer cell lines.</p><p><b>METHODS</b>The expression of SHH, PTCH, SMO, SUFU and GLI1 in seven cell lines were tested by RT-PCR. siRNA targeting GLI1 mRNA was transfected into MKN28 cells. Cell proliferation and in vitro tumorigenicity were examined by CCK8 and soft agar colony formation test.</p><p><b>RESULTS</b>SHH in six gastric cancer cell lines was up-regulated. Expression of PTCH in KATOIII cell lines and expression of SUFU in MKN28 and KATOIII were reduced. GLI1 siRNA significantly inhibited the expression of GLI1 in MKN28 cell line. Growth rate and colony formation rate of MKN28 cells treated with GLI1 siRNA were significantly lower than those of control cells (all P <0.001).</p><p><b>CONCLUSIONS</b>HH signaling pathway is widely activated in gastric cancer cell lines. The activation of HH signaling pathway promotes the growth of MKN28 cells.</p>

Expression and intracellular localization of FRZB gene in gastric cancer and its significance / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study the expression and intracellular localization of FRZB gene in gastric cancer tissue, and to explore its significance in gastric cancer.</p><p><b>METHODS</b>The expression of FRZB in tumor tissues from 90 patients with gastric cancer and in normal gastric mucous as control were analyzed by immunohistochemistry in tissue array. FRZB expression in gastric cancer cell lines and immortalized gastric epithelial cell line GES-1 were detected by quantitative real-time PCR(Q-PCR) and Western blot. The intracellular localization of FRZB was observed by immunofluorescence staining.</p><p><b>RESULTS</b>The positive expression rate of FRZB in gastric cancer was 92.2%. FRZB expressed in gastric cancer with well differentiation was higher than that with poor differentiation.The positive rate in normal gastric mucous was 10.0% (one out of ten). By confocal microscope, FRZB localized both in cytoplasma and nucleus, especially on the nuclear membrane. The Q-PCR and Western blot results also showed that the expression of FRZB in gastric cancer cell lines was higher than that in GES-1.</p><p><b>CONCLUSIONS</b>The expression of FRZB in gastric cancer is correlated with tumor cell differentiation and tumor Lauren classification. The nuclear localization of FRZB may contribute to its function in gastric cancer formation and progression.</p>

Inhibition of the proliferation of human gastric cancer cells SGC-7901 in vitro and in vivo using Bcl-2 siRNA / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Bcl-2, the anti-apoptotic protein is overexpressed in the majority of gastric cancers and associated with its pathogenesis. To better understanding of the role of Bcl-2, RNA interference (RNAi) was used to inhibit Bcl-2 expression in the human gastric cancer cells in vitro and in vivo.</p><p><b>METHODS</b>Bcl-2 small interfering RNA (siRNA) was transfected into human gastric cancer cells SGC-7901, and Bcl-2 expression was monitored by real-time polymerase chain reaction (PCR) and Western blot. Cell proliferation, apoptosis, and telomerase activity were examined by MTT, flow cytometry, and TRAP assay, respectively. Gastric cancer cells treated with 100 nmol/L Bcl-2 siRNA were subcutaneously transplanted into nude mice and tumor growth was assessed.</p><p><b>RESULTS</b>Bcl-2 siRNA significantly inhibited the expression of Bcl-2 in human gastric cancer cells at both mRNA and protein levels in a time- and dose-dependent manner. Bcl-2 siRNA also decreased telomerase activity (by 78.76%) and increased the rate of apoptosis (by 37.47%). SGC-7901 cell growth was also significantly suppressed in vivo and in vitro.</p><p><b>CONCLUSIONS</b>Bcl-2 expression knockdown suppressed the growth of gastric cancer cells. Thus, Bcl-2 may play a very important role in carcinogenesis of gastric cancer and its knockdown may offer a new potential gene therapy approach for human gastric cancer in future.</p>

Identification of biomarkers in the serum of the patients with poorly differentiated gastric cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the application of proteomics in the field of serology,and to screen the differential expression proteins related with poorly differentiated gastric carcinoma.</p><p><b>METHODS</b>Two-dimensional electrophoresis (2-DE) was applied to segregate the total proteins in the serum form gastric cancer patients and health volunteers. After staining,the differential expression proteins were analyzed using PDQuest software,and identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS).</p><p><b>RESULTS</b>Electrophoresis figures with high resolution and reproducibility were obtained. Six differential expression proteins were found only in the serum from gastric cancer patients, while four other proteins from healthy volunteers.</p><p><b>CONCLUSIONS</b>Protein expression is differential in the serum from the gastric cancer patients and health volunteers. It is hopeful to find the biomarkers related with poorly differentiated gastric carcinoma using proteomics.</p>

Expression of Ezrin in gastric carcinoma and its significance / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of Ezrin in gastric cancer, its role in tumor metastasis.</p><p><b>METHODS</b>Ezrin expression in tumor tissues from 90 gastric cancer cases and in normal gastric mucosa from 12 cases with benign disease was examined by immunohistochemical staining. Ezrin expression in gastric cancer cell lines was also detected by Western blot, and in vitro invasion assay was used to examine the invasive ability of the cell lines.</p><p><b>RESULTS</b>The expression rate of Ezrin was significantly higher in gastric cancer tissues than that in normal tissues (P< 0.05), and significantly correlated with lymph node metastasis (P< 0.05). Western blot showed that MKN-45 cell line had the highest expression of Ezrin among 5 gastric cancer cells. MKN-45 possessed highest invasion ability.</p><p><b>CONCLUSION</b>Ezrin expression is up-regulated, and may be associated with lymph node metastasis in gastric cancer.</p>

Expression and Immune Effect Study of Therapeutic Recombinant Protein Vaccine VP22?-mE6?/mE7 / 中国生物工程杂志

In order to investigate the biological effects of the VP22?-mE6?/mE7 built-in adjuvant fusion protein vaccine on the tumor associated with HPV-16 infection.HSV-1 VP22? and HPV-16 mE6?/mE7 genes were cloned,and the pET28a-VP22?-mE6?/mE7 recombinat prokaryotic expression vector was constructed.Vector was transformed into Rosetta(DE3)E.coli string and expressed under the induction of IPTG.The re-naturalized protein was then purified via Ni2+ affinity adsorbent column and identified by SDS-PAGE and Western blot.Purified protein was immunized BalB/C and C57BL/6 mice to evaluate the immunogenicity and anti-tumor activity.The expressed recombinant protein formed as inclusion body with a prediction MW about 34kDa and contained approximately 45% of total somatic protein.The VP22?-mE6?/mE7 immunization induced higher titer of specific IgG against HPV,higher level of lymphocyte proliferation and better effect on suppressing HPV16 positive TC-1 tumor growth than the mice immunized with mE6?/mE7 alone.The results showed that the recombined built-in adjuvant vaccine could induce specific cellular immune response in vitro and inhibit the TC-1 tumor proliferation in vivo,that would be a foundation for further studies and developments of inner adjuvant vaccines.

Expression and Purification of a Novel Influenza Virus Subunit Vaccine / 中国生物工程杂志

The influenza A virus matrix protein2 gene(M2)which deleted transmembrane region was amplified by overlap extending PCR,and the multiepitope gene of hemagglutinin(HA)was PCR amplified with seven continuous synthesized segments by designing primer.The two gene segments were separately cloned into pMD18T vector to sequence analysis and prokarytic expression vector pET28a+ to construct the recombinant plasmid pET28a+M2dHA.The recombinant plasmid was transformed into E.coli BL21(DE3),and the high expression strain was obtained by screening monoclones.The recombinant protein existed as inclusion bodies,which accounted about 45% of the total cellular protein.The inclusion bodies were washed with 1% Triton X100 solution twice,and dissolved in 8 mol/L urea solution.The solution protein was purified by Ni+2 affinity chromatography,and refolded by dilution renaturation,then purified by Q Sepharose FF cation exchange column.The purity of the protein was over 90% by HPLC analysis.The result of Western blot showed it has good antigenicity and specificity.These results strongly supported for the further study of the broadspectrum influenza virus subunit vaccine.

Expression and clinical significance of cancer-related gene MPS-1 in gastric cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of cancer-related gene MPS-1 in gastric cancer and to evaluate its significance in clinical diagnosis and therapy.</p><p><b>METHODS</b>The mRNA expression of MPS-1 was determined by polymerase chain reaction after reverse transcription (RT-PCR) in cancer tissues and adjacent non-cancerous tissues from 42 cases with gastric cancer. The expression levels of MPS-1 in 6 gastric cancer cell lines (AGS, MKN-45, SGC 7901, KATO III, N-87 and SNU-1) were also determined by RT-PCR and Western blot.</p><p><b>RESULTS</b>The MPS-1 mRNA was expressed in all tissues and cell lines. The mRNA expression level of MPS-1 in cancer tissues were 1.37+/- 0.87, significantly higher than 0.99+/- 0.67 in adjacent normal gastric mucous tissues (P< 0.01). The expression of MPS-1 was correlated with TNM stage (P< 0.05), but not with age, gender, tumor size and differentiation. The expression level of MPS-1 mRNA in the primary lesions was hig her in the patients with TNM stages III, IV than those with TNM stages I, II. Meanwhile, RT-PCR and Western blot showed the same results that MPS-1 expression was higher in the six gastric cancer cell lines as compared with that in the normal gastric cell line GES-1.</p><p><b>CONCLUSION</b>The high expression of MPS-1 in gastric cancer indicates that MPS-1 might play an important role in gastric carcinogenesis,which may provide a new target in immunotherapy for gastric cancer.</p>