miR-29b Reduces Cisplatin Resistance of Gastric Cancer Cell by Targeting PI3K/Akt Pathway / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the regulatory effect of miR-29b on gastric cells' resistance to cisplatin.</p><p><b>METHODS</b>The expression of miR-29b in gastric cancer cell line treated with cisplatin concentration gradient was detected using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. CCK8 was used to measure the cell viability after cisplatin treatment in condition of miR-29b knock-down and overexpression.</p><p><b>RESULTS</b>The expression of miR-29b was significantly upregualted by cisplatin treatment,while its target gene AKT2 was downregulated. The up-regulation of miR-29b enhanced the sensitivity of gastric cancer cells to cisplatin,while the knock-down of miR-29b enhanced the cisplatin resistance. Rescue experiments demonstrated that the miR-29b might regulate cisplatin resistance of gastric cancer cell by targeting PI3K/Akt pathway. The expressions of the other two members of miR-29 family, miR-29a/c, were promoted by cisplatin treatment,but they had no significant effect on gastric cancer cell's resistance to cisplatin.</p><p><b>CONCLUSION</b>miR-29b can enhance the sensitivity of S gastric cancer cell by directly targeting PI3K/Akt pathway.</p>

Establishment and preliminary application of dengue virus envelope domain III IgG antibody capture enzyme-linked immuno-absorbent assay / 中华预防医学杂志

<p><b>OBJECTIVE</b>To establish a highly sensitive and specific assay to detect dengue virus (DENV) envelope protein domain III (EDIII) IgG antibody, and to explore its value in the diagnosis and seroepidemiological survey of dengue.</p><p><b>METHODS</b>The DENV EDIII IgG antibody capture ELISA was developed using the recombinant full-length DENV EDIII, which was prepared by Pichia yeast expression system as the capture antigen. The serum samples were collected from the same group of 35 DENV-1 patients of primary infection during disease period in 2006 and their follow-up phase in 2010; and the sensitivity of the assay was compared to that of the commercial Panbio DENV IgG ELISA.</p><p><b>RESULTS</b>The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from disease period and follow-up phase was 87% (20/23) and 94% (33/35), respectively; whereas the sensitivity of Panbio DENV IgG ELISA was 71% (25/35) and 0, respectively. The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from both periods was similar, without statistical significance (χ(2) = 0.946, P = 0.331). For serum samples from disease period, the sensitivity of DENV EDIII IgG ELISA was comparable with that of Panbio DENV IgG ELISA (χ(2) = 1.924, P = 0.165). However, DENV EDIII IgG ELISA demonstrated a significantly higher sensitivity than Panbio DENV IgG ELISA in detecting the serum samples from follow-up phase (χ(2) = 62.432, P = 0.000).</p><p><b>CONCLUSION</b>DENV EDIII IgG capture ELISA is highly sensitive in detecting IgG in the serum samples from either disease period or follow-up phase. This method might be a promising alternative for diagnosis and seroepidemiologic survey of dengue.</p>

Bidirectional effect of MnSOD overexpression on the proliferation of esophageal cancer cells in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct a recombinant lentiviral vector for manganese superoxide dismutase (MnSOD) gene expression, and observe its effect on the proliferation of esophageal cancer cells in vitro.</p><p><b>METHODS</b>Chemical methods were employed for synthesis of the MnSOD cDNA sequence sections, along with the attB sites. Target gene fragment was constructed on the pMD-18T vector, and the recombinant plasmid pDONR221 was obtained after BP recombination reaction. Sequencing was followed by LR recombination reaction between the plasmid and DEST to obtain the lentiviral vector, which worked with helper plasmid for co-transfection of human embryonic kidney epithelial cells (293T cells). Amplification was done to determine its titer, and both transfection and selection procedures were made to get two stable transfected esophageal cancer TE-1 cell lines with medium MnSOD expression (TE-1Mm cells) and high MnSOD expression (TE-1Mh cell), and empty vector cell (TE-1Mn cells). Reverse transcription polymerase chine reaction (RT-PCR), immunofluorescence, immunocytochemistry and Western blot were used to detect the target gene with respect to its expression in the TE-1 cells. Additionally, colorimetric 3-[4,5-dimethy thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, agar colony formation assay, annexin V-FITC/PI staining and flow cytometry experiments were also conducted as to observe the influence of the medium and high MnSOD overexpressions on the proliferation of esophageal cancer cells.</p><p><b>RESULTS</b>RT-PCR indicated that the transfected TE-1 cells showed positive MnSOD expression at different levels. Immunofluorescence, immunocytochemistry and Western blot suggested that TE-1Mm cells and TE-1Mh cells had MnSOD protein expression at different levels. MTT assay indicated that TE-1Mm cells had a significantly decreased survival rate compared with that of the two control cells (TE-1 cells and TE-1Mn cells), and TE-1 Mh cells had an significantly increased survival rate (P<0.05). The colony formation ability of TE-1Mm cells was (23.0 +/- 2.7)%, and that of TE-1Mh cells was (45.3 +/- 4.5)%, significantly different form the (34.7 +/- 4.2)% in TE-1 cells and (33.7 +/- 4.7)% in TE-1Mn cells (P<0.05). Annexin V-FITC/PI double staining experiment of the stably transfected cells cultured for 48 h showed that the early apoptosis rate in TE-1Mm cells was (10.6 +/- 1.0)%, significantly higher than (2.6 +/- 0.2)% in the TE-1 cells, (2.5 +/- 0.6)% in the empty vector cells and (1.0 +/- 0.1)% in the TE-1Mh cels (P<0.05). The fluorescence index (FI) of mitochondrial apoptosis of TE-1Mm cells was 0.948 +/- 0.019, significantly lower than that of TE-1 cell (1.000 +/- 0.022) and empty vector The fluorescence index of TE-1Mn cells (0.997 +/- 0.023) and TE-1 cells (1.000 +/- 0.022) were significant different from that of 0.948 +/- 0.019 in TE-1Mm cells and 1.076 +/- 0.022 in TE-1Mh cells, indicating a significant difference of mitochondrial apoptosis between the cell groups. FCM results indicated that the ROS fluorescence index of TE-1Mm cells was 0.859 +/- 0.040, that of TE-1Mh cells was 0.763 +/- 0.039, significantly lower than that of TE-1 cells (1.000 +/- 0. 042) and empty vector cells (1.002 +/- 0.047) (P<0.05).</p><p><b>CONCLUSIONS</b>Stably transfected cell lines with MnSOD expression have been successfully established. MnSOD overexpression shows bidirectional effect on the proliferation of esophageal cancer cells.</p>

Expression of MnSOD mRNA and protein in esophageal squamous cell carcinoma and its clinical significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of manganese superoxide dismutase (MnSOD) and to determine the relationship between MnSOD expression and clinicopathological features, biological behaviors in esophageal carcinoma.</p><p><b>METHODS</b>Immunohistochemistry (SP) and RT-PCR were respectively used to detect the expression of MnSOD in 45 specimens of esophageal carcinoma tissues and normal esophageal mucosa (5 cm distant from the margin of cancer).</p><p><b>RESULTS</b>The positive rate of MnSOD protein expression was 31.1% in esophageal carcinoma tissues, significantly lower than 86.7% in the normal tissues (P < 0.05). The expressions of MnSOD mRNA and protein were significantly correlated with the lesion length, depths of invasion and histological grade (P < 0.05), but not with lymph node metastasis, lesion site and gross type of the tumor (P > 0.05). The relative content of MnSOD mRNA was (0.310 ± 0.036) and (0.482 ± 0.053) in the cancer and normal tissues, respectively, with a significant difference between the two groups (P < 0.05). The relative content of MnSOD mRNA was significantly related to lesion length, depths of invasion and histological grade (P < 0.05), but not correlated with lymph node status, lesion site and gross type of the tumor (P > 0.05).</p><p><b>CONCLUSION</b>The expression of MnSOD protein and mRNA is decreased in esophageal carcinoma, suggesting that MnSOD gene may be closely associated with the carcinogenesis and the degree of malignancy. Detection of MnSOD expression may be useful in diagnosis, treatment and prognosis of esophageal carcinoma.</p>

Expression of E-cadherin in nasopharyngeal carcinoma and its relationship with cervical lymph node metastasis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of E-cadherin in nasopharyngeal carcinoma (NPC) and its relationship with cervical lymph node metastasis.</p><p><b>METHODS</b>The expression of E-cadherin in 80 patients with NPC was detected by immunohistochemistry.</p><p><b>RESULTS</b>Lower expression of E-cadherin was associated with advanced N-stage of the tumor (P = 0.018). There was no significant correlation between the expression of E-cadherin and lymph node size (P = 0.435). The expression of E-cadherin was higher in patients with cervical lymph node metastasis limited to a single area than that distributing in some scattered areas (P = 0.000). There was a trend that the expression of E-cadherin in the cases with the tumor and lymph nodes in the same side was higher (56.5%) than that in the patients with bilateral lymph node metastases (32.6%), however, the difference was not significant (P = 0.059). The expression rates of E-cadherin in patients with lymph node metastasis in levels II, III and Va were higher than that in levels I, IV, Vb and VI, but with a non-significant difference (P = 0.059).</p><p><b>CONCLUSION</b>The expression of E-cadherin has influence on the lymph node metastasis in nasopharyngeal carcinoma. E-cadherin expression is negatively correlated with the numbers of the lymph node metastases and the metastasis distance, i.e. a lower expression of E-cadherin leads to an advanced N-stage. The lymph node metastasis of nasopharyngeal cancer from above to below is more considerably influenced by E-cadherin expression than the metastasis towards contralateral lymph nodes.</p>

Association of single nucleotide polymorphism of MnSOD gene with carcinogenesis and development of esophageal squamous cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the association of single nucleotide polymorphism (SNP) of manganese superoxide dismutase (MnSOD) gene with carcinogenesis and progression of esophageal squamous cell carcinoma.</p><p><b>METHODS</b>The MnSOD9 T-->C SNP was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 103 patients with esophageal squamous cell carcinoma and 195 healthy controls.</p><p><b>RESULTS</b>A significant difference was observed in the MnSOD allelotype distribution among esophageal squamous cell carcinomas and healthy controls (chi(2) = 4.645, P < 0.05). Individuals with the 9 C allele had a significantly higher risk to develop esophageal squamous cell carcinoma compared with those with the TT allele. The frequency of C allelotype among patients with lesions of different lengths (</= 5 cm and > 5 cm) was 16.3% and 36.7%, respectively. A significant difference was observed in the MnSOD allelotype distribution between patients with lesions of different lengths (chi(2) = 5.147, P < 0.05). No significant association of the MnSOD polymorphism at 9 T-->C with the tumor site, maximal length and clinical staging was found in esophageal squamous cell carcinoma.</p><p><b>CONCLUSION</b>Single nucleotide polymorphism (SNP) of MnSOD gene may be correlated with the susceptibility and disease progression of esophageal squamous cell carcinoma, and may become a tumor marker for prediction of this cancer.</p>

Endostar reduces the growth and metastasis by inhibiting angiogenesis and lymphangiogenesis in nude mouse models of human cervical cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To observe the influence of endostar alone or in combination with cisplatin on tumor growth and metastasis, as well as the inhibition of angiogenesis and lymphangiogenesis in nude mouse models of human cervical cancer.</p><p><b>METHODS</b>HeLa cells were inoculated subcutaneously into the hind flank region of female nu/mice to establish xenograft models. The nude mice were randomly divided into 5 groups: (1) sodium chloride (as control); (2) cisplatin alone; (3) endostar alone; (4) cisplatin plus endostar (10 mg/kg); (5) cisplatin plus endostar (20 mg/kg). The course of all the treatments lasted for 4 weeks. The tumor growth and lymph node metastasis were observed. Immunohistochemical staining was employed to detect the angiogenesis and lymphangiogenesis.</p><p><b>RESULTS</b>(1) Either endostar alone or endostar with cisplatin inhibited the tumor growth significantly than cisplatin and NS (P < 0.05). (2) The rates of lymph node metastasis in the endostar (20 mg/kg) with cisplatin, the endostar (10 mg/kg) with cisplatin, the endostar, the cisplatin and the NS groups were 0 (0/8), 12.5% (1/8), 12.5% (1/8), 62.5% (5/8) and 75.0% (6/8) (P = 0.002), respectively. (3) The MVD of tumor tissue in these five groups were 10.88 +/- 1.38, 10.25 +/- 1.22, 10.83 +/- 2.29, 15.58 +/- 2.31 and 22.08 +/- 1.93, respectively (P < 0.05). The MLD were 5.00 +/- 0.63, 5.17 +/- 0.75, 6.00 +/- 0.63, 14.33 +/- 1.63 and 13.67 +/- 1.21, respectively (P < 0.05).</p><p><b>CONCLUSION</b>Endostar can reduce the tumor growth and metastasis by inhibiting angiogenesis and lymphangiogenesis in nude mouse model of human cervical cancer.</p>

Expression and identification of H5 subtype hemagglutinin of avian influenza A virus in insect cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone and express avian influenza A virus [A/Hong Kong/482/97(H5N1)] H5 subtype hemagglutinin in baculovirus-insect cell expression system and investigate the antigenicity and bioactivity of the recombinant protein.</p><p><b>METHODS</b>H5 gene of influenza A virus was amplified by PCR. The recombinant bacmid was obtained by cloning the gene to the donor plasmid of pFastBacHTB and transformed into DH10Bac competent cells. The recombinant baculovirus stock was prepared by transfecting the recombinant bacmid DNA into the insect cell line for protein expression after amplification. Immunofluorescene assay (IFA) and Western blotting were performed to identify the antigenicity of the recombinant protein, and hemagglutination assay was used to identify its bioactivity.</p><p><b>RESULTS</b>The recombinant his-H5 protein was expressed in the insect cells with a relative molecular mass of 64,000, which showed erythrocyte-agglutinating activities with the red blood cells of guinea pig. Western blotting and IFA demonstrated that the recombinant his-H5 could be recognized and bound by standard anti-H5 sera.</p><p><b>CONCLUSION</b>The recombinant his-H5 with a post-translation modification is successfully obtained in insect cells, which may provide a potential source for further study of the antigen's biological function and for production of the subunit vaccine or monoclonal antibodies.</p>

Antigenicity analysis of nucleocapsid proteins of 3 human coronaviruses SARS-CoV, 229E and OC43 with their monoclonal antibodies / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare and characterize monoclonal antibodies (mAbs) against the recombinant nucleocapsid (N) protein of 3 human coronaviruses SARS-CoV, 229E and OC43 and study the antigenic relationship between the 3 N proteins.</p><p><b>METHODS</b>BALB/c mice were immunized with the recombinant N proteins of SARS-CoV, 229E and OC43 to obtain the mAbs by means of hybridoma. Screening and identification of the mAbs were performed using indirect enzyme-linked immunosorbent assay (ELISA), Western blotting and indirect immunofluorescence assay. Cross-reactivity between the N proteins of the 3 coronaviruses was analyzed with the prepared mAbs.</p><p><b>RESULTS</b>The mAbs against the recombinant N proteins of SARS-CoV, 229E and OC43 were obtained, which reacted specifically with the corresponding viral N protein as shown by indirect ELISA, Western blotting and indirect immunofluorescence assay. No cross-reactivity was found between the 3 N proteins.</p><p><b>CONCLUSION</b>The prepared mAbs against the recombinant N proteins may provide valuable assistance in studying antigenic relationships of N proteins between the 3 human coronaviruses.</p>

Development and application of triple antibodies-based sandwich ELISA for detecting nucleocapsid protein of SARS-associated coronavirus / 中华流行病学杂志

<p><b>OBJECTIVE</b>To prepare and characterize monoclonal antibodies (mAb) and polyclonal antibodies against nucleocapsid (N) protein of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and to establish antibodies-based sandwich ELISA for detecting N protein of SARS-CoV, which might apply to early diagnosis of patients with SARS-CoV infection.</p><p><b>METHODS</b>BALB/c mice were immunized with purified recombinant N protein of SARS-CoV for producing mAbs, and New Zealand white rabbits were immunized for producing polyclonal antibodies. The identification of antibodies was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA), and Western immunoblotting. Capturing and detecting antibodies were selected by pairing the mAbs and polyclonal antibodies one by one and an antibodies-based sandwich antigen capture ELISA was used for detecting N antigen of SARS-CoV.</p><p><b>RESULTS</b>Nine mAbs and hyperimmune rabbit polyclonal antibodies, specifically against SARS-CoV nucleocapsid protein were obtained. Using paired ELISA assay, three mAbs N1E8, N8E1 and N10E4 were selected as capturing antibody and rabbit polyclonal antibodies as detecting antibody then triple antibodies-based sandwich ELISA was established following horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G. The recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml with this assay. When tested with 420 serum specimens from serologically confirmed SARS patients, the positive rates of serum N protein were 90.1%, 23% and 0%, in which sera collected from 1 to 10 days, 11 to 20 days and beyond 21 days respectively after the onset of symptoms. The specificity of the assay was 99.86% in 715 control serum specimens. There was no cross-reaction with other respiratory viruses and coronaviruses.</p><p><b>CONCLUSION</b>Specific and high affinity mAbs and rabbit polyclonal antibodies were obtained. By paired and optimized sandwich ELISA, a sensitive and specific antigen capture ELISA was established for detecting N antigen of SARS-CoV, which might apply to early diagnosis, source tracing and epidemiological studies of SARS.</p>

Cloning, purification, and antigenic characterization of three recombinant fragments derived from SARS-CoV S1 domain / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The present study aimed to clone and express three fragments of genomic RNA derived from SARS associated coronavirus (SARS-CoV) S1 domain and to study its immunogenicity.</p><p><b>METHODS</b>The S1 domain gene was amplified by PCR with specific primers and was inserted into the prokaryotic expression vector pQE-30. Three fragments (40-751, 746-1344 and 746-2001 bp) derived from S1 domain produced after the recombinant plasmid (pQE-30/S1) was digested by restriction endonucleases. The three fragments were cloned into pQE-30 and expressed in M15 strains of Escherichia coli. The expression products, designated S1a, S1b and S1c respectively, were purified by Ni affinity chromatography. The immunogenicity was analyzed by Western Blot and ELISA using serologically confirmed sera from SARS patients and the sera from healthy donors was used as control at the same assay.</p><p><b>RESULTS</b>Three recombinant plasmids (pQE-30/S1a, pQE-30/S1b, pQE-30/S1c) were constructed.Fusion proteins with relative molecular mass of 26,700, 22,500 and 46,000 dalton were successfully expressed with amounts of 35%, 35% and 30% of total cell protein and purified by Ni affinity chromatography, respectively. Western Blot and ELISA analysis showed that the S1c protein could be specifically recognized by the sera from SARS patients.</p><p><b>CONCLUSION</b>The recombinant S1c protein was a good immunogen and has the potential to be used as a vaccine against SARS-CoV infection.</p>