Biomedical subjects

Sen Zhong

Publications and source records attributed to Sen Zhong.

9 recordsLinked to original sources

PubMed2006

[Construction, expression and immunogenicity study of a chimeric MS/hIL-12 eukaryotic expression plasmid].

OBJECTIVE: To construct and express a chimeric Mtb8.4 with signal peptide (MS)/hIL12 eukaryotic expression plasmid, and to study the immunogenicity of the MS/hIL-12 chimeric genetic vaccines. METHODS: The MS/hIL-12 chimeric gene was amplified by polymerase chain reaction (PCR) and cloned into the eukaryotic expression vector pCI-neo. The correct pCI-neo-MS/hIL12 (pMSI) recombinant plasmid was identified by PCR, restricted enzyme digestion and DNA sequencing. COS-7 cells were transfected with pMSI constructs by cationic liposome. After 48 hours, mRNA of the target gene was detected by RT-PCR, and hIL-12 protein in culture supernatant and cell lysates was detected by Western blot. C57BL/6N mice were vaccinated with MS/hIL-12 chimeric gene vaccine for three times at 3 week intervals. Four weeks after the final inoculation, three mice were sacrificed for measurement of the cytokine response and cytotoxic T lymphocyte (CTL) induction. RESULTS: The accuracy of plasmid construction was confirmed by a number of molecular biological techniques. Transfection of COS-7 cells with plasmids pMSI lead to transient expression of fusion proteins. The IFN-gamma and IL-2 titers were (1,521 +/- 48) ng/L and (755 +/- 41) ng/L in MS/hIL-12 chimeric gene vaccine group, (820 +/- 50) ng/L and (297 +/- 31) ng/L in MS gene vaccine group, (1,487 +/- 40) ng/L and (767 +/- 50) ng/L in BCG group, (121 +/- 16) ng/L and (62 +/- 10) ng/L in vacant vector group, and (48 +/- 16) ng/L and (32 +/- 17) ng/L in PBS group respectively. The levels of IFN-gamma and IL-2 in MS/hIL-12 chimeric gene vaccine group were higher than those of MS gene vaccine group, vacant vector group and PBS group (P < 0.01) and was similar to the BCG group (P > 0.05). The level of IL-4 in BCG group [(91 +/- 11) ng/L] increased significantly as compared to other groups (P < 0.01). When effector-cell-to-target-cell ratio (E:T ratio) were 100:1, 50:1, and 10:1 respectively, the CTL activity was 77.5%, 51.2%, 30.3% in MS/hIL-12 chimeric gene vaccine group, 56.2%, 37.8%, 11.5% in MS gene vaccine group, 28.9%, 21.4%, 9.8% in BCG group. The cytotoxicity in MS/hIL-12 chimeric gene vaccine group was higher than that of other groups (P < 0.01). CONCLUSION: When used to construct the chimeric gene vaccine, hIL-12 could improve the immunogenicity of MS gene vaccine.

Animals↗

PubMed2006

[Screening and their anti-tumor activity of antisense oligodeoxynucleotides targeting KDR mRNA in breast cancer MCF-7 cells].

OBJECTIVE: To screen the antisense oligodeoxynucleotides (asONs) which could hybridize with KDR (kinase insert domain-containing receptor) mRNA in an effective and specific way and to explore their anti-tumor effects on breast cancer MCF-7 cell line in vitro. METHODS: The asONs were firstly selected using oligodeoxynucleotides library hybridization or computer prediction, then their hybridization ability with KDR mRNA was further tested with oligonucleotide microarray. The asONs with strong hybridization intensity were selected. Their inhibitory effects on MCF-7 cells proliferation and KDR expression were assayed by MTT, RT-PCR and Western blotting assay, respectively. RESULTS: In 13 asONs selected with oligodeoxynucleotides library hybridization, 8 (8/13, 61.5%) showed strong hybridization signals, while such was only 1 in 17 asONs designed by computer prediction. 9 asONs with strong hybridization intensity were selected and synthesized with phosphorothioated modification. All these asONs inhibited the MCF-7 cells proliferation in a dose-dependent manner, in which asON4 and asON7 screened by oligodeoxynucleotides library in combination with oligonucleotide microarray were the most effective, with inhibitory rates of 51.6% and 62.2% at 0.8 micromol/L, respectively. The KDR expression at mRNA and protein levels was reduced by both the two asONs, in a dose-dependent manner. CONCLUSION: asONs screened by oligodeoxynucleotides library hybridization are well consistent with that chosen with oligonucleotide microarray. The combination of oligodeoxynucleotides library with oligonucleotide microarray is an effective approach of asONs screening. The asONs targeting KDR mRNA showed prominent anti-tumor activity on breast cancer MCF-7 cells.

Breast Neoplasms↗

PubMed2006

[Immunogenicity of chimeric gene vaccine Mtb8.4/hIL12].

AIM: To construct chimeric gene vaccine Mtb8.4/hIL-12, express it in COS-7 cells and study its immunogenicity. METHODS: Chimeric gene Mtb8.4/hIL-12 was amplified by PCR and cloned into the eukaryotic vector pCI-neo to construct the recombinant plasmid pCI-neo-Mtb8.4/hIL12. After the recombinant plasmid was identified by restriction enzyme digestion analysis, PCR and DNA sequencing, COS-7 cells were transfected with pCI-neo-Mtb8.4/hIL12 through cationic liposome. 48 hours later, the expression of mRNA was detected by RT-PCR and the level of hIL-12 in culture supernatant and cell lysates were detected by Western blot. C57BL/6N mice were vaccinated with chimeric gene vaccine Mtb8.4/hIL-12 three times at the interval of 3 weeks each time. Four weeks after the final inoculation, three mice were sacrificed to assess the cytotoxicity of CTLs and response to cytokine. RESULTS: The recombinant plasmid pCI-neo-Mtb8.4/hIL12 was constructed successfully. After COS-7 cells were transfected with pCI-neo-Mtb8.4/hIL12, chimeric gene Mtb8.4/hIL12 was expressed in COS-7 cells. The chimeric gene vaccine could induce strong antigen-specific immune response. With the increase of IFN-gamma and IL-2 secretion and the decrease of IL-4 secretion, the cytotoxicity of specific CTLs was heightened. CONCLUSION: Recombinant plasmid pCI-neo-Mtb8.4/hIL12 has been successfully constructed and expressed in COS-7 cells. The constructed chimeric gene vaccine Mtb8.4/hIL12 is of strong immunogenicity and can obviously induce the cytotoxicity of CTLs.

Animals↗

PubMed2005

The immunogenicity and protective efficacy of Mtb8.4/hIL-12 chimeric gene vaccine.

DNA vaccination has emerged as a powerful approach in the search for a more efficacious vaccine against tuberculosis (TB). In this study, we evaluated the immunogenicity and protective efficacy of Mtb8.4/hIL-12 chimeric gene vaccine. The Mtb8.4/hIL-12 chimeric gene was amplified by PCR and cloned into the eukaryotic expression vector pCI-neo. C57BL/6N mice were vaccinated with Mtb8.4/hIL-12 chimeric gene vaccine for three times at 3 weeks intervals. Four weeks after the final inoculation, three mice per group were sacrificed to assess cytokine response and CTL induction and the other five mice per group were challenged intravenously in a lateral tail vein with 1 x 10(6) CFU of virulent Mycobacterium tuberculosis H37Rv. Spleen and the left lung were harvested from each mouse at 4 weeks after infection and homogenized in sterile saline. Serial dilutions of organ homogenates were plated on L-J agar and incubated 37 degrees C until colonies were visible 4 weeks later. Protective efficacies in each experiment were expressed as reduced CFU and were compared with the negative control group. The right lung was obtained from each mouse and immediately inflated with and stored in 10% formalin saline. Tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Mtb8.4/hIL-12 chimeric gene vaccine induced the secretion of more of Th1 cytokines, but not IL-4 and enhanced CTL activity. Mice immunized with Mtb8.4/hIL-12 chimeric gene vaccine had fewer and smaller tubercles than control groups. As expected, control mice had the highest bacterial loads in both lung and spleen. Immunization with Mtb8.4/hIL-12 chimeric gene vaccine could remarkably reduced CFU counts in organs. When it was used to construct the chimeric gene vaccine, hIL-12 could improve the immune efficacy of Mtb8.4 gene vaccine.

Animals↗

PubMed2005

Clinical analysis of primary malignant melanoma of the cervix.

OBJECTIVE: To investigate the clinical and pathological characteristics of primary cervical malignant melanoma, and its prognosis. METHODS: The clinical and pathological data of four patients with primary malignant melanoma of the cervix were analyzed retrospectively. Nerve tissue protein S-100 and monoclonal antibody to melanoma (HMB-45) were measured in all cases by immunohistochemical method. All four patients received radical hysterectomy. Three of them received chemotherapy preoperation or postoperation, and one of them received biotherapy with interferon-gamma and interleukin-2 at the same time. All the cases were followed up. RESULTS: The average age of four patients was 45 years. Clinical symptoms presented with irregular vaginal bleeding, postcoital bleeding, or increase of vaginal discharge. Gynecologic examination showed polypus papilla cauliflower-shaped or nodulated black-brown or black-blue mass on the cervix. All the four cases were pathologically diagnosed with cervical malignant melanoma. S-100 and HMB-45 were positive in all patients. Two patients died at 6 and 41 months postoperation, respectively. The other two patients survived for 3.5 and 7 years postoperation, respectively. CONCLUSIONS: S-100 protein and HMB-45 play very important roles in the diagnosis of primary malignant melanoma of cervix. Radical hysterectomy, chemotherapy combined with dimethyl triazemo imidazole carboxamide and biological therapies may improve the prognosis of the primary malignant melanoma of cervix if the disease could be diagnosed in an early stage.

Adult↗

PubMed2004

[The anti-cancer effect of siRNA targeting human telomerase reverse transcriptase in SMMC-7721 cells].

OBJECTIVE: To explore the anti-cancer effects of siRNAs targeting hTERT in SMMC-7721 cells. METHODS: Two siRNAs targeting hTERT mRNA were designed and synthesized by T7 transcription system in vitro. MMT, RT-PCR and Western blot were applied to evaluate effects on inhibiting cell growth, hTERT mRNA and protein expression in SMMC-7721 cells. RESULTS: siRNAs decreased cell proliferation in a dose-dependent manner. At a concentration of 100 nmol/L, siRNAs exhibited obvious effects on inhibiting hTERT mRNA and protein expression in SMMC-7721 cells. CONCLUSION: siRNAs targeting hTERT have significant inhibitory effects on hTERT gene expression in SMMC-7721 cells. siRNA has the possibility to become a new anti-cancer agent

DNA-Binding Proteins↗

PubMed2003

Distribution and anti-HBV effects of antisense oligodeoxynucleotides conjugated to galactosylated poly-L-lysine.

AIM: To describe distribution of the phosphorothioated antisense oligodeoxynucleotides (PS-asODNs) conjugated to galactosylated poly-L-lysine (Gal-PLL) in mice, and to observe their effects on expression of HBV gene in the 2.2.15 cells and transgenic mice. METHODS: According to the result of direct sequencing of PCR amplified products, a 16 mer phosphorothioate analogue of the antisense oligodeoxynucleotides (PS-asODNs) directed against the HBV U(5)-like region was conjugated to the hepatotropic Gal-PLL molecules. Its distribution was demonstrated using asODNs labeled with (32)P at the 5' terminus with a T4-polynucleotide Kinase. Its inhibition effect on HBV expression was observed in the transfected 2.2.15 cells and transgenic mice. RESULTS: The Gal-PLL and asODNs could form stable complex at a molar ratio of 2:1. As shown in the HBV-transfected 2.2.15 cells, the inhibition effects of asODNs alone and asODNs conjugated to Gal-PLL, at 10 micromol/L for both, on HBsAg and HBeAg production were different,the former being 70 % and 58 %, respectively, and the latter being 96 % and 82 %, respectively. A more pronounced reduction was also observed in viral DNA load in the culture supernatant for the test with Gal-PLL-asODNs. Among many mouse organs, livers retained more asODNs molecules after administration. The preferential concentration in liver was found to be 52.14 % for Gal-PLL-asODNs, as high as 2.38-fold of that for asODNs (21.9 %). Both elements decreased gradually in liver, with 2.9 % of the former, 5.99 % of the latter retained 24 hours after the administration. The injection interval, therefore, was recommended to be 24 hours. In the transgenic mice, serum HBsAg decreased significantly (P<0.01) at the 12th day after administrating Gal-PLL- asODNs, the serum HBV DNA turned negative in 4 of the 6 mice. CONCLUSION: Antisense oligodeoxynucleotides conjugated to Gal-PLL can be concentrated in liver and intaked by hepatocytic cells. This may result in specific inhibition of expression and replication of HBV in vitro and in vivo.

Animals↗

PubMed2002

Prolongation of functional life-span of neutrophils by recombinant verotoxin 2.

OBJECTIVE: Verotoxin-producing Escherichia coli (VTEC) strains of serotype O157 : H7 have been implicated in a wide spectrum of diseases, including blood diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To further explore the pathological role of verotoxin (VT) in HUS and other VTEC associated diseases, we investigated the effects of recombinant verotoxin 2 (rVT2) on the biological activity of neutrophils. METHODS: The technique of flow cytometry, a fluorescent probe 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM), and the assay of reduced cytochrome c to detect superoxide production were used in this study. RESULTS: gammaVT2 significantly inhibited spontaneous apoptosis in neutrophils. Neutrophils with prolonged survival due to gammaVT2 maintained various biological functions, such as the expression of adhesion molecules (shading CD62L and raising CD11b/CD18), adherence to human umbilical vein endothelial cells (HUVECs), and generation of superoxide (O(2)(-)). CONCLUSION: Prolongation of the functional life-span of neutrophils by gammaVT2 may accelerate inflammatory responses at sites of inflammation. This may play a crucial role in neutrophil-mediated tissue injury in HUS and other VTEC-associated diseases.

Apoptosis↗

PubMed2002

[In vivo inhibition of hepatitis B virus replication and gene expression by targeted phosphorothioate modified antisense oligodeoxynucleotides].

OBJECTIVE: To investigate the antiviral effect of targeted antisense oligodeoxynucleotides (asODN) in HBV transgenic mice. METHODS: asODN phosphorothioated (5'-CATGCCCCAAAGCCAC-3') targeted to HBV pre-C/C region was synthesized. Gal15-PLL was used as drugs carrier which targeted asODN to mice liver. Twelve mice with positive serum HBsAg, HBV-DNA were divided into the Gal15-PLL-asODN-treated group or the control group randomly. In Gal15-PLL- asODN-treated group, each mouse was injected i.v. asODN 15mug/g weighty/day via tail vein for 12 days successively; while in the control group, each mouse received the same volume normal saline by the same way. RESULTS: In the Gal15-PLL- asODN-treated group, serum HBsAg decreased at the 6th day (P<0.05), and decreased significantly at the 12th day vs pretreatment (P<0.01). The serum HBV DNA of 4/6 mice became negative. Immunohistochemistry test showed lowered HBsAg, HBcAg content in the liver. In contrast, the control group showed no apparent changes. CONCLUSIONS: Gal15-PLL-asODN targeted to pre-C/C region could inhibit HBV replication and gene expression.

Animals↗