Biomedical subjects

Jian-Min Wang

Publications and source records attributed to Jian-Min Wang.

At least 19 recordsLinked to original sources

PubMed2006

Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system.

Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.

Angiogenesis Inhibitors↗

PubMed2006

Cell death-inducing DFF45-like effector may take part in neuronal apoptosis of the lumbar spinal cord after sciatic nerve injury caused by a firearm.

OBJECTIVE: To investigate neuronal apoptosis and gene regulation after peripheral nerve injury caused by a firearm. METHODS: A rabbit model of sciatic nerve injury caused by a firearm was used. Neuronal apoptosis was determined by using terminal transferase-mediated dUTP nick end-labeling and flow cytometry. Differentially expressed genes were screened with mouse_8192S GeneChips. Cell death-inducing DFF45-like effector (CIDE-B) gene expression in spinal cord after sciatic nerve injury was detected with reverse transcription-polymerase chain reaction. RESULTS: The size of the injury caused by the firearm was widely extended. Neuronal apoptosis was found in days 1, 3, and 7 groups. The expression of apoptosis-related gene CIDE-B was high according to analysis of the GeneChips and reverse transcription-polymerase chain reaction detection. The trend of CIDE-B expression was consistent with neuronal apoptosis after nerve injury. CONCLUSIONS: Neuronal apoptosis is an important pathological change after peripheral nerve injury caused by firearms, and CIDE-B may be an important regulator.

Animals↗

PubMed2006

[Migration and distribution of allogeneic T lymphocytes in organs of graft-versus-host disease mouse model].

This study was aimed to investigate the migration and distribution processes of allogeneic donor T lymphocytes in the organs of recipient mice. GVHD model was established by transfusion of the splenocytes of eGFP transgeneic C57BL/6 mice together with born marrow cells harvested from C57BL/6 mice into BALB/c mice underwent 8.0 Gy total body irradiation. The migration and homing of eGFP(+) cells were tracked by stereo-fluorescent microscopy or inverted fluorescent microscopy and flow cytometry. The enzyme linked immunosorbent assay (ELISA) was performed on supernatants from the tissue homogenates to detect the amount of MIP-1alpha. The results indicated that GVHD clinical manifestation and pathological changes of organs appeared on day 8 post transplantation. eGFP-positive donor T cells in recipient organs were observed by inverted fluorescence microscope in frozen section, or by stereo-fluorescence microscopy in living organs, such as liver, spleen, skin, lungs, bowels, and tongue. The highest expression of MIP-1alpha was on day 7 post transplantation in the liver (491.3 +/- 32.1 pg/ml), and day 3 post transplantation in the spleen (881.5 +/- 45.2 pg/ml), respectively (P < 0.05). It is concluded that GVHD was induced by splenocytes of eGFP transgeneic C57BL/6 mice. eGFP(+) cells in the organs can be observed by fluorescent microscopy. In this GVHD model, donor T cells proliferate and infiltrate in liver, skin, bowels, as well as lungs and tongue. MIP-1alpha may be in relation with the infiltration of T lymphocytes in liver and spleen.

Animals↗

PubMed2006

[Effects of human mesenchymal stem cells and fibroblastoid cell line as feeder layers on expansion of umbilical cord blood CD34(+) cells in vitro].

To investigate the effects of human mesenchymal stem cells (MSC) and human fibroblastoid cell line (HFCL) as feeder layer on expansion of umbilical cord blood CD34(+) cells in vitro, (60)Co gamma-ray irradiated MSC and HFCL were used as feeder layer to expand cord blood CD34(+) cells in culture. The efficiencies of MSC and HFCL on expansion of CD34(+) cells in culture with or without cytokines were compared. The results showed that no matter whether cytokines (rhFL, rhSCF, rhTPO) were added, the proliferation of nucleated cells after expansion for 12 days in HFCL group was statistically higher than that in MSC group, i.e. with cytokines (9797 +/- 361)% vs (7061 +/- 418)%; without cytokines (5305 +/- 354)% vs (1992 +/- 247)%, when the cell numbers at day 0 was accounted as 100%), P < 0.01. The proliferation of propagated CD34(+) cells between MSC group and HFCL without addition of cytokines was not statistically different (820 +/- 191)% vs (825 +/- 305)%, P > 0.05. However, in the presence of cytokines, the propagating rate of MSC group was lower than that of HFCL group (939 +/- 212)% vs (1617 +/- 222)%, P < 0.01. MSC was better than HFCL in maintaining the LTC-IC of UCB CD34(+) cells, i.e. the number of CFU-GM colonies in the fifth week was (129.95 +/- 8.73) /10(5) seeded cells vs (89.81 +/- 10.29) colonies/10(5) cells, P < 0.05; with addition of cytokines, the effect was more obvious, i.e. the number of CFU-GM colonies in the fifth week (192.93 +/- 4.95)/10(5) seeded cells vs (90.47 +/- 14.28) colonies/10(5) seeded cells, P < 0.01. MSC mixed with a certain proportion of HFCL facilitated maintaining the LTC-IC of UCB CD34(+) cells. When the proportion was 4:1, the number of CFU-GM colonies was the highest (186.89 +/- 11.11)/10(5) seeded cells, which was higher than that of both 3:2 group [(138.92 +/- 14.84) colonies/10(5) seeded cells] and MSC only group, i.e. (64.63 +/- 6.11) colonies/10(5) seeded cells, both P < 0.01. It is concluded that HFCL is better than MSC in maintaining the expansion of CD34(+) cells and cytokines can enhance this effect, while MSC are stronger than HFCL in maintaining the LTC-IC of UCB CD34(+) cells in vitro. MSC with addition of a certain proportion of HFCL can significantly enhance the efficiency of CD34(+) cell expansion.

Antigens, CD34↗

PubMed2005

[Preparation of human recombinant kringle 1-5 and its bioactivity].

To investigate antiangiogenesis activity and effects on endothelial cell proliferation of human recombinant K1-5 expressed in E.coli BL21, the cDNA of human K1-5 obtained from a cloning vector pUC57K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E.coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed and purified to a purity of 96% by the nickel affinity chromatography. Refoled K1-5 protein was tested on chicken CAMs, and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, human recombinant K1-5 potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrated that human recombinant K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.

Angiogenesis Inhibitors↗

PubMed2005

[Immunomodulation of bone marrow mesenchymal stem cells in hematopoietic stem cell transplantation--review].

Bone marrow mesenchymal stem cells (BM-MSC) have the characteristics of self-renewal and multipotency. They secrete a variety of cytokines and provided and ideal microenvironment for the division, proliferation and differentiation of hematopoietic stem cells (HSC) through the interaction with other stromal cells. Previous studies indicated that MSC could enhance engrafment and alleviate GVHD in allo-HSC and MSC co-transplantation. This effect on transplantation immunity may associate with escaping MHC compatible HSC from antigen recognition and suppressing activation and proliferation of nonspecific lymphocytes. In this paper, the characteristics of BM-MSC influencing transplantation immunity and its mechanism were reviewed.

Bone Marrow Cells↗

PubMed2005

[Recipient lymphopenia state enhances the expansion and anti-leukemia effect of leukemia specific cytotoxic T lymphocytes].

OBJECTIVE: To determine the role of recipient lymphopenia state in the expansion and function of leukemia specific cytotoxic T lymphocytes (CTLs). METHODS: C57BL/6 mice were induced to lymphopenia with 6 Gy total body irradiation. Spleen T cells or leukemia specific T cells from EGFP+ transgenic C57BL/6-EGFP mice were adoptively transferred by intravenous injection. The mice were challenged subcutaneously with 1 x 10(6) FBL3 leukemic cells at day 2 after irradiation. The peripheral WBC count, percentage of EGFP+ cells, subsets of T cells and tumor sizes were monitored. RESULT: Both of the spleen T cell and leukemia specific CTL proliferated efficiently with the percentage of EGFP+ cells of 28. 81% and 42.24%, respectively, after infused into lymphopenic recipients. However, spleen T cells had no anti-leukemia effect regardless of its expansion. In contrast, leukemia specific CTLs showed a more rapid and extensive expansion under the condition of lymphopenia and a enhanced anti-leukemia immunity. CONCLUSION: Transfusion of leukemia specific CTLs under lymphopenia state could be a feasible strategy to expand leukemia specific CTLs and generate favorable anti-leukemia effect in vivo.

Animals↗

PubMed2005

[Pulmonary fungal infection in malignant hematological diseases: an analysis of 14 cases].

The objective of this study was to evaluate the etiological factors, diagnosis and therapy of pulmonary fungal infection in hematological malignancies, 14 cases of malignant hematological disease with pulmonary fungal infection were collected and analyzed. The results showed that 11 out of 14 cases had the respiratory manifestations, sputum was sticky and difficult to be expectorated in 7 cases, X rays in chests showed shadows with features of stigma and sheet in 11 cases, Candida albicans and aspergillus infection were observed in 10 and 2 cases respectively, the numbers of neutrophil were below 0.5 x 10(9)/L in 8 cases and below 1.0 x 10(9)/L in 3 cases respectively, fluconazole was used for 12 cases and clinical symptoms of 11 cases were improved within two weeks. In conclusion, the occurrence of pulmonary fungal infection in malignant hematological diseases is associated with intensive chemotherapy, decrease of neutrophil counts and using of broad-spectrum antibiotics, the diagnosis at early stage is difficult and clinicians should pay more attention to its clinical and laboratory examinations, and give them therapy in time.

Adolescent↗

PubMed2005

Nuclear matrices and matrix attachment regions from Green alga: Dunaliella salina.

Nuclear DNA of eukaryotic organism attaches to the proteinaceous nuclear matrices via specific matrix attachment regions (MARs). In order to investigate the interactions between chromosomal DNA and nuclear matrices,we isolated the MARs from unicellular alga Dunaliella salina. As the first step,a random MAR library was set up and then the binding affinity of the selected clones to nuclear matrices was tested in this study. Three DNA fragments were found to bind specifically to the nuclear matrices in vitro,of which two were strong binders and all contained known consensus motifs and a hairpin loop structure of MAR.

Base Sequence↗

PubMed2004

Recombinant mouse canstatin inhibits chicken embryo chorioallantoic membrane angiogenesis and endothelial cell proliferation.

Human canstatin, a 24 kD fragment of the alpha2 chain of type IV collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth. To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan. The recombinant mouse canstatin efficiently expressed in E. coli M15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93%. The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.

Angiogenesis Inhibitors↗

PubMed2004

Identification of differentially expressed genes from spinal cord after firearm injury to rabbit sciatic nerves.

OBJECTIVE: Peripheral nerve regeneration depends on gene regulation by central neurons. To search for more effective treatment methods to improve the regeneration of wounded peripheral nerves, gene expression profile of spinal cord after firearm injury to rabbit sciatic nerves are studied with DNA micro-array technique. METHODS: A total of 54 rabbits were randomly divided into 4 groups: Groups d1, d3, d7 and normal control group. Lumbar spinal cords were sampled. RNA and mRNA were extracted, labeled by Cy3 and Cy5, and analyzed by mouse_8192S gene chips. RESULTS: A total of 1367, 923, and 61 genes with differential expression were found on day 1, day 3, and day 7 after trauma respectively. Five expressed sequence tag (EST) sequences demonstrated differential expression during 7 days after trauma. CONCLUSIONS: There is complex gene profile with differential expression after firearm nerve injury, among which AW701496, U84291, W13926, X04017 and AW822394 EST sequences may be important regulation factors that involved in regeneration of peripheral nerve injury.

Animals↗

PubMed2004

[Establishment of K562 cell lines resistant to STI571 and a preliminary biological study].

To produce leukemic STI571-resistant cell lines and to explore the molecular mechanism of STI571-resistance, cell lines K562-n and K562-n/VCR were induced by exposing cells to gradually increasing STI571 concentration of culture medium to have STI571-resistance and major biological characteristics between these subclones and the parent cells were compared. The results showed that a STI571-resistant cell line based on multidrug-resistance was established, which exhibited 23.41-fold resistance to STI571, 662.26-fold resistance to VCR and cross-resistance to HHT. K562-n/STI was generated from K562-n and had some characteristics of MDR. The intracellular accumulation of DNR in K562-n/STI and K562-n/VCR/STI were 33.24 and 18.76 respectively. Transcription of mdr-1 gene in both K562-n/STI and K562-n/VCR/STI was positive. Cell doubling time of K562-n/STI and K562-n/VCR/STI was significantly longer than that in their parent cells (P <0.05). And proliferation index was also higher than that in parent cells (P <0.05). It is conclusion that the tolerance of K562-n cells to STI571 can be augmented by adding low-dose of STI571 into the culture medium repeatedly. K562-n/STIs expressed MDR at some extent, and transcription of mdr-1 gene in K562-n/STIs was positive. As K562-n is a cell line used to develop human leukemia in nude mice, K562-n/STI and K562-n/VCR/STI 571 will contribute to the study of mechanism of STI571-resistance as in vitro and in vivo experimental models.

Antineoplastic Agents↗

PubMed2004

[Expression of mouse canstatin and its N-domain in E. coli BL21].

The mouse canstatin and its N-domain cDNA were amplified from total RNA of mouse liver by RT- PCR and cloned into vector pMD18-T for sequencing. Prokaryotic expression vectors pET/Can and pET/Can-N were constructed and expressed in E.coli BL21(DE3) with induction of IPTG.. Mouse canstatin cDNA is 684bp in length encoding 227 amino acids. The sequences of both cDNA and amino acids share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. N-domain of mouse canstatin is the same amino acid sequence as that of human canstatin. In the present study, prokaryotic expression vector pET/Can and pET/Can-N were expressed in E.coli BL21 with amount of 35% and 18% of the total bacterial proteins after being induced by IPTG for 4h. The expressed products existed mainly as inclusion bodies. This work has laid down the basis for further study of its angiogenic activity and potential application for tumor dormancy therapy.

Amino Acid Sequence↗

PubMed2004

Expression and cytotoxicity of EGFP-labeled D-amino acid oxidase in HeLa cells.

To observe the expression of R. gracilis D-amino acid oxidase (DAAO) gene labeled by EGFP in HeLa cells and how it mediates cytotoxicity, DAAO cDNA and EGFP gene were cloned into Eukaryotic expression vector pIRES to construct DAAO gene expression vector pIRES-DAAO. HeLa cells were transfected with pIRES-DAAO in vitro. Expression of EGFP gene in HeLa-D cells was observed under a fluorescent microscope. The efficiency of transfection was analyzed by flow cytometry (FCM). The fluorescent cells were screened by FCM and were named HeLa-D. The activity of HeLa-D cells was detected by MTT colorimetric assay after they were treated with D-Ala at various concentrations. The results indicted that expression of EGFP gene in HeLa-D cells was seen under fluorescent microscope and HeLa-D cells were screened by FCM. Apparently,the prodrug D-Ala killed HeLa-D cells. These results demonstrate that EGFP gene can be regarded as a reporter gene to screen the cells transduced with DAAO quickly, and DAAO/D-Ala suicide gene system may prove helpful in gene therapy of cancer.

Alanine↗

PubMed2003

[Application of SCID mouse-human leukemia model for leukemia research].

SCID mouse-human leukemia model is an important and useful tool for study on proliferation, differentiation and modulation of leukemic cells. In this article, the establishment of the model, advances in research and application in studies of pathogenesis, cell biology, clinical diagnosis, therapy and assessment of prognosis of leukemia patients are reviewed. The limitations of the model are also commented.

Animals↗

PubMed2003

GPCEG-A database for genomic polymorphism of Chinese ethnic groups.

This paper reports the construction of the database for Genomic Polymorphism of Chinese Ethnic Groups (GPCEG). GPCEG contains denomination and basic information of Chinese 56 ethnic groups, with introduction of their in geographic distribution, population quantity, spoken and written language, religious belief and physical characteristics. GPCEG collects the data of genomic polymorphism, cell lines, reference and links of other international related databases. The visualization, query and update system were also available. GPCEG laid the foundations of establishing a national database with Chinese characteristics.

Cell Line↗

PubMed2003

[The reverse effect on drug-resistance against tyrosine kinase inhibitor STI571 in mdr1 and bcr-abl positive leukemic cells].

To explore the possibility of leukemia cell line of both bcr-abl and mdr-1 positive were cross-resistant to tyrosine kinase inhibitor STI571 and its reversal way, the inhibitory effect of STI571 on K562-n/VCR cells was detected with MTT method and reverse effects of CsA, TAM, IFN-alpha and CsA cominated with IFN-alpha were observed. The results showed that K562-n/VCR cell line expressing bcr-abl and mdr1 positive was resistant to STI571, and could be reversed by 5.18, 1.82 and 1.67-fold respectively, when treated with CsA, TAM, and IFN-alpha. It could be reversed by 34.87-fold with combination of half-dose CsA and IFN-alpha. In conclusion, amplification of mdr1 gene may contribute to drug-resistance of bcr-abl positive leukemic cells against STI571. The reversal agents, CsA, TAM and IFN-alpha show obviously reverse effects on drug-resistance. The combination of half-dose of both CsA and IFN-alpha display stronger effect than the full dose of either.

Antineoplastic Agents↗

PubMed2003

[Cloning and characterization of Hsp70a cDNA fragment of Dunaliella salina].

The present study is to obtain heat shock protein 70a cDNA fragment from Dunaliella salina. Two pairs of degenerate primers were designed according to conserved motifs of DIDLGTT,DQGNRTTP,PAYFNDS and ATKDAG of the homologous amino acid sequences and used to amplify hsp70a cDNA fragment from heat-shock-treated Dunaliella salina by nest PCR technique. The resulting PCR products were inserted into T-vector then transformed into JM109. Ten colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequences were performed by BLAST and subsequently compared with GenBank data. Three of ten nucleotide sequences were obtained,of which there was 372 bp coding 126 amino acids. The sequences shared high homology with hsp70a,with identity 96% to Chlamydomonas reinhardtii, 94% to Petunia, 93% to Pisumsativum, 92% to tomato, 92% to human,90% to Drosophila and 89% to yeast respectively. It can be concluded that the cloned sequence is putatively hsp70a cDNA fragment from Dunaliella salina.

English Abstract↗