Establishment of orthotopic implantation model of human U87-MG brain glioma cell line in nude mice / 中国药理学通报

Aim To establish human U87-MG glioma model in nude mice brain and to observe the characteristics of the tumor growth. Methods Human U87-MG glioma cells were cultured in vitro. 5 μL of cell suspension containing 3.0 ×1010·L-1, 4.0×1010·L-1and 5.0×1010·L-1respectively was inocula-ted into the right caudate nucleus of 18 male nude mice brain un-der the guidance of stereotaxic apparatus, separately, whereas another 6 nude mice as the control group, were inoculated into the same volume of Hanks solution. The moving and survival state of rats with gliomas were observed. The examinations of the tumors formation, volumes, metastasis and histopathology were performed and the obtained brain samples were stained with HE and immunohistochemistry. Results All the tested rats of dif-ferent inoculation doses developed brain tumors without extracra-nial metastasis. The mean survival time of three groups was (46.50 ± 3.27) d,(38.50 ± 3.28) d and (30.67 ± 3.51) d,respectively. The tumors showed the similar morphological fea-tures and immunophenotype to human glioma. There was positive expression of GFAP and S-100 in the tumors. Conclusions The orthotopic implantation model of human U87-MG glioma, by in-oculating quantitative U87-MG cells stereotaxically into the brains of the nude mice, is successfully established with 100 yield of intracranial tumor and no extracranial growth extension. It resembles the histopathological and morphological features of human glioma,which can be used as a reliable animal model for the study of the tumorigenesis, pathogenesis, biological charac-teristics and therapy of glioma.

Effect of electromagnetic pulse exposure on permeability of blood-testicle barrier in mice / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the effect of electromagnetic pulse (EMP) exposure on the permeability of blood-testicle barrier (BTB) in mice.</p><p><b>METHODS</b>Adult male BALB/c mice were exposed to EMP at 200 kV/m for 200 pulses with 2 seconds interval. The mice were injected with 2% Evans Blue solution through caudal vein at different time points after exposure, and the permeability of BTB was monitored using a fluorescence microscope. The testis sample for the transmission electron microscopy was prepared at 2 h after EMP exposure. The permeability of BTB in mice was observed by using Evans Blue tracer and lanthanum nitrate tracer.</p><p><b>RESULTS</b>After exposure, cloudy Evans Blue was found in the testicle convoluted seminiferous tubule of mice. Lanthanum nitrate was observed not only between testicle spermatogonia near seminiferous tubule wall and sertoli cells, but also between sertoli cells and primary spermatocyte or secondary spermatocyte. In contrast, lanthanum nitrate in control group was only found in the testicle sertoli cells between seminiferous tubule and near seminiferous tubule wall.</p><p><b>CONCLUSION</b>EMP exposure could increase the permeability of BTB in the mice.</p>

Expression and immunogenicity of recombinant Mycobacterium bovis Bacillus Calmette-Guérin strains secreting the antigen ESAT-6 from Mycobacterium tuberculosis in mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guérin (BCG) is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis.</p><p><b>METHODS</b>Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG (rBCG). BALB/c mice were immunized subcutaneously on the back with 100 microl normal saline containing 10(6) CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses.</p><p><b>RESULTS</b>There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1:8000, which was significantly higher than that in BCG group (1:1400, P < 0.05). The elicited IFN-gamma level of rBCG group was (1993 +/- 106) pg/ml, which was also significantly higher than that in BCG group ((1463 +/- 105) pg/ml, P < 0.05). The splenocyte proliferation index of rBCG group reached 4.34 +/- 0.31, which was higher than that of BCG group (3.79 +/- 0.24, P < 0.05).</p><p><b>CONCLUSION</b>rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.</p>

Inhibition of growth and angiogenesis of U251 cell xenograft in vivo by short hairpin RNA targeting survivin gene / 中华外科杂志

<p><b>OBJECTIVE</b>To observe the impact of specific short hairpin RNA (shRNA) targeting survivin gene on tumorigenesis and angiogenesis of human brain glioblastoma U251 cells in vivo of nude mice.</p><p><b>METHODS</b>U251 cells, U251-SR cells transfected stably with shRNA eukaryotic expression vector pWH1-SR targeting survivin gene, and U251-P cells transfected stably with blank pWH1 vector, were inoculated respectively into subcutaneous tissue in flank of 15 nude mice (each group 5 mice), and the tumor growth status was observed and measured. Protein expressions of survivin, proliferating cell nuclear antigen (PCNA) and factor VIII related antigen (F VIII RAg) were investigated by immunohistochemistry SABC method, apoptotic cells were screened by TUNEL method, furthermore proliferative index (PI), apoptotic index (AI) and microvessel density (MVD) were measured respectively in each group of tumor specimens.</p><p><b>RESULTS</b>Comparing with those in U251 and U251-P groups, in U251-SR group, the tumorigenesis time delayed, tumor grew slowly, both tumor volume and tumor weight decreased significantly (P < 0.01 for both); Survivin protein expression was down-regulated markedly; PI and MVD decreased significantly, whereas AI increased remarkably (P < 0.01 for all).</p><p><b>CONCLUSIONS</b>The specific shRNA targeting survivin gene can inhibit significantly tumorigenesis and angiogenesis of U251 cells in vivo.</p>

Construction and bio-activity of the chimeric protein of BMP2-EGFP / 中南大学学报(医学版)

OBJECTIVE@#To construct the recombined retroviral expression vector of BMP2/pLEGFP and investigate the bio-activity of the expressed chimeric protein.@*METHODS@#The recombinant vector constructed by gene recombinant technology was analyzed by restriction enzyme digestion and PCR. BMP2/pLEGFP was transfected into COS-7 cells with liposome transfection reagents for transient expression. The expression of chimeric protein BMP2/EGFP was identified by fluorescent microscope and Western blotting. The bio-activity was examined by the cellular activity and animal heterotopic osteogenesis experiment.@*RESULTS@#The recombinant plasmid proved successful by restriction enzyme digestion and PCR. The expression of the chimeric protein was shown by fluorescent microscope and Western blotting. The chimeric protein had the double bio-activities of BMP2 and EGFP identified by the cellular activity and animal heterotopic osteogenesis tests.@*CONCLUSION@#The recombinant vector of BMP2/pLEGFP is successfully constructed by the gene recombinant technology and its chimeric protein has double bioactivities of BMP2 and EGFP.

Change of the cell cycle after flutamide treatment in prostate cancer cells and its molecular mechanism / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To explore the effect of androgen receptor (AR) on the expression of the cell cycle-related genes, such as CDKN1A and BTG1, in prostate cancer cell line LNCaP.</p><p><b>METHODS</b>After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction (RT-PCR) tests were carried out to confirm the results of the chips.</p><p><b>RESULTS</b>After AR antagonist flutamide treatment, three hundred and twenty-six genes (3.93%) expressed differentially, 97 down-regulated and 219 up-regulated. Among them, eight up-regulated genes might be cell cycle-related, namely CDC10, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, CDKN1A and BTG2. The CDKN1A and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, while p53 mRNA expression had no significant changes.</p><p><b>CONCLUSION</b>Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control.</p>