Objective To serially examine virus carrying condition of clinical specimens collected from SARS patients, in order to evaluate the dynamic changes of virus in the bady and its clinical significance. Methods A total of 494 samples, including plasma, urine, feces, sputum and throat swab, obtained from 84 cases of clinically diagnesed SARS patients at different time-points were examined for the study. Nested RT-PCR was employed for the detection of SARS-CoV using 2 pairs of primers targeting P and N region of the viral genome. The amplified viral genomic fragments were confirmed by DNA sequence analysis. Results Specific bands for SARS-CoV were amplified from various specimens of some SARS patients. Highest positive rate was found in sputum compared with the others. However, simultaneous examination of more than two specimens increased detectable rate of the virus. Moreover, dynamic examination revealed that the highest positive rate occurred in the 2nd week after the onset of the disease and next to the highest during the 3rd to 4th week. However, dynamic detectable rates for different specimens were not identical. Conclusion Nested RT-PCR is a practicable method for the detection of SARS-CoV and helpful for the early diagnosis or confirmation of the disease. Dynamic examinations for SARS-CoV by combined employment of the N and P primers and by involving multiple specimens may significantly increase the positive detectable rate, therefore would be helpful in diagnosis and eratuation of therapeutic effect of SARS.

The specific anti-rat LH in the peroxidase anti-peroxidase (PAP) immunohistochemical method was used to demonstrate the pituitary gonadotropic cells in the pregnant rats, and then the morphometric analysis method was used to study the change of the number, size and nucleo-cytoplasmic ratio parameters of the gonadotropic cells in pregnant rats after being injected by a high dose of LHRH-A.It was observed that on 24h after injection the number of gonadotropic cells decreased, their size became smaller and their nucleo-cytoplasmic ratio larger: the cell number per mm~3 pituitary tissue was from (4.25?0.21)?10~4 to (2.49?0.14)?10~4; the cell size from 1295?35?m~3 to 895?31?m~3; the nucleo-cytoplasmic ratio from 0.144?0.005 to 0.229?0.010. On 48h after injection these changes were in the process of recovery. On 168h after injection all parameters were recovered.Our observations suggest that after being injected by a high dose of LHRH-A the change of gonadotropic cells in pregnant rats results from the exogenous high dose of LHRH-A which has stimulated gonadotropic cells to release a great amount of LH. It is believed, however, that the changes of gonadotropic cells caused by a high dose of LHRH-A can still return to normal.

Objective To study the relationship between genetic polymorphisms of cytochrome P450 2E1(CYP2E1) and susceptibility to gastric cancer. Methods Genotype of CYP2E1 was determined by polymorphism (PCR-RFLP) analysis on DNA in 92 patients with gastric cancer and 92 controls in case-control study. Results The results showed that the frequency of the wild-type genotype (C1/C1) detected by RsaⅠ digestion was 66.3% and 48.9% in gastric cancer group and controls, respectively (P

Objective:To investigate the ways of pharmaceutical care performed by clinical pharmacists for the patients with severe infection. Methods:Through deciding the anti-infection therapeutic regimen, providing drug counselling and pharmacy education and focusing on adverse drug reactions, pharmacists offered suggestions for one patient with severe legionella pneumonia complicated with AECOPD. Results:The pharmaceutical care performed by clinical pharmacists could solve the problems and improve the compliance, safety, effectiveness and rationality in the drug treatment of the patient. Conclusion:According to the individual condition of patients, clinical pharmacists can realize their own values through looking for the breakthrough points of pharmaceutical care and participating in clinical practice.

Objective: To provide an effective hGM CSF gene transferring vector mediated by gene gun and a basis for study of hGM CSF gene modified tumor cell vaccines. Method: The gastric tumor cell line (SGC) was transfected with eukarytic expression plasmid deoxyribonucleic acid containing the human granulocyte macrophage colony stimulating (hGM CSF) gene using the gene gun. The SGC cell clones (SGC GM CSF 1～5)secreting high hGM CSF level were obtained after G418 resistance selection. The hGM CSF gene had been integrated into chromatosome of SGC by the assay of RT PCR.Results: There was hGM CSF production whose lane was about 30 kD in the culture medium of SGC GM CSF by the assay of SDS PAGE and Western blot. SGC GM CSF had the ability of the high level of GM CSF for a long time(mean 247ng/(10 6 cell?24 h).Conclusion: The hGM CSF gene transferring vector mediated by gene gun was effective and safe. These results provide a basis for study of GM CSF gene therapy for cancer.

AIM Taurine can regulate Asp、 Glu、 GABA from rat cortical synaptosomes. But its mechanism is unclear. The release regulated effects were investigated through analysis of GABA receptors.METHODS Bicuculline、Phaclofen、Baclofen were added in a Krebs Ringer buffer with resuspended synapotomes.Endogenous Asp、 Glu and GABA release during the 5 min superfusion were measured by high perfusion liquid chromatography using percolumn durivatization with Dans Cl. RESULT Phaclofen,but not bicucullion baclofen, counteracted the inhibition of GABA overflow,although the inhibition of Asp and Glu overflow was not attenuated. CONCLUSION Taurine inhibits the depolarization evoked release of GABA through the activation of presynaptic autoreceptors and taurine also acts on presynaptic sits of Asp Glu nerve terminals to inhibit their evoked release in rat cerebral cotex.

Objective:To investigate the antitumor effect of TKD-activated NK cells on tumor growth inhibition of human pancreatic carcinoma in nude mice .Methods:CD3-CD56+NK cells were obtained from human peripheral blood mononuclear ( PBMC) in stem cell growth medium SCGM , 2 μg/ml TKD was added to the medium on day 10.The activating receptor CD 94/NKG2C expression levels on NK cells was detected with FAC after 4 days.The cytolytic activity of TKD-activated NK cells against human pancreatic carcinoma subline with HSP 70 expression on their cell surface was analyzed by MTT assay .Established a new model of orthotopic-transplantation tumor of human pancreas .NK cells were injected i.v.into the tail vein of tumor-bearing mice on day 15,the antitumor activity of the NK were evaluated .The capacity to infiltrate Colo 357 tumors in SCID/beige mice was detected with Immunohis-tochemistry.Results:Flow cytometry analysis showed that CD3-CD56+NK cells could expanded in SCGM medium ,and the average percentage of NK cell was (87.50 ±1.35 )%.CD94 expression levels on NK cells increased obviously ,the mean fluorescence intensity of CD94 was(220.56±1.82),compared to (68.72±1.85)of control group cell.The cytolytic activity against HSP70 membrane-positive pancreatic carcinoma sublines Colo 357 cells was high and there was significantly statistical difference between TKD-activated NK cells and unactivated NK cells.The cytolytic activity was(68.72±2.55)%when ratio of effector cells and target cell was 40:1.TKD-activated NK cells had a stronger suppressive effect on tumor growth in BALB /c nude mice bearing Colo 357 cells in vivo ,Median inhibitory rates was ( 61.3 ±1.5 )% .There was significant statistical difference compare to control group ( P <0.01 ) .The result of Immunohistochemistry indicated that predominantly NK cells induced with TKD had the capacity to infiltrate Colo 357 tumors in SCID/beige mice.Conclusion: TKD-activated NK cells are highly efficient cytolytic effector cells which have a stronger significant suppression against pancreatic carcinoma growth in vivo .

Objective:To investigate the Migration ability toward human pancreatic carcinoma cell line and human colon carcinoma cell line with difference HSP 70 plasma membrane expression .Methods: CD3-CD56+NK cells were obtained from human peripheral blood mononuclear(PBMC)in stem cell growth medium SCGM,2μg/ml TKD was added to the medium on 10th day,the ac-tivating receptor CD94/NKG2C expression levels on NK cells was detected with FAC after 4 days.The human pancreatic carcinoma cell line Colo357 and the human colon carcinoma cell line CW 2 were separated into Colo+and CW2+with high HSP70 expression and Colo-and CW2-with low HSP70 expression;Migration assays of NK to the four difference cell lines were performed in a Transwell cell culture system.The cytolytic activity of TKD-activated NK cells against the four subline with HSP 70 expression on their cell surface was analyzed by MTT assay.Results:Flow cytometry analysis showed that CD 3-CD56+NK cells could expanded after 2 weeks in SCGM medium,and the largest percentage of NK cell was (92.50 ±1.25 )%.CD94 expression levels on NK cells increased obviously after TKD inducement the cell surface HSP 70 expression of Colo+, Colo-were ( 78.2 ±2.2 )% and ( 27.3 ±1.2 )% separately , the cell surface HSP70 expression of CW2+,CW2-were (91.1±2.5)%and (18.2±1.0)%separately after FACS;the Migration of NK cells toward Colo+was (68.6±2.8)%,higher than the migration toward Colo-with (22.8±1.5)%;the Migration of NK cells toward CW2+was(73.5±2.7)%,higher than the migration toward CW2-with (18.2±1.3)%;the cytolytic activity of NK against Colo +was(61.2± 3.0)%compared to (24.5 ±1.5)%against Colo-when the ratio of effector cells and target cell was 20 ∶1,the cytolytic activity of NK against CW2+was (63.8±3.2)%compared to (22.4±1.8)% against CW2-when the ratio of effector cells and target cell was 20∶1.Conclusion:TKD-activated NK cells are highly efficient cytolytic effector cells which have stronger significant migration toward HSP70-positive tumor target cells on their cell surface in vitro .

Objective: : To explore the effect of PD-1 gene knockout by CRISPR/Cas9 system on the proliferation and IFN-γ secretion in human T cells. Methods: : The sequence of sgRNA targeting PD-1 was designed. The PD-1-sgRNA and Cas9 mRNA were synthesized by T7 RNApolymerase in vitro, and then the mixture of PD-1-sgRNAand Cas9 mRNAwas delivered into activated T cells by nucleofection. The efficiency of gene knockout was confirmed by sequencing. The phenotypes of T lymphocytes and the expression of PD-1 after gene knockout were analyzed by Flow cytometry. The proliferation of T lymphocytes was calculated by trypan blue counting. The level of IFN-γ secreted by T lymphocytes was detected by ELISA. Results： ：PD-1-sgRNA and Cas9 mRNA were successfully synthesized in vitro and delivered into T cells by nucleofection. Sequencing technology confirmed that the PD-1 gene sequence was edited and the editing efficiency was 58.3%. The expression of PD-1 on T lymphocyte surface was down-regulated successfully by CRISPR/Cas9 system [(9.6±1.85)% vs (16.2±2.05)%, P<0.05]. The knockout of PD-1 gene did not affect the proliferation and phenotype of T lymphocytes(P<0.05); However, compared with the control group, the level of IFN-γ secreted by T lymphocytes in the PD-1sgRNA group was significantly increased (P<0.01). Conclusion: : CRISPR/Cas9 system can successfully ablate PD-1 gene in human T lymphocytes, which could block the negative regulation of PD-1／PD-L1 and further promote the IFN-γ secretion in T cells.

Objective To establish C6 glioma model in rat brain and to study its biological behavior(such as the incidence of tumor development, the process of cell invasion pathological characteristics of C6 and neoangiogenesis, the spontaneous regression of experimental gliomas and the best experimental time window).Methods C6 tumor cells and DMEM were implanted into the right caudate of 50 male Wistar rats. 9 rats implanted DMEM is the control group. The animals were examined by MRI and pathological staining at postoperative day (POD) 3, 7, 14, 21,28, 35, and 50. Matrix metalloproteinases-2 (MMP-2) and CD31 immunohistochemistry staining were used to study the histopathological features of the developed tumor. Methodology, physical findings and biological behavior were also discussed. Results 45 Wistar rats survived after surgery and tolerated MRI procedures well. On POD 7, there was a focal signal at the implantation site. The C6 cells sprout to the surroundings along the nerve fiber. During the day 14～28, the tumor exhibited a marked increase in size with focal mass effect, and immunohistochemical-staining shows MMP-2 and CD31 is overexpression; C6 cells were aggregated and blood brain barrier were destroyed greatly. Most of the tumor bearing rats died within 30 days. But, C6 cells in the two rats retrogress spontaneously after more leucocytes rounded 28 days. HE staining shows tumors.Conclusion The characteristics of rat C6 brain tumor model mimicked the human tumor with respect to its development, progression, and invasion. Although, part of C6 tumor spontaneously regressed, it is a useful animal model of glioblastoma for pre-clinical evaluation of various therapeutic strategies for the management of glioblastoma. The best experimental time window is 14 to 28 days.