Objective To investigate the effect of bortezomib alone and in combination with imatinib on apoptosis of human chronic myeloid leukemia cells of the line K562 ,what is more, to explore the mechanism. Methods K562 was cultured and treated with bortezomib and (or) imatinib in different concentrations for 12, 48, 72 hours. Cell proliferation was analyzed by MTT assay, the apoptosis of K562 cells was observed by flow cytometry, and the expression of Livin mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR). Results MTT assay and flow cytometry showed that bortezomib could inhibit K562 cell proliferation and induce its apoptosis, and it can strengthen cells' lethal effect by imatinib. The expression of Livin mRNA was decreased by bortezomib or imatinib to some extent, and was downregulated significantly when combined treatment was given. Conclusion Bortezomib alone and in combination with imatinib can induce K562 cells apoptosis, in which decrease of the expression of Livin mRNA is the possible mechanism.

Purpose:To evaluate two different methods of radiotherapy, which affect the results of cervical cancers treated and complications of the rectum or bladder. Methods:From July 1991 to December 1996, 124 cases of cervical cancer were randomly allocated into two groups. Group A (59 cases:stage Ⅰ 1, Ⅱa 12, Ⅱb 30, Ⅲa 11, Ⅲb 5) received 45—55 Gy of external radiation(ER) to the parametrium and 50—65 Gy of intracavitary irradiation (4—5 Gy per fraction, 2 fractions per week). Group B(65 cases: stage Ⅰ 2, Ⅱa 15, Ⅱb 29, Ⅲa 10, Ⅲb 9) received 26—40 Gy ER to the whole pelvic and 30—40 Gy of intracavitary irradiation (4—5 Gy per fraction, 2 fractions per week), an additional 15—29 Gy ER to the parametrium. Results:In group A, 3 year and 5 year survival rates were 81.4% and 71.7%. In group B, they were 84.6% and 70.8%. The rates of complication for the rectum were 25.4%(3 year) and 21.7%(5 year) in group A, 10.8%(3 year) and 8.3%(5 year) in group B. The rates of rectal complication( P

Objective To establish human renal cell carcinoma (RCC) cell lines,and to investigate the cell phenotypes and expression of tumor associated antigens. Methods Fresh tumor tissues from pathologically proven human RCCs were primary cultured and passed generation to generation until a stably grow in vitro.The cell cloning form,chromosome and graft into nude mice in vivo were examined for cell lines.Immunofluorescent stain and flow cytometric analysis were carried out for cell's phenotype,RT PCR examination for MN/CA9 gene expression. Results Six human RCC cell lines have been established,including four of the cell lines derived from clear cells,one from mixed clear granular cells and one from papillary cells.All the cell lines showed the characteristics of malignant cells.All the lines highly expressed HLA ClassⅠ and HER2/neu.Three lines weakly expressed HLA ClassⅡ and one cell line highly expressed CD86 but all the lines did not express CD80.RT PCR showed that three cell lines have the expression of MN/CA9 gene. Conclusions These newly established RCC cell lines would provide a useful in vitro model for studies related to biological characteristics,tumor associated antigen,immunogenity and immunotherapy of human RCC.

ObjectiveTo provide experimental proof for the specific immunotherapy in patients with renal cell carcinoma (RCC).MethodsThe expression of c-erbB-2 and HLA-A2 molecules in RCC were examined by flow cytometery and HLA-A2 cDNA RT-PCR.Tumor lysatic antigens (Tuly) was used to load DCs (DC-Tuly) to induce the generation of c-erbB-2 specific CTLs derived from peripheral blood mononuclear cells of RCC patients in vitro and CTLs was used to kill RCC cells which express or not express c-erbB-2 and HLA-A2.The cytotoxic activity of induced CTLs was further studied by antibodies (anti-c-erbB-2 and anti-CD 8) blocking assay.Resultsc-erbB-2 protein is tumor associated antigens for RCC.DC-Tuly induced specific CTLs highly killed c-erbB-2+ HLA-A2+ autologous and allogenic RCC,but non-specific ones lowly killed c-erbB-2- HLA-A2+ or c-erbB-2+ HLA-A2-RCC cells. The cytotoxic activity against tumor cells was blocked by anti-c-erbB-2 and anti-CD 8 monoclonal antibodies.ConclusionsThese results suggest that this specific CTLs adoptive immunotherapy for RCC patients with c-erbB-2+ HLA-A2+ could be a novel approach for clinical use.

Objective:To optimize the ethanol precipitation technique for Fufang Shenqi soft capsules. Methods: An orthogonal design was used to optimize the technique with the relative density of the concentrated solution, ethanol concentration, standing time, temperature of ethanol precipitation as the influencing factors and the yield of dry extract and the content of total polysaccharides as the indices. Results:The best ethanol precipitation technique was as follows:the relative density of the concentrated solution was 1. 10, 95% ethanol was used to obtain 60% ethanol concentration, and the standing time was 48 h under the temperature of 10-30℃. Con-clusion:The optimized ethanol precipitation technique for Fufang Shenqi soft capsules is simple and practicable, and suitable for prac-tical production.

Objective To analyze inhibitory effect on Wistar rattish hepatoma by endostatin, angiostatin,hyper-liquid hpiodol and a combination of these agents. Methods The rattish hepatoma models were randomly divided into 8 groups, including control group, lipiodol group, endostatin gene group, angiostatin gene group, endostaitin gene and angiostatin gene group, endostatin gene and lipiodol group, angiostatin gene and lipiodol group and endostatin gene, angiostatin gene and lipiodol group. Following treatment on Wistar rots with these agents or a combination of these agents, we measured the changes in tumor volume growth rate, metastases quantity rote, rattish life span, micmvessel density (MVD) value, apoptotic index(AI), vascular endothelial growth factor. Single factor variance test and Chi-square test were used for statistics. Results On the sixth day after treatment, the tumor volume growth rate of all the therapy groups were smaller than the control group. The therapeutic effect of the above mentioned two genes in combination with lipiodol was the best and the tumor volume growth rate was the smallest. On the eighteenth day after treatment, the numbers of pulmonary metastasis were(37.2±7.2)、(26.2±5.0)、(25.5±4.7)、 (26.2±3.9)、(14.9±2.6)、(19.1.4-2.8)、(20.2±2.7)and(6.1±1.2); the life span was(23.3± 4.4),(35.2±4.9),(28.2±3.6), (29.4±3.4), (38.3±6.7),(37.7±5.8), (36.4±5.5) and (59.8±9.4)days for each group respectively. The difference of the numbers of pulmonary metastases between the therapeutic groups and the control group was statistically significant(P<0.O1). The difference of the life span between the control group and the single gane group was not statistically significant, but the difference of the life span between the control group and the combination groups is statistically significant. MVD[(63.4±8.7)strip/high power field] in the lipiodol group was higher than the other therapeutic groups. However, the difference of MVD between the lipiodol group and the control group was not statistically significant. The apoptotie indexes in all the therapeutic groups were higher than the control group [(4.2±1.6)% ], the index in the two genes in combination with lipiodol group was the highest[ (19.6± 2.4)%]. The expression rate of VEGF in the control group was the highest(100%)and the rate in the two genes in combination with lipiodol group was the lowest(12.5%). Conclusions The inhibitory effect on Wistar rattish hepatoma of a combination of the above-mentioned two genes and lipiodol via transhepatic artery infusion is obviously superior to that of each individual agents.

Background To understand the distribution and development of corneal nerve in animal or human has an important significance for clinical and basic research of corneal diseases.At present,some studies on cornea nerve development and location have been performed.However,the quantified study on innervation and distribution of corneal nerve fibers as embryonic development has not been reported.Objective This study attempted to understand the distribution of corneal nerve fibers in the development of chick embryo,and to evaluate the changes of the length and density of corneal nerve fibers with aging of chick embryo.Methods Whole chick corneas with limbus were obtained from chick embryo aged 6-20 days (E6-E20),and corneal nerve fiber was labeled using immunofluorescence technique by anti-neuron-specific β-Ⅲ tubulin antibody.The corneas were radially cut into 4 parts,and the integrate corneal flat mounts were prepared with the upward epithelium and mounting with anti-fade fluorescent quenching buffer glycerin containing DAPI.Fluorescence microscope was used to capture the nerve fiber images in cornea,and cornea area and the number of nerve fiber bundles were exhibited by using Photoshop CS4.Cornea nerve fiber density and total length were measured by Imaris x64 7.4.2 software.Results Total cornea flat mounts showed that the nerve bundles grew from temporal scleral forward cornea limbus at E6-E8,and the nerve fibers formed the ring surrounding by limbus during E9-E10.Then the fibers extended forward the central cornea in E11 to E15 and developed into nerve fiber plexus on the whole cornea in E16 to E20.During the period of E6-E20,the corneal surface area,the length and density of corneal nerve fibers were gradually increased with the aging of chick embryo,showing statistically significant differences among different time points (F =127.007,227.051,67.748,all at P＜0.01).The increase of the corneal area of the chick embryo presented a strong positive correlation with the extending of length of the corneal nerve fiber (r =0.863,P＜0.01).Chick corneal nerve fiber bundle appeared at E13,with a number of (59.00 ± 1.14)/mm2 and then increased to a peak of (576.75 ±29.16)/mm2 at E 18 and reduced to (299.67± 25.46)/mm2 at E20,with a significant difference among them (F =13.759,P=0.000).Conclusions Corneal nerve starts to develop in E9 of chick embryo,and the corneal surface area,the total length of the corneal nerve fibers and the density rapidly increase concurrently with the development of chick embryo.

Objective To investigate the secrection of Polytuftsin, a macrophage activator in Pichia pastoris expression system. Methods The recombinant plasmid encoding polytuftsin was transformed into the yeast by chemical method, and the positive clones were screened and expressed under the induction of methanod. It was observed that the influence of polytuftsin on macrophage in vivo and in vitro. Results It was verified that the recombinant products was confirmed to be our target protein by SDS-PAGE analysis and PCR assay. The expressed product could activate macrophage to secret cytokine and enhance the capacity of macrophages to kill L1210 tumor cells, the maximum killing rate is up to 40 %. In vivo the leukemia therapeutic trial, the time of tumor arise was delayed; the survival time of L1210 tumor-bear mice treated with Polytuftsin was extended, which is about 20 day. Conclusion The recombination protein polytuftsin, which could activate macrophage and has anti-tumor activity.

Objective To establish a human adrenocortical carcinoma (ACC)cell line ACC-LWL, and investigate its cell phenotypes and expression of tumor associated antigens, to detect sensitivity of ACC-LWL against adoptive immune cells. Methods Fresh tumor tissues from the resection of a human ACC were primary cultured and passed generation to generation so as to achieve stable growth in vitro. For analyzing its biological characteristic, the cell cloning formation in soft agar, chromosome and tumorigenesis were tested. Flow cytometric analysis was carried out for phenotype analysis and RT-PCR examination for MN/CA9 and HLA-A2 genes expression. PBMC from 4 healthy donors (2 HLA-A2+ or 2 HLA-A3+) co-cultured with IL-2 (200 U/ml)and the tumor cell lysate of ACC-LWL (20 μg/ml) in vitro to generate CTL. After 14 day stimulation, CTLs were incubated with ACC-LWL with a E/T ratio of 10:1 for 4 hours at 37 ℃. Cytotoxicity was measured with MTr assay. Results One human ACC cell line had been established that showed the characteristics of malignant cells. The cells grew a solid tumor in nude mice. ACC-LWL had a high expression of MHC-Ⅰ, weakly expression of Her2/neu and MHC-Ⅱ, no expression of CD80 or CD86. RT-PCR showed that ACC-LWL expressed MN/CA9 and HLA-A2 genes. CTL, either HLA-A2+ or HLA-A3+, had the capability of killing ACC-LWL in vitro. Conclusion This newly established ACC would provide a useful target in vitro and in vivo for investigations related to human ACC.

Objective: To observe the expressions of NMDAR2 and GLAST in the hippocampal gyrus and supraoptic nucleus(SON) under sleep deprivation in young rats.Methods: The changes in expressions of NMDAR2 and GLAST in SON and hippocampal gyrus under sleep deprivation in younger rats were observed by immunohistochemistry method. Results:The expressions of NMDAR2 and GLAST in SON and the hippocampal gyrus were significantly increased on the 3rd day of sleep deprivation,even more sigificantly on the 5th day,decreased gradually on 7th day,and became similar to those of control group on the 14th day.Conclusion: Sleep deprivation can affect the expressions of NMDAR2 and GLAST in the hippocampal gyrus and supraoptic nucleus,but the effect diminisshes gradually with prolonged time,which may be associated with self-regulation and self-protection of the central nervous system.