Objective:To explore the availability for tumor-derived gp96 to induce specific CTL activity of splenocytes in vitro.Methods:gp96 purified by the techniques for protein extraction and identified by SDS-PAGE gel electrophoresis and Western blot method; CD8+T cell induced by gp96 and CTL activity detected by flow cytometry, immunofluorescence technic and CCK-8 assay.Results:gp96 was identified by SDS-PAGE and Western blot; Analysis with FCM showed that the number of CD8+T cells(nearly 70%) was obviously increased after pulsed with gp96-peptide complexes as compared with that of control groups(35%,26%, P

AIM:To study the expressing variation of TNF-? and IFN-? mRNA in mouse splenocytes induced by H22 tumor cells derived heat shock protein gp96-peptide complexes in vitro,and to observe the morphologic change of H22 tumor cells which treated with the culture supernatant.METHODS:H22 tumor cells derived HSP gp96 was obtained by the techniques for protein extraction and purification and was identified by Western blotting method.The expression values of TNF-? and IFN-? mRNA in spleen lymphocytes were detected by semi-quantitative RT-PCR.The morphologic changes of H22 tumor cells induced by the culture supernatant were observed by laser scanning confocal microscopy(LSCM)and transmission electron microscope(TEM).RESULTS:Purified heat shock protein gp96 was identified by Western blotting.The expression value of TNF-? and IFN-? mRNA in activated spleen lymphocytes induced by gp96-peptide complexes was higher than that in control groups(P

AIM:To investigate the immunomodulatory effect of astragalus saponin(AS)on macrophages and explore the mechanism of its immunomodulation.METHODS:By adding different concentrations of AS into cultured mouse peritoneal macrophages in vitro,the influence of AS on the synthesis of nitro oxide(NO)was detected by NO kit(enzymatic method).MTT assay was used to determine the cytotoxicity of macrophages induced by AS.The morphological changes of macrophages were observed under transmission electron microscope.LSCM and specificity fluorescent probe Fluo-3/AM were applied to observe the change of Ca2+ in macrophages induced by AS.RESULTS:AS significantly increased NO synthesis,enhanced the effects of mouse peritoneal macrophages on killing carcinoma cells.Cell surface projection exhibited multiplication,becoming thickening and growth longer via transmission electron microscope.Increase in intracellular Ca2+ in macrophages was also observed.CONCLUSION:AS enhances the immune function of macrophages by increasing NO synthesis and enhancing cytotoxicity.The increased intracellular Ca2+ may be the mechanism of immunomodulatory effect of AS.

Objective To prepare the chitosan-Mg membranes and to explore the biocompatibility of membranes with PC12 cells and its usage as biomaterials in tissue engineering.Methods The appearance of the chitosan-Mg membranes (CM) was observed under scanning electromicroscope (SEM) and the element of the membranes was analyzed by x-ray energy spectromete. PC12 cells were co-cultured with CM in vitro,the morphological changes of PC12 cells on membranes were observed under scanning electron microscope (SEM) and light microscope (LM),the cell vitality was detected by MTT assay. Results The surface of chitosan membranes (CS) was smoother than that of CM;but the CM groups were full of pore observed under SEM;The content of Mg element was related with the dosage of MgSO4 added into chitosan solution. Morphology observation showed that PC12 cells grew well on CM as compared with CS group. The cells were rich of microvilli and long processes on 7th day,and synapses-like structure was formed in many PC12 cells. In addition,the cell viability of experiment group was higher than that of control group(P

Objective To investigate the effects of up?regulated miR?206/miR?1 on the proliferation of breast cancer stem cells and the effect mech?anism. Methods Breast cancer stem cells(BCSCs)were isolated from breast cancer cell line MCF?7 by fluorescence?activated cell sorting. Cells in the experiment were divided into the blank control group,the negative control group,the miR?206 group and the miR?1 group. The BCSCs were transfected by negative control mimic,hsa?miR?206mimic and hsa?miR?1mimic in all groups except the blank control group. MiR?206and miR?1 expression levels as well as the transcription factor EVI?1 gene were detected by real time PCR. The expression levels of the transcription factor EVI?1 protein were detected by Western blot. MTT method was used to detect the effects of miR?206 and miR?1 on the proliferation of BCSCs. Results The BCSCs(CD44+/CD24-/low cells)isolated from MCF?7 cell lines were successfully cultured in serum?free medium for subsequent studies. After transfection of hsa?miR?206mimic and hsa?miR?1mimic for 48 hours,miR?206and miR?1relative expression levels increased. EVI?1mRNA ex?pression levels significantly decreased. The results of Western blot and MTT showed that up?regulated expression levels of miR?206 and miR?1 could significantly reduce the expression of EVI?1 protein and inhibited the proliferation of BCSCs. The differences in levels of miR?206,miR?1 and EVI?1 protein were statistically significant(P<0.05). Conclusion Up?regulated miR?206 and miR?1 expression can inhibit the proliferation ability of BCSCs,which may be related to the down?regulation of EVI?1.

Objective:To study the difference of the development between mandibular condylar cartilage and femoral head cartilage in vitro.Methods:Mandibular condyles and femoral heads were sampled from 1 2 neonatal mice and cultured in vitro.The samples before culture and after 6-week culture were examined by gross observation,HE staining,Alizarin Red staining and PCNA immunohistochem-istry respectively.Results:After in vitro culture,abnormal changes were observed in condyle cartilage,but the surface area of condyle cartilage was not changed(P >0.05).HE staining showed partial cartilage layer structure disappearance and the Alizarin Red staining confirmed calcification in the cartilage matrix.However,calcification was not found in femoral head cartilage,and the surface area of femoral head cartilage increased(P <0.05).HE staining showed the hypertrophied layer was thicker after culture than before and the Alizarin Red staining showed there was no calcification in the femoral cartilage matrix.The immunohistochemistry displayed PCNA posi-tive expression in both cartilage after culture.Conclusion:In vitro,the mandibular condylar cartilage matrix can be spontaneously cal-cificated.

Objective: To evaluate the cytotoxic effect of PEM? induced by HSPgp96 on anti-tumor in vitro. Methods; PEM? separated from mice induced by thioglycolate were divided into three groups randomly: Culture medium in control; LPS-induced group; HSPgp96-induced group. The production of NO, the cytotoxic effect to H22 cells and the morphologic change of PEM? were investigated separately by enzyme method, MTT assay and scanning electron microscope. Results: In vitro, HSPgp96 can increased NO production from PEM? of mice and significantly enhance the cytotoxic effect of PEM? to H22 cells as well as LPS. Conclusion: HSPgp96 can effectively induce the cytotoxic effect of PEM? on anti-tumor in which NO is one of the capital effective molecules in vitro.

AIM: To investigate the CdTe quantum dots coated with MPA and explore its biocompatibility with living cells. METHODS: CdTe quantum dots coated with MPA were prepared in aqueous phase and MPA CdTe QDs were Characterized with TEM, fluorospectrophotometer and ultraviolet spectrophotometer. QDs were Modified with with avidin, purified and prepared as flurescent probe. LSCM was used to observe the expression of MHC Ⅱ antigen on PMφ cells, which was labeled by QDs. Cell culture and MTT assays were used to determine the biocompatibility of MPA coated CdTe quantum dots with the B-16 cells as target cells. RESULTS: The particle diameter of CdTe quantum dots prepared in aqueous phase was well distributed. They had good photological performance and greater stability after coated with MPA. MHC Ⅱ antigen on PMφ was labeled with the QDs-Avidin fluorescent probe showed great fluorescence intensity, which was easy to be detected by fluorescence microscope and LSCM. MPA CdTe QDs showed cytotoxicity when its density was very high, but they showed little cytotoxicity during the normal use of influence label density limit. CONCLUSION: MPA CdTe QDs can be used as new fluorescent lable as they are of even size, not easy to bleach or quench, have good photological performance and stability and good biocompatibility.

Objective To sequence and analyze the near full-length genome of an HIV-1 subtype G strain identified in Guangxi,China.Methods The demographic information of an individual infected with HIV-1 subtype G strain was investigated,whose peripherial blood was collected.Viral RNA in plasma was extracted.The near full-length genome of HIV was amplified in two halves using RT-nested-PCR.The PCR products were purified and sequenced.Phylogenetic analysis was made using MEGA6 software.Results A near full-length genome of 8847 bp was obtained.In the neighbor-joining tree,the strain clustered with subtype G references,as supported by the high Bootstrap value (100%).The closest phylogenetic relationship was found between our strain and another subtype G strain (JN106043)previously identified in Guangxi,which was supported by the genetic distance (5%)and high Bootstrap value (100%).Conclusion The strain identified in the study might have originated from subtype G strains in Guangxi,suggesting that subtype G has spread locally in Guangxi.Further surveillance of subtype G epidemic in Guangxi is necessary.The near full-length subtype G strain genome will provide information for the surveillance of HIV in Guangxi.

ObjectiveTo evaluate the sensitivity and accuracy of an in-house detecting method of HIV-1 genotypic drug resistance system. MethodsTotally 130 serum specimens from Henan and Guangxi province were collected from April 2004 to October 2008 and tested in the Military HIV Testing Center of China. ViroSeqTM v2.0 (Abbott, Switzerland), a US FDA approved HIV genotypic drug resistance detecting system was utilized as the reference method. All the specimens were detected by the novel in-house method and the reference method to validate the difference in amplifying efficiency, drug resistance mutation detection and drug resistance report. ResultsConcerning the 14 850 known drug resistance mutation sites,14 752 (99. 3％ ) mutations can be detected by both of the two methods. Rates of concordance of detection in the regions of protease inhibitors-, reverse transcriptase inhibitors- and both two classes inhibitors-resistance were99.7％ ( Kappa =0. 909 9 , P ＜0. 01 ) , 99. 0％ (Kappa=0.952 1, P＜0. 01) and99.3％ (Kappa=0. 948 8, P ＜ 0. 01 ) respectively. Drug resistance reports from these two systems showed similar results (Kappa = 0. 637 4, P ＜ 0. 01 ). The in-house detecting system identified 34 novel mutations besides the ViroSeqTM drug resistance mutation database ( ViroSeqTM software v2. 7). Two mutations, V179F and K238T,had significant effect on HIV drug resistance. ConclusionsThe in-house genotyping system is an accurate,cost-effective method and has a high concordance with commercial ViroSeqTM genotyping system. Database from the in-house assay was superior to this of the ViroSeqTM assay.