Objective To prepare the PLGA nanoparticles( NPs) loading tetrandrine, investigate in vitro drug release behavior. Methods Tetrandrine loaded PLGA NPs were prepared by the emulsion solvent diffusion method and the preparation process was optimized by changing the stabilizer concentration and emulsion energy. The drug loading and entrapment efficiency were studied to evaluate the drug-loading property.The influence of particles size and pH value of release media on drug release behavior was investigated. Results Nanoparticles in the mean size of(203.4±2.8)nm had spherical shape and showed negative surface charge.Drug loading and entrapment efficiency was(2.17±0.10)% and(67.88±4.27)%, respectively.Tet-PLGA NPs retarded drug release in vitro, the cumulative release percentage was increased with the particle size increasing and it in acidic release medium showed a higher drug release amount. Conclusion Tetrandrine loaded PLGA nanoparticles have good entrapment efficiency, uniform particle size and can retard drug release in vitro.

Objectives To investigate HPV infection and its correlati on with the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibito r of metalloproteinase-1 (TIMP-1) in vulvar squamous cell carcinoma (VSCC). Meth ods HPV6/11 and 16/18 DNA were detected using in situ hybridization in 26 case s of VSCC, 21 cases of vulvar intraepithelial neoplasia (VIN) and 10 specimens o f normal skin. The expression of MMP-9 and TIMP-1 was measured by immunohistoche mical method in these three groups. Results The infection rates of HPV6/11 and 16/18 in VSCC and VIN were 69.23% (18/26) and 38.46% (10/26), 42.86% (9/21) and 28.57% (6/21) respectively, and no HPV6/11 or 16/18 DNA was discovered in norma l skin epidermis. The expression rates of MMP-9 and TIMP-1 in VSCC, VIN and norm al skin epidermis were 92.31%(24/26) and 76.92% (20/26), 90.45% (19/21) and 80.9 5% (17/21), and 80.00% (8/10) and 50.00% (5/10) respectively. MMP-9 expression in HPV positive lesions was higher than that in HPV negative lesions, but no sig nificant difference of TIMP-1 expression was observed in HPV positive and negati ve lesions. Conclusions HPV infection may play a role in the development of VS CC. The higher expression of MMP-9 comparing with that of TIMP-1 may be an early marker of tumor progression in VSCC, and HPV infection may facilitate the invas ion and metastasis of VSCC.

Objective　To investigate the molecular regulation of G1 arrest of mouse thymocytes induced by ionizing radiation. Methods　Cell cycle was analyzed by flow cytometry (FCM) following staining of cells with proidium iodide. Fluorescent staining and flow cytometry analysis were employed for measurement of protein expression. Results　It was demonstrated that G1 phase of mouse thymocytes increased significantly at 12h after whole body irradiation (WBI) with the doses of 0.5, 1.0 and 2.0 Gy, and at 24h following 2.0Gy exposure, measured by FCM. In the time course experiment, it was found that G1 phase of thymocytes increased significantly at 4h, reached a peak level at 24h and came down toward 48h after WBI with 2.0Gy X-rays. The results also showed that after 2.0Gy exposure, the expression of proteins in mouse thymocytes increased significantlty from 1h to 8h for p53, for p21 from 4h to 48h, and for MDM2 at 4h and 8h, measured by FCM. But no change was found for GADD45 protein expression. Conclusion　These results suggest that G1 arrest could be induced by a single dose of 0.5 Gy, 1.0Gy or 2.0Gy, and its molecular control might be established through the p53-p21 pathway.

Objective To explore the possibility of basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF)combined with striatal conditioned medium promoting the directional differentiation of rat bone marrow mesenchymal stem cells(BMMSCs)into dopaminergie neurons.Methods 1.Separation and culture of BMMSCs:BMMSCs were harvested from healthy adult Wistar rats for serial subcultivation.2.Preparation of Striatal conditioned medium:newborn Wistar rats within 24 hours were selected,and their brain tissues were removed to prepare striatal conditioned medium.3.Induced differentiation of BMMSCs:the 5th passage BMMSCs were collected and pre-induced in low glucose-Dulbecco's modified eagle medium(L-DMEM)containing bFGF and EGF.Twenty-four hours later,pre-induction liquor was replaced with striatal conditioned medium for further induced differentiation.4.Result assessment:the morphological changes of stem cells were observed under inverted phase microscope.The expression of neuron specific enolage(NSE)and tyrosine hydmxylage(TH)were identified by immunocytochemical technique.Results The cell body of rat BMMSCs contracted into round and spindle shape after induction by bFGF and EGF combined with striatal conditioned medium.Partial neuron-like cells with prominence could be found.Immunocytochemieal detection showed that the percentages of NSE and TH positive cells were(72.70±14.81)% and(34.50±15.93)%,respectively.Conclusion BMMSCs can be induced directionally into dopaminergiC neurons by bFGF and EGF combined with striatal conditioned medium in vitro.

Objective To evaluate the effect of different preparation processes on preparation of the glial cell line-derived neurotrophic factor(GDNF)loaded microspheres and observe the biological activity of GDNF.Methods With polylactide-co-glycolide(PLGA)as the coating material,the GDNF-loaded microspheres were prepared by using double emulsion(W1/O/W2).Two-factor factorial design variance analysis was done to analyze the effects of the composition proportion of lactic acid(LA)and glycolic acid(GA)in PLGA and the stirring speed of multiple emulsion on particle size,entrapment efficiency,burst release and in vitro release characteristics of the GDNF-loaded microspheres.PC-12 bioassay was employed to detect the biological activity of the released GDNF so as to determine the optimal preparation process.Results The composition proportion of PLGA could affect the microspheres'burst release(P ＜ 0.05),with no effect on particle size and entrapment efficiency.with the higher.With higher proportion of GA,the release speed of GDNF in the microspheres was increased.When the stirring speed of multiple emulsion was increased from 1 000 r/min to 3 000 r/min,the particle size of the microspheres was decrease significantly(P ＜ 0.01),the burst release was increased markedly(P ＜ 0.01)and the in vitro release rate was accelerated.The activity of GDNF in the microspheres could last for about 20 days at 37℃,which was 10 days longer than that of single GDNF.Conclusions Double emulsioncan prepare the GDNF-loaded microspheres with high entrapment efficiency and suitable in vitro release time.In the meantime,the microspheres can extend the validity of GDNF.

Objective To study the expression of interleukin-1β in aortic dissections and aneurysms. Methods Aortic specimens were obtained from patients with type Ⅰ thoracic aortic dissection (11 cases),ascending thoracic aortic aneurysms (10 cases),and healthy organ donors (7 cases).Expression of interleukin-1β,matrix metalloproteinase-9,and signal transduction factors phospho-p38 and phospho-JNK were detected by real time RT-PCR,Western blot,and immunohistochemistry,respectively.TUNEL staining was performed to detect apoptosis of media cells. Results Apoptosis in the media of thoracic aortic dissection and ascending thoracic aortic aneurysms was dramatically higher than control group.Expression of interleukin-1β gradually increased in an order of control group,thoracic aortic dissection to ascending thoracic aortic aneurysms ( P ＜ 0.01,respectively).Expression of matrix metalloproteinase-9significantly increased in the media of thoracic aortic dissection and ascending thoracic aortic aneurysms compared with control group (P ＜ 0.01,respectively).There were positive correlations between interleukin1 β and matrix metalloproteinase-9,interleukin-1β and phospho-p38 in thoracic aortic dissection ( P ＜ 0.01,respectively),interleukin-1β and apoptosis in ascending thoracic aortic aneurysms (P ＜ 0.01 ).Conclusions Interleukin-1β and interferon-γ might effect the formation of thoracic aortic dissection and ascending thoracic aortic aneurysms possibly through the up-regulation of matrix metalloproteinase-9 and apoptosis of media cells in humans.

AIM:To investigate the impact of long-term administration of pentoxifylline (PTX) on morphology and inflammation of the lung in mouse models with chronic exposure of cigarette smoke. METHODS: Male BALB/c mice were randomized into the following four study groups: smoke-exposure only, shamed smoke-exposure, smoke-exposure and PTX administration, shamed smoke-exposure and PTX administration. Animals assigned to smoke-exposure were put inside a chamber twice a day for cigarette smoke exposure. The oral dose of PTX allocated to each mouse was about 20 mg?kg-1?d-1. Animals were sacrificed anaesthetically at day 120. Slices of lung were stained with H&E for pathological analysis. Modified ashcroft pulmonary fibrosis score (mAPFS) was estimated, and IFN-? (a Th1 cytokine), IL-4 (a Th2 cytokine) in broncho-alveolar lavage fluid (BALF) and hydroxyproline in mouse lung tissue were measured by commercial kits of ELISA assay. RESULTS: Lungs in smoke-exposure only group exhibited emphysema-like morphology, low mAPFS (median 1.50, 95%CI 1.25-3.75), lowest hydroxyproline (2.43?0.11) mg/L and lowest ratio of IL-4 to IFN-? (20.3?25.5), whereas lungs in smoke-exposure and PTX interference group exhibited interstitial fibrosis-like morphology, highest mAPFS (4.75, 4.09-5.71), highest hydroxyproline (5.57?0.55) mg/L and highest ratio of IL-4 to IFN-? (70.7?59.9) among the four study groups (P

AIM: To study the effect of vascular endothelial growth factor(VEGF) on hematopoietic differentiation from mouse embryonic stem cells(ESC) in vitro.METHODS: ES-D3 was allowed to grow on mouse fetal fibroblast feeder layer,and then was developed into embryoid bodies(EB).EB cells were transferred into medium supplemented with different concentration of VEGF and VEGF+SCF for 1 week.Six groups,including.VEGF 5 ?g/L,VEGF 10 ?g/L,VEGF 20 ?g/L, VEGF 5 ?g/L+SCF,VEGF 10 ?g/L+SCF and VEGF 20 ?g/L+SCF,were designed.The group of spontaneous differentiation without cytokine(s) was used as control.Hematopoietic transcription factor GATA-2 and early hematopoietic differentiation genes(c-kit and ?-H1) were detected by RT-PCR.The content of CD34~+ cells in each group were measured by flow cytometry.The cells derived from ESC were incubated in semisolid methycellulose cultures.The numbers of total colony-forming units in culture(CFU-C) were counted by reverse microscope.RESULTS: ES-D3 grew and formed EB at day 4.VEGF had a stimulatory effect as a single factor on the expression of genes associated with early hematopoietic differentiation(GATA-2,c-kit and ?-H1),the generation of CD34~+ cells and CFU-C in EB.The effects of VEGF+SCF were the most potent in the experimental groups according to the percent of CD34~+ cells and the numbers of hematopoietic colonies.The most highest inducing efficacy was achieved in VEGF 20 ?g/L or VEGF 10 ?g/L combined with SCF.CONCLUSION: VEGF promotes the differentiation of ESC into hematopoietic cells in vitro.The strongest effect was achieved when VEGF was combined with SCF.

OBJECTIVETo study the effect of gene radiotherapy combining injection of recombinant plasmid pNEgr-mIL-12 with local X-irradiation on cancer growth and to elucidate the mechanisms of tumor inhibition.

METHODSAlkaline lysis was used to extract the plasmid and polyethylene glycol 8000 (PEG 8000) was applied for further purification of plasmids. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of IL-12 protein. C57BL/6J mice were subcutaneously inoculated with B16 melanoma cells and the plasmid was injected directly into the tumor. Gene-radiotherapy combining pNEgr-mIL-12 recombinant plasmid with X-irradiation was given three times to C57BL/6J mice bearing B16 melanoma. Changes in immunologic parameters of tumor-bearing mice were detected with relevant immunologic assays.

RESULTSResults showed a significant decrease in tumor growth rate (P<0.05-0.001) after 3 times of gene-radiotherapy with IL-12 and X-irradiation. Immunologic studies showed a significant increase in CTL and NK cytolytic activity (P<0.05-0.001) and an up-regulated secretion of IFN-gamma and TNF-alpha (P<0.01-0.001). Moreover, the expression of mIL-12 in B16 melanoma cells of the treated tumor-bearing mice was found to be higher than that of control.

CONCLUSIONpNEgr-mIL-12 plasmid combined with X-irradiation can increase tumor control and the mechanism of increased tumor inhibition is related to the enhancement of anticancer immunity in tumor-bearing mice.

Animals ; Combined Modality Therapy ; Enzyme-Linked Immunosorbent Assay ; Female ; Genetic Therapy ; Interferon-gamma ; immunology ; Interleukin-12 ; biosynthesis ; genetics ; therapeutic use ; Killer Cells, Natural ; immunology ; Macrophages ; immunology ; Melanoma, Experimental ; immunology ; radiotherapy ; therapy ; Mice ; Mice, Inbred C57BL ; Plasmids ; Spleen ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Necrosis Factor-alpha ; immunology ; X-Rays

AIM To observe the effects of Qibao Meiran Oral Liquid (Polygoni multiflori Radix Praeparata,Angelicae sinensis Radix,Psoraleae Fructus,etc.) on learning and memory function,hippocampus tissue pathological morphology,SOD activity and carbonyl protein content in SAMP8 mice.METHODS Twenty-seven SAMP8 mice were randomly and equally divided into model control group,donepezil hydrochloride group and Qibao Meiran Oral Liquid group.Another nine SAMR1 mice were selected as normal control group.Mice were given successive intragastric administration for 60 days.On the 56th day,the passive avoidance test was adopted,and the learning and memory capacities were determined after 5 d;The pathological morphology was observed by HE staining;ELISA assay was used to detect the activity of SOD and the content of carbonyl protein in brain tissue.RESULTS Compared with the model control group,the escape latency of mice in the Qibao Meiran Oral Liquid group was significantly prolonged,and the number of errors decreased significantly (P ＜0.01);the pathological morphology of hippocampus tissue was significantly improved;SOD activity increased significantly,and carbonyl protein content decreased significantly (P ＜ 0.01).CONCLUSION Qibao Meiran Oral Liquid can not only improve the learning and memory function of SAMP8 mice,but also reduce the degree of hippocampus tissue degenerative disease.