This study aimed at preparing paeonol thermosensitive gel and preliminary exploring its properties in vitro.Tube inversion method was adopted to investigate the effects of concentrations of poloxamer 407 and poloxamer 188 on gelation temperature.Then,viscosity of the gel was detected by rotary viscometer,and in vitro erosion and drug release characteristics of the gel by no film stripping method.As a result,the gelation temperature of poloxamer 407 decreased with the increase of its concentration,while gelation temperature of poloxamer 407 increased with the accelerating concentration of poloxamer 188.The cumulative drug release of paeonol thermo sensitive gel was up to 70％ in 320 rin.Gel dissolution and drug release were simultaneously performed without burst release phenomenon.It was concluded that the preparation process of paeonol thermo sensitive gel was simple and easy to use with the overt effect of sustained-release.

Objective To investigate the correlation of epidermal distribution of lamellar bodies and expression of ceramidase with skin barrier dysfunction in polymorphous light eruption.Methods Forty-seven patients with polymorphous light eruption and 40 healthy volunteers were recruited into this study.Noninvasive instruments were used to measure skin sebum content,transepidermal water loss(TEWL)and water content in stratum corneum in all of the subjects.Then,tissue specimens were obtained from the lesions at sunexposed sites in the patients and normal skin of the healthy volunteers.The ultrastructure and distribution of lamellar bodies were observed with transmission electron microscopy in five lesion and control specimens.Immunohistochemistry was performed to detect the expression of ceramidase in the tissue specimens.Results Compared with the normal skin from healthy volunteers,the lesions from patients showed decreased number of lamellar bodies in the granular layer and prick cell layer with a disorganized arrangement.Ceramidase was positively expressed in 20 lesion specimens and 36 normal control specimens,weakly expressed in 21 lesion specimens and 4 normal control specimens,and negative in 6 lesion specimens; there was a significant difference in the expression of ceramidase between the lesion specimens and normal control specimens(P ＜ 0.01).The lesions also showed high TEWL(34.2191 ± 12.70 vs.16.8350 ± 6.50,P ＜ 0.01),lower water content in stratum corneum(22.7319 ± 8.71 vs.29.4250 ± 5.08,P ＜ 0.01)and similar skin sebum content compared with the normal skin.Conclusions There is a disturbance in the synthesis of ceramide in patients with polymorphous light eruption,which may contribute to the impairment of skin barrier.

Objective To study the function of vascular endothelial growth factor (VEGF) in type 2 diabetes model rats and its effect on focal cerebral ischemia induced by middle cerebral artery occlusion in these rats. Methods Focal cerebral ischemia was induced by middle cerebral artery occlusion for 6 hours in type 2 diabetes rats and normal control rats.Blood vessels morphology was examined by ink perfusion,infarct size was measured by TTC and expression of VEGF and CD34 were evaluated by immunohistochemistry staining. Results Ink perfusion revealed increased number of small vessels in type 2 diabetes rats. Infarct size was significantly smaller in type 2 diabetes rats ( ( 80. 07 ± 11.21 ) mm3 ) than that in normal controls ((98. 91 ± 14. 86) mm3,t = 2.48,P = 0. 0326). There were more hemorrhage lesions in the ischemic hemisphere in type 2 diabetes rats when comparing with the controls. VEGF and CD34 showed significantly higher expression in type 2 diabetes rats than in normal controls. Conclusions High expression of VEGF and CD34 are found in type 2 diabetes rats after middle cerebral artery occlusion. There is cerebrolvascular remodeling in diabetes rats. While this diabetes-induced remodeling appears to prevent infarct expansion,the changes also increase the risk of hemorrhagic transformation. The latter may result in poor prognosis.

This study was aimed to optimize dihydromyricetin liposome formulations by orthogonal design in order to study its characterization. Dihydromyricetin liposomes were prepared with thin-film ultrasonic dispersion technology. Formulations of dihydromyricetin liposomes were optimized with entrapment efficiency as the optimized index. The formulation and technology were evaluated by the single factor. Based on this, orthogonal design was made on the screening and optimization of dihydromyricetin liposome. Its morphology was observed by transmission electron micro-scope. Its in vitro drug-release behavior was studied by equilibrium dialysis. The preliminary stability was studied. The results showed that the optimized formulation and technology of dihydromyricetin liposome was when the molar ratio of lecithin and cholesterol was 1:1, the dosage of dihydromyricetin was 4.0 mg. The pH of PBS was 5.0, ultra-sonic time was 3 min. The encapsulation efficiency of dihydromyricetin liposome was 58.1%. Its morphology was small spherical or nearly spherical vesicle with observation in transmission electron microscope. It showed that the in vitro release of dihydromyricetin liposome arrived 76.29% in 48 h. The drug content was still 96.57% of dosage for 30 days at 4oC in the dark place. It was concluded that the optimized formulation and preparation technology of di-hydromyricetin liposome were simple, replicable and stable.

Objective To construct a recombinant Bacillus Calmette-Guerin ( BCG ) vaccine strain, rBCG::Rv3478-pMV261, expressing the Rv3478 protein of Mycobacterium tuberculosis and to inves-tigate its immunogenicity.Methods The gene fragments encoding Rv3478 antigen were amplified by PCR and then respectively cloned into pMV261 and pET-28a vectors to construct the recombinant expression plas-mids (Rv3478-pMV261 and Rv3478-pET-28a).The Rv3478-pMV261 plasmids were transformed into the BCG cells to construct the rBCG vaccine strains, while the Rv3478-pET-28a plasmids were transformed into Escherichia coli BL21 strains for the expression of Rv3478 protein.Polyclonal antibodies were induced in mice upon the immunization with Rv3478 protein.The rBCG vaccine strains overexpressing Rv3478 protein were screened out with Western blot assay.The C57BL/6 mice were divided into four groups including the PBS treated group, BCG treated group, rBCG::pMV261 ( R0) treated group and rBCG::Rv3478-pMV261 ( R3) treated group.All mice were sacrificed in 4 or 12 weeks after immunization.Enzyme-linked immunos-pot assay ( ELISPOT) , ELISA and flow cytometry analysis were performed to evaluate the induced humoral and cell-mediated immune responses in mice.Results The Rv3478 protein was successfully expressed and could induce polyclonal antibodies in mice.High levels of IFN-γand TNF-αwere detected in mice treated with R3, indicating that the immunization with R3 enhanced the cellular immunity.Moreover, the ratios of CD4+to CD8+T cells and the percentages of CD44+CD62L+T cells were increased in mice upon the immuni-zation with R3.Conclusion The recombinant BCG vaccine strain overexpressing Rv3478 protein could in-duce stronger cell-mediated immune responses in mice.It might be have a great significance as a new tuber-culosis( TB) vaccine strain against TB infection in the future.

Objective To study the induction of integrin molecules mediated by CYR61 in human peripheral blood mononuclear cells(PBMC). Methods A recombinant expression plasmid containing full length of human CYR61 labeled with human IgG Fc fragment was constructed and identified by DNA sequencing.COS7 was used as host cell for identification of secretary CYR61 expression,confirmed by Western blot method.The commercial lipofectin was adopted for recombinant plasmid transfection into PBMC.Real-time PCR was ultilized to analyze expression patterns of CYR61 and integrin molecules in transfected PBMC. Results The insert sequence was correct in the recombinant plasmid.Western blot test showed that CYR61 protein secreted into culture supernatant or was in COS7 cytoplasm.The recombinant plasmid was transfected into PBMC stimulated with phytohemagglutinine(PHA) to induce CYR61 expression and secretion.The results demonstrated that exogenous CYR61 transcribed rapidly after being incubated with PHA and reached the peak after 24 h.But the expression dropped down quickly to a very low level after 48 h.Simultaneously,integrin molecules expressed just after CYR61 transcription.In the set of integrin molecules tested in the study,?v,?M,?3 and ?5 expression were higher than the other integrin molecules(P

OBJECTIVETo discuss the mechanism of traditional Chinese medicines reducing phlegm and resolving masses in treatment of iodine deficiency-induced goiter by observing the expression of growth factors and the balance-regulating mechanism of proliferation and apoptosis.

METHOD180 four-week-old Wistar rats were selected to establish the iodine deficiency model. After the modeling, the rats were randomly divided into six groups: the normal control group, the model control group, the iodine group, the phlegm compound group, the L-T4 group and the phlegm compound and L-T4 group. At the 21st day and 77th day after administration, 15 rats in each group were killed to collect specimens. Doses were calculated and adjusted according to body surface area and body weight. TT3, TT4 radioimmunoassay, TSH, immunoradiometric method were adopted. Fas, FasL and PCNA protein expressions are detected using immunohistochemical methods.

RESULTCompared with the normal group and the model group, the expressions of fas and FasL in the phlegm Group significantly increased, the expressions of fas and FasL in the phlegm and L-T4 group were also increased significantly. The expression of fas in the L-T4 Group was significantly lower than that of the L-T4 group and the phlegm compound and L-T4 group. Compared with the normal group, the expression of PCNA of the phlegm group and the phlegm and L-T4 group was significantly lower. Compared with the model group, the expression of PCNA of the iodine group, the phlegm groups and the phlegm and L-T4 group were significantly lower. Compared with the normal group, the expression of VEGF in the iodine group significantly decreased after treatment. Compared with the iodine group, the expression of VEGF in the phlegm group and the L-T4 group significantly reduced. Compared with the normal group, the expression of TGF-beta1 in the model group and the phlegm group significantly increased. Compared with model group, the expression of TGF-beta1 in the iodine group significantly reduced. Compared with the phlegm group, the expression of TGF-beta1 in the phlegm compound and L-T4 group was significantly reduced.

CONCLUSIONTraditional Chinese medicines reducing phlegm and resolving masses can completely recover goiter by promoting apoptosis of thyroid cells, inhibiting their proliferation and the expression of growth factors and enhancing the expression of TGF-beta, without causing injury on thyroid cells.

Animals ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Gene Expression ; drug effects ; Goiter ; drug therapy ; genetics ; metabolism ; Humans ; Male ; Proliferating Cell Nuclear Antigen ; genetics ; metabolism ; Rats ; Rats, Wistar ; Thyroid Hormones ; secretion ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Microgravity is known to produce a number of neurological disturbances during space flight; however, the underlying mechanism of these disturbances is yet to be elucidated. There have been some reports about the increased oxidative stress under microgravity or simulated microgravity. In the present study, we investigated the process of oxidative stress induced by simulated microgravity in different areas of rat brain, which may shed light on the mechanism of neurological disturbances and further neuroprotective research in spaceflight. After adaption for 7 d, 40 healthy male Sprague-Dawley rats were matched for body weight and randomly assigned to control groups (7, 14, 21 and 28 d) and tail-suspended simulated microgravity groups (7, 14, 21 and 28 d). The tail-suspended groups were treated with 30 angels of tail suspension and the control groups were treated similarly to the tail-suspended groups but without tail suspension. After the required times, different structures of rat brain, including cerebellum, cerebral cortex and hippocampus, were harvested and frozen for the further determination. Griess assay, thiobarbituric acid reactive substance (TBARS) assay, competitive ELISA and ferric reducing ability of plasma (FRAP) assay were used for the observation of the changes of reactive nitrogen species (RNS), malondialdehyde (MDA), nitrotyrosine (NT) and total antioxidant capacity (TAC), respectively. As shown in the results, there were different changes in various brain regions after tail suspension compared with control groups. (1) In cerebellum, NT increased after 7 d tail suspension, decreased after 14 d and increased again after 28 d; MDA increased after 14 d; RNS increased and TAC decreased after tail suspension for 21 d; (2) Increase of NT after14 d tail suspension, increase of MDA and decrease of TAC after 21 d were found in cerebral cortex; (3) In hippocampus, RNS increased after tail suspension for 7 d, decreased after 14 d and increased again after 28 d; MDA increased after 21 d; NT increased after 28 d; TAC increased after 7 d and recovered after 21 d. These results suggest that simulated microgravity induced by tail suspension increases the level of oxidative stress in rat brain; however, there are different features in different areas of rat brain. During the response to simulated microgravity, rat brain tissues present a similar process from adaptive response to irreversible oxidative damage.
Animals ; Antioxidants ; metabolism ; Brain ; physiopathology ; Hindlimb Suspension ; Male ; Malondialdehyde ; metabolism ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Weightlessness Simulation

High mobility group A2 protein (HMGA2), an architectural factor, is highly expressed in various cancer types including lung cancers. It is a candidate target for cancer therapy. RNAi is an effective gene silencing method with low cost and less time-consuming. It is possible to exploit this technology in therapy. Here, 5 siRNAs targeting Hmga2 gene (HMGA2 siRNA1-5) were designed and synthesized. MTT assay, colony formation assay, transwell assay and flow cytometry were used to evaluate the effects of these siRNAs on lung cancer cell lines (NCI-H446 and A549). Results from cell proliferation, clone formation, migration and apoptosis showed that HMGA2 siRNA1, 3, 5 could affect these aspects for both lung cancer cell lines. Among the five siRNAs, HMGA2 siRNA5 showed the greatest inhibition effects. The inhibition effects of HMGA2 siRNA5 are sequence specific and are not due to the induction of interferon response. Taken together, siRNAs targeting Hmga2 gene are potential candidates for lung cancer gene therapy.
Apoptosis ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colony-Forming Units Assay ; Gene Silencing ; Genetic Therapy ; HMGA2 Protein ; genetics ; metabolism ; Humans ; Interferons ; metabolism ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Point Mutation ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

BACKGROUND: Short-term (1 week) vitreous cryopreservation avoiding the formation of ice crystals has been achieved in preserving tissue-engineered bone composed of adipose-derived stem cells (ADSCs)/demineralized bone matrix (DBM) compound through adjusting particular composition of cryopreservation fluid. However, whether vitreous cryopreservation can be utilized to cryopreserve tissue-engineered bone for long term (12 weeks) and maintain cellular viability and osteogenic function after rewarming remains unclear. OBJECTIVE: To investigate the effects of vitreous cryopreservation on viability and osteogenic function of ADSCs for short-term (1 week) and long-term (12 weeks) cryopreservation. METHODS: ADSCs were isolated from New Zealand rabbits and expended to passage 3. Cells at passage 3 were seeded onto DBM derived from porcine trabecular bone and followed by 1 week osteogenic induction. The tissue-engineered bone was transferred to freezing vials of 2 mL containing vitreous cryopreservation fluid and then directly quenched into liquid nitrogen. The composition of cryopreservation fluid was 30% dimethyl sulfoxide, 70% low glucose-Dulbecco's modified Eagle medium (L-DMEM), 0.8 mol/L trehalose. Following vitrification for 1 week or 12 weeks, the composite of ADSCs/DBM was removed and thawed. After rewarming, ADSCs viability were viewed under confocal laser microscope by staining viable cells with the green fluorescent dye Calcein AM and the red fluorescent dye Propidium iodide at days 1, 3, 7, 11 and 13. The number of cells seeded onto the DBM was assayed by Hochest33258 at days 1, 3, 5, 7, 9, 11 and 13. Meanwhile, alkaline phosphatase (ALP) activity was also assayed by PNP microplate method at days 1, 4, 7, 10, 14 and 21. Osteogenic gene expression including Runx2, OCN, ALP, COL-1 was detected by real- time PCR at days 1, 4, 7, 10, 14 and 21. RESULTS AND CONCLUSION: After cryopreservation of 1 week or 12 weeks, it was found that more red-staining live cells was observed at 1 day post-rewarming by live/dead double staining, and the green-staining live cells increased at 3 days. By Hoechst 33258 assay, it was found that the cell number decreased at 1 and 3 days post-rewarming, compared with pre-cryopreservation. However, a constant increase in the cell number was observed beginning at 3 days, reaching the pre-cryopreservation level at 5 days post-rewarming. By PNP microplate method, it was found that ALP activity reduced at 1 and 4days post-rewarming, but compared with the level of pre-cryopreservation there were no significant difference. However, a constant increase in ALP activity was detected since 4 days. By real-time PCR, osteogenic gene expression including Runx2, OCN, ALP, COL-1 reduced at days 1 and 4, but compared with the level of pre-cryopreservation there was no significant difference. However, a constant increase in the osteogenic gene expression was since 4 days. The cell viability and osteogenic function were observed without significant difference at each time point after rewarming of cells that had undergone vitreous cryopreservation for 1 or 12 weeks. Preliminary findings indicate that vitreous cryopreservation can maintain cellular viability and osteogenic function of tissue-engineered bone. Cryopreservation time (1 and 12 weeks) has no significant effect on the cell viability and osteogenic function of the tissue-engineered bone after rewarming.