Objective To select an optimal non-specific antigen blocking method by using immuno-infiltration assay so as to suit protein chip preparation. Methods Human papillomavirus type 16 L1 protein expressed by insect-baculovirus espressin system was incubated with skimmed milk powder, calf serum, bovine serum albumin (BSA) combinations of five kinds of methods to block the non-specific antigen. PBS was used as control. The effect of eliminating non-specific stain was detected by immuno-infiltration assay. Results After repeated tests, the results showed that the stability and repeatability of blocking effects were poor for the fixing up antigen first and then blocking method, and the blank control was prone to false positive. The infiltration rate of NC membrane would be affected by using skimmed milk powder as a blocking agent because the pore of NC membrane was easily plugged by milk powder particles. The use of calf serum as a blocking agent made it very difficult to determine the result because the calf serum absorbed by NC membrane produced the background; however, when 20g/L BSA was used to blocking before fixing up antibody, the results became satisfactory. Conclusion Fixing up antibody after blocking in immuno-infiltration assay showed that the blocking effect against non-specific antigen was satisfactory, stable and repeatable, indicating this method is a novel optimal blocking method compared with others.

Objective To investigate the prevalence of children dental fluorosis in drinking-water-borne endemic fluorosis areas in Liaoning province and to evaluate the disease prevention effects of water improvement measures.Methods Haicheng city,Linghai city,Faku county,Fumeng county,Liaoyang county and Jianping county six endemic fluorosis diseased counties (cities) of Liaoning province were included in the study in September 2011.In each county,3 water improvement villages were selected as survey points.In each surveyed village,children aged 8-12 were generally surveyed the prevalence of dental fluorosis.One tap water sample was collected in each surveyed village for determination of water fluorine.level.Results A total of 786 children aged 8-12 were examined; 110 of them were found with dental fluorosis; detection rate of dental fluorosis was 14.0％ ; no defect case of dental fluorosis was found,and dental fluorosis index was 0.27.Among the cases,very mild were 76 cases,and the detection rate was 9.7％; mild 33 cases,the detection rate was 4.2％; moderate 1 case,the detection rate was 0.1％; and no severe case.Four cases were in the eight-year-old group,accounting for 3.6％ of the total number of cases; 20 in the 9-year-old group,accounting for 18.2％ ; 26 in the 10-year-old group,accounting for 23.6％; 32 in the 11-year-old group,accounting for 29.1％; and 28 in the 12-year-old group,accounting for 25.5％.The differences of detection rates of dental fluorosis of children in each age groups were statistically significant (x2 =13.57,P ＜ 0.05).Water fluoride of the 18 water samples ranged from 0.34 to 2.03 mg/L; 77.8％ (14/18) of the water improvement areas reached the control standard for endemic fluorosis areas.Conclusion The prevalence of dental fluorosis in the areas with high fluoride in drinking water remains high,the measures of water improvement should be strengthened.

Objective To evaluate the role of calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in the up-regulation of expression of Cav3.2 T-type calcium channels in spinal cord in a rat model of neuropathic pain (NP).Methods Forty-eight male Sprague-Dawley rats,aged 3 months,weighing 220-250 g,were randomly divided into 6 groups (n =8 each)∶ sham operation group (group S),group NP,dimethyl sulfoxide group (group D) and different concentrations of a specific CaMK Ⅱ inhibitor KN93 groups (groups K1-3).NP was produced by chronic compression of dorsal root ganglion.The rats in groups D and K1-3 received a single intrathecal injection of dimethyl sulfoxide and KN93 15,30,60 nmol/L (10 μl),respectively,on 5th day after NP.Paw withdrawal threshold to von Frey filament stimulation (MWT) and paw withdrawal latency to thermal nociceptive stimulus (TWL) were measured before NP,before intrathecal injection on 5th day after NP,and at 30 and 60 min and 3,6 and 8 h after intrathecal injection on 5th day after NP (T1-7).The rats were sacrificed after the measurement of pain threshold at T7 and their lumbar enlargements were removed to detect the expression of Cav3.2 mRNA and protein using Western blot and RT-PCR.Results Compared with group S,MWT was significantly decreased,TWL was shortened and the expression of Cav3.2 mRNA and protein was up-regulated in NP,D and K1-3 groups (P ＜ 0.05).Compared with NP group,MWT was significantly increased,TWL was prolonged and the expression of Cav3.2 mRNA and protein was down-regulated in a concentration-dependent manner in K1-3 groups (P ＜ 0.05),and no significant change was found in the parameters mentioned above in group D (P ＞ 0.05).Conclusion CaMK Ⅱ is involved in the development and maintenance of chronic NP by up-regulating the expression of Cav3.2 T-type calcium channels in rat spinal cord.

BACKGROUND:The osmotic tissue expander is a self-fil ing device consisting of an osmotic active hydrogel which is made of vinylpyrrolidone and mehtylmethacrylate. It can absorb body fluids and swel up gradual y after embedded.
OBJECTIVE:To explore the short-term and long-term regular patterns as wel as histocompatibility of the osmotic tissue expander in vivo.
METHODS:A self-control design was carried out in Wistar rats by embedding the osmotic tissue expander and high-density polyethylene into each side of their spinal column subcutaneously. Wound healing, tissue expansion and inflammatory reaction were detected and compared at different periods after operation.
RESULTS AND CONCLUSION:Al the wounds got primary healing. The device expanded fastest at week 1 after the implantation. After being enlarged to about nine times that of the initial size at week 4, the expander slowed down its swel ing. It reached its ultimate volume at week 12 which was about 10 times as big as that of the initial one. Then it remained almost the same size until the end of our design. Pathological sections showed that the inflammatory reaction of osmotic-tissue-expander-group had no significant difference from that of the control group (P>0.05). These findings suggest that osmotic tissue expander has a slow-lasting swel ing ability and good histocompatibility.

Objective To investigate clinical manifestations and clinical diagnosis and treatment and pathological and immunohistochemical features in gastrointestinal stromal tumors. Methods The clinical data of fifty-two cases with gastrointestinal stromal tumors were collected, whose clinical diagnosis and treat-ment and pathological features were retrospectively analyzed from January 1995 to December 2007. Results All patients received operation therapy, only forty-five cases with complete surgical resection. The immu-nohistochemical staining showed that the cases with CD117 positive accounted for 100% (52/52) and CD34 positive accounted for 88.5% (46/52). Conclusions Surgery was necessary for all patients, especially complete surgical resection. Gastrointestinal stromal tumors were poor in preoperative diagnosis, which diag-nosis was based on the immunohistochemical staining of the tumor tissue. CD117 and CD34 tumor markers may help to diagnose gastrointestinal stromal tumors.

Aim To investigate the protective effect of cinnamic aldehyde ( CA ) on hormone-induced osteo-clasts proliferation and bone resorption in vitro and its molecular mechanisms. Methods RAW264. 7 cells induced into osteoclast were treated with RANKL and M-CSF and then were divided into control group, dexa-methasone ( DEX ) group and different doses of CA (11. 6, 23. 2, 46. 4 μg·L-1 ) groups. OCs were ob-served after tartrate resistant acid phosphatase( TRAP) staining. The cell proliferation was determined by MTT assay at different time points. The expression levels of TRACP5 b in cell cultured supernatants were measured by ELISA. RT-PCR technique was applied to examine the transcriptional levels of RANK and NFATc1 . Re-sults In MTT assay, the proliferation of osteoclasts stimulated by dexamethasone was promoted seriously compared with negative control group ( P < 0. 05 ) . Meanwhile, DEX could strengthen the content of TRACP5 b and up-regulate the expressions of RANK and NFATc1 mRNA. After administration of CA, the proliferation was inhibited, while the enhanced expres-sion of TRAP5b was reversed,and the over-expressions of RANK and NFATc1 mRNA were obviously down-regulated in a time-and-dose-dependent manner ( P <0. 05 ) . Conclusion The results suggest that CA in-hibits proliferation and bone resorption of osteoclast in-duced by DEX, which may be mediated by down-regu-lation of RANK and NFATc1 mRNA.

Objective To investigate the effects of repetitive ultraviolet A(UVA) radiation on the endocytosis and intracellular degradation of extracellular elastin by human dermal fibroblasts(HDFs), and to explore the mechanism responsible for elastin accumulation in photoaging skin. Methods Cultured HDFs were divided into two groups:repetitive UVA radiation group treated with UVA radiation(9.9 J/cm2)once a day for seven consecutive days to establish a chronic photodamage model, and blank control group receiving no treatment. For the verification of the model, flow cytometry was performed to detect cell cycle, and senescence-associated β-galactosidase staining to determine the degree of cell senescence. Fluorescent tracer technique and conjugated polymer-based fluorescence quenching technique were conducted to observe the endocytosis and intracellular degradation of extracellular elastin in culture media by HDFs in these groups. Results Compared with the blank control group, the repetitive UVA radiation group showed increased proportions of G1-phase cells and senescent cells, which confirmed the successful establishment of chronic photodamage model in the repetitive UVA radiation group. After coculture with elastin for 24, 48 and 72 hours, the endocytosis rate of elastin was 10.0% ± 1.4%, 27.8% ± 4.2% and 39.9% ± 4.1% respectively in the blank control group, 9.9% ± 1.6%, 28.3% ± 5.1% and 42.0% ± 5.7% respectively in the repetitive UVA radiation group, with no significant difference between the two groups at the three time points (all P > 0.05). The percentage of cells showing intracellular degradation of extracellular elastin was significantly lower in the repetitive UVA radiation group than in the blank control group (4.2% ± 1.1% vs. 7.7% ± 0.9% at 24 hours, 16.5% ± 2.4% vs. 22.8% ± 1.8% at 48 hours, 26.7% ± 2.6% vs. 33.9% ± 3.1% at 72 hours, all P < 0.05). Conclusions UVA radiation at 9.9 J/cm2 for 7 consecutive days can decrease the intracellular degradation of extracellular elastin by HDFs, but has no obvious effect on endocytosis of elastin.

Objective To observe the effect of electroacupuncture on back three-acupoints ( namely bilateral Dazhu, Fengmen, Feishu) on the protein and mRNA expression of transforming growth factor-beta 1 (TGF-β1) and Smad3 in asthmatic rat airway remodeling model, and to explore its therapeutic efficacy and molecular mechanism. Methods Rat asthma model was established by inhalation of ovalbumin. After sensitization for 6 weeks, rats were killed. And then the airway morphological parameters of rats were measured by image analysis. The protein and mRNA expression of TGF-β1 and Smad3 in lung tissues were detected by immunohistochemistry and real-time quantitative polymerase chain reaction (qRT-PCR) respectively. Results Compared with the blank group, the standardized values of muscle cross-sectional area including airway smooth muscle area (WAm) /perimeter of the basement membrane (Pbm), and bronchial inner wall area (WAi)/Pbm were increased in the model group. The protein and mRNA expression levels of TGF-β1 and Smad3 were also increased in the model group. In electroacupuncture group, the above observation indexes were decreased, and the differences were statistically significant ( P<0.05 compared with the model group). Conclusion The experimental results indicated that back three-acupoint electroacupuncture has an effect on remodeling airway, and one of the mechanisms is probably associated with the regulation of TGF-β1/Smad3 signaling pathway.

[ ABSTRACT] AIM: To investigate the differential expression of human leukocyte antigen-G ( HLA-G) isoforms and its receptors in human monocyte line THP-1 after human cytomegalovirus ( HCMV) infection for exploring the role of HLA-G in HCMV escaping the immune response of the organism .METHODS: THP-1 cells were infected with HCMV Towne strain.The expression of HLA-G isoforms at mRNA and protein levels was determined by RT-PCR and Western blot, respectively.The surface expression of HLA-G and its receptors ILT2/ILT4 and the cell viability were analyzed by flow cytometry.The levels of soluble HLA-G (sHLA-G) and IL-10 were measured by ELISA.RESULTS:After infection of the THP-1 cells with HCMV , no obvious apoptosis in the cells was observed , and the viability of the cells was high .A significant up-regulation of HLA-G1,-G3,-G4 and-G5 at mRNA expression level 1 d after infection was found , while the protein expression of HLA-G1 and HLA-G5 isoforms was mainly detected .The expression of HLA-G/ILT2/ILT4 was evi-dently up-regulated 1 d after infection .The level of sHLA-G was significantly increased 1 d after infection as compared with control group (P<0.01).The expression of IL-10 was obviously up-regulated 1 d post-infection as compared with control group.CONCLUSION:The differential expression of HLA-G isoforms and secretion of the receptors ILT 2/ILT4 and IL-10 in the THP-1 cells are induced after HCMV infection .This study provides experimental evidence for evaluating the immune mechanism of HCMV infection .

ObjectiveTo study a fast and feasible system in clinical application of computer aided socket design and manufacture. MethodsThe biomechanical index were compared of traditional hand-made prosthetic sockets and of computer aided design and manufacture ones based on 3D scanning and reverse engineering. ResultsThe index from 3 cases wearing the computer aided design and manufacture socket prostheses appeared similar or better in static mechanical parameters, walking kinetic parameters and stump-socket interface pressure than they wearing traditional hand-made ones. ConclusionThis computer aided socket design and manufacture system can meet patients' needs in using their prostheses.