Objective:To compare the effects of 3 different crystalloid fluids at different osmotic concentrations on blood-brain barrier(BBB) and brain edema in severe hemorrhagic shock rats.Methods: A total of 150 male SD rats were equally randomized into a lactated Ringers(LR) group,a 7.2% hypertonic saline(HS) group and a plasmalyte A(PA) group.LR,PA and HS were administered after an hour of severe hemorrhagic shock induced by drawing out about 40% of total blood and maintaining MAP at 35-45 mmHg.Serum S100B,cerebra1 Evans Blue(EB) and water content were determined before(T_0) and 1 h after bleeding(T_1) and immediately(T_2),1 h(T_3) and 2 h(T_4) after administration.The changes of BBB in the hippocampus CA1 area were observed by electron microscopy.Results: The serum S100B level was obviously higher at T_1,T_2,T_3 and T_4than at T_0 in all groups(P0.05).The cerebra1 water content was significantly increased at T_1,T_2,T_3 and T_4in the LR group,at T_1in the HS and at T_1,T_2 and T_3 in the PA as compared with T_0(P

Objective To explore the mechanism of protecting cells from hypoxia/ reoxygenation (H/R) injury by constructing specific small interference RNA (siRNA) to inhibit NALP3 expression in rat renal tubular epithelial cells (NRK-52E).Methods (1) To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition,the cells were cultured with hypoxia for 1 h and then with reoxygenation for 1 h,2 h,4 h,8 h,16 h and 24 h.The activity of lactae dehydrogenase (LDH) in the culture medium,cell count and cell viability,the expression of NALP3 were determined by biochemical method,trypan blue exclusion and Western blotting.(2) The siRNA was transfected into NRK-52E.The irrespective siRNA transfected group wasused as control.NALP3 expression was examined by Western blotting.(3) The cells were divided into 4 groups:control group,H/R group,irrespective siRNA transfected group and NALP3-siRNA transfected group.To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition,the cells were cultured with hypoxia for 1 h and then with reoxygenation for 4 h.And the expression of NALP3 was determined by Western blotting.(4)Cellular apoptosis was examined by Annexin V/PI staining and flow cytometry.NF-κB DNA binding activity,IκB-α,Bcl-2 and Bax expression were examined by EMSA and Western blotting.Results (1)Compared with the control group,the activity of LDH significantly increased,cell count and cell viability significantly decreased (all P＜0.05).The expression of NALP3 significantly increased and peaked at 4 h after H/R.(2)The specific siRNA could efficiently inhibit NALP3 expression in NRK-52E.Compared with the irrespective siRNA transfected group,the protein expression of NALP3 was significantly down-regulated in NALP3 siRNA transfected group (P＜0.05).(3)After hypoxia 1 h and reoxygenation 4 h,the activity of LDH and the expression of NALP3 increased.Compared with the irrespective siRNA transfected group,LDH concentration in media and the expression of NALP3 significantly decreased in NALP3-siRNA transfected group.(4)After hypoxia 1 h and reoxygenation 4 h,NF-κB DNA binding activity was increased,IκB-α phosphorylation and degradation,Bcl-2 and Bax were significantly up-regulated.However,compared with the irrespective siRNA transfected group,NF -κB DNA binding activity,IκB-α degradation and Bax/Bcl-2 were significantly decreased (P＜0.05) in NALP3-siRNA transfected group.At the same time,the ratio of apoptosis was significantly increased in three groups than that in control.Compared with the irrespective siRNA transfected group,the ratio of apoptosis in NALP3-siRNA transfected group was significantly decreased (P＜0.05).Conclusions H/R induces the expression of NALP3 in NRK-52E.The synthesized siRNA can inhibit the expression of NALP3 and protect NRK-52E from hypoxia/reoxygenation injury.The mechanism may be via inhibiting the activation of NF-κB,modulating expression of Bcl-2 and Bax,as well as decreasing cell apoptosis.

A fragment containing amino acid residues 561～578 of HSV-2 glycoprotein G(gG2) was obtained by PCR assembling technique,and doubly cloned into vector pET-KDO.The recombinant plasmid was transformed to BL21(DE3)plysS.Fusion protein,of molecular weight about 39kDa was highly expressed by induction of IPTG.Western blot result showed the fusion protein had good antigenicity.After putification and digestion,the purity reached 95%.The digested purified protein was analysed by ELISA and showed good sensitivity and specificity.The recombinant protein should be useful for type-specific serodiagnosis of HSV-2.

Objective To investigate the diagnostic value of internal septation for differentiating benign from malignant breast lesions.Methods A total of 26 patients were included in the study,in which 12 patients had 20 lesions of breast carcinoma and 14 patients had 25 lesions of fibroadenoma diagnosed either pathologically or clinically.The differential diagnoistic value of the hypointensive internal septation was analyzed.Results The signal intensity of fibroadenomas and malignant lesions on T_2-weighted fat- suppressed images could be classified as iso- to hyper- intensity,hypointensity and mixed intensity. According to the signal intensity classification,there were 5,11 and 4 cases in patients with breast carcinoma respectively,while 11,10,4 cases in patients with fibroadenoma respectively.There was no statistical difference in the distribution between the two patient groups(?~2=1.764,P=0.414).The shape of fibroadenomas and malignant lesions could be classified as irregular、roundish or lobulated.According to the morphological classification,there were 12,7 and 1 case in patients with breast carcinoma respectively, while 1,7,17 cases in patients with fibroadenoma respectively.There was statistical difference in the distribution between the two patient groups(?~2=23.262,P=0.000).The typical features of fibroadenomas were as follows:lobulated shape,hypointensive internal septations on T_2-weighted or postcontrast images. The diagnostic sensitivity of the three imaging features for fibroadenoma was 68%(17/25),52%(13/25), and 72%(18/25)respectively;and the diagnostic specificity was 95%(19/20),90%(18/20),95% (19/20)respectively.Conclusion The internal septation is a rather specific sign for diagnosis of fibroadenomas.

AIM: To investigate therapeutic efficiency of amniotic extraction on dry eye in rabbit model induced by topical benzalkonium chloride (BAC).
METHODS: Totally 26 rabbits (26 right eyes) with dry eye model were studied and divided into two groups:group A (control group with PBS eye drops, n = 13) and group B ( amniotic extraction group, n = 13). Another two rabbits were chosen as normal control. The SchirmerⅠ tests ( S Ⅰ t) and corneal fluorescein staining ( FL) were made, and the tear total protein content, amylase activity, lactoferrin, lysozyme contents, goblet cell density were performed in two groups before treatment and 1, 2, 4 and 8 wk after treatment.
RESULTS: There were significant differences in SIT, FL scores, lysozyme activity and goblet cell density among different groups at different time points (P<0. 05). But, there was no significant differences in SⅠt, FL scores, lysozyme activity and goblet cell density between two groups before treatment (P>0. 05). After 8wks' treatment with PBS, the mean differences of the group A showed great changes in SⅠt, lysozyme and goblet cell density compared with those before treatment ( P < 0. 05); but there was no significant differences in FL scores compared with those before treatment (P>0. 05). As for group B, 8wks after treatment, there were statistical changes in SⅠt, FL, lysozyme (P<0. 05); but there was no significant differences in goblet cell density compared with those before treatment ( P > 0. 05). It was evident that statistical differences were observed in S Ⅰ t, FL scores, lysozyme activity and goblet cell density between two groups at each time point (P<0. 05). However, there were no significant differences in total protein, lactoferrin, amylase activity at different time points (P>0. 05). Meanwhile there was no significant differences in total protein, lactoferrin, amylase activity between two groups before treatment ( P > 0. 05 ). But there were significant differences in total protein, lactoferrin, amylase activity between two groups after 4 and 8 wks'treatment (P<0. 05).
CONCLUSION: Amniotic extraction has significant therapeutic effect on the dry eye in rabbit model.

OBJECTIVETo explore the relationship between genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), the central enzymes in folate metabolism that affects DNA methylation and synthesis, and the risk of Down syndrome in China.

METHODSGenomic DNA was isolated from the peripheral lymphocytes of 64 mothers of children with Down syndrome and 70 age matched control subjects. Polymerase chain reaction and restriction fragment length polymorphism were used to examine the polymorphisms of MTHFR 677C-->T, MTRR 66A-->G and the relationship between these genotypes and the risk of Down syndrome was analyzed.

RESULTSThe results show that the MTHFR 677C-->T polymorphism is more prevalent among mothers of children with Down syndrome than among control mothers, with an odds ratio of 3.78 (95% confidence interval (CI), 1.78 approximately 8.47). In addition, the homozygous MTRR 66A-->G polymorphism was independently associated with a 5.2-fold increase in estimated risk (95% CI, 1.90 approximately 14.22). The combined presence of both polymorphisms was associated with a greater risk of Down syndrome than the presence of either alone, with an odds ratio of 6.0 (95% CI, 2.058 approximately 17.496). The two polymorphisms appear to act without a multiplicative interaction.

CONCLUSIONMTHFR and MTRR gene mutation alleles are related to Down syndrome, and CT, TT and GG gene mutation types increase the risk of Down syndrome.

Alleles ; Case-Control Studies ; China ; Down Syndrome ; diagnosis ; ethnology ; genetics ; Female ; Ferredoxin-NADP Reductase ; genetics ; Folic Acid ; metabolism ; Genetic Predisposition to Disease ; Genotype ; Homozygote ; Humans ; Lymphocytes ; metabolism ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Polymorphism, Genetic ; Risk Factors

OBJECTIVETo evaluate the efficacy and safety of irinotecan combined with xeloda (CAPIRI regimen) in patients with metastatic colorectal cancer after failure of chemotherapy with oxaliplatin.

METHODSTotally 38 patients with metastatic colorectal cancer after failure of chemotherapy with oxaliplatin were enrolled. Patients received xeloda 1 000 mg/m2 orally twice daily on day 1 to 14 and intravenous irinotecan 100 mg/m2 on day 1 and 8 every 3 weeks.

RESULTSThe median age of 38 patients was 58.5 (27-77) years. CAPIRI regimen was used 11.0 (3.0-24.0) months after the diagnosis of metastatic colorectal cancer (CAPIRI regimen as second-line chemotherapy in 33 patients, third-line in 4 patients, and fourth-line in 1 patient). A total of 121 cycles of chemotherapy (median 3.0) were administered. Thirty-four patients were evaluable for response. The overall response rate and disease control rate were 5.9% and 61.8%, respectively. The median time to progression and overall survival were 4.5 months (95% CI, 3.4-5.6 months) and 11.0 months (95% CI, 10.2-11.8 months), respectively. All 38 patients were evaluable for safety. The most common adverse events were leukopenia (73.7%), neutropenia (65.8%), nausea and vomiting (60.5%), and diarrhea (28.9%). The occurrence rates of these grade 3-4 events were 10.5%, 13.2%, 10.5%, and 7.9%, respectively. All adverse events were tolerable.

CONCLUSIONCAPIRI regimen is effective and well-tolerated in Chinese patients with metastatic colorectal cancer after failure of chemotherapy with oxaliplatin.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; therapeutic use ; Camptothecin ; administration & dosage ; analogs & derivatives ; Capecitabine ; Colorectal Neoplasms ; drug therapy ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; Female ; Fluorouracil ; administration & dosage ; analogs & derivatives ; Humans ; Male ; Middle Aged ; Neoplasm Metastasis ; Organoplatinum Compounds ; therapeutic use ; Treatment Outcome

The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A(*)1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A(*)1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3'terminal of the HLA-A(*)1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A(*)1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A(*)1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A(*)1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A(*)1101-T2A-EGFP plasmid was successfully constructed; after G418 selection for 2 months, two sublines of K562 cells (HLA-A(*)1101(+)K562, pEGFP-N3(+)K562) expressing GFP were constructed. The expression of HLA-A*A1101 gene could be determined in HLA-A(*)1101(+)K562 cell line by RT-PCR, while the pEGFP-N3(+)K562 cells could not express HLA-A*A1101 gene. HLA-A(*)1101 protein and GFP double positive HLA-A(*)1101(+)K562 cells were up to 88.5%, which was obviously higher than pEGFP-N3(+)K562 cells (0.698%) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A(*)1101(+)K562 cells has been established and a subline of K562 cell expressing HLA-A(*)1101 protein on its cell membrane was successfully constructed, which provides the tool cells for further studying the specific cellular immunity against-CML.
Genetic Vectors ; HLA-A11 Antigen ; genetics ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukocytes, Mononuclear ; Plasmids ; Transfection

OBJECTIVETo explore the effects of cofilin on the actin cytoskeleton reorganization in osteoblasts induced by fluid shear stress.

METHODSFluid shear stress (1.2 Pa) was applied to osteoblasts for 0 (control group), 15, 30, 45, 60, 120 min in vitro. Cells were stained with fluorescein isothiocyanate (FITC)-phalloidin for fiber-actin, and confocal laser scanning microscope(CLSM) was used to observe the fluorescence of fiber-actin. Western blotting was used to detect the expression of the cofilin and the phospho-cofilin.

RESULTSActin filaments became organized into stress fibers that were thicker and more abundant than those in non-flowed cells. The fluorescence intensity (38.00 ± 6.88) of fiber-actin after 120 min (42.93 ± 6.41) loading it was 2.8 times as much as that in control group (15.41 ± 3.60, P < 0.05). Additionally, the level of phospho-cofilin protein was dramatically elevated after loading. Fluid shear stress induced an initial decrease of cofilin at 60 min. However, at 120 min cofilin (0.254 ± 0.026) increased to 1.5 times as much as that at 60 min (0.162 ± 0.004).

CONCLUSIONSThe results indicate that cofilin phosphorylation mediates fiber-actin reorganization in the osteoblasts induced by fluid shear stress.

Actin Cytoskeleton ; ultrastructure ; Actin Depolymerizing Factors ; biosynthesis ; Humans ; Osteoblasts ; ultrastructure ; Phosphorylation ; Stress, Mechanical