Objective To study the expression of P16 ,Ki‐67 in cervical squamous epithelial lesions and the significance of their detection combined with human papillomavirus(HPV) .Methods 188 cases of cervical tissues from surgical excision were collected , including 48 cases of cervical cancer ,100 cases of cervical intraepithelial neoplasia(CIN) ,and 40 cases of chronic cervicitis ,which were set as cervical cancer group ,CIN group and cervicitis group ,respectively .Expression of P16 ,Ki‐67 in cervical tissue were de‐tected by using immunohistochemistry ,and the HPV infection were detected by using PCR technology .Results The positive ex‐pression rates of P16 and Ki‐67 in cervical cancer ,CIN group was significantly higher than those in cervicitis group(P<0 .05) ,and the positive expression rates of P16 and Ki‐67 in cervical cancer group were higher than those in CINⅠ and CINⅡ group (P<0 . 05) ,but between the cervical cancer group and CIN Ⅲ group there were no statistically significant difference(P>0 .05) .With the increase of CIN level ,the positive expression rates of P16 and Ki‐67 increased ,among the three CIN groups there were significant difference(P<0 .05) .In cervical cancer group and CINⅢ group ,the expression rates of P16 and Ki‐67 were both significantly high‐er than the other 3 groups(P<0 .05) .In CINⅠ - Ⅲ groups ,the over expression rates of P16 and Ki‐67 were statistically different (P<0 .05) .The expression of P16 positively correlated with the positive rates of HPV infection (rs =0 .706 ,P=0 .011);the ex‐pression of Ki‐67 positively correlated with the positive rates of HPV infection(rs=0 .695 ,P=0 .021) .Conclusion CIN and cervi‐cal cancer of early stage could be diagnosed and the pathological progress of CIN could be predicted by using the combined detection of P16 ,Ki‐67 and HPV .

Objective To investigate the location of CYLD in the neurons and explore the expression of CYLD in OGD/reperfusion-induced neuronal necroptosis.Methods Primary cortical neurons were cultured for 6days and neuronal purity was observed by double staining immunofluorescence of β3-tubulin and DAPI.The location of CYLD was identified by double staining immunofluorescence of NeuN,DAPI and CYLD using primary cortical neurons cultured for 14 days.Then,primary cortical neurons were divided into 8 groups:Control,EBSS,DMSO,OGD/reperfusion(0 h,2 h,6 h,8 h,12 h).Neurons were pretreated with zVAD-fmk for 30 min,OGD for 2 h and the levels of CYLD were evaluated after reoxygenation at different time points.The peak value(8 h) was chosen as reoxygenation time point.Neurons were divided into two groups as Control and OGD.The levels of CYLD were determined in both cytoplasm and nucleus after OGD 2 h and reoxygenation 8 h.Results The double staining immunofluorescence showed that neuronal cultured purity was about 70％ and the CYLD strongly expressed in nucleus but weakly in cytoplasm.The levels of CYLD increased gradually with different reoxygenation time and arrived at peak value after reoxygenation for 8 h (P ＜ 0.05),which was in accordance with the change of LDH (P ＜0.05) (Control (1.00±0.00),EBSS (1.07 ±0.03),DMSO (1.09 ±0.03),0h (1.40±0.12),2 h (1.74±0.08),6 h (2.25 ± 0.12),8 h (2.97 ± 0.15),12 h (3.01 ± 0.08)).The level of cytoplasm CYLD increased significantly in the OGD group (reoxygenation for 8 h)than that in control group (P＜0.05).But the level of nucleus CYLD had no difference between OGD and control group (P ＞ 0.05),which was in accordance with the results of immunofluorescence.Conclusion The CYLD in neurons cytoplasm is involved in necroptosis induced by OGD/deprivation and downregulating of CYLD has a protective effect on the brain injury resulted from ischemia/ reperfusion.

Objective To establish a new inhibitory kinetic spectrophotometric method for the determination of trace Sb3+ in water sample. Methods A new inhibitory kinetic spectrophotometric method for the determination of trace Sb3+ was established based on the inhibitory effect of Sb3+ on the oxidation of thionine by H2O2 in a buffer solution of NH3-NH4Cl at pH=10.50. Results The linear range of this method was 0.004-0.40 ?g/ml, r=0.999 2,the limit of detection was 1.71?10-3 ?g/ml, and RSDs were 1.4%～ 2.1% , the recovery rates were 96.2% -101.9% . Conclusion This method is accurate, fast, simple and applicable to the determination of antimony content in environment water samples.

levels are elevated in patients with NAFL and correlated with HOMA-IR, suggesting RBP4 may participate in the development of IR and NAFL.

Objective To prepare a detective membrane strip for detection of food allergen-specific IgE in serum samples and estimate its clinical application value in allergic diseases. Methods The crude extracts of the food allergens were prepared. Nitrocellulose membrane as the solid support was selected and the coating and the detecting conditions were optimized. The membrane strips were used to detect serum samples in 210 patients with allergic diseases and the results were compared with German Allergy Screentesting system. Results The optima] experimental conditions were as follows: The NC membrane was adopted as the solid support. After being spotted, the food allergens were incubated for 2 hours at room temperature, followed by 2% PVA blocking for 1 hour. After serum samples were diluted (1: 10) and incubated for 2 hours at room temperature, the concentration of anti-human IgE was 2 μg/mL Compared with the German Allergy Screen-testing system, their positive detectical coincidence was 63.6%, and negative detectical coincidence was 94. 6%. The two methods had no difference in detecting the majority of food allergens such as egg white, milk, peanut, soybean, crab and shrimp (X2 2.53, 2.40, 2.08, 2.38, 0.17,1.13, P>0.05). Conclusions The advantages of our method for detecting allergic diseases are little serum needed, multiple detective allergens, simple manipulation and low cost. This method has obvious clinical application value, which should be a new detective method for the allergic diseases with broad perspectives.

Objective To silence the expression of CTGF by small interfering RNA technology,to observe the influence on fibroblast?like synovial cell apoptosis and several apoptosis?related genes,and to explore the mechanism of action of CTGF in rheumatoid arthritis synovial lesions. Methods Effective CTGF siRNA was screened through real?time PCR. The influence of CTGF siRNA on FLS apoptosis was detected with FITC?PI double staining by flow cytometry. bax,bcl?xl and survivin were detected using real?time PCR when CTGF mRNA has been silenced. Results Compared with other 2 groups of oligo and NC oligo,H1 oligo exhibited the strongest interfering action to CTGF(inhibition ratio>70%),so that it is selected as the effective target gene sequence for the following experiment. Apoptosis of FLS induced by serum deprivation was significantly decreased in the presence of exogenous CTGF. When expression of the CTGFgene was knocked down in FLS,FLS apoptosis was significantly increased,and expres?sion levels of survivin mRNA were decreased significantly(P<0.01). Conclusion FLS survival is positively regulated by CTGF,which may through the sustaining the expression of survivin.

Objective To investigate the correlation between hypoglycemia and brain injury of newborns.Methods Medical records and follow-up data of 110 newborns with hypoglycemia (blood glucose level≤2.2 mmol/L) who admitted into neonatal department of Peking University First Hospital from December 2006 to December 2009 were studied.All patients were divided into 3 groups:no brain injury group,mild and severe brain injury group according to their clinical manifestation,cerebral radiological characteristics and cerebral functional tests.By using receiver operating characteristic (ROC) curve and x2 test,the potential optimal blood glucose level and duration of hypoglycemia for predicting brain injury were confirmed.Multivariate Logistic regression was taken to determine independent predictors for brain injury.The analyzed factors included gender,preterm/small for gestational age,hyperbillirubinemia,fetal distress,asphyxia,infection,seizures and maternal hypertensive disorder complicating pregnancy and hyperglycemia.Results Among the 110 hypoglycemia newborns,33 (30.0％) infants suffered from brain injury,of which 23 were mild and 10 were severe.Blood glucose ≤1.7 mmol/L had high specificity (73％) and sensitivity (60％)for predicting brain injury.When blood glucose≤ 1.7 mmol/L,the incidence of brain injury and severe brain injury was 43.6％ (24/55) and 18.2％ (10/55),which was higher than those [16.4％(9/55) and 0.0％ (0/55)] of patients whose glucose level ＞1.7 mmol/L(x2 =9.74 and 11.00,P＜0.01 respectively).Blood glucose ≤ 1.2 mmol/L had high specificity (100％) and sensitivity (81％) for predicting severe brain injury.When blood glucose ≤1.2 mmol/L,the incidence of severe brain injury was higher than that of the patients whose glucose level was higher than 1.2 mmol/L [34.5％ (10/29) vs 0.0％ (0/81),x2 =30.72,P＜0.01].Duration of hypoglycemia ≥12 h had specificity (100％) and sensitivity (36 ％) for predicting brain injury.When duration of hypoglycemia ＜12 h,the incidence of brain injury was lower than that of the patients whose duration of hypoglycemia≥12 h [21.4％ (21/98) vs 6/6,x2 =27.69,P＜0.01].Multivariate Logistic regression showed that fetal distress (OR=4.69,95％CI:1.47-14.97,P=0.009),glucose level≤1.2 mmol/L (OR =5.16,95％CI:1.56-17.03,P=0.007),duration of hypoglycemia≥12 h (OR=8 885 220 297.12,95％CI:0.00-∞,P =0.000) and maternal hyperglycemia (OR =3.34,95％CI:1.01-11.02,P=0.048) were independent risk factors for neonatal brain injury.Conclusions Low blood glucose level and prolonged hypoglycemia might induce injury of neurol system.Fetal distress and maternal hyperglycemia might increase the incidence of brain injury in newborns with hypoglycemia.

Objective To explore the possible roles of PI3K pathway and MAPK pathway in mediating the survival and differentiation-promoting effect of glial cell line-derived neurotrophic factor(GDNF) on midbrain dopaminergic neurons.(Methods Midbrain) slices of early postnatal rats were cultured,the slices were divided into four groups according to the different substances added into the culture medium: i.e.blank control group,GDNF group,Wortmanin plus GDNF group,and PD98059 plus GDNF group.On the 6th day,some slices were fixed,embedded and sectioned.The sections were processed for tyrosine hydroxylase(TH) immunohistochemistry,and then examined under microscope to determine the morphologic index for statistic analysis(n=6).Meanwhile,some slices were taken for Western blot to examine the expression of TH protein in midbrain slices(n=4). Results GDNF group slices showed the morphologic maturity,the density and the diameter of TH positive neurons,as well as the level of TH expression,were all significantly higher than that of the control groups.The survival effect of GDNF on DA neurons was almost abolished when the PI3K pathway was blocked with Wortmannin;while the MAP kinase and ERK kinase(MEK) inhibitor PD 98059 was added to block the MAPK pathway,the diameter of TH positive neurons was decreased significantly.Conclusion The PI3K pathway might mediate the survival effect of GDNF,while the MAPK pathway seems to be involved in the differentiation process.

Objective To observe the proliferation and differentiation of the neural progenitors in the midbrain substantia nigra(SN) of adult rat when dopaminergic neurons(DA neurons) were destroyed. Methods The Parkinson disease model(PD model) was induced in adult SD rats by steriotaxic injection of 6-OHDA into the right striatum,and then assessed by behavioral analysis to screen the qualified models.After varied survival period,the brain of model rats were perfused and fixed by 4% paraformaldehyde,moved the segment of SN,embedded with paraffin and coronally sectioned continuously.The microsections were processed by immunohistochemistry labeling separately the neural progenitors with anti-nestin monoclonal antibody,the dividing cells with anti-PCNA(proliferation cell nuclear antigen),the neuronal precursors with anti-Tuj1(?-tubulin isotype Ⅲ),and DA neurons with anti-TH(tyrosine hydroxylase).The labeled cells were counted under microscope and analyzed statistically.(Results It) was found in the right SN of PD model rats that: 1.Nestin positive(Nestin~+) cells appeared 10d after 6-OHDA injection,became abundant on 14d,declined in number on 17d,and disappeared on 21d.2.Weakly positive PCNA(PCNA~+) cells appeared on 7d.PCNA~+ cells were abundant on 14d,decreased in number from 21d,with only a few positive cells noticed on 28d.3.Tuj1 positive cells appeared in small number on 10d,became abundant on 14d,decreased in number from 17d and dropped nearly to zero on 21d.4.The number of TH positive neurons was significantly less than the normal control(by 24%) on 7d,and became even less as time elapsed.Conclusion When 6-OHDA is injected into the striatum of adult rats to cause degeneration and death of the DA neurons there,there would be a certain period of time in which a number of neural progenitors will be induced to proliferate actively and differentiate toward neuronal cells(except DA neurons).

Objective To investigate the effects of parathyroid hormone (PTH) on the synthesis and secretion of collagen Ⅲ and fibronectin (FN), and the expressions of plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA in cultured human renal tubular epithelial cells (HK-2).Methods HK-2 cells were cultured in DMEM-F12 medium supplemented with 5% FBS. Cells were exposed to different concentrations of PTH (0, 10-12, 10-11, 10-10, 10-9, 10-8 mol/L) for 48 h, or 10-8 mol/L PTH at different time (0, 12, 24, 48, 72 h). The gene expressions of collagen Ⅲ,FN, PAI-1, MMP-1, and TIMP-1 were detected by semi-quantitative RT-PCR. The protein expression of collagen Ⅲ was detected by Western blotting. The level of FN in the supernatant was assayed by enzyme linked immunosorbent assay (ELISA). Results PTH increased gene expressions of collagen Ⅲ, FN, PAI-1 and TIMP-1 in a dose- and time-dependent manner, but decreased MMP-1 gene expression. Then the ratio of MMP-1/TIMP-1 was decreased. PTH increased the collagen Ⅲ protein expression in cultured HK-2 cells and the level of FN in the supernatant of cultured HK-2 cells in a dose- and time-dependent manner. Conclusion PTH can up-regulate PAI-1, TIMP-1 gene expressions, and down-regulate MMP-1 gene expression,resulting in elevation of extracellular matrix (ECM) synthesis and reductim of degradation.