Objective To investigate the effect of N-methyl-N-nitrosourea (MNU) on expressions of interphotoreceptor retinoid-binding protein (IRBP) and recoverin in rat retinal.Methods Thirty female Sprague-Dawley (SD) rats were randomly divided into five groups,including normal control group,and MNU treatment one day,three days,seven days and 10 days groups,each group had six rats.Rats in MNU-treatment groups received a single intraperitoneal injection of 40 mg /kg MNU,while rats in the normal control group received intraperitoneal injection of saline 5 ml/kg.Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence were used to detect the retinal expression of IRBP and recoverin.Results RT-PCR results indicated that retinal IRBP levels decreased after MNU injection compared with normal control (P＜0.01),while recoverin levels increased (one day:P＞0.05; three days:P＜0.05; seven days:P＜0.5; 10 days:P＜0.05).Immunofluorescence assays demonstrated that normally IRBP protein is mainly in the inner and outer segments of photoreceptors.After MNU treatment,IRBP protein was detected in all retinal layers but much faint.Recoverin was expressed in the inner nuclear layer,inner plexiform layer and ganglion cell layer,and its expression was increased after MNU injection.Conclusion MNU suppresses IRBP expression and promotes recoverin expression in rat retina.

BACKGROUND: In the central nervous system(CNS) of normal adults there are neural stem cells or neural progenitor cells, which are capable of self-renewing and multiple differentiating. In normal physiological conditions, intrinsic neural stem cells are in a resting state. What state will they be in during cerebral ischemia?OBJECTIVE: To observe the distribution, proliferation and differentiation of intrinsic neural stem cells in focal transient ischemia in rats.DESIGN: A randomized controlled exploratory trial based on the rats.SETTING: Neurobiological department, histological and embryological department of a medical college.MATERIALS: The experiment was conducted in the Neurobiological Department of Luzhou Medical College from July 2001 to July 2002. Altogether 41 healthy adult SD rats of either gender, weighting 250 g - 300 g, were selected.INTERVENTIONS: The focal ischemia model was made by blocking middle cerebral artery(MCA) and reperfusing for 0.5 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days and 10 days. Sham-operation group was treated by the same method, but the filament was not long enough to block MCA, and normal rats served as control group. The rats were sacrificed at given time points, and their brains were made into cerebral slices. The single-and double-labeled immunohistochemical staining was employed to detect the proliferation, distribution and differentiation of intrinsic neural stem cells.MAIN OUTCOME MEASURES: The distribution, proliferation and differentiation of intrinsic neural stem cells.RESULTS: Immunoreactivity of proliferating cell nuclear antigen(PCNA)was present in most ependymal cells in ventricular zone(VZ), and PCNA-positive cells were sparsely distributed in the parenchyma in normal and sham-operation groups. At 3 hours of reperfusion, PCNA-labeled cells were first detected in rostral subventricular zone. At 12 hours of reperfusion and onward, PCNA-positive cells appeared in some choroid plexus cells in bilateral lateral VZ. At day 3 to day 10 of reperfusion, PCNA-labeled cells significantly increased in infarct boundary in preoptic area, striatum and deep layer of frontoparietal cortex. PCNA-labeled cells were first detected in subgranular zone of dentate gyrus 3 days after reperfusion, and increased with time. A very small number of double-positive cells expressed with PCNA and glial fibrillary acidic protein(GFAP) were first detected in infract boundary in preoptic area on day 3 and onward. No double-PCNA and NF-positive cells were detected within 10 days of reperfusion.CONCLUSION: Focal cerebral ischemia activates intrinsic neural stem cells, which proliferate and differentiate, and migrate toward ischemic striatum and frontoparietal cortex. This may help clarify the mechanism of functional recovery after ischemia.

Objective To analyze the relationship between HBsAg level and HBV DNA level as well as illness severity in patients with HBV-related acute-on-chronic liver failure (ACLF).Methods One hundred and nineteen patients with chronic hepatitis B (CHB group) and 98 patients with ACLF(ACLF group) were enrolled.HBsAg and HBeAg were assayed with Roche electrochemical luminescence method.HBV DNA was quantified using a real-time polymerase chain reaction (PCR) assay.HBsAg and HBV DNA levels were compared between two groups and between HBeAg-positive and HBeAg-negative patients in ACLF group respectively,also the correlationbetween HBsAg and HBV DNA was studied.Results The proportions of HBeAg-negative patients were 68.4％(67/98) and 42.9％(51/119) in ACLF group and CHB group respectively,and there was significant difference between two groups (P ＜0.01).There was no significant difference in HBV DNA between two groups (P ＞ 0.05).HBV DNA in HBeAg-positive patients was higher than that in HBeAg-negative patients in two groups(P ＜ 0.05).There was no significant difference in HBsAg between HBeAg-positive patients and HBeAg-negative patients in ACLF group (P ＞ 0.05 ),but they were higher than that in HBeAg-positive patients in CHB group (P＜ 0.05).HBsAg was correlated to ALT,AST in HBeAg-positive patients (P ＜ 0.05).No significant correlation was found among HBsAg and HBV DNA as well as biochemical changes (P＞ 0.05).There was no significant difference in the ratio of different HBsAg levels among the patients of different HBV DNA in ACLF group (P＞ 0.05).Conclusion The level of HBsAg does not directly correlate with serum HBV DNA level,and has no directly correlation with the severity of the disease in patients with HBV-related ACLF.

Aims To establish the methods of primary culture of fibroblast-like synoviocytes in rats with adju-vant arthritis(AA-FLS)and analyze the feature and to investigate the possibility of AA-FLS as the model for the RA in vitro.Methods The synovial cells obtained from the SD rats were immunized by the Mtb and iden-tified by morphology and immunocytochemistry.The viability of AA-FLS was assessed by Cell Counting Kit-8.ELISA was applied to detect TNF-αand IL-1 βin cell media. Apoptosis was measured by Hochest 33258.The expressions of mitochondrial apoptosis-re-lated molecules,including Bcl-2,Bax,Pro-caspase-3 and Cleave-caspase-3 were determined by Western Blot.Result In isolated primary synovial cells,more
than 95% of AA-FLSs were fusiform.Immunocyto-chemistry result showed a positive expression of vimen-tin and a negative expression of CD68 in AA-FLS.Cell proliferation of AA-FLS was higher than FLS and cell apoptosis of AA-FLS was curbed.Western blot data demonstrated that the protein expressions of Bcl-2,Bax were regulated and the expression of caspase-3 was ac-tivated in AA-FLS.Conclusions AA-FLS is biologi-cally characterized by high level proliferation activity and inflammatory cytokines and apoptosis suppression. AA-FLS can be used as the model for the RA in vitro.

We report a spectrophotometic method for the determination of cyanogen chloride (CNCl) in air pollution.Experimental parameters for the stability and absorbility of CNCl gas in the different media were studied.The experimental conditions were optimized for CNCI as follows:absorbent:1%isonicotinic acid-barbituric acid;reaction acidity:pH 5.8 in phosphate buffer at room temperature;masking agent:0.01mol/L EDTA;λmax:598nm. The apparent molar absorptivity was found to be 1.17×105L·mol-1·cm-1,the linear range was within 0～5μg of CNCl in 25 mL solution; the regression equation of the curve has given by A=-0.0040+1.935C(r=0.9999).The method has been applied to the determination of CNCl in chimney gas,workshop air and production area air of the cyanuric trichloride plant,chemical works and pharmaceutical factory air.The relative standard deviation was below 2.7%(n=4)for the cyanogen chloride amount within 13～64mg/m3,and when CNCl amounts were within 0.1～1.0mg/m3,RSD was below 6.9%(n=6).The recovery was 98.7%.

Objective To investigate the correlation of the expression of KAI1/CD82 and laminin receptor (LNR) in cholangiocarcinoma, and study its role in the invasion and metastasis of cholangiocarcinoma. Methods The expressions of KAI1/CD82 and LNR in 48 cholangiocarcinoma tissue samples were detected by SP immunohistochemistry, and their relationships with clinicopathological factors were analyzed. Results The positive expression rates of KAI1/CD82 and LNR in cholangiocarcinoma were 31% (15/48) and 54% (26/48), respectively. In highly differentiated cholangiocarcinoma, the positive expression rate of KAI1/CD82 was high (χ2=3.911, P<0.05), while that of the LNR was low (χ2=6.970, P<0.05). The positive expression rate of KAI1/CD82 in cholangiocarcinoma with metastasis was significantly lower than that in cholangiocarcinoma without metastasis (χ2=5.765, P<0.05), while the positive expression rate of LNR in cholangiocarcinoma with metastasis was significantly higher than that in cholangiocarcinoma without metastasis (χ2= 9.952, P<0.05). The expression level of KAI1/CD82 was negatively correlated with that of the LNR ( r = -0.462, P < 0.01 ). Conclusions The up-regulated expression of LNR in cholangiocarcinoma correlates with the decreased expression of KAI1/CD82, and plays an important role in the invasion and metastasis of cholangio-carcinoma.

Objective To observe and scale knee joint space of the patients who suffered adults' Kaschin-Beck disease (KBD) in Shangzhi City of Heilongjiang Province in order to acquire basic knowledge for upcoming intervention trial. Methods Clinical test was proceeded in adults aged above 40 in Guanghui Village of Shangzhi City in 2007. In typical patients, bilateral knee X-ray was taken and knee joint space was scaled. Three points were chosen on inner and outer epicondyle of each knee and results were recorded. Results There were 307 peoples over the age of 40 in the village. Two hundred eighty-two accepted clinical test, in a rate of 91.9%. Eighty out of 282 suffered KBD, accounting for 28.4%. When the patients' condition became more serious, measurements from flank, middle, inside points on outer epicondyle of joint space presented a narrowing trend [degree Ⅰ: (5.85±0.17), (4.84±0.17), (4.36±0.18)mm; degree Ⅱ: (5.11±0.43), (4.24±0.34), (3.48±0.28)nun; degree Ⅲ: (3.59± 0.78), (3.10±0.56), (2.14±0.62)mm; Fflank,middle,inside= 6.547,5.372,10.302, all P < 0.05], but those on inner epicondyle of joint space did not[degree Ⅰ:(3.66±0.17), (3.47±0.17), (3.73±0.18)mm; degree Ⅱ:(3.55± 0.34), (3.54±0.29), (4.35±0.35)mm; degree Ⅲ: (3.19±0.72), (3.92±0.66), (4.51±0.72)ram; Finside,middle,flank= 0.351,0.356,1.883, all P > 0.05]. Joint space < 5.00 mm were found in 618, among which 363 were measured on inner epieondyle, obviously greater than those measured on outer epicondyle of joint space(255), the difference being statistically signifieant(χ2=9.59, P<0.05). Conclusions Adult cases of KBD occur in Guanghui Village. Knee joint space of adult patients narrows obviously, the more serious, the narrower, especially occurring more obviously on outer epicondyle, but frequently on inner epicondyle.

Objective To analyze the treatment effect and factors affecting the prognosis in young people of papillary thyroid carcinoma (PTC).Methods A total of 69 PTC patients were reviewed retrospectively in Sun Yatsen University Cancer Center from Dec 1996 to Oct 2005.The prognostic status were compared using the Kaplan-Meier methods,the factors were analyzed by Log-rank test and the multiple factors were estimated by the Cox regression models.Results There were 26 males and 43 females.The median age was 24 years (mean:23.1 years).The average follow-up time was 113 months ranging from 73 to 174 months.20.3％ patients (14/69) experienced recurrence during follow-up time and 1.4％ patient (1/69) died.All the patients received levothyroxine after operation.The 10 year disease-free survival (DFS) rate of patients with or without extra thyroidal extension (ETE) of PTC was 29.6％ and 80.1％,respectively (P ＜ 0.01).Multivariate analysis showed that young PTC people with ETE had a worse prognosis (P =0.004).Conclusions Young papillary thyroid carcinoma patients had a favorable outcome.Surgical procedure was the first choice for this kind of patients.ETE was an independent factor for the recurrence of young PTC patients.

AIM:Long bone growth plate injury induced by wound and infection may cause limb reduction or angular deformity. Tissue engineering provides a promising treatment of growth plate injuries. In this study,we investigated the feasibility of establishing tissue-engineered growth-plate by allograft demineralized bone matrix(DBM) co-cultured with rabbit iliac growth-plate cells. METHODS:The experiment was performed at Department of Orthopaedics,Fourth Military Medical University of Chinese PLA from June 2005 to June 2006. ①Two 3-week-old New Zealand rabbits irrespective of gender(clean grade,2.0-2.5 kg) were selected. Growth plate cells were harvested from iliac crest epiphyseal cartilage of the rabbits by dissection and digestion with type Ⅱ collagenase. The third passage cells cultured were collected and incubated on allograft demineralized bone matrix. ② Histology,immunohistochemical staining and electronic scanning microscope(SEM) examinations were performed to observe cell growth on DBM 24 hours,7,14 and 21 days after culture. RESULTS:①Growth plate chondrocytes exhibited polygonal in monolayer culture,and immunohistochemical staining for type Ⅱ collagen was positive. ②SEM examination showed that twenty-four hours after coculture,the cells adhered to DBM scaffolds;Seven days after culture,the growth-plate chondrocytes rapidly proliferated and began to secret extracellular matrix;cells covered the whole scaffold and became overlapped on the 21st day. ③HE staining showed after 14 days of culture,growth plate cells adhered DBM scaffold in spherical shape with abundant cytoplasm. CONCLUSION:Tissue-engineered growth-plate is successfully constructed by allograft mineralized bone matrix co-cultured with rabbit iliac growth-plate cells.

Objective We increase the soluble expression of artificial tandem hybrid ubiquitin binding domains ( ThUBD) in prokaryotic cytoplasm of Escherichia coli BL21 ( DE3 ) , which offer an effective and special profiling for ubiquitin conjugates( UbC) .Methods Codon optimization of the ThUBD was performed, followed by analysis of codon relative adaptiveness based on relative frequency of synonymous codon ( RFSC) of E.coli.Further induced expression and yeast ubiquitin conjugate enrichment quantified the soluble ThUBD-S and tested the ability to bind UbC.Results The statistical result showed that the percentage of codon of the highest usage frequency was increased from 48%to 75%, and codon adaptation index( CAI) was increased from 0.63 to 0.88 after codon optimization, which might suggest a higher expression of the ThUBD in E.coli BL21 (DE3).The subsequent SDS-PAGE indicated that the soluble target protein was increased four times, which accounted for 13.06%of total cell lysis.Further ubiquitinated proteome of yeast demonstrated that the ability to bind and enrich UbC of optimized ThUBD-S did not change compared with original ThUBD.Conclusion The expression of ThUBD-S can quadruple after codon optimization.At the same time, codon optimization does not impact its soluble expression and the ability to bind UbC.