OBJECTIVE:To establish the method for assaying bacterial endotoxins in levofloxacin hydrochloride injec?tion.METHODS:The interference test of3batches of levofloxacin hydrochloride injection with2kinds of limulus test agent was studied.RESULTS:The interference between the samples and limulus test agents was eliminable(below1.0mg/ml).The detection results were up to standard.CONCLUSION:The bacterial endotoxin in the sample can be examined by limulus test instead of pyrogen test in rabbits.

Objective To determine the frequency of Toll-like receptor 4 (TLR4)polymorphisms Asp299Gly and Thr399Ile in a cohort of hematopoietic stem cell transplant recipients and their donors in China.Method We examined the polymorphisms in 208 peripheral blood samples collected from 104 recipients and their donors in a single center between 2007-2012 in Zhejiang Province,China,and Asp299Gly and Thr399Ile TLR4 gene polymorphisms were detected using the sample DNA amplification products direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.Result Both methods didn't demonstrate TLR4 polymorphisms Asp299Gly or Thr399Ile base mutation in our samples.Conclusion The TLR4 Asp299Gly and Thr399Ile polymorphisms are very rare in our part of the population of hematopoietic stem cell transplantation.

OBJECTIVETo study the effect of Zhusha Anshen pill, cinnabar, HgS, HgCl2 and MeHg on the gene expression of renal transporters in mice.

METHODHealthy male mice were given equivalent physiological saline, Zhusha Anshen pill (1.8 g · kg(-1), containing 0.17 g · kg(-1) of mercury), cinnabar (0.2 g · kg(-1), containing 1.7 g · kg(-1) of mercury), high dose cinnabar (2 g · kg(-1), containing 1.7 g · kg(-1) of mercury), HgS (0.2 g · kg(-1), containing 0.17 g · kg(-1) of mercury), HgCl2 (0.032 g · kg(-1), containing 0. 024 g · kg(-1) of mercury), MeHg (0.026 g · kg(-1), containing 0.024 g · kg(-1) of mercury), once daily, for 30 d, measuring body mass gain. 30 days later, the mice were sacrificed. The mercury accumulation in kidneys was detected with atomic fluorescence spectrometer. Expressions of Oat1, Oat2, Oat3, Mrp2, Mrp4, Urat1 were detected with RT-PCR.

RESULTCompared with the normal control group, a significant accumulation of Hg in kidney in HgCl2 and MeHg groups was observed (P <0.05), but these changes were not found in other groups. Compared with normal control group, mRNA expressions of Oat1 and Oat2 were evidently lower in HgCl2 and MeHg groups, but mRNA expressions of Mrp2 were apparently higher in HgCl2 group (P <0.05), mRNA expression of Mrp4 was significant higher in HgCl2 and MeHg groups, and mRNA expression of Urat1 was apparently lower in MeHg group.

CONCLUSIONHgCl2 and MeHg groups show significant difference from the normal group in mercury accumulation in kidneys and gene expression of kidney transporters, but with no difference between other groups and the normal group. Compared with HgCl2 and MeHg, cinnabar and its compounds could cause lower renal toxicity to mice.

Animals ; Carrier Proteins ; genetics ; Drugs, Chinese Herbal ; toxicity ; Gene Expression ; drug effects ; Kidney ; drug effects ; metabolism ; Male ; Mercuric Chloride ; toxicity ; Mercury Compounds ; toxicity ; Methylmercury Compounds ; toxicity ; Mice ; Multidrug Resistance-Associated Proteins ; genetics ; Organic Anion Transport Protein 1 ; genetics ; Organic Anion Transporters, Sodium-Independent ; genetics

Objective To investigate the possible protective effect of adenoviral vector expressing interleukin-1β (IL-1β) small hairpin RNA (shRNA) on spinal cord injury (SCI) and its mechanism in rats.Methods Forty-eight adult male Sprague-Dawley rats were randomly assigned to 4 groups including the Sham, the Vehicle,the Ad-GFP and the Ad-shIL-1β groups.SCI was induced by epidural compression.Motor function of hind limbs was evaluated by Basso-Beattie-Bresnahan (BBB) score, the expressions of green fluorescence in injured spinal cord tissue were observed by fluorescence microscope.Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were also performed.Results The expressions of green fluorescence in injured spinal cord tissue were observed in the Ad-GFP and Ad-shIL-1β groups one day after SCI.Significant functional improvement was observed in the Ad-shIL-1β group (8.17 ± 1.17, 10.17 ± 0.98 and 11.33 ± 0.82, respectively) compared to the Vehicle (4.00 ± 0.89, 5.67 ± 1.03 and 6.17 ± 1.17, respectively) and Ad-GFP (3.83 ± 0.98, 5.33 ± 1.21 and 5.67 ± 1.03, respectively) groups at 7, 14 and 21 days after SCI (P ＜ 0.05).Rats in the Ad-shIL-1β group had less neuronal loss 21 days after SCI.In addition, IL-1β downregulation significantly decreased IL-1β, tumor necrosis factor-or (TNF-α) and IL-6 levels (138.83 ± 7.96,143.38 ± 10.20 and 120.43 ± 9.79 in Ad-shIL-1β group;169.33 ± 11.45, 172.33 ± 8.26 and 163.00 ± 9.57 in Vehicle group;172.83 ± 10.85,167.48 ± 8.19 and 159.48 ± 10.98 in Ad-GFP group, respectively) one day after SCI (P ＜ 0.05).Conclusion This study demonstrated that the IL-1β downregulation may have potential therapeutic benefits for improving the outcomes after SCI.

Objective To investigate the feasibility of using low volume of low concentration isotonic contrast medium for 320 row coronary CT angiography.Methods 64 patients whose heart rate 70 beats per minute or less,normal cardiac rhythm,BMI≤24 kg/m2 were scanned using a 320 row dynamic volume CT with the tube voltage of 100 kVp and injection of contrast medium with a concentration 270 mg I/mL.Prospective ECG gating technique and adaptive iterative dose reduction algorithm reconstruction were used.In group A,22 patients were injected 50 mL contrast medium by a rate of 5.0 mL/s.In group B,21 patients were injected with the dosage of contrast medium calculated by 0.7 mL/kg and injection rate was 4.5 mL/s.In group C,21 patients were injected with the dosage of contrast medium calculated by 0.6 mL/kg and the injection rate was 4.0 mL/s.The attenuation value,signal-to-noise(SNR),con-trast-to-noise ratio(CNR),image quality and iodine intake between three groups were compared using One-Way ANOVA .Results There was no significantly statistical difference of age,sex ratio,BMI,heart rate between the three groups (P >0.05).However,the dosage of the contrast agent and different injection time had statistical significance (P <0.05).The attenuation value from group A to group B and then to group C was on the decline,the CT value of group A was obviously higher than that of group B and group C,and the differences were statistically significant (P <0.05),and there was no significantly statistical difference between the group B and group C (P >0.05).The image quality,SNR and CNR in three groups did not have significant difference (P >0.05).The total iodine and iodine injection rate were lowest in group C.Conclusion In 320 row coronary CT angiography with 100 kVp tube voltage and iterative reconstruction algorithm,the patients whose heart rate 70 beats per minute or less,BMI≤24 kg/m2 injected the low concentration of contrast medium by 0.6 mL/kg dose injection can give a good image quality which can meet the diagnostic requirement.Mean-while,it can also reduce the iodine intake and the risk of contrast induced nephrology (CIN).

Objective To explore the incidence and risk factors of bacterial infection after othtotopic liver transplantation (OLT). Methods Altogether 56 OLT recipients from January 2005 to October 2007 were included in the study. The incidents and the related variables of the infection were analyzed retrospectively. The related variables were evaluated using multivariate logistic regression model to identify the significant risk factors. Results Bacterial infection was confirmed in 29 recipients (51.8%). Among them, the lung infection was the most common site (53.7%). The Gram-positive cocci were 46.3%, while the Gram-negative bacilli were 53.7%. The risk factors for bacterial infection included duration of the operation and detained respirator using. Conclusion Bacterial infection is a major complication following OLT. Surveillance for the risk factors, enhancement the skill of operation, and improving the recovery of respiratory function is the key to decreasing the incidence of bacterial infection after transplantation.

Lipase gene from Penicillium expansum(lip07) was cloned and over-expressed in Pichia pastoris.a random mutant named ep8,which contained a single amino acid substitution,was obtained by using the lip07 as an error-prone PCR template in previous study.ep8 shows higher thermostability than that of lip07,To further improve the thermostability of the lipase,the Lys of wild-type(lip07) and mutant(ep8) in 202 were substituted by Ala using the Overlap extension PCR technique respectively.The mutant genes(lip07-K202A and ep8-K202A) were subcloned into pAO815,and then transformed into the Pichia pastoris GS115 for extracelluar expression,respectively.15% SDS-PAGE analysis indicated that the molecular mass of PEL-ep8-K202A and PEL-lip07-K202A are both about 28kDa,which is same with the wild-type lipase.The Tm of PEL-ep8-K202A is 41.66℃,2.63℃ higher than that of the wild-type(39.03℃) and 1.21℃ higher than the random mutant(PEL-ep8:40.45℃);the Tm of single mutant(PEL-lip07-K202A) is 37.08℃,2℃ lower than that of the wild-type lipase.

Objective To study the biodistribution of anti-HER-2/neu monoclonal antibody Herceptin labeled by 131I(131I-Herceptin) in healthy KM mice and nude mice bearing human ovarian cancer xenografts and radioimmunoimaging (RII) of the nude xenografts-bearing mice.Methods 131I-Herceptin was prepared using Iodogen method.The labeling efficiency, radiochemical purity, stability and immunocompetence were measured.The percentage activity of injection dose per gram of tissue (%ID/g) and the radioactivity ratio of tumor to non-tumor tissue (T/NT) were calculated for each time point.The optimal time for imaging was investigated by comparing the 131I-Herceptin SPECT for the nude mouse models bearing ovarian cancer xenografts at different time points.Results The labeling efficiency and radiochemical purity of 131I-Herceptin were 89.8% and 98.4%, respectively.The labeling was stable and had good immunocompetence.131 I-Herceptin was cleared rapidly mainly through liver, spleen and kidneys, consistent with first order two-compartment model.The uptake of 131I-Herceptin in the tumors bearing human SKOV-3 xenografts was much higher than that in nontumor tissue.The% ID/g was 18.08 in the tumor at 24 h post injection.The T/NT ratio increased with time and was 27.27 at 72 h post injection.The tumors in nude mice bearing SKOV-3 xenografts could be visualized on 131I-Herceptin SPECT imaging 2 h post injection; definitely identiffed 48 h post injection and the radioactivity ratio of tumor to contralateral tissue was 11.44 at 120 h post injection.However, the tumor in nude mice bearing HO-8910 xenografts did not show abnormal uptake of 131 I-Herceptin at each time point.Conclusions 131 I-Herceptin is a good radiopharmaceutical targeting SK-OV-3 xeuografts and it may be useful in imaging carcinoma of ovary and target therapy of its metastases with high HER-2/neu expression.

Objective To explore the feasibility of imaging and treatment of cervical cancer xenograft model using 131I mediated by hNIS gene transfection. Methods The cervical cancer xenograft models were established with Hela-NIS( +) cells and Hela cells, respectively. Five Hela-NIS( +) xenograft models and five Hela xenograft models were dynamically imaged at 0.5, 1, 2, 4, 8, 16 and 20 h postinjection of 131I(7.4 MBq). Five Hela-NIS( +) xenograft models were imaged at 0. 5,1,2,4,8,16, 20 and 25 h postinjection of 99TcmO4-(11.1 MBq). Twenty Hela-NIS( +) cervical cancer xenograft models were randomly divided into four groups: Three 131I treating groups and one control group. The therapeutic effects of 131I at threelevels (74,111,148 MBq) were investigated following intraperitoneal injection. Results Hela-NIS( +)human cervical cancer xenografts were established successfully in nude mice. The Hela-NIS( +) xenografts significantly accumulated radioactivity after intraperitoneal injection of 131I, and the radioactivity was persistently present until 20 h postinjection, but Hela xenografts had no radioactive accumulation. The T/B value of the Hela-NIS( +) xenografts reached 17.34 at 8 h postinjection. The imaging with 99TcmO4- showed that the radioactivity was persistently present in Hela-NIS( +) xenografts for almost 25 h. The Hela-NIS( +)xenografts shrinked after 131I treatment. The inhibition ratios of tumor growth in 111 MBq and 148 MBq groups were both significantly higher than that of 74 MBq group (t: 2.74-5.75, P ＜0.05). Conclusions Hela-NIS( +) cervical cancer xenografts in nude mice could persistently accumulate 131I and 99TcmO4- and could be treated successfully with 131 I. 131 I treatment mediated by hNIS gene transfection could be a promising cancer treatment method.

Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.