BACKGROUND: The implanted cartilage calls can synthesize cartilage matrix as cartilage in cartilage tissue enginsedng, and the density of implanted cells is the key point.OBJECTIVE: To evaluate the effect of cell seeding concentration on the chondrogenic differentiation of the adipose dadved sromal cells (ADSCs).DESIGN, TIME AND SETTING: The in vitro cellular-scaffold observation was performed at the cytobiological laboratory of China Medical University from November 2007 to July 2008.MATERIALS: Six male SD rata with clean grade were supplied by the Experimental Animal Center of China Medical University.METHODS: Totally 5 g/L type ; collagen solution and 20 g/L chitosan was mixed in a mould with volume ratio of 7:3, after lyophillization, it was cut into pieces with 5 mm ～ 5 mm x 2 mm, followed by crosslinking with ethanol contained of 2% chondroitic acid at room temperature. After washing with double distilled water and freeze drying, the chitosan-collagen-chondroitin sulfate copolymar matrices scaffolds were harvested. ADSCs isolated from rat inguinal fat pads were digested with collagenase and trypsase. The prepared scaffolds were randomly divided into 3 groups, and the third passage cells with density of 2×10 9/L,2×10 109/L, and 2×10 11/L were seeded into chitosan-coflagen-chondroitin sulfate scaffolds, and cultured in chondrogenic medium for 3 weeks.MAIN OUTCOME MEASURES: The expression of cartilage specificity gene was detected by hematoxylin-eosin staining, type Ⅱ collagen immunohistochemical staining and RT-PCR.RESULTS: Hematoxylin-eosin staining showed that after 3 weeks of culture, the cell proliferated and differentiated well, especially in 2x101～/L group, more extrocelluer matrices were produced and cartilage lacuna-structure could be seen. The type Ⅱ collagen was positive expressed in each group, which showed a gradually increasing tendency with the cell seeding concentration increasing. RT-PCR showed that the expression of proteoglycen and type Ⅱ collagen mRNA were slowly increased. However the expression of Ⅹ collagen mRNA was decreased with increasing cell seeding concentration.CONCLUSION: The chitosan-collagen-chondroitin sulfate copolymer matrices can provide an appropdate environment for the generation of cartilage-like tissues and high call seeding concentration of 2×1010/L facilitate ADSCs to differentiate into cartilage.

Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.

Purpose:To study whether the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway could sensitize the response of tumor cells to some chemotherapyeutic agents. Methods:The different tumor cells has been treated with the combination of isoform specific Akt inhibitor and either adriamycin or camptothecin; the quantitation of the induction of apoptosis by drugs has been estimated with caspase 3 assay; immunoprecipitation western blotting has been used to evaluate the inhibition of the phosphorylation of different isoforms of Akt after the treatment. Results:①The inhibitors could reduce the phosphorylation of Threonine 308 and Serine 473 of isoform specific Akt. ②The inhibition of any one isoform specific Akt could not reverse the resistance of tumor cells tested to chemotherapeutic drugs, but it is not the same case if blocking of two isoforms of both Akt1 and Akt2 was done at the same time. ③The synergistic effects of Akt inhibitors is maybe relative to the level of endogenous PTEN(phosphatase and tensin homolog deleted on chromosome 10) expression. Conclusions:It is required to inhibit two isoforms of both Akt1 and Akt2 in order to maximally sensitize the tumor cells to chemotherapy.

Objective:To study the clinical significance of β2-microglobin(β2-MG)and fibronection (Fn) contents in patients with nasopharyngeal carcinoma.Methods:The contents of serum β2-MG and Fn were detected by radioimmunoassay and agar-diffusion methods in 60 patients with nasopharyngeal carcinoma before and after radiotherapy,also in 60 normal subjects as control group.Results:The contents of serum β2-MG and Fn were 2.99±1.11mg/L(β2-MG)and 134.60±28.93mg/L(Fn) in 60 patients with nasopharyngeal carcinoma,2.16±0.50mg/L(β2-MG)and 196.16±34.65mg/L(Fn)in 60 health subjects,respectively.Those results showed that the content of β2-MG in patients group was higher than that in control group (P＜0.05),and the content of Fn in patients group was lower than that in control group(P＜0.05).After radiotherapy of patients group,the content of β2-MG were lower(2.16±0.55mg/L)than that before radiotherapy(P＜0.05).Conversely,the content of Fn was higher(180.53±34.66mg/L)than after radiotherapy(P＜0.05).Conclusions:The detection of serum β2-MG and Fn have clinical significance on the evaluation prognosis of patients with nasopharyngeal carcinoma.

ObjectiveTo study the effects and molecular mechanisms of Shuanglong Pill on angiogenesis of ischemic myocardium. Methods63 Wistar rats were ligated left anterior descending branch to be made into models of acute myocardial infarction(AMI).All rats were randomly divided into large-dosage group of Shuanglong Pill(LDG),small-dosage group of Shuanglong Pill(SDG),The control of acute MI(MIC).Other 11 rats were set as normal control(NC).Rats in every group had been treated half for 2 weeks and half for 4 weeks. New vessels' quantity in ischemic myocardium of acute MI rats were detected by immunohistochemistry double steps assay;the expressions of vessel endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) on ischemic myocardium were detected by immunohistochemistry SP assay;the expressions of VEGF mRNA and bFGF mRNA on ischemic myocardium were detected by reverse transcription polymerase chain reaction(RT-PCR) assay.ResultsThere were more new capillaries on ischemic myocardium of LDG, SDG compared with that in MIC at every stage,moreover,there was significantly increase at the end of 4 weeks compared with that at 2 weeks.At the end of 2 weeks, the expression of VEGF in LDG were significantly elevated compared with that in MIC; and the expression of bFGF in LDG were significantly elevated compared with that in SDG and MIC.Whereas the expression of VEGF in LDG were significantly decreased at the end of 4 weeks compared with 2 weeks. There were significant differences of above comparison (P<0.05-0.01).At every stage the expression of VEGF mRNA in LDG, SDG were significantly elevated compared with that in MIC; but its expression was significantly decreased at the end of 4 weeks compared with that 2 weeks. The expression of bFGF mRNA was significantly elevated in LDC at the end of 2 weeks compared with that in other groups.ConclusionsShuanglong Pill could promote angiogenesis in ischemic myocardium,application of large dose of Shuanglong Pill could significantly upregulate the expression of VEGF, bFGF, VEGF mRNA and bFGF mRNA.

Objective The purpose of this study was to investigate the correlation of high sensitivity C-reactive protein (hsCRP) and fibrinogen with carotid artery arteriosclerosis of patients with cerebral infarction. Methods One hundred and thirteen patients with cerebral infarction were assigned as study group, and 102 healthy persons as control group. The levels of serum hsCRP and Fib in the two groups were measured. The carotid artery arteriosclerosis and carotid intimal-medial thickness (IMT) were examined by color Doppler and B-ultrasound. Results The value of IMT between study group and control group was statistically significant. The positive rates of carotid artery arteriosclerosis plaque and vulnerable plaque in study group were significantly higher than those in control group (all<0.05) . The level of serum hsCRP was significantly higher in study group than that of control ( <0.05) . The level of serum Fib between study group and control group was not statistically significant ( >0.05) . Conclusion The level of hsCRP was closely related to the degree of carotid artery arteriosclerosis and the occurrence and development of cerebral infarction. But the level of Fib was not closely related to the degree of carotid artery arteriosclerosis.

The paper is to report the study of pharmacokinetics of transdermal administered nicotine in the brain of freely moving rat by using microdialysis with stable labeled isotope as internal standard. The pharmacokinetic behavior of nicotine in Sprague Dawley rat brain was investigated after intranasal administration (3.75 mg). Brain fluid samples were collected by intracerebral microdialysis with DL-nicotine as internal standard. Concentrations of nicotine and DL-nicotine in the sample were measured by HPLC-MS/MS. Main pharmacokinetic parameters were calculated and analyzed by Das 2.0 pharmacokinetic software. The recovery of nicotine and the delivery of DL-nicotine were the same. The fate of absorption and distribution was two compartment model and the values of t1/2alpha was 170.31 min, t1/2beta was 263.30 min and the AUC(0-infinity) was 2.75 x 10(5) microg x L(-1) min separately. DL-nicotine can be used to calibrate the recovery of nicotine, and the new method of stable isotope microdialysis can be used to study the pharmacokinetics of freely moving rat. It will make sense for the treatment of addiction of tobacco and provide a new thought for the research of pharmacokinetics-pharmacodynamic combination.

OBJECTIVE:To establish a method for the simultaneous determination of acetic acid and succinic acid in Banxia syrup. METHODS:RP-HPLC was performed on the column of GL InterSustain-C18 with mobile phase of 0.03 mol/L ammonium di-hydrogen phosphate buffer solution(pH2.0)-methanol(gradient elution)at a flow rate of 0.8 ml/min,the detection wavelength was 210 nm,column temperature was 30 ℃,injection volume was 10 μl. RESULTS:The linear range was 0.020 98-0.209 8 μg/ml for acetic acid(0.999 9)and 12.04-120.4 μg/ml for succinic acid(r=0.999 9);limits of quantification were 0.15,18.24 ng,limits of detection were 0.045, 5.53 ng;RSDs of precision, stability and reproducibility tests were lower than 2%;recoveries were 97.45%-101.68%(RSD=1.39%,n=9) and 98.31%-101.08%(RSD=1.01%,n=9). CONCLUSIONS:The method is accurate and rapid,and suitable for the simultaneous determination of acetic acid and succinic acid in Banxia syrup.

Objective:To clone the RC-RNase gene and prepare its recombinant prokaryotic construct, and then to express RC-RNase protein using Escherichia coli system. Methods: RC-RNase cDNA was obtained by RT-PCR from liver of Rana catesbeiana, and cloned into pUCm-T plasmid for nucleotide sequencing. Its expression construct was prepared using the 6?His vector pRSET-A, and induced to express by IPTG in Escherichia coli BL21(DE3). Western blotting identified the expression product. Results: A 380 bp long cDNA was obtained from liver of Rana catesbeiana, restriction sites and sequence being consistent to those reported for RC-RNase. After introducing the gene into Escherichia coli and through the induction by IPTG, it was observed a new peptide at the expected position (Mr 16000) on SDS-PAGE gel. This product was proved to be the target protein via Western blotting. It existed in a form of inclusion body and its efficiency reached 12.5% of total bacterial proteins. Conclusion: RC-RNase gene was cloned and expressed in Escherichia coli. The protein could be used for characterizing the biological activities and function of RC-RNase.

Objective To study the relationship between the expression of inducible nitric oxide synthase(iNOS)and neural cell apoptosis after chronic cauda equina compression. Methods Totally 30 male adult SD rats were randomly divided into 2 groups as the control group and the experi?mental group. The control group received sham operation with single laminectomy of L5 lumina. In the experimental group,the silicon sheet was in?serted into the spinal canal of L4 to cause single level compression of cauda equina. The L4 level of spinal cords were harvested at 2 weeks,4 weeks,8 weeks,and 12 weeks after operation in the experimental group,and at 4 weeks in the control group respectively,and then immunohistochemistry and image analysis were performed to observe the expression of iNOS in spinal cord and the TUNEL method was applied to observe cell apoptosis. The morphology of cells was observed by transmission electron microscope. Results There was few amount of iNOS expressed in the control group. The expression of iNOS was slight at 4 weeks in the experimental group and was higher at 8 weeks and 12 weeks compared with the control group. Small amount of neural cell apoptosis was evidenced in the control group,while neuron apoptosis appeared remarkably in the experimental group since 4 weeks and increased with the extension of time. Transmission electron microscopy found apoptosis changes in neurons in the experimental group. Conclusion The expression of iNOS increases in corresponding spinal cords after chronic compression of cuada equine and neural cell apoptosis oc?curs,indicating that iNOS is positively correlated with neural cell apoptosis.