Abstract:Objective：To construct a recombinant retroviral vector harboring MafA and to establish a liver epithelial progenitor cell (LEPC) line for stable expression of MafA .Methods: MafA was amplified by PCR and suncloned into pBMN-Z-IRES-Neo vector to obtain pBMH-MafA-Neo vector.After introducing the pBMN-MafA-Neo into Phoenix package cells,the cell culture supernatant was used to infect LEPCs.LEPCs stably expressing MafA gene were screened out.RT-PCR was used to detect the influence of MafA on the phenotype of LEPCs.Results: We successfully constructed pBMH-MafA-Neo vector and obtained LEPCs which stably expressed MafA.Expression of GK and GLUT2 genes in LEPCs-MafA cells was higher than that in the LEPCs.Conclusion: We have successfully obtained LEPCs which can stably express MafA,laying a basis for studying the differentiation of LEPCs into pancreas cells.

Abstract:Objective：To observe the expression of miR-451 during erythroid differentiation of K562 cells and investigate the role of miR-451 in erythroid differentiation.Methods: MiR-451 expression was analyzed in different tissues (the liver,bone marrow,erythrocytes,and white blood cells) of mice,human erythrocytes,and chicken red blood cells by Northern blotting.Erythroid differentiation of K562 cells was assessed by DAB staining and RT-PCR of heamoglobin mRNA before and 36 h after hemin induction (50 μmol/L).The expression of miR-451 in K562 cells was further explored by Northern blotting and stem-loop RT-PCR before and 24,48,72,and 96 h after hemin induction (50 μmol/L).Results: Expression of miR-451 was only identified in the erythrocytes,not in white blood cells,hepatic cells or bone marrow of mice.MiR-451 expression was also detected in human erythrocytes and chicken erythrocytes with nuclei.Two bands were detected in the human and mouse erythrocytes by Northern blotting,indicating that,in addition to the one reported by Sanger’s miRBase,there was another miR-451 sequence which had additional uracil residue in 3′ terminal.Hemin induced differentiation of K562 cells and DAB staining showed more positive cells after induction (P<0.05); the expression of γ, δ and ε-globin mRNA was also increased after treatment with hemin（P＜0.05）.Although Northern blotting revealed no changes in miR-451 expression in K562 cells after hemin induction,more sensitive stem-loop RT-PCR showed that miR-451 expression, which maintained at lower level in un-induced K562 cells, was increased during erythroid differentiation 24 h after hemin induction.With the upregulation of δ-globin protein,the expression of miR-451 reached its peak 72 h later.Conclusion: miR-451 is specifically and highly expressed in erythroid terminal differentiation.Two different sequences of miR-415 (one with and additional uracil residue) are present in the human and mouse erythrocytes,and their expression is elevated during the erythroid differentiation of K562 cells.

Abstract:Objective：To express Tumstatin 183-230-TRAIL fusion protein and to observe its biological functions. Methods: SOE-ing PCR was employed to amplify the recombinant sequence of Tumstatin 183-230 and TNF-related apoptosis-inducing ligand (TRAIL114-281). An expression vector pMAL-Tu-T was constructed by inserting Tu-T sequence into pMAL-c2; the vector was used to transfect E. coli BL21 (DE3) and expression of MBP-Tu-T fusion protein was induced by IPTG.Amylose Resin columns were employed to purify the fusion protein. The biological functions of MBP-Tu-T protein was examined by inhibitory test of endothelial cell proliferation, standard tumor cell cytotoxic assay, in vitro tube formation inhibition, and electron microscopic observation (apoptosis). Results: The expression rate of MBP-Tu-T fusion protein in E.coli was about 20%. Purified recombinant protein obviously inhibited endothelial cell proliferation (IC50 12.5 μg/ml ), induced apoptosis of pancreatic cancer cells, and inhibited tube formation. Conclusion: Constructed MBP-Tu-T fusion protein is bifunctional, which lays a solid foundation for further investigation of antitumor effect of Tumstatin 183-230-TRAIL in vivo.

Abstract:Objective：To establish clear cell renal cell carcinoma (ccRCC) cell lines from clinical ccRCC specimens of Han nationality in China and to characterize the biological features.Methods: From 2005 to 2007,fresh surgical samples of ccRCC were obtained from 43 patients; the samples included primary tumor in situ，osseous metastasis,lymph node metastasis,and cancerous embolus.The samples were cultured in vitro using explant-culture method within 30-60 min after surgery.Analysis on cell growth and colony-forming efficiency was recorded for the lines which were passaged for over 50 generations.Chromosome examination,pathological examination and tumorigenesis in NOD-SCID mice were used to determine their malignancy.Flow cytometry was used to determine expression of CA9 and CD133.Results: Most of the primary cells could only be passaged for less than 5 generations; 5 lines could be serially passaged for over 5 passages,3 lines for over 10 passages, and only 2 lines could be stably passaged.One line,named RCC05-TXJ, was from osseous metastatic ccRCC and had been serially passaged for 110 generations in 21 months; the average doubling time was 19.2 h,average chromosome number was 75,and colony forming efficiency was 41%.Another line,named RCC05-ZYJ,was from primary ccRCC specimen and had been serially passaged for 160 generations in 18 months; the average doubling time was 16.5 h,average chromosome number was 55,and the colony forming efficiency was 37%.Immunohistological analysis demonstrated that both lines expressed CA9 and CD133.Flow cytometry analysis found that expression levels of CA9 and CD133 increased with the passages.Both RCC05-ZYJ and RCC05-TXJ lines were able to form tumor and to metastasize in NOD-SCID mice; however,their metastatic ability was obviously different.Conclusion: We have established 2 ccRCC cell lines with different metastatic potentials from the clinical ccRCC specimens of Han nationality in China.The ratio of tumor stem cells increases with the passages.

Abstract:Objective：To screen for methylated biomarkers for the early diagnosis and prediction of clear cell renal cell cancer (ccRCC) metastasis.Methods: Highly and lowly metastatic ccRCC cell lines,which were established using fresh surgical specimens in our laboratory,were used in this study.Genomic DNA was extracted after pathological identification.Methylated genomic fragments were enriched by 3 cycles of NotⅠ genomic DNA subtractive hybridization and were linked to vectors,which was then used for transformation,followed by α selection.Forty positive clones were randomly selected for DNA sequencing.Bioinformatics analysis was used to identify methylation of CpG islands and to predict the function of unknown genes.Results: DNA sequencing revealed 27 independent clones with different methylations between the highly and lowly metastatic ccRCC.Five of the 27 clones contained CpG islands,and 2 of the 5 fragments contained CpG islands in the promoter regions of genes MYADM and LOC646024.MYADM was associated with maturation of hematopoietic cells and regeneration of stem cells.LOC646024 shared 85% homology with UL16 binding protein 1; the latter was related to tumor killing function of NK cells.Conclusion: Novel methylated sequences have been discovered from Chinese ccRCC cells with different potentials for metastasis.Methylation of 2 candidate genes,MYADM and LOC646024,is indicated to be involved in ccRCC metastasis.Our findings are valuable for the biomarker exploration to predict metastasis as well as molecular epidemiological research.

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Abstract:Objective：To prepare NIH3T3 cells harboring microencapsulated VEGF gene and investigate the proliferation,activity and metabolic function of the modified cells.Methods: Microencapsulated VEGF modified NIH3T3 cells were prepared through an alginate-BaCl2 process.Morphological appearance of the microencapsulation and the cell morphology were observed under inverted phase microscope; untreated NIH3T3 cells served as control.The concentrations of VEGF in the culture supernatant (collected every 48 hours) were measured by ELISA; the proliferation and vitality of the cells were examined by MTT assay and flow cytometry with PI staining.Results: The microcapsules were round in shape and the cells grew well.There was no significant difference in the concentrations of VEGF，MTT values and vitalities of cells between the 2 groups.Conclusion: The growth and metabolic functions of NIH3T3 cells are not influenced by microencapsulated NIH3T3 cells harboring VEGF gene.The bio-properties of modified cells are similar to those of the control cells,which lays a foundation for transplantation of microencapsulated VEGF modified NIH3T3 cells in vivo.

Abstract:Objective：To observe the influence of ischemia-reperfusion (I/R) on hepatoma growth and on the expression of genes associated with tumor metastasis and recurrence (VEGF and MMP-9) in the adjacent tissues of cancer in nude mice. Methods: BALB/c nude mouse model bearing Hep3B-tumor in the liver was established and the model mice were evenly randomly into 5 groups: sham group and ischemia/reperfusion 1 h, 6 h, 5 d, and 7 d groups (n=8). I/R models were established by blocking porta hepatic; the sham group underwent the same treatment as the I/R model group except for blocking of porta hepatic. ALT and AST were detected in I/R 1 h and 6 h groups. Real-time-PCR was employed to detect the change of VEGF and MMP-9 in the adjacent tissues of cancer and the results were compared with that of the control group (n=6). Histopathological changes of liver were studied by H-E staining and necrotic areas were calculated in I/R 5 d and 7 d groups(n=6). The remnant tumor bearing mice were sacrificed 2 weeks after I/R to measure the volume and mass of the tumors. Results: Two weeks later, the tumor volume and mass in I/R group were increased compared with those in the sham group (\[209.6±25.74\] mm3 vs \[330.6±32.01\] mm3,\[0.214±0.036\] g vs \[0.374±0.045\] g, P<0.01). Levels of ALT and AST were significantly elevated in I/R 1 h and 6 h groups compared with those in the sham group (P<0.01). H-E staining showed that the infiltration of inflammatory cells around the tumors and the areas of necrosis became more prominent 5 days after I/R compared with the sham operation group (P<0.05). The necrosis area was reduced 7 days after I/R compared with 5 days after I/R (P<0.05); however, the necrosis area was replaced by invasion of tumor cells (P<0.05). It was found that the expression of VEGF and MMP-9 mRNA was higher in the adjacent tissues of cancer after I/R than that in the sham group (P<0.01), and the expression of the 2 was correlated with each other (r=0.418, P<0.01). Conclusion: I/R induced by blocking porta hepatic can accelerate tumor cell growth and promote expression of VEGF and MMP-9 in the adjacent tissues of cancer.

Abstract:Objective：To compare the hepatic damaging effect in mice exposed to arsenic of different valence states (iAs3+ and iAs5+ ) and to probe into the related mechanisms.Methods: Sixty mice were divided into control group(distilled water),sodium arsenite group（iAs3+ ,300 mg/L）and sodium arsenate group（iAs5+ 300 mg/L）at random.The mice were sacrificed 10 months after exposure in the 3 groups.The liver function and total arsenic content in the liver were examined in each group.H-E staining and Masson staining were employed to observe the pathologic changes of the livers.Real-time RT-PCR were used to examine the expression of TNF-α,IL-6 ,GSH-Px,MMP-8,ColⅠand Col ⅢmRNA.Results: The accumulation of arsenic in iAs3+ group was higher than that in the iAs5+ group; the liver function of animals in iAs3+ group was worse than that in the iAs5+ group (P<0.05).H-E and Masson staining showed that the hepatic injury and fibrosis in iAs3+ group were severer than those in the iAs5+ group (P<0.05).The expression of TNF-α,IL-6,Col Ⅰand Col Ⅲ mRNA was higher and the expression of GSH-Px,MMP-8 mRNA was lower in arsenic exposed mice compared with those in the normal mice (P<0.05).Compared with iAs5+,animals in iAs3+ group had higher expression of TNF-α,IL-6,and Col ⅠmRNA (P<0.05).Conclusion: Long-term exposure to oral drinking with arsenic can result in hepatic injury and hepatic fibrosis by stimulating secretion of mediators of inflammation and ECM.And the effect of arsenic is related to the arsenic accumulation and the valence states of arsenic.

Abstract:Objective：To investigate the expression of HIF-1α and its relationship with angiogenesis in osteosarcoma. Methods: Osteosarcoma MG-63 cells were cultured in vitro under hypoxia and mimic hypoxia conditions.Thirty paraffin-embedded osteosarcoma tissues and 20 fresh frozen osteosarcoma specimens were collected.The mRNA and protein expression of HIF-1α and VEGF were detected by RT-PCR,Western blotting,ELISA,and immunohistochemistry methods.The mean vessel density(MVD) were also calculated.Results: The mRNA level of HIF-1α had no change under hypoxia and minic hypoxia conditions,whereas the protein expression was increased dramaticaly.The mRNA and protein expression of VEGF was significantly increased under hypoxia and minic hypoxia conditions.The positive rate of HIF-1α mRNA (90%) and VEGF(100%) in 20 fresh frozen tissues were higher than those of the para-tumor tissues(P<0.05).The positive rates of HIF-1α and VEGF protein in paraffin-embedded osteosarcoma were 86.7% and 93.3%, respectively, significantly higher than those in the para-tumor tissues (6.7%,26.7%,P<0.05).Spearman related analysis demonstrated that the expression of HIF-1α(P=0.005,r=0.767) and VEGF(P<0.002,r=0.701) had a positive relation with MVD.Conclusion: HIF-1α is overexpressed in osteosarcoma and might be closely associated with tumor angiogenesis.Overexpression of HIF-1α might indicate a poor prognosis of osteosarcoma.

Abstract:Objective：To study the change of immunological response and cell proliferation in breast tissues augmented by polyacrylamide hydrogel injection(PHI).Methods:The expression of CD68,CD25 and PCNA in 20 breast tissues with indurations,12 without indurations after breast augmentation by PHI,and 10 normal breast tissues was examined by immunohistochemistry P-V6000; analysis was also done by H-E staining.Results:Hyperplasia of fibrous tissue and infiltration of inflammatory cells and macrophages were found in the breast and adjacent tissues 3-8 years after PHI.Positive cells of CD68 ,CD25 and PCNA hardly existed in the normal tissues,but the breast tissues around the polyacrylamide hydrogel had many positive cells of CD68 and PCNA,especially in cases with indurations;there were significant differences between the 3 groups(P<0.01); and the numbers of cells positive of CD25 were similar between the 3 groups.Conclusion:Long-term implantation of polyacrylamide hydrogel can cause immunological reaction and cell proliferation in the local adjacent tissues.

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Abstract:Objective：To observe polyethylenimine-mediated BMP-7 gene transfection in promoting fracture healing in elderly rats. Methods：Male SD rats (20-month-old) were randomly divided into 3 groups: group A received BMP-7 gene therapy mediated by polyehtylenimine, group B was treated with normal saline and group C was treated with BMP-7 plasmid without polyethylenimine. Right femoral shaft fracture model was established in all rats. Animals in group A received transdermal injection of pcDNA3.1-BMP-7/PEI; those in group B and C received the same volume of normal saline and pcDNA3.1-BMP-7, respectively. X-ray photography, histological observation (H-E staining), immunohistochemical staining of collagen type Ⅰ, biomechanical and bone mineral density test were employed to assess the healing of fracture 2, 4, and 8 weeks after treatment. Results: The results of X ray and H-E staining showed no fracture healing in the 3 groups during the 8th week after fracture; however, the growth of coloboma in group A was better than that of the other 2 groups, with partial bone union between the fracture ends. Staining of collagen type Ⅰ showed deeper, wider staining in group A compared with the other 2 group. Anti-bending intensity and bone mineral density tests showed that the parameters in group A were better than those of the other 2 groups (P<0.05 or 0.01). Conclusion: Polyethylenimine-mediated hBMP-7 gene transfer can effectively promote fracture healing in elderly rats.

Abstract:Objective：To explore a new evaluation method for hospital intangible assets through a case study on evaluation of hospital “Know-how”.Methods: Using the contingent valuation method(CVM) we designed a questionnaire to survey the willingness of patients to pay for hospital “Know-how”.The data was analyzed by econometrics methods and a model was established for evaluating the “Know-how” in hospitals.Results: Under three scenarios,the effect of “Know-how” increased gradually; the percentages of patients who were willing to pay were 58.48%,65.50% and 98.54%,respectively.The mean maximal payments were 4,5,and 5 folds that of the basal level.The annual family income was the most important factors influencing the patients’ willingness to pay. According to the model established,the value of the hospital “Know-how” was between 85.014 95 million to 252.229 million RMB in 2006.Conclusion: CVM may serve as a new method to evaluate hospital “Know-how” and should be further studied in the future.

Abstract:Objective：To evaluate the clinical value of TruviewTM EVO2 optic laryngoscope by comparing it with the Macintosh laryngoscope in patients receiving cervical vertebral surgery.Methods: One hundred patients scheduled for elective cervical vertebral surgery were enrolled in this randomized crossover study.After induction,the patients’ glottis in group A (n=50) was displayed by Macintosh laryngoscope and the Cormack-Lehane (C/L) grade was recorded,and then optic laryngoscope was employed to display the laryngeal structure.The order of laryngoscopy attempts was reversed in group B (n=50). Parameters recorded included demographics,airway assessment features (BMI,thyromental distance,mandibular size,mouth opening,mallampati oropharyngeal scale,and neck movement),C/L grade,laryngoscopic force applied,duration of intubation, difficulties of laryngeal view and injury of upper airway.Results: There were no significant difference in demographics,airway assessment features,C/L grade and duration of intubation between the 2 groups,whereas the laryngoscopic force in group A was significantly lower than that in group B(P<0.05).The C/L grade obtained with optic laryngoscope was 69265 (ⅠⅡⅢ）,which was significantly better than that with Macintosh laryngoscope (264628,P<0.001).A poor laryngeal view of 5% was obtained with optic laryngoscope,which was significantly better than that with Macintosh laryngoscope (28%,P<0.001).There was significant association between airway assessment features and C/L grade or laryngoscopic force (P<0.05).Conclusion: The TruviewTM EVO2 optic laryngoscope has better glottic view and lighter laryngoscopic force than those of Macintosh laryngoscope,and may have potential advantages for managing the difficult airways for cervical vertebral surgery.

Abstract:Objective：To observe the 99mTc-MIBI myocardial perfusion images in patients with myocardial infarction before/after percutaneous coronary intervention (PCI),so as to screen for the reliable predictors for the therapeutic effect of PCI.Methods:Forty-eight patients with myocardial infarction receiving PCI were included in this study.The rest,stress and nitroglycerin myocardial perfusion imaging tests were performed before PCI.The stress and rest myocardial perfusion imaging tests were repeated 1-2 weeks after PCI.The value of（rest 99mTc-MIBI imaging in myocardial perfusion scores after PCI-stress 99mTc-MIBI imaging in myocardial perfusion scores before PCI）/ stress 99mTc-MIBI imaging in myocardial perfusion scores before PCI was taken as the curative effect of PCI; the value of（rest 99mTc-MIBI imaging in myocardial perfusion scores before PCI-stress 99mTc-MIBI imaging in myocardial perfusion scores before PCI）/ stress 99mTc-MIBI imaging in myocardial perfusion scores before PCI was taken as the functional viable myocardium; the value of (stress 99mTc-MIBI imaging in myocardial perfusion scores before PCI-nitroglycer 99mTc-MIBI imaging in myocardial perfusion scores before PCI）/ stress 99mTc-MIBI imaging in myocardial perfusion scores before PCI was taken as potential functional viable myocardium; and the value of（rest 99mTc-MIBI imaging in myocardial perfusion scores before PCI-nitroglycer 99mTc-MIBI imaging in myocardial perfusion scores before PCI）/ rest 99mTc-MIBI imaging in myocardial perfusion scores before PCI was taken as drug relative viable myocardium. To screen the proper predictors for therapeutic effects, linear correlation analysis was performed between the curative effect of PCI and the latter 3 factors.Results:Stress and rest myocardial perfusion defects were significantly improved after PCI (P<0.01).Correlation analysis showed that the curative effect of PCI was correlated with the latter 3 factors,with the correlation coefficients being r1=0.63,P<0.01,r2=0.94,P<0.000 1,and r3=0.92,P<0.000 1,respectively,indicating that the potential functional viable myocardium had the best correlationship with PCI.Conclusion: It is necessary to perform the rest,stress and nitroglycerin myocardial perfusion imaging tests in patients with cardiac infarction.The functional viable myocardium is a reliable index in screening the patients suitable for PCI and in predication of the therapeutic effect.

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Abstract:Objective：To investigate the therapeutic efficacy of double-J catheter in treatment of renal tuberculosis(TB) and in rescuing the structure and function of the kidney. Methods: Thirty-four patients with renal TB (22 combined with single side hydronephrosis) were divided into 2 groups randomly. Group A were treated with antituberculous therapy and group B with antituberculous therapy combined with pre-treatment with double-J catheter. All 34 patients were followed up for 3 months and were re-examined. Results: The results of B ultrasound, intravenous urogram (IVU), CT and isotope nephrogram were comparable between the 2 groups before treatment, and the results were significantly different between the two groups after 3 months’ drug treatment (P<0. 05). The results of routine urine tests, ESR and the positive rates of urine ABF were not significantly different between the 2 groups (P<0. 05). Nephrectomy was performed in 21 cases, with 15 cases（88.2%） in group A and 6 cases（35.3%）in group B (P<0. 05). Conclusion: Pre-treatment with double-J catheter in the diseased side of patients with renal TB can better preserve the structure and function of the kidney, and can help to lower the rate of nephrectomy.

Abstract:Objective：To prepare stable, small-sized injectable sustained-release nanoparticles harboring brain-derived neurotrophic factor (BDNF) and to evaluate its drug releasing process.Methods: The nanoparticles were prepared using poly(D,L-lactic-co-glycolic acid) (PLGA) as the carrier by w/o/w double emulsion-solvent evaporation method.The formula and technique were optimized; the shape,size and the distribution of the diameters of the particles were observed; and recovery rate,precision,repeatability,encapsulation efficiency,and drug releasing characteristics were assessed.Results: With the optimized formula,the drug loading rate was 1%,the polymer concentration was 3.3 mg/ml, and the ultrasound time was 40 s; mannitol was used as the supporting agent.BDNF nanoparticles were round,homogenous in size,with a mean diameter of 156.7 nm.The prepared particles had high recovery rate,precision,repeatability,and encapsulation efficiency.The drug release was characterized by slow corrosion and the process lasted for 30 days.Conclusion: We have successfully prepared slow-release nanoparticles harboring BDNF,which are stable and have high encapsulation efficiency.

Abstract:Objective： To study the relationship of anxiety with personality and family factors in servicemen and search for factor influencing the anxiety of servicemen,so as to provide evidences for effective intervention. Methods: The personality character,family factors and anxiety statuses were assessed by Eysenck Personality Questionnaire (EPQ),State-Trait Anxiety Inventory (STAI) and information scale in 2 001 male soldiers. Results: There was a negative association between P,N scores of EPQ and the trait- and state-anxiety scores,and a positive association between L,E scores of EPQ and trait- and state-anxiety scores (all P<0.01). Trait-anxiety score was correlated to the parental relationship and economic status; state-anxiety score was related to the parental relationship,the structure of family and economic status(P<0.05). The results of Multi-regression analysis suggested that the factors of trait-anxiety included parental relationship and economic status; the factor of state-anxiety included economic status only(P<0.01). Conclusion: Personality and family factors are the most important factors of the anxiety in servicemen. The four dimensions of EPQ all contribute significantly to the anxiety status. Also the parental relationship and economic status in the family factors contribute significantly to anxiety status. \[KEY WORDS\]military men；state-anxiety；trait-anxiety；personality；family

Abstract:Objective：To construct the eukaryotic expression vectors of two NF2 mutants (neurofibromatosisⅡ) and to study their expression in rat schwannoma cell line (RT4).Methods: Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Lys42 in NF2 into Pro in pcDNA3.1-NF2ΔLys42Pro， and convert the codons for Phe 47 into that of Leu.RT4 cells were transiently transfected with the 3 kinds of plasmids containing the mutations and wide-type NF2 via lipofectin separately,then the expression levels of NF2 mRNA and protein were determined by RT-PCR and Western blotting in 3 groups.The cell proliferation was determined by the MTT after transfection.Results: DNA sequence analysis demonstrated the two-step mutagenesis was successful and the two plasmids of pcDNA3.1-NF2ΔLys42Pro and pcDNA3.1-NF2ΔPhe47Leu were obtained,both of the transfectants could produce merlin protein and mRNA.The two mutants had a significantly lower inhibitory rate for RT4 cells compared with wide-type NF2 (P<0.05).Conclusion: The recombinant plasmids pcDNA3.1-NF2ΔLys42Pro and pcDNA3.1-NF2ΔPhe47Leu have been successfully constructed and they can be efficiently expressed in RT4 cells.

Abstract:Objective：To construct subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury in cats.Methods: Fifteen adult cats were randomly divided into 3 groups (n=5): control group，4-w compression group and 8-w compression group.The chronic optic nerve injury was produced by an inflatable balloon implanted under the optic chiasm.The total RNA was prepared from optic nerves of each group by TRIzol method.Double-stranded cDNA was produced by SMART PCR cDNA synthesis protocol.Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes in the optic nerves after 4-w and 8-w compression.The cDNA fragments were directly inserted into T/A cloning vector to establish the subtractive library，followed by amplification of the libraries through E.coli transformation with calcium chloride and screening of blue and white clones．Three hundred positive bacterial clones were randomly picked in each library and identified by colony PCR.Results: Analysis of the white clones by PCR showed that 80％ clones contained 200-800 bp inserts in each library.Conclusion: Four subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury have been successfully constructed by SSH and T/A cloning techniques，which lays a solid foundation for screening and cloning specific differentially expressed genes associated with chronic optic nerve injury.

Abstract:Objective:To evaluate the long-term safety of living kidney donors after nephrectomy. Methods:Twenty-six living donors of kidney were followed up for 2-8 years, with a mean of (4.27±2.11) years. The serum creatinine, blood pressure, proteinuria and psychological stress were observed in the donors during follow-up.Results: The serum creatinine had a significant increase after nephrectomy（P＜0.01）,but all had normal serum creatinine levels and remained stable thereafter. There was no significant change in blood pressure or proteinuria after nephrectomy;only one case developed microalbuminuria after nephrectomy.One of the donors regretted donation and felt more mental stress after nephrectomy. All of the donors were alive without complications.Conclusion: Pre-operation assessment and selection of donors, along with standard intra-operation management and long-term post-operation follow-up, can guarantee the safety of the donors.

Abstract:Objective：To examine the feasibility of using cerebral state index (CSI) for monitoring the sedation depth during target-controlled infusion (TCI) with propofol and remifentanil．Methods: Forty-four consenting ASA Ⅰ or Ⅱ patients (aged 18-60 years) undergoing elective surgery under general anesthesia were randomly divided into 4 groups (n=11 each) according to the target effect-site concentrations of remifentanil administered by TCI during induction of anesthesia.The target effect-site concentrations of remifentanil of R0,R2,R4,and R6 groups were 0,2 ng·ml-1,4 ng·ml-1,and 6 ng·ml-1,respectively.Anesthesia was induced by TCI with remifentanil and propofol.CSI and bispectral index (BIS) were used to measure the sedation depth.The initial effect-site propofol concentration(PCe) was 1.5 μg·ml-1,which was increased by 0.5 μg·ml-1 every 4 min．The modified OAA/S score (5=alert，1=does not respond to prodding)，loss of eyelash reflex (LOR eyelash) and loss of response to electric tetanic stimulation (LOR tetanic) were compared against CSI,BIS and PCe(calculated effect-site propofol concentration).Correlation coefficients were calculated between CSI and other parameters．Results: The 4 groups were comparable with respect to the ages and bodyweights．CSI and BIS values were higher but PCe value were lower at LOR eyelash and LOR tetanic in R2,R4,and R6 than those in the R0 group ( P<0.05 or 0.01)．CSI was positively correlated with modified OAA/S score and BIS,but negatively with PCe (All P<0.01).Conclusion: CSI can be used for monitoring the depth of sedation produced by TCI with propopo1 and remifentanil.

Abstract:MicroRNA (miRNA) is a small non-coding RNA that contains 21 to 23 nucleotides and can down-regulate gene expression by translational repression.Recent studies found that some miRNAs might function both as oncogenes and tumor suppressors;its role in the tumorigenesis may complement and enrich the mechanisms of tumorigenesis.Liver cancer is a great threat to human health,whose pathogenesis is still not completely understood.MiRNAs can influence the tumorigenesis,differentiation and treatment of liver cancer.Here we summarizes the related progression in research of miRNA and liver cancer.

Abstract:Osteoblasts are the most important mechanoreceptive and osteogenic cells.The loss of bone in microgravity is mainly due to the dysfunction of osteoblasts.Research on osteoblast cytology under simulated weightlessness has made great progress in recent years. Current experiments are focusing on the changes in the cellular proliferation,differentiation,apoptosis,function and signal transduction in osteoblasts.This paper reviews the progress of the studies in this field.

Abstract:目的：观察骨质疏松大鼠胫骨骨缺损应用硫酸钙骨水泥填充后骨密度（BMD）的变化，探讨骨质疏松病理条件下应用骨水泥修复骨缺损的意义。方法：36只成年SD雌性大鼠随机分为3组：空白对照组，仅行输卵管结扎术；骨质疏松组，卵巢切除术后3个月胫骨近端形成骨质缺损；骨水泥治疗组，卵巢切除术后3个月胫骨近端形成骨质缺损后应用硫酸钙骨水泥填充。卵巢切除术后3个月测定大鼠全身BMD判断骨质疏松模型制备情况。胫骨近端骨质缺损形成后2、4、8、12周取缺损处标本进行BMD检测。结果：空白对照大鼠全身BMD（g/cm2）明显高于卵巢切除大鼠（0.399±0.035 vs 0.302±0.042，P＜0.05），证实骨质疏松模型建立成功。骨水泥治疗组大鼠硫酸钙骨水泥填充后2周和4周骨质缺损处样本BMD明显高于骨质疏松组，差异有统计学意义（P＜0.01），填充后8周和12周时BMD差异无统计学意义。结论：骨质疏松大鼠骨缺损填充硫酸钙骨水泥早期能增加骨密度，但其在体内降解较快，成骨效应受到骨质疏松条件的限制，在使用骨水泥治疗的同时，应针对骨质疏松症进行积极治疗。

Abstract:目的：对食物频度问卷的信度和效度进行考察。方法：对101名同济大学在校大学生进行两次食物频度问卷调查和1次3日膳食记录,两次食物频度问卷调查的时间间隔为3周。采用Wilcoxon符号等级检验和Spearman等级相关系数进行统计学分析。结果：两次食物频度问卷个体营养素摄入量的中位数差别小于10%；二者显著相关，相关系数在0.382和0.727之间(P<0.001)。食物频度问卷与3日膳食记录所测各营养素的中位数除胆固醇(31.32%)外差别不大；相关系数校正后为0.232~0.646,除胆固醇(P=0.064)外,其余均有统计学意义(P<0.05)。结论：食物频度问卷具有较好的信度和效度,但明显高估了胆固醇的摄入量,故需要对问卷作进一步的改进,才可以更好地评估研究对象的膳食结构。

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