AIM:To study the chemical constituents of leaves of Streblus asper Lour. METHODS:The constituents were isolated by column chromatography and their structures were elucidated through spectroscopic analysis. RESULTS:Seven compounds were isolated and identified as salicylic acid(1),?-sitosterol ( 2 ),?-daucosterol(3),oleanolic acid(4),magnolol(5),quercetin(6),taxifolin(7). CONCLUSION:The compounds are all obtained from the leaves of Streblus asper for the first time.

BACKGROUND:Large-scale expansion of undifferentiated and multipotential adipose-derived stem cells using serum-free culture system is a difficult issue to be resolved. OBJECTIVE:To establish an in vitro culture system combined with the extracellular matrix in order to investigate the efficiency, effectiveness and security of extracellular matrix on expanding adipose-derived stem cells. METHODS:In vitro isolated adipose-derived stem cells were seeded in traditional two-dimensional plastic plates and extracellular matrix-coated plates supplemented with serum-free medium respectively. After in vitro expansion, total cellnumber, expression of cellsurface markers, cellsenescence degree and multipotent differentiation ability (adipogenic, osteoblastic and chondrogenic differentiation) of adipose-derived stem cells cultured under both conditions were detected and compared. Moreover, the clinical safety of adipose-derived stem cells expanded in extracellular matrix-coated plates was investigated. RESULTS AND CONCLUSION:Total cellnumber of passage 5 adipose-derived stem cells cultured in extracellular matrix-coated plates was 10 times more than that in traditional two-dimensional plastic plates. Flow-cytometric analysis showed that adipose-derived stem cells cultured with extracellular matrix expressed stem cellsurface markers. cellular senescence examination showed that almost al of passage 15 adipose-derived stem cells cultured with extracellular matrix showed no aging, while most passage 5 adipose-derived stem cells cultured by the two-dimensional system aged and lost their proliferation ability. Multidirectional induction of adipose-derived stem cells showed that passage 15 adipose-derived stem cells cultured with extracellular matrix could stil differentiate into adipocytes, osteoblasts and chondrocytes as passage 5 adipose-derived stem cells did, which performed much better than the induced differentiations of passage 5 adipose-derived stem cells cultured by the two-dimensional system. Karyotype analysis and in vivo invasion experiment insured the clinical safety of adipose-derived stem cells expanded with extracellular matrix. Al above results suggest a safe and more efficient expansion system of extracellular matrix for clinical application using the serum-free culture system combined with extracellular matrix.

This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.

AIM:To study the mechanism of the unique export of one of human basic fibroblast growth factor (hbFGF) forms lacking the N-terminal nuclear localization signal (NLS),we high expressed and purified this hbFGF form in E.coli strain BL21(DE3).METHODS:The cDNA fragment of the hbFGF amplified by polymerase chain reaction (PCR) was cloned into the expression vector pET3c and expressed in BL21(DE3) by IPTG induction.The expressed hbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate.The mitogenic activity was measured by MTT.RESULTS:The expression level of hbFGF in E.coli was about 20% of the total cellular protein.The appreciable mitogenic activity of the purified hbFGF was comparable to that of hbFGF standard.CONCLUSION:The BL21(DE3)/ pET3c expression system could be used to high express hbFGF lacking NLS.The purified recombinant hbFGF was prepared and sufficient for further study.

AIM:To synthesize the human platelet factor-4(hPF4) gene with a convenient and effective approach, and high express the hPF4 gene in E. coli BL21 (DE3). METHODS: According to the primary structure of hPF4, the nucleotide sequence was synthesized using touch-down PCR method. The resultant gene fragment containing EcoR Ⅰ and Xho Ⅰ overhangs at 5' and 3' ends was cloned into the expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX-4T-1-hPF4,which was then transformed into the E. coli strain BL21 (DE3). RESULTS: hPF4 gene was successfully synthesized by touchdown PCR method. A fusion protein composed of glutathione S-transferase (GST) and the recombinant hPF4 was expressed in BL21(DE3) by IPTG induction. The expression level of the fusion protein in E. coli was about 30% of the total cellular protein. CONCLUSION: Touch-down PCR may provide a convenient and effective approach to obtain other target genes. The expressed fusion protein forms the inclusion bodies, providing sufficient material for further purification and biological activities process.

AIM: To observe the effects of the human acidic fibroblast growth factor mutant (mhaFGF), lacking 27 amino acids at N-terminal, on the proliferation and signal transduction of the hepatocarcinoma cells. METHODS: The hepatocarcinoma cells were treated with human acidic fibroblast growth factor (haFGF) and mhaFGF, respectively. The expression levels of the signal proteins, Grb2 and Erk1/2, in the hepatocarcinoma cells were detected by semi-quantitative Western-blotting after treated for 15 min. The mitogenic activity of both haFGF and mhaFGF was detected by MTT method and the cell cycle was analysed by flow cytometer (FCM) after treated for 48 h. RESULTS: The mitogenic activity and the ratio of G 1 and S phase cells in mhaFGF-treated cells were markedly lower than that of the haFGF, and close to that of the control group. The expression level of both Grb2 and Erk1/2 in the mhaFGF-treated cells were lower than those in the haFGF- treated cells. CONCLUSION: The decrease in the mitogenic activity of mhaFGF is probably associated to its down-regulating the expression of the signal molecular, MAPK-ERK1/2 and Grb2.

In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broadspectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.

Objective To explore the sensitivity and specificity of colposcopy directed biopsy (CDB) and the value of loop electrosurgical excision procedure (LEEP) for the diagnosis and treatment of microinvasive cervical cancer (MCC). Methods One hundred and thirty five patients with MCC were diagnosed with LEEP in Obstetrics and Gynecology Hospital, Fudan University from April 2008 to November 2010, and were retrospectively analyzed on CDB diagnoses and following treatment after LEEP. According to patient′s desire for preservation of fertility and cone margin status, following strategies after LEEP included follow-up, second LEEP, hysterectomy, modified radical hysterectomy and radical hysterectomy. Single and multiple factors related to residual lesions after LEEP were analysed with Pearson Chi-square test and logistic regression model, respectively. Results CDB diagnosed MCC with a sensitivity of 4.4%(6/135), specificity of 100.0%(4 680/4 680), and false negative rate of 95.6%(129/135). Among the 135 patients, 29 did not receive further treatment in our hospital and lost contact. One hundred and six patients had secondary treatment or follow-up in our hospital, 4 of among which were closely followed up;one hundred and two received further treatment, which included 6 cases with second LEEP (3 received extrafascial hysterectomy after repeat LEEP), 59 cases hysterectomy, 14 cases modified radical hysterectomy and 26 cases radical hysterectomy. For factors related to residual lesions after LEEP, single factor analysis showed that the ratio of residual lesion in patients aged 27-39, 40-49 and 50-65 years were respectively 19.0%(11/58), 15.4%(10/65) and 5/12 (χ2=4.505, P=0.105). Residual lesions occurred in 24.7%(23/93) of patients with positive LEEP margins, which was more than that 7.1%(3/42) of patients with negative LEEP margins (χ2=5.756, P=0.016). The ratio of residual lesions in patients with positive endocervical, ectocervical and deep stromal margins were respectively 29.6%(8/27), 17.1%(7/41)and 30.6%(11/36;χ2=2.275, P=0.321). Residual lesions in patients with or without lymph vascular space invasion (LVSI) were 2/7 and 18.8%(24/128), respectively (χ2=0.412, P=0.521). The ratio of residual lesions in patients with invasion depth of<1 mm was 17.1%(7/41), 1-<3 mm was 19.0%(16/84), and 3-5 mm was 3/10, with no significant difference among three groups (χ2=0.870, P=0.647). Logistic regression analysis showed positive cone margin (OR=5.069, P=0.014) and age (OR=1.080, P=0.024) were the independent risk factors of residual lesions after LEEP conization. Conclusions CDB alone is not adequate for the diagnosis of MCC. For young patients who desire to preserve fertility with a negative cone margin, close follow-up is acceptable. Cone margin status and age are two independent risk factors for residual lesions after LEEP.

Objective To discover the mutation of ADAR gene in a pedigree with dyschromatosis symmetrical hereditaria(DSH). Methods We investigated this family and collected blood samples of the individuals in this family. Mutation screening was carried out by PCR and direct sequencing. The allele specific primer was designed for the mutation point, and allele-specific PCR was carried out on the patients, normal family members and 40 normal individuals. Results A single nucleotide deletion (c.1642 delC) was identified in exon3 of ADAR gene in the patients of this family. This mutation was not detected in the normal family members and in any of the control individuals. Conclusion This single nucleotide deletion was responsible for the disease in the family.

AIM:The present study was designed to establish H2O2-induced NRK52E cell apoptotic model and to investigate the effects and the mechanism of nmhaFGF on the NRK52E cell apoptosis induced by H2O2. METHODS: In vitro experiment, a apoptotic model of normal rat kidney epithelium (NRK52E) was made by MTT method, Hoechst33342 dyeing and flow cytosorting (FCR). NRK52E cells were cultured with different concentrations of nmhaFGF and haFGF for 24 h before H2O2 was added. The apoptotic rate was detected with FCR method. RESULTS: H2O2 at concentration of 0.4 mmol/L, the optimal condition to establish the apoptotic model, was used to treat NRK52E cells for 18h. Different doses of nmhaFGF (0.01, 0.03, 0.10, 0.30, 1.00 mg/L) reduced the apoptotic rates with the dose rising. However, the decreasing tends of apoptotic rates with dose increasing for haFGF was not so obvious. CONCLUSION: nmhaFGF protects the NRK52E cells against apoptosis. The mechanism might be connected with its non-mitogenic property.