Objective To establish the human telomerase reverse transcriptase (hTERT) gene viral transferring system. Methods By means of liposome mediation, a retroviral vector pLNC-hTERT carrying a selectable marker neomycin resistance genes (Neo r) and a target gene (hTERT) was transferred into the ectropic package cells ?-2, and by using the supernatant of ?-2 cells to infect the amphotropic producer clone PA317, established PA317/hTERT package cell line. In order to get higher-titer virus, the supernatants of ?-2 infected PA317 repeatedly. Human endothelial cells were used to detect the effect of gene transfer. Results We established a PA317/hTERT package cell line which produced high-titer virus. The viral titer produced by PA317 cells was raised 20 times by repeated infection method, and with higher-titer virus the exogenous genes were successfully transferred into endothelial cell. Conclusion This method can be used to establish higher-titer viral transferring system.

Objective To construct a full-length cDNA library of human normal kidney tissues and identify the quality of the library. Methods By using the template-switching mechanism at 5′end of mRNA technique to construct the library, a powerscript reverse transcriptase was used to transcribe, and a 5′-oligo fragment as an extended template was added to 5′ end of mRNA to enrich full-length cDNAs. After amplification, the ds cDNAs digested by sfi I and size-fractionated by columns were recombined into ?TripIEx 2 vectors. After package, the recombinant vectors were titered and the recombinant rate (blue/white) was determined,then the library was amplified. We identified the library using PCR reaction to determine the size of the inserts. Results The titer of cDNA library was 2.6?10 6pfu?mL -1, the rate of recombinant was above 95%, and the titer of amplified library was 9?10 11pfu?mL -1. The insert size ranged from 0.7 to 2 kb. Conclusion The cDNA library of human normal kidne we constructed is a highly efficient one and can be used for screening by probe and antibody to find the genes related to kidney diseases.

Objective To examine the differently expressed in vasion-related genes in two anchorage-independent uterine cervical carcinoma c ell lines derived from the same patient using a cDNA array. Methods Two human uterine cervical carcinoma subclonal cell lines CS03 and CS07 derived from a single donor line CS1213 were established by limited diluting procedure and their cloning efficiency was counted in soft aga r. The expression of cellular adhesive molecules CD44, E-cadherin and Fibroneti n were studied by flow cytometry and immunohistochemical staining. The two cDNA samples retro-transcribed from total RNA derived from CS03 and CS07 cells were screened by a cDNA microarray carrying 1011 human signal transduction and membra ne receptor-associated genes and scanned with a ScanArray 3000 laser scanner. Results The cloning efficiency was 38.5% in CS07 cells and 0 in CS03 cells, respectively. CD44 were 35.1% and 13.7% in CS03 and CS07,respe ctively. The cDNA microarray analysis showed that 12 genes in CS03 were upregula ted compared with CS07, and 21 genes in CS07 were upregulated. The function of a number of differently expressed genes is consistently associated with cell prol iferation, migration, apoptosis, signal transduction and tumor metastasis, inclu ding p34 cdc2, TSC22, plasminogen activator inhibitor I (PAI-1)and desmoso me associated protein (Pinin). Conclusion Multiple genes are differently expressed in uter ine cervical carcinoma cell lines even in the same patient. It is suggested that these genes are involved with the phenotypic characteristics and development of cervical carcinoma.

Objective To establish a human cervical carcinoma cell line. Methods A primary culture was initiated from malignant tissue collected by dissection of cervical biopsy specimens.Characterizing cells in culture which included morphological observation,biological and karyotypic analysis,experimental tumorigenesis and the expression of p53,bcl-2 and Ki67 genes was carried out. Results The new established cervical carcinoma cell line (CS1213) had been maintained in culture for over 170 generations.The cells which were nonadherent had a common,rounded appearance with a cell cycle time of 25-hour and a 19 colony formation rate in soft agar.Electron micrographs demonstrated abundant tonofilaments in the cytoplasm.The karyotype showed a hyperdiploid feature with a main chromosome stem number ranged from 80 to 88.The culture was not contaminated by mycoplasma and had a distinct lactic acid dehydrogenase isozyme pattern.High expression level of p53(31.9%),bcl-2(89.3%) and Ki67(33.7%) proteins was detected by flow cytometry.The xenogeneic tumors were grown in nude mice with the histological structure of the original one. Conclusions The novel CS1213 cells have the characteristics of human cervical squamous cells and could be used as an appropriate cellular model system for studying tumor invasion and metastasis.

AIM　To study the effects of quercetin (Que) on cell cycle and nitric oxide in H2O2-induced the human umbilical vein endothelial cell line(ECV-304). METHODS　The experiment were performed in culture of H2O2-induced human umbilical vein endothelial cell line(ECV-304) in vitro. Cell viability was assessed by MTT assay and cell cycle was observed by flow cytometry. Nitroxide(NO) of ECV-304 was monitored as NO2- with colorimetry. RESULTS　H2O2 inhabited the ECV-304 prolifteration. Preincubation of ECV-304 with Que for 24 h before H2O2 exposure significantly increased the cell viability and S-phase and G2M-phase cells. Que reduced lactate dehydrogenase(LDH) and increased the level of nitric oxide in H2O2-induced ECV-304. CONCLUSION　These results demonstrate that Que can produce the protective action on H2O2-induced cultured ECV-304 and its effect of action may be related to level of nitric oxide.

ABSTRACT:Objective To observe whether N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP)can inhibit rat silicotic fibrosis by regulating stimulatory G proteinα(Gαs)/inhibitory G proteinα(Gαi)signal.Methods Male Wistar rats were randomly divided into three groups (n=1 0 ):control 1 6-w group,silicosis 1 6-w group,and Ac-SDKP pre-treatment group.The pathological changes of the lung tissue was observed by HE staining;the expressions ofα-smooth muscle actin (α-SMA),Collagen Ⅰ,fibronectin (Fn),Gαs,Gαi2 ,Gαi3 and cAMP were detected by Western blot.Immunofluorescence was performed on lung tissue sections to detect the coexpression ofα-SMA/Gαi3 . Results Within silicosis 16-w group,HE staining showed that the silicotic nodule volume increased,nodule fusion and the formation of interstitial fibrosis could be seen,and cell fibrous nodules were visible.Immunofluorescence staining showed the enhanced coexpression ofα-SMA/Gαi3 in fibrosis area.Compared with those in control group, the expressions ofα-SMA,Collagen Ⅰ,Fn,Gαi2 and Gαi3 significantly increased in silicosis 1 6-w group,but the expressions of Gαs and cAMP decreased.Compared with silicosis 1 6-w group,Ac-SDKP pre-treatment group had alleviated lung injury and decreased coexpression ofα-SMA/Gαi3 .The expressions ofα-SMA,Collagen Ⅰ,Fn,Gαi2 and Gαi3 protein significantly decreased in Ac-SDKP pre-treatment group,while the expressions of Gαs and cAMP increased obviously.Conclusion Ac-SDKP can regulate the expressions of Gαs and Gαi and promote the formation of cAMP,thus playing an effective role against silicotic fibrosis.

Objective To investigate the effect of tripterygium glycosides (TG) contained serurn on the pathological boneforming related inflammatory markers and miR-21.Methods Previous isolated and cultured AS fibroblasts were stimulated using IL-1 of 1ng/ml for 24h,different concentrations of blank serum (5％,10％,and 15％) and TG contained serum (5％,10％,and 15％) were added for 48h.PGE-2,IL-17,IL-22,IL-23,CCL19 and CCL21 proteins were examined by Western blot.The osteogenesis marker BMP and microRNA-21 mRNAs were tested.Results 48 h after intervention,the expressions of inflammatory markers were obviously inhibited by TG contained serum;the boncforming related inflammatory markers,expressions of BMP-2 and miR-2t were all inhibited in a dose-dependent manner.Conclusions TG could inhibit the expressions of boneforming related inflammatory markers,BMP-2 and miR-21,thus providing theoretical basis to treat AS pathological boneforming.

Objective To explore the correlationship between level of bFGF (basic fibroblast growth factor) in serum and synovia and X-ray severity (Kellgren-Lawrence Grading System) in patients with knee OA. Method 68 patients with knee OA were enrolled into this study. Knee OA grading was evaluated according to the Kellgren-Lawrence classification. bFGF levels in both serum and synovia were examined using enzyme-linked immunosorbent assay. Results The level of bFGF in serum and synovial fluid in patients with knee OA were positively correlated with radiographic severity (r = 0.619, P < 0.001 and r = 0.603, P < 0.001, respectively). Further analysis revealed that there was a positive correlation between the level of bFGF in serum and in synovia fluid (r = 0.688, P < 0.001). Conclusions Levels of bFGF in serum and synovial fluid were significantly increased in patients with OA, and levels of bFGF were positively correlated with radiographic severity. These findings indicate that bFGF levels may be a biomarker of disease severity and could play an crucial part in the pathophysiology of degenerative process in OA.

AIM To study the effects of quercetin (Que) on cell cycle and nitric oxide in H2O2-induced the human umbilical vein endothelial cell line(ECV- 304). METHODS The experiment were performed in culture of H2O2-induced human umbilical vein endothelial cell line(ECV-304) in vitro. Cell viability was assessed by MTT assay and cell cycle was observed by flow cytometry. Nitroxide(NO) of ECV304 was monitored as No2- with colorimetry. RESULTS H2O2 inhabited the ECV-304 prolifteration. Preincubation of ECV-304 with one for 24 h before H2O2 exposure significantly increased the Cell viability and S-phase and G2M-phase cells. one reduced lactate dehydrogenase(LDH) and increased the level of nitric oxide in H2O2-induced ECV-304. CONCLUSION These results demonstrate that one can produce the protective action on H2O2-induced cultured ECV-304 and its effect of action may be related to level of nitric oxide.

Objective To screen and identify differentially expressed genes in two new human urothelial carcinoma cell lines, BLS-211 and BLX. Methods Suppression subtractive hybridization (SSH) was used to createa subtracted library, and clones were sequenced. Results Totally 13 over-expressed genes in BLX and 9 in BLS-211 cells were obtained, respectively. Among them, 18 were known genes and 4 were new ESTs (Expressed Sequence Tag), and were collected by GenBank dbEST database (The access number was EB390424-7). Conclusion SSH is a powerful method for the identification of differentially expressed genes. The differential expression of some BCG-associated genes in different cells may be related to the different responses to clinical BCG therapy. The identified new ESTs can be cloned for full length to further study their functions.