The effect of heat stress on proliferation, synthesis of steroids, and gene expression of duck granulosa cells.

Granulosa cells (GCs) play an important role in the development of follicles. In this study, we investigate the impact of heat stress at 41°C and 43°C on duck GCs' proliferation and steroids secretion. And, the transcriptomic responses to heat treatment were examined using RNA-sequencing analysis. Digital gene expression profiling was used to screen and identify differentially expressed genes (fold change ≥ 2 and Q value < 0.05). Further, the differential expression genes (DEGs) were classified into GO categories and KEGG pathways. The results show that duck GCs blocked in the G1 phase were increased on exposure to heat stress. Meanwhile, the expression of proliferative genes, which were essential for the transition from G1 to S phase, was inhibited. At the same time, heat stress inhibited the estradiol synthesis of GCs by decreasing CYP11A1 and CYP19A1 gene expression. A total of 241 DEGs including 181 upregulated and 60 downregulated ones were identified. Transcriptome result shows that heat shock protein and CXC chemokines gene were significantly activated during heat stress. While collagenases (MMP1 and MMP13) and strome lysins (MMP3) were downregulated. And, the hedgehog signaling pathway may be a prosurvival adaptive response under heat stress. These results offer a basis for better understanding the molecular mechanism underlying lay-eggs-less in ducks under heat stress.

Reference gene selection for expression studies in the reproductive axis tissues of Magang geese at different reproductive stages under light treatment.

In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels. In order to screen the reference genes suitable for detecting gene expression in tissues of the reproductive axis, a total of 420 (males and females = 1:5) 3-year-old Magang geese were selected and subjected to light treatment. The hypothalamus, pituitary and testicular tissues were subsequently collected at different stages. Ten genes including HPRT1, GAPDH, ACTB, LDHA, SDHA, B2M, TUBB4, TFRC, RPS2 and RPL4 were selected as candidate reference genes. The expression of these genes in goose reproductive axis tissues was detected by real-time fluorescent quantitative PCR. The ΔCT, geNorm, NormFinder and BestKeeper algorithms were applied to sort gene expression according to stability. The results showed that ACTB and TUBB4 were the most suitable reference genes for the hypothalamic tissue of Magang goose in the three breeding stages; HPRT1 and RPL4 for pituitary tissue; and HPRT1 and LDHA for testicular tissue. For all three reproductive axis tissues, ACTB was the most suitable reference gene, whereas the least stable reference gene was GAPDH. Altogether, these results can provide references for tissue expression studies in geese under light treatment.

[Study of production of sesquiterpenes of Aquilaria senensis stimulated by Lasiodiplodia theobromae].

To investigate the mechanism of agarwood formation in Aquilaria sinensis induced by Lasiodiplodia theobromae, the fermentation liquor of L. theobromae was analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry (GC-MS). JAs were detected in the fermentation liquor. The effect of the fermentation liquor on the abundance of sesquiterpenes in the callus of A. sinensis was analyzed by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). And the fermentation liquor stimulated alpha-guaiene, alpha-humulene and delta-guaiene biosynthesis in calli. It was inferred that L. theobromae produced JAs, which resulted in a significant increase of sesquiterpenes in A. sinensis.

Simultaneous recovery of dual pathways for ammonia metabolism do not improve further detoxification of ammonia in HepG2 cells.

BACKGROUND: Key enzyme deficiency in the dual-pathway of ammonia metabolism leads to low detoxification capacity of HepG2 cells. Previously, we established a HepG2/AFhGS cell line with overexpression of human glutamine synthetase (hGS) in pathway 1 and a HepG2/(hArgI+hOTC)4 cell line with overexpression of human arginase I (hArgI) and human ornithine transcarbamylase (hOTC) in pathway 2. The present study aimed to investigate whether simultaneous recovery of the two pathways contributes to the further improvement of ammonia detoxification in HepG2 cells. METHODS: We adopted a recombinant retrovirus carrying the hGS gene to infect HepG2/(hArgI+hOTC)4 cells and selected a new recombinant HepG2 cell line. The capacities of ammonia tolerance and detoxification in cells were detected by biochemical methods. Cell cycle PCR chip was used to assess the changes of gene expression. RESULTS: Introducing hGS into HepG2/(hArgI+hOTC)4 cells did not lead to hGS overexpression, but inhibited hArgI expression. The levels of synthetic glutamine and urea in HepG2/(hArgI+hOTC+AFhGS)1 cells were significantly lower than those in HepG2/(hArgI+hOTC)4 cells when cultured in the medium with 10 and 15 mmol/L glutamate (Glu) and with 60 and 180 mmol/L NH4Cl, respectively. In addition, the comparison of different cell growth showed that HepG2/AFhGS cells significantly lagged behind the other cells by the 5th and 7th day, indicating that introduction of hGS impedes HepG2 cell proliferation. Analysis of the mechanism suggested that the decreased expression of BCL2 played an important role. CONCLUSIONS: This study demonstrated that the recovery of two ammonia metabolic pathways in HepG2 cells is not helpful in increasing ammonia metabolism. The reinforcement of the pathway of urea metabolism is more important and valuable in improving the ammonia metabolism capacity in HepG2 cells.

Therapeutic evaluation of a microbioartificial liver with recombinant HepG2 cells for rats with hepatic failure.

BACKGROUND: HepG2/(ArgI+OTC)4 (previously constructed) is a recombinant human liver cell line with a strong ability to reduce ammonia in vitro. However, its application value ex vivo has not been investigated. OBJECTIVES: To examine the efficacy of HepG2/(ArgI+OTC)4 cells in a micro-bioartificial liver (micro-BAL) device for application ex vivo. METHODS: A simple micro-BAL device containing a microbioreactor and a small-type peristaltic pump was installed. The rats with hepatic failure were randomly divided into three groups (n = 10) and were treated with different micro-BAL loaded HepG2/(ArgI+OTC)4 cells, HepG2 cells and control (without cells), respectively. Changes in the liver and kidney function of the rats were determined before and after the treatment. The lifespan of the rats were observed and recorded. RESULTS: Despite the difference in survival time between experimental groups of rat model was not statistically significant, the capacity of HepG2/(ArgI+OTC)4 cells treatment group for tolerance and detoxifying ammonia was increased much more than that of HepG2 cells (p < 0.05), and other biochemical indicators of HepG2/(ArgI+OTC)4 cells treatment group were also better than that of HepG2 cells treatment group (p < 0.05). CONCLUSIONS: HepG2/(ArgI+OTC)4 cells can provide a better biological support for rats with hepatic failure in a short period of time, and they may be used as a convenient and useful choice for further cell material research of BAL.

N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells.

BACKGROUND: Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved. METHODS: The MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively. RESULTS: Both of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvß3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, ß3 and ß1 expression as well as the reduction of MMP-9 activity. CONCLUSIONS: Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.

[Removing nitrate-nitrogen from wastewater using rotten wood as carbon source].

In this research, COD release of rotten wood was studied and rotten wood was investigated as the sole carbon source as well as biofilm carrier to remove nitrate from wastewater in up-flow laboratory reactor. The experimental results indicated that rotten wood could release carbon source continuously. COD released of rotten wood inoculated with humus was 2. 3 times higher than that of sterilized rotten wood, and VFA was 5 times. The research of denitrification was carried out at 25 degrees +/- 1 degrees C, 30 mg/L of initial NO3(-) -N concentration and 12 h of hydraulic retention time. Nitrate removal efficiency was above 80%. A time-dependent decrease in nitrate removal efficiency was observed after 46 days of operation. The results showed that rotten wood could be used as an effective carbon source for denitrification.

[Expressions of VEGF-C and VEGF-D and their correlation with lymphangiogenesis and angiogenesis in gallbladder carcinoma].

OBJECTIVE: To investigate the expression of VEGF-C and VEGF-D and their correlations with lymphangiogenesis and angiogenesis in gallbladder carcinoma. METHODS: Fifty cases of gallbladder carcinoma with complete clinical and pathological data were analyzed. The expression of VEGF-C and -D, D2-40, CD31 was assayed by immunohistochemical staining, with 10 samples of normal gallbladder tissues away from cancer and 19 samples of chronic cholecystitis as controls, and their correlation with clinicopathological findings were analyzed retrospectively. RESULTS: Thirty-two (64.0%) of the 50 gallbladder cancers were positive for VEGF-C protein expression by immunohistochemistry and the positive rate of VEGF-D protein expression was 62.0% (31/50). The protein expression of VEGF-C and VEGF-D in tumor tissues was significantly higher than that in normal gallbladder tissues away from the tumor (P < 0.05), but no correlation with that in chronic cholecystitis (P < 0.05). The VEGF-C expression correlated with the patient age and lymph node metastasis (both P < 0.05). The VEGF-D expression only correlated with lymph node metastasis (P < 0.05). In the 50 gallbladder cancers, the MLVD was 6.9 + or - 3.6 and the MVD was 36.1 + or - 12.8. The MLVD in both VEGF-C and -D positive groups was significantly higher than that in the negative groups (P = 0.000), and the lymph node metastasis also increased. MVD in both VEGF-C and -D positive groups was higher than that in the negative groups (P < 0.05), and it was also correlated with tumor differentiation (P < 0.05). A significant positive correlation was also found between VEGF-C and VEGF-D expression (r = 0.498, P < 0.01). CONCLUSION: VEGF-C and VEGF-D are involved in the lymphangiogenesis and angiogenesis in gallbladder carcinoma, promote lymph node metastasis of the tumor, and both are important in the regulation of lymphangiogenesis and angiogenesis in this cancer. VEGF-C and VEGF-D are of clinical significance in evaluating lymph node metastatic potency and estimation of prognosis in gallbladder carcinoma.

Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase.

BACKGROUND: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxification. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS: hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and amplified. Then hGS mRNA was measured by semi-quantitative RT-PCR; hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH4+ were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS: The expression of hGS mRNA in HepG2/pLNChGS cells (8.306+/-0.336) and HepG2/pLNAFhGS cells (21.358+/-1.716) was much stronger than in control cells (P<0.05), and that in HepG2/pLNAFhGS cells was markedly stronger than in HepG2/pLNChGS cells (P<0.05). The hGS enzymatic activities of HepG2/pLNChGS cells (3.279+/-0.328 U/mg prot) and HepG2/pLNAFhGS cells (4.557+/-0.253 U/mg prot) were higher than those of control cells (P<0.05), and those of HepG2/pLNAFhGS cells were also higher than the activities of HepG2/pLNChGS cells (P<0.05). In addition, the effect of hGS introduction on HepG2 cell proliferation was not significant. The amount of glutamine synthesis in HepG2/pLNChGS or HepG2/pLNAFhGS cells in three different concentrations of NH4+ was higher than in the two control cells (P<0.05). The amount of glutamine synthesis and cell proliferation in the higher concentrations of NH4+ (5 or 10 mmol/L) in HepG2/pLNAFhGS cells increased more than those in HepG2/pLNChGS cells (P<0.05). NH4+ at a high concentration (10 mmol/L) was toxic to HepG2 and HepG2/pLNCX cells, but less toxic to HepG2/pLNChGS and HepG2/pLNAFhGS cells. CONCLUSION: The constructed hepatocytes (HepG2 cells) with specific high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.

[Effect of different extractants on leaching characteristics of the fly ash from municipal solid waste incinerator].

The effect of three extractants, which are HNO3/NaOH, CH3COOH/NaOH, and HNO3-H2SO4 , on leaching characteristics of the fly ash from municipal solid waste incinerator (MSWI) was investigated. The results showed that: (1) different extractants had different buffering capacities for the leaching solutions of the fly ash from MSWI, which are in the order of HAC > HNO3-H2SO4 > HNO3. (2) HAC showed better dissolvability to Zn, Cd, and Cr than HNO3-H2SO4, under more acidic condition, but the leaching concentration of Pb was not affected by extractant types obviously. (3) The leaching concentrations of Pb and Zn reached their maximal values at the ratio of liquid to solid of 40, while Cd leaching concentration did at the ratio of 20, and the leaching concentrations of Zn, Pb, Cd, and Cr decreased gradually with the increase of the ratios of liquid/solid when HNO3 and HNO3-H2SO4 extractants were used.

[Transfection of GFP mRNA in dendritic cells and analysis of some factors involved].

AIM: To investigate the transfection of green fluorescent protein (GFP) mRNA in dendritic cells (DC) and analyze some factors which influence the transfection efficiency. METHODS: GFP (as a report gene) mRNA with cap was synthesized, in vitro, with mMESSAGE RNA Transcription Kit containing T7 RNA polymerase, and then the poly(A) was added to the GFP caped-mRNA by yeast poly(A) polymerase. DC were generated from the monocytes isolated from human peripheral blood by stimulation of GM-CSF and IL-4. The GFP mRNA was transfected into DC mediated by transfection reagent. The transfection efficiency and the expression levels were measured by flow cytometry. RESULTS: GFP expression in DC has been obtained by transfecting its mRNA synthesized in vitro. The transfection reagents, mRNA concentrations and cell densities have the significant effects on the transfection. The high level of transfection efficiency (up to 27%) was obtained using Transmessenger Transfection Kit with 1 microg gfp mRNA in 200 microL X-VIVO-15 serum-free medium at the cell density of 2.5x10(9)/L. CONCLUSION: The high-level transfection efficiency of gfp gene in DC could been achieved by using GFP mRNA in the optimum transfection conditions.

[Study on spatial site selection assessment of urban medical waste treatment facility].

Based on the principle of regional differentiation and gravity model, this paper proposes a GIS-based urban medical waste treatment facility spatial site selection assessment method. And the method is implemented by use of the third generation GIS database model, Geodatabase. Taking a city in Pearl River Delta as a case, based on its 46 basic units, two scenarios for medical waste central treatment are designed and analyzed by means of scenario analysis (SA) and then the better scenario is recommended. The assessment result of traditional cost model shows the same conclusion. Further spatial analysis shows that the distribution of medical waste quantity and density are both related to the better scenario spatially.

High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells.

AIM: To develop a highly efficacious method for preparation of soluble SARS S-protein using adenovirus vector to meet the requirement for S-protein investigation. METHODS: The human adenovirus vector was used to express the soluble S-protein (corresponding to 1 approximately 1190 amino acids) fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells. RESULTS: Under the conditions of infection dose (MOI of 50) and expression time (48 h), the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 microg/10(6) cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells,demonstrating that the soluble S-protein could remain the biologic activity in the native molecule. CONCLUSION: The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.

[Influence of additive on characteristic of slag during the process of melting fly ash from municipal solid waste incinerator].

A kind of compound additive was studied and prepared in the paper, which associated with many functions. Using typical fly ash of Shanghai and Fuzhou, We studied the effect of the additive on evaporation rate, flowing temperature, fixed rate of heavy metals and its leaching characteristics in melting slag in the process of melting fly ash. The result showed that the flowing temperature and the evaporation rate of fly ash could be respectively reduced approximately 150 degrees C and 10% - 20% by adding 10% additive, and the fixed rate of heavy metals Cu and Pb increased 10% - 20%, especially Zn, which could increase 40%. The amount of leaching heavy metals in melting slag were both under corresponding standard limits using either national toxicity leaching methods or TCLP of EPA.

[Characteristics of melting and solidification process of fly ash from refuse incinerator].

This study was conducted to investigate the migration patterns of main compositions of the fly ash from refuse incinerator during melting and solidification process. The experiment was performed in a high temperature melting furnace with temperature controlled. X-ray fluorescence spectroscopy (XRF) and X-ray diffractometer (XRD) were used to analyzed the fly ash treated. The parameters investigated included main compositional contents, phase constituents, alkalinity, vaporation rate, volume reduction rate. The results show: (1) During the melting and solidification process, the contents of CaO, Al2O3, and SiO2 in fly ash increased as the temperature went up, but element Cl and S decreased from initial 20.59%, 10.74% to final 0.15%, 0.22%, respectively. This suggests that higher amount of Cl and S in original fly ash could lead to more vaporation in the form of chloride and calcium salfate in the process, which was further verified by XRD analyzing result. (2) The alkalinity decreased as melting temperature increased, but tended to be stable and maintained around 0.95 after temperature reached flowing temperature. (3) The decomposition and vaporation of the salts in the fly ash mainly occurred in the temperatures between 1150 degrees C and 1260 degrees C, which was approximately 100 degrees C lower than melting temperature.

Transfection and expression of hepatitis B virus x gene and its effect on apoptosis in HL-7702 cells.

AIM: To investigate the effects of hepatitis B virus x gene and its protein product HBxAg on apoptosis in hepatocyte line HL-7702. METHODS: The reconstituted plasmid pcDNA3-x was established through recombination DNA technique; pcDNA3-X was transfected into HL-7702 cells by lipid-mediated trasfection. Positive clones were screened by G418, and HL-7702/HBx cells were analysed by the RT-PCR to confirm the steady expression of X gene in HL-7702 cells. The apoptosis rate in HL-7702 cells was determined by flow cytometry, TUNEL technology, electronic microscope. At the mean time, pcDNA3-X was transfected transiently into HL-7702 cells, and total RNA from HL-7702 cells was extracted 24, 48, 72, 96 and 120 h after the transient transfection, and semi-quantitative analysis was performed by RT-PCR to detect the expression of HBV X gene. Furthermore, apoptosis rate in HL-7702 cells was determined by flow cytometry analysis at the different times. RESULTS: RT-PCR analysis showed that HBV X gene could be expressed stably in HL-7702 cells. Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/HBx cells were much higher than those of HL-7702/pcDNA3 and HL-7702 cells. Furthermore, the apoptotic phenomena and apoptotic body were observed in HL-7702/HBx cells under electronic microscope, but not in HL-7702/pcDNA3 and HL-7702 cells. In the experiment of transient transfection, RT-PCR reveals that X gene was expressed most at 72 h after transfection; and the apoptosis rate reached the highest at the same time. After that, the apoptosis rate was reduced with the decrease of the X gene expression. CONCLUSION: HBV X gene and X protein can promote the apoptosis in hepatocyte. And there exist a quantity-effect relationship between the X gene expression and apoptosis rate in hepatocyte.

Cooperative inhibitory effects of antisense oligonucleotide of cell adhesion molecules and cimetidine on cancer cell adhesion.

AIM: To explore the cooperative effects of antisense oligonucleotide (ASON) of cell adhesion molecules and cimetidine on the expression of E-selectin and ICAM-1 in endothelial cells and their adhesion to tumor cells. METHODS: After treatment of endothelial cells with ASON and/or cimetidine and induction with TNF-alpha, the protein and mRNA changes of E-selectin and ICAM-1 in endothelial cells were examined by flow cytometry and RT-PCR, respectively. The adhesion rates of endothelial cells to tumor cells were measured by cell adhesion experiment. RESULTS: In comparison with TNF-alpha inducing group, lipo-ASON and lipo-ASON/cimetidine could significantly decrease the protein and mRNA levels of E-selectin and ICAM-1 in endothelial cells, and lipo-ASON/cimetidine had most significant inhibitory effect on E-selectin expression (from 36.37+/-1.56% to 14.23+/-1.07%, P<0.001). Meanwhile, cimetidine alone could inhibit the expression of E-selectin(36.37+/-1.56% vs 27.2+/-1.31%, P<0.001), but not ICAM-1 (69.34+/-2.50% vs 68.07+/-2.10%,P>0.05) and the two kinds of mRNA, either. Compared with TNF-alpha inducing group, the rate of adhesion was markedly decreased in lipo-E-selectin ASON and lipo-E-selectin ASON/cimetidine treated groups(P<0.05), and lipo-E-selectin ASON/cimetidine worked better than lipo-E-selectin ASON alone except for HepG2/ECV304 group (P<0.05). However, the decrease of adhesion was not significant in lipo-ICAM-1 ASON and lipo-ICAM-1 ASON/cimetidine treated groups except for HepG2/ECV304 group (P>0.05). CONCLUSION: These data demonstrate that ASON in combination with cimetidine in vitro can significantly reduce the adhesion between endothelial cells and hepatic or colorectal cancer cells, which is stronger than ASON or cimetidine alone. This study provides some useful proofs for gene therapy of antiadhesion.

Construction of IL-2 gene-modified human hepatocyte and its cultivation with microcarrier.

AIM: To construct interleukin-2 gene-modified human hepatocyte line (L-02/IL-2) and investigate the changes of the function of liver cells and IL-2 secretion in culture with microcarrier,laying the foundation for further experimentation on hepatocyte transplantation. METHODS: hIL-2 gene was transduced into L-02 hepatocytes by recombinant retroviral vector pLNCIL-2, and the changes of morphology and clonogenicity rate of the transduced cells were observed, the secretion levels of hIL-2 in cultural supernatant were detected by ELISA and NeoR gene was amplified by PCR. The growth of L-02/IL-2, the special biochemistry items and the levels of IL-2 were detected after cultivation with microcarrier. RESULTS: The clonogenicity rate of the L-02/IL-2 cells was lower than that of L-02/Neo cells and L-02 cells. The levels of hIL-2 could reach 32,000 pg/10(6) cells per day and kept secreting for more than ten weeks. NeoR gene segment was respectively obtained by PCR from both L-02/IL-2 and L-02/Neo cell's genomic DNA. At the 6(th) day in culture with microcarrier, the matrix-induced liver cell aggregates were formed, the number of alive L-02/IL-2 cell were 16.8+/-0.53 x 10(6)/flask and the levels of ALB and UREA were 52.54+/-1.28 mg/L and 5.29+/-0.17 mmol/L, respectively. These data had not significantly changed as compared with those of L-02 cells (P>0.05); However, the levels of IL-2 in IL-2/L-02 cells remarkably exceeded that in L-02 cells in the whole culture process (P<0.001). CONCLUSION: The IL-2 gene-modified hepatocyte line has been successfully constructed. The L-02/IL-2 cellular aggregates cultured with microcarrier have a high capacity of IL-2 production as well as protein synthesis and amino acid metabolism.

[Expression of adenovirus-mediated pGH cDNA with first intron in CHO cells].

The recombinant adenoviruses containing pGH cDNA and pGH cDNA with the first intron under the control of CMV promoter were constructed respectively by homogenous recombination method. The results showed that the recombinant adenoviruses could mediate pGH cDNA expression in CHO cells infected with the recombinant adenoviruses. The expression level of pGH cDNA with the first intron increased by 117% compared with pGH cDNA without intron. This indicate that the first intron of pGH gene have the function of improving the expression of the pGH gene.