Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling.

The motility of mesenchymal stem cells (MSCs) is highly related to their homing in vivo, a critical issue in regenerative medicine. Our previous study indicated copper (Cu) might promote the recruitment of endogenous MSCs in canine esophagus defect model. In this study, we investigated the effect of Cu on the motility of bone marrow mesenchymal stem cells (BMSCs) and the underlying mechanism in vitro. Cu supplementation could enhance the motility of BMSCs, and upregulate the expression of hypoxia-inducible factor 1α (Hif1α) at the protein level, and upregulate the expression of rho family GTPase 3 (Rnd3) at messenger RNA and protein level. When Hif1α was silenced by small interfering RNA (siRNA), Cu-induced Rnd3 upregulation was blocked. When Rnd3 was silenced by siRNA, the motility of BMSCs was decreased with or without Cu supplementation, and Cu-induced cytoskeleton remodeling was neutralized. Furthermore, overexpression of Rnd3 also increased the motility of BMSCs and induced cytoskeleton remodeling. Overall, our results demonstrated that Cu enhanced BMSCs migration through, at least in part, cytoskeleton remodeling via Hif1α-dependent upregulation of Rnd3. This study provided an insight into the mechanism of the effect of Cu on the motility of BMSCs, and a theoretical foundation of applying Cu to improve the recruitment of BMSCs in tissue engineering and cytotherapy.

[Comparison Between Different Mitochondrial Staining Methods in Cell and Tissue Samples].

OBJECTIVE: To compare the effects of mitochondria staining between specific mitochondrial fluorescent probes and anti-mitochondrial protein antibody in cell and tissue samples. METHODS: The HepG2 cells fixed by 4% paraformaldehyde were stained with MitoTracker Deep Red (100 nmol/L) or anti-Grp75 antibody (75 nmol/L or 100 nmol/L). The human healthy liver tissue samples fixed by 4% paraformaldehyde were stained with 150 nmol/L MitoTracker Deep Red or anti-Grp75 antibody. The above stained cell and tissue samples were observed using confocal microscopy. RESULTS: We found non-specific staining in HeLa cells and obscure mitochondrial image using MitoTracker Deep Red probes, while clear tubular and punctate distribution using anti-Grp75 antibody. In contrast, we observed more specific and better effects of MitoTracker Deep Red probes-stained liver tissue samples as compared to the antibody. CONCLUSION: To visualize mitochondria, the anti-Grp75 antibody staining worked better on cells and the MitoTracker Deep Red probes are more suitable for tissue samples.

[Expression and Significance of HIF-3α in Mitochondria].

OBJECTIVE: To investigate the mitochondrial translocation of hypoxia inducible factor-3α (HIF-3α) under normoxia and hypoxia and its physiological and pathological meanings. METHODS: ① After hypoxic (1%O2) or DMOG, CoCl2 treatments mimicking the hypoxic treatment, Western blot and immunofluorescence were used to examine the HIF-3α expression in mitochondria of HeLa and ACHN cells, respectively. ②The protease sensitivity experiment was used to explore the sub-organelle localization of HIF-3α in mitochondria. ③Western blot was used to examine mitochondrial HIF-3α in the normal mouse tissues and human liver carcinoma tissues. RESULTS: ① In HeLa and ACHN cells, HIF-3α translocated to mitochondria under normoxia and hypoxia, and its mitochondrial expression was higher under hypoxia; ②The protease sensitivity of HIF-3α was similar to proteins locating in the mitochondrial outer membrane; ③Mitochondrial HIF-3α expressed in multiple normal mouse tissues; The expression of mitochondrial HIF-3α was higher in human liver carcinoma tissues than the normal and adjacent tissues. CONCLUSIONS: HIF-3α translocated to mitochondrial outer membrane under both normoxia and hypoxia, and hypoxia could up-regulated HIF-3α mitochondrial translocation. Meanwhile, the phenomenon may be involved in the process of liver carcinoma.

One new linear C14 polyacetylene glucoside with antiadipogenic activities on 3T3-L1 cells from the capitula of Coreopsis tinctoria.

Based on characteristic UV spectrum of the ene-diyne chromophore, one new polyacetylene glucoside and three known polyacetylene glucosides have been isolated from the EtOH extract of Coreopsis tinctoria. Their chemical structures were determined by detailed spectroscopic analysis and by comparison with literature data. Compounds 1-2 were tested for their antiadipogenic effects on 3T3-L1 adipocytes, and both of them reduced lipid accumulation dose-dependently in 3T3-L1 adipocytes.

[Allogenic BMSCs reverse chimerism inducing the tolerance of xeno-skin graft in mouse model].

OBJECTIVE: To investigate the feasibility of bone marrow mesenchymal stem cells (BMSCs) in inducing immune tolerance and to establish the mouse model of reverse chimerism in xeno-skin transplantation. METHODS: The mouse model of bone marrow-chimerism was established with immuncompromised BALB/C-nu/nu female mice by receiving the transplantation of BMSCs from green fluorescent protein (GFP)-C57BL/10 male mice, the optimized chimeric time was identified by RT-PCR testing of SRY gene and immunohistochemistry measurement of GFP expression. In the experiment group, GFP-C57BL/10 male mice received the transplantation of the skin from immuncompromised BALB/C-nu/nu female mice with BMSCs bone marrow-chimerism. In the rejection group, GFP-C57BL/10 male mice received the transplantation of the skin from immuncompromised BALB/C-nu/nu female mice without BMSCs bone marrow-chimerism. In the control group, allo-transplantation of skin was performed in GFP-C57BL/10 male mice. Histological study was performed to investigate the survival rate and angiogenesis of the transplanted skin. RESULTS: The bone marrow chimeric model was established, the expressions of SRY gene and GFP protein reached the highest level at four weeks (1.22 +/- 0.10; 458.0 +/- 3.4) post-transplanted with BMSCs (10(6)), which was significantly different in comparison with those at one week, two weeks and six weeks posttransplantation(P < 0.05). Four-weeks after transplantation was further confirmed as the optimized chimeric time. The mean survival time of donor skin graft > 14 d in the experimental group, while it only was 5 d in the rejection group. CONCLUSION: The bone marrow-chimerism can be formed in the recipient by donor BMSCs transplantation, which can further induce tolerance of mice xeno-skin transplantation by reverse chimerism.

[Differentiation of human bone marrow mesenchymal stem cells into Leydig or steroidogenic cells in vitro].

OBJECTIVE: To investigate the possibility of differentiating mesenchymal stem cells (MSCs) into steroidogenic or testicular Leydig cells in vitro. METHODS: The 3rd-passage cells of MSCs were divided into 4 groups to be induced and cultured. The experimental groups were cultured with conditional medium which consisted of luteinizing hormone (LH), human chorionic gonadotrophin (hCG), platelet-derived growth factor (PDGF) and interlukin-1alpha (IL-1alpha) as follows. Group A: LH 0.75 U/mL, hCG 40 U/mL,PDGF 10 ng/mL, IL-1alpha 0.0005 ng/ mL; Group B: LH 0.375 U/mL, hCG 20 U/mL, PDGF 10 ng/mL, IL-1alpha 0.0005 ng/mL; Group C: LH 0.1875 U/ mL,hCG 10 U/mL, PDGF 10 ng/mL, IL-1alpha 0.0005 ng/mL. Meanwhile, the control group was cultured in basal medium with normal sodium. The results were analysed by microscopic observations, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) immunocytochemistry and immunofluorescence on the 7th, 14th and 21st day of induction respectively. The function of the induced cells was characterized by testosterone ELISA. RESULTS: 10-14 days after induction, the induced cells with a typical reticular intercellular connection possessed the morphologic characteristic of Leydig cells. 3beta-HSD immunocytochemistry and immunofluorescence showed that Group A at 21th day had the most positive cells (P < 0.05). 21 days of induction revealed a higher testosterone production than others (P < 0.05). CONCLUSION: MSCs from human bone marrow are able to differentiate into steroidogenic or testicular Leydig cells in vitro. Human bone marrow-derived MSC may become important cells for studies of steroidogenic differentiation and offer a potential clinical stem cell source for diseases of steroidogenic organs.

Enhancement of cytotoxicity of antimicrobial peptide magainin II in tumor cells by bombesin-targeted delivery.

AIM: To investigate whether the conjugation of magainin II (MG2), an antimicrobial peptides (AMPs), to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells. METHODS: A magainin II-bombesin conjugate (MG2B) was constructed by attaching magainin II (MG2) to bombesin at its N-terminus. The peptides were synthesized using Fmoc-chemistry. The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 cell counting kit. Moreover, the in vivo antitumor effect of the peptide was determined in tumor xenograft models. RESULTS: The IC(50) of MG2B for cancer cells (10-15 µmol/L) was at least 10 times lower than the IC(50) of unconjugated MG2 (125 µmol/L). Moreover, the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2. In contrast, conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2, suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding. Indeed, MG2B selectively induced cell death in cancer cells in vitro with the IC(50) ranging from 10 to 15 µmol/L, which was about 6-10 times lower than the IC(50) for normal cells. MG2B (20 mg/kg per day, intratumorally injected for 5 d) also exhibited antitumor effects in mice bearing MCF-7 tumor grafts. The mean weights of tumor grafts in MG2B- and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g, respectively. CONCLUSION: The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy.

[Separation, culture and purification of rat brain microendothelial cell].

OBJECTIVE: To develop a separation method for brain microendothelial cells with the comparison of other ones. METHODS: Twice enzymatic digestion and twice gradient centrifugation were applied to separate rat brain microendothelial cells. Then, immunomagnetic beads and Thy1.1 antibody were used respectively to purify the cultured cells. RESULTS: Twice enzymatic digestion and twice gradient centrifugation could separate the cell successfully. High purification but low cell yield was obtained with immunomagnetic beads. The cells handled with Thy1.1 antibody had both higher purify coefficient and higher yield. CONCLUSION: The developed method could separate the brain microendothelial cells successfully.

Creating rat hepatocyte organoid as an in vitro model for drug testing.

BACKGROUND: Liver organoids have recently been applied as models for liver disease and drug screening, especially when combined with liver-on-a-chip technologies. Compared to hepatocyte-like cells, primary hepatocytes have high functionality but cannot maintain their function when cultured in vitro. Mesenchymal stem cells (MSCs) enhance hepatocyte function and maintain hepatocyte metabolism when co-cultured with hepatocytes. MSCs can help induced pluripotent stem cells to generate an organoid structure via the MSC-based traction force triggered by extracellular matrix (ECM) proteins. In this study, primary hepatocytes were co-cultured with MSCs on a liver-derived ECM to generate liver organoids within a short duration. AIM: To create hepatocyte organoids by co-culturing primary hepatocytes with MSCs on a porcine liver extracellular matrix (PLECM) gel. METHODS: Perfusion and enzymatic hydrolysis were used to form the PLECM gel. Rat hepatocytes and human MSCs were mixed and plated on pre-solidified PLECM gel in a 48-well plate for 48 h to generate organoids. Generated organoids were evaluated through hematoxylin and eosin, periodic acid-Schiff, immuno-histological, and immunofluorescence staining, and quantitative PCR for alb, CYP450 gene markers, and urea cycle genes. Culture medium was collected to detect albumin (ALB) and urea production on days 2, 4, 6, 8, 14, and 20. RESULTS: The whole porcine liver was perfused and enzymatically hydrolyzed to form a PLECM gel. The structural components and basement membrane composition of the ECM, such as collagen type I, collagen type IV, fibronectin, and laminin, were demonstrated to be retained. Through interaction of human MSCs with the liver-derived ECM, primary hepatocytes and human MSCs assembled together into a 3D construction and generated primary hepatocyte organoids for 48 h. The mRNAs of the gene alb, the CYP450 gene markers cyp1a1, cyp1a2, and cyp3a2 as well as urea cycle genes arg-1, asl, ass-1, cps-1, nags were highly expressed in hepatocyte organoids. Long-term survival of the primary hepatocyte organoids, as well as stable functionality, was demonstrated via ALB and urea production in vitro. CONCLUSION: Our new method of creating primary hepatocyte organoids by co-culturing hepatocytes with MSCs on liver-derived ECM hydrogels could be used to develop models for liver disease and for drug screening.

Identification and characterization of two morphologically distinct stem cell subpopulations from human urine samples.

Urine-derived stem cells (USCs) have shown potentials for the treatment of skeletal and urological disorders. Based on published literature and our own data, USCs consist of heterogeneous populations of cells. In this paper, we identify and characterize two morphologically distinct subpopulations of USCs from human urine samples, named as spindle-shaped USCs (SS-USCs) and rice-shaped USCs (RS-USCs) respectively. The two subpopulations showed similar clone-forming efficiency, while SS-USCs featured faster proliferation, higher motility, and greater potential for osteogenic and adipogenic differentiation, RS-USCs showed greater potential for chondrogenic differentiation. POU5F1 was strongly expressed in both subpopulations, but MYC was weakly expressed. Both subpopulations showed similar patterns of CD24, CD29, CD34, CD44, CD73, CD90 and CD105 expression, while a higher percentage of RS-USCs were positive for CD133. SS-USCs were positive for VIM, weakly positive for SLC12A1 and UMOD, and negative for KRT18, NPHS1, AQP1 and AQP2, indicating a renal mesenchyme origin; while RS-USCs are positive for VIM, partially positive for KRT18, NPHS1, AQP1, SLC12A1 and UMOD, and negative for AQP2, indicating a nephron tubule origin. The above results can facilitate understanding of the biological characteristics of subpopulations of USCs, and provide a basis for further research and applications of such cells.

[Effects of cardiotrophin-1 on differentiation of cardiomyocyte-like cells induced from rat bone marrow mesenchymal stem cells].

OBJECTIVE: To investigate the effects of cardiotrophin-1 (CT-1) on differentiation of induced rat bone marrow mesenchymal stem cells (BMMSCs) in vitro with 5-azacytidine (5-aza) for the purpose of elucidation of the cellular biological mechanisms. METHODS: BMMSCs isolated from femur of rats were divided into four groups: untreated group as control (group A); 0.1 nmol/L CT-1 added to medium (Group B); induced with 10 micromol/L 5-aza only (Group C); induced with 10 micromol/L 5-aza combined with 0.1 nmol/L CT-1 added to medium (Group D). After 4 weeks of induced culturing, the differentiation of induced myocyte like cells were estimated, levels of cardiac troponin-T (cTnT) by immunohistochemical staining and ultrastructure of induce-cultured BMMSCs were determined and mRNA expression of alpha-actin, beta-myosin heavy chain (beta-MHC), Nkx2.5, GATA4 were analyzed by real time polymerase chain reaction (RT-PCR). RESULTS: After 4 weeks of induced culturing, morphological characteristics of myocyte like cells with the expression of cTnT were observed in group C and D cells. Higher level expression of GATA4, Nkx2.5, alpha- actin and beta-MHC mRNA in group D was observed by comparing with those of group C and the differentiated BMMSCs with formations of myofilaments distinctly were also existed in 5-aza combined with CT-1 treatment group. CONCLUSION: This study suggests that induced culturing of BMMSCs in the presence of 5-aza combined with CT-1 can enhance cardiomyocytic characteristics. CT-1 upregulates the expression of GATA4, Nkx2.5, alpha-actin and beta-MHC mRNA, and rapidly promotes the differentiation and maturation of cardiomyocyte-like cells differentiated from BMMSCs induced with 5-aza.

HIF-1α protects against oxidative stress by directly targeting mitochondria.

The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates adaptive responses to oxidative stress by nuclear translocation and regulation of gene expression. Mitochondrial changes are critical for the adaptive response to oxidative stress. However, the transcriptional and non-transcriptional mechanisms by which HIF-1α regulates mitochondria in response to oxidative stress are poorly understood. Here, we examined the subcellular localization of HIF-1α in human cells and identified a small fraction of HIF-1α that translocated to the mitochondria after exposure to hypoxia or H2O2 treatment. Moreover, the livers of mice with CCl4-induced fibrosis showed a progressive increase in HIF-1α association with the mitochondria, indicating the clinical relevance of this finding. To probe the function of this HIF-1α population, we ectopically expressed a mitochondrial-targeted form of HIF-1α (mito-HIF-1α). Expression of mito-HIF-1α was sufficient to attenuate apoptosis induced by exposure to hypoxia or H2O2-induced oxidative stress. Moreover, mito-HIF-1α expression reduced the production of reactive oxygen species, the collapse of mitochondrial membrane potential, and the expression of mitochondrial DNA-encoded mRNA in response to hypoxia or H2O2 treatment independently of nuclear pathways. These data suggested that mitochondrial HIF-1α protects against oxidative stress induced-apoptosis independently of its well-known role as a transcription factor.

[Identification and clonogenicity of side-population cells in human decidua of first trimester pregnancy].

OBJECTIVE: To explore the existence of side-population cells (SP cells) in human decidual tissues in early pregnancy, and its biological characteristics. METHODS: Decidual cells were obtained by enzymatic digestion from the decidual tissues of human early pregnancy. The cells then were stained with Hoechst33342 dye either alone or in combination with verapamil. Fluorescence-activated cell sorter (FACS) analysis was used to identify and isolate SP cells. The isolated cells were cultured in conditioned media culture to observe the proliferation and ability to form colony. RESULTS: Stem cells were found in the decidual tissues of early pregnancy with a percentage of 0.78% +/- 0.71% (0% - 3.20%). These cells further proliferated and formed colonies during long-time culture in vitro. No significant statistical difference was found in contents of SP cells among different gestational week (P > 0.05). CONCLUSION: SP cells exist in human decidua that can further proliferate and have the clonogenicity in culture in vitro.

[Experimental study on thermochemotherapy combined with verapamil for reversing the multidrug resistance responsible to breast cancer cell].

OBJECTIVE: To reverse the multidrug resistance of MCF-7/ADM cell line by thermochemotherapy combined with verapamil (VRP). METHODS: The drug resistance cells MCF-7/ADM were treated with 3 microg/mL Adriamycin and different concentration VRP (0-5 micromol/L), and then cultured with 37-43 degrees C for 30 minutes. Confocal microscopy was used to demonstrate the fluorescence adriamycin in cells, and the cell apoptosis rate was analyzed with flow cytometry. RESULTS: With the increases of thermochemical temperature and VRP concentration, the cell adriamycin fluorescence intensity and apoptosis rate of MCF-7/ADM were raised. Thermochemotherapy showed marked synergistic effect with VRP in MCF-7/ADM. CONCLUSION: It is suggested that thermochemotherapy combined with Verapamil could be used as a way to overcome the multidrug resistance.

[The study on oral carcinoma-associated fibroblast promoting the proliferation of lingual carcinoma cell line].

OBJECTIVE: The aim of this study was in vitro to investigate the effects of oral carcinoma-associated fibroblasts (CAFs) on the proliferation of lingual carcinoma cells. METHODS: The interaction model between primary oral CAFs and a lingual carcinoma cell line Tca8113 was established for this study project. The effects of CAFs on the viability and cell cycle of Tca8113 were investigated through morphological observation, MTT assay and flow cytometry. RESULTS: After oral CAFs interacted with Tca8113 directly, Tca8113 showed the cellular shape changes in morphology; compared with normal fibroblasts (NFs), CAFs enhanced the viability of Tca8113 (P<0.05), and increased the percentage of carcinoma cells in S phase and G2 phase (48.1% vs 40.0%). CONCLUSION: Oral CAFs can promote the proliferation of the lingual carcinoma cell line Tca8113 in vitro, and may play a key role in the progression of oral squamous cell carcinoma (OSCC).

An engineered multidomain bactericidal peptide as a model for targeted antibiotics against specific bacteria.

We constructed a peptide consisting of a staphylococcal AgrD1 pheromone fused to the channel-forming domain of colicin Ia and named it pheromonicin. This fusion peptide had bactericidal effects against methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA, respectively), but not against Staphylococcus epidermidis or Streptococcus pneumoniae. Growth rates, vital staining and colony forming unit (CFU) counts showed that pheromonicin did not merely suppress growth but killed S. aureus cells. The specificity of pheromonicin was shown by the absence of bactericidal effects against an accessory gene regulator (agr) locus knockout of S. aureus, and a dose-dependent inhibition of the bactericidal effects of pheromonicin by competition with corresponding free AgrD pheromone. In vivo, all pheromonicin-treated mice survived administration of MRSA that was lethal to controls. No toxicity was detectable in human liver or renal cells in culture, or in livers, kidneys or spleens of pheromonicin-treated mice. The results suggest that these types of chimeric peptides may be of value as antibiotics against specific bacterial infections.

Expressed sequence tags analysis of a liver tissue cDNA library from a highly inbred minipig line.

BACKGROUND: Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore, we investigated the liver expression profile of a highly inbred minipig line. METHODS: A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme. RESULTS: Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences. CONCLUSION: These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.

[Signaling pathway of BDNF protecting embryonic cortical neurons from hypoxia].

OBJECTIVE: To investigate the changes of TrkB and Ras-MAPK in brain-derived neurotrophic factor (BDNF) protecting the embryonic rat cerebral cortical neurons against hypoxia-induced neurotoxicity. METHODS: The immunofluorescence technique, laser scanning confocal microscope and half-quantitative analysis were done to explore the changes of the intracellular levels of tyrosine kinase B (TrkB), phosphorylated TrkB and the mitogen-activated protein kinase (MAPK) in rat embryonic cortical neurons cultured in different time groups of hypoxia with or without BDNF pretreatment. RESULTS: The fluorescent intensity of TrkB and phosphorylated TrkB in the cytoplasm and the fluorescent intensity of MAPK in both cytoplasm and cell nucleus of the neurons were significantly increased in the presence of BDNF (P < 0.05), the fluorescent intensity of neurons pretreated with BDNF 24 h before the hypoxia culture was stronger than those with BDNF just before the hypoxia culture (P < 0.05). CONCLUSION: The Ras-MAPK approach may be the major signal transferring way of BDNF in protecting the cortical neurons from hypoxia-induced neurotoxicity. The approach of signal transferring begins with tyrosine phosphorylation of TrkB receptors.

[Construction of a cDNA library from liver tissue of rhesus monkey, Macaca mulatta].

OBJECTIVE: To screen the target rhesus genes and give some basic genetic evidences to its value as one of the most important animal model in biomedical study, we constructed a cDNA expression library from liver tissue of a healthy rhesus monkey. METHODS: With Trizol reagent, the total RNA was extracted from healthy rhesus liver tissue. By mutant Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), the first-strand cDNA was synthesized from purified mRNA, and subsequently the second-strand cDNA was generated via E. coli DNA polymerase I . Then, the EcoR I adapter was added to the synthesized double-strand cDNA, which was subsequently digested by Xho I restriction enzyme and fractionated with CHROMA APIN-400 column. The fractionated cDNA fragments to be longer than 0. 5 kb were ligated into lambda ZAP express vector to form the phagemid cDNA recombinants, which were further packaged into the lambda ZAP cDNA library according to the standard protocol with phage lambda Gold packaging extract. In order to get more stable clones with larger quantity, the primary library was amplified through infecting the host strain XL1-Blue MRF'. Then, the library titre, recombinant rate and length of inserted cDNA were measured, respectively. RESULTS: The capacity of the primary stand or unamplified library was 1. 2X 10(6) pfu. The titers of the unamplified library or the amplified library was 1.1 X 10(6) mixture, pfu/mL or 7. 7 X 10(9) pfu/mL respectively, the percentages of recombinants were 99. 3% and 98. 2%, and the average lengths of the inserts were 2.0 kb and 2. 3 kb, respectively. CONCLUSION: An excellent cDNA expression library has been constructed successfully, which would lay solid foundation for transplantation study and pre-clinic evaluation of related drugs.

[Hepatitis B virus surface antigen presenting function of peripheral blood dendritic cells from liver transplant receipients].

OBJECTIVE: To test the hepatitis B virus (HBV) antigen presenting capacity of peripheral blood dendritic cells (DCs) from liver transplant recipients who were in the condition of postoperative HBV clinical clearance. METHODS: Blood samples were obtained from 18 postoperative liver transplant recipients and 6 healthy adults. The peripheral blood mononuclear cells (PBMC) were isolated and cultured in vitro. The DCs were induced by recombainant human interleckin-4 (rhIL-4) and recombainant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and collected on day 7 of the culture. The DCs were then incubated with hepatitis B virus surface antigen (HBsAg) for 3 hours, and cocultured with allogeneic PBMC for 5 days. The 3HTdR was added with 1microci per well 18 hours before the end of the culture. The cpm values were identified for all of the cells. RESULTS: Under the same culture condition, the proliferation of the cells from the liver transplant recipients was poorer than those from the healthy adults (cpm value 4310.8 +/- 1820.3 vs 19002.5 +/- 13357.8, P < 0.001). The capacity of antigen presenting of DCs from the liver transplant recipients was significantly weaker than those from the healthy adults (cpm value 4974.9 +/- 2414.7 vs 39258.4 +/- 5554.9, P < 0.001). CONCLUSION: Antigen presenting capacity of DCs from liver transplant recipients, who are in the condition of postoperative HBV clinical clearance, is weakened. It might be the reason of the deficient immune response to HBV and hepatitis B vaccine of patients with liver transplant. The antigen presenting function is associated with the preoperative HBV replication, length after operation, prophylaxis of HBV re-infection/HB recurrence, and postoperative PBMV HBV DNA.