BACKGROUND: There is no accepted treatment for liver fibrosis recently. Bone marrow meaenchymal stern cells (BMSCs) used in the treatment of liver fibrosis has been reported as an effectively treatment, but the mechanism is unclear.OBJECTIVE: To study the regulation of hepatic stellate cells mediated by human BMSCs in vitro.DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Center for Stem Cells and Tissue Engineering of Sun Yat-sen University and the Central Laboratory of Third Affiliated Hospital of Sun Yat-sen University from June to December 2008.MATERIALS: Human bone marrow masenchymal stem cells were collected from normal youth volunteers; Human hepatic stellate cells and normal liver call line L-O2 were supplied by the Animal Experimental Center of Sun Yat-sen University.METHODS: The purified human BMSCs and hepatic stellate calls were set up in Transwell co-culture system. The incubation density was 2×104cells/well. L-O2 was set up instead of human BMSCs as negative control. Hepatic stellate cells cultured alone served as blank control group. The culture was performed for 72 hours.MAIN OUTCOME MEASURES: Morphology of hepatic stellate cells and results of immunocytochemical staining. Apoptosis of hepatic stellte calls was determined by flow cytometry. Western blot were used to assay the expression of α-actin.RESULTS: Activated hepatic stellate cells presented fiat and thin shape under an inverted microscope. Fat drop was lack in cytoplasm, a -actin located in hepatic stellate calls, with the presence of high tension fibers. Compared with the L-O2 + hepatic stellate cell and hepatic stellate call groups, the apoptotic rate of hepatic stellate cells was significantly increased in the BMSC + hepatic stellate cell group (P < 0.05). α -actin expression was significantly down-regulated.CONCLUSION: Human BMSCs can inhibit activation of hepatic stellate ceils and promote them apoptosis, which may be the anti-hepatic fibrosis mechanism of BMSCs.

Objective To construct the bioartificial liver by bone mesenchymal stem cell (BMSC) and alginate scaffold. Method Alginate scaffold was used as the cell carrier for the cultivation of BMSC and the differentiation from BMSC into hepatic like cells was induced by the cell factors of HGF, EGF and FGF-4 in the scaffold in vitro. The compatibility of the cells and the scaffold was observed by microscopy and the function of the differentiatd cells was tested. The gene of AFP and ALB was detected by RT-PCR. The secretion of ALB and the urea synthesis of the cells were tested by ALB kit and urea kit respectively. The glycogen synthesis and the CK-18 was tested by the glycogen stanning method and the immunofluorescence test. Results BMSC was able to attach, grow and proliferate well in the alginate scaffold, the well compatibility was observed by microscopy. ALB and urea were detected in the cultivating medium, the gene of ALB and AFP was identified by RT-PCR. The glycogen synthesis ability and the expression of CK-18 were induced during the differentiation. Conclusion The three dimensional atginate scaffold exhibited well compatibility with BMSC, BMSC could be differentiated into the hepatic like cell in the scaffold. BMSC and the alginate scaffold could be used to construct the bioartificial liver for the hepatic tissue engineering.

Objective To observe the expression of Na+/I- symporter(NIS) in cultured lactating mammary cells with different levels of iodine and the effect of tumor necrosis factor-α(TNF-α). Methods Original generation of mouse lactating mammary cells cultured in vitro were divided into low iodine group Ⅰ (LI-Ⅰ), low iodine group Ⅱ (LI-Ⅱ), adequate iodine group(AI), high iodine group Ⅰ(HI-Ⅰ), and high iodine group Ⅱ(HI-Ⅱ). Cells were cultured in DEME/F12 culture medium for 24 h with different concentrations of iodine (0,5,50,3000 and 10 000 μg/L, respectively), and TNF-α( 10-2 mg/L) was added to some of cultured cells for 24 h. The expression of NIS mRNA of lactating mammary cells was determined by real-time quantitative PCR and the expression of NIS protein was detected by In-Cell Western. Results In iodine alone group, the expression of NIS mRNA in LI-Ⅰ group [ (64.66 ± 14.99) x 10-4] was higher than that of AI group[ (22.76 ± 7.36) × 10-4, P ＜ 0.01 ]; HI-I group[ (10.18 ±3.53) × 10-4] and HI-Ⅱ group[ (8.59 ± 2.89) × 10-4] were lower than that of AI group(all P ＜ .0.05); With increased iodine concentration, the expression of NIS mRNA decreased. The expression of NIS mRNA in LI-Ⅰ group [(2.72 ± 0.45) × 10-4], LI-Ⅱ group[ (2.69 ± 0.68) × 10-4] and AI group[(1.80 ± 0.67) × 10-4] with iodine plus TNF-o were all lower than that of LI-Ⅰ group, LI-Ⅱ group[ (29.82 ± 4.47 ) × 10-4], and AI group without TNF-α (all P ＜ 0.01). In iodine plus TNF-α, the expression of NIS mRNA in HI-Ⅰ group[(6.58 ± 2.87) × 10-4] and HI-Ⅱ[(7.04 ± 1.36) × 10-4] group were all higher than that of AI group(all P ＜ 0.05); With increased iodine deficiency or iodine excess, the expression of NIS mRNA increased. With increased iodine concentration, the expression of NIS protein decreased in iodine alone group. The expression of NIS protein in iodine plus TNF-α was all lower than that in iodine alone group. In iodine plus TNF-α, the expression of NIS protein increased in both iodine deficiency and iodine excess conditions. Conclusions Iodine may decrease the expression of NIS mRNA and protein of lactating mammary cells. The expression of NIS mRNA and protein of lactating mammary cells was inhibited by TNF-α under different levels of iodine.

ObjectiveTo study the morphological and functional changes of thyroid in lactating rats and their offspring in iodine deficiency and iodine excess animal models.MethodsOne hundred and twenty Wistar rats(30 males and 90 females) were selected.Based on their body weight,the 90 females were stratified and randomly divided into five groups( 18 in each group):low iodine group 1 and group 2(fed with low iodine feed and deionized water containing iodine of 0,5 μg/L) ; high iodine group 1,group 2 and control group(feed with normal diet and deionized water containing iodine of 3000,10 000,50 μg/L).After fed for 3 month,all female rats were mated with males in a ratio of 3 ∶ 1.After birth for 10 days,8 female rats and their offspring in each group were sacrificed.Changes of thyroid were observed by naked eyes.The thyroid weight was measured and pathological changes of thyroids were observed under light microscope.Results①Absolute and relative weight of lactating rats thyroid in low iodine group 1 and group 2 [ (92.02 ± 24.40 ),(77.11 ± 23.32 )mg,(0.509 ± 0.072),(0.384 ± 0.089) mg/kg] were much higher than that of control group[ (17.41 ± 9.25)mg,(0.102 ± 0.016)mg/kg,all P＜ 0.05].Absolute and relative weight of lactating rats thyroid in high iodine group 1 and group 2[(8.22 ± 0.41 ),(9.42 ±0.43)mg,(0.047 ± 0.006),(0.035 ± 0.005)mg/kg] were lower than that of control group(all P ＜ 0.05).Absolute and relative weight of lactating rats and their offspring thyroid was decreased with increase of iodide intake in the diet.②Thyroid enlargement of lactating rats in low iodine group 1 and group 2 was evident,but that of high iodine group 1 and group 2 was not.③Epithelial cell hyperplasia and smaller follicular cavity were observed in low iodine group 1 and group 2 under light microscope.Epithelial cell deformation and mostly flat were observed in high iodine group 1 and group 2.ConclusionsThyroid morphology is changed with iodide intake in the lactating rats and their offspring,and the changes are consistent between female rats and their newborns.

OBJECTIVETo explore the effect of combined application of psoralen (PSO, an extract from psoralea) and ultraviolet A (UVA) for inducing the apoptosis of human leukemic cell line NB4 and its impact on the Fas/FasL gene expressions.

METHODSAccording to factorial design, changes of apoptosis rate and ultrastructure of NB4 cells, as well as the gene and protein expressions of Fas/FasL were observed after cells were treated with PSO in different concentrations and irradiated by UVA of 360 nm wavelength for different times, using flow cytometry, transmission electron microscopy and quantitative polymerase chain reaction (PCR), and the outcomes were treated with variable analysis.

RESULTS(1) After treatment of PSO in concentration of 10, 20, 40, 80 microg/mL combined with a 5-min exposure of UVA, the NB4 cells apoptosis rate induced were 26.57% +/- 0.42%, 30.67% +/- 0.11%, 34.90% +/- 0.30% and 24.63% +/- 0.38% respectively. The effects were dose- and time-dependent, and an interaction was shown between the two actors. (2) After being treated by PSO plus UVA, obvious ultrastructure changes with apoptosis characteristics were shown in NB4 cell under electron microscope. (3) PSO plus UVA showed up-regulatory effect on gene and protein expressions of Fas, and down-regulatory effect on gene and protein expressions of FasL in a dose- and time-dependent manner, with the interaction between the two actors in altering Fas gene expression, also in altering FasL gene and protein expressions.

CONCLUSIONCombined application of PSO and UVA can induce the apoptosis of NB4 cells, and the Fas/FasL system is one of the pathways for apoptosis inducing.

Apoptosis ; drug effects ; radiation effects ; Cell Line, Tumor ; Fas Ligand Protein ; metabolism ; Ficusin ; pharmacology ; Gene Expression ; Gene Expression Regulation, Leukemic ; Humans ; Ultraviolet Rays ; fas Receptor ; metabolism

OBJECTIVETo observe the therapeutic effect of Kangyi Zhongyu Decoction (KZD) combined with gonadotropin releasing hormone-a (GnRH-a) on infertile patients with severe endometriosis.

METHODSSeventy-five infertile patients with the diagnosis of endometriosis confirmed by laparoscope who were scheduled to receive in vitro fertilization and embryo transfer (IVF-ET) were randomly assigned to three groups, they were treated respectively with KZD (A), GnRH-a (B) alone and combined of both (C), and IVF-ET were applied in the patients after 3 months of treatment. The clinical efficacy and adverse reactions in the three groups were observed and the changes of serum cancer antigen 125 (CA125) and endometrial antibody (EMAb) levels before and after treatment were tested.

RESULTSScore of dyspareunia in Group A and C was obvioushy lower than that in Group B after treatment (P <0.01). Pregnancy rate in Group C was higher than that in Group A and B (P <0.05), with the adverse reactions less than in Group B (P <0.01). The positive rate of plasma EMAb was reduced obviously after treatment in Group C with the level lower than that in the other two groups (P<0.05).

CONCLUSIONThe combined use of KZD and GnRH-a is a new method in treating infertile patients with severe endometriosis with ideal effectiveness and fewer adverse reactions, and it could advance the successful rate of reproductive assistant technique.

Adult ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Embryo Transfer ; Endometriosis ; complications ; drug therapy ; Female ; Fertilization in Vitro ; Gonadotropin-Releasing Hormone ; therapeutic use ; Humans ; Infertility, Female ; drug therapy ; etiology ; Integrative Medicine ; methods ; Phytotherapy ; Treatment Outcome ; Young Adult

BACKGROUND:It is reported that bone marrow mesenchymal stem cell(BMSC)transplantation might be a promising treatment for liver fibrosis.But the mechanism is still unclear.OBJECTIVE:To observe the hepatic stellate cells apoptosis induced by BMSC transplantation,and to study the mechanism of BMSC in treating hepatic fibrosis in vivo.METHODS:CCl_4 subcutaneous injection was performed to induce rat liver fibrosis.After 8 weeks of CCU injection,20 rats which underwent successful model establishment were randomly divided into experimental group and control group,10 in each group.The experimental group received MSC transplantation via tail vein injection,and the control group were given DMEM instead.The rats were killed and the livers were harvested at three time point,the day of MSC transplantation,3 days after transplantation,and 7 days after transplantation.The hydroxyproline content was detected by HE and Masson staining,and the expression changes of α-smooth muscle actin(α-SMA)proteins were determined using immunohistochemistry.The apoptosis of hepatic stellate cells were determined by α-SMA and TUNEL(terminal dUTP nick-end labeling)dual-staining.RESULTS AND CONCLUSION:After 8 weeks of CCU injection,the hydroxyproline content increased and histology indicated progress of liver fibrosis.At 7 days after MSC transplantation,the hydroxyproline in the liver was decreased,and the liver fibrosis was alleviated in the experimental group but aggravated in the control group.Immunohistochemistry indicated that α-SMA positive cells were increased at 8 weeks after CCU injection.At day 7 after transplantation,α-SMA positive cells in the experimental group were significantly less than control group(P ＜ 0.05).At 3 days after transplantation,the hepatic stellate cells apoptosis in the experimental group was significantly aggravated compared with control group(P ＜ 0.05).This suggested that MSC transplant was an effective treatment for liver fibrosis.MSC inducing hepatic stellate cells apoptosis may be one of the mechanisms.

Designed the degenerate primers of alcohol dehydeogenase and L-lactate dehydrogenase to aug- ment Ethanoligenens harbinense B49 genomic DNA, and obtained about 780 bp and 610 bp PCR product respectively. Augmented flank sequences of the two PCR fragments with the Cassette PCR method. Similar- ity alignment showed that the products of the cloned DNA were very high similar to those of alcohol dehy- drogenase genes and L-lactate dehydrogenase genes respectively. One of the two sequences was 1902 bp long, and the ORF of adh was 1101 bp long and encoded 366 amino acids. Its putative molecular weight was about 39.71 kD, its calculational isoionic point was pH 5.93. The maximal identity and positive was 51% and 73% with Clostridium thermocellum ATCC 27405 adh. The other one was 2490 bp long, and the ORF of adh was 951 bp long and encoded 316 amino acids. Its putative molecular weight was 34.23 kD, its calcula-tional isoionic point was pH 6.09. The maximal identity and positive was 55% and 74% with Bacillus megaterium L-ldh. Successfully cloning these two genes would not only enrich the gene resources of L-lactate dehydrogenase and alcohol dehydrogenase genes, but also give the scientific warrant for the meta- bolic engineering research and the construction of the gene-engineering bacteria.

Incidence of acute closed Achilles tendon rupture is high in clinic. Accurate diagnosis of this injury depends on a combination of physical signs and imaging examinations. Although conservative and surgical interventions are common for acute closed Achilles tendon rupture, its optimal treatment remains controversial. This literature review focuses on the research progress concerning this injury so as to analyze its risk factors, diagnostic methods, treatments, early functional rehabilitation, postoperative complications and prognosis evaluation.

Objective The relationship was analyzed between clinic and the expression intensity of HBsAg and HBcAg with in the hepatic tissue from the serum HBeAg negative group and the positive group.Methods A total of 317 liver biopsy specimens were divided into the HBeAg negative group and the positive group,and the relationship was analyzed between the expression inten sity of HBsAg and HBcAg within the hepatic tissue and their age,gender,ALT level,serum HBV-DNA load,hepatic inflammatory activity grading and fibrosis staging in the two groups.Results Age,ALT level,hepatic inflammatory activity grading and fibrosis of the serum HBeAg negative patients were greater than those of the serum HBeAg positive patients,while their serum HBV-DNA load and the expression intensity of HBcAg within the hepatic tissue were lower than those of the serum HBeAg positive patients (P＜0.05).The expression intensity of HBsAg within the hepatic tissue between the serum HBeAg patients and the serum HBeAg positive patients was not significantly different,and it was not correlated with age,ALT level,hepatic inflammatory grading and fi brosis staging (P＞0.05).After the serum HBeAg turned negative,the expression intensity of HBcAg within the hepatic tissue was decreased (P=0.00,t=12 349.0),and it became positively correlated with the serum HBV DNA load(P=0.007,r=0.251) and its negative correlation with the hepatic inflammatory activity and fibrosis was weakened.Conclusion After the serum HBeAg turned negative,other antigenic components of HBV may still maintain the adequately active immune status within the hepatic tis sue of organisms.After the serum HBeAg turned negative,the expression intensity of HBcAg within the hepatic tissue was de creased and became positively correlated with the serum HBV DNA,while its negative correlation with the hepatic inflammatory activity grade and fibrosis stagings was weakened.