Objective　To study chemotherapy sensitivity and apoptosis in tumor cell line.Methods　Two different human colon carcinoma cell lines, LoVo and Ls-174-t were incubated with DDP,MMC,5-FU，EPI at various peak plasma concentrations (PPC), 1/10PPC，1/5PPC，5PPC and 10 PPC. Apoptosis was detected by FACScan and TUNEL technique after 24 hours and 48 hous. Cell growth inhibition rates were assessed by MTT, DNA ladder were examined by agarose electrophoresis.Results　At 48 hours, the highest cellular apoptosis rates were observed with PPC which was assessed by FACScan technique. There was a positive linear correlation between apoptosis assessed by TUNEL technique and growth inhibition rates of cells determined by MMT(P＜0.05).Conclusion　Using Annexin-V-Fluos staining FACScan technique to assess apoptosis of tumor cells is an exploring and useful method which can be used to choose both kinds and doses of chemotherapeutic drugs.

The present study was designed to investigate the effects of mifepristone on apoptosis of the decidual tissue cells and apoptotic correlated gene Fas/FasL expression in human early gestational period. The extent of DNA fragmentation of apoptotic cells were assessed by using an in situ terminal transferase reaction to label free 3′ ends of the DNA.The expression of Fas, FasL in the deciduas was examined by in situ hybridization with cDNA probe and immunohistochemical staining with polyclonal antibodies, respectively. The results showed that there were evident apoptosis in decidual tissues at about 40 days of gestation. Fas and FasL were strongly expressed in these decidual tissues. At about 50 days of gestation, there were a few apoptotic cells in the decidual tissues. Expression of Fas, FasL decreased at the same time. More apoptotic cells were observed in decidual tissues after adiministration of Mifepristone (RU486) in about 50 days of gestation. Fas, FasL mRNA and protein were up regulated after administration of Mifepristone. The results suggested that the induction of apoptosis by Mifepristone in decidual tissues might be one of the major mechanisms with regards to contraceptive and antigestational effects of mifepristone. Fas pathway might participate in the process of decidua apoptosis induced by RU486.

Objective To evaluate the reliability of colony PCR in identifying the recombinant clones selected from phage antibody libraries. Methods The digested pComb3 vector and Lc fragments, Lc library plasmid, and Fd fragments were ligated successively. The ligation product was transformed into Escherichia Coli strain XL 1-blue bacilli by electroporation and thus murine Fab phage antibody library was constructed.The transformed recombinant clones selected randomly from libraries were identified simultaneously by colony PCR, plasmid PCR and restriction enzyme digestion. The identification consistency was analyzed. The interference was excluded by control PCR using the relevant constituents as template. Results The educed library content of Lc library was 1.175?10 6 CFU and the content of Fab antibody library was 1.02?10 6 CFU. Different recombinant percentages were obtained through three different identification methods (the Lc positive recombinant percentages by colony PCR, plasmid PCR and enzyme digestion were 100%, 78% and 78%, respectively; the Fd positive recombinant percentages by three methods were 90%, 66% and 66%, respectively; the dual positive recombinant (Fd and Lc insert simultaneously) percentages by three methods were 90%, 50% and 50%, respectively). A high frequency of false-positives appeared in colony PCR identification. Nonspecific amplification of control PCR was presumably induced by some intracellular components in XL 1-blue bacilli. Conclusion The identification of the recombinant clones selected from phage antibody libraries by colony PCR remains ambiguous. So it is our assertion that the traditional identification methods such as plasmid PCR or enzyme digestion are more accurate and will less false positive results.

Objective To construct human-mouse hybrid Fab phage antibody libraries and to select target Fab genes. Methods With Fd repertoire genes of PBMC from hepatoma patients and the chemeric light chain gene of cFab-HAb18, two hybrid Fab phage antibody libraries(pComb3 and pComb3X) were constructed. The GST fusion and non-fusion HAb18GE were used as antigens to select the target antibodies with the optimized strategies of amplifying and screening. Results When pComb3 displaying library was cultivated at 37℃, the target fragments could not be cut out with the restriction endonucleases from most clones of the library, which was an indication of “recombinant-deletion”. But no “recombinant-deletion” was found in pComb3X displaying library. Because of the serious cross-reaction, the phage-display Fab ELISA could not be used for the selection of positive clones. But positive clones could be identified in the supernatant of lysate with ELISA afterbeing induced by IPTG. Conclusion Through the construction of HuMFab phage antibody libraries and the optimization of screening strategies, we get the desired HuMFab genes for subsequent research.

Objective To explore the impact of recombinant human hepatocyte growth factor (rhHGF) in Celsior (CS) solution on the expression of INF-γ, IL-4 and IL-10 in a rat liver transplan-tation model. Methods After flushed with CS solution with addition of rhHGF (experimental group) or saline (control group), NHBD livers were stored at 4℃; for 16 h.then they were transplanted using the two-cuff technique with arterial reconstruction. The serum levels of INF-γ, IL-4 and IL-10 at lh after reperfusion were detected using ELISA. The INF-γ, IL-4 and IL-10 mRNA in the corresponding liver tissue were determined by RT-PCR. The 7-day survival rate was calculated and the histopatho-logical examination results were analyzed by hematoxylin and eosin staining. Results Compared with the control group, the experimental group showed lower INF-γ level and higher IL-4 and IL-10 levels in serum at 1 h after reperfusion (P<0. 05). The level of INF-γ mRNA in liver tissue was significant decreased at 1 h after reperfusion (P<0. 05) , and the level of IL-4 and IL-10 mRNA was significantly increased in the experimental group (P<0. 05). In experimental group, recipients got a better survival rate and histopathological examination showed a well-preserved hepatic architecture without hepatocyte necrosis, milder sinusoidal and portal congestion. Conclusion Adding exogenous rhHGF in CS solu-tion can protect NHBD livers from ischemia-reperfusion injury and prolong the survival in rats, which might be due to down-regulation of TNF-γ and up-regulation of IL-4 and IL-10.

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18. Methods: The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAblSGE, extracellular region of HAblSG, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with p Ⅲ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2?107 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CHI belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions: Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.

Purpose:To study the relationship between Fas/FasL expression and apoptosis of colon adenocarcinoma cell lines induced by chemotherapeutic drugs and to screen out hypersensitive drugs. Methods:Colon adenocarcinoma cell lines LS174T and LoVo were incubated respectively in cultural media with DDP,MMC,5 FU and EPI at concentrations of PPC(plasma peak concentration),1/10 PPC,1/5 PPC,5 PPC and 10 PPC. Fas/FasL expression and apoptosis were assessed by FACScan, and DNA ladder was examined by agarose gel electrophoresis. Results:In LS174T cells, Fas expression and apoptosis of DDP group, MMC group and EPI group reached their highest peaks at 1/5 PPC or PPC,and Fas expression and apoptosis were positively correlated by relativity analysis. There was no significant correlation between Fas expression and apoptosis in LoVo cells. Conclusions:We could find out hypersensitive drugs and their appropriate dosage according to apoptosis, partly according to Fas expression. The different apoptosis paths between LS174T cells and LoVo cells demonstrate the importance of individualized selection of chemotherapeutic drugs.

Objective To assess the effectiveness of repeated trans-cranial magnetic stimulation ( rTMS) in relieving post-stroke depression ( PSD). Methods PubMed, Embase, the Cochrane library, Web of Science, CNKI, WANFANG, and VIP were searched for reports of randomized, controlled trials of rTMS treatment of PSD published before June 2015. Crude standardized mean differences ( SMDs) and odds ratios with 95% confidence in-tervals ( CIs) were calculated for depression intensity and effectiveness rate after treatment using random or fixed effects models. Results Twenty-four studies involving 856 rTMS-treated patients and 802 control patients were in-cluded in the meta-analysis. The results showed that compared with the control group, PSD patients showed significant reductions in depression after rTMS treatment ( SMD=-1.36;95% CI-1.6 to-1.12;P≤0.05) . The total effective-ness rate in the treated group was 85% with a reduction in NIHSS score ( SMD=-0.82;95% CI-1.2 to-0.44;P≤0.05) . Subgroup analysis showed that neither the frequency of rTMS stimulation, the site stimulated, nor time after stroke had a significant influence on the effectiveness of rTMS. Additionally, a few studies reported adverse reactions after rTMS. Conclusion rTMS appears to be a safe and effective therapy for PSD. Further well-controlled trials may elucidate the mechanism underlying the placebo effects of the sham rTMS observed among PSD patients.

OBJECTIVETo study the antiproliferative effects of beta-sitosterul and its mechanism in hepatoma HepG2 cells.

METHODCell proliferation was assessed by MTT assay. Cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by high content screening (HCS). The protein expression of caspase-3, caspase-8, caspase-9, Bcl-2, Bax, tBid and cytochrome c in the HepG2 cells were evaluated by Western Blots.

RESULTbeta-Sitosterul exerted significant antiproliferative effects in HepG2 cells. Furthermore, beta-sitosterul also induced HepG2 cells apoptosis, lost mitochondrial membrane potential, activated caspase-3, caspase-8 and caspase-9, up-regulate Bax, tBid protein, down-regulation Bcl-2 protein. However, beta-sitosterul had hardly any effects on QSG7701 cells.

CONCLUSIONbeta-Sitosterul exerted antiproliferative effects and induced HepG2 cells apoptosis via mitochondrial pathway and membrane death receptor pathway.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Sitosterols ; pharmacology

Objective To explore the effects of injection of wnt3a gene-modified bone marrow mesenchymal stem cells (MSCs) on acute graft-versus-host disease (aGVHD) in a murine allogeneic bone marrow transplantation (allo-BMT) model.Methods C57BL/6 mice were used as the donors and Balb/c mice as the recipients in the murine allo-BMT model.The recipient mice were divided into four groups by random number table method: transplantation control group (group A) (infusion of 5 × 106bone marrow cells via the tail vein of recipient mice); aGVHD group (group B) (infusion of 5 × 106bone marrow cells and 5 × 106 splenocytes via the tail vein of recipient mice); aGVHD + empty vector group (group C) (infusion of 5 × 106 bone marrow cells,5 × 106 splenocytes and 1 × 106 pAd-GFP-transfected MSCs via the tail vein of recipient mice) ; experimental group (group D) (infusion of 5 ×106 bone marrow cells,5 × 106 splenocytes and 1 × 106 wnt3a gene-modified MSCs).The general performance and survival were monitored,the occurrence of aGVHD was observed,the changes of donor T lymphocyte quantity present in the spleen,and interleukin-2 (IL-2) and interferon-γ (IFN γ)levels of the recipient mice were detected in each group after transplantation.Results The survival time of recipient mice in group A was all more than 60 d,and that in groups B,C and D was (19.1 ±6.19),(32.6 ± 19.6) and (47.2 ± 15.6) d,rcspcctivcly.The survival time in group D was significantly longer than in groups B and C (P＜0.05).After the transplant,the aGVHD score points in groups B,CandDwere (8.0±0.41),(6.7±0.29) and (4.0± 1.0),respcctively.The aGVHD score points in group D were significantly less than in groups B and C (P＜0.05),and the pathological grade in group D was significantly reduced.The number and proliferation rate of T lymphocytes were reduced significantly in group D as compared with groups B and C at 3rd and 5th day after transplantation (P ＜ 0.05).The levels of IL-2 and IFN-γ in peripheral blood were decreased significantly in group D as compared with those in groups B and C at 7th,14th,21st and 28th day after transplantation (P＜0.05).The chimeric rate of the murine H-2Kb cells in the bone marrow cells of long-term survival mice was all in the range of 95％ to 100％ 60 d after transplantation.Conclusion The injection of wnt3a gene-modified MSCs can more effectively alleviate aGVHD in murineallo-BMT model,which may be correlated with the Wnt3a overexpression which activating the Wnt/β-catenin signaling pathway of MSCs,thereby inhibiting the early activation and amplification of donor T lymphocytes and the IL-2 and IFN-γ expression.