Objective To observe the influence of bone marrow mesenchymal stem cells (BMSCs) transplantation on the expression of alpha-amino hydroxymethyl-oxazole propionic acid (AMPA) receptors GluR1 and GluR2 in rats with spinal cord injury (SCI) so as to investigate the potential anti- chronic stress mechanism of BMSCs transplantation in treatment of rats with spinal cord injury.Methods A total of 48 adult male SD rats were equally divided into three groups:control group,treatment group and model group.The rats in the model and treatment group underwent lower thoracic SCI with the modified Allen' s method,and the rats in control group received only laminectomy.At day 7 after thoracic SCI,100 μl of Hank's buffered saline solution containing 1.0 × 106 BMSCs was injected into the subarachnoid space from L4-L5 intervertebral space in the treatment group and control group,and the same amount of Hank' s buffered saline solution was injected in the model group.The motor function of the rat posterior limbs was assessed by Basso-Beattie-Bresnahan (BBB) scale before and after operation.Half of the rats were anesthetized at days 14 and 28 postoperatively to harvest brains which were frozen and cut in a cryostat to detect the expressions of GluR1 and GluR2 proteins by immunohistochemistry.Results After BMSCs transplantation,the motor function of the posterior limbs in the treatment group was improved progressively.At day 14 after transplantation,the number of GluR1-positive cells of the model and treatment group was higher than that of the control group in the hippocampus CA1 region (P ＜0.05,P ＜0.01 respectively) ; GluR2-positive cells had the similar tendency,without significant difference(P ＞ 0.05 ).At the 28th day after transplantation,GluR1 positive cells of the model group were higher than those of the control group in CA1,CA3,DG regions and those of the treatment group in CA1,CA3 regions (P ＜0.05,P ＜0.01,respectively) ; GluR1 positive cells of the model and treatment group were higher than their counterpart at day 14 after grafting procedure,with significant difference (P ＜0.05,P ＜0.01,respectively).GluR2 positive cells of the treatment group were higher than those of the control group in the basolateral amygdale (BLA) (P ＜0.05 ) and had similar tendency with GluR1 expression in other regions ( P ＞ 0.05 ).Conclusion BMSCs transplantation implies a potential antichronic stress mechanism of SCI rats,since it can improve the motor function of posterior limbs in rats with lower thoracic SCI and regulate the expressions of AMPA receptor GluR1 and GluR2.

Objective To investigate the antitumour effects of transfected gut-enriched Krüppellike factor(GKLF) on human gastric carcinoma cell line SGC-7901 in vitro and in vivo. Methods The expression of GKLF mRNA and protein in human gastric carcinoma cell line SGC-7901 were detected before and after transfection by real-time fluorescence quantitative PCR and Western blot,respectively. Proliferation and invasion in SGC-7901 were measured respectively by MTT assay, flow cytometry, colony formation assay and cell invasion assay after transfected with GKLF. The growth of xenograft was observed, the microvessel density(MVD) of xenograft tissue was determined by immunohistochemistry. Results The GKLF mRNA and protein in SGC-7901 were overexpressed after transfected with GKLF(P＜0.05). The proliferative speed of SGC7901-pcDNA3.1-GKLF group was markedly lower than that of SGC-7901 and SGC7901-pcDNA3.1 groups (P＜0.05). Transfected with GKLF caused part of the G0/G1 arrest, decreased clone formation rate and the invasion ability (P＜0.05). The growth speed of xenograft in SGC7901-pcDNA3.1-GKLF group was lower, the weight and MVD of xenograft tissue in SGC7901-pcDNA3. 1-GKLF group were less (P＜ 0. 05).Conclusion Transfected with GKLF maysuppress proliferation and invasion in human gastric carcinoma cell line SGC-7901, inhibit the growth and the angiogenesis of xenograft in nude mice.

Background:Galectin-3 is a member of the galectin family that participates in a variety of physiological and pathological events including cell growth and apoptosis,cell adhesion,angiogenesis,as well as tumor invasion and metastasis,and has been reported to be overexpressed in many human cancers.Aims:To investigate the effect of galactin-3 targeted RNA interference on proliferation,apoptosis and chemosensitivity of human gastric cancer cell line SGC-7901. Methods:Galectin-3 targeted siRNA was constructed and transfected into SGC-7901 cells.Efficacy of RNA interference was evaluated by real time PCR and Western blotting,while cell proliferation was assessed by CCK-8 assay and cell apoptosis by flow cytometry.Results:The transfection efficiency at 24 hours after transfection was 83.8%;expression of galectin-3 in SGC-7901 cells was significantly inhibited at mRNA and protein levels with a decreasing of 87.8% and 90.4%,respectively (P <0.01).Proliferation inhibition rates of SGC-7901 cells in galectin-3 siRNA group at 24,48 and 72 hours after transfection were 15.57% ±1.45%,32.90% ±0.76% and 57.35% ±1.05%,respectively,and the apoptosis rate at 72 hours after transfection was 46.17% ±2.39%;all were significantly higher than those in blank control,liposome control and negative siRNA control groups at the same time points (P <0.01).Proliferation inhibition of SGC-7901 cells induced by oxaliplatin,a chemotherapeutic agent,was also markedly increased in galectin-3 siRNA group (P <0.01).Conclusions:Expression of galectin-3 in SGC-7901 cells can be inhibited successfully by RNA interference;cell proliferation is decreased,cell apoptosis is increased and sensitivity to chemotherapeutic agent is augmented,which indicates that galectin-3 is a promising target for gastric cancer gene therapy.

To study the role of BK virus(BK polyomavirus)in the development of the hemorrhagic cystitis(HC)after hematopoietic stem cell transplantation(HSCT)and analyze the risk fators for BK viruri4a and HC.Methods From August 2006 to November 2007,blood and urine samples were collected from 80 patients undergoing HSCT.BK virus DNA was detected with PCR.Cytomegalovirus (CMV)antigen was detected with immunofluorescence histochemical examination.A control group including 20 healthy individuals was established.Results Late-onset HC occurred in 15 of the 80 HSCT patients with an incidence of 18.8%.The median onset time of HC was 44(13-150)days after transplantation.BK viruria was detected in 30 of the 80 HSCT patients(37.5%)and the positive rate of viruria in the HC patients was 86.7%(13/15).The median time of BK viruria detection in HC patients wag 23(0-56)days after transplantation,being earlier than the onset time of HC.The persistence time of BK viruria was 7(2-14) weeks,being much longer than that of HC(11 days).CMV antigen viremia was detected in 12 of the 80 transplanted patients.with a positive rate of 36.7% in patients with BK viruria and 40.0% in HC patients.Nine of the 30 HC patients developed acute graft versus host disease(Agvhd)of grade Ⅱ-Ⅳ(30.0%).BK virus was not detected in the urine of the remainimg two HC patients and the 20 control subjects as well as in all the blood samples.Univariate analysis indicated that CMV viremia and Agvhd of grade Ⅱ-Ⅳ were agsociated with the occurrence of BK viruria.Condusions BK viruria is the main cauge of the late-onset HC after HSCT.CMV infection and Agvhd may contribute to the occurrence of HC agsociatieg with BK virus.

Objective To detect the gene expression of PCA3 and PSA in peripheral blood and urine simultaneously to investigate whether PCA3 combining PSA gene could become new markers for diagnosis of Pca. Methods From June 2009 to December 2009,the initial urine after prostatic massage and the peripheral blood specimens were collected from 37 patients with PCa and 68 patients with BPH that were pathologically confirmed,g patients with urinary stone were used as normal control,the expression of PCA3 and PSA mRNA of mononuclear cells in urine sediments and peripheral blood were detected by fluorescence real-time quantitative PCR,with β-actin mRNA as internal control. Results The sensitivity and specificity of the expression of PCA3 mRNA in peripheral blood for diagnosis of prostate cancer were 48.6％ and 100％ respectively.ROC curve analysis was performed for the PCA3 score and the area under the ROC curve was 0.908.Using 64.6 as the cutoff,the sensitivity was 81.1％ and the specificity was 86.8％.In group with serum tPSA value ＜4 pg/L,the positive rate and negative rate of urinary PCA3 score for diagnosing prostate cancer were 80％ (4/5) and 89.4％ (20/22) respectively.In group with serum tPSA value 4 - 10 μg/L,the positive rate and negative rate of urinary PCA3 score were 66.7％ ( 2/3 ) and 84.2％(16/19) respectively.In group with serum tPSA value ＞ 10 μg/L,the positive rate and negative rate of urinary PCA3 score were 82.8％ (24/27) and 81.5％ (22/27) respectively.The sensitivity of simultaneous detection of PCA3 mRNA in peripheral blood and urinary PCA3 score was 86.5％. Conclusions The expression of PCA3 mRNA in peripheral blood was a specific marker for the diagnosis of PCa.The simultaneous detection of PCA3 mRNA in peripheral blood and urinary PCA3 score could increase the sensitivity for the diagnosis of PCa.

Objective To develop allele specific oligonucleotide(ASO) -PCR assay based on TCR βgene rearrangements and provide a screening method for minimal residual disease (MRD) in adult patients with T-lineage acute lymphoblastic leukemia (T-ALL).Methods DNA samples from newly diagnosed 20 adult T-ALL patients were obtained.The TCR β gene rearrangements were detected by multiplex PCR,which included 38 paired of primers in 3 reaction tubes.Gel electrophoresis and two-color Gene Scanning was also applied for clonality analysis of TCR β followed by sequencing and subsequent blasting for monoclonal PCR products in four patients.ASO primers were designed based on the sequence of junction regions.MRD were detected in the bone marrow by RQ-PCR with ASO upstream primers, consensus Jβprobes and downstream primers.Results The detection rate of the clonal TCR β rearrangements was 85.0% (17/20).At least one complete Vβ-Jβ rearrangement could be detected at the time of diagnosis in 16 out of 17 patients(94.1%, 16/17).Incomplete Dβ-Jβ rearrangement could be detected in 7 patients (41.2% ,7/17).The positivitity rate of Vβ-Jβ to Dβ-Jβ was 2∶1 (94.1% versus 41.2% ).Two-color Gene Scanning analysis showed the Jβ2 family was used more frequently than the Jβ1 family (73% versus 27% ).The slopes of the standard curves ranged from - 3.60 to - 3.27.The correlation coefficients of all four standard curves were more than 0.99.The detection sensitivity of ASO-PCR was 4 × 10 -5 μg/μl.The fluorescence background were detected at a low level.Quantitative MRD values of TCR β rearrangement in sequential BM specimens of 4 adult T-ALL patients were monitored during the treatment, including complete remission after induction and after consolidation therapy. RQ-PCR showed the MRD values of TCR β rearrangement were gradually decreased in response to the treatment.Conclusions The quantification of TCR β rearrangement by ASO-PCR approach is sensitive, specific and reliable for the accurate evaluation of malignant clones.It is suitable for the monitoring of minimal residual disease of adult T-ALL patients.

AIM: To explore the effects of cholestoral and mevalonate synthesis inhibitor lovastatin (LOV) on the proliferation of NB4 cells and elucidate the mechanisms. METHODS: Cell proliferation was analyzed by MTT assay;the expression of H, K, N- ras oncogenes in NB4 cells at different time point after LOV treatment were determined by semi-quantitative RT-PCR. Both total p21 Ras protein and p21 Ras protein on the cellular membrane were examined by flow cytometry. RESULTS:①LOV inhibited the proliferation of NB4 cells. ②All three kinds of ras were expressed in NB4 cells. ③LOV caused no increase in H, K, N- ras mRNA level. Amount of total p21 Ras protein did not change as the time varied. Concomitantly,p21 Ras protein localized on the cellular membrane decreased. CONCLUSION:LOV inhibits the proliferation of NB4 cells. Targeting HMG-CoA reductase, LOV blocks the isoprenylation of p21 Ras protein which affects its anchorage on the cellular membrane. No change in the H, K, N- ras mRNA and total p21 Ras protein expression is detected.

AIM: To investigate the protective effect of the bone marrow cells transfected with human multidrug resistance gene (MDR1) on the reconstruction of murine hematopoietic function.METHODS: The mononuclear cells of the bone marrow from donors, BALB/C mice, treated with 5-Fu previously, were isolated and transfected with human multidrug resistance gene in vitro , then transplanted to the tertiary recipients. After lethal irradiation(8.5 Gy) and bone marrow transplantation, the recipients were selected with Taxol 7 mg/kg intraperitoneal injection, VCR 5 mg/kg or DNA 5 mg/kg intravenous injection. The survival rate and blood pictures of mice as well as the integration and expression of target gene MDR1 were studied. RESULTS: The lethal irradiated murine hematopoietic function could be reconstructed and protected from toxicity of high doses Taxol, VCR and DNR selection after reinfusing the hematopoietic progenitor cells containing human multidrug resistance gene (MDR1). The survival rate and survival time of experimental mice were higher than that in the control group. The integration and expression of MDR1 gene in recipients were confirmed by PCR, RT-PCR and FCM. CONCLUSION: The integration and expression of human multidrug resistance gene in recipients may play an important role in the reconstruction and protection of murine hematopoietic function.

Objective To investigate the inhibitory effects of RNA interference on expression of matrix metalloproteinase-2(MMP-2)gene and invasiveness of human pancreatic cancer cell line PANC-1 in vitro.Methods Small interference RNA targeting MMP-2 gene was designed and constructed to plasmid pGCsi-U6.Recombinant plasmids were transfected to pancreatic carcinoma PANC1 cells with Lipofectamine 2000.The efficiency of transfection was evaluated by flow cytometry.RQPCR was used to detect the expression of MMP-2 mRNA.The expression of MMP-2 protein was determined by ELISA.The invasiveness of PANC-1 cells was measured by transwell chamber experiment.MTT assay was used to detect the proliferation and growth of PANC-1 cells.Results Sequencing confirmed that the MMP-2 siRNA plasmid was successfully constructed.The best efficiency of transfecting recombinant plasmid was 82.1%.After transfection of the MMP-2 siRNA plasmid, the MMP-2 gene expression of PANC-1 cells was suppressed to 71.74 %(P＜0.05),and protein expression of MMP-2 fell to 49.82%(P＜0.05).The corresponding inhibition ratio of invasiveness was 33.0%(P＜0.05).There was no marked difference in proliferation rate measured by MTT assay among different groups(P＞0.05).Conclusions RNAi targeting MMP-2 can suppress invasiveness of PANC-1 cells in vitro.This suggests that MMP-2 could be a target for gene therapy of pancreatic carcinoma.RNAi is expected to open up a new prospect for tumor therapy.

Objective To investigate the effect of low dose radiation on the expression of p16 gene in chronic myelogenous leukemia.Methods Leukemic stem cells(LSCs)which expressed CD34+,CD38- and CD123+ were isolated from bone marrow cells obtained from twenty patients newly-diagnosedas chronic myeloid leukemia with EasySepTM magnet beads.Hematopoietie stem cells(HSCs) which expressed CD34+ and CD38- were isolated from human cord blood cells obtained from twenty full-term deliveries with EasySepTM magnet beads as control.HSCs vs LSCs samples were further divided into three dose groups,including 0,12.5 and 50 cGy,respectively.RT-PCR and real-time quantitative reverse transcription-polymerase chain reaction methods were used to detect mRNA expression of p16 gene in HSCs and LSCs after irradiation.Cells were harvested at different time for detection of cell cycle and apoptosis by flow cytometer.Results p16 mRNA level in CML-LSCs was increased slightly at 12.5 cGy,and significantly increased at 50 cGy(Z=-3.39,P<0.01),but ho significant change was found in HSCs.The percentage of CML-LSCs cell in G0/G1 stagewas increased 48 h after 12.5 cGy irradiation,and 72 h post-irradiation with 50 cGy.The apoptosis rate of CML-LSCs was gradually raised after LDR,especially at 72 h post-irradiation of 50 cGy[(17.75±11.76)%vs(6.13±4.71)%,Z=-2.37,P<0.01 ].Conclusions p16 gene transcription could be up-regulated by low dose radiation,which might provide a theoretical evidence for CML therapy and LDR in leukemic clinical application.