Objective To determine whether ultrasound mediated microbubble destruction could increase naked human wild type p53 plasmid delivery to ovarian cancer in rats by intratumoral injection. Methods Forty rats with ovarian cancer successfully induced by chemical intoxicant were divided into four groups.The wild type p53 gene was cloned into a high expressing efficiency eukaryotic plasmid pcDNA 3.1+ and the mixture of naked human wild type p53 plasmid was carefully injected directly into the center of ovarian neoplasms with or without echo contrast agent, and it was exposed to ultrasound for twenty minutes once a week.Two months after p53 gene transfection, semiquantitative RT-PCR was used to detect wild type p53 mRNA expression and western blot was to evaluate p53 protein expression level. Results Recombinated eukaryotic expression plasmid DNA encoding pcDNA 3.1+ /p53 was successfully constructed and confirmed by sequence analyzing and endonuclease cutting. The ovarian cancer model of rat was obtained by exposure to intoxicant. Expression of human wild type p53 mRNA was significantly higher in rats transfected with wild type p53 plasmid by means of ultrasound and contrast agent than others(P

BACKGROUND:Operation is an important measure to improve the function of spinal cord and to stop the pathological progress of multilevel cervical spondylotic myelopathy. There are controversies how to select the optimum operation mode, to reduce postoperative complications and to elevate clinical curative effects.
OBJECTIVE:To systematical y review patients’ profiles of multilevel cervical spondylotic myelopathy, and to evaluate the effects of simple anterior approach, simple posterior approach and one stage posterior anterior combined approach on cervical spinal curvature index and functional recovery in patients.
METHOD148 sample profiles of patients, who received multilevel cervical spondylotic myelopathy operation in The Affiliated Hospital of Qingdao University and Qingdao Municipal Hospital from February 2000 to February 2008, and met the inclusion and exclusion criteria, were selected. They were divided into simple anterior approach group, simple posterior approach group and one stage posterior anterior combined approach group. The differences in the functional recovery were assessed after treatment using different therapeutic methods.
RESULTS AND CONCLUSION:Cervical spinal curvature index was highest in the simple posterior approach group before treatment (P<0.01). Cervical spinal curvature index was highest in the one stage posterior anterior combined approach group after treatment (P<0.01). Changes in cervical spinal curvature index were most obvious in the simple anterior approach group before and after treatment (P<0.01). No significant difference in Japanese Orthopaedic Association Scores was detected among three groups after treatment (P>0.05).
Significant differences in improvement rate of Japanese Orthopaedic Association Scores were detectable after treatment between the one stage posterior anterior combined approach group and simple anterior approach and simple posterior approach groups (P<0.001). Significant differences in cervical dysfunction index and SF-36 scores were detectable among the three groups before and after treatment (P<0.05). Results indicated that compared with the simple anterior approach and simple posterior approach, decompression through one stage posterior anterior combined approach is a reliable and effective operative procedure for treatment of multilevel cervical spondylotic myelopathy.

Objective: To clone and express the preferentially expressed antigen of melanoma (PRAME) gene in E.coli. Methods: The cDNA encoding human PRAME gene was extracted from human AML cell line HL-60 and amplified by RT-PCR, then the PRAME gene was inserted into plasmid PGEM-T-easy. After sequenced, it was cloned into the prokaryotic expression vector pGEX-4T-2 to construct a clone with high level expression and the recombinant plasmid was cloned in E.coli. Results:The protein product reached the highest level at 5 h after IPTG induction. Conclusion: Since PRAME is only expressed at low levels in a few normal tissues and encodes an antigen recognized by autologous cytotoxic T lymphocytes, while expressed at high levels in patients with leukemia, it might be a new targeting candidate for tumor immunotherapy.

Objective:To explore the possibility of inducing cell mediated immune response with HSP70 antigenic peptide complex in vitro.Methods:HSP70 peptide complex was reconstituted in vitro.Granulocyte/macrophage colony stimulating factors and interleukin 4 were used to cultivate DC from peripheral blood of HLA A2 positive healthy donors.HSP70,HSP70 peptide complex or peptide was used to activate the DC individually,which will initiate homogenize T lymphocyte to form cytotoxic T lymphocyte(CTL).The cytotxicity of the CTL was detected by MTT assay.Results:It was found that peptide specific CD8 + CTL responses were readily elicited by HSP70 peptide complex or peptide.The CTL response primed by HSP70 peptide complex was more potene than peptide alone.Conclusion:The results suggest that HSP70 peptide complex is immunogenic and HSP70 can lead to great efficient CTL response,antigenic peptides and HSP70 complex may be used as peptide vaccines for cancer immunotherapy.

Objective To detect the expression of survivin protein in intramedullary gliomas and evaluated the clinical significance of the survivin expression. Method Seventeen cases of intramedullary gliomas were removed by using microsurgical technique.It composed of 8 cases of ependymomas and 9 ones of astrocytomas.The patients were followed up by MRI scanning periodically.The survivin protein expression of the intramedullary gliomas were examined by immunohistochemical stain (SP). Results Total resection of the tumor was obtained in 7 cases of ependymomas and subtotal resection was undertaken in the other one case.For 9 cases of astrocytomas,total resection of the tumor achieved in 3 cases,subtoal resection in 5 cases and partial resection in one case.Survivin expression was detected in 2 samples of ependymomas and 6 ones of astrocytomas.2 astrocytoma cases were moderate positive staining,who suffering from intracranial and vertebral subarachnoid dissemination of the tumor. Conclusion The result of microsurgical treatment for intramedullary ependymomas is satisfactory.The survivin expression in astrocytoma samples is significantly higher than that in ependymoma.Moderate positive staining may correlated with subarachnoid dissemination of the tumor.

AIM: To investigate the inhibitory effect of vector-based RNA interference(RNAi) on the expression of melanoma associated antigen A3(MAGEA3) protein in hepatocellular carcinoma cells and on apotposis of hepatocellular carcinoma cells.METHODS: A vector for transcribing specific small hairpin RNA(shRNA) targeting MAGEA3 gene was constructed,introduced into hepatocellular carcinoma MEL-ED1 cells by Lipofectamine 2000.The MAGEA3 protein and mRNA expression levels of MEL-ED1 cells were detected by Western blotting and RT-PCR, respectively.The cell apoptosis was studied by DNA fragmentation,electron microscopy,TUNEL assay,and annexin V/PI staining.RESULTS: The vector of RNA interference was successfully constructed and MAGEA3 expression was descreased significantly in MEL-ED1 cells.After the shRNA expression vector was transfected into the MEL-ED1 cells,the expression of MAGEA3 gene was inhibited significantly(by 90%).DNA fragmentation,electron microscopy and TUNEL assay showed classic apoptosis characters in the MEL-ED1 cells transfected with pSilencer-MAGEA3 plasmid with an apoptosis rate of 21.41% ?1.98%,significantly higher than those in the negative control group transfected with pSilencer-neo and in the non-transfected group(both P

Objective To discuss the role of DNA-dependent protein kinase catalytic subunit (DNA-DPKCS) in human lung adenocarcinoma cell line (A549) by using small interfering RNA (siRNA) to specifically knockdown DNA-DPKCS expression and its effects on cell proliferation, cell cycle and radio-sensitivity. Methods The DNA-DPKCS-siRNA expression vector was constructed and transfected into A549 cell line. The transformed clones were randomly selected and isolated. The cell cycle distribution and apop-tesis were analyzed by flowcytometry analysis. Cell survival was detected by using clonogenic formation as-say. Results With specific inhibition of DNA-DPKCS expression, stable transfected cell line 549pRNA-DNA-DPKCS was constructed by RNA interference technique. The 549pRNA-C and 549pSUPER cell lines were the control cell lines tansfected with control and blank plasmids, respectively. Compared with A549 cells, the expression levels of DNA-DPKCS mRNA (0.110: 1. 000), protein (0. 870: 2.967) and activity of DNA-DPKCS (0.004: 0.266) in 549pRNA-DNA-DPKCS cells were significantly lower (F = 80.55 ,P < 0.01;F=63.96, P<0.01;F=51.62,P<0.01, respectively). The analysis of SF_2(0.25:0.76), D_0 (1.42:1.62) and D_q (0.06: 1. 00) showed significant difference between 549pRNA-DNA-DPKCS and A549 cells (F = 996.86, P < 0.01 ; F = 17.41, P < 0.05 ; F = 68.92, P < 0.01). The number of 549pRNA-DNA-DPKCS cells in S (24.5%: 35.5%) and G_2 (10.7%: 11.0%) phases was significantly decreased (F = 4.83, P<0.05 and F=32.04, P <0.01, respectively). Conclusions In A549 cells, inhibit of DNA-DPKCS gene expression can enhance the radiosensitivity and affect cell cycle distribution.

AIM: To investigate the possible mechanism of PRL-2 in invasive metastasis of tumors.METHODS: The PRL-2 vector was transfected into CL1 cell with lipofectamine reagent,the transfectants were selected by growth in the medium supplemented with G418.Zymographic analysis of metalloproteinases(MMPs) activity was performed,RT-PCR was used to determine the mRNA levels of(MMP-2),MMP-9,TIMP-1 and TIMP-2,the protein levels of(MMP-2),MMP-9,TIMP-1 and TIMP-2 were analyzed by Western blotting.The effects of the special inhibitor of PRL-2 on transfected cells were also observed.RESULTS: The stable cell line selected by G418 was identified by RT-PCR and Western blotting.More abundance of MMP-2,MMP-9 and its activated type were secreted by the CL-1-PRL-2 cells than untransfected cells and transfected vector cells(P

Objective To establish a HRM assay to screen for KRAS mutations in clinical colorectal cancer patients.Methods The sensitivity of HRM was analyzed by detecting somatic mutations in exon 2,notably codons 12 and 13 of the KRAS gene in the serial plasmid mixture samples which were mixed using the different proportions mutation plasmid and wide type plasmid of KRAS.HRM analysis was performed for KRAS on DNA insolated from a panel of 60 colorectal cancer samples derived from fresh tissues.The results were compared with the direct sequencing data.Results After the PCR amplification,the mutation results could be available by performing HRM analysis in the same tube on a real time PCR machine with HRM capability.HRM detection could identify KRAS mutation in a proportion of 10% of mutation plasmid DNA.All 60 samples identified the KRAS mutation by HRM and sequencing.17 samples were positive(28.3%) by HRM for KRAS exon 2 mutations,and 15 samples were confirmed the presence of codon 12 or 13 mutations(25.0%) and the other 2 samples were wild type by sequencing.The 60 samples detected by HRM were given 100% sensitivity with 96% specificity.Conclusions HRM is a sensitive intube methodology to screen for mutations in clinical samples.HRM will enable high-throughput screening to gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.

AIM:To construct recombinant adenovirus vector carrying human miR-133a and study its expression in human mesenchymal stem cells(hMSCs).METHODS:The PCR product containing miR-133a was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrack-CMV.Then the recombinant shuttle plasmid linearized by pmeⅠwas cotransformed into competent E.coli.BJ5183 with the adenoviral backbone plasmid pAdEasy-1 to generate the recombinant adenovirus vector rAd-mir-133a.rAd-mir-133a was then packaged and amplified in human embryonic kidney 293(HEK293) cells.The purified rAd-miR-133a was used to infect the hMSCs and the expression of miR-133a was detected by non-quantitative RT-PCR and real-time PCR.RESULTS:The recombinant adenovirus shuttle vector pAdTrack-CMV-miR-133a was constructed and verified by restriction endonuclease analysis and DNA sequence analysis.rAd-miR-133a was successfully packaged and amplified in HEK293 cells.The transcriptions of primary miR-133a and mature miR-133a were over-expressed in the hMSCs infected with rAd-miR-133a.CONCLUSION:The recombinant adenovirus vector carrying human miR-133a is successfully constructed,which lay a foundation for miR-133a function study.