iFlow in the auxiliary diagnosis of cerebral vascular disease / 中国介入影像与治疗学

ObjectiveTo evaluate the impact of iFlow in the auxiliary diagnosis of cerebral vascular diseases.Methods Totally 65 patients with cerebral vascular diseases underwent whole brain 2D DSA.Then the data were converted to color coded images with iFlow.The vascular anatomy,the shape and size of lesions,the blood dynamics on 2D DSA and iFlow images were evaluated by junior and senior physicians,who then reported whether iFlow images had supplementary diagnosis value.ResultsiFlow images displayed whole information of lesions more clearly than those of 2D DSA in brain vascular dynamic sequence,including vascular anatomy,hemodynamic changes and tissues perfusions.The evaluation of 2D DSA and iFlow images with junior and senior physicians had statistical difference (x2 =6.032,P=0.014).ConclusionCompared to single DSA,iFlow can facilitate the diagnosis and evaluation of cerebral vascular diseases,especially for junior physicians.

Retroviral endostatin gene transfer inhibits human colon cancer cell growth in vivo / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the therapeutic effect of retroviral endostatin gene transfer on the human colon cancer cell line, LoVo.</p><p><b>METHODS</b>A retroviral vector pLESSN expressing secretable endostatin was constructed and packaged with a titer of 8.2 x 10(5) CFU/ml. A LoVo cell line was subjected to retrovirus-mediated endostatin gene transfer. The proviral integration of endostatin was analyzed with PCR. The function of endostatin was tested by MTT assay in vitro and a mouse xenograft model in vivo.</p><p><b>RESULTS</b>After transfection and superinfection, amphotropic retrovirus was collected, and transduction with amphotropic retroviruses resulted in endostatin proviral integration. The endostatin secreted by transduced LoVo cells markedly inhibited cell growth up to 67% (P<0.001), compared with the control cells. The gene expression of endostatin in LoVo colon tumor cells significantly inhibited tumor growth in vivo. There was an 86% reduction in tumor size in the endostatin-transduced group, accompanied by a reduction in vessels, compared with the control group (P<0.01).</p><p><b>CONCLUSION</b>Retroviruses can allow functional expression of the endostatin gene in human colon tumors, showing promise for an antitumor strategy using antiangiogenesis.</p>

Angiogenesis in colorectal cancer and its relationship with apoptosis and proliferation / 中华消化杂志

Objective To study the expression of vascular endothelial growth factor(VEGF), microvessel density (MVD) and the relationship among angiogenesis,proliferation,and apoptosis in colorectal cancer (CRC). Methods Expression of VEGF mRNA was evaluated by semi quantitative RT PCR in 52 CRC samples and 48 adjacent normal colorectal tissue samples. The PCR product was sequenced to verify the desired results. Expressions of VEGF protein, MVD, proliferating index (PI), and apoptotic index (AI) were detected by immunohistochemical methods in 52 human CRC. Results Expression of VEGF mRNA was detected in a significantly greater proportion of colorectal carcinoma samples than that adjacent normal colorectal tissues (76.9% vs. 52.1%; P 0.05). The expression of VEGF protein was positively correlated with PI ( r =0.409, P 0.05). Conclusions VEGF may play an important role in the angiogenesis of colorectal carcinoma and cell proliferation, but may not directly correlate with cell apoptosis.

The inhibitory effects of endostatin gene transfer on the growth of breast cancer cells in vivo and in vitro / 中国病理生理杂志

AIM: To investigate the therapeutic action of secreted endostatin (ES) on breast cancer cells. METHODS: Retroviral-mediated endostatin gene was transferred to breast cancer cell line MDA-MB-231. The ES biological properties and function were evaluated by polymerase chain reaction (PCR), MTT and a murine xenograft model. RESULTS: After retroviral transduction, endostatin genetically modified breast tumor cells were confirmed by PCR, and the integration and durative expression of endostatin gene was successfully committed. Compared with controls, endostatin secreted by genetically modified cells markedly inhibited endothelial cell proliferation (P0.05). The results of the transplanted subcutaneous tumor model in nude mice suggested that the subcutaneous growth of MDA-MB-231 was significantly inhibited by the expression of endostatin gene (P

Cloning and expression of the metalloproteinase domain of human von Willebrand factor-cleaving protease / 中国病理生理杂志

AIM: To clone and express the metalloproteinase domain of human von Willebrand factor-cleaving protease (vWF-cp). METHODS: The metalloproteinase domain of human vWF-cp, amplified from the plasmid containing the vWF-cp cDNA gene by using polymerase chain reaction, was cloned into pUC18, and its accuracy was verified by sequencing. Then the domain was inserted into the multiclone site of pET28a(+) and included a 6?His Tag at its amino terminal. After induced by IPTG, the recombinant protein was purified by using a Ni-NTA column and confirmed by Western blot. RESULTS: Comparison of the nucleotide sequence of our cloned domain with the GenBank sequence revealed no difference. High-level expression of the recombinant protein was yielded after 5-hour induction, which amounted to 28% of total bacteria protein in inclusion body. Western blot demonstrated that it possessed high specificity. CONCLUSION: The metalloproteinase domain of vWF-cp was high efficiently expressed in Escherichia coli. This might contribute to the further study of the relationship between its structure and function. [

Construction and Expression of Retroviral Vector Encoding Enhanced Green Fluorescent Protein / 中国实验血液学杂志

Retroviral vectors are wildly used as vehicles for gene transfer into hematopoietic cells based on its potency for efficient gene delivery and integration of transgene in host genome. The development of better transduction protocols depends on gene markers that allow a rapid detection and effective selection of genetically transduced cells. In this study, the enhanced green fluorescent protein (EGFP), a gene that is optimized for detection and expression in mammalian cells, was firstly amplified and cloned by high-fidelity PCR. The vector LGSN carrying EGFP gene was then constructed and the retroviral producer cell lines that yield high titers of LGSN vector in supernatants were developed by liposome-mediated transfection in combination with cross infection. Both GP + envAm12 murine fibroblasts and K562 leukemic cells transduced with EGFP virus demonstrated a stable green fluorescence signal readily detectable by flow cytometry or fluorescence microscopy in up to 97% and 86% of examined cells, respectively. The integration of LGSN provirus in transduced cells was confirmed by PCR analysis. These results indicate EGFP is a suitable reporter molecule for gene transfer and expression in hematopoietic cells. Therefore, the bright and long-term expression of EGFP in living cells will advance the study of gene therapy in vitro and in vivo, particularly for human applications.

Expression of the Wilms' Tumor Gene WT1 and Detection of Minimal Residual Disease in Acute Leukemia / 中国实验血液学杂志

The WT1 gene encoding a zinc finger DNA-binding protein was identified as a tumor suppressor gene being responsible for Wilms' tumor. Recently, aberrant expression of WT1 gene and an inverse correlation between its expression levels and prognosis have been demonstrated in acute myeloid leukemia (AML), suggesting it is a novel tumor marker for leukemic blast cells. To explore whether the WT1 may be used as a marker for detection of minimal residual disease (MRD) in acute leukemia, we examined the sensitivity of the nested reverse transcriptase-polymerase chain reaction (RT-PCR) by using WT1 gene primers in comparison with tumor-specific marker genes, such as PML/RARalpha gene in NB4 cells or bcr-abl gene in K562 cells. In all samples, the integrity of RNA was confirmed by amplification of the c-abl gene as an internal control. The limits in amount of leukemic cells detected by two-step RT-PCR with primers for WT1 or tumor specific fusion gene were 10(-4) and 10(-5) in NB4 cells and 10(-3) to 10(-4) and higher than 10(-6) for K562 cells, respectively. None was WT1 positive in peripheral blood mononuclear cells (MNC) from 29 blood donors, while bone marrow MNCs from eight of 21 cases (38.1%) of nonmalignant patient WT1 gene expression were found. Our results suggested that monitoring of WT1 expression makes it possible to rapidly assess the effectiveness of treatment and follow up MRD in AML cases regardless of the presence or absence of tumor-specific markers.

Protective effect of the bone marrow cells transfected with multidrug resistance gene on the reconstruction of murine hematopoietic function / 中国病理生理杂志

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.

Effects of lovastatin on ras expression and p21~(Ras) membrane localization in human promyelocytic leukemia NB4 cells in vitro / 中国病理生理杂志

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.