Nianfeng Sun

The research of nanoparticles as gene vector for tumor gene therapy

With the development of molecular biology, the application of the gene therapy becomes a tendency in the development of oncotherapy. The gene therapy has been acknowledged as the major progress of modern medicine, also a focus in the oncotherapy research. Commonly vectors of the gene therapy mainly include two categories, namely, viral vectors and nonviral vectors. Nanoparticles gene vector of various different kinds of materials, which belong to non-viral carriers. It presents excellent abilities of adsorption, concentration and protection of DNA, which can be attributed as a main reason of the adsorption and operation of nano-gene vector on exogenous genes. In this article, we mainly reviewed the recent studies of the characteristics of nanoparticles, characteristics and transport mechanism of nanoparticles as gene vector, the progress on nanoparticles as gene vector in tumor gene therapy. Nano-gene vectors, as new drug and gene carriers, present characteristics such as the controlled-release, targeting, and the improvement of bioavailability. Nanoparticles for cancer imaging and therapy have evolved rapidly during the last decade and it is expected that more and more will become clinical practise. In the near future, as a new nanometer gene delivery vector will be in medical research and treatment play a bigger role.

Expressions of the anti-apoptotic genes Bag-1 and Bcl-2 in colon cancer and their relationship

BACKGROUND The aims of this study were to investigate the expressions and significance of the antiapoptotic genes Bag-1 and Bcl-2 in colon cancer and to evaluate their relationship. METHODS The expressions of Bag-1 and Bcl-2 were examined in 128 colon cancer and 20 normal colon tissue samples by reverse-transcription polymerase chain reaction and immunohistochemical technique (streptavidin-biotin-peroxidase complex method). RESULTS Bag-1 and Bcl-2 were expressed in colorectal cancer tissues but not in normal colorectal tissues by reverse-transcription polymerase chain reaction. The expression of Bag-1 in colon cancer was closely correlated with pathologic grade, distance metastasis, Duke stage, and prognosis, but it had no effect on the pathologic type, tumor diameter, depth of invasion, and lymphoid node metastasis of the cancer. By contrast, Bcl-2 had no significant correlation with all the clinical and pathologic factors. There was a positive correlation between Bag-1 and Bcl-2 in the development of colon cancer. CONCLUSIONS High expressions of Bag-1 and Bcl-2 proteins in colon cancer were found. They might be regarded as biomarkers for the diagnosis of the early stage of colon cancer. In addition, they have significant relevance for the prognosis of colon cancer.

Treatment of Budd-Chiari syndrome with inferior vena cava thrombosis

The aim of this study was to evaluate the initial results of 41 patients with Budd-Chiari syndrome (BCS) with inferior vena cava (IVC) thrombosis, with regard to the clinical safety and feasibility of the therapeutic approaches selected according to the classification of the condition. Forty-one patients with BCS and IVC thrombosis were admitted for retrospective analysis. All 41 patients were classified as having one of three types of BCS. Interventional therapy was used successfully in 28 patients (68.3%), 7 patients (17.1%) were given conservative treatment and 6 patients (14.6%) were treated with surgical shunts. The interventional approach was used in 29 patients in total and was successful in 28 patients (all those of types I and II, and 3 of the 4 patients of type III with acute thrombosis; 96.6%). None of these 28 patients had pulmonary embolism, pericardial tamponade or intra-abdominal bleeding. After 1–5 years, 4 patients (9.8%) had a second dilation of the IVC. In the 7 cases treated in a conservative manner, 2 cases succumbed to upper gastrointestinal bleeding and 1 case succumbed to liver and kidney failure. This study indicates that the classification of BCS patients with IVC thrombosis is helpful in selecting a therapeutic approach. Interventional therapy is the first therapeutic choice for BCS patients with IVC thrombosis of type I, type II or type III with acute thrombosis. For the patients of type III with an obsolete thrombus, surgical shunts or conservative treatment are the main therapeutic methods.

Nanoliposome-mediated FL/TRAIL double-gene therapy for colon cancer: In vitro and in vivo evaluation

OBJECTIVE To investigate the therapeutic effects of cationic nanoliposome-mediated gene therapy combined with immunotherapy for colon cancer treatment. METHODS Recombinant plasmids containing green and red fluorescent protein reporter genes were constructed using gene cloning methods. Gene-carrying cationic nanoliposomes were prepared based on the electrostatic adherence principle and then transfected into dendritic cells (DC), which were transplanted into colon cancer cells. RESULTS Recombinant plasmids containing green or red fluorescent protein reporter genes were successfully constructed by gene cloning and confirmed by restriction enzyme digestion and sequencing. Gene-carrying cationic nanoliposomes were transfected into colon cancer cells, and good gene expression was detected. A better level of apoptosis was observed in the combined group of tyrosine kinase receptor 3 ligand (FL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), while the lowest level was detected in the control group. The parameters in the FL and TRAIL groups were between the above-mentioned combined group. CONCLUSION Cationic nanoliposomes have the advantage of being gene carriers. The joint therapeutic effects of the two genes are superior to those of a single gene. Gene therapy combined with immunotherapy has significant implications for cancer treatment.

Coexpression of recombinant adenovirus carrying GDNF and EDNRB genes in neural stem cells in vitro

Gene therapy and nerve stem cells isolated from the developing human enteric nervous system (ENS) are significant. They may open the route for the cell therapy of Hirschsprungs disease (HD). We have constructed the recombinant adenovirus‐carrying glial cell line‐derived neurotrophic factor (GDNF) and endothelin receptor B (EDNRB) gene, and investigated the exosomatic coexpression in neural stem cells. The recombinant adenovirus Ad‐GE coexpressing GDNF and EDNRB gene was constructed by the AdEasy system and confirmed by the reverse transcription polymerase chain reaction (RT‐PCR) method. Expression of exogenous genes in neural stem cells after transfection was confirmed by the flow cytometry and real‐time fluorescence quantitative PCR. Fragments of pAd Track‐CMV‐GE were consistent with GDNF and EDNRB. The green fluorescence of the positive cells was followed by fluorescence microscopy at 24 h after NSCs had been transfected, reaching a peak at 72 h after transfection. Flow cytometry showed that the efficiency of transfection was 15.0, 23.6, and 25.4% at 24, 48 and 72 h respectively. Real‐time fluorescence quantitative PCR showed the expression levels of mRNA of GDNF and EDNRB in 48 and 72 h groups were obviously higher than that in 24 and 96 h groups. Recombinant adenovirus carrying GDNF and EDNRB genes are coexpressed in neural stem cells, which may offer the possibility of a novel approach to local combination gene therapy for Hirschsprungs disease.

Antiapoptotic gene BAG-1 vector structure of RNA interference and endogenous targeted screening in colon cancer cell lines

The purposes of the present work were to construct the shRNA plasmids for BAG-1 gene of human and test the expression of mRNA and protein of BAG-1. Recombinant plasmids containing green fluorescent protein reporter genes are constructed using gene cloning methods. The shRNA plasmids for the BAG-1 gene are constructed by RNA interference technology. We applied fluorescent plasmid-transfected target cells in the cell transfection experiments and monitored the transfection rate of plasmids by observing the fluorescence amount. We transfected three synthesized shRNA in target screening cell and adopted RT-PCR and Western test to identify the difference of target gene transfection and translation level in cells. The specific shRNA plasmid for the BAG-1 gene was successfully recombined, and stably transfected colon cancer Lo Vo cell lines were obtained. The results present that the constructed shRNA plasmids significantly inhibited the expression of mRNA and protein of Lo Vo cell BAG-1, and can maintain the effect for a long term. pGPH1/GFP/Neo-BAG-1-homo-825 was screened as the optimum sequence of interference so as to lay a solid foundation to explore into the research on the BAG-1 gene and the biological behavior of colon cancer cells. It showed the remarkable advantage of RNAi in the generation of posttranscriptional gene silencing.

Magnetic gold nanoparticle-mediated small interference RNA silencing Bag-1 gene for colon cancer therapy

Bcl-2-associated athanogene 1 (Bag-1) is a positive regulator of Bcl-2 which is an anti-apoptotic gene. Bag-1 was very slightly expressed in normal tissues, but often highly expressed in many tumor tissues, particularly in colon cancer, which can promote metastasis, poor prognosis and anti-apoptotic function of colon cancer. We prepared and evaluated magnetic gold nanoparticle/Bag-1 siRNA recombinant plasmid complex, a gene therapy system, which can transfect cells efficiently, for both therapeutic effect and safety in vitro mainly by electrophoretic mobility shift assays, flow cytometric analyses, cell viability assays, western blot analyses and RT-PCR (real-time) assays. Magnetic gold nanoparticle/Bag-1 siRNA recombinant plasmid complex was successfully transfected into LoVo colon cancer cells and the exogenous gene was expressed in the cells. Flow cytometric results showed apoptosis rate was significantly increased. In MTT assays, magnetic gold nanoparticles revealed lower cytotoxicity than Lipofectamine 2000 transfection reagents (P<0.05). Both in western blot analyses and RT-PCR assays, magnetic gold nanoparticle/Bag-1 siRNA recombinant plasmid complex transfected cells demonstrated expression of Bag-1 mRNA (P<0.05) and protein (P<0.05) was decreased. In further study, c-myc and β-catenin which are main molecules of Wnt/β‑catenin pathway were decreased when Bag-1 were silenced in nanoparticle plasmid complex transfected LoVo cells. These results suggest that magnetic gold nanoparticle mediated siRNA silencing Bag-1 is an effective gene therapy method for colon cancer.

Correlation between the expression of the BAG-1 gene and clinicopathologic factors in colorectal cancer.

ObjectiveThe present study aims to investigate the expression and significance of the anti-apoptotic gene Bag-1 in colorectal cancer and to evaluate the relationship between the gene and the disease.MethodsBag-1 expression was examined in 320 colorectal cancer and 30 normal colorectal tissue samples using reverse transcriptase polymerase chain reaction (RT-PCR) and the immunohistochemical staining (streptavidin–biotin-peroxidase complex method.ResultsUsing RT-PCR, Bag-1 was observed to be expressed in colorectal cancer tissues, but not in normal colorectal tissues. The expression of Bag-1 in colorectal cancer was closely correlated with pathologic grade, distant metastasis, Dukes stage, and prognosis, but it was not correlated with the pathologic type, tumor diameter, depth of invasion, and lymph node metastasis.ConclusionBag-1 protein was found to be overexpressed in colorectal cancer. They might be regarded as a biomarker for the diagnosis of the early stages of colorectal cancer. In addition, they have particular significance for the prognosis of colorectal cancer.

Preparation and Characterization of Nano-Liposome-Mediated FL Gene in the Lovo Cells

AIMS The purpose of the present work was to formulate and evaluate cationic nano-liposomes as novel nonviral gene delivery for colon cancer treatment. METHODS Recombinant pEGFP-c1-Fms-like tyrosine kinase receptor 3 ligand (FL) plasmids containing human FL gene and green fluorescent protein (GFP) reporter genes were constructed. FL and GFP Gene-carrying cationic nano-liposomes were prepared based on the electrostatic adherence principle and then transfected into Lovo cells. The morphology, particle size, and zeta potential of gene-carrying cationic nano-liposomes were observed using an electron microscope. GFP expression was observed by fluorescence microscopy to assay the transfection efficiency. The cytotoxicity of FL/nano-liposomes was evaluated by the MTT method. RESULTS Recombinant plasmids pEGFP-c1-FL are successfully constructed using gene cloning methods and confirmed by restriction enzyme digestion and sequencing. The cationic nano-liposomes carrying pEGFP-cl-FL were observed by an electron micrograph and showed uniform spherical or elliptical shapes and many pores. The fluorescence microscopy images of gene-carrying cationic nano-liposomes showed good expression of GFP in pEGFP and pEGFP-cl-FL groups. The MTT assay of cell death indicated a significantly higher level of cell death between the FL group and the control group at 24, 48, and 96 hours after transplantation. CONCLUSION Cationic nano-liposomes show safe and high-performance transfection as gene carriers. Gene therapy has significant implications for colon cancer treatment in future.

Surgical and endovascular treatment of thoracic aortic dissection combined with aberrant right subclavian artery aneurysm

Aortic dissection combined with aberrant right subclavian artery (ARSA) is very rare. The endovascular treatment of aortic dissection is less invasive than conventional open‐heart surgery because of lower surgical risk and faster recovery. In the present work, the origin of ARSA was dilated, and the aorta was also dissected down to the abdominal aorta. The aortic true lumen became narrow, but the visceral arteries had good perfusion. The patient was treated on the entry of dissection with endovascular isolation and adjunctive surgical bypass after controlling blood pressure for 2 weeks.