ABSTRACT
Objective To detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand (hTRAIL) in vivo,by using a novel double expressing adenoviral vector encoding hTRAIL and firefly luciferase ( luc ) gene ( ad -luc-hTRAIL),in which luc was used as reporter gene.Methods Lung cancer A549 cell xenografts in 16 nude mice models were established in subcutaneous inoculation way,the adenovirus vectors ( ad-luc-hTRAIL,ad-hTRAIL,ad-luc) and phosphate buffer saline (PBS) (n =4) as control were injected into tumor respectively.The size of the tumor was measured at different time points (4,7,10,14,21,28 d)after injection.The activity of luciferase in surface of the tumor was detected in vivo by using high-sensitivity cooled-charged coupled device(CCD) camera.The expression of hTRAIL was demonstrated by immunohistochemistry staining after sacrificing the animals at different time points,and immunohistochemical scores (IHS) were measured. The apoptosis rate of tumor cells was detected by using TUNEL and calculated. Analysis of variance,the paired t test and linear correlation analysis was used for the statistics. Results The growing speed of tumour xenografts was more slowly in ad-luc-hTRAIL and ad-hTRAIL groups than PBS group (t =2.71,2.72,P ＜ 0.05 ).The tumor volumes of ad-luc-hTRAIL,ad-hTRAIL,ad-luc and PBS groups 28 days after injection were (208.4 ± 42.3 ),( 181.5 ±23.9),( 403.1 ± 54.0 ) and ( 427.0 ± 59.3 ) mm3, respectively. There was no significant difference between ad-luc group and PBS group(t =2.07,P ＞ 0.05).The expression of luciferase in ad-luc-hTRAILgroup reached its peak at 7th day ( 1.37 ± 1.04),and then decreased quickly.The IHS and apoptosis rate in ad-luc-hTRAIL and ad-hTRAIL groups reached their peaks at 7th day,the peak values of IHS were 6.25 ±2.06 and 6.5 ± 2.89,the peak values of apoptosis rate were (60.75 ± 8.06 ) ％ and ( 61.50 ± 8.47 ) ％,respectively.The amount of luciferase expression ( absolute number of photons detected by CCD camera)was linear positive correlated with IHS and apoptosis rate ( rphotons/IHs =0.942,rph /rate =0.842,rIHs/rate =0.887,P ＜ 0.05 ).Conclusion The target gene hTRAIL can be transfected into lung cancer A549 cell xenografts nude mice models efficiently with a high level expression,and the therapeutic effect of hTRAIL can be monitored by detecting the expression of luc.
ABSTRACT
Objective To investigate the expression of exogenous gene transferred by piggyBac (PB) transposon in various gynecological malignant cell lines and reveal its potential application of gene therapy in gynecological cancer.Methods Amplified herpes simplex virus thymidine kinase (HSV-tk) gene coding region by PCR and integrated it into PB expression vector, PB[Act-RFP]DS, for reconstructing PB[Act-RFP, HSV-tk]DS (pPB/TK).By using different transfection reagents: FuGENE HD, jetPEI, lipofectamine 2000, pPB/TK together with helper plasmid Act-PBase were cotransfected into four mostly common gynecological malignant tumor cell lines HeLa, JEG-3, SKOV3 and HEC-1B.The mRFP1 report gene expressions was observed and detected by fluorescence microscope and flow cytometry to analyze transfection efficiency.The expressions of HSV-tk and mRFP1 gene were detected by reverse transcription PCR (RT-PCR).The cytotoxic effect of various concentration of pro-drug ganciclovir (GCV) for transfected cells was detected by methyl thiazole tetrazolium assay.The transfected cells were positive sorted by flow cytometry and limiting diluted to obtain the stable transfected cell line.The insertion sites of foreign gene tranferred by PB transposon in genome were analyzed by inverse PCR.Results (1) Double digests analysis and sequences test demonstrated that pPB/TK vector was reconstructed successfully.(2) Using three different transfective reagents, PB trausposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1 B, SKOV3 and JEG-3 cell efficiently, and the transfection efficiency of pPB/TK for the same cell was different by using different transfective reagents; in Hela cell, the transfection efficiency of FuGENE HD [(78.7 ± 9.2) %]was higher than that of lipofectamine 2000 [(54.1 ± 11.4) %]and jetPEI [(46.5 ± 7.4) %, all P < 0.05] ; using the same transfective reagent, the transfection efficiency of pPB/TK was also different on various cell lines, using FuGENE HD, the transfeetion efficiency of pPB/TK on HeLa, JEG-3 and SKOV3 cell was (78.7 ± 9.2) %, (74.4 ± 8.9) % and (83.2 ± 9.7) % respectively, which all were higher than that on HEC-1B [(39.5 ± 8.7) %, P < 0.05] .(3) RT-PCR showed that there were the mRNA expression of HSV-tk and mRFP1 in all cell lines.(4) 50% inhibitory concentration of GCV for transfected cells, HeLa, JEG-3, SKOV3 and HEC-1B, was 1.29, 3.35, 0.09 and 13.28 μg/ml respectively.Inhibitory effect of GCV (10 μg/ml) on SKOV3 transfected with pPB/TK was (86 ± 9) %, which was superior to that transfected with pORF-HSVtk alone [(52 ± 12)%, P < 0.05] .(5) The insertion sites of PB transposon in the target cells genome were located at TTAA sites, mRFP1 expression still could be detected in three months after transfected.Conclusions PB transposon could transfer exogenous gene into various gynecological malignant cells, which could integrated into genome and obtain a long-term and stable expression.It is expected that PB transposon may supply a more efficient and safer transgene technology platform for gene therapy in gynecological cancer.
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Objective To develop transgenic mice harboring the fusion gene of mutant amyloid precursor protein and two types of fluorescent protein for the future study on Alzheimer's disease.Methods The fusion gene CFP-54 bp-YFP-C99 was introduced into mice by mieroinjection.The presence of CFP-54 bp-YFP-C99 was confirmed by PCR in the founders.Results CFP-54 bp-YFP-C99 gene was injected into pronucleus of 2202 zygotes and 1806 injected eggs were implanted into 56 foster mothers, 13 of which were pregnant.There were 13 foster mothers who borne 52 offspring and 32 of them survived.Recipient mouse pregnancy rate was 23.2% (13/56) and the integration rate was 3.9% (2/52).Conclusion CFP-54 bp-YFP-C99 transgenic mice is obtained, but the transgenic efficiency is low.
ABSTRACT
Objective To demonstrate the superiority and feasibility of using bioluminescent image to monitor the islet graft after islet transplantation.Methods Diabetic models were established by intraperitoneal injection of streptomycin into mature male C57BL/6 mice.Islets were harvested from the pancreas of C57BL/6 and Bclb/c mice by digestion and purification,and transfected with Lueiferase gene.The mouse diabetic models were divided into iso-transplantaion group (n=20) and allo-transplantation group (n=7).The islets of C57BL/6 were transplanted into iso-transplantaion mice with different doses (10,50,110 and 200,n=5 in every dose),and Bclb/c mouse islets were transplanted into allo-transplantation group.The islets were transplanted into the subcutaneous fat tis- sue near left scapula.The receptor mice were scanned with CCD camera to get bioluminescent images at different scheduled time points,and the changes in random blood glucose of allo-transplantation group were observed.Results On day 6 after transplantation,the scanning image showed that the pi- xel intensity from the region of interest (ROI) was increased with the increased number of islet grafts and they had a positive correlation.The random blood glucose was reduced to the normal level in the first 2 days,and then increased again to the diabetic level on 11 days averagely,while pixel intensity from the ROI reached the peak on day 6-7,and then reduced rapidly after islet transplantation in allo- transplantation group.The beginning of pixel intensity reduction occurred on day (6.14?0.90), while that of the random blood glucose raise occurred on day (10.00?0.82) after transplantation,and the former alteration occurred obviously earlier than the latter (P