ABSTRACT
Objective The study is to present a novel assay for rapid detection of fetal aneuploidies in chorionic villus for spontaneous abortion.Methods Fetal chorionic villus samples were collected from 60 cases of women diagnosed with recurrent spontaneous abortion (RSA) before 13 weeks gestation.All samples were analyzed using CNVplex (copy numbcr variations multiplex) assay and fluorescence in situ hybridization (FISH) in addition to chromosome analysis.All villi specimens were cell cultured and karyotyped to confirm the fetal chromosomal status.Results Among 48 successfully cultured and karyotyped samples,the chromosomal abnormality rate was 60.42 ％.The results of karyotyping and the CNVplex assay were identical,both yielding 20 cases of euploidies,23 autosomal aneuploidies,3 triplodies and 2 × monosomies(Tumer Syndrome).However,FISH obtained only 38 results identical to karyotyping.Two cases of deletion and duplication of chromosome were also identified by CNVplex but not always by karyotyping.As for non-mosaic and non structural abnormity samples,the concordance between cytogenetics and genoty ping was 100％ in CNVplex and 79.17％ in FISH.Conclusion With CNVplex combined with STR(short tandem repeat) assay,we can detect the aneuploidy abnormalities as effectively as routine karyotyping without the need for cell culture,while also analyzing deletions and duplications(larger than 5 Mbp) that are not always detected by karyotype analysis.Our study demonstrates that CNVplex assay is an efficient,convenient,and accurate method to explore the etiology of miscarriage.
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Objective To evaluate the therapeutic effect of hepatocyte growth factor(HGF) gene modified placenta-derived mesenchymal stem cells( PMSCs) on limb ischemia in a rabbit model.Methods The placental tissue was digested with enzyme, cultured and passaged.The PMSCs were characterized by surface marker expression.These cells were infected with adenoviral( Ad)-HGF and intramuscular injected for treatment of limb ischemia in a rabbit model.The blood supply of the limb was detected by digital subtraction angiography ( DSA ) and the vessel number was evaluated in histopathological HE staining.Results The results showed that Ad-HGF gene transduction increased the vascular endothelial growth factor ( VEGF ) , basic fibroblast growth factor, bFGF ( bFGF ) and HGF expression in PMSCs. Transplantation of HGF-transduced PMSCs resulted in the increase in vessel density and improvement of blood supply in the rabbit limb ischemia model.Conclusion The therapeutic effect of HGF gene engineered PMSCs on ischemia by enhancing angiogenesis in a rabbit model is evaluated.Transplantation of PMSCs with HGF gene therapy may be a promising strategy for the treatment of ischemia diseases.
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Objective To investigate the effect of gemcitabine on the cell cycle and apoptosis of the human pancreatic cancer cell line MiaPaCa-2 with Beclinl gene silencing.Methods siRNA targeting at Beclinl gene was constructed,then it was inserted into an expression vector and transfected into MiaPaCa-2 cells.The Beclinl mRNA and protein expression were detected by RT-PCR and Western blot.Gemcitabine was used to treat MiaPaCa-2 with Beclinl gene silencing,then the cell cycle and apoptosis were detected by flow cytometry.Results The MiaPaCa-2 cells with Beclinl gene silencing were successfully constructed,and the expression of Beclin1 mRNA was decreased from 1.0 in control group to 0.295,and number of cells in S and G2 phase was decreased,but number of cells in G1 phase was increased,and there was no change in apoptosis.After gemcitabine treatment,number of cells in S phase was further decreased,but number of cells in G1,G2 phase was increased,and apoptosis was inhibited.Conclusions Beclinl gene silencing can change the cell cycle of pancreatic cancer cells MiaPaCa-2,and influence the effects of gemcitabine on cell cycle and apoptosis.
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Objective To study the effect of heavy ion radiation on proliferation and apoptosis of human peripheral blood derived T lymphocytes and the mechanism .Methods T lymphocytes were isolated from heparinized whole blood samples by density gradient centrifugation using Ficoll before being irradiated with heavy ion beams 12 C.The accumulated absorbed dose (dose-rate values=0.5 Gy/min, and meanLET=29 keV/μm).12 h and 24 h post-infection, total RNA of T lymphocytes was isolated , and the apoptosis related gene expression , including Bcl-2, Bax, Caspase3, Caspase8 and Caspase9, was detected by RT-RT-PCR.24 h and 48 h after irradiation, the proliferation was analyzed by CCK 8 kit.The cell apoptosis was detected by flow cytometry after being labeled with AnnexinV-PE/7-AAD or AnnexinV-FITC/PE.The expression of Bcl-2, Bax and Caspase3 was also assayed by RT-PCR.Results Data showed that heavy ion radiation could inhibit the proliferation of T lymphocytes obviously , and the inhibition ratio in cells that received 2 Gy dose was much high-er than in cells that received 1 Gy dose.Furthermore, heavy ion radiation promoted the apoptosis of T lymphocytes signifi-cantly.The results of RT-PCR showed that the mRNA expression of Bcl-2 was down-regulated in heavy ion radiation T lym-phocytes while the expression of Bax and Caspase 3 was up-regulated.Conclusion Heavy ion radiation can inhibit the pro-liferation and promote the apoptosis of human peripheral blood derived T lymphocytes .
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Objective To construct a prostate cancer specific oncolytic adenovirus armed with a fusion protein gene , PSA-IZ-CD40L, and to evaluate its oncolytic efficiency and immune activation ability in vitro.Methods Prostate Specific Antigen (PSA) gene, CD40L-N and CD40L-C genes were obtained from cDNA of LNCaP cells and Jurkat cells using poly-merase chain reaction (PCR) or nested-PCR, respectively.PSA,Linker,CD40L-N and CD40L-C were linked sequentially to generate fusion protein gene PSA-IZ-CD40L (PL) by overlapping PCR.Then, prostate specific oncolytic adenovirus PL-carrying gene, Ad-PL-PPT-E1A,was constructed using the oncolytic adenovirus system , which was based on Adeasy sys-tem.PC3M cells were infected by Ad-PL-PPT-E1A at serial multiplicity of infection (MOI), and the apoptosis was detec-ted by flow cytometry at several time points post-infection.For immune activation detection , PC3M cells were infected with Ad-PL-PPT-E1A at a MOI of 50, and the cell lysate was collected at 48 h post-infection.Peripheral blood mononuclear cells derived (PBMCs) from healthy donors were stimulated by the lysate from PC 3M cells or Ad-PL-PPT-E1A infected PC3M cells before proliferation was assayed using cell counting kit-8 (CCK8).Results Fusion protein gene, PSA-IZ-CD40L, was successfully constructed and cloned into the prostate cancer specific adenovirus to generate Ad -PL-PPT-PL. The expression of E1A and PL protein could be detected by reverse transcription PCR and Western-blotting.Cytopathic effect was observed in PC3M cells infected with Ad-PL-PPT-E1A.Furthermore, the apoptosis rate reached 70.67% ± 2.98%at 48 h post-infection with 200 MOI Ad-PL-PPT-E1A.Compared with the lysate of PC3M cells, that from Ad-PL-PPT-E1A infected cells could promote the proliferation of PBMCs .Conclusion We have constructed a prostate cancer spe-cific oncolytic adenovirus armed can fusion protein gene PL , Ad-PL-PPT-E1A, which could kill PC3M cells effectively and enhance the proliferation of PBMCs in vitro.