Fluorescent supermagnetic nanoparticles-labeled adipose-derived mesenchymal stem cells in the three-dimensional culture system / 中国组织工程研究

BACKGROUND:Fluorescent magnetic nanoparticles with the properties of quantum dots and magnetic particles have good biocompatibility, and can label cels effectively through endocytosis. OBJECTIVE:To validate the feasibility of fluorescent magnetic nanoparticles in labeling human adipose-derived mesenchymal stem cels. METHODS:Healthy human adipose tissue was extracted and adipose-derived mesenchymal stem cels were isolatedin vitro by type I colagenase digestion. Passage 6 cels were incubated with the fluorescent magnetic nanoparticles overnight. Prussian blue staining and laser scanning confocal microscope were used to observe labeled adipose-derived mesenchymal stem celsin vitro after co-culturing with fluorescent magnetic nanoparticles. The tracing effect of labeled adipose-derived mesenchymal stem cels in vivo was detected by fluorescence imaging system. RESULTS AND CONCLUSION:Prussian blue staining showed that the fluorescent magnetic nanoparticles dispersed in the cytoplasm of adipose-derived mesenchymal stem cels in the form of blue particles. Under the laser scanning confocal microscope, the nuclei of adipose-derived mesenchymal stem cels were dyed blue by Hoechest33258, and the cytoplasm was dyed green. The fluorescence imaging results showed that labeled human adipose-derived mesenchymal stem cels had good imaging results. Therefore, as an efficient tracer, the fluorescent magnetic nanoparticles can label human adipose-derived stem cels in vitro and provide a new method for transplantation and transformation of adipose-derived mesenchymal stem cels.

Systematic establishment for directed differentiation of induced pluripotent stem cells into hematopoietic progenitor cellsin vitro / 中国组织工程研究

BACKGROUND:There have been a large number of reports on establishing induced pluripotent stem celllines, but studies concerning large-scale in vitro induced differentiation of induced pluripotent stem cels into hematopoietic progenitor cels stil have a lack of in-depth discussion. OBJECTIVE:To develop methods to induce differentiation of induced pluripotent stem cels into hematopoietic progenitor cels in vitro. METHODS: Using the method of infection with lentivirus particles containing four transcriptionfactor genes, which are Oct4, Sox2, Nanog and Lin28, human skin fibroblasts are transduced into induced pluripotent stem cels. In the induced differentiation system, Y-27632, a kind of tyrosine kinase inhibitor-ROCK (p160-Rho-associated coiled-coil kinase), was added, which obviously suppressed apoptosis of cels. Based on conditioned medium with OP9 cels, a differentiation system of inducing induced pluripotent stem cels differentiating into hematopoietic progenitor cels was established. RESULTS AND CONCLUSION:(1) Apoptosis of induced pluripotent stem cels at the first three passages was very obvious, and the cels were difficult in a large-scale expansion. After Y-27632 was added, the apoptosis of embryonic stem cels was obviously inhibited. (2) During embryoid body differentiation, induced pluripotent stem cels cultured in OP9 conditional growth medium differentiated into hematopoietic progenitor celsin vitro that were positive for CD34.

Fluorescent signal detection of chromatographic chip by algorithms of pyramid connection and Gaussian mixture model / 中国医疗器械杂志

We proposed a new algorithm for automatic identification of fluorescent signal. Based on the features of chromatographic chips, mathematic morphology in RGB color space was used to filter and enhance the images, pyramid connection was used to segment the areas of fluorescent signal, and then the method of Gaussian Mixture Model was used to detect the fluorescent signal. Finally we calculated the average fluorescent intensity in obtained fluorescent areas. Our results show that the algorithm has a good efficacy to segment the fluorescent areas, can detect the fluorescent signal quickly and accurately, and finally realize the quantitative detection of fluorescent signal in chromatographic chip.

Influence of the inhibitory oligonucleotides on the immune status of BXSB lupus mice / 中国基层医药

Objective To research whether the inhibitory oligonucleotides could improve the immune status of the BXSB mice. The purpose was to provide a valuable direction for treating systemic lupus erythematosus. Methods 3-month-old BXSB lupus mice were divided into three groups (including inhibitory oligonucleotides group, saline group and blank control group). The 24 hours urine proteins were determined before treatment. After treatment,the urine protein, anti-dsDNA, peripheral blood lymphocytes apoptosis and immune complex in renal glomeruli were measured. Results Before treatment,the urine proteins had no statistical differences among the three groups ( P > 0.05 ).After treatment,the urine protein, anti-dsDNA levels in inhibitory oligonucleotides group were significantly lower than that of control group(P < 0.01 ). Apoptosis of peripheral blood lymphocytes in inhibitory oligonucleotides group was significantly higher than that of control group( P < 0.05 ) ,immune complex in renal glomeruli in inhibitory oligonucleotides group was significantly lower than that of the other groups ( P < 0.05 ). Compared with saline group, inhibitory oligonucleotides had prolonged life period of BXSB mice ( P < 0.01 ). Conclusion The inhibitory oligonucleotides could improve immune status of BXSB, and could put off the disease progression.

Preparation of human malignant melanoma ganglioside ScFv antibody-conjugated quantum dot nanoprobe and its specific binding with human malignant melanoma cells / 中国肿瘤生物治疗杂志

Objective: To prepare a nanoprobe, anti-human melanoma ganglioside single chain variable fragment (GD/ScFvMEL) antibody conjugated with CdTe quantum dot, and to observe its ability to specifically bind human malignant melanoma cells. Methods: The GD/ScFvMEL gene was cloned into pET32a (+), and the plasmid was then transformed into E. coli BL21 (DE3) for GD/ScFvMEL protein antibody expression. The expressed GD/ScFvMEL antibody was purified by denaturing method and further refolded by modified dialysis method. The purified GD/ScFvMEL antibody was analyzed by SDS-PAGE. The GD/ScFvMEL-QDs nanoprobe was prepared by conjugating GD/ScFvMEL antibody with CdTe quantum dot, and its specificity was observed by incubating with MGC-803 cells and melanoma A375 cells. Results: The recombinant pET32a-GD/ScFvMEL was constructed and confirmed by PCR, restriction endonuclease analysis and DNA sequencing. The proportion of expressed GD/ScFvMEL antibody in total bacteria proteins was about 40% as detected by SDS-PAGE. The purified- and refolded-GD/ScFvMEL antibody was effectively conjugated with CdTe quantum dot, and the resulting GD/ScFvMEL-QDs nanoprobe was successfully prepared. The GD/ScFvMEL-QDs nanoprobe could specifically bind melanoma A375 cells, but could not bind stomach cancer MGC-803 cells. Conclusion: We have successfully prepared an anti-human melanoma ganglioside single-chain antibody-CdTe quantum dot nanoprobe, which can specifically bind melanoma cells.

Cloning, expression and purification of novel gene NBEAL1 and its relationship with pathological grades of glioma / 中国肿瘤生物治疗杂志

Objective: To construct the expression plasmid of a novel gene human NBEAL1 (neurobeachin like 1), and to study its relationship with the pathological grades of glioma. Methods: Total RNA of human glioma cell line U251 was extracted. NBEAL1 expression plasmid pGEX-KG/NBEAL1 was constructed and transferred into E. coli BL21. Recombinant NBEAL1 protein was induced by IPTG and further purified by GST affinity chromatographic column. The purity of recombinant NBEAL1 protein was examined by Western blotting analysis. A NBEAL1 protein specific monoclonal antibody was prepared and was used to study the relationship of NBEAL1 expression with pathological grades of glioma. Results: The NBEAL1 gene fragment was successfully cloned into pGEX-KG expression plasmid and verified by DNA sequencing. The recombinant NBEAL1 protein was expressed in inclusion bodies, with a yield of more than 30% of total bacterial proteins; the purity of purified NBEAL1 protein was above 95%. Western blotting analysis confirmed that the purified protein containing GST tag and NBEAL protein. NBEAL1 protein was lowly expressed in normal brain tissues and highly expressed in low grade glioma tissues; and the expression of NBEAL1 decreased with the increase of glioma malignancy. Conclusion: The NBEAL1 protein has been successfully cloned, expressed and purified. NBEAL1 protein expression in glioma tissues is negatively associated with the pathological grades of glioma.

Antitumour effects of survivin antisense oligonucleotide carried by nano-high-polymer on colorectal cancer / 中国组织工程研究

BACKGROUND: In recent years, nano-carriers have been regarded as the most promising technologies for breakthrough the bottleneck of gene transfer. Polyamidoamine dendrimer (PAMAM) is a kind of new nanometer material. PAMAM can transfer target gene to the cell with high efficiency and lower toxic both in vivo and in vitro.OBJECTIVE: To evaluate the antitumour effects of survivin antisense oligonucleotide (Survivin-asODN) carried by PAMAM on colorectal cancer transplanted subcutaneously in nude mice.DESIGN, TIME AND SETTING: An in vivo experiment regarding tumor gene therapy was performed from February to August in 2008 at the Laboratory of Bionanometer Engineering, Research Institute of Micro/nanometer Science & Technology of Shanghai Jiao Tong University and Central Laboratory of Zhujiang Hospital of Southern Medical University.MATERIALS: Human colorectal cancer cells SW620 were from Shanghai Cell Institute of Chinese Academy of Sciences.PAMAM dendrimer was offered by the Bionanometer Engineering Laboratory, Research Institute of Micro/nanometer Science & Technology, Shanghai Jiao Tong University. Lipofectamine ~(TM)2000 was purchased from Invitrogen, USA. Survivin-asODN was synthesized by Shanghai Bioengineering Company.METHODS: The PAMAM and cation liposome were respectively mixed with Survivin-asODN to generate the transfection complex carrying antisense gene. The shape of the complex was observed by transmission electron microscope, the particle size was determined by laser particle size analysator and the zeta potential was measured by an analytical tool. The encapsulating efficiency and release progress in vitro were determined by ultraviolet spectrophotometer in centrifuging method. Human colorectal cancer cells SW620 at logarithmic phase were inoculated into the abdominal region of 18 Blab/C nude mice subcutaneously to produce transplanted tumor models in colorectal cancer nude mice, which were randomly divided into 3 groups: liposome, PAMAM and blank control groups. They were injected respectively with Hposome-survivin-asODN complex,PAMAM-survivin-as ODN transfection complex and seroculture liquid. The volumes of tumor were surveyed in the 2 groups.Western blotting method was used to determine the survivin gene expression in the transplanted tumor tissue.MAIN OUTCOME MEASURES: Particle size, zeta potential, gene loading level, encapsulation efficiency, release rate of cationic liposome-survivin-asODN complex and PAMAM-survivin-asODN complex, as well as survivin expression rate and apoptosis rate after transfection, inhibition rate of the transplanted tumor growth, Survivin protein expression and activity in the transplanted tumor cells.RESULTS: The particle size of PAMAM-survivin-asODN complex was smaller (P < 0.01), but the zeta potential was greater (P < 0.05), compared with liposome-survivin-asODN. There was no significant difference between PAMAM and liposome groups in terms of gene loading rate and transfection efficiency. DNA release lasted for 14 days for PAMAM, but only 5 days for liposome.After colorectal cancer cell transfection, survivin protein expression was lower, but apoptosis rate was higher, in the PAMAM-survivin-asODN complex than in the liposome-survivin-asODN complex (P < 0.05).CONCLUSION: PAMAM facilitates delivery of Survivin-asODN into transplanted colorectal cancer cells SW620. As a result,survivin protein expression was decreased, and apoptosis rate was increased in vivo which inhibited transplanied tumour growth.

Inhibitory effects of BRCAA1 gene silencing on gastric cancer MGC-803 cells and its possible mechanism / 中国肿瘤生物治疗杂志

Objective:To investigate the inhibitory effect of breast cancer-associated antigen 1(BRCAA1)gene silencing on gastric cancer MGC-803 cells and the related mechanism.Methods:Plasmid shRNA-BRCAA1 and shRNA-N were constructed and transfected with FuGene HD into gastric cancer cell line MGC-803.The transfection efficiency was examined using fluorescent microscope 24 h later.The total RNAs was extracted 48 h 'after transfection and the expression of BRCAA1 and GAPDH gene were analyzed by real-time PCR.The cell proliferation was assessed by MTT assay 24 h,48 h,and 72 h after transfection.The cell apoptosis was determined by Annexin V-PE/TAAD.The expression of Rb,Bax, Bcl-2 and BRCAA1 proteins was analyzed by Western blotting 48 h after transfection.Results:We found that the transfection efficiency of shRNA-BRCAA1 was(81.2?2.6)%24 h after transfection.Forty-eight hours after transfection with shRNA-BRCAA1 the expression of BRCAAI mRNA decreased by 61.4%;the inhibition rate of MGC-803 cells growth was 45.0%.The cell apoptosis rate of shRNA-BRCAA1 transfection group was significantly higher than those of untransfected group and mock plasmid transfected group([14.4?1.6]%vs[5.4?2.0]%,[4.4?2.5]%,P

Advance and prospects of nanoscale diagnosis and therapy technology for tumors / 中国肿瘤生物治疗杂志

Nanomaterials,endowed with unique properties,bring new chance for early diagnosis and therapy of tumors and show a bright future.In recent years,research in in vitro detection techniques of nanoparticle-based early tumor biomarkers,in vivo dynamic multimodel molecular imaging techniques,nanoscale effects of nanopartilce-based nano-imaging and treatment integrated technology,nanoscale slow-release drugs,and nanoscale drug delivery devices has made great progress.However,the long term interaction between nanomaterials and human body still remains poorly understood,the application of nanoscale therapy technology in clinical medicine still faces new challenges.In this paper,we review the main advances of tumor nanoscale diagnosis and therapy,with the aim to improve the development of tumor nanoscale diagnosis and therapy technology in China.

Nanocomoposite probes composed of fluorescent magnetic nanoparticles and PSA ScFv antibody for targeted imaging and therapy of prostate cancer / 中国肿瘤生物治疗杂志

Objective:To investigate the feasibility of targeted imaging and therapy of prostate cancer using nanocomposite probes composed of fluorescent magnetic nanoparticles(FMCNPs) and single chain Fv(ScFv) antibody specific for gama-seminoprotein.Methods:The nanocomposite probes(FMCNPs-ScFv) were prepared by conjugating fluorescent magnetic nanoparticles with singlegama-chain Fv antibody specific gama-seminoprotein,and were characterized by high resolution transmission electron microscopy,fluorescent spectrum and magnetic spectrum.Nanocomposite probes were incubated with prostate cancer LNCaP cells,and the targeting results of nanocomposite probes were observed by fluorescent microscopy.The cytotoxicity effect of the nanocomposite probes was measured by MTT.Nude mice models of prostate cancer were established and identified by immunohistochemistry method.The nanocomposite probes were injected into nude mice via tail vein.The distribution of nanocomposite probes in the nude mice was observed by Micro-animal imaging system,targeted imaging of the prostate cancer was observed by MR instrument.The nude mice with prostate cancer were irradiated with 100 W magnetic field for 30 min,and the changes of tumor sizes were observed.Results:The FMCNPs-ScFv nanocomposite probes were successfully prepared.Nanocomposite probes entered into the cytoplasm of cancer cells and exhibited low cytotoxicity effect.Nude mice model with prostate cancer were successfully fabricated;the nanocomposite probes distributed quickly in the main organs of mice,and gradually concentrated on the tumor tissues within 24 h.MR images showed that the tumor images were gradually enhanced from 6 h to 24 h after injection of the nanocomposite probe.Four days after magnetic irradiation,the tumors in the nude mice grew slower compared with the control nude mice(P

Overexpressed Gene YA61 Cloned from Human Gastric CarcinomaCell SGC-7901 and Its Sequencing / 生物化学与生物物理进展

To clone overexpressed gene from human gastric carcinoma cell SGC-7901, DDRT-PCR technique is used with human gastric epithelial cell GES-1 as control. After cloned into pGEM -T vector, YA61, one of the overexpressed genes, was analyzed by dot blot and was sequenced then. The sequence gotten was then compared to GenBank data and analyzed by NCBI ORF Finder. Dot blot results showed that the gene YA61 was overexpressed in human gastric carcinoma cell SGC-7901. NCBI's sequence similarity search indicated that the gene YA61 was a new gene sequence. Open reading frame analysis demonstrated that the gene YA61 had one complete open reading frame. In conclusion, the gene YA61 was a new gene sequence that was overexpressed in human gastric carcinoma cell SGC-7901.

Apoptosis in Human Osteosarcoma Cell Line SOSP-9607 Induced by Caspase-6 / 中国肿瘤生物治疗杂志

Objective:To explore caspase-6's effect on the apoptosis of osteosarcoma cell line SOSP 607. Methods: The expression level of caspase-6 in the osteosarcoma cell line SOSP-9067 was examined by RT-PCR method. The adeno-virus adv5 vector with caspase-6 gene was constructed and transferred into the osteosarcoma cell line SOSP-9607 by lipo-fection. The cell survival rate after transfection was assayed by MTT method. The cell morphological changes were observed by microscope and electron microscope, the apoptosis of transferred cells were examined by gel electrophoresis. Results:No expression of caspase-6 was examined in the osteosarcoma cell line SOSP-9607. A high expression of caspase-6 was identified by RT-PCR after the transfection. The cell growth curve declined after transferring caspase-6. Electrophoresis of DNA displayed the apoptosis ladder. Conclusion: Transferring caspase-6 into the osteosarcoma line SOSP-9607 may inhibit the growth of the osteosarcoma cell line SOSP-9607 and this effect might be achieved by inducing apoptosis.

Effect of blocking BRCAA1 gene with siRNA on proliferation of MCF-7 cells and expression of Rb gene / 中国肿瘤生物治疗杂志

Objective:To investigate the effect of blocking BRCAA1 gene expression with siRNA on the proliferation of tumor cell line MCF-7 and Rb gene expression.Methods:RNAi was employed to specifically knock down BRCAA1 expression.MCF-7 cells were transfected with complexes constructed with lipids and chemically synthesized Pre-designed anti-BRCAA1 siRNAs.The total RNA was isolated and reversely transcribed after 48 h.The expressions of BRCAA1 and Rb mRNA were determined by Real-Time PCR.Results:Compared with negative control,transfected MCF-7 cells had a 42.3% decrease in expression of BRCAA1 mRNA and an 11.1% increase in Rb mRNA expression.The inhibitory rate of MCF-7 cells proliferation was(81.6?6.1)%.Conclusion:There may be some antagonistic effect between BRCAA1 gene and Rb gene in proliferation of tumor cells,which provides a potential target for anti-tumor gene therapy.

Inhibitory effects of carbon nanotube-PAMAM-anti-survivin oligonucleotide compounds on proliferation of HepG2 cells / 中国肿瘤生物治疗杂志

Objective:To investigate the efficiency of carbon nanotube(CNT)-PAMAM mediated entrance of anti-survivin oligonucleotide into HepG2 cells,and its effects on the proliferation of HepG2 cells.Methods:CNT-PAMAM-anti-survivin oligonucleotide compounds were prepared and characterized by AFM and 1% agarose gel electrophoresis analysis.TEM was used to observe the distribution of CNT-PAMAM-ASODN compounds in HepG2 cells.CNT-PAMAM-ASODN compounds were added into the medium and co-cultured with HepG2 cells for 24 h,48 h,72 h,and 96 h at 37℃,5% CO_2.MTT method was used to detect the effects of ASODN and CNT-PAMAM-ASODN on the proliferation of HepG2 cells.Results:CNT-PAMAM-ASODN compounds were successfully synthesized via AFM and agarose gel electrophoresis.TEM showed that the compounds were located in the cytoplasm.When CNT-PAMAM-ASODN(1.0 ?mol/L)and ASODN(1.0 ?mol/L)were used for a 48 h culture,the inhibitory rates of HepG2 cells were(45.97?4.28)% for CNT-PAMAM-ASODN compounds group,(9.33?0.85)% for ASODN group,and(6.37?0.69)% for CNT-PAMAM group.CNT-PAMAM-ASODN compounds at 1.5 ?mol/L inhibited HepG2 cells by(70.22?7.25)%,and the inhibitory effects were in a time-and concentration-dependent manner.There was statistical difference between experiment group and control group(P