This study was aimed to construct lentivirus-mediated shRNA expression vector targeting Bmi-1 and establish a stable cell line U266-li, so as to pave the way for further research on function of Bmi-1 and application of shRNA to gene therapy. One pair of oligonucleotide sequences targeted at human Bmi-1 mRNA were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pLVTHM vector. Virus particles were collected after the control or shRNA vectors were co-transfected with the psPAX2 packaging plasmid and the plasmid pMD2.G was enveloped into HEK-293T cells by using Lipofectamine2000. The U266 cells were transduced with 5 × 10(6) recombinant lentivirus-transducing units plus 6 µg/ml of polybrene. Real-time PCR and Western blot were used respectively to detect the expression of Bmi-1 and P14 after lentivirus transduction. DNA sequencing demonstrated that the lentivirus RNAi vector of Bmi-1 was constructed successfully and the virus was packaged in 293T cells. The titer of virus was 5 × 10(7) TU/ml. Stable transfected U266 cell line was established. As was expected, the mRNA and protein levels of Bmi-1 was reduced significantly in U266 cells after lentivirus transduction, whereas the mRNA and protein levels of P14 was upregulated. It is concluded that the lentiviral RNAi vector of Bmi-1 is constructed, and U266 stable cell line is established.
Cell Line, Tumor ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Polycomb Repressive Complex 1 ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

This study was aimed to construct miRNA sponge targeting miR-20a and to establish a stable cell line Jurkat-S, paving the way for further research on function of miR-20a and application of RNAi in gene therapy. One pair of two-repeated oligonucleotide sequences containing bulged sites that are mispaired opposite miR-20a positions 9-12 was designed and synthesized with enzyme cutting sites. The annealed oligonucleotide fragments were subcloned into pCDNA3.0-L expressing vector. After double-enzyme cutting, the vector was ligated to the annealed oligonucleotide fragments again. Enzyme cutting and luciferase activity assay were performed for identification after four repeats. Then the ligated fragment was subcloned to lentivirus expressing vector. Virus particles were collected after the control or sponge vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pMD2.G into HEK-293T cells using Lipofectamine 2000. The Jurkat cells were transfused with recombinant lentivirus-transfusing units plus 6 µg/ml of Polybrene. Real-time PCR and Western blot were used to detect the mRNA and protein expression of P21 and E2F1 after lentivirus transfusion respectively. As a result, luciferase activity assay demonstrated that the sponge targeting miR-20a was constructed successfully and the virus was packaged in 293T. The titer of virus was 5×10(7) TU/ml. Stable transfected Jurkat-S cell line was established. As was expected, the mRNA and protein level of P21 and E2F1 was upregulated significantly in Jurkat-S cells. It is concluded that the miR-20a sponge is constructed successfully, and Jurkat-S stable cell line is established, in which the expression of miR-20a is inhibited stably.
Gene Expression ; Genetic Vectors ; Humans ; Jurkat Cells ; MicroRNAs ; genetics ; Plasmids ; RNA, Small Interfering ; genetics ; Transfection

Objective To discuss the value and experience of the percutaneous vertebroplasty (PVP)in the treatment of vertebral body compression fracture(VCF)in aged osteoperosis.Methods PVP was performed in 44 cases with VCF including 28 with single vertebral compressed fracture,12 with double compressed fracture and four with triple compressed fracture,with 67 vertebrae,for clinical and radiologieal evaluation.Results The mean follow-up was 15 months(4-23 months).There could be seen immediate relief of pain in 40 cases,out-of-bed activities at operation day in 19 and out-of-bed activ- ities at second day after operation in 25.Postoperative X-ray showed uniformly distributed bone cement in the vertebral,without leakage.Conclusion PVP is a recommendable method for VCF,for it has ad- vantages of pain relief,vertebrae stabilization,minimal invasion and minor complications.

This study was aimed to quantitatively detect the expression level of lysophosphatide acid acyltransferase β (lpaat β) mRNA in leukemia patients so as to provide theoretical basis for the target therapy of lpaat β in leukemia. Real-time fluorescently quantitative PCR was used to detect the relative expression level of lpaat β mRNA to analyze its expression change in various type of leukemia. The results showed that the lpaat β mRNA expression level in acute leukemia (AL) patients was significantly higher than that in normal controls (p < 0.05); lpaat β mRNA expression level in acute myeloid leukemia (AML) patients was significantly higher than that in normal controls (p < 0.05) and was positively correlated with white blood cell count (≥ 20.0 × 10(9)/L) (p < 0.05) and CD34 expression level of leukemia, but was not related with extramedullary infiltration. Except for acute promyelocytic leukemia (APL), the lpaat β mRNA expression level was negatively correlated with chemotherapy sensitivity in chronic myeloid leukemia (AML) patients. lpaat β mRNA expression level in chronic myeloid leukemia (CML) patients was significantly higher than that in normal controls (p < 0.05). lpaat β mRNA expression level in acute lymphoid leukemia (ALL) patients was not higher than that in normal controls (p > 0.05). It is concluded that the lpaat β gene overexpression exists in both AML and CML patients. lpaat β produced by AML cells probably plays an important role in abnormal proliferation and drug-resistance of AML cells.
Acyltransferases ; genetics ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Child ; Female ; Gene Expression ; Humans ; Leukemia ; genetics ; pathology ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Young Adult

This study was aimed to quantitatively detect the levels of april mRNA expression in leukemia patients so as to provide theoretical basis for the target therapy directing at april in leukemia. Real time fluorescent quantitative PCR was used to detect the relative expression level of april mRNA in newly diagnosed leukemia patients and to analyze the changes of its expression level in various type of leukemia. The results showed that the april mRNA expression level in acute leukemia (AL) patients was significantly higher than that in normal controls, there was statistical difference between them (p < 0.05); april mRNA expression level in acute myeloid leukemia (AML) patients was significantly higher than that in normal controls (p < 0.05) and positively correlated with white blood cell count ≥ 20.0 × 10(9)/L (p < 0.05), but not related with extramedullary infiltration and the expression of CD34. Except for acute promyelocytic leukemia (APL), april mRNA expression level was negatively correlated with sensitivity of patients to chemotherapy. april mRNA expression levels in acute lymphoid leukemia (ALL) and chronic myeloid leukemia (CML) patients were not higher than that in normal controls, there was no statistical difference between them (p > 0.05). It is concluded that april gene overexpression exits in AML patients. APRIL protein produced by AML cells probably plays an important role in abnormal proliferation and drug-resistance of AML cells.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Female ; Humans ; Leukemia ; genetics ; therapy ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; genetics ; Young Adult

This study was aimed to investigate the relationship between Richter's syndrome (RS) transformation and clinical characteristics as well as karyotype of patient with chronic lymphocytic leukemia (CLL). By the follow-up of a patient with CLL, the clinical characteristics, karyotype, treatment pattern and its effect, as well as disease progression were monitored regularly with serological test, flow cytometry and FISH technique. The results indicated that the patient typically presented with history of CLL at initial diagnosis, with expression of CD5(+), CD19(+) and CD23(+), Binet stage C, as well as karyotype aberration of trisomy 12, and poorly responded to 4 cycles of standard chemotherapy of FCR regimen. The disease progression was confirmed at 5 months with the symptoms of fever in the absence of infection, elevated lactate dehydrogenase level and rapidly enlarging lymphnodes which showed typically diffuse large B cell lymphoma by the biopsy. It is concluded that karyotype aberration of trisomy 12 is one of the risk factors for RS transformation, and treatment pattern of the patient with CLL may be associated with the transformation of RS.
Cell Transformation, Neoplastic ; genetics ; Chromosomes, Human, Pair 12 ; Female ; Humans ; Karyotype ; Karyotyping ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; Middle Aged ; Trisomy

This study was aimed to establish a high-throughput luciferase reporter system, through which to screen and identify miRNAs directly targeting p21, and to explore the biological function and significance of these miRNAs. Molecular cloning technique was used to construct and identify two lentivirus-expressing vectors-pWPXL-Luc and pWPXL-Luc-P21-3'UTR, virus particles were collected after the pWPXL-Luc or pWPXL-Luc-P21-3'UTR vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pDM2G into HEK-293T cells. Furthermore, two stable cell lines expressing luciferase singly or co-expressed luciferase and P21-3'UTR were established by transducing HEK-293 cells with recombinant lentivirus; the former was used as control in the following experiments. Finally luciferase activity of the latter stable cells was measured by using the luciferase reporter assay system. The results showed that high-titre recombinant lentivirus was produced and two stable cell lines were constructed, also to some certain, the luciferase activity was in direct proportion to the number of cells. In conclusion, the high-throughput luciferase reporter system is established successfully; using this system, the 28 miRNA that directly target P21 Cip1/Waf1 are screened experimentally.
Genes, Reporter ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Luciferases ; genetics ; MicroRNAs ; genetics ; Plasmids ; Transduction, Genetic

To evaluate the adjuvant effect of recombinant enterovirus 71 (EV71) subunit vaccine formulated with chitosan, rabbits were orally immunized with recombinant VP1 (rVP1) or rVP1 mixed with chitosan adjuvant. Levels of virus-specific IgG and IgA antibodies in sera, mucosal wash buffer (intestine, nasal cavity, and lung), and feces were determined by indirect enzyme-linked immunosorbent assay (ELISA). The titers of neutralizing antibodies against EV71 were determined using cytopathic effect-based neutralizing assay, and levels of cytokines (IFN-gamma and IL-4) secreted from in vitro-cultured rabbit splenic lymphocytes under antigen stimulation were also determined by ELISA. Results showed that immunization with rVP1 alone could only induce low levels of serum IgG and mucosal IgA, while rVP1 combined with chitosan adjuvant were able to induce significantly higher levels of antibodies, rVP1 can only induce neutralizing antibodies when used in combination with chitosan. Levels of IFN-gamma and IL-4 in the group immunized with rVP1 plus chitosan were significantly higher than those in the group immunized with rVP1 only or those in the control groups. Our study lays the foundation for development of oral VP1 vaccine against EV71 infection.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Antibodies, Viral ; immunology ; Chitosan ; administration & dosage ; immunology ; Enterovirus A, Human ; genetics ; immunology ; Enterovirus Infections ; immunology ; prevention & control ; virology ; Female ; Humans ; Rabbits ; Vaccination ; Vaccines, Subunit ; administration & dosage ; genetics ; immunology ; Viral Proteins ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

OBJECTIVETo observe clinical therapeutic effect of head point-through-point electroacupuncture (EA) on poststroke depression (PSD) and to study the mechanism.

METHODSOne hundred and eight cases of PSD were randomly divided into a point-through-point EA group (n = 38), a non point-through-point group (n = 36) and a western medicine group (n = 34). After treatment of 28 days, their therapeutic effects, scores of HAMD depression scale and SDS self-rating scale, and plasma 5-HT contents were compared before and after treatment among the 3 groups.

RESULTSThe effective rate of 86.84% in the point-through-point EA group was better than 63.89% in the non point-through-point group and 67.65% in the western medicine group (P < 0.05 or P < 0.01). Plasma 5-HT content in the point-through-point EA group increased significantly, with a very significant difference as compared with that of the non point-through-point group (P < 0.01).

CONCLUSIONHead point-through-point therapy can obviously increase plasma 5-HT content of the patient with PSD, so as to cure poststroke depression, with a better therapeutic effect than other two groups.

Adult ; Aged ; Depression ; therapy ; Electroacupuncture ; Female ; Humans ; Male ; Middle Aged ; Scalp ; Serotonin ; blood ; Stroke ; complications

This study was aimed to explore the synergistic effect of 2-methoxyestradiol (2-ME2) and bortezomib (Bor) on the proliferative inhibition and apoptosis of U266 cell line and its possible mechanism. The cells were treated with 2-ME2, Bor alone and 2-ME2 combined with Bor, respectively. The cell viability and proliferative curve were detected by CCK8, the cell apoptosis was detected by caspase 3/7 activity test, cell cycle status was analyzed by flow cytometry, and real-time PCR was used to detect the mRNA expression of P21, BAX and BCL-2. The results showed that compared with cells treated with 2-ME2 or Bor alone, the proliferative potential of cells in combination group was significantly inhibited (p < 0.05), and apoptosis rate markedly increased (p < 0.05), cell cycle was arrested at G(1)-S phase, the mRNA expressive level of P21 and BAX increased, while the expression of BCL-2 decreased. It is concluded that 2-ME2 combined with Bor synergistically inhibits cell proliferation and induces apoptosis in U266 cell line. The possible mechanism may be associated with its effect of up-regulating P21 and BAX expressions.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Drug Synergism ; Estradiol ; analogs & derivatives ; pharmacology ; Humans ; Pyrazines ; pharmacology