Increased expression of serum gelsolin in patients with osteosarcoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Identification of potential serum biomarkers of osteosarcoma to aid in its early diagnosis and in the discovery of possible therapeutic targets is an area of increasing interest.</p><p><b>METHODS</b>Two-dimensional difference-in-gel electrophoresis was used to assess multiple serum samples in patients with osteosarcoma. In addition, differential expression of protein biomarkers was characterized in osteosarcoma serum by using matrix-assisted desorption/ionization time-of-flight mass spectrometry coupled with database interrogation. Serum samples from four individuals with osteosarcoma and four age- and sex-matched healthy control subjects were compared.</p><p><b>RESULTS</b>Fifty-eight significant protein spot features in the osteosarcoma sera were found. These spot features were excised, digested with trypsin, and analyzed with mass spectrometry. Gelsolin was down-regulated only in osteosarcoma. Furthermore, Western blotting and enzyme linked immunosorbent assay (ELISA) confirmed decreased levels of gelsolin in the osteosarcoma serum samples.</p><p><b>CONCLUSIONS</b>These results indicated that gelsolin might have great potential as a biomarker of osteosarcoma and as a potential target for gene therapy.</p>

Construction and expression of recombinant adeno-associated virus vector containing HSV1-TK gene / 中南大学学报(医学版)

OBJECTIVE@#To construct the recombinant adeno-associated virus(rAAV) vector plasmid pSNAV2.0-TK containing HSV1-TK gene, to produce recombinant adeno-associated virus rAAV2/HSV1-TK, and to detect the integration and expression of HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV1-TK, and to provide foundation for gene therapy of posterior capsular opacification.@*METHODS@#The recombinant vector plasmid constructed by gene recombinant technology was analyzed by PCR and restriction enzyme digestion. The cell strain BHK-21/TK was screened by G418 after the plasmid was transfected into BHK-21 cells,with the helper virus HSV1-rc/UL2 to produce the recombinant virus rAAV2/HSV1-TK. The purity of rAAV2/HSV1-TK was detected by SDS-PAGE and HPLC, and the titre of rAAV2/HSV1-TK was observed by dot blot hybridization. The HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV-TK was investigated by PCR and RT-PCR.@*RESULTS@#The recombinant plasmid proved successful by PCR and restriction enzyme digestion. The recombinant virus rAAV2/HSV1-TK was produced successfully and its titre was 1 x 10(12) v.g./mL by dot blot hybridization. The HSV1-TK gene was integrated and expressed in lens epithelial cells.@*CONCLUSION@#The recombinant adeno-associated virus vector plasmid containing HSV1-TK gene is successfully constructed, and high titre recombinant adeno-associated virus (rAAV2/HSV1-TK) is obtained. The HSV1-TK gene in lens epithelial cells is expressed after being transfected by rAAV2/HSV1-TK.

Construction of rAAV2-GPIIb/IIIa vector and test of its expression and function in vitro / 中国实验血液学杂志

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.

Expression of coagulation factor IX in human CD34 + hematopoietic stem cells by adeno-associated virus 2 / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of human coagulation factor IX (hFIX) gene in human umbilical cord blood (CB) CD34+ cells which was transfected with recombinant adeno-associated virus 2 (rAAV-2).</p><p><b>METHOD</b>The CD34+ cells were transfected with rAAV-2/hFIX and cultured for 21 days for inducing differentiation into myeloid, erythroid and megakaryocytes, respectively. The expression of hFIX was studied at mRNA, protein and biological activity levels. The cytotoxicity of rAAV-2 to CD34+ cells was evaluated by cell proliferation, cell vitality, CD antigen expressions and CFU yields.</p><p><b>RESULTS</b>The hFIX mRNA was expressed in the cultured cells which was verified by RT-PCR and DNA sequencing. An elevated level of hFIX expression and biological activities were detected with a maximum amount of 14.10 ng/10(6) cell x 24 h. During the period of 21 day culture, the cell vitality, cell proliferation, CD antigen expression and CFU yields between the transfected and un-transfected groups had no difference(P > 0.05).</p><p><b>CONCLUSION</b>The human CB CD34+ cells are able to produce functional hFIX after transduction by rAAV-2/hFIX. The cell proliferation and differentiation capacities of the host CD34+ cells were not affected by rAAV-2.</p>

Preparation of rAAV2/hFIX and experimentally application to gene therapy for hemophilia B / 中华血液学杂志

<p><b>OBJECTIVE</b>To prepare the rAAV2/hFIX and evaluate the efficiency of the preparation on gene therapy of hemophilia B model mice.</p><p><b>METHODS</b>The rAAV-2/hFIX was prepared by "one helper virus-one vector cell line" strategy and transfected both BHK-21 and C2C12 cells in vitro. The hFIX antigen level in cell culture supernatant was assayed. The rAAV-2/hFIX was injected into muscles of hemophilia B model mice and assayed the serum hFIX levels, hFIX clotting activity, bleeding time, 5 min bleeding volume.</p><p><b>RESULTS</b>The hFIX antigen could be detected from 24 h till 120 h after BHK-21 and C2C12 cells were transfected with highest levels at 24 h reaching (51.0 +/- 6.5) ng/10(5) cells and (68.0 +/- 7.2) ng/10(5) cells, respectively. The rAAV2/hFIX injected mice could efficiently express hFIX and peaked at three weeks after injection, then slowly decreased but low level hFIX antigen was still detectable till 10 weeks after injection. There were significant differences between the high, middle and low dose groups of rAAV2/hFIX and the control group (P < 0.01), the plasma FIX clotting activities in the model mice were improved remarkably, bleeding time was greatly shortened and bleeding in 5 min was decreased. The hFIX expression level and FIX clotting activity of the high dose of rAAV2/hFIX group (1.6 x 10(13) v.g./kg) reached about (387.0 +/- 12.5) ng/ml plasma in contrast with the normal levels of (30.0 +/- 5.5)% at the third week after injection. No rAAV2 vector DNA was detected in the organs except for injected muscle tissue.</p><p><b>CONCLUSION</b>The rAAV2/hFIX transfected BHK-21 and C2C12 cells could efficiently express hFIX antigen and was of therapeutic effects for the hemophilia B model mice by intramuscularly injection.The results provide the basis for clinical trial of rAAV2 gene therapy for hemophilia B.</p>

Transduction efficiency of recombinant adeno-associated virus 2 in human umbilical cord blood CD34+ hematopoietic stem/progenitor cells / 中国实验血液学杂志

To investigate the transduction efficiency of recombinant adeno-associated virus 2 (rAAV-2) in human umbilical cord blood CD34(+) hematopoietic stem/progenitor cells, the CD34(+) cells sorted by the method of magnetic cell sorting from human cord blood were infected with the rAAV-2 expressing the green fluorescent protein (GFP) gene. After transduction for 19 hours, the expression of GFP was detected under fluorescence microscope. The results showed that 43% CD34(+) cells expressed the GFP gene at a multiplicity of infection of 2 x 10(5). It is concluded that the rAAV-2 can transduce human cord blood CD34(+) hematopoietic stem/progenitor cells efficiently.

Construction of a tRNAVal promoter plasmid expressing shRNAs in mammalian cells which mediates RNA interference / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Constructing a plasmid containing tRNAVal promoter to express shRNA which mediates RNA interference.</p><p><b>METHODS</b>A tRNAVal gene was amplified from human genomic DNA by PCR and replaced the last several bases of 3' end by a linker. The tRNAVal promoter after artificial mutation followed a shRNA sequence to luciferase was cloned into pUC18, Puc-tRNAVal, lucRi Cotransfected with pMAMneoLuc into BHK-21 cell to detect the effect of luciferase expression.</p><p><b>RESULTS</b>pUC-tRNAVallucRi suppressed the luciferase expression from pMAMneoLuc by 97.9%-9.5%.</p><p><b>CONCLUSION</b>The results showed that the tRNAVal shRNA plasmid could efficiently suppress luciferase expression in BHK-21.</p>