Objective To construct the recombinant co-expression vector carrying antisense RNA to dual-target BAG-1 and Bcl-2 genes and recombinant single expression vector carrying antisense RNA to target BAG-1 and Bcl-2 gene respectively,then to pre-liminarily investigate their effect on the proliferation of gastric cancer cell SGC-7901 in order to lay the foundation for further study the effect of this recombinant vector on the tumor cells.Methods RT-PCR was used to amplify the full length of BAG-1 and Bcl-2 cDNA from total RNA of gastric cancer cell line SGC-7901.The BAG-1 cDNA fragment and the Bcl-2 cDNA fragment were insert-ed into pMD18-T simple vector respectively.The pMD18-T-BAG-1 was digested with BamH Ⅰ and Cla Ⅰ and the pMD18-T-Bcl-2 was digested with EcoR Ⅰ and Nhe Ⅰ.Then the BAG-1 cDNA fragment and the Bcl-2 cDNA fragment were inserted into the mcs1 and mcs2 of the eukaryotic co-expression vector pVITRO2 in the antisense orientation respectively.The construction of the single expression vector pVITRO2-AsBAG-1 and pVITRO2-AsBcl-2 was confirmed by restriction endonuclease treatment and se-quence identification.Then the Bcl-2 cDNA fragment was inserted into the mcs2 of the recombinant vector pVITRO2-AsBAG-1 in the antisense orientation to construct the co-expression vector pVITRO2-AsBAG-1-Bcl-2,and the recombinant vector was also iden-tified by restriction endonucleases digestion and sequence identification.Then the recombinant vector was transfected into SGC-7901 cell respectively.The proliferation of the cell was determined by the MTT assay.The level change of BAG-1 and Bcl-2 mRNA in SGC-7901 cell was detected by the semi quantitative RT-PCR and the change situation of cell cycle was detected by the flow cytom-etry(FCM).Results The restriction endonucleases digestion and sequencing identification indicated that the eukaryotic co-expres-sion vector pVITRO2-AsBAG-1-Bcl-2 and the single gene expression vector pVITRO2-AsBAG-1 and pVITRO2-AsBcl-2 were con-structed successfully.The MTT assay method demonstrated that compared with the control group,the recombinant vector could in-hibit the proliferation of the cells in time dependent manner,and the inhibiting effect was most notably in the 72 h transfection group(P <0.01);inhibition ratio of recombinant vector groups was significantly higher than that of control group and pVITRO2 group(P <0.01).BAG-1 and Bcl-2 mRNA expression in recombinant vector groups were significantly decreased compared with that in the control group and pVITRO2 group(P <0.01),The RT-PCR results showed that the expression level of BAG-1 mRNA and Bcl-2 mRNA was significantly decreased(P <0.01),moreover the co-expression vector group was more notably than the single expression vector groups(P <0.05),but the pVITRO2-AsBcl-2 had no obvious effect on the expression of BAG-1 mRNA(P >0. 05);the FCM detection results showed that the apoptosis rate of the recombinant vector groups was significantly higher than that of the control group and the pVITRO2 group with statistical difference(P <0.01),and the co-expression vector group was more nota-bly than single expression vector groups(P <0.01).In addition,the effect of the co-expression recombinant vector group was more significant than that of the single expression co-expression vector(P <0.01 ),the apoptosis rate was increased from 0.57% to 15.75%.Conclusion The co-expression recombinant vector pVITRO2-AsBAG-1-Bcl-2 and single expression vector pVITRO2-As-BAG-1 and pVITRO2-AsBcl-2 are successfully constructed and they can inhibit the proliferation of SGC-7901 cell and induce cell apoptosis,moreover the pVITRO2-AsBAG-1-Bcl-2 vector is most notably.

AIM: To investigate the effect of antisense oligodeoxynucleotides against platelet-derived growth factor B-chain(PDGF-B) in the proliferation of cultured rat hepatic stellate cells and collagen-Ⅰ production.METHODS: The phosphorothioate antisense oligodeoxynucleotides and missense oligodeoxynucleotides were synthesized.The rat hepatic stellate cells(HSCs) were treated with these oligodeoxynucleotides at different concentrations,respectively.The inhibition of the proliferating of cultured rat hepatic stellate cells was assayed by MTT method.The expressions of PDGF-B and collagenⅠ mRNA were detected by means of reverse transcription-polymerase chain reaction(RT-PCR).The expression of endogenous PDGF-B was determined by flow cytometry(FCM).Collagen-Ⅰin the supernatant was determined by ELISA.RESULTS: The results showed that the ASODN against the PDGF-B at a final concentration of 10 ?mol/L inhibited the HSCs proliferation,the expressions of PDGF-B and collagen-ⅠmRNA.FCM and ELISA demonstrated that the expression of endogenous PDGF-B and the production of collagen-Ⅰin HSCs were significantly lower than those in controls,whereas the controls(missense oligodeoxynucleotides) had no effect at the same concentration.CONCLUSION:PDGF-B ASODN inhibits the proliferation of cultured rat hepatic stellate cells,the endogenous expression of PDGF-B and the production of collagen.PDGF-B ASON might be useful for gene therapy in liver fibrosis.

Purpose:To investigate the effect of in vitro antisense oligodeoxynucleotide of cyclin D 1 on the cyclin D 1 gene expression and cell proliferation of human stomach adenocarcinoma cell BGC 823 cell line.Methods:Phosphorothioate cyclin D 1 ASODN and random oligodeoxynucleotide (RODN) were synthesized and transfected into BGC 823 Cells. Their effects on cell proliferation were examined by MTT method,RT PCR method,immunohistochemical study.Results:Cyclin D 1 ASODN could significantly inhibit the growth of BGC 823 Cell lines.The RODN showed no such effect.The inhibition peaked at 48 hour after transfection by MTT method and was dose dependent.ASODN could downregulate the expression levels of cyclin D 1 mRNA and protein by RT PCR method and immunohistochemical study respectively.Conclusions:The data suggested that ASODN could specifically inhibit the expression of cyclin D 1 mRNA and protein and regulate cell cycle and cell proliferation of BGC 823 cells. [

Objective To investigate the effects of c-myc promoter binding protein(MBP-1)gene expression silencing on the pro-liferation in vitro in human gastric cancer cell line SGC-7901.Methods The cells divided into three groups:blank control group (cells without transfecting gastric cancer cell),negative control group(cells transfecting missense sequence)and experimental group (cells transfecting MBP-1 shRNA).Two MBP-1 shRNA sequences and one negative control shRNA sequence were designed,syn-thesized and cloned into pSIREN-retroQ plasma.Then the recombinant plasmids were constructed and transfected into human gas-tric cancer SGC-7901 cells by Lipofectamine 2000.After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was obtained.The expressions of MBP-1 mRNA and protein in SGC-7901 were deter-mined by the real time PCR and Western blot,respectively.The effects of altered expression of MBP-1 on the cell proliferation were measured by MTT cell proliferation assay.Results PCR and sequencing indicated that the recombinant plasmids pSIREN-retroQ was constructed.Then the recombinant plasmids were transfected into human gastric cancer SGC-7901 cells by Lipofectamine 2000. After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was ob-tained.The relative expression level MBP-1 mRNA in the MBP-1 siRNA transfection group was significantly decreased compared with the blank control group(P <0.05).Compared with the blank group,the expression levels of MBP-1 protein in the experimental group also significantly decreased.The proliferation abilities of SGC-7901 cells at 48,72,96,120 h after MBP-1 siRNA transfection were significantly increased compared with the blank control group (P < 0.05 ).Conclusion Down-regulating the expression of MBP-1 can obviously promote the proliferation of human gastric cancer cell line SGC-7901.MBP-1 gene may become the new target of gene therapy for gastric cancer.

Objective: To observe and evaluate the clinical effect of cement reinforced pedicle screw fixation in the treatment of osteoporotic thoracolumbar fractures and its effect on the quality of life. Methods: From April 2015 to June 2017, 60 patients with osteoporotic thoracolumbar fractures were selected from Shanxi Coal Center Hospital.The patients were divided into two groups according to different treatment method, with 30 cases in each group.Routine percutaneous vertebroplasty was performed in the control group, and bone cement reinforced pedicle screw fixation was performed in the observation group.The scores of health and function, life satisfaction score, the scores of social and economic factors, self-concept score, visual analogue scale (VAS) and Cobb's angle were calculated and evaluated before operation and 6 months after operation in the two groups. Results: The scores of health and function, life satisfaction, social and economic factors, self-concept in the observation group at 6 months after operation were (60.2±6.0)points, (39.8±4.0)points, (32.0±3.5)points, (47.7±4.0)points, respectively, which were significantly higher than those in the control group[(48.0±5.5)points, (32.4±3.0)points, (29.3±2.0)points, (44.0±3.3)points], the differences were statistically significant (t=8.988.106, 3.668, 3.908, all P<0.05). The VAS score in the observation group was significantly lower than that in the control group at 6 months after operation[(1.5±0.4)points vs.(2.3±0.8)points](t=4.898, P<0.05). The Cobb angle of the observation group[(5.3±1.0)°] was significantly lower than that of the control group[(14.0±2.2)°] at 6 months after operation (t=19.718, P<0.05). Conclusion Treatment of osteoporotic thoracolumbar vertebrae fracture with bone cement reinforced pedicle screw fixation has better clinical effect and can obviously improve the quality of life.

An in vitro synthesized random ssDNA library was subjected to 12 rounds of selection against anti-screening cells and sieving cells by SELEX. Normal and inflammatory cervical exfoliation cells were selected as anti-screening cells, and the cervical exfoliation cells of low-grade squamous intraepithelial lesion (CIN1), high-grade squamous intraepithelial lesion (CIN2, CIN3) and cervical carcinoma were selected as sieving cells during the screening process. Then, the highly specific aptamer CIN-Ap4 was established by the analysis of the specificity, affinity and cell immunofluorescence, which can be used as biomarker for Cervical Intraepithelial Neoplasia. Prime Premier 5.0 was applied to design a random ssDNA library. According to the fixed sequence at both ends of the library, a pair of primers were designed and synthesized. At the same time, the optimal annealing temperature, cycle times and primer concentration ratio of PCR procedure were selected. The results under the optimal condition are shown as follows. In the 50 μL reaction system, the optimum reaction conditions of symmetry PCR are as follows: annealing temperature is 49.5 ℃, number of cycles is 15. The optimal reaction conditions of indirect asymmetric PCR are as follows: the primer concentration ratio is 80:1, and the number of cycles is 35. The experiment proves that the oligonucleotide library is constructed successfully, and the highly specific dsDNA and ssDNA can be obtained under optimal PCR conditions with good repeatability, which establishes the foundation for the further exploration and experimentation.