BACKGROUND:Matrix attachment region(MAR)are DNA elements that bound to the nuclear matrices after chromatin digested with restriction endonuclease.Plenty of studies have shown that MAR considered as initiaI point of DNA replication or transcription of regulatory gene.Thereby,construction of MAR expression vector can elevate the overall level of transgene expression,enhance stability of exogenous gene.as welI as increase frequency of stable transfectant cells.OBJECTIVE:To construction pLXSN-CAT recombinant retrovirus vector that containing chloramphenicol acetyltransferase(CAT)via cloning MAR sequence of human.and to explore the influence of MAR on the gene expression.METHODS:An open experiment was performed at the Department of Biochemistry and Molecular Biology.Xinxiang Medical College from September 2007 to December 2007 The PLXSN-CAT vector of CAT was constructed by the laboratory.TaqDNA polymerase,T_4 DNA ligase,DNA Marker,restriction enzyme BamH I,agarose gel DNA purification kit,as well as plasmid purification kit were purchased fromTakara Biotechn0Iogy(Dalian)Co.,Ltd.The sequence of csp-B MAR was amplified by polymerase chain reaction(PCR)method applied to human DNA.The fragment was inserted into retrovirus vector PLXSN-CAT plasmid.The recombinant plasmid was verified by double digestion and DNA sequencing.RESULTS AND CONCLUSION:The length of specific fragment applied by PCR was 931 bp,and the recombinant plasmid PLXSN-CAT-MAR presented two bands:5.9 kb and 931 bp using respective restriction enzymes BamH I The sequence of MAR was confirmed by blasting to Genbank(serial numobr:M6271 6).It suggested that MAR had been cloned into PLXSN-CATR vector correctly.The recombinant retrovirus vector PLXSN-MAR was successfully constructed.

BACKGROUND: Matrix attachment region (MAR), a DNA sequence, is still bound to the nuclear matrices after chromatindigested with restriction endonuclease, not only affects expression of endogenous gene, but also overcames transgenic silence andimproves transcription and expression of exogenous gene. OBJECTIVE: To investigate the influence of ?-interferon MAR of CHO cells on the transgenic expression of chloramphenicolacetyltransferase (CAT). DESIGN, TIME AND SETTING: The opening experiment was performed at the Department of Biochemistry and MolecularBiology, Molecular Institute, Xinxiang Medical College from October 2006 to April 2007. MATERIALS: CHO cell lines were obtained from China Center for Type Culture Collection. The pCATG vector of CAT and G418screening markers were constructed by this laboratory. METHODS: Human ?-interferon MAR by PCR was digested with SacI/KpnI and BamHI/SalI, and was inserted into pCATGvector, which was propagated in Escherichia coli JM109, then extracted and purified followed by enzyme digestion analysis. Vectorof CAT expression cassette and human ?-interferon MAR by the two sides was successfully constructed, and christened aspCAT-MAR. Two methods were compared between CHO cells of pCATG transformation and CHO cells of pCATG-MARtransformation. After G418 selecting, genome DNA of cell lines of G418 was extracted, then primers for PCR to amplify the CATtarget gene fragment was designed. MAIN OUTCOME MEASURES: The activity of CAT was analyzed by ELISA method. It was also tested to see if thepCATG-MAR was stably integrated into genomic DNA in the transfected cells. RESULTS: CHO cells of pCATG transformation was screened to have 16 strains of positive cell, and CHO cells of pCATG-MARtransformation was screened to have 17 strains of positive cell. Human ?-interferon MAR could increase the CAT gene expressionby 2.8 fold. The coefficient of variation of CHO cells of pCATG transformation was 2.065 0, and coefficient of variation of CHOcells of pCATG-MAR transformation was 0.813 1. Genome DNA of stable transformation cell lines was amplified by a fragment of437 bp. The results confirmed the pCAT-MAR vector was stably integrated into genomic DNA. CONCLUSION: Human ?-interferon MAR can increase transgenic expression in CHO cells and decrease the transgenicexpression variation in different transfected cells.

BACKGROUND:Some patients with cervical spondylosis have not been fully corrected sagittal position balance after cervical surgery,and this continuous sagittal position imbalance may be an important reason for the poor long-term clinical outcome of patients. OBJECTIVE:To analyze the correlation between the cervical sagittal position balance parameters and their changes and the clinical efficacy of patients in the unbalanced state after anterior cervical decompression and fusion and to explore the necessity of surgical correction of sagittal balance in order to improve the clinical effect in the later stage. METHODS:A retrospective analysis was performed on 125 patients with cervical spondylosis who underwent anterior cervical decompression and fusion in the Department of Spinal Surgery of Affiliated Hospital of Southwest Medical University from July 2019 to July 2022.Follow-up patients had good postoperative recovery(neck disability index score less than 10%one week after surgery)and had complete follow-up data.According to the axial vertical distance(C2-7 SVA)in sagittal position one week after surgery,patients were divided into type I imbalance group(C2-7 SVA loss≤5 mm,n=27),type Ⅱ imbalance group(C2-7 SVA loss>5 mm,and≤10 mm,n=19),and type Ⅲ imbalance group(C2-7 SVA loss>10 mm,n=12),and non-unbalanced group(C2-7 SVA in the normal range,n=67).The changes of visual analog scale score and neck disability index were compared among groups postoperatively and the last follow-up,as well as the changes of imaging sagittal balance parameters C2-7 cobb angle,C2-7 SVA value,neck inclination angle,T1 inclination angle,and thoracic entrance angle.The correlation between the late clinical effect and postoperative cervical sagittal disequilibrium was explored. RESULTS AND CONCLUSION:(1)There was no statistical difference in general data among the four groups(P>0.05).All patients underwent successful surgery without serious complications and postoperative wound infection.The follow-up time was more than 1 year.(2)There was no significant difference in preoperative symptom score and clinical efficacy one week after surgery(P>0.05).At the last follow-up,pain visual analog scale score,neck disability index and C2-7 SVA were lower than those before surgery but higher than those one week after surgery(P<0.05).C2-7 cobb angle was increased compared with those before operation(P<0.05).T1 inclination angle was decreased compared with those before operation(P<0.05).(3)Pearson correlation test showed that the change of neck disability index was positively correlated with the change of C2-7 SVA(P<0.05).(4)It is indicated that anterior cervical decompression and fusion is effective in the treatment of cervical spondylosis,and can effectively relieve the symptoms of patients.Patients with more severe cervical sagittal disequilibrium after surgery had worse curative effect in the later period.Continuous sagittal disequilibrium in patients with cervical spondylosis after surgery is an important cause of poor curative effect in the later stage.Clinicians should pay more attention to the correction of cervical sagittal balance before and during surgery,formulate surgical strategies and plans according to sagittal balance parameters before surgery,and correct C2-7 SVA intraoperatively to the normal range.