Objective To clone the human β-globin matrix attachment region(MAR) and construct the mammalian animal expression vector pCAT-MAR, which contains the MAR and CAT reporter gene.Methods The human genomic DNA was extracted through phenol/chloroform and precipitated with ethanol, followed the MAR was amplified through PCR using the primers designed according to the GenBank sequence. After identified by agarose gel electrophoresis, sequenced and analyzed by the software, the PCR products were cut with restriction enzymes and ligated into the pCAT3-control vector to construct the pCAT-MAR vector. Results About 770bp band appeared in the agarose gel electrophoresis, the similarly compared with the published MAR sequence was 99.9%, the DNA fragment has the MAR typical features. The pCAT-MAR vector was demonstrated to be right through digestion with the corresponding enzymes and agarose gel electrophoresis. Conclusion The humanβ-globin MAR is cloned through PCR, and the expression vector pCAT-MAR containing the MAR is successfully constructed.

Objective Study the relationship between the component of medium and spore formation of Bacillus sp WTFY. Methods Alter the carbon source、nitrogen source、inorganic concentration and kinds in turns, observe the sporulation circumstances of Bacillus sp WTFY.Results When glucose concentration was higher over 0.1%, spore did not emerge;Nitrogen source、phosphorus、magnesiumions concentration and kinds have some degree influence,but not significantly.Conclusion Carbon source concentration is a key factor that influence spore formation.

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.

Objective To investigate the effect of different promoters on the expression level of transgene containing MAR expression vector in recombinant CHO cells.Methods The CMV promoter and 3-globin MAR were amplified by PCR,then CMV promoter was replaced the SV40 promoter in pCAT1 for constructing the expression vector droved by CMV promoter.The control vectors of pCAT1 and pCAT2 without containing MAR were simultaneously transfected into the CHO cells.Then the stably transfected cell line was screened by G418.The CAT gene expression level was analyzed by ELISA.Results The expression level of CAT enzyme in the cells transfected with MAR-containing vectors was increased compared with the cells transfected by pCATG and pCAT3 vectors without containing MAR,which were increased by 1.75 and 1.25 times respectively(P＜0.05);but CAT enzyme expression level in the pCAT1 transfected cells droved by SV40 promotor with the MAR-containing expression vectors was 1.4 times higher than that in the pCAT2 vector droved by the CMV promoter(P＜0.05).Conclusion MAR can enhance the transgene expression level in stably recombinant CHO cells,and the promoting efficiency of SV40 promoter and MAR combination is superior to that of CMV promoter and MAR combination.

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.

Objective To study the effect of human ?-globin matrix attachment region(MAR) on transgene expression in stably transfected CHO cells.Methods Expression vector was constructed,which contained the ?-globin MAR in both sides of Chloramphenicol acetyltransferase(CAT) reporter gene expression cassette in cis,then transfected into CHO cells.The CAT reporter gene expression was analyzed by ELISA method.Results The ?-globin MAR enhanced the CAT gene expression 5.5-fold in stably transformed CHO cells,while the transgene expression variation among individuals of transformants was decreased.Conclusion MAR increase transgene expression in stably transfected CHO cells.

Objective To analyze the effect of GC-rich DNA fragments on the level of transgenic expression in Chinese hamster ovary (CHO) celts and its position effect.Methods The synthetic DNA fragment with GC-rich was cloned into the 5'or 3'or both 5'and 3'ends of expression cassette of expression vector.Three new expression vectors (pIRES-G1,pIRES-G2 and pIRES-G3) which was inserted with the GC-rich DNA fragments in different position were transfected CHO ceils,respectively,and then was observed under fluorescence microscope;the control vector was pIRES-EGFP.Stable transfected cell lines were screened under G418,and enhanced green fluorescent protein(EGFP) expression was analyzed by flow cytometry and the transgenic copy number was detected by quantitative real-time quantitative polymerase chain reaction (qRT-PCR).Results Three expression vectors with a GC-rich DNA fragments in different position were constructed successfully.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector could obviously improve the expression level of vector in CHO cells;and the expression level of the stably transfected CHO cells increased 1.39 fold and 1.32 fold compared to the control vector,respectively;the transgene copy number increased 1.32 fold and 1.24 fold compared with the control vector.While the insertion of GC-rich DNA fragments at 5'end of expression cassette had no obvious effect on the level of gene expression.Conclusion The role of DNA fragment with GC-rich in improving the transgenic expression of CHO cells is related to its position in the vector.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector can improve transgenic expression.

BACKGROUND:With the development of surgical techniques and reconstruction material technology, joint replacement has also been widely used in the dislocation of the shoulder;especial y al kinds of custom-made or assembled prosthesis make replacement indications improved evidently. OBJECTIVE:To investigate the long-term effects of arthroscopy or arthroplasty for treating recurrent anterior dislocation of the shoulder joint with severe bone defects. METHODS:144 patients with recurrent anterior dislocation of the shoulder joint with severe bone defects were enrol ed in this study. They were divided into treatment group and control group based on a random drawing (n=72). The control group was given arthroscopic surgery, and the treatment group was subjected to arthroplasty. The 3 years of fol owed-up were done by telephone investigation and further consultation. Neer shoulder score, shoulder activity and complications were recorded.
RESULTS AND CONCLUSION:After 3-year fol owed-up, the excel ent and good rate of shoulder function was significantly higher in the treatment group (90%) than in the control group (81%) (P<0.05). The magnitude of the flexion in the 3-year fol owed-up was apparently increased, while the lateral margin external rotation was decreased, which showed significant differences after intragroup comparison (P<0.05). Simultaneously, the magnitude of the flexion and the lateral margin external rotation in the treatment group had statistical y significant differences compared to the control group in the 3-year fol owed-up (P<0.05). The complications of wound infection, shoulder dislocation, and implant loosening in the treatment group during fol ow-up were significantly lower than in the control group (P<0.05). These findings verified that compared with arthroscopic surgery, arthroplasty for treating recurrent anterior dislocation of the shoulder joint with severe bone defects in long-term fol ow-up can effectively restore shoulder function and range of motion, and it has few complications, thereby effectively rebuilds shoulder joint.

BACKGROUND:Traditionaly, non-surgical treatment was used to treat degenerative femoral intercondylar fossa impingement syndrome, but it can cause function loss of cruciate ligament, or knee instability. With the development of medical technology, more and more views believed that ligament damage and combined with other knee structural damage should receive surgery as soon as possible.
OBJECTIVE: To investigate the repair effect of knee arthroplasty for treating degenerative femoral intercondylar fossa impingement syndrome, and compare with AO cannulated screw fixation.
METHODS:A total of 72 patients with degenerative femoral intercondylar fossa impingement syndrome were equaly and randomly divided into treatment group and control group. Patients in the control group were treated with open AO cannulated screw fixation, and patients in the treatment group were subjected to knee arthroplasty. At 7 days after treatment, repair effect was evaluated. Before treatment and 7 days after treatment, knee function was assessed by using Lysholm knee Scoring Scale and the international knee documentation committee knee evaluation form. Al patients were folowed up for 6 months after treatment to investigate the occurrence of complications.
RESULTS AND CONCLUSION:The surgery was successfuly completed in al patients. At 7 days after treatment, the excelent and good rate was 94% in the treatment group and 75% in the control group. The excelent and good rate was significantly higher in the treatment group than in the control group (P < 0.05). Lysholm scores were significantly greater, but the international knee documentation committee knee evaluation form scores were significantly lower at 7 days after treatment compared with that before treatment in the treatment group (P < 0.05). Simultaneously, Lysholm scores and the international knee documentation committee knee evaluation form scores were significantly better in the treatment group than in the control group at 7 days after treatment (P < 0.05). During 6-month folow-up, wound infection, intra-articular infection, joint pain, and deep vein thrombosis were significantly less in the treatment group than in the control group (P < 0.05). These findings indicate that knee arthroplasty for degenerative femoral intercondylar fossa impingement syndrome can improve short-term efficacy, effectively restore knee function and reduce the incidence of postoperative complications.

BACKGROUND:Arthroscopy can observe the involutive relation of patelofemoral joint directly and dynamicaly, which can be used to judge whether the patelofemoral joint abnormalities can be completely corrected. OBJECTIVE:To analyze the clinical effect of lateral retinacular release and ligament reconstruction under arthroscope for patela recurrent dislocation. METHODS: A total of 58 patients diagnosed as having recurrent patelar dislocations were divided randomly into control and experimental groups, with 29 cases in each group. Patients in the control group received lateral retinacular release and ligament reconstruction under common operation and those in the experimental group received lateral retinacular release and ligament reconstruction under arthroscopy. RESULTS AND CONCLUSION:There was no significant difference in the Lysholm and Kujala scores before treatment in the two groups (P > 0.05), but at 12 months after treatment, the Lysholm and Kujala scores were both increased in the two groups, especialy in the treatment group (P < 0.05). No difference was found in the congruence angle and lateral patelofemoral angle with CT value at 30° of knee flexion (P > 0.05), and CT measurement values of the congruence angle and lateral patelofemoral angle were both decreased in the two groups, especialy in the experimental group, at 12 months after treatment. In addition, the operation time, healing time, and total effective rate were better in the experimental group than the control group (P < 0.05). These results indicate that the lateral retinacular release and ligament reconstruction under arthroscopy has a better effect on recurrent patelar dislocation.