Protective effect of diacerein on MIA-induced injury in rat osteoarthritis chondrocytes / 重庆医学

Objective To investigate the protective effect of diacerein on monosodium iodoacetate (MIA) induced injury in rat osteoarthritis chondrocytes .Methods The experiment was divided into five groups ,including the normal group ,model group (4μM MIA) ,diacerein low ,middle and high doses groups (1 ,10 ,100μM) .The viability of chondrocytes was detected by MTT assay . The activity of cysteinyl aspartate specific proteinase‐3 (Caspase‐3) was measured by spectrophotography .The activation of nuclear factor kappa B (NF‐κB) signaling pathway and expression level of downstream target molecule cell Bax ,Bcl‐2 ,matrix metalloprotei‐nase‐9 (MMP‐9) and MMP‐13 were detected by Western blot .Results 1 ,10 ,100μM diacerein could increase the viability of MIA‐induced chondrocytes and reduce the activity of Caspase‐3(P<0 .05) .10 ,100μM diacerein could decrease the phosphorylation level of IκBαand NF‐κB p65 ,furthermore downregulated the level of Bax ,MMP‐9 and MMP‐13 protein ,and upregulated the level of Bcl‐2 protein (P<0 .05) .Conclusion Diacerein could inhibit cell apoptosis and degradation of extracellular matrix in MIA‐induced rat chondrocytes ,which might be related to the NF‐κB signal pathway .

Biomechanical properties of retrograde interlocking intramedullary nail versus less invasive stabilization system plate in the repair of distal femoral fractures / 中国组织工程研究

BACKGROUND:Distal femoral fractures are mainly treated with less invasive stabilization system (LISS) plate or retrograde interlocking intramedul ary nail fixation, but choosing which method is controversial, and studies on their biomechanical properties are few. OBJECTIVE:To compare the biomechanical stability of retrograde interlocking intramedul ary nail and LISS plate fixation in the treatment of distal femoral fractures. METHODS:Twelve male cadaveric femurs were col ected, and the injured, with abnormal bone density and osteonosus specimens were excluded through X-ray examination, fol owed by randomly divided into two groups. Models of AO type A3 supracondylar fracture were prepared, and were fixed with LISS plate and retrograde interlocking intramedul ary nail, respectively. The compressive stiffness and displacement values under axial compression and loading of 100, 300 and 500 N, as wel as the bending strength of the specimens under bending load were observed. RESULTS AND CONCLUSION:(1) The resistance to axial deformation capacity (compressive stiffness) of the LISS plate was superior to the retrograde interlocking intramedul ary nail (P0.05). (2) Under axial compressive loading of 100, 300 and 500 N, the displacement values of LISS plate were significantly less than those of the retrograde interlocking intramedul ary nail (P<0.05). (3) In conclusion, in the distal femoral fracture fixation, the stiffness of the retrograde interlocking intramedul ary nail is low. While the LISS plate not only has certain deformation, but also has strong rigidity with firm internal fixation, which provides excel ent biological environment for fracture healing;thus it is a reliable treatment for distal femoral fractures.

Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai / 生物医学工程学杂志

This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

Eukaryotic expression and DNA immunization of hemolysin HlyX gene from Leptospira serovar Lai / 中华微生物学和免疫学杂志

Objective To study on the expression of the eukaryotic recombinant vector carrying HlyX gene of Leptospira serovar Lai in mammalian cell and explore the humoral immune response in BALB/c mice immunized with the recombinant plasmid. Methods The HlyX gene was amplified from Leptospira serovar Lai genomic DNA by PCR and inserted into pcDNA3.1 vector. After transformed into E. coli DH5α,the recombinant plasmid was assayed for identification by PCR analysis,restriction nuclease enzyme digestion and sequencing. The recombinant plasmid was transfected into COS-7 cells,then RT-PCR and Western blot were performed to test the expression of the target gene. The recombinant plasmid was injected intramuscularly into BALB/c mice for three times at intervals of two weeks,and the antibody titer was measured by ELISA. Results PCR showed the full length HlyX gene was about 1100 bp. PCR analysis,restriction nuclease enzyme digestion and sequencing indicated the recombinant vector was constructed successfully. After the plasmid Was transfected into COS-7 cells,a fragment about 1100 bp was found by RT-PCR and a specific band relative molecular mass(Mr)about 40×103,which was consistent with the expected size of the target proteins was showed by Western blot. ELISA showed the antibody titer in BALB/c mice immunized by the HlyX gene of Leptospira serovar Lai can elicit high-titer antibody in BALB/c mice,which has laid the foundation for the application of the DNA vaccine.

Construction of the recombinant integrating shuttle plasmid with cfpl0-esat6 fusion gene of Mycobacterium tuberculosis and its expression in BCG / 生物医学工程学杂志

This study was conducted to amplify the cfpl0-esat6 fusion gene by SOE and insert into the integrating shuttle plasmid pMV361 to form the recombinant plasmid. Then another recombinant plasmid was constructed by insertinga-A g signal sequence of BCG. The two recombinant plasmids were introduced into BCG and the induced products from recombinant BCG were analyzed. In conclusion,the successful construction of rBCG expressing the fusion protein CFP10-ESAT6 will be the base of the development of novel Mycobacterium tuberclosis vaccines.

Expression and immunity reaction of a novel gene OmpL17 of the strong virulent L. interrogans serovar Lai in China / 生物医学工程学杂志

This study was conducted to potentiate the expression of outer membrane protein OmpL17 of the strong virulent L. interrogans serovar Lai and investigate its immunogenicity in rabbits. The OmpL17 was cloned into prokaryotic expression vector pGEX-1lambdaT. The recombination expression plasmid pGEX-OmpL17 was transformed into E. Coli JM109. The GST fused protein GST-OmpL17 was expressed after induction by IPTG, then GST-tag was by thrombin and purified using Bulk GST purification Modules. SDS-PAGE and Western blotting analysis indicated that the molecular weight of GST-OmpL17 and OmpL17 was about 54 KDa and 28 KDa respectively. The outer membrane protein OmpL17 was subcutaneously injected into rabbits and high titre anti-OmpL17 antibody was obtained (1:4896) which could conjugate specifical with OmpL17. In conclusion, OmpL17 and specifical anti-OmpL17 antibody were obtained, which provided an experimental basis for researching pathogenic effect and immunity functions of OmpL17.

Ability of reproduction and maintaining characteristics of hypertension and stroke in prone-stroke spontaneously hypertensive rats / 中国组织工程研究

BACKGROUND: Stroke-prone spontaneously hypertensive rat(SHRsp) is an animal model of hypertensive stroke commonly used. The species is hard to be maintained due to hypertension and easily affected by environmental factors, therefore the characteristics of stroke are always aberrated.OBJECTIVE: To observe the abilities of reproduction and maintaining the characteristics of hypertensive stroke in SHPsp.DESIGN: Observational controlled study based on rats.SETTING: Cardiovascular Disease Institute and Fuwai Hospital of Chinese Academy of Medical Sciences.MATERIALS: The experiment was conducted Cardiovascular Disease Institute and Fuwai Hospital of Chinese Academy of Medical Sciences during September 1999 to December 2001. Totally 93 pairs(186) strain SHRsp, 48stroke-prone spontaneously hypertensive rats of 8 weeks age as well as 98pairs(196) rats of normal blood pressure [Wistar Kyoto rat (WKY) ] were selected.METHODS: Brother sister mating method was used. And the conception rate, number of litter size(only calculating the litter size of mother rat not eating neonatal rats), rate of eating neonatal rats by mother rats were recorded and compared with those of WKY. The systolic pressure and heart rate of strain rat were measured when they were 12, 16 and 20 weeks old. In addition, 488-week SHRsp were loaded 10 g/L salty water to accelerate the occurrence of stroke and hypertension and executed when they naturally dead or 12 weeks after salty water load. The brain tissue was processed by H-E staining and observed under microscope to detect the incidence of stroke.MAIN OUTCOME MEASURES: Conception rate of female rats within 2years; litter rate of pregnant rats; eating, rate of eating neonatal rats by mother rats; systolic pressure of strain rat; heart rate; detection rate of stroke.RESULTS: Totally 93 pairs(186) strain rats of SHRsp, 98 pairs(196) WKY and 48 SHRsp of 8 weeks old entered the final analysis. In the first year there were 2 generations delivered, the average conception rate( 100% ) and average litter number (10.3 rats) of SHRsp were higher than those of WKY of the same term(90%, P ＜ 0. 001; 6. 5 rats, P ＜ 0. 001) . In the second year, there were 2 generations of which the conception rate and average litter number(89%, 8.2 rats) of SHRsp were higher than those of WKY(59%, P ＜ 0. 001; 4. 3 rats, P ＜ 0. 001). There were 87 SHRsp female rats and 67normal blood pressure rats pregnant within 2 years, the rate of eating neonatal rats within 4 weeks postnatal was 6% (5/87) which was lower than that of WKY(18%, 12/67), P ＞ 0.05. The systolic pressure of 12weeks old male rats and female rats was 191.6-223.8 mm Hg and 174. 2-196. 3 mm Hg respectively, while that of 16-week old and 20-week male rats were 219.0 -224. 9 mm Hg and 232.0 -242.6 mm Hg respectively. The blood pressure of SHRsp increased with the advancing of age. The heart rate of 12-week old male and female rats was 388-428 times per minute and 373-417times/minute respectively while that of 16-week and 20-week male rats were 392 -410 times per minute and 404-425 times per minute respectively. The pathological detection rate of 48 SHRsp was 81% (39/48).CONCLUSION: The reproduction ability of SHRsp is similar to normal rats. The blood pressure, heart rate and pathological examination of brain tissue of them all maintain the characteristics of hypertension and stroke so that they can be used as qualified experimental model.

Cloning expression and toxic effect on the host bacterial viability of an outer envelope protein from the strong virulent leptospira lai in China / 中国病理生理杂志

AIM: Construction of an eukaryote- E. coli shuttle expressing recombinant plasmid which expresses OmpL1 envelope protein of pathogenic Leptospira, serovar Lai strain 017. METHODS: The OmpL 1 gene was amplified by PCR from the leptospiral genome. Then it was cut with restriction enzymes and ligated to the plasmid pBK-CMV. The correct recombinant plasmid was screened out with analysis of restriction enzymes and PCR. After inducing the E. coli baring recombinant plasmid with IPTG,the complete protein of the bacteria was extracted for SDS-PAGE. At the same time, OD600 of the host bacteria was examined at different time after inducing or uninducing with IPTG. RESULTS: Five strains E. coli containing proper recombinant plasmids were screened out. Four strains E. coli expressed a new protein with a weight of 37 kD among them. With the expression of the heterogenous protein,the OD600 of the host bacteria decreased. CONCLUSION: The shuttle expressing plasmid of the OmpL 1 gene of strong virulent Leptospira strain 017 was successfully constructed. Furthermore,the recombinant plasmid expressed the expected OmpL 1 fusion protein in E. coli and the expression of the heterogenous protein had toxic effect on the host bacteria. This work was important for the future research of OmpL1 protein which relates to the diagnosis,new vaccine preparing and the pathogenic mechanism of leptospirosis.

Preliminary study on detecting drug-resistance mycobacterium tuberculosis by using DNA microarray hybridization / 中国病理生理杂志

AIM:To detect the drug-resistance mycobacterium tuberculosis by using DNA microarray hybridization.METHODS: DNA microarray for detecting drug-resistance of mycobacterium tuberculosis was prepared; Clinical isolated strains were cultivated and their drug-resistance sensitivity was detected. The genome DNA of mycobacterium tuberculosis was prepared and the drug-resistance genes of the mycobacterium tuberculosis were amplified by PCR. Then the gene chip was hybridized, washed, detected and analyzed. RESULTS: Results of cultivating mycobacterium tuberculosis and detecting the drug-resistance sensitivity: one strain was drug-sensitive; four strains were multi-drug-resistant; The detecting results of the drug-resistance was consistent with the results of diagnosis therapy of the 5 clinical patients. The detecting results of gene chip confirmed the above facts. CONCLUSION: Detecting drug-resistance mycobacterium tuberculosis by the gene chip is precise, fast and highly-efficient.

Screening and sequencing of the novel differential gene in leptospira serovar lai / 中国病理生理杂志

AIM: To amplify and analyze the differential DNA fragment between pathogenic leptospira serovar lai and nonpathagenic leptospira serovar patoc I. METHODS: The previously subtractive DNA fragment only existing in leptospira serovar Lai was amplified by cassette ligation and semi-nested PCR.The obtained gene was sequenced and searched homologically. In addition, the deduced amino acid was analyzed and the secondary stracture of protein was predicted. RESULTS: The 580 bp DNA fragment, which deposited in GenBank (AF495587), was cloned, and four overlapping open reading frames (ORF) was contained. The high homology with conserved hypothetical protein streptococcus pyogenes was found. CONCLUSION: This study lays foundation for deeply exploring biological actions of new gene and pathogenic mechanism of leptospira serovar lai.