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 (P<0.05);while the resistance to bending deformation capacity (flexural strength) of the retrograde interlocking intramedul ary nail was not significantly greater than that of the LISS plate (P>0.05). (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.

In order to investigate the effect of sequence-specific small interfering RNA on suppressing cyclin D1 expression and proliferation and cell cycle and expression of G1 phase regulators of fibroblasts derived from keloid, the plasmid expression vector of siRNA targeted against cyclin D1 was constructed and transfected into fibroblasts with LipofectamineTM 2000. The changes of cyclin D1 expression were detected by fluorescent quantitative PCR(FQ-PCR), semi-quantitative RT-PCR. The effect of sequence- specific small interfering RNA in suppressing the proliferation of fibroblasts was detected by MTT assay. Flow cytometry were used for evaluation ofceU cycle. The expression of cyclin D1, CDK4, pRb and P16 was detected by immunohistochemical method. The results showed that: (1) The sequence- specific siRNA effectively suppressed cyclin D1 expression at both mRNA levels with inhibition rate of 63.68% and 92.83% (P<0.01). (2) Significantly inhibited the proliferation of fibroblasts, and changed cell cycle in percentage of G0/G1 phase cells was increased compared with the controls groups in fibroblasts(P < 0.05). (3) 72 h after transfection, the expression of cyclin D1, CDK4 and pRb decreased, and the expression of P16 increased. It can be concluded that the plasmid expression vector for the RNAi against cyclin D1 constructed in the study can effectively and specifically suppress cyclin D1 expression, and progression of G1/S is effected by G1 phase related regulatory protein, and suppresses proliferation of fibroblasts derived fiom keloid.

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.

BACKGROUND: Both density gradient centrifugation and adherence method arc frequently used to isolate bone marrow mesenchymal stem cells (BMSCs).OBJECTIVE: To investigate the approaches to isolate, culture and identify the rabbit BMSCs in vitro by the combination of den,ity gradient centrifugation and adherence method. DESIGN, TIME AND SETTING: Contrast cytological study, which was performed in Central Laboratory of Shanghai 6th People's Hospital between October 2007 and March 2008.MATERIALS: Six 2-week-old rabbits were selected for BMSCs preparation and primary culture; Percoll separating medium (1.073 kg/L) was also used for this study.METHODS: BMSCs were separated and purified with Percoll separating medium by density gradient centrifugation and adherence method. The three-, five-, seven-, and nine-passage BMSCs were counted for growth curve. MAIN OUTCOME MEASURES: Morphological features and growth states of primary and passage cells were observed under inverted microscope. Indirect immunofluorescence of CD44 and CD34 antibodies were used to examine the stem cells. CD44 staining was positive, and CD34 staining was negative, suggesting the extracting and purifying cells were BMSCs. RESULTS: The passage BMSCs were uniformly distributed like fusiform shape, which were more uniform than primary cultured cells. The BMSCs grew productively and proliferated rapidly; meanwhile, the nucleolus was clear, caryopla.sm was in a large proportion, morphological features were uniform, ceils like bostrychoid or whirlpool were arranged parallelly, and the five-pa.ssage cells were not changed remarkably. Proliferation was decreased gradually with the passage increasing; especially, the proliferation of three-five-passage cells was the strongest. The separated cells expressed CD44 but not CD34. CONCLUSION: High-purified rabbit BMSCs are obtained by both density gradient centrifugation and adherence method.

AIM:To investigate the effects of irbesartan on the expression of hepatocyte growth factor (HGF) at mRNA and protein levels in rats with myocardial infarction (MI), and to explore the mechanisms of irbesartan attenuating myocardial fibrosis.METHODS:The male Wistar rat model of MI was successfully established.The surviving rats 24 h after the operation were randomly divided into 3 groups:model group,irbesartan group and sham group, with 9 rats in each group.The rats in irbesartan group were treated with the solution of irbesartan (50 mg·kg-1·d-1) by intragastric administration, while the rats in model group and sham group received the equal volume of saline by the same way.The body weight and left ventricle mass (LVM) of the rats were measured at the 4th week after operation, and the pathological changes of the ischemic myocardium were observed with HE staining.Meanwhile, the expression of HGF at mRNA and protein levels was detected by RT-qPCR and Western blot.RESULTS:HE staining showed that the myocardial cells in sham group were in neat arrangement, while the cardiac structure in model group and irbesartan group was in disorder.The pathological changes in irbesartan group were less than that in model group.No difference in the body weight at the 4th week after operation was observed, while the LVM was significantly different among the 3 groups (P<0.01).The LVM in model group was higher than that in sham group (P<0.01), and that in irbesartan group was higher than that in sham group (P<0.05).The LVM in irbesartan group was lower than that in model group (P<0.05).The expression of HGF at mRNA and protein levels was detected in each group.The expression of HGF at mRNA and protein levels in irbesartan group was higher than that in sham group (P<0.05), and that in model group was higher than that in sham group (P<0.01).Moreover, the mRNA and protein levels of HGF in irbesartan group were lower than those in model group (P<0.05).CONCLUSION:The LVM of MI rats with the treatment of irbesartan was reduced obviously at the 4th week after operation, and the pathological changes were also improved.At the 4th week after the operation, the treatment of irbesartan inhibited the expression of HGF at mRNA and protein levels.

Objective To compare the short and long term effect of knee osteoarthritis treatment with two therapeutic methods which are simple high cut of fibula and arthroscopic lavage debridement combined with fibular osteotomy.Methods Data of 45 consecutive patients with knee osteoarthritis were retrospectively observed and selected,and then they were divided into two groups according to the operation method.23 patients with simple high cut of fibula were selected as the control group,and 22 patients with arthroscopic lavage debridement combined with fibular osteotomy were selected as the observation group.Results The VAS and AKS scores of the control group at the time of 3m,6m,1 2m were (4.1 3 ±0.76)points,(1 07.04 ±21 .53)points;(2.70 ±0.64)points,(1 41 .1 7 ±1 2.57)points;(2.43 ±0.79)points,(1 48.26 ±5.81 )points;and the scores of the observation group were (3.45 ± 0.60)points,(1 26.64 ±1 4.24)points,(2.70 ±0.73)points,(1 46.45 ±7.26)points,(2.41 ±0.85)points and (1 48.26 ±5.81 )points.The differences between the two groups were statistically significant at the time of 3 months after surgery(t =3.32,-3.62,all P <0.05),the effect of observation group was better than the control group;but the other indices had no statistically significant differences (t =0.51 ,-1 .72,0.1 1 ,-0.20,all P >0.05 ). Conclusion The simple high cut of fibula with small trauma,less cost,less surgical difficulty,and long -term clini-cal curative effect is not less than the arthroscopic lavage and debridement combined with fibular osteotomy.There-fore,it should be carried out in the primary hospitals widely.

Objective To express the novel fibronectin-binding protein Fba of group A streptococcus(GAS)and analyze its immunogenicity,so that to evaluate the immune responses to GAS infection.Methods fba gene was amplified by polymerase chain reaction(PCR)and confirmed by sequencing.Then it was cloned into pGEX4T-2 vector and Fba protein was expressed in E.coli BL21.The protein expression was identified by enzyme-linked immunosorbent assay(ELISA)and Westernblot.The sera from mice infected with GAS and anti-streptolysin-O positive patients were detected using microtiter plates coated with purified Fba protein as antigen.Afterward Balb/C mice were immunized with this purified protein and the sera were collected after the third immunization for the detection of IgG titer.Results It was confirmed by ELISA and Western blot that the recombinant Fba protein had a specific affinity with anti-Fba sera of rabbit.The anti-serum IgG titer of mice imrnunized with Fba protein was up to 1:4800.Conclusions GAS infection or Fba protein immunization are able to induce high serum titer of anti-Fba which could react specifically with the recombinant Fba protein.It indicates that Fba protein has good immunogenicity and antigenicity.So Fba protein could be a GAS candidate vaccine and an important tool to detect anti-GAS titer in GAS infected patients.

Objective The truncated fibronectin-binding proteins A (Fba protein) were cloned and expressed, then animals were immunized with Fba protein and subsequently challenged with group A streptococcus (GAS) to further investigate protective antibodies induced by each domain and determine the most immunogenic domain. Methods Fba proteins, which were divided into four overlaps based on the structural domains, were truncated and expressed. The fba truncated genes were amplified by polymerase chain reaction (PCR) with SSI-9 of GAS as template, and cloned into prokaryotic expression plasmid pGEX-2T and expressed in E. coli BL-21. The products were confirmed by Western blot and purified by affinity chromatography column. Female BALB/c mice were immunized with the four truncated proteins respectively, with phosphate buffered solution (PBS) as control. The serum IgG of mice was detected by enzyme-linked immunosorbent assay (ELISA). After the third immunization, mice were challenged with fatal dose of GAS (M+ Fba+ ) to evaluate the protective rates in each group. The data were compared by analysis of variance and Fisher's exact test.Results Prokaryotic expression plasmids of pGEX-2T/FbaAl, pGEX-2T/FbaA2, pGEX-2T/FbaA3 and pGEX-2T/FbaA4 were successfully constructed and the four truncated proteins were expressed and purified successfully. Serum levels of IgG in each experimental group gradually increased with immunization with Fba protein more times. After the third immunization, the IgG titer against FbaA2 1290.2, P<0. 01). After GAS challenge, four out of eight mice were protected in FbaA2 protein group, while two out of eight mice in FbaAl protein, FbaA3 protein and FbaA4 protein groups,respectively (P<0. 05). Conclusions Four truncated Fba proteins are constructed and expressed successfully. Truncated FbaA2 protein could be able to induce strongest protective immune response.

Objective:To identify monoclonal antibody McAb2 recognizing epitope of Fba of GAS.Methods:The overlapped peptides were synthesized and their abilities to bind McAb2 were detected by dot-ELISA.The predominance amino acids specific for McAb2 were screened using phage 7 peptide library.Results:The result by dot-ELISA analysis demonstrated that the synthetized peptide,amino-acid residues 100-112~(th),could bind McAb2 with high affinity.The predominance amino acids specific for McAb2 were ITPDL,which was located in 100-110~(th)aa of Fba by panning with phage 7 peptide library.Conclusion:The domain and the predominance amino acids of Fba recognized by McAb2 is determined.The results would contribute to the research of the role of Fba on the pathogenic mechanism of GAS,the identification of function of McAb2,and the development of epitope-peptide vaccine.

Objective:To observe cellular,humoral and mucosal immune responses induced by DNA-or protein-based of FimH of UPEC type 1.Methods:After mice were immunized respectively with recombinant plasmid pcDNA3.1/fimH or pcDNA3.1/fimC,and the combinant FimH and FimC protein,the anti-FimH protein IgG of sera and SIgA in bladders were detected by ELISA.The lymphocyte phenotypes of CD3,CD4 and CD8 were analyzed by FCM.Results:The titers of IgG in sera and SIgA in the bladders were all low in the group immunized by recombinant FimH plamid,but the percentage of CD4+T cells in spleen was high,which revealed that recombinant FimH plamid was able to trigger better cellular immune response.The titers of IgG were very high in the group immunized by FimH protein,which suggested that the FimH protein was able to trigger better humoral immune response,but SIgA in the bladders was not detectable.Conclusion:The DNA for FimH can induce humoral,mucosal and cellular immune response.FimH protein can only induce humoral immune response.FimC protein is able to enhance the immunogenicity of FimH protein.