[Objective]To study the techqiue and method of using in-situ replantation of dead bone to treat infected nonunion of femur. [Method] Debridement and removal of dead bone were carried out in 2 patients who had a chunk of dead bone and multiple sinus without involucrum formation.Sterilized sequester was replanted in-situ,tibia traction was performed and continual Kawashima's irrigation was applied with sensitive antibiotic after surgery.[Result]The time of bone healing in 1 case was ten months.The patient was followed up for 42 months,there was no recurrence of osteomyelitis.The time of bone healing in another case was about 12 months,he was followed up for 30 months,there was also no recurrence in this patient.[Conclusion]Traumatic osteomyelitis complicated with a massive sequester and nonunion could be cured by process which included debridement,in-situ replantation of sterilized death bone,continual washing as well as comprehesive treatment.It could achive the two primary purposes,inflammation cure and bone healing.Among these methods,improved Kawashima's irrigation is the most important method for inflammation cure and bone union.

Lesion was made individually to the various cortical areas in 14 rats.The cortico- fugal fibers terminating into cochlear nuclei were traced in sections by means of the modified Nauta and Fink-Heimer silver staining method.The results were as follows: 1.After damaging the auditory area or somato-sensory area 1(SI)of the cerebral cortex unilaterally,degenerating fibers were found bilaterally in the ventral and dorsal cochlear nuclei.This result showed that the above cortical areas have direct descending connection with the cochlear nuclei. 2.No degenerating fibers were found in the cochlear nuclei after the ablation of the motor or visual area of the cerebral cortex,which demonstrated that the cochlear nuclei do not receive descending fibers from the above cortical areas. 3.The pathway from the cerebral cortex to the cochlear nuclei was:The descending fibers which originated from the auditory area or SI area ipsilaterally passed through the capsula interna and basis pedunculi,and part of these fibers passed dorsally and downward to the lateral portion of the reticular formation of the midbrain;and from there the fibers passed through the lemniscus lateralis and dorsal acustic stria to the cochlear nuclei of the ipsilateral side.The cochlear nuclei in contralateral side also received the corticofugal fibers by way of the dorsal acustic stria.These fibers might cross to the contralateral side at the region in the posterior commissure,superior colliculus commissure and inferior colliculus commissure.

Objective To construct the recombinant plasmid of fusion protein containing respiratory syncytial virus (RSV) cytotoxicity T lymphocyte (CTL) epitope M2:81-95 and heat shock protein (HSP) 70L1, and to investigate its immunogenicity after prokaryotic expression. Methods HSP70L1 gone was cloned from SMMC7721 cells. The M2:81-95 gene fragment was amplified by polymerase chain reaction (PCR) method. Plasmid pET-HSP70L1-M2 : 81-95 (pET-HSP70L1-M2) was constructed, identified and transferred into E. coli BL21 (DE3). Expression of HSP70L1-M2 : 81-95(HSP70L1-M2) was induced by isopropy-β-D-thiogalaetosidc (IPTG). The expressed protein was purified by affinity chromatography and renatured by gradient dialysis. The BALB/c mice were immunized with this fusion protein. IgG antibodies and the subtypes were detected by enzyme-linked immunosorbent assay (ELISA), and CTL responses were determined by methyl thiazolyl tetrazolium (MTT). Results The recombinant plasmid pET-HSP70L1-M2 was successfully constructed. The fusion protein HSP70L1-M2 was expressed in E. coll. The purified protein induced strong RSV-and CTL epitope-specific CTL responses and high titer of protein specific lgG antibody 4.87±0.35. The subtypes were IgG1 (5.53±0.28) and lgG2a (4.40±0.21) and IgG1/ IgG2a ratio was balanced. The titers of lgG, IgG1 and IgG2a in PBS control group were 0.33±0.17, 0.51±0.21 and 0, respectively, which werc significantly lower than those in immunized group (t = 3.512, 3.681, 5.856; P<0.05). Conclusions The recombinant plasmid pET-HSP70L1-M2 is successfully constructed and the fusion protein is expressed and purified. HSP70L1-M2 induced strong RSV-and CTL epitope-specific CTL responses and mixed T helper cell (Th)1/Th2 response in BALB/c mice.

Objective: To investigate the regulation of respiratory syncytial virus CTL epitope fused with G protein antigen fragment G1 to the specific immunoresponses. Methods: The recombinant plasmid pET-DsbA-G1 or pET-DsbA-G1F/M2 was transferred into E.coli BL21(DE3) and the fusion protein DsbA-G1F/M2 or DsbA-G1 was expressed.The expressing product was induced and purified by affinity chromatography. The two proteins were used to immunized BALB/c mice i.p, respectively. Serum and spleen cells were collected regularly. RSV-specific CTL responses were measured by MTT, IgG and IgG1 and IgG2a antibodies by ELISA, neutralizing antibodies by plaque reduction assay. Results: The recombinant proteins were expressed successfully and the purity was over 90% after purified by affinity chromatography. The protein DsbA-G1F/M2 induced significant RSV-specific CTL responses, while DsbA-G1 without CTL epitope did not induce detctable CTL responses. Strong IgG antibody responses were elicited,indicated by both. IgG1 and IgG2a titers induced by DsbA-G1F/M2, while only IgG1 was induced by DsbA-G1.The ratio of IgG1/IgG2a was downregulated significantly. Both antigens induced high level of neutralizing antibodies, but the latter was a little lower. Conclusion: DsbA-G1F/M2, the fusion protein of a CTL epitope and G protein fragment G1 can induce both cellular immunity and humoral immunity. The activation of CTLs downregulates the ratio of IgG1/IgG2a.The more balanced immunoresponse is advantageous for improving safety of the candidate vaccine.

AIM To analyze the toxicity and inhibitory mechanism of extracts from cultured cells (F 4 cell line) of Taxus chinensis on cancer cell lines SMMC 7721 and HEp 2. METHODS MTT assay for cell viability and flow cytometry for cell cycle analysis. RESULTS IC 50 of SMMC 7721 and HEp 2 were 0 161 4 g DCW?L -1 and 0 275 6 g DCW?L -1 respectively,tumor cells in G 2～M stage all increased with higher concentration and longer incubation of extracts from Taxus chinensis cells. CONCLUSION Extracts from cultured cells of Taxus chinensis could have cytotoxic effect on SMMC 7721 and HEp 2 and could induce apoptosis of both two cancer cells.

Objective To investigate the pathologic mechanism of radiofrequency ablation ( RFA ) combined with intravenous infusion of thermosensitive liposome encapsulated vinorelbine (TL-Vin) in treating liver tumors, and to analyze the effect of combination therapy on the long-term survival rate. Methods H22 liver adenocarcinoma tissue was subcutaneously implanted into ICR mice to establish the animal models. At the first experimental period, 40 mice were randomly and equally divided into 5 groups to receive different therapeutic scheme (using different TL-Vin concentrations). Twenty-four hours after the treatment the tumor specimens were collected, the necrotic areas were measured separately, and the optimal TL-Vin concentration was determined. At the second experimental period, 13 mice were randomly selected to receive treatment. Half an hour after the treatment the tumor tissues were collected and the TL-Vin concentration within the tumor was determined. At the third experimental period, 32 mice were randomly and equally divided into 4 groups, and 90 days after treatment the tumor growth curve was drawn. The survival rate was compared between each other of the groups. Results Compared with pure RFA group, TL-Vin + RFA significantly increased tumor coagulation extent (P<0.01). But free-VIN+RFA had similar tumor necrotic extent as that produced by RFA alone (P>0.05). Tumor coagulation area in TL-Vin + RFA group was bigger than that in free-VIN + RFA group at the concentration of 10 mg/kg [(341.8 ± 65.4)mm2 vs (225.3 ± 25.4)mm2, P < 0.01]. In TL-Vin group the coagulation margin was clear. The mean intratumoral Vinorelbine accumulation in TL-Vin + RFA group was 10 folds of that in free-Vin group [(1 156.5 ± 158.3)ng/ml vs (194.5 ± 52.3)ng/ml, P = 0.005]. TL-Vin +RFA had better survival result than that of RFA alone (37.6 ± 20.1 days vs. 23.4 ± 5.0 days, P=0.015), as well as than that of free-Vin + RFA [(37.6 ± 20.1)days vs (23.3 ± 1.2)days, P = 0.016]. Conclusion Thermosensitive liposomal chemotherapies (Vinorelbine) can be selectively delivered at the edge of RFA coagulation area and thus effectively increase RFA-induced tumor coagulation and prolong the end-point survival in experimental mice.

Objective:To investigate whether His-tag to change the immunogenicity of recombinant protein G1F/M2 of respiratory syncytial virus.Methods:The G1 and F/M2 gene fragments were amplified by PCR method and then ligated into the expressing vector pET-His or pET-DsbA-His.Each recombinant plasmid was transferred into E.coli BL21(DE3) and the expression was induced by IPTG.The expressed His-G1F/M2 or DsbA-His-G1F/M2 was purified by affinity chromatography.The latter was digested with thrombase and G1F/M2 was purified by affinity chromatography.His-G1F/M2 or G1F/M2 was used to immunize BALB/c mice.Anti-RSV antibody was measured by ELISA and RSV-specific CTL responses by MTT.Results:No significant difference was observed between the level of anti-RSV antibody or RSV-specific CTL response induced by G1F/M2 and that by His-G1F/M2.Conclusion:His-tag does not change the immunogenicity of recombinant protein G1F/M2 of respiratory syncytial virus.

Objective To establish a quick method to analyze vinorelbine ( NVB) in plasma of beagle dogs and study its pharmacokinetics of long-circulation and thermosensitive liposome loaded vinorelbine bitartrate.Methods The plasma was treated with liquid-liquid extraction after precipitation in methanol.The analysis was perfomed on a Venusil XBP C18 column(2.1 mm ×50 mm, 3 μm) at 35℃,the mobile phase consisted of methanol and water( containing 10 mmol/L ammonium acetate, 1%acetonitrile) 80∶20 and injection volume was 10μl.The type of mass spectrum was multireactive monitoring(MRM) in a positive mode.The monitor transitions were m/z 779.4-765.4 for vinorelbine and m/z 825.4-122 for vincristine.Results The concentration range from 10 to 2500 ng/ml had a good linearity ( r=0.0994).The precision, accuracy and extraction efficiency were acceptable.The plasma samples were stable for 10 days at -20℃ and 24 h at room temperature.Pharmacokinetic study in beagle dogs showed that the main parameters for injection and liposome were Cmax(833.51 ±150.42) and (1397.95 ±443.05)ng/ml, AUC(0-t) (577.16 ±223.57) and (1059.82 ±408.27) ng/ml· h, Cl(3014.64 ±1049.17)and 1633.10 ±551.77 ml/(h· kg), respectively.Conclusion A reliable HPLC-MS/MS method for vinorelbine analysis is established and can be applied to the pharmacokinetics study of liposome.The results show that liposome has a higher AUC, Cmax and longer Cl than injection.Meanwhile, liposome has a lower irritability.

An on-line solid phase extraction ( SPE ) coupled with HPLC-MS/MS method was developed to determine S-ammuxetine and R-ammuxetine in rat plasma. The sample preparation consisted of the following steps:A protein precipitation extraction used methanol and acetonitrile ( 50:50 , V/V ); an on-line SPE treatment to remove most matrixes in plasma;an enrichment and separation step used a C18 analytical column. S-and R-ammuxetine were determined by tandem mass spectrometry. The SPE column was a Retain PEP Javelin (10 mm × 2. 1 mm × 5 μm), while the chromatographic separation was achieved using a ZORBAX SB-C18 (50 mm × 2. 1 mm × 3. 5 μm) analytical column with an isocratic mobile phase composed of acetonitrile-water-formic acid (40:60:0. 1, V/V/V, 0. 3 mL/min). The selected reaction monitoring mode of the positive ion was performed and the precursor to the product ion transitions of m/z 292 . 1/154 . 0 and m/z 260. 4/116. 2 were used to measure S-ammuxetine, R-ammuxetine and internal standard (propranolol). The method was linear over a concentration range from 0 . 2 to 1000 μg/L with the correlation coefficients of 0 . 9903 and 0 . 9951 . The average intra-day precision values were 1 . 2% -12 . 0% for S-ammuxetine and 0. 4%-11. 2% for R-ammuxetine, respectively. The average recoveries were 94. 2%-101. 6% for S-ammuxetine and 94. 3% -109. 4% for R-ammuxetine. Compared to the literature, the sensitivity of this method increased dramatically. The present method has been successfully applied to the preclinical rat research of ammuxetine isomers following intragastric administration.