Objective To study the clinical efficacy of acupuncture combined with medication plus joint mobilization in the treatment of lumbar intervertebral disc protrusion accompanied with secondary lumbar spinal stenosis.MethodsTotally 66 cases of lumbar intervertebral disc protrusion accompanied with secondary lumbar spinal stenosis were collected randomly and divided into treatment group (34 cases) and control group (32 cases). The control group was given treatment of simple acupuncture and TCM medication, while the treatment group was given joint mobilization treatment besides acupuncture and TCM medication. Functions of lumbar vertebra were evaluated by ODI scale and the degrees of pain were evaluated by VAS. The clinical efficacy in the two groups was compared. Results ODI in both groups were significantly improved after treatment compared with that before treatment. However, the changing range of the ODI of the treatment group was more significant than that in control group (P<0.01). After treatment, VAS scores were relieved (P<0.05) in both groups, and treatment group was more significant than that in control group (P<0.05). The total clinical efficacy was 97.06% (33/34) in the treatment group, and 84.38% (27/32) in the control group, with statistical significance (P<0.05).Conclusion Acupuncture combined with medication plus joint mobilization in the treatment of lumbar intervertebral disc protrusion accompanied with secondary lumbar spinal stenosis has good efficacy.

Objective To separate and purify intrahepatic macrophages from Microtus fortis Mf and identify its phagocy?tosis. Methods The intrahepatic macrophages from Mf were separated and purified by perfusion collagenase digestion and density gradient centrifugation. The function of the cells was identified by FACS analysis and ink phagocytosis activity. Results The macrophage cells from the liver of Mf were obtained. These cells were bright and circular and grew adhering to the wall. The proportion of the living cells was 95%. The binding rate of these cells from Mf with anti?mouse CD14 antibody Clone Sa2?8 was about 50%of the rate of macrophage from C57BL/6 mice with this monoclonal antibody. The result of ink?phagocytosis ex?periment of macrophage cells from the liver of Mf was positive. Conclusion The method above mentioned is useful to separate and purify macrophage from the liver of Mf. The study builds the foundation for further research on macrophages of Mf against Schistosoma japonicum.

Objective To observe the destroyed architecture of splenic lymphoid follicles in mice infected with Schistosoma japonicum by immunohistochemistry. Methods The mice infected with S. japonicum(20 cercariae/mouse)for 8 weeks were sacrificed,and the splenic samples were paraffin embedded and sliced. The sections were first stained by hematoxylin and eosin to observe the massive structure of splenic lymphoid follicles,and then B cells,follicular dendritic cells(FDC)and germinal center cells were labeled with anti-B220,anti-CD21 or anti-Ki67 antibodies respectively by immunohistochemistry to observe the distribution of the specific cells of lymphoid follicles. Results The results of HE staining showed that the structure of lym-phoid follicles in spleens of infected mice was blurred,the number and area of follicles were significantly reduced compared to those of the normal mice. The immunohistochemical staining showed that the splenic T/B lymphocyte segregation ,FDC network and germinal centers of the infected mice all disappeared. Conclusion The structure of splenic lymphoid follicles in the mice infected with S. japonicum is obviously damaged.

Objective To compare the effect of ibuprofen and glucosamine on synoviocyte proliferation and cartilage oligomeric matrix protein (COMP) expression in human knee osteoarthritis. Methods Human synoviocytes were isolated from synovium (earlier stage and late stage of OA) by tissue culture and were cocultured with ibuprofen and glucosamine. The concentration of COMP was determined by MTS/PMS method and hCOMP kit. Two-tailed t-test was used for statistical analysis. Results The observation time of tissue culture was determined at 5～7 day by the MTS/PMS method. The A values of glucosamine [ late stage group (0.054±0.021), early stage group (0.777±0.034)] were less than the normal serum control group (P＜0.05).Both ibuprofen [late stage group (35.4±1.9), early stage group (46.0±2.2)] and glucosamine [late stagegroup (36.6±1.3), early stage group (48.8±1.3) ] could decrease the concentration of COMP in synoviocyte secretion in vitro (P＜0.05). Conclusion Glucosamine can inhibit the synoviocyte proliferation of human knee osteoarthritis (both early stage and late stage) in vitro. Both ibuprofen and glucosamine can inhibit the COMP secretion of synoviocyte in vitro.

Objective To clone and express the partial encoding sequence of Mr 70 000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. Methods Total RNA was extracted from oocysts of C.andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. Results The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70 000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43 000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. Conclusion The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.

Objective To study the structural features and characteristics of a novel gene Schistosoma japonicum 79(Sj79), and observe its effect of RNA interference(RNAi),so as to provide the experimental basis for its further function study and mechanism study of anti reproductive development of schistosome. Methods The gene structure and characteristics of Sj79 were analyzed by bioinformatics methods. Then the expressions of Sj79 messenger RNA(mRNA)during the different develop?mental stages of schistosome were analyzed and the effects of RNAi silencing were observed by the soaking method. The tran?scriptional levels of Sj79 after RNAi were detected by real time PCR. Results The open reading frame of Sj79 contained 696 base pairs with an exon structure. The gene had obvious stage specificity,and its transcriptional level in mature female worms was the highest. After soaking for 3 d,the Sj79 mRNA level[(41.0 ± 12.3)%]in the siRNA?1 group with low dosage(20 nmol/L) was lower than that in the siRNA?NC group[(103.2 ± 14.4)%],the difference was statistically significant(t=3.28,P<0.05). When with high dosage(200 nmol/L ),both the Sj79 mRNA levels in the siRNA?1 group[(15.8 ± 10.9)%]and siRNA?2 group [(11.1 ± 8.8)%]were significantly lower than that in the siRNA?NC group[(100.1 ± 6.3)%](t=13.44,27.84,both P<0.01). After soaking for 7 d,only the Sj79 mRNA levels in the siRNA?1group[(43.4 ± 4.5)%]and siRNA?2 group[(62.5 ± 5.4)%]with low dosage were lower than that in the siRNA?NC group[(100.4 ± 5.2)%],and the differences had statistical sig?nificance(t=8.33,5.07,both P<0.01). Conclusion Through this study,we have improved the mRNA sequence and genom?ic information of Sj79 gene,and understood its structural features,as well as selected out two effect fragments siRNA?1and siR? NA?2 which will provide the basic evidences for the further study on egg laying interference of the female adult worm of schisto?some in vitro.

Objective To compare the effect of glucosamine (Virtral-s) on the cartilage oligomeric matrix protein (COMP) secretion of chondrocytes and synoviocytes in vitro.Methods Chondrocytes and synoviocytes isolated from knee cartilage of osteoarthritic patients were cultured by phased enzymatic digestion.Sera containing Virtral-s of the experimental animals were obtained after orally administrated Virtral-s at the dosages that equal to human.Cells were cultured in the medium with Virtral-s containing sera.Super-natant COMP level was tested by enzyme-linked immunoabsorbent assays (ELISA).Results COMP conceu-tration of synoviocytes cultured in vitro was significantly higher than that of chondrocytes (P＜0.05).Virtral-s could significantly increase COMP secretion in cultured chondrocytes in vitro (P＜0.05),however,it had a weaker role on synoviocytes,ie,it could only mildly reduce COMP secretion of synoviocytes.Conclusion Glucosamine (Virtral-s)-containing serum can promote COMP secretion of chondrocytes in vitro,and it has no significant effect on synoviocytes in vitro.

Objective To explore the mechanism of intrathoracic pressure(ITP) influncing cardiac function and facilitate noninvasive determination of ventricular pressure theoretically.Methods With Valsalva and Mueller maneuver,two-dimension images of standard long axis views and the cross-sectional views were recorded in 20 volunteers,aged from 18 to 45 years,at the specific ITP levels(including-20 mmHg、-10 mmHg、0 mmHg、+ 10 mmHg and + 20 mmHg).The subjects were instructed to mantain for at least 10 s,and three successive measurements were recorded and averaged.The stroke volume(SV) and radius of curvature(R) were obtained from further off-line analysis.Results With the ITP maintaining at -20 mmHg,-10 mmHg,0 mmHg(end expiration),+ 10 mmHg and + 20 mmHg respectively,the corresponding radiuses of curvature were (2.35 ±0.24)cm,(2.25 ± 0.23)cm,(2.14 ± 0.21)cm,(2.02 ± 0.21) cm,(1.93 ± 0.19) cm,there were statistically significances between two groups (P =0.006,0.031,0.005 and ＜0.001,respectively].When the ITP were at 0 mmHg(end expiration),+ 10 mmHg and + 20 mmHg,the stroke volume were (71.54±8.81)ml,(73.20±9.52)ml and (78.81± 14.61)ml (P =0.674,0.135).When the intrathoracic pressure decreased from 0 mmHg to-20 mmHg,the stroke volume were (78.81±14.61)ml,(68.28 ±9.28)ml and (59.69±7.52)ml(P =0.029,0.037).Conclusions The ITP has different effects on the two ventricles,and subsequently generates a pressure gradient across the IVS which can alter its shape and position at end-diastole.With the IVS shifting,the preload and filling function of left ventricle gets changed acorrding to the Frank Starling principle.

Objective To perform the cloning of the gene encoding Schistosoma japonicum Chinese-strain hypoxanthine-guanine phosphoribosyltransferase(HGPRT)and its expression in Escherichia coli.MethodsA couple of primers were designed with the BamHI restriction endonuclease site introduced in forward primer and SalI in reverse primer.Total RNA was isolated from adult worms of S.japonicum Chinese-strain(Anhui-strain,Sjc-A)and the SjcHGPRT gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR).The PCR product and the prokaryotic expression vector pET28a were digested by both restriction endonucleases BamHI and SalI.The target DNA fragments were purified and cloned properly into pET28a.After identification by en-donucleases digestion,PCR and sequencing,the recombinant plasmid pET28a-SjcHG PRT was transformed into competent E.coli BL21 and expressed in the presence of IPTG.Results pET28a-SjHGPRT was sequenced and shown to be 99% and 83% identical in deduced amino acid sequence to that of S.japonicum Chinese-strain(Hunan-strain,Sjc-H)and S.mansoni HGPRT,respectively.The results of SDS-PAGE and Western blot revealed that the molecular weight of expressed protein was around 30 kDa and could be recognized by anti-His-G-HPR antibody and sera from mice and human with schistosomasis japonica.Conclusion The recombinant plasmid containing SjcHGPRT cDNA is successfully constructed and its expression protein(reSjcHGPRT)is also successfully purified.

Objective To clone and express the gene encoding Schistosoma japonicum myophilin-like protein (SjcMLP) and to study the antigenicity of the recombinant protein. Methods The SjcMLP gene was amplified by PCR. The PCR product was cloned into T vector, and then subcloned into expression vector pQE30. The recombinant plasmid of pQE30-SjcMLP was transformed into E.coli M15, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analysed by SDS-PAGE. The recombinant protein (reSjcMLP)was purified with the Ni-NTA resin, and analysed with SDS-PAGE and Western blot. The titers of sera from C57BL/6 mice immunized subcutaneously with reSjcMLP were detected by ELISA. Results The results of SDS-PAGE and Western blot showed that the molecular weight of expressed fusion protein was around 24.8 kDa and was recognized by the sera from the mice infected with Schistosoma japonicum. The purified protein of reSjcMLP was coated for ELISA test and the IgG titers in the sera from the mice immunized with reSjcMLP were as high as 1∶12 800 reacted with. However, no significant difference was found in worm reduction rates between the immunized mice and control mice. Conclusions The fused recombinant protein of reSjcMLP is successfully ex-pressed and purified. The recombinant protein in this experiment fails to induce significant protection against the challenge infection in C57BL/6 mice.