Objective To study the aglycone of the saponins from Mensamaria intercedens.Methods Complete acid hydrolysis of the glycoside mixture of the sea cucumber with 15%(H_(2)SO_(4))afforded an aglycone product that was separated by multichromatography and their chemical structures were elucidated by chemical and spectral analyses.Results Two compounds were obtained and identified as 16?-acetoxy-9(11),22(23) E,24(25)-3?-hydroxyholost(Ⅰ),16?-acetoxy-8(9),22(23) E,24(25)-3?-hydroxyholost(Ⅱ).Conclusion Two compounds are new triterpenoids.

AIM: To provide experimental evidence for gene therapy of thrombophilia disease, we constructed the eukaryotic expression plasmid with human thrombomodulin (hTM) gene and observed the alteration of hTM expression on the surface of human umbilical vein endothelial cells (HUVECs) with and without the reconstructive plasmid. METHODS: The whole expressive fragment of hTM gene was amplified by PCR from human genome. Both hTM gene and pcDNA3.1(+)/neo empty vector was digested by HindⅢ and EcoRⅠ. Two digested fragments were ligated into pcDNA3.1/hTM with T_4DNA ligase. After identifying, the reconstructive plasmid transfected into HUVECs using lipofectin. The hTM antigen on the HUVECs was detected by immunohistochemistry. RESULTS: The hTM reconstructive plasmid was confirmed by double endonuclease redigesting and sequencing. About 10% HUVECs were transfected by pcDNA3.1/hTM plasmid with lipofectin and the high-level hTM was detected on the transfected cells. CONCLUSION: We constructed the pcDNA3.1/hTM plasmid successfully, and it could be expressed on the HUVECs. [

Objective:To screen the proteins interacting with the human cytomegalovirus(HCMV)UL132 protein from the human fetus brain cDNA library by using Yeast Two-Hybrid System, and to elucidate the possible mechanism of UL132 protein in congenital cytomegalovirus infection.Methods:The HCMV UL132 fragment was amplified by polymerase chain reaction,the amplified HCMV UL132 fragment and expression vector pGBKT7 were digested and purified,and the HCMV UL132 fragment was linked to the vector pGBKT7.The pGBKT7-UL132 was constructed and transformed to yeast AH109, then the Human Fetal Brain DNA Library DNA was transformed into AH109 yeast.Using HCMV UL132 as abait, a human fetus brain cDNA was screened and the proteins interacting with UL132 protein were searched, the positive clone was sequenced and analyzed by bioinformatics methods.Results:The bait expression vector pGBKT7-UL132 was successfully constructed.The results of double enzyme digestion showed that there were two visible bands of 800 and 7 000 bp, respectively.After transformation of library plasmid, the transformation efficiency was calculated, and the transformation efficiency was 6.6×103 cfu· μg-1.There were 95 blue clones by X-gal coloration reactionsequencing and there were 10 clones interacting with the protein encoded by UL141 protein.The BLAST analysis showed that 7 of them were highly homologous with CAML.Conclusion:CAML might be one interaction protein with HCMV UL132 in Human Fetus Brain cDNA Library,suggesting that the interaction may be associated with the invasion and proliferation of the HCMV.

Objective To screen a human fetal brain cDNA library for proteins that can interact with HCMV UL145 using a yeast two-hybrid system.Methods A bait plasmid (pGBKT7-UL145) was constructed.Using HCMV UL145 as bait,a human fetal brain cDNA library was screened and proteins interacting with UL145 were identified using bioinformatic methods to sequence and analyze the positive clones.Results Three clones interacting with HCMV UL145 were found,and identified as FOXG1.Conclusion Several proteins interacting with HCMV UL145 in the human fetal brain cDNA library were identified as FOXG1,indicating that this protein may play an important role in the course of HCMV infection.

Objective To investigate the effect of small interfering RNA (siRNA) targeting cytoplasmic domain of tissue factor on apoptosis of vascular endothelial cells. Methods Specific siRNA targeting cytoplasmic domain of tissue factor were designed, and synthetic oligos were inserted into plasmid DNA. The siRNA constructs were transfected into human umbilical vascular endothelial cells (HUVEC) with liposome. The HUVEC were transfected with the constructs encoding siRNA Ⅰ, siRNA Ⅱ and pcDNA~(TM)6.2 GW/-miR plasmid separately. The transfected HUVEC were mixed with CD8~+ T lymphocytes. The apoptotic rate of tranfected HUVEC mixed with lymphocytes was analyzed by flow cytometry. Magnetic beads were used to measure PT of the supematant in the mixed lymphocytes culture. Results The siRNA constructs were confirmed by DNA sequence analysis. The apoptotic rate of HUVEC transfected with siRNA Ⅰ and Ⅱ plasmids was decreased significantly as compared with the empty control group (P<0.01). The apoptosis rate of HUVEC transfected with siRNA Ⅰ plasmid was lower than that of HUVEC transfected with siRNA Ⅱ plasmid (P<0.05). APTT of the culture supernatants in the three transfection groups was lower in the control groups (P <0.05), but there was significant difference among the three transfection groups. Conclusion The siRNA targeting cytoplasmic domain of tissue factor were successfully constructed, siRNA can protect HUVEC, and reduce the apoptotic rate of endothelial cells in mixed lymphocyte reaction without influencing the coagulation function.

The expression of paired immunoglobin-like receptors A (PIR-A) and B (PIR-B) and their relationship with tolerogenic dendritic cells (T-DC) in mice were investigated. The mouse DCs line, DC2.4 cells were cultured with the recombinant murine interleukin-10 (IL-10) and recombinant human transforming growth factor beta1 (TGF-beta1) respectively to develop the T-DC and stimulated with lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small interfering RNAs (siRNA) molecule for PIR-B was chemically synthesized and transfected into DC2.4 cells (Si-DC) by lip2000. The expression of PIRs on DC2.4 cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), flow cytometry (FCM) and Western blot. Realtime reverse transcriptase-polymerase chain reaction (Realtime-PCR) was applied for measurement of PIR-A and CD80, CD86, MHC-II mRNA expression. The allogeneic stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR) using (3)H-thymidine incorporation test. The concentration of IFN-gamma in supernatants of MLR from distinct groups was analyzed by ELISA. The results showed that PIR positive rate was (28.65+/-8.12)% examined by FCM on DC2.4 cells. PIR positive rate was increased dramatically to (54.21+/-6.34)%, (58.78+/-4.70)%, (48.24+/-6.75)% respectively for IL-10, TGF-beta1 and LPS induction (P<0.01), but there was no significantly different among the three groups (P>0.01). The semi-quantitative RT-PCR and Western blot revealed that IL-10 and TGF-beta1 induced the higher PIR-B level and lower PIR-A level. On the contrary, the LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. Realtime PCR examination demonstrated that PIR-A and co-stimulating molecules such as CD80, CD86 and MHC-II were increased significantly after stimulation with LPS. Compared with the DC2.4 cells and the LPS-DC, the T-DCs inhibited alloactivated T cell proliferation and down-regulated the IFN-gamma secretion in MLR supernatant. Si-DC promoted the T cell proliferation (P<0.01) and enhanced the IFN-gamma secretion (P<0.01). It was concluded that up-regulating the PIR-B and down-regulating the PIR-A expression were the general feature of phenotype and constructed the new targets for dendritic cells to acquire immune tolerance in mice. Overexpression of PIR-B can inhibit the up-regulation of the PIR-A, CD80, CD86 and MHC-II expression, which might be the molecular mechanism for the T-DC.

To investigate the option of treatments for bone defect in femoral revision after total hip replacement (THR), 29 patients (29 hips) who had undergone hip replacement 13.7 years previously were hospitalized again to undergo revisions. 3 patients were given cement protheses and the other 24 patients were given non-cement protheses. In 3 cases were used Synergy+Reflection protheses, and in the others were used CLS +ALLOFIT. All of the patients have got primary healing without early infection symptoms post-operatively. We have been following up 25 cases (25 hips) for 58 months averagely. All the hip joints functioned well. Harris score increased from preoperative 36.8 to postoperative 93.2. In our experience, if there exist significant bone defect in acetabular bone and the coverage of the cup by the graft is more than 80%, bone graft is not needful. If the coverage is between 50% and 80%, bone grains combining bone segment grafts are needful. If the coverage is less than 50%, bone segment grafts be likely to give good effect. In summary, compacted bone grains grafts combining allograft osintegumentale plate could gain satisfactory outcome for femoral bone defect.
Acetabulum ; surgery ; Aged ; Arthroplasty, Replacement, Hip ; methods ; Bone Transplantation ; methods ; Female ; Hip Prosthesis ; Humans ; Male ; Middle Aged ; Osteolysis ; Prosthesis Failure ; Reconstructive Surgical Procedures ; methods ; Reoperation

Objective To evaluate the diagnostic value of fluorescent quantitative polymerase chain reaction(FQ-PCR) assay in human cytomegalovirus (HCMV) infection by detecting quantitatively HCMV DNA in peripheral blood mononuclear cell ( PBMC) of newborns,to evaluate the choice of detection methods for neonatal HCMV infection,and to provide a reasonable diagnosis basis for the clinic. Methods The urina-ry HCMV-DNA levels in 102 neonates with suspected HCMV infection were detected by FQ-PCR. The HCMV-DNA in PBMC was detected by FQ-PCR,and serum HCMV-IgM antibody was detected by chemilu-minescence immunoassay ( CLIA) . Then the sensitivity, specificity, coincidence rate and other indicators in the three kinds of detection methods were compared. Results Among 102 cases of suspected HCMV-infec-ted newborns,56 cases were symptomatic and 46 cases were non-symptomatic. The positive rate of HCMV-DNA in urine[87. 3％(89/102)] was significantly higher than that of PBMC HCMV-DNA [58. 8％ (60/102)] and serum HCMV-IgM antibody [40. 2％ (41/102)](all P<0. 01). For symptomatic HCMV-infec-ted newborns, PBMC HCMV-DNA quantitative detection sensitivity ( 71. 4％) was higher than serum HCMV-IgM antibody (57. 1％), and the specificity (56. 5％) was higher than urine HCMV-DNA quantifi-cation (8. 7％). The area under receiver operating characteristic(ROC) curve of PBMC HCMV-DNA quan-tification and HCMV-IgM antibody detection were 0. 642 (P=0. 014) and 0. 659 (P=0. 006),respectively;therefore PBMC HCMV-DNA and HCMV-IgM antibodies were of great importance in diagnosing symptom-atic HCMV infection in neonates. The area under the ROC curve of urinary HCMV-DNA quantification was 0. 461 ( P =0. 496 ) , and there was no significant difference between symptomatic and non-symptomatic HCMV infections in neonates. Conclusion HCMV-DNA detection in PBMC has higher sensitivity compared with HCMV-DNA detection in urine and higher specificity compared with IgM antibody detection in serum. It can be used to detect the early infection of HCMV in newborns. The rate of detection of HCMV infection can be improved by combination of the three methods.

The expression of paired immunoglobin-like receptors A (PIR-A) and B (PIR-B) and their relationship with tolerogenic dendritic cells (T-DC) in mice were investigated. The mouse DCs line, DC2.4 cells were cultured with the recombinant murine interleukin-10 (IL-10) and recombinant human transforming growth factor β1 (TGF-β1) respectively to develop the T-DC and stimulated with lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small interfering RNAs (siRNA) molecule for PIR-B was chemically synthesized and transfected into DC2.4 cells (Si-DC) by lip2000. The expression of PIRs on DC2.4 cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), flow cytometry (FCM) and Western blot. Realtime reverse transcriptase-polymerase chain reaction (Realtime-PCR) was applied for measurement of PIR-A and CD80, CD86, MHC-Ⅱ mRNA expression. The allogeneic stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR) using 3H-thymidine incorporation test. The concentration of IFN-γ in supernatants of MLR from distinct groups was analyzed by ELISA. The results showed that PIR positive rate was (28.65±8.12)% examined by FCM on DC2.4 cells. PIR positive rate was increased dramatically to (54.21±6.34)%, (58.78±4.70)%,(48.24±6.75)% respectively for IL-10, TGF-β1 and LPS induction (P＜0.01), but there was no significantly different among the three groups (P＞0.01). The semi-quantitative RT-PCR and Western blot revealed that IL-10 and TGF-β1 induced the higher PIR-B level and lower PIR-A level. On the contrary, the LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression.Realtime PCR examination demonstrated that PIR-A and co-stimulating molecules such as CD80,CD86 and MHC-Ⅱ were increased significantly after stimulation with LPS. Compared with the DC2.4 cells and the LPS-DC, the T-DCs inhibited alloactived T cell proliferation and down-regulated the IFN-γ secretion in MLR supernatant. Si-DC promoted the T cell proliferation (P＜0.01) and enhanced the IFN-γ secretion (P＜0.01). It was concluded that up-regulating the PIR-B and down-regulating the PIR-A expression were the general feature of phenotype and constructed the new targets for dendritic cells to acquire immune tolerance in mice. Overexpression of PIR-B can inhibit the up-regulation of the PIR-A, CD80, CD86 and MHC- Ⅱ expression, which might be the molecular mechanism for the T-DC.

At present,ultrasonic treatment technology develops rapidly and has been applied in many medical fields.Focusing ultrasound (FUS) technology can focus ultrasound,which penetrates the skull into the lesions to play therapeutic role.In this paper,the mechanism of FUS therapy and its application in the treatment of central nervous system diseases,such as epilepsy,are summarized as follows.