Objective To establish the amplified luminescent proximity homogeneous assay(AlphaLISA) for the detection of Sendai virus.Methods The antigen concentration,serum concentration and the donor beads/acceptor beads ratio used in the AlphaLISA method were optimumized by the phalanx experiments, then the antibodies of Sendai Virus in 40 rat sera were detected by the established AlphaLISA method and ELISA detection method.The results were compared and the differentia between the two methods was confirmed by IFA.Results The optimum concentration of SV bio-peptide in AlphaLISA assay was 250 nmol/L, the best proportion of donor beads/acceptor beads ratio was 1 ∶1, using the concentration of 20 μg/mL and the serum dilution was 1∶10000.7 of the 40 rat sera were detected SV positive by ELISA, the positive rate was 17.5%, 8 of the 40 rat sera were determined SV positive by AlphaLISA, and the positive rate was 20.0%, the AlphaLISA positive serum was confirmed by IFA.Conclusions We preliminary established the Amplified Luminescent Proximity Homogeneous Assay(AlphaLISA) for the detection of Sendai virus.The sensitivity of the method is comparable to classical ELISA method, but this method use less serum samples and without washing steps.The method has some advantages in degeneracy and accuracy.

Objective To investigate the effects of small interfering RNA(siRNA)on the expression of CD147 on peripheral blood T-lymphocytes from patients with psoriasis vulgaris,and its effect on the proliferation and activation of these cells.Methods Peripheral blood monouclear cells(PBMC)were obtained from 10 patients with psoriasis vulgaris,and T lymphocytes were isolated.CD147 siRNA was chemically synthesized,then electroporated into the peripheral T-lymphocytes.Untransfected cells,blank-transfected cells and unspecifically transfected cells served as the control.After 24-,48-,72-and 96-hour incubation,RT-PCR was used to detect the mRNA expression of CD147 in these cells.MTT assay and flow cytometry were utilized to assess the proliferation of these cellas,and the expression Of CD25 at 24,48,and 72 hours after the transfection.Results Compared with untransfected cells,the mRNA expression of CD147 declined significantly in CD147 siRNA-transfected cells at 24 hours(P＜0.05),reached to the minimum at 48 hours (P＜0.01):there was no significant difierence in the expression of CD147 between the two groups of cells at 96 hours after the transfection(P＞0.05).There was a decrease of cell proliferation level by(44.5±3.13)%,(50.7±3.5)%and(53.98±4.15)%in CD147 siRNA-transfected cells 24,48 and 72 hours following the transfection,respectively;the corresponding decrease in blank-transfected cells was (37.28±3.56)%,(33.73±3.29)%,and(28.80±1.49)%,respectively,and that in unspecifically transfected cells,(31.29±2.46)%,(36.1±2.62)%and(32.08±2.78)%,respectively.A significant decrease was observed in the proliferation of CD147 siRNA-transfected cells compared with that of blank-transfected cells and unspecifically transfected cells at these three time points(P＜0.05,0.01,0.01 respectively).The expression rate of CD25 at 24,48 and 72 hours was(47.23±3.65)%,(31.50±4.22)%and(23.05±4.15)%,respectively,on CD147 siRNA-transfected cells,and,(80.2±4.8)%,(81.6±3.35)%and(83.5±4.1)%,respectively,on untransfeeted cells;the differences between the two groups at the three time points were statistically significant(all P＜0.01).Conclusion CD147 is correlated with cell proliferation and activation of peripheral T lymphoeytes from patients with psoriasis vulgaris,and may serve as a new treatment target for psoriasis.

Objective To investigate the effects of donor-specific regulatory T cells (Treg) transfusion on islet allograft survival. Methods Allogeneic fresh islets from Balb/c mice were transplanted to streptozotocin-induced diabetic C57 mice. The survival of islet allografts was observed. The experiment was divided into 3 groups: control group, nothing had been done to the recipients; simple islet transplantation group, the recipients received the islet transplantation only; experimental group, the recipients were given 1 ×106 Treg, then received islet transplantation. Results Blood glucose (BG) was above 16. 7 mmol/L after islet transplantation in control group; In simple islet transplantation group,BG level returned to normal level 1 to 2 days after transplantation, and hyperglycemia appeared 7 to 11 days after transplantation and maintained as the same as that before transplantation; In experimental group, BG level returned to normal level 2 days after transplantation and maintained at a low level,and at the 21st day after transplantation BG level was over 16. 7mmol/L in some recipients. Islet allograft survival in experimental group was significantly prolonged as compared with simple islet transplantation group. Conclusion Donor-specific Treg transfusion could prolong the islet allograft survival,and maybe have positive effect on tolerance induction of islet transplantation.

Objective To study the changes of behavior and depression-like classic indicators after hippocampal microinjection of K252a,and to establish a new animal model of depression.Methods SD rats were randomly divided into five groups,namely the control group,sham group,chronic stress depression model group,hippocampal of K252a microinjection group,and hippocampal microinjection K252a plus chronic stress group.Open field experiments,sucrose consumption test,and Morris water maze behavioral assay were used to assess the behavioral changes in the rats.ELISA was used to detect the plasma monoamine neurotransmitter,radioimmunoassay was used to determine the plasma CRH,ACTH,CORT contents,and western-blotting was performed to observe the protein expression of BDNF,CREB,ERK1/2,and BCL-2 in the hippocampus.Results Compared with the control group,the amount of activity,sugar consumption,learning and memory abilities were decreased(P<0.05 or P<0.01),also the serum monoamine neurotransmitters were decreased (P<0.01),HPA axis function was improved (P<0.01),and the expression of BDNF,CREB,ERK1/2,BCL-2 decreased in the CUMS group(P<0.05 or P<0.01),but there was no significant difference in the DMSO group.Compared with the DMSO group,the activity,consumption of sucrose,learning and memory ability were significantly decreased(P<0.05 or P<0.01),while the HPA axis function was increased (P<0.05 or P<0.01),the serum monoamine neurotransmitters decreased(P<0.05 or P<0.01),and the BDNF,CREB,ERK1/2,BCL-2 expressions in the hypocampus were significantly decreased(P<0.05 or P<0.01) in the K252a group and K252a + CUMS group.Compared with the CUMS group,the K252a group and K252a + CUMS group did not show significant changes in these parameters.Compared with the K252a group,these indicators were not significantly changed in the K252a + CUMS group.Conclusions The results of behavior,hematology,and molecular biology analysis show that this model has a great similarity to the classical model of CUMS in surface validity,construct validity,and functional validity.It may provide an alternative investigative technology platform for basic research and antidepressant drug screening.

Objective:A questionnaire for the compliance with moxibustion was designed based on the 4-item Morisky scale, and the reliability and validity of the questionnaire was investigated.Methods:A modified Morisky scale was designed based on the 4-item Morisky scale for the compliance with moxibustion, and 146 patients having received moxibustion for over 2 weeks were investigated using this scale to evaluate their compliance with moxibustion; the internal equity and the construct validity of the scale were statistically analyzed.Results:The analysis of reliability showed that in item of internal consistency, the Cronbach's α was 0.72 and the split-half coefficient was 0.71, the correlation coefficients between the 4 component scores and the total score ranged 0.67-0.80, and the between-component correlation coefficients ranged 0.24-0.56; the exploratory factor analysis (EFA) totally extracted 1 common factor, and the explicable variation was 55.02%, and the loads of the 4 items were respectively 0.82, 0.81, 0.74 and 0.58.Conclusion:The reliability and validity of the modified Morisky scale for the compliance with moxibustion are acceptable, while several items need further modification and improvement in the expression and content.

Objective To observe the long term effects of arthroscopic knee debridement and reconstructing operation for treating osteoarthritis in patients with Kaschin-Beck disease. Methods Thirty-one cases of patients with Kaschin-Beck disease were followed for 6 years after operation of articular clearing by arthroscope. Index of pain, symptoms of self-evaluation, range of motion, walking distance, standing test by affected leg when bending at 30° or 60° were recorded and compared with the preoperative results. Results Twenty-four cases were followed up for 6 years. Six years after operation the pain index(3.38 ± 2.87) was dramatically decreased compared to that before operation (6.88 ± 1.45, t = 5.30, P ＜ 0.05). Patients symptoms markedly improved by subjective self-evaluation was 70.83% (17/24), the effective rate was 100% (24/24). The number of cases that could stand up when leg bending at 30° or 60° were 21,18 cases, respectively, compared with that of preoperative of 14, 11 cases, respectively, the difference was statistically significant(x2 = 5.17,4.27, all P ＜ 0.05). Six years after operation the walking distance(3 cases ＜ 1 km, 11 cases 1 - 5 km and 10 cases ＞ 5 km) were greatly improved compared to the results before operation (12 cases ＜ 1 km, 9 cases 1 - 5 km and 3 cases ＞ 5 km, U = 2.88, P ＜0.05). Six years after operation the knee activity[(132.25 ± 14.52)°] remained at the same level, compared with that of preoperative [(131 .58 ± 14.68) °], the difference was not statistically significant (t = 0.16, P ＞ 0.05) .Conclusions The method of arthroscopic joint debridement to cure Kaschin-Beck disease knee osteoarthritis can significantly reduce pain, improve function and walking distance, with more stable long-term satisfactory outcome.

OBJECTIVETo isolate and identify human periodontal ligament stem cells (PDLSC) by improved methods and assess the characteristics of PDLSC ex vivo.

METHODSThe periodontal ligament cells were obtained from the healthy impacted third molars and teeth extracted for orthodontic purposes and used to isolate PDLSC by limiting dilution assay. PDLSC were cultured and expanded in alpha-MEM supplemented with 10% FBS. Colony-forming assay, immunohistochemistry, flow cytometry, osteogenic and adipogenic induction were used to identify PDLSC.

RESULTSThe obtained cells had high colony-forming efficiency and were positive staining for vimentin and negative for pancytokeratin. Flow cytometry revealed that the isolated cells were positive for STRO-1 and CD146 antibodies and most were in the G0/G1 phase of cell cycle. Under specific conditions, they could differentiate to the osteoblast and adipocyte lineages in vitro.

CONCLUSIONLimiting dilution assay is an effective method to isolate PDLSC and the single-cell-derived colonies demonstrate the properties of stem cells in vitro.

Cell Differentiation ; Cell Separation ; Humans ; In Vitro Techniques ; Osteoblasts ; Periodontal Ligament ; Stem Cells

OBJECTIVETo explore the mechanism of cake-separated moxibustion in treatment of hyperlipemia.

METHODSSixty rabbits were randomly divided into 4 groups, a blank group,a model group, a direct moxibustion group and a cake-separated moxibustion group. Hyperlipemia model was developed by high fat diet of cholesterol. Changes of ultrastructures of endothelial cells of the aorta of the rabbit were observed with electron microscope.

RESULTSThe endothelial cells in the cake-separated moxibustion group were more intact, most of them were normal in forms, internal elastic membrane was continuous, their thickness was even, the cells of smooth muscles in the medial membrane were relatively normal, which are similar to those in the blank control group. But the structure of endothelial cells of the aorta in the model group disappeared, in cytoplasm the sedimentation of a great number of lipids can be seen, internal elastic membrane was interrupted, the thickness was uneven, with focal dissolution, the cells of smooth muscle in the medial membrane had sedimentation of lipids, with frothy change.

CONCLUSIONCake-separated moxibustion has a certain protective action on endothelial cells of the aorta in the rabbit of hyperlipemia.

Animals ; Aorta ; Endothelial Cells ; Hyperlipidemias ; Lipids ; Moxibustion ; Rabbits

OBJECTIVETo investigate the effect of post-remission therapy mainly with Compound Huangdai Tablet (CHDT) on long-term survival of patients with acute promyelocytic leukemia (APL).

METHODSOne hundred and twelve APL patients were treated after remission mainly with CHDT administered alternately with chemotherapeutic projects such as HACP, HAOP, HAEP and HAMP. The relapse rate and relapse-free survival (RFS) rate in them were estimated by bone marrow examination.

RESULTSThe total relapse rate was 14.29% (16/112), and the median time of relapse was 12.5 (4-67) months. Patients were followed up for 1-72 months, the median follow-up time being 59 months. The actual RFS rate of 1-, 2-, 3-, 4-, 5- and 6-year was 92.86%, 89.29%, 88.39%, 87.50%, 86.61% and 85.71%, respectively, while the estimated RFS rate (%) of corresponding year was 92.45 +/- 2.57, 88.25 +/- 3.20, 87.09 +/- 3.36, 85.89 +/- 3.52, 84.44 +/- 3.75 and 82.78 +/- 4.03 respectively; the relapse rate in patients who received treatment after complete response for <10 courses group was 34.29%, while in those treated for > or = 10 courses was 5.19%; and the RFS rate in them was 65.71% and 94.81% respectively, the difference between groups was statistically significant (P<0.01).

CONCLUSIONThe post-remission therapy mainly with CHDT is an effective and feasible program for the treatment of APL.

Adolescent ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Arsenicals ; administration & dosage ; Child ; Child, Preschool ; Disease-Free Survival ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; Male ; Middle Aged ; Phytotherapy ; Remission Induction ; Survival Analysis ; Young Adult

OBJECTIVETo explore method of recombinant gene lentivirus containing LIM mineralization protein-1 (LMP-1) in transfecting bone marrow mesenchymal stem cells (BMSC), and to observe the effect of gene LMP-1 on proliferation effect and expression of BMSC.

METHODSSix clean SD rats aged 4 weeks were selected, bone marrow mesenchymal stem cells were extracted under sterile conditions and cultured to the third generation, then divided into three groups:control group (the third generation of BMSC), lentiviral vector transfection group (PGC-FU-GFP and Polybrene were injected into the third generation of BMSC) and recombinant gene transfection group (PGC-FU-LMP-1-GFP and Polybrene transfection were injected into the third generation of BMSC). After 48 hours' transfection, fluorescent expression were detected under immuno-fluorescence microscopy; lentiviral transfection efficiency were detected by flow cytometry; effect of lentiviral transfection on BMSC were evaluated by MTT; gene expression of transfected cells were determined by Western Blot.

RESULTS1) The third generation of BMSC was cultured successfully,and transfected with MOI:100. After 48 hours, green fluorescent expression were detected and transfection efficiency was 67% under immuno-fluorescence microscopy; 2) Compared to control group, there were no statistical differences between control group and other two groups; 3) Western blot teast showed that 72KDa specific band was observed in recombinant gene transfection group and its size was similar to LMP-1 fusion protein (50 kDa+28 kDa=78 kDa).

CONCLUSIONThere is no effect of recombinant gene lentivirus containing LIM on BMSC, and can effectively influence the expression of LMP-1.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Cell Proliferation ; Cells, Cultured ; Cytoskeletal Proteins ; genetics ; metabolism ; Female ; Genetic Therapy ; Genetic Vectors ; genetics ; metabolism ; Humans ; LIM Domain Proteins ; genetics ; metabolism ; Lentivirus ; genetics ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; virology ; Osteoporosis ; genetics ; physiopathology ; therapy ; Rats ; Rats, Sprague-Dawley