Objective To discuss the diagnosis and treatment of a variety of multiple nerve entrapment in the brachial plexus and around anterior and middle scalene muscles in 179 cases.Methods From July 2003 to January 2006,179 outpatients in our department were diagnosed as thoracic outlet syndrome(TOS)with a variety of symptoms of the nerve entrapment in the brachial plexus and around anterior and middle scalene muscles.They were treated with drugs,local block injections,or operations,depending on their severity of the condition. Results Of the 128 cases who were followed up for one to 29 months,the symptoms were significantly relieved with drugs and massage in 55 cases,with local block injection in 58 cases of whom 24 received the second in- jection,with operation in 15 cases of whom 10 returned to work.One of the operated cases reported symptoms of injury to the long thoracic nerve which responded to further therapy.The visual analogue scale(VAS)evaluations after the first injection were one point in two cases,two points in 16 cases,three points in 20 cases,four points in 12 cases,five points in three cases,six points in three cases,and seven points in two cases.The VAS scores after the second injection were two points in five cases,three points in 16 cases,and four points in three cases. Conclusions The entrapment of the anterior anti middle scalene muscles can affect many nerves around the muscles,and cause clinical symptoms other than the TOS ones.The conservative treatment,such as drug,massage, and local block injection,can work well.When the non-operative methods do not work,operation can be consid- ered.

Objective To explore the effects of injection of wnt3a gene-modified bone marrow mesenchymal stem cells (MSCs) on acute graft-versus-host disease (aGVHD) in a murine allogeneic bone marrow transplantation (allo-BMT) model.Methods C57BL/6 mice were used as the donors and Balb/c mice as the recipients in the murine allo-BMT model.The recipient mice were divided into four groups by random number table method: transplantation control group (group A) (infusion of 5 × 106bone marrow cells via the tail vein of recipient mice); aGVHD group (group B) (infusion of 5 × 106bone marrow cells and 5 × 106 splenocytes via the tail vein of recipient mice); aGVHD + empty vector group (group C) (infusion of 5 × 106 bone marrow cells,5 × 106 splenocytes and 1 × 106 pAd-GFP-transfected MSCs via the tail vein of recipient mice) ; experimental group (group D) (infusion of 5 ×106 bone marrow cells,5 × 106 splenocytes and 1 × 106 wnt3a gene-modified MSCs).The general performance and survival were monitored,the occurrence of aGVHD was observed,the changes of donor T lymphocyte quantity present in the spleen,and interleukin-2 (IL-2) and interferon-γ (IFN γ)levels of the recipient mice were detected in each group after transplantation.Results The survival time of recipient mice in group A was all more than 60 d,and that in groups B,C and D was (19.1 ±6.19),(32.6 ± 19.6) and (47.2 ± 15.6) d,rcspcctivcly.The survival time in group D was significantly longer than in groups B and C (P＜0.05).After the transplant,the aGVHD score points in groups B,CandDwere (8.0±0.41),(6.7±0.29) and (4.0± 1.0),respcctively.The aGVHD score points in group D were significantly less than in groups B and C (P＜0.05),and the pathological grade in group D was significantly reduced.The number and proliferation rate of T lymphocytes were reduced significantly in group D as compared with groups B and C at 3rd and 5th day after transplantation (P ＜ 0.05).The levels of IL-2 and IFN-γ in peripheral blood were decreased significantly in group D as compared with those in groups B and C at 7th,14th,21st and 28th day after transplantation (P＜0.05).The chimeric rate of the murine H-2Kb cells in the bone marrow cells of long-term survival mice was all in the range of 95％ to 100％ 60 d after transplantation.Conclusion The injection of wnt3a gene-modified MSCs can more effectively alleviate aGVHD in murineallo-BMT model,which may be correlated with the Wnt3a overexpression which activating the Wnt/β-catenin signaling pathway of MSCs,thereby inhibiting the early activation and amplification of donor T lymphocytes and the IL-2 and IFN-γ expression.

Objective To establish neutralization assay based on H5N1 avian influenza pseudotyped virus in vitro and to evaluate neutralizing titer of convalescent serum from 2 patients with H5N1 avian influenza.Methods pHR-Luc,pCMV△8.2 and CMV/R-SH or CMV/R-TH were cotransfected into 293T cell by co-precipitation with calcium phosphate.Pseudotyped virus supernatant was harvested 72 h posttranofection and identified the expression of HA and P24 by Western blot,and then we analyzed infective activity of 200 μL supernatant of pseudotyped virus.293T cell integrated HA was prepared and anti-HA antibodies in convalescent serum were measured with FACS assay.Neutralizing titers of convalescent serums against Shenzhen and Thailand pseudotyped virus were determined based on calculating IC50 with neutralizing assay.Results Pseudotyped virus involved P24 and HA,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Pseudotyped virus possessed better infective activity,and RLA value was about 2 × 104 with 200 μL supernatant.Both convalescent serums contained anti-HA antibodies and had cross-reactivity against different virus clades with FACS assay.Both convalescent serums had neutralizingactivity and could cross-neutralize different virus clades.However,both serums'neutralizing titers against Shenzhen virus were higher than Thailand.Conclusion We successfully constructed infectious pseudotyped virus which integrated HA of Shenzhen or Thailand virus,and it could be used for evaluation of serum neutralizing activity fast,efficiently and safely with broadly application prospect.

Objective To construct pseudotype retrovirus which integrates hemagglutinin(HA)of H5N1 avian influenza virus(AIV)isolated from human in Shenzhen.Methods AIV HA gene was amplified bv RT-PCR,then it was ligated with pGEM-T vector,and identified by restriction enzyme digestion and sequenced.HA gene was cloned into CMV/R vector at the site of Sal Ⅰ and BamH Ⅰ.pHR-Luc,pCMV&8.2 and CMV-HA were co-transfected into 293T cell by co-precipitation with calcium phosphate.The pseudotype virus supernatant was harvested 72 h post-transfection and ultracentrifugation,and the HA and P24 expression on the surface of pseudotype virus was analyzed by western blot.Meanwhile.the infection activity of HIV-HA pseudotype virus was identified in different kinds of cell lines,including MDCK,HeLa,CHO and 293T.Results A/Shenzhen/406H/06 belonged to subclade2.3 with open reading frame(ORF)of HA gene encoded 567 amino acides,whose accession number was EF137706 in GenBank.HA gene was cloned into CMV/R successfully.After co-transfection of above vectors,it revealed that HA protein could integrate pseudotype virus by western blot,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Finally.HIV-HA pseudotype virus could infect 4 kinds of cell lines,which indicated its property of infectivity and catholicity.Conclusion The pseudotype retrnvirns wassuccessfully constructed,which can integrate HA protein of A/Shenzhen/406H/06 and had property of infectivity.It call be used in the further research,including selection of neutralizing antibodies and epitope analysis.

Objective:To valuate the treatment value and analyse the effect on the cellular immune functions by studying the differences of T-lymphocyte subsets and CD4+CD25+Treg cells in peripheral blood after adoptive immunotherapy ( dendritic cells and cytokine-induced killer cells,DC-CIK) combined with chemotherapy on MM.Methods:50 patients with MM were randomly divided into two groups.24 patients in chemotherapy group were treated by chemotherapy only,26 patients in joint group were treated by adoptive immunotherapy( DC-CIK) combined with chemotherapy,and the clinical outcomes and the levels of T-lymphocyte subsets and CD4+CD25+Treg cells in peripheral blood between two groups were compared.Moreover,the differences of cellular immune indicators (Th1/Th2,the ratio of AgNOR,and TGF-β)between two groups were also compared.Results: After treatment,quality of life,clinical index and survival in joint group were better than in chemotherapy group( P<0.05);the proportion of CD3+CD8+,the ratios of CD4+CD25+,CD4+CD25+/CD4+and the level of TGF-βof joint group wes clearly lower than chemotherapy group(P<0.05),and the ratios of CD3+CD4+/CD3+CD8+, Th1/Th2 and AgNOR of joint group wes clearly higher than chemotherapy group .Conclusion: DC-CIK combined with chemotherapy could be an effective and promising treatment to patients with MM,and it maybe strengthen the anti-tumor action of bodies by regulating the balance between Th1 and Th2 reaction.

OBJECTIVETo establish a method for isolating and culturing human amniotic epithelial cells (hAECs) in a serum-free medium and investigate their transdifferentiation ability into islet-like cells.

METHODSThe culture condition of hAECs was optimized using DMEM with different supplements. The genetic stability of the tenth-passage cells was assessed by chromosome analysis and G-banding method. The stem cell characteristics of the cells were identified by examination of the surface markers using immunofluorescence methods. The endocrine-related genes and hormones of the cells were tested after induced differentiation into islet-like cells.

RESULTSThe hAECs allow stable passaging in the presence of 10 ng epidermal growth factor (EGF) in the culture medium. After 10 passages, the cells maintained a normal karyotype and G-banding profile. The hAECs expressed many multi-potent stem cell markers, including SSEA4, TRA-1-60, and TRA-1-81. After induced differentiation, the endocrine-related genes were expressed in the islet-like cells, including PDX1, ngn3, insulin and glucagon. Insulin secretion increased in the differentiated islet-like cells in response to high glucose exposure.

CONCLUSIONWe established a method for isolating and expanding the hAECs in a serum-free medium. hAECs possess stem cell characteristics and can be induced to differentiate into islet-like cells in vitro.

Amnion ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; cytology ; secretion ; Humans ; Insulin ; secretion ; Islets of Langerhans ; cytology ; Stem Cells ; cytology ; secretion

Objective To explore the effects of mesenchymal stem cells ( MSC ) treatment on platelet counts in mice with immune-mediated thrombocytopenia ( ITP) and the possible mechanism .Meth-ods ITP was induced by daily intraperitoneal injection of anti-platelet membrane CD 41 antibody (MWReg30) into BALB/c mice.The mice were then divided into experiment and control groups with 20 mice in each.Each mouse in experimental group was injected with 2×107 mesenchymal stem cells (MSC) through the tail vein .The numbers of blood platelets in mice from two groups were counted on days 5, 7 and 14 after MSC injection .Reverse transcriptase polymerase chain reaction ( RT-PCR) was performed to meas-ure T-bet and GATA-3 gene expression in peripheral blood mononuclear cells ( PBMCs ) at mRNA level on day 14.The levels of IFN-γ, IL-2, IL-4 and IL-10 in serum were detected by ELISA .Results The platelet counts in experimental group were significantly higher than those in control group on days 7 and 14 after MSC injection [(588.0±81.6)×109/L and (623.0±78.9) ×109/L vs.(317.0±90.1) ×109/L and (288.0± 87.8)×109/L ] (P<0.05).On day 14 after MSC injection, the T-bet expression at mRNA level in PBMCs from mice in experimental group was significantly lower than that in control group [(0.04±0.03) vs.(0.27 ±0.05)] (P<0.05), while the GATA-3 expression at mRNA level was higher than those in control group [ (0.14±0.04) vs.(0.07±0.05)] (P<0.05).Compared with control group, the concentrations of Th1 type cytokines such as IFN-γand IL-2 were remarkably down-regulated in experimental group [(3.1±1.7) pg/ml and (3.2±2.1) pg/ml vs.(10.3±4.8) pg/ml and (16.3±5.7) pg/ml](P<0.05), while the con-centrations of Th2 type cytokines such as IL-4 and IL-10 were up-regulated in experimental group [(88.6± 15.2) pg/ml and (38.3±11.8) pg/ml vs.(32.7±5.7) pg/ml and (22.1±3.4) pg/ml ] (P<0.05). Conclusion MSC treatment can effectively increase platelet counts in mice with immune-mediated thrombo-cytopenia, which may be associated with the suppression of Th 1-dominant response mediated by abnormal ex-pression of T-bet and GATA-3.

Objective To investigate the clinical characteristics,diagnosis,treatment and prognosis of human swine streptococcosis occurred in some areas of Jiangsu Province from late summer to autumn since 1998.Methods The epidemiologic and clinical features of 42 cases were collected and analyzed.The bio- chemical features of strains isolated from patient's blood or cerebrospinal fluid(CSF) were tested,and the homogeneity were compared among 15 Streptococcus suisⅡ.Results All patients had acute infection toxe- mic symptoms such as chill,fever,headache and malaise etc.Toxic shock syndrome or meningitis syndrome were the major clinical manifestations.Forty two cases of human swine streptococosis were classified into 3 types:the rates of general,shock and meningitis type were 7.1% (3/42),38.1% (16/42) and 54.7%(23/42),respectively.Ten patients were died of shock type,32 were cured.Strain isolated from patients was identified as Streptococcus suisⅡby API-Strep,the biochemical reactional code was 0641473,and appraised result was 99.9%.There was highly homogeneity in the strains of Streptococcus suisⅡisolated from patients and sick pigs identified by genomic fingerprinting.Com- bined therapy of large doses of penicillin G and ceftriaxone was effective in these patients.Conclusions Human swine streptococosis is zoonosis caused by Streptococcus suisⅡand the clinical manifesta- tions are variable.In the cases of shock type,the onset of disease is stormy and the fatality rate is very high.While the prognosis of general and meningitis type is good and the majority of the cases are cured by effective antibiotic therapy.

Background TLR-4 is a natural immunity receptors in immunity,and it plays an important role in the repair of central nervous system damage.But its effect in glaucoma optic nerve injury is unclear.Objective This study was to investigate the expression of TLR-4 in retina with high intraocular pressure(IOP)in genetic and Drotein level and therefore explore the mechanism of TLR-4 on retinal ganglion cells(RGCs)injury. Methods Chronic ocular hypertension models were established in the right eyes of 150 clean purebred Sprague-Dawley rats by cauterizing the 3 sallow sclera veins.IOP was measured before and after 2 h,1 day,3,7,14,28,56 days after operation by PEN Ⅱ TONO-type pen tonometer.The expression of TLR-4 protein in rat retina was detected by immunohistochemistry and Western blot,and expression of TLR-4 mRNA was assayed by real time-PCR.This experimental procedure foliowed the Statement of Association for Research in Vision and Ophthalmology. Results The IOP was elevated in various time points after operation in experimental group,showing significant differences in comparison with control group(P<0.01).The immunohistochemistry revealed that the expression of TLR-4 protein in rat retina with chronic hypertension in 2 h,1 day,3,7,14,28,56 days after operation with the high A298 values in comparison with control eyes(P<0.05-0.01).Increased levels of TLR-4 mRNA in rat retinas were detected by RTPCR in high IOP eyes compared with control eyes in all time points after operation,presenting statistically significant differences between two groups(P<0.05-0.01).Western blot detection displayed the high expression of TLR-4 in retina in high IOP eyes early after operation with statistically significant results between model group and control group (P<0.05-0. 01). Conclusion TLR-4 is up-regulated in rat retina with chronic high IOP,suggesting that TLR-4 plays an immunoregulatory effect in glaucomatous eye.

OBJECTIVETo investigate the best injection method of the bone marrow mesenchymal stem cells (BM-MSCs) transplantation for the treatment of a rat model with hind limb ischemia.

METHODSTwenty four SD rats with hind limb ischemia were randomly divided into four groups: control group, model group, acupoint BM-MSCs injection group (API group) and thigh muscle BM-MSCs injection group (TMI group). The acupoints of "Sanyinjiao" (SP 6), "Housanli" (ST 36), "Zhaohai" (KI 6), "Huantiao" (GB 30) and "Yanglingquan" (GB 34) were selected for API group, and five non-acupoints were selected on gastrocnemius and adductor of ischemic hind limb for TMI group. Both groups were accepted BM-MSCs transplantion. Model rat with hind limb ischemia was established with the method of blocking the femoral artery and its branches. The changes of blood flow (perfuse unit, PU) was monitored with laser Doppler flowmetry (LDF). In order to describe the visual changes in blood flow, the PU index (PUI) was determined as the ratio of ischemic to non-ischemic hind limb blood perfusion. And also, the levels of VEGF,bFGF in serum were tested to analyze the immunohistochemical expression quantity of VEGF and bFGF.

RESULTSComparing with the model and the TMI groups, the PUI value on 3rd, 14th and 21th days after BM-MSCs transplantation were significantly increased in the API group (P < 0.05, P < 0.01). In contrast to the model group, the VEGF,bFGF levels in serum and the immunohistochemical expression quantity of VEGF and bFGF in the API and TMI groups were significantly increased (all P < 0.01).

CONCLUSIONTransplantation of BM-MSCs through the acupoint can more significantly and quickly increase the blood flow and cause the greater improvement on hind limb ischemia than that of through the way of muscle injection.

Acupuncture Points ; Animals ; Bone Marrow Transplantation ; Disease Models, Animal ; Female ; Humans ; Ischemia ; physiopathology ; therapy ; Lower Extremity ; blood supply ; Male ; Mesenchymal Stem Cell Transplantation ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Regional Blood Flow