The second exons of HLA-DRB genes of Chinese homozygous cell lines were amplified with polymerase chain reaction (PCR), followed by digestion of the amplified DNA segments with the allele-specific restriction endonucleases Cfo 1 and Hinf I. The resulted patterns of restriction fragment length polymorphism (PCR-RF LP) in polyacrylamide gel electrophoresis were used for HLA-DR genotyping. With advantages such as short time-consuming, accuracy and no usage of radioisotope to label oligonucleotide probes for hybridization, the technique has been proved to be capable of subtyping five Chinese HLA-DR 5 cell lines to DRw11 and DRw12 when compared with the PCR-RFLP patterns of reference cell lines. In addition, three Chinese DRw12 cell lines Showed their differences with the reference DRw12 cell BM16 in the genotyping, suggesting that the Chinese DRw12 cell lines might be categorized as new DR5 subtypes or variants related to DRw12.

Objective To evaluate tubal pateney by hysterosalpingography after treatment of tubal pregnancy.Methods 80 patients with tubal pregnancy underwent hysteresalpingography after clinical treatment.Of them,30 were treated with methotrexate of 50mg/m2 intramuscularly( study group,n=30) and 50 were followed up expeetandy (control group,n=50).Results Ipsilateral and contralateral tubal pateneies in the study group were 84% and 97% ,respectively,whereas in the control group were 78% and 92% ,respeetively.There were no statistically significant differences between the two groups.Conclusion Appropriate options for unruptured tubal pregnancy includes either expectant management or methotrexate treatment,both of which result in similar tubal patency rate.

Objective To investigate the effect of Fas ligand gene on vascular remodeling in rat carotid artery after balloon injury. Methods The rat carotid artery model after balloon injury was performed .Twelve SD rats were randomizely assigned to ① control group Ad ?gal ② FasL group . Fourteen days after balloon injury in situ perfusion fixation was performed with 10% formalin and the carotid artery specimens were obtained . Morphometric analysis were performed. Results FasL significantly reduced intimal area(IA) , increased lumen area (LA) and external elastic lamina area (EEL) Conclusions FasL has the role of promoting favorable vascular remodeling of injuied vessel.

OBJECTIVE:To investigate the clinical effects of pidotimod in the bronchial asthma complicated with recurrent re-spiratory tract infection,and its effects on immunoglobulin and related indexes. METHODS:A total of 120 bronchial asthma pa-tients with recurrent respiratory tract infection selected from our hospital during Mar. 2011-May 2013 were divided into trial group and control group according to random number table,with 60 cases in each group. Control group received routine corticosteroid therapy,and trial group was additionally given Pidotimod oral solution 0.4 g,po,bid,for 14 d,on the basis of control group. Clinical indexes(the times of respiratory infection,the duration of fever,cough,wheezing attack and antibiotics use),serum in-dexes [β-defensin-1(hBD-1),immunoglobulin A(IgA),IgG,IgM,UREA,ALT],the results of pharynx test before and after treatment,and the occurrence of ADR were observed in 2 groups. RESULTS:Before treatment,there was no statistical signifi-cance in clinical indexes,serum indexes,the results of pharynx test between 2 groups(P>0.05). After treatment,clinical indexes of trial group were significantly lower or shorter then before treatment or control group,while serum levels of hBD-1,IgA and IgG were significantly higher than before treatment or control group,with statistical significance(P<0.05). There was no statistical sig-nificance in clinical indexes and serum indexes of control group,serum levels of IgM,UREA and ALT in trial group before and af-ter treatment(P>0.05). The types and number of pathogenic bacteria of respiratory tract infection were decreased significantly in 2 groups,and the trial group was significantly less then the control group,with statistical significance(P<0.05). No obvious ADR was found in 2 groups. CONCLUSIONS:Pidotimod shows good clinical effects on bronchial asthma complicated with recurrent re-spiratory tract infection,can improve immunity and reduce the types and number of pathogenic bacteria with good safety.

Objective To prepare polyclonal antibodies of anti Nogo-66, the extracellular region of one central nervous system neurite regeneration inhibitor Nogo, which could be used to further identification and functional study of Nogo molecule.Methods Preparing rabbit anti rat Nogo-66 polyclonal antibodies with a purified Nogo-66 fusion protein expressed in E.coli system. Studying its specificity by Western-blot and immuno-histochemical techniques and identifying its biological activity in PC12 cells.Results The high titer (1∶[KG-*2]10 000) anti rat Nogo-66 polyclonal antibodies were obtained.This antibody could specifically recognize the Nogo protein expressed in E.coli system.Immuno-histochemical staining indicated that the Nogo was widely expressed in rat spinal cord neurons and oligodendrocytes.It could effectively block the neurite extensioninhibition of Nogo protein in PC12.Conclusion Successful preparation of anti rat Nogo polyclonal antibodies provides a useful tool in identification or further functional study of Nogo molecule.

Objective To study the effect of hypothermia on cerebral mitochondrial respiratory function and ultrastructure after traumatic brain injury(TBI). Methods Sprague-Dawley rats were subjected to moderate brain injury by using lateral fluid-percussion(LFP)and randomly divided into sham operation group,normothermic TBI group(rectal temperature for 36-37℃)and hypothermic TBI group(rectal temperature for 31-32℃ lasting for two hours).The ipsilateral brains were dissected and homogenized brain tissues were extracted to obtain mitochondfia by density-centrifugation and speed-centrifugation at 2,24 hours and at days 3 and 7 after TBI.The mitochondrial uhrastructure was studied by electron microscope.The indices of respiratory control rate(RCR)and P/O ratio of mitochondrial respiratory function were measured after oxygen consumption was determined with a Clark-type electrode.Results The mitochondrial uhrastructure of normothermic TBI group was damaged severely while that of hypothermic TBI group kept relatively integrated.The RCR and P/O ratio were markedly decreased two hours after TBI and reached the lowest level at the 24th hour(P＜0.01).At day 7,RCR kept at a lower level compared with sham operation group but P/O ratio recovered to normal.Change of RCR was similar in hypothermie TBI group and normothermic TBI group.However,RCR of the hypothermic TBI group was significantly higher than that of the normothermic TBI group within three days after TBI.In the meantime,P/O ratio recovered to normal three days after TBI. Conclusion Hypothermia can improve cerebral mitochondrial respiratory function and protect the mitochondrial structure after TBI.

Objective To observe the morphologic protection effect of intra-articular injection of osteoprotegerin (OPG)on articular cartilage in a rabbit model of osteoarthritis (OA).Methods Sixty male New Zealand rabbits were randomly divided into 3 groups:OPG group (n=20),sham-operated group (n=20) and PBS group (n=20).In OPG group and PBS group,each rabbit underwent anterior cruciate ligament transection (ACLT) in left knee joint,then 0.1 ml OPG solution or PBS were injected into the left knee for 8 weeks (5 times a week) in OPG group and PBS group,respectively.In sham-operated group,the anterior cruciate ligament was just exposed without transection,and then the incision was closed.All rabbits were sacrificed 12 weeks after operation,and the left knee joints were obtained.The Pelletier score and Mankin score were used to evaluate the macroscopic and microscopic cartilage morphology.Results The score of femoral condyle cartilage and tibial plateau cartilage was 1.80±0.89 and 1.80±0.77 in OPG group,respectively,and 3.10±0.97 and 3.20±0.77 in PBS group.However,there was no statistical difference in Pelletier score between the sham-operated group and the OPG group.Consistent with the macroscopic data,the OPG group has a significantly lower Mankin score involving cartilage structure (2.65±0.88),chondrocyte (1.35±0.71),Safranin O staining (1.83±0.83),tidemark integrity (0.30±0.47) and total score (6.13±1.97) compared with the PBS group (4.52±1.09,1.85±0.63,2.80±0.75,0.65±0.49,and 9.83±1.98,respectively).The OPG group has a significantly higher total Mankin score compared with the sham-operated group (4.80±1.25),however,there were no statistical differences in four subitem scores between the two groups.Moreover,the cartilage thickness in OPG group was 371.84±38.94 μm,255.09±74.82 μm in PBS group,and 404.68±15.97 μm in the sham-operated group,and the differences were statistically significant between three groups.Conclusion OPG has a protective effect on articular cartilage and can slow the progression of OA by reducing the grade of synovitis,decreasing the volume of osteophytes,and suppressing the loss of cartilage thickness.

Objective To study the effect of intra-articular injection of dehydroepiandrosterone (DHEA) on the balance of ADAMTS/TIMP-3 system in a rabbit osteoarthritis models.Methods Sixty rabbits were underwent bilateral anterior cruciate ligament transection (ACLT).Rabbits were randomizedn to the following treatment:one knee of each rabbit was treated with 100 μmol/L DHEA dissolved in dimethylsulphoxide (the experimental group) and the other knee was treated under the same schedule using dimethylsulphoxide (the control group) 4 weeks after transection,once a week for eight weeks.Twelve weeks after ACLT,all rabbits were killed after X-ray assessment and the knee joints were evaluated by gross morphology and histology.The concentration of hydroxyproline and glycosaminoglycan in the cartilage were analyzed.The mRNA expression of ADAMTS-4,ADAMTS-5,tissue inhibitor of metalloproteinases-3 (TIMP-3),transforming growth factor (TGF)-β1.Aggrecan and Collagen Ⅱ in the cartilage were analyzed using reverse transcription polymerase chain reaction (RT-PCR).The protein expression of aggrecan ARGxx and Collagen Ⅱ in the cartilage were analyzed by Western blot.Results By Mann-Whitney test,Gross morphologic scores on femoral condyle and tibial plateau in the control group were significantly higher than the experi-mental group (Z=-3.517,P＜0.01 ; Z=-2.518,P＜0.05).By unpaired Student's t test,histological evaluation showed that the grade of cartilage damage in the experimental group [(5.3±1.2) μg/ml] were less severe than that in the control group (10.1 ± 1.3,P＜0.01).The concentration of hydroxyproline [(5.7±0.3,23.6± 1.7) μg/ml] and glycosaminoglycan (30±4) in the experimental group increased significantly when compared with the control group [(4.6±0.5),(18.5±1.4),(24±4) μg/ml,P＜0.01].The mRNA expression of ADAMTS-4 (0.15±0.03)and ADAMTS-5 (0.10±0.04) in the experimental group decreased significantly compared with the control group (0.29±0.08,0.15±0.05; all P＜0.05).The mRNA expression of TIMP-3 (0.85±0.10),TGF-β1(1.2±0.4),Aggrecan (0.87±0.31) and Collagen Ⅱ (2.74±0.59) in the experimental group increased significantly when compared with the control group (0.70±0.13,0.8±0.4,0.49±0.16,2.2±0.5; all P＜0.05).The protein expression of Aggrecan ARGxx (0.53±0.10) in the experimental group decreased significantly when compared with the control group (0.81±0.12,P＜0.01).The protein expression of Collagen Ⅱ (2.3±0.7) in the experimental group increased significantly when compared with the control group (1.7±0.5,P＜0.05).Conclusion DHEA protects against cartilage degradation and inhibits the progression of OA,TGF-β1,Aggrecan and Collagen Ⅱ in cartilage may be the mechanism of the protective effect of DHEA on OA.

Objective To evaluate a new method for the isolation of rat pancreatic stellate cells (PSCs) and to investigate the influence of adipose derived stem cells (ADSCs) on PSCs in vitro.Methods Normal rat PSCs was isolated by collagenase and Optiprep density gradient centrifugation.The coculture system of ADSCs and PSCs was set up by transwell insert.The proliferation of PSCs was tested by CCK-8 test kit.Smoothmuscle α-actin (α-SMA) expression of PSCs were tested by Western blot.The apoptosis of HSCs was tested by flow cytometer.The cytokines in the culture solution were tested by ELISA kit.Results The quantity of PSCs was above 5 × 106 cells per rat.The purity and the viability of the cells were about 90-97 percent.After coculture for 72 h,the proliferation and activation of PSCs was inhibited by ADSCs (F =223.27,P ＜ 0.05 ; F =52.97,P ＜ 0.05) and the apoptosis of PSCs was promoted by ADSCs (F =43.62,P ＜ 0.05).more NGF and less TGF-β1 was secreted by ADSCs than by PSCs (NGF:14.68 ± 0.94 vs.8.31 ±0.86,t =4.67,P ＜0.05;TGF-β1:10.65 ±0.46 vs.70.47 ±0.99,t =21.72,P＜0.01).Conclusions ADSCs inhibit the proliferation and activation of PSCs by ADSCs secreting cytokines.

AIM: To investigate the effect of Fas ligand on reendothelialization in the rat carotid artery with balloon withdrawl injury. METHODS: Balloon withdrawal injury was used to establish the denuded carotid artery rat model in twelve SD rats and then six SD rats were treated with ?-gal virus supernatant (?-gal group), and the others were treated with FasL virus supernatant (FasL group). After fourteen days, Evans blue dying was conducted via injection through rat tail vein at 30 min before the animals were killed, then fixation perfusion in situ was performed with 10% formalin and specimens were obtained. Histologic observation and morphometric analysis were made. RESULTS: The reenothelialization area (RA) of arterial balloon injury and the ratio of the reenothelialization area to the total area ((RA/TA)) significantly increased in FasL group compared with those in ?-gal group (P