Objective To investigate the proliferation and the change of antigen on the cord blood CIK cells. Methods Cord blood CIK cells were generated by culture cord blood mononuclear cells in the presence of ?-IFN on day 0 and rhIL-2, antiCD3 MCAb on day 1. CIK cultures were analysed on different time point by FACS(Fluorescence activated cell sorter). The killing efficacy in tumor cell lines and primary leukemia cells were detected by MTT assay. Results After 2~3 weeks of culturing cord blood CIK cells expanded about 64.14?16 folds and CD+3 CD+56 cells which were the major cytolytic effector in CIK cells proliferated about 900 folds. Cord blood CIK cells have great cytotoxity against tumor cell lines as well as primary leukemia cells. Conclusions Cord blood MNCs can be cultured to CIK cells and the times of expansion are very high. Cord Blood CIK cells are highly efficient cytolytic effector cells.

Objective:To investigate the neuroprotective effect and possible mechanism on rats with low dose Lipopoly -saccharide ( LPS) preconditioning after spinal cord injury.Methods:120 female SD rats were randomly divided into the empty virus (EV) group,LPS+empty virus (LPS+EV) group,Nrf2 interference virus (NIV) group,LPS+Nrf2 interference virus (LPS+NIV) group.The model of traumatic spinal cord injury ( TSCI) was established by the modified Allen′s method,motor function of the rat hind limb was assessed by the Basso Beattie and Bresnahan (BBB) score at 1,3,7,14 and 28 d after the operation.The injured spinal cord tissue samples were harvested at each time ,and the pathological changes of rat spinal cord were observed by HE staining ,the Nissl body and neuron survival index were observed by Nissl staining ,the expressions of Nrf2 and GCLC protein level were detected by immunohis-tochemical staining and Western blot.Results:The rat BBB score of LPS+EV group increased significantly than EV group at 7,14,28 d after operation ( P<0.05 ,P<0.01 );The NIV group between LPS+NIV group have no statistical significance at each time.As compared with EV group:the Nrf2 protein of LPS+EV group was expression increased significantly and Nissl staining showed that the neurons survival index was increased at 1,3 and 7 d(P<0.05,P<0.01);The GCLC protein of LPS+EV group was expression increased significantly at 1-14 d( P<0.05 );HE staining showed that the injured spinal cord pathological changes of LPS +EV group was obviously improved.Conclusion:Low dose lipopolysaccharide preconditioning can accelerate the nerve function recovery on rats with traumatic spinal cord injury ,the mechanism may be regulated by activating the Nrf 2 antioxidant stress pathway.

BACKGROUND:The cryopreservation of single tissue has achieved great advancement and is gradual y applied in clinics. However, the cryopreservation of complex tissue is rarely reported.
OBJECTIVE:To investigate the morphological change in rabbit limb tissue after replantation through different rewarming methods, find the best rewarming methods of compound textured blood vessels, and provide theoretical basis for the feasibility of limb replantation after long-term cryopreservation.
METHODS:Thirty New Zealand white rabbits were randomly divided into control group, slow freezing-slow thawing group, and slow freezing-rapid thawing group. The right posterior limbs of al the rabbits were cut off 1 cm above the knee joint. Except control group, the latter two groups were given limb replantation after thawing, and then the right posterior limb was again cut off after the replanted limbs were survived for 6 hours. For al groups, the histological changes and gross observation in aorta tissue were observed by light microscopy and transmission electron microscope, and the results were analyzed with statistical methods.
RESULTS AND CONCLUSION:In the slow freezing-slow thawing, slow freezing-rapid thawing groups, the pathological changes (gross specimen, light microscope, electron microscope) of rabbit limbs 6 hours after replantation were worse than those in control group. Compared with slow freezing-rapid thawing group, better integrity of endothelial cells and less damage of the organel es were found in slow freezing-slow thawing group. Through deep cryogenic freezing-thawing process, rabbit limb blood vessels can maintain the structural integrity after replantation and survived at 6 hours. Slow freezing-slow thawing is better than slow freezing-rapid thawing for the preservation of severed limbs, providing evidences for the long-term survival fol owing a deep cry ogenic treatment after the severed limb replantation.

Objective To observe the effects of intermedin (IMD) on nitric oxide synthetase (NOS) in renal ischemia-reperfusion (IR) rat models and the action mechanism.Methods A total of 24 rats were divided into four groups (n =6 each).Group Ⅰ underwent right nephrectomy one week prior to the exposure of left renal pedicles,but did uot receive any I/R.Group Ⅱ underwent right nephrectomy one week prior to left renal I/R surgery.Group Ⅲ underwent right nephrectomy and left renal IMD-pCDNA3.1 ( + ) transfection by ultrasound-mircobubbles and renal I/R surgeries were performed one week after gene transfection.Group Ⅳ was treated with the same way as group Ⅲ except that empty control vector was transfected.All the animals were killed at the end of 24 h of reperfusion.The expression and site of IMD were determined by using immunohistochemistry.Serum levels of BUN and creatinine were determined.The kidney formaldehyde-fixed and paraffin-embedded sections were stained with HE and PAS by standard methods and then histological changes were analyzed semiquantitatively.The mRNA expression levels of endothelial NOS (eNOS),inducible NOS (iNOS) and neuronal NOS (nNOS) in the kidneys of the four groups were detected by using RT-PCR.The protein expression levels of the three NOS mentioned above in the kidneys were semiquantitatively analyzed by Western blotting.Results IMD was weakly expressed in the plasma of tubulointerstitial cells in sham-operated group; whereas IMD expression in the kidneys subject to I/R was increased.Moreover,as compared with I/R group,IMD expression levels were obviously increased (P＜0.01 ).The degree of morphological changes as well as renal dysfunction in group Ⅲ was obviously lessened as compared with group Ⅱ.The mRNA and protein expression levels of eNOS in group Ⅲ were notably increased as compared with group Ⅱ,while the mRNA and protein expression levels of iNOSin group Ⅲ were obviously reduced as compared with I/R group not transfeeted with IMD (P＜0.05).Meanwhile,there were no significant differences in the mRNA and protein expression levels of nNOS among groups Ⅱ,Ⅲ and Ⅳ.Conclusion IMD gene in the kidneys of rats can promote the expression of eNOS and attenuate over-expression of iNOS in the kidneys following I/R,thus protecting against tubulointerstitial damages and renal dysfunction in rat I/R models.

Objective To investigate the effect of intermedin (IMD) on renal ischemia/ reperfusion (I/R) injury after the up-regulation of IMD. Methods A total of 24 healthy Wistar male rats were randomly divided into four groups,sham-operated group,I/R group,IMD gene transfection +I/R group and empty plamid +I/R group.All the animals were killed at the end of 24 h of reperfusion.Histological changes and renal function were estimated.The expression and site of IMD were determined by Immunohistochemistry method,semi-quantitative RT-PCR and Western blotting.The protein expressions of endothelin 1 (ET-1),tumor necrosis factor αt (TNF-α) were detected by Western blotting. Results Compared with sham-operated group,tubulointerstitial pathological injury was significant aggravated in I/R group (7.6±2.3) and empty plamid +I/R group (7.0±1.8),and such injury was improved in IMD+I/R group (1.5±0.8) (P＜0.05).Compared with I/R group and empty plamid +I/R group,the renal dysfunction of IMD +I/R group was obviously lessened [BUN:(7.73±1.03) mmol/L vs (10.13±2.14) mmol/L,(9.77±1.92) mmol/L; Scr:(58.50±3.27) μmol/L vs (80.33±7.15) μmol/L, (75.67±7.58) μmol/L,all P＜0.05].IMD expression was weak in the plasma of tubulointerstitial cells in sham-operated group,and was up-regulated in I/R group. Compared with I/R group, immunohistochemical IMD expression increased obviously (262.03±67.89 vs 175.57±48.06,P＜0.01).The mRNA expression of IMD in IMD+I/R group was up-regulated significantly by 60.7％,66.1％ and the protein expression of IMD in IMD+I/R group increased significantly by 51.4％,55.9％ as compared to I/R and empty plasmid +I/R group.Meanwhile,the protein expressions of ET-1 and TNF-αt in IMD+I/R group were obviously lower compared with those in I/R group (ET-1:0.08±0.02 vs 0.17±0.02; TNF-α:0.21±0.04 vs 0.35± 0.02,all P＜0.05). Conclusion IMD gene transfected into kidneys of rats prior to I/R surgery can attenuate the over-expressions of both ET-1 and TNF-o in I/R injured rat kidneys as well as the damages to the structure and function of the kidneys.

Objective To investigate the protective effect of intermedin(IMD)on renal ischemia reperfusion injury(IRI)and its mechanism. Methods A total of twenty-four male Wistar rats were randomly divided into four groups: control group, IRI group, empty plasmid group and IMD group. After remove of right kidney, plasmid was transfected into the kidney by ultrasonic microbubbles technology, and IRI model was made after 1 week. Renal pathology was observed by PAS staining. Renal tissue superoxide dismutase(SOD), myeloperoxidase(MPO), caspase-3 activity, and malondialdehyde(MDA)content were detected by colorimetric method. The intercellular cell adhesion molecule-1(ICAM-1), endothelin 1(ET-1)and P-selection expression of renal tissue were detected by immunohistochemical method. Apoptosis of renal tubular cell was detected by TUNEL.Results Compared with control group, tubulointerstitial pathological injury was significant aggravated in IRI group(P<0.01);compared with IRI group, IMD pretreatment significantly alleviated the degree of renal injury(P<0.01). Compared with control group, in IRI group, SOD activity was significantly decreased(P<0.05), MPO activity, caspase-3 activity, MDA production and the expression of ICAM-1, P-selection, ET-1 were increased significantly(all P< 0.01). Compared with IRI group, IMD pretreatment significantly increased SOD activity(P <0.05), decreased the MPO activity, caspase-3 activity, MDA production and the expression of ICAM-1, P-selection, ET-1 (all P<0.01). The apoptosis rate of renal tubular epithelial cells in IRI group was significantly higher than that in control group(34.83%±8.75% vs 3.33%±0.47%, P<0.01), while the apoptosis rate of IMD group(20.67%±7.71%)was significantly lower than that of IRI group. There was no difference of above indexes between empty plasmid group and IRI group. Conclusions IMD pretreatment protects against renal IRI. The mechanism may be at least partly related to the clearance of oxygen free radicals, the improvement of lipid peroxidation, inflammatory cell infiltration and cell apoptosis, leading to the decrease of the production of reactive oxygen species caused by oxidative stress.

Objective To study the topical application of Tuhuai decoction and its disassembled prescriptions on skin function of mice with epidermal hyperplasia, and to provide a theoretical basis for the treatment of psoriasis. Methods According to the Denda method, skin barrier function in mice was destructed with tape, to produce epidermal hyperplasia model. Tuhuai decoction and its disassembled prescriptions with different concentrations of water and alcohol extracts were external applied on the mice. Since the beginning of the treatment, loss of skin moisture of both sides the trunk was detected with Multi-functional instrument at every day before using the medicine. Results Tuhuai decoction had function of reducing the amount of water loss of skin. The disassembled prescriptions, such as Angelica group, safflower group, Danshen root group, and water and ethanol extracts of Prtmella group can reduce the amount of water loss of skin effect. Conclusion Water and ethanol extracts of Tuhuai decoction has the functions of reducing water loss of skin and resisting hyperplasia,suitable for treating psoriasis by external application, setting a foundation for clinical treatment of psoriasis.

Objective To construct eukaryotic expression vector encoding rat IMD gene and deliver it into rat renal tissue via ultrasound-mircobubbles. Methods IMD gene was inserted into pCDNA3.1 ( + )between Hind Ⅲ and EcoRI enzyme sites. The recombinant plasmid designated as IMD-pCDNA 3.1 wasconfirmed by restrictive enzyme digestion and sequencing. Eighteen male Wistar rats were randomized into 3groups, which were treated with no transfection, empty vector transfection and IMD transfection, respectively, in renal tissue via ultrasound-microbubbles. RT-PCR and Western blotting were applied to detect the expression level of IMD. Results Enzyme- digestion and sequencing data showed that IMD-pCDNA 3.1 was correctly constructed. The differences in ALT, AST, BUN and SCr were not significant; No obvious damage in the glomerular, tubular and interstitial was observed in all the treated groups;Compared with non-transfection group and empty vector-transfection group, IMD mRNA and protein expression in IMD transgenic renal tissue were significantly increased. Conclusion IMD-pCDNA 3.1 expression vector was successfully constructed and well expressed in rat kidney.

The Her-2 proto-oncogene encodes a 185kDa transmenbrane glycoprotein p185 which has intrinsic tyrosine kinase activity. It is overexpressed in several malignant human tumors like breast cancer. A chimeric antibody by assembling a single-chain Fv antibody and a human IgG1 Fc fragment was constructed. This chimeric antibody reacts with tumor surface antigen p185c-erbB-2 specifically. In order to put the antibody into clinical application, two steps purification method was used to attain the antibody’s purity more than 95%. Both the lyophilized pharmaceutical formulations of the antibody were found. The formulations can keep the stability and activity of the antibody for at least one year. These results were the foundation of the chimeric antibody for cancer therapy.

Objective:To observe the effect of internal and external use of Chinese material medica with syndrome differentiation on acne vulgaris.Methods:200 patients with acne vulgaris were randomly divided into treatment group(110 cases)and control group(90 cases).Treatment group were given acne facial mask,oral medicine decoction,and the control group were treated with13-cis-Retinoic Acid Capsules,topical Clindamycin Gel,7 weeks for a course of treatment,the clinical effect were compared.Results:The effect on facial lesion in treatment group and control group had significant difference(P