Purpose To prepare streptavidin-tagged mouse interleukin-4(mIL-4-SA)bifunctional fusion protein and to study on its bioactivity.Methods The mIL-4 gene was cloned by RT-PCR and cloned into pET21 vector to get mIL-4-SA-pET21 expression plasmid.The mIL-4-SA fusion protein was expressed in BL21 (DE3)host bacteria and purified through the Ni-NTA affinity chromatography and refolded by dilution and dialysis.The effect of mIL-4-SA fusion protein on mouse thymocytes proliferation was evaluated by MTY.Flow cytometric analysis was performed to detect the mIL-4-SA fusion protein on the biotinylated B16F10 tumor cells.Results The mIL-4-SA-pET21 vector was successful by constructed and the mIL-4-SA fusion protein was expressed in BL21(DE3)at about 35%of total bacterial proteins.The purity of mIL-4-SA Was about 95% through Ni-NTA.The mIL-4-SA fusion protein exhibited bifunctional activities,i.e.,stimulative effect for mouse thymocyte proliferation and SA-mediated high-affinity binding to biotinylated cell surfaces(anchoring modified rate Was about 96.69%).Conclusion The mIL-4-SA fusion protein was expected to be developed for the treatment of tumors.

Objective:To study the expression of IP-10 and its mRNA in liver biopsy of patients with cirrhosis.Methods:The typical patients with cirrhosis after HBV infection were selected,and their serum IP-10 concentrations were evaluated with ELISA,the content of IP-10 mRNA in liver biopsy was measured by Real-time PCR.The level of IP-10 mRNA was represented by lg cDNA/lg GAPDH.The expression of IP-10 in liver biopsy tissue was detected by SP immunohistochemistry stain.Results:The levels of IP-10 in serum and its mRNA in liver biopsy of patients with cirrhosis were (299.3?77.2)pg/ml and (0.856 3?0.150 6),lg cDNA/lg GAPDH respectively,which were significantly higher than those of control groups(P

OBJECTIVE To study the expression of matrix metalloproteinase 2 (MMP-2) and its correlation with microvascular density(MVD) and tumor metastasis in nasopharyngeal carcinoma. METHODS The expression of MMP-2 and MVD were detected in the specimens of 50 cases with nasopharyngeal carcinoma and 15 cases with nasopharyngeal inflammation by immunohistochemical technique. RESULTS The MVD in nasopharyngeal carcinoma(21.92?7.80) was significantly higher than that in inflammation nasopharyngeal tissues (9.23? 1.84, P

Objective To explore the effects of mental practice on upper extremity function after stroke. Methods Thirty sub-acute stroke patients were randomly divided into a treatment group ( n=15 ) and a control group (n=15). The patients in the control group were treated with conventional therapy. The patients in the treat-ment group were treated with motor imagery therapy in addition. All patients were assessed using the Fugl-Meyer mo-tor assessment (FMA) and the motor assessment scale (bIAS) before treatment and after 2, 4 and 8 weeks of treat-ment. Results After 2 weeks of treatment, average MAS scores in the treatment group improved significantly com-pared with before treatment, but there was no significant difference between the two groups. After 4 weeks, FMA and MAS scores in the two groups had improved, and the FMA scores in the treatment group were significantly higher than those of the control group. After 8 weeks, the FMA and MAS scores of both groups had further improved significant-ly, but the average FMA and MAS scores in the treatment group were now significantly higher than those in the control group. Conclusions Mental practice can improve the functional performance of the upper extremities of stroke pa-tients.

Objective To study the content of IL-8 in the peripheral blood and the expression of its mRNA in liver biopsy tissue of patients with hepatocirrhosis.Methods The patients with typical hepatocirrhosis resulted from HBV infection were selected.The IL-8 concentrations in peripheral blood of the Datients were evaluated with ELISA.The content of IL-8 mRNA in liver biopsy was measured by real-time PCR.The level of IL-8 mRNA was surrogated by lg cDNA/lg GAPDH.Reslllts The levels of IL-8 in the serum and its mRNA in hepatic tissue of patients with hepatoeirrhosis were(43 1.39±97.39)μg/L and 1.3647±0.2203,respectively,which were significantly higher than those of control group.MoreoCer,the levels of IL-8 in the serum and IL-8 mRNA in hepatic tissue of patients with positive HBV-DNA were (502.43-4-102.24)μg/L and 1.51424±0.2245,respectively,and both ofwhich were higher than those of patients without HBV-DNA replication(P<0.0l,P<0.05).And the levels of IL-8 in the selqlm and its mRNA in hepatic tissue of patients with higher ALT.AST also increased significantly.compared with those in control group(P<0.01).Conclusion The levels of IL-8 in the peripheral blood and the IL-8 mRNA in liver biopsy tissue of patients with hepatocirrhosis increase.which are related to the degree of HBV-DNA replication and the damage of hepatocytes.The IL-8 plays a key role in local inflammatory infihration of liver and the progression of post-hepatitis cirrhosis.

Objective To study the expression and clinical significance of miR-183-5p, TβRⅠ and TβRⅡ in esophageal squamous cell carcinoma (ESCC). Methods The mRNA and protein expression of miR-183-5p, TβRⅠ and TβRⅡ were examined in ESCC cell lines ECA-109, TE-1, normal esophageal epithelial cells, tumor tissues and tumor-free tissues from 72 ESCC patients. Their clinical significance and the relationship between miR-183-5p and the latter two were analyzed. The effects of miR-183-5p on the expression of TβRⅠand TβRⅡ in ECA-109 cells and the cell functions of ECA-109 were also investigated. Results Compared with the normal esophageal epithelia cells, ESCC cell lines TE-1 and ECA-109 were statistically characterized by a high expression of miR-183-5p (all P<0.05) and low expression of TβRⅠand TβRⅡ(all P<0.05). The expression of miR-183-5p in ESCC tissues was higher than that in adjacent normal tissues, while the expressions of TβRⅠ and TβRⅡ were lower (all P< 0.05). The expression of miR-183-5p was closely related to sex, tumor differentiation, tumor staging, distant metastasis, lymphatic metastasis, and tumor location (all P<0.05). TβRⅠlevel was associated with sex, lymph node metastasis and tumor size (all P<0.05). Experimental data showed the negative correlation between the expression of miR-183-5p and TβRⅠin ESCC tissues (r= -0.521, P< 0.05). Over expression of miR-183-5p significantly inhibited the expression of TβRⅠ in ECA-109 cells (P< 0.05) and promoted the growth, invasion and metastasis of ECA-109 cells (P< 0.05). Low expression of miR-183-5p significantly promoted the expression of TβRⅠ in ECA-109 cells (P< 0.05), and suppressed the growth, invasion and metastasis of ECA-109 cells (P< 0.05). There was no significant change in the expression of TβRⅡ in the transfection experiments. Conclusion MiR-183-5p is closely related to the abnormal expression of TβRⅠ, which may exert an important role in the progression of lymphatic metastasis.

Intrathecal (I. T.)administration of K opioid dynorphin A (1-17) is used as a model of neurological dysfunction which lnvolved in spinal cord injury and secondary affection according to several previous reports. 5-amlno-2-phosphoveroleric acid (APV ), an NMDA receptor antagonist, can significantlly prevent the hindlimb paralysis in rats produced by I. T. dynorPhin A (1-17). To further investigate the mechanisms, we establis11 a nlodel of [3H]L,-Glu release in rats spinal slices influenced by dynorphin A (dynA ) (1-17). [3H]L-Glu release evoked by high [K+] (5Ommol/L,)is a Ca2+-dependent process. DynA (1-17) slgnificantly inhibited [3H]L,-Glu release at 1O-8mol/L,, but very significantly enhanced [3H]L-Glu release at 10-6 mol /L. The synthetic k agonist U50, 488H, which has no neurotoxic effect, inhibited [3H]L-Glu release at both high and low concentrations and did not have any enhancing effect. The results suggest that the analgesic effect of dynA (1-17) at physiological dosage may be rnediated by presynaptic K opioid receptor through the inhibition of Ca2+ influx and L-Glu release;but dynA (1-l7)enhanced L-Glu release through a non-opioid pathway and induced hindlimb paralysis at high neurotoxic dosage.

AIM: To investigate the effects of human umbilical cord-derived mesenchymal stem cells ( hUC-MSCs) on the proliferation and migration of osteosarcoma cells ( Saos-2 ) and the underlying molecular mechanism. METHODS:hUC-MSCs were isolated and cultured by tissue explants adherent method.The cell surface markers on hUC-MSCs were identified by flow cytometry.The effects of conditioned medium ( CM) from hUC-MSCs ( hUC-MSCs-CM) , re-combinant human interleukin-6 (rhIL-6) and IL-6 neutralizing antibody on the proliferation of Saos-2 cells were detected by CCK-8 assay and cell counting.IL-6 secretion of hUC-MSCs was assayed by ELISA.RT-PCR was used to assess the tran-scription level of proliferation-related genes proliferating cell nuclear antigen ( PCNA) , cyclin D1 and survivin.The migra-tion potential of hUC-MSCs and Saos-2 cells was measured by Transwell assay.RESULTS:hUC-MSCs migrated to Saos-2 cells.hUC-MSCs-CM contained a high concentration of IL-6, up to (1 835.5 ±134.1) ng/L.hUC-MSCs-CM and rhIL-6 promoted the proliferation and migration of Saos-2 cells.Addition of neutralizing antibody against IL-6 in the hUC-MSCs-CM impaired this proliferation and migration of Saos-2 cells.The mRNA expression of PCNA, cyclin D1 and survivin was up-regulated by hUC-MSCs-CM and rhIL-6, while this effect was dramatically attenuated by treatment with IL-6 neutralizing antibody.CONCLUSION:hUC-MSCs migrate to osteosarcoma cells and promote the proliferation and migration of osteo-sarcoma cells through secreting IL-6 in vitro.

Objective During pregnancy , exosomes can be released from the placenta into maternal circulation and play im-portant roles in normal pregnancy or placenta-related diseases .We aimed to establish a simple and efficient method for isolating and i-dentifying placental exosomes from maternal serum and lay a foundation for the studies of pregnancy -related diseases . Methods Using sucrose gradient centrifugation with 8% PEG6000 precipitation twice , we isolated and purified placenta-derived exosomes from normal maternal serum and detected their molecular markers CD 63 , CD81 and PLAP by Western blot , followed by silver staining anal-ysis of the protein profile of the exosome pellet .We identified the morphology of the placenta-derived exosomes by transmission electron microscopy ( TEM) and measured the size and distribution of the particles by dynamic light scattering ( DLS) . Results Silver stai-ning of the protein profiles of the exosomes after sucrose gradient centrifugation clearly revealed the bands of the protein molecules . Western blot showed the expressions of CD 63, CD81, and PLAP in the 21-34%density layer, which demonstrated the presence of serum placental exosomes mainly in the 1.09-1.16 g/mL density layer.TEM exhibited that the placenta-derived exosomes were round or oval cup-shaped, specifically expressing PLAP, and the particles were uniform in size, with a mean diameter of (41.79 ±11.94) nm. Conclusion A simple, fast, and efficient method was successfully established for isolating placenta-derived exosomes from ma-ternal serum, which provides a basis for studying the roles of placental exosomes in normal pregnancy and placenta -related diseases.

Objective:To detect the expression of oncogene c-myc and explore its role on human aryngeal cancer.Method:The mRNA and of oncogene c-myc levels were detected in human laryngeal cancer tissues and iaryngeal normal tissues by the means of real-time PCR and immunohistochemistry,respectively.Result:Compared with normal laryngeal tissues,the mRNA and protein levels of oncogene c-myc were both upregulated in laryngeal cancer tissues of all differentiation degree(P＜0.05).Moreover,the level of e-myc was inverse correlated with the differentiation degree in human laryngeal cancer tissues.Conclusion:The oncogene c-myc is overexpressed in human laryngeal cancer tissues,and c-myc may play an important role in carcinogenesis and progression of human laryngeal cancer tissues.It may provide a new target for gene therapy of human laryngeal cancer.