Objective To establish a nested-real-time-PCR for the detection of IL-21R mRNA in peripheral blood mononuclear cells(PBMC)of patients with rheumatoid arthritis,and explore its clinical significance.Methods The outer primers of span intron and inner primers were designed for nested-real-time-PCR.To increase specific template,the concentration of primers and enzyme,cycle times were decreased in the first amplification,and the real-time fluorescent quantity assay was performed in the second amplification.The ?-actin gene was used as internal reference,and the results were presented as the ratios of IL-21R mRNA to ?-actin mRNA.Results IL-21R mRNA was detected in 60 patients with RA and 30 healthy controls.The expression of IL-21R mRNA gene in the patients with RA was increased,and the statistical analysis has significant differences(P0.05),but a significant difference was found in the patients of grade III compared with those of grade I and II(P

Objective To investigate the lateralizing value of head deviation(HD) during complex partial seizures (CPS) in patients with refractory mesial temporal lobe epilepsy (mTLE).Methods Presurgical videotypes of 43 patients who were seizure-free for at least one year after temporal lobectomy were retrospectively reviewed.Attention was paid to the relationship between time and type of HD and the side of epileptogenic zone.Results HD was seen in 88 CPS from 43 patients who had total 206 CPS with or without secondary generalization.Both versive and non-versive HD displayed high positive predictive value (83％ (33/40) and 88％ (22/25)) for localization of an ipsilateral and contralateral seizure onset,respectively.Conclusion Both non-versive HD and versive HID during CPS in patients with mTLE are reliable lateralizing signs that can complement other diagnostic modalities in presurgical evaluation.

Objective:To detect the effect of EphrinA1-Fc on the phosphorylation of EphA2 and extracellular signal-regulated ki-nase (ERK) in 786-O renal carcinoma cells (RCCs). Methods:The soluble ligand EphrinA1-Fc was used to inhibit the 786-O RCCs in vitro. Western blot analysis was used to examine the phosphorylation of EphA2 and ERK1/2 in the 786-O RCCs at different time points. Results:After the intervention with EphrinA1-Fc for 5, 10, 30, and 60 min, the expression of p-EphA2 increased (F=9.392, P=0.025) as well as that of p-ERK (F=4.428, P=0.041). No p-EphA2 and p-ERK expression was observed in the pre-intervention group. Conclusion:One of the possible mechanisms of the inhibitory effect of EphrinA1-Fc on tumor metastasis and recurrence involves the phosphorylation of EphA2 by EphrinA1-Fc, leading to the degradation of EphA2.

Objective To establish the pyrogen removing effect of activated carbon in the technics of traditional Chinese medicine (TCM) injections. Methods The content of bacterial endotoxin concentration was detected by kinetic turdimetric assay to evaluate the effect of removing pyrogen before and after using activated carbon in concentrated solution of TCM injection. Results The activated carbon adsorption rate of Shuanghuanglian concentrated solution≥70%and the activated carbon adsorption rate of Danshen concentrated solution>95%. Conclusion Pyrogen manufacturing process is scientific and rational by adding activated carbon adsorption in powder injection of TCM. The bacterial endotoxin of large doses can't be removed fully by activated carbon adsorption.

Objective To explore the effect of different pH values on calcification of rat vascular smooth muscle cells (VSMCs) through bone morphogenetic protein (BMP)-2 signaling pathway. Methods Healthy male SD rats aged 5-8 weeks were selected in the study. VSMCs from rat thoracic aorta were cultured in vitro, and then identified by immunocytochemistry. The VSMCs were randomly divided into 4 groups by random sampling method:normal group (pH 7.4), pH7.4+high phosphorus group, pH 7.1+high phosphorus group, and pH 7.7+high phosphorus group. Calcium deposition and alkaline phosphatase (AKP) activity were measured by alizarin red staining and enzyme linked immunosorbent assay. The expressions of BMP-2, Smad1 and Runx2 mRNA were detected by RT-PCR. Results Compared with the control group, the calcification staining was increased in pH 7.4+high phosphorus group, calcium content was increased and expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were also increased (P<0.05). While compared with the pH 7.4+high phosphorus group, calcification staining, calcium content, expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were decreased in pH 7.1+high phosphorus group (P<0.05). The calcification staining, calcium content, expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were increased in pH 7.7+high phosphorus group (P<0.05). Conclusion The extracellular acidic environment (pH 7.1) can inhibit high-phosphotus-induced VSMCs calcification, whereas extracellular alkaline environment (pH 7.7) induce high-phosphotus-induced VSMCs calcification. The mechanism is presumably that VSMCs calcification is induced by influencing BMP-2 pathway, which may be mediated by VSMCs phenotype transdifferentiation of BMP-2 signaling pathway.

Objective To explore the effects of L?type calcium channel (LTCC) α1C and β3 subunits on that magnesium inhibited thoracic aortic calcification induced by β?glycerophosphate (β?GP). Methods Vascular smooth muscle cells (VSMCs) and aortic rings from rat aortic were cultured, then divided into control group, high phosphorus group (10 mmol/L β?GP), magnesium group (10 mmol/L β?GP+3 mmol/L MgSO4) and 2?APB (an inhibitor of magnesium transporter) group (10 mmol/L β?GP+3 mmol/L MgSO4+0.1 mmol/L 2?APB). Calcium deposition of VSMCs and aortic rings were respectively measured by alizarin red staining and Von Kossa staining, meanwhile the quantification of their calcium was tested by OCPC. The mRNA expressions of Runx2, LTCCα1C andβ3 in VSMCs were detected by RT?PCR, and their protein expressions were detected by Western blotting. Intracellular calcium ion of VSMCs was tested by fluorescence probe and alkaline phosphatase (ALP)activity was measured by ELISA. The Runx2 expression of aortic rings was detected by immunohistochemistry. Results After VSMCs stimulated for 7 days, calcium, ALP, mRNA and protein expressions of LTCCα1C, LTCCβ3 and Runx2, and intracellular calcium ion in high phosphorus group were higher than those in control group (all P<0.05). Moreover, calcium, ALP, mRNA and protein expressions of LTCCα1C, LTCCβ3 and Runx2, and intracellular calcium ion were decreased in magnesium group as compared with those in high phosphorus group (all P<0.05). In aortic rings, magnesium group had lower calcium and protein expression of Runx2 than high phosphorus group. No statistical difference between 2?APB group and high phosphorus group was observed in above indexes (all P>0.05). Conclusion Magnesium may down?regulate expressions of LTCCα1C andβ3 subunit, prevent calcium influx and then inhibit osteogenic differentiation so as to reduce β?glycerophosphate?induced VSMCs calcification.

Objective To observe the role of intermediate conductance calcium?activated potassium channels (KCa3.1) in alkalinization and β?glycerophosphate induced vascular calcification. Methods Vascular smooth muscle cells (VSMCs) and aortic rings were obtained from rat thoracic aorta, and then randomly divided into control group (pH was provided into 7.4, 8.0), high phosphorus groups (pH was provided into 7.4, 7.7 and 8.0, VSMCs in three groups were treated with 10 mmol/L β?glycerophosphate; HCl and NaHCO3 were used to adjust the pH) and TRAM?34 group (20 nmol/L was added into pH8.0 high phosphorus dulbecco's modified eagle's medium). Calcium deposition and alkaline phosphatase (ALP) activity were measured by Alizarin red staining, calcium content and enzyme linked immunosorbent assay after cells were simulated for 12 days. Intracellular free Ca2 + was measured by ELISA. The expression of KCa3.1, runt?related transcription factor 2 (Runx2) were detected by RT?PCR and Western blotting 4 days after cells were stimulated. Calcium deposition was measured by von Kossa staining and calcium content after aortic rings were cultured for 12 days. The expressions of KCa3.1 and Runx2 were detected by immunohistochemistry after aortic rings were cultured for 4 days. Results Compared with control group, calcification in VSMCs and aortic rings were significantly increased in high phosphorus group (P<0.05) while decreased in TRAM?34 group (P<0.05). Compared with control group, the expressions of KCa3.1, Runx2 and the activity of ALP in high phosphorus groups were increased (P<0.05) while decreased in TRAM?34 group (P<0.05). Besides, expressions of Runx2 and KCa3.1 were augmented as the pH was higher (P<0.05). The expression of Runx2 in aortic rings was the same situation. Besides, the Ca2+ influx was blocked by TRAM?34 (P<0.05). Conclusions Alkalinization contributes to β?glycerophosphate induced VSMCs calcification through increase of Ca2 + influx, up?regulation of KCa3.1 and promotion of osteogenic/chondrogenic differentiation.

Objective To explore the effect and mechanism of magnesium on calcification induced by hyperphosphate.Methods Vascular smooth muscle cells (VSMCs) were primarily cultured in vitro and induced calcification by β-glycerophosphate (β-GP).VSMCs were randomly divided into control group,high phosphorus group (10 mmol/L β-GP),magnesium intervèntion group(10 mmol/L β-GP + 3 mmol/L MgSO4) and 2-aminoethoxy-diphenylborate (2-APB,an inhibitor of magnesium transporter) intervention group(10 mmol/L β-GP+3 mmol/L MgSO4+ 10-4 mol/L 2-APB).Calcium deposition and alkaline phosphatase (ALP) activity were measured by alizarin red staining,quantification of calcium and euzyme linked immunosorbent assay.RT-PCR and Western blotting were used to observe the expression of core binding factor α-1 (Cbfα-1) mRNA and protein,respectively.In vivo,male Sprague-Dawley rats (n=24) were randomly divided into control group (methylcellulose+high phosphorous diet),vascular calcification group (adenine suspension + high phosphorous diet),high magnesium intervention group(adenine suspension+high phosphorous and magnesium diet).The aortic pulse wave velocity (PWV) was measured,and vascular calcification was determined by von Kossa stain and quantification of calcium.Cbfα-1 in aortic was measured by immunohistochemistry.Results In vitro,compared with high phosphorus group,calcification,ALP activity (P ＜ 0.05) and Cbfα-1expression in VSMCs were significantly decreased in magnesium intervention group after incubation for 14 days,but the addition of 2-APB might inhibit the protective effect of magnesium on VSMCs.Dynamic observation of Cbfα-1 showed that magnesium significantly inhibited the expression of Cbfα-1 (P ＜ 0.05) on the third day and the inhibitory role was obviously increased in a time-dependent manner.Consistent with the findings in vitro,the aortic PWV,calcification were all significantly reduced (P ＜ 0.05) in high magnesium intervention group with high serum magnesium level,when compared with vascular calcification group.Immunohistochemistry showed that hypermagnesemia downregulated obviously the expression of Cbfα-1 induced by hyperphosphatemia(P ＜ 0.05).Conclusion Magnesium protects against vascular calcification by inhibiting osteogenic differentiation of VSMCs.

Objective To explore the effect of vitamin K2 on β-glycerophosphate(β-GP)-induced rat vascular smooth muscle cells (VSMCs) calcification and and the mechanism.Methods VSMCs were obtained from rat aortic,and identified by immunocytochemistry,then randomly divided into control group,high phosphorus group,vitamin K2 group (the group was settled three subgroups according to the concentration of vitamin K2 based on the high phosphorus medium,namely 10 μmol/L,25 μmol/L,50 μmol/L) and noggin (bone morphogenetic protein pathway inhibitor) group.Calcification was visualized by Alizarin red staining,calcium load in cells was quantified by o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured after stimulating 14 days,gene expressions of bone morphogenetic protein-2 (BMP-2),SMAD1,SMAD7 and Runx2 mRNA were detected by RT-PCR,Runx2 protein levels was detected by Western blotting after stimulating 3 days.Results Compared with the cells in control group,high phosphorus induced cell calcification,increased ALP activity,up-regulated the expression of BMP-2,SMAD1,Runx2 mRNA (P ＜ 0.05) and down-regulated the expression of SMAD7 (P ＜ 0.01),while compared with high phosphorus group,the calcium deposition,ALP activity and the expression of BMP-2,SMAD1,Runx2 mRNA were remarkably reduced in a dose-dependent manner by treatment with vitamin K2 (P ＜ 0.05) and the expression of SMAD7 was increased (P ＜ 0.01).Compared with high phosphorus group,SMAD1 and Runx2 expression in noggin group were remarkably reduced(P ＜ 0.01).Conclusion Vitamin K2 inhibits β-glycerophosphate-induced VSMCs calcification which correlates with the suppression of the expression of osteoblast markers through the down-regulation of bone morphogenetic protein pathway.

Objective To investigate the relationship between polymorphisms in mitochondrial displacement-loop (mtDNA D-loop) and renal cell carcinoma. Methods Fifty-nine patients with clear cell renal cell cancer (renal cancer group) and 68 healthy control (control group) were selected in this study. The mtDNA D-loop region was amplified and se-quenced using polymerase chain reaction (PCR). Data were compared and analysed with the Revised Cambridge Reference Sequence (rCRS) in library of mitochondria. The difference in frequency analyses of mtDNA D-loop region was compared be-tween two groups. Results A total of 143 single nucleotide polymorphisms (SNP) of mitochondria D-Loop region were de-tected in renal cancer group and control group. Compared with control group, there were significantly higher frequencies of 262T and 16293G alleles in mitochondria D-loop region, and significantly lower frequencies of 16298C and 16319A alleles, in renal cancer group (P<0.05). Conclusion The analysis of genetic polymorphisms in the D-loop can be used as predic-tors of renal cell carcinoma and contribute to the early detection in patients of renal cell carcinoma.