Objective To construct the recombinant plasmid of fusion protein containing respiratory syncytial virus (RSV) cytotoxicity T lymphocyte (CTL) epitope M2:81-95 and heat shock protein (HSP) 70L1, and to investigate its immunogenicity after prokaryotic expression. Methods HSP70L1 gone was cloned from SMMC7721 cells. The M2:81-95 gene fragment was amplified by polymerase chain reaction (PCR) method. Plasmid pET-HSP70L1-M2 : 81-95 (pET-HSP70L1-M2) was constructed, identified and transferred into E. coli BL21 (DE3). Expression of HSP70L1-M2 : 81-95(HSP70L1-M2) was induced by isopropy-β-D-thiogalaetosidc (IPTG). The expressed protein was purified by affinity chromatography and renatured by gradient dialysis. The BALB/c mice were immunized with this fusion protein. IgG antibodies and the subtypes were detected by enzyme-linked immunosorbent assay (ELISA), and CTL responses were determined by methyl thiazolyl tetrazolium (MTT). Results The recombinant plasmid pET-HSP70L1-M2 was successfully constructed. The fusion protein HSP70L1-M2 was expressed in E. coll. The purified protein induced strong RSV-and CTL epitope-specific CTL responses and high titer of protein specific lgG antibody 4.87±0.35. The subtypes were IgG1 (5.53±0.28) and lgG2a (4.40±0.21) and IgG1/ IgG2a ratio was balanced. The titers of lgG, IgG1 and IgG2a in PBS control group were 0.33±0.17, 0.51±0.21 and 0, respectively, which werc significantly lower than those in immunized group (t = 3.512, 3.681, 5.856; P<0.05). Conclusions The recombinant plasmid pET-HSP70L1-M2 is successfully constructed and the fusion protein is expressed and purified. HSP70L1-M2 induced strong RSV-and CTL epitope-specific CTL responses and mixed T helper cell (Th)1/Th2 response in BALB/c mice.

Objective To explore a feasible and effective method of purse-string suture and traction in proce- dures for prolapse and hemorrhoids(PPH)with circular stapler.Methods Width of resected mucosal band and ex- tent of residual hemorrhoid mucosal lifting were compared between two groups of patients with hemorrhoids receiv- ing PPH with“two-point retraction”method(group one,110 cases)and other retraction methods(group two,40 cas- es).Results For group one of 110 cases with“two-point retraetion”,resected mucosal band of all cases were intact and and average width ranged from 3.5 to 5cm.There was a completely residual hemorrhoid mucosal lifting(com- plete return of prolapsed hemorrhoid into the anal canal)in 100 cases of group one(91%).But in group two with other retraction methods,average width of resected mucosal bands ranged from 0.2 to 3.5cm and there was an in- complete band in 10 cases(25%).The rate of mucosal lifting in this group was only 62.5%(19/40).Conclusion Two-point retraction is a feasible,effective and simple method in PPH,resulting in an intact resected mucosal band and sufficient hemorrhoid mucosal lifting.So the two-point retraction PPH is worth to be extended for application.

Objective: To investigate the regulation of respiratory syncytial virus CTL epitope fused with G protein antigen fragment G1 to the specific immunoresponses. Methods: The recombinant plasmid pET-DsbA-G1 or pET-DsbA-G1F/M2 was transferred into E.coli BL21(DE3) and the fusion protein DsbA-G1F/M2 or DsbA-G1 was expressed.The expressing product was induced and purified by affinity chromatography. The two proteins were used to immunized BALB/c mice i.p, respectively. Serum and spleen cells were collected regularly. RSV-specific CTL responses were measured by MTT, IgG and IgG1 and IgG2a antibodies by ELISA, neutralizing antibodies by plaque reduction assay. Results: The recombinant proteins were expressed successfully and the purity was over 90% after purified by affinity chromatography. The protein DsbA-G1F/M2 induced significant RSV-specific CTL responses, while DsbA-G1 without CTL epitope did not induce detctable CTL responses. Strong IgG antibody responses were elicited,indicated by both. IgG1 and IgG2a titers induced by DsbA-G1F/M2, while only IgG1 was induced by DsbA-G1.The ratio of IgG1/IgG2a was downregulated significantly. Both antigens induced high level of neutralizing antibodies, but the latter was a little lower. Conclusion: DsbA-G1F/M2, the fusion protein of a CTL epitope and G protein fragment G1 can induce both cellular immunity and humoral immunity. The activation of CTLs downregulates the ratio of IgG1/IgG2a.The more balanced immunoresponse is advantageous for improving safety of the candidate vaccine.

Objective To observe the effect of bone marrow mesenchymal stem cells (BMSCs) transplantation on the expression of growth associated protein-43 (GAP-43) in rats after spinal cord injury (SCI). Methods The SCI models were made by impacting at the level of T11 segment. After modeling, the rats were randomly and equally divided into control group (n=18) and observation group (n=18). 1 week after injury, DMEM/F12 medium 0.1 ml was injected into tail vein of rats in the control group, and an equal volume of BMSCs suspension was injected in the observation group. 1, 2, 4, 8 weeks after SCI, the hindlimb function was evaluated by the Basso-Beattie-Bresnahan (BBB) score. 2, 4, 8 weeks after SCI, the expression of GAP-43 mRNA was detected by RT-PCR, and the expression of GAP-43 was detect-ed by the immunohistochemistry. Results 2, 4, 8 weeks after SCI, compared with the control group, The BBB score increased, the expres-sion of GAP-43 mRNA and GAP-43 increased in the observation group (P<0.05). Conclusion BMSCs transplantation can improve the ex-pression of GAP-43 in gene and protein level, and then promote the repair of SCI.

Objective To evaluate the effect of fingolimod(FTY720) on angiotensin Ⅱ(AngⅡ)-induced renal oxidative stress in rats and its possible mechanisms.Methods Thirty-six male Sprague-Dawley rats were divided into three groups through random number tables:AngⅡ infusion group(twelve rats,infusion of AngⅡvia subcutaneous osmotic pumps),0.9% sodium hydrochloride solution infusion group(twelve rats,infusion of equal volume of 0.9% sodium hydrochloride solution via subcutaneous osmotic pumps) and FTY720 intervention group(twelve rats,intra-gastric administration of FTY720 besides AngⅡ infusion).Urine samples were collected for 24 hours every week.Blood samples and kidneys were collected when rats were sacrificed at day 14 and 28 d respectively.MDA and T-SOD assay Kits were used to detect the concentrations of MDA and T-SOD in serum and kidney homogenates.Renal pathological changes were observed by light microscope.The expression of Nox4 in the kidneys was evaluated by immunohistochemistry.Results Compared with 0.9% sodium hydrochloride solution infusion rats of the same period,AngⅡ-infused rats developed significant proteinuria.The concentration of serum creatinine,urea nitrogen,AngⅡ and MDA were increased(P<0.05),T-SOD was significantly decreased on days 14 and 28(P<0.05),the serum albumin was significantly increased on day 28(P<0.05),while FTY720 partially reversed these changes.The renal tissue of AngⅡ-infused rats presented mesangial cells proliferation,mesangial matrix deposition,tubular epithelial cells effacement,sclerosis and atrophy in part of the glomerular.FTY720 can alleviate these changes.Immunohistochemistry showed that Nox4 was primarily located in the distal tubule.The expression of Nox4 was significantly increased in AngⅡ-infused rats as compared with that of 0.9% sodium hydrochloride solution infused group rats,and FTY720 administration prevented the change effectively.Conclusion AngⅡ-induced renal injury could be attenuated by FTY720 via preventing oxidative stress.

Objective To investigate the effects of MG132 on diabetic nephropathy (DN) rats induced with streptozocin. Methods Seventy-two male SD rats were randomly divided into three groups: normal control group (NC, n=24), DN group (n=24) and DN treated with MG132 group (DN+MG132, n=24). At the end of 4, 8 and 12 weeks, 24 hour urinary protein excretion rate (UPER) was detected. Morphology of kidney was examined by special staining of periodic acid-schiff (PAS). Renal 26S proteasome activity was determined by quantifying the hydrolysis of S-LLVY-AMC in a fluorescence reader. Urinary malondialdehyde (MDA) level and renal SOD and GSH-PX activity were detected by commercial kits. Renal SOD, GSH-PX and p47phox mRNA expressions were determined by real-time fluorescence PCR. Renal p47phox protein expression wasdetermined by Western blotting. Results Compared with NC group, the DN group showed a significant increased of UPER at week 4, 8, 12 (all P<0.05), of mesangium proliferation and mesangial matrix expansion at week 12. In DN+MG132 group, UPER was significantly decreased compared with DN group at the end of 4, 8 and 12 weeks (P<0.05, respectively), and the glomeruler pathological alteration induced by diabetes was attenuated. Increased renal 26S proteasome activity in DN rats was significantly inhibited after MC132 administration (P<0.05). Moreover, renal p47phox mRNA expression in DN group was 155%, 149% and 120% more than those in NC group at 3 time points (all P<0.05), and so was the renal p47phox protein expression, 139%, 152% and 186% more (all P<0.05). Urinary MDA levels in DN group were 1.95-, 2.04-and 2.62-folds more than those in NC group (all P<0.05). In addition, compared with NC group at 3 time points, in DN group, renal SOD activity was decreased by 23.09%, 33.59% and 53.31% (all P<0.05); renal GSH-PX activity was decreased by 28.57%, 33.06% and 48.76% (all P< 0.05); renal SOD mRNA was decreased by 38.09%, 61.44% and 76.53% (all P<0.05); renal GSH-PX mRNA group was decreased by 29.16%, 37.26% and 62.40% (all P<0.05). Compared with DN group, renal p47phox mRNA and protein expression, and urinary MDA levels were significantly lower in DN+MG132 group (all P<0.05); renal SOD and GSH-PX activity as well as mRNA expression were significantly increased in DN+MG132 group (all P<0.05). Conclusions MG132 treatment can provide renoprotection for DN rats effectively maybe through enhancing renal anti-oxidative ability.

Objective To evaluate left atrial appendage regional function and movement changes of systolic and diastolic in patients with atrial fibrillation by speckle tracking imaging(STI) and real-time three-dimensional transesophageal echocardiography(RT-3D TEE).Methods Sixty-seven patients underwent RT-3D TEE was divided into 24 controls,22 paroxysmal atrial fibrillation,21 persistent atrial fibrillation.Left atrial appendage was divided into basal,middle and apical segment.Left atrial appendage emptying fraction of the overall (LAA-EF),basal emptying fraction (B-EF),middle emptying fraction (M-EF) and apical emptying fraction (A-EF) was measured by three-dimensional volume measurement.Each segment systolic strain rate (SRS) and diastolic strain rate (SRD) was measured by STI.Results LAA-EF,B-EF,M-EF,A-EF of paroxysmal atrial fibrillation and persistent atrial fibrillation group were lower than those of the control group,and persistent atrial fibrillation group was the lowest in the three groups.Compared with the control group,SRS and SRD of left atrial appendage basal,middle,apical segment was lower in paroxysmal atrial fibrillation group and persistent atrial fibrillation group,and persistent atrial fibrillation group decreased more significantly.Conclusions Paroxysmal atrial fibrillation and persistent atrial fibrillation can lead to left atrial appendage global or local systolic and diastolic function to reduce,and persistent atrial fibrillation decreased more significantly.

Objective To investigate the effect of 4-phenylbutyric acid(4-PBA)on the renal pathogenesis of rats with streptozotocin-induced diabetes and its mechanism. Methods Fifty-four male SD rats were randomly divided into three groups:normal control group(NC group,n=18),diabetic nephropathy group(DN group,n=18),diabetic nephropathy plus 4-PBA treatment group(4-PBA group,n=18).At the end of 4,8 and 12 weeks,index of kidney weight/body weight ratio(KI)were measured and calculated.Serum creatinine (Scr),blood urea nitrogen(BUN),urinary MDA levels,urinary SOD activity,and 24 hour urinary protein excretion ram(UAER)were detected by HITACHI automatically.Morphology of kidney wag examined by special staining of periodic acid-schitt (PAS).The p47phox and nitrotyrosine (NT) expression in kidney were determined by real-time fluorescence PCR and Western blotting. Results Compared with the NC group, the DN group rats showed a significant increase of KI(P＜0.05), UAER(mg/24 h) (4.92±0.70 vs 0.26±0.07, 5.29±0.83 vs 0.28±0.08, 5.54±0.81 vs 0.29±0.04,respectively, P＜0.05]for indicated time, mesangial cells proliferation and mesangial matrix expansion at 12 week. However,4-PBA treatment could significantly inhibit the increase of KI (P＜0.05), decrease UAER (mg/24 h) (3.71±0.37, 3.47±0.36, 3.28±0.40, respectively, P＜0.05]for indicated time, and prevent the glomeruler pathological alteration induced by diabetes. Moreover, the mRNA expression of p47phox in the kidney of DN group was 154.72%, 148.60% and 91.95% more than that of NC group (all P＜0.05) for indicated time. The protein expression of p47phox was 118.00%, 140.10% and 177.82% more than that of NC group (all P＜0.05), and the protein expression of NT was 45.29%,59.13% and 89.28% more than that of NC group (all P＜0.05). In addition, urinary MDA levels in DN group were 2.05-, 2.26- and 2.43- folds of NC group, and urinary SOD activities were decreased by 64.78%, 71.29% and 79.32% of NC group. Compared with the DN group, the mRNA and protein expression of p47phox, and protein expression of NT in 4-PBA group were decreased markedly (all P＜0.05) at the end of 8 and 12 weeks. The urinary MDA level was decreased, and the urinary SOD activity was increased significantly in rats with diabetes after 4-PBA treatment for indicated time (all P＜0.05). Conclusion 4-PBA treatment can significantly inhibit the renal pathogenesis of rats with diabetes through inhibition of oxidative stress.

OBJECTIVE: To select specific DNA aptamer for determining ketamine by FluMag-SELEX. METHODS: Based on magnetic beads with tosyl surface modification as solid carrier and ketamine as target, a random ssDNA library with total length of 78 bp in vitro was compounded. After 13 rounds screening, DNA cloning and sequencing were done. Primary and secondary, structures were analyzed. The affinity, specificity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity. RESULTS: Two ssDNA aptamers (Apt#4 and Apt#8) were successfully selected with high and specific abilities to bind ketamine as target with Kd value of 0.59 and 0.66 μmol/L. The prediction of secondary structure was main stem-loop and G-tetramer. The stem was the basis of stability of aptamer's structure. And loop and G-tetramer was the key of specific binding of ketamine. CONCLUSION FluMag-SELEX can greatly improve the selection efficiency of the aptamer, obtain the ketamine-binding DNA aptamer, and develop a new method for rapid detection of ketamine.
Aptamers, Nucleotide/metabolism* ; DNA ; DNA, Single-Stranded/genetics* ; In Vitro Techniques ; Ketamine/metabolism* ; Oligonucleotides ; SELEX Aptamer Technique/methods*

The excipient processing is an essential part of traditional Chinese medicine processing, and understanding its scientific connotations is a critical scientific issue that urgently needs resolution. Building upon a foundation where the composition of traditional Chinese medicine substances is fundamentally clear, this paper applies the techniques and methods of chemoinformatics to the study of the excipient processing mechanism. Relevant information on traditional Chinese medicines processed with four kinds of excipients (wine, vinegar, salt and honey) was collected, including properties, taste, meridian tropism, chemical components, etc. Molecular descritors and skeletons corresponding to each chemical component were calculated using chemoinformatics to characterize the properties and structural features of the components. Characteristic components associated with the four excipients (wine, vinegar, salt and honey) were explored through multivariate statistical analysis and Murcko skeleton analysis. Further analysis, taking honey-processed Astragali Radix as an example, the focus was on the impact of the processing excipient honey on the solubility and permeability of its characteristic pharmacologically active components (isoflavones). It was discovered that the excipient honey can increase their solubility and permeability, emphasizing that the impact of processing excipients on the pharmacological classification properties is a key breakthrough in elucidating the mechanism of excipient processing. In summary, this study analyzed the characteristic components associated with the processing excipients "wine, vinegar, salt and honey" based on their composition characteristics, providing data support for the research on the mechanism of excipient processing from a biopharmaceutical perspective.