Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice, Inbred BALB C ; Cats ; Mice ; Allergens/genetics* ; Glycoproteins/genetics* ; Enzyme-Linked Immunosorbent Assay ; Hybridomas/immunology* ; Recombinant Proteins/genetics* ; Female ; Antibody Specificity

AIM To prepare the hydroxypropyl-β cyclodextrin inclusion compound of volatile oil from Houttuyniae Herba.METHODS In the content determination of volatile oil by GC,2-undecanone was taken as a reference substance,and inclusion rate and drug loading were calculated.After the determination of optimal method for preparing the inclusion compound among techniques of mixing,grinding and ultrasonic methods.the preparation process was optimized by orthogonal test with ratio of volatile oil to hydroxypropyl-β cyclodextrin,inclusion temperature,mixing speed and inclusion time as influencing factors,and inclusion rate and drug loading as evaluation indices.The characterization was performed by TLC,infrared spectroscopy and differential scanning calorimetry.Then high temperature,high humidity and strong light tests were applied to investigating the inclusion compound stability.RESULTS Mixing method brought forth the maximal yield,inclusion rate and drug loading for the inclusion compound.And under the optimal conditions of 1 ∶ 25 for ratio of volatile oil to hydroxypropyl-β cyclodextrin,50 ℃ for inclusion temperature,420 r/min for mixing speed,and 80 min for inclusion time,the inclusion rate and drug loading were identified to be 77.35％ and 4.48％,respectively.The obtained inclusion compound was found to be white powder with loose texture and significantly increased solubility,the in vitro accumulative release rate reached 80.85％.Deliquescence and agglomeration,as well as obviously decreased inclusion rate and drug loading,were observed at a relative humidity of more than 75％,despite the good stability at high temperature (60 ℃) and strong light (3 000 lx),and yet the rehydration character was good.CONCLUSION It is possible that the hydroxypropyl-β cyclodextrin inclusion compound of volatile oil from Houttuyniae Herba generates a new phase rather than a simple mixture,which should be kept dry and sealed at the time of storage.

Objective The aim of this study was to explore the effect of low density lipoprotein ( LDL) on the proliferation and differentiation of MC3T3-E1 osteoblasts, as well as the expression of low density lipoprotein receptor-related protein 5(LRP5) and dickkopf-1(DKK1) mRNA of MC3T3-E1 osteoblasts. The possible mechanisms of the treatment of atorvastatin on LDL expression in MC3T3-E1 osteoblasts were also investigated. Methods Proliferation, osteocalcin expression, LRP5, and expression of DKK1 mRNA of MC3T3-E1 with interaction of LDL at 0. 05, 0. 10, 0. 20 mg/ml levels after 24 h, 48 h, 72 h were detected by CCK8, ELISA, and fluorescence quantitative PCR. Furthermore, proliferation, osteocalcin expression, LRP5 and DKK1 mRNA of MC3T3-E1 after the treatment of atorvastatin of 10-6 mol/L and 10-5 mol/L were also be studied, respectively. Results The effect of LDL on proliferation, expression of osteocalcin and expression of LRP5 and DKK1 mRNA in MC3T3-E1 osteoblasts was the most obvious under LDL with 0. 20 mg/ml level. Under that level, atorvastatin (10-6 mol/L or 10-5 mol/L) was able to make the proliferation of MC3T3-E1 osteoblasts in 48 h and 72 h be decreased, while significantly caused upregulation of osteocalcin, LRP5 mRNA expression; and down regulated DKK1 mRNA expression ( all P<0. 05). Conclusions Atorvastatin can reduce the inhibitory effect of LDL on the proliferation and differentiation of osteoblasts. The mechanisms of atorvastatin on osteoblasts are possibly related to the osteoblast proliferation and expression of LRP5 mRNA and DKK1 mRNA of osteoblasts of wnt signal pathway.

Objective Cerebral microbleeds (CMBs) are important indicators of cerebral small vessel disease .However, it is still unclear whether endothelial dysfunction is involved in CMBs .The aim of this study is to investigate the correlation between CMBs and soluble E-selectin (sE-selectin) in patients with acute ischemic stroke . Methods Based on the results of MRI (3.0 T) susceptibility weighted imaging , we divided patients with first acute ischemic stroke into a CMBs group ( n=63 ) and a non-CMBs group (n=63), and recruited another 45 volunteers with normal MRI findings as controls .We collected and conducted comparative a-nalysis on the demographic data , biochemical variables ( including the sE-selectin level ) , vascular risk factors , and the number of CMBs of the patients . Results Ordinal logistic regression analysis showed a significantly positive correlation between sE -selectin and the number of CMBs (OR=1.062, 95%CI:1.023-1.103, P=0.002), higher systolic blood pressure associated with more CMBs (OR=1.014, 95%CI:1.002-1.025, P=0.021). Conclusion Serum sE-selectin is significantly positively correlated with and can be used as a biological marker for the severity of CMBs .

Transcranial Doppler (TCD) is a non-invasive,convenient and inexpensive method for cerebral hemodynamic assessment widely used in clinical practice.Monitoring of cerebrovascular reserve capacity with TCD can provide relevant information for clinical decision making in the treatment of carotid stenosis.During carotid angioplasty and stenting,TCD can be used to evaluate effect of stenting on hemodynamics,and predict major complications,particularly postoperative hyperperfusion.

Objective To investigate the enlarged perivascular space (EPVS) and its clinical significance in patients with cerebral small vessel disease (CSVD).Methods One hundred seventy-four patients with CSVD and 86 patients without CSVD admitted to Jinling Hospital,Clinical School of Nanjing University School of Medicine from October 2011 to February 2012 were recruited.All patients underwent cranial MRI examination (including diffusion-weighted imaging and fluid attenuated inversion recovery sequences).The numbers of EPVS and anatomic distribution in all the subjects of both groups were analyzed.The receiver operator characteristic (ROC) curve was used to investigate its diagnostic critical value of anatomic distribution.Results Multivariate logistic regression analysis showed that EPVS in basal ganglia region (odds ratio [OR] 1.491,95％ confidence interval [CI] 1.165-1.909; P =0.002) and EPVS in centrum semiovale (OR 1.279,95％ CI 1.022-1.601;P=0.032) were independently associated with CSVD.EPVS in the basal ganglia region and the centrum semiovale in patients with CSVD was significantly more than that in patients with non-CSVD (all P ＜0.001).Its corresponding diagnosis cut-off points of CSVD were 4 and 6 respectively.The area under the ROC curve and the diagnostic sensitivity and specificity were 0.859,72.4％,93.0％ and 0.808,65.5％,95.3％,respectively.Conclusions EPVS contributes to the diagnosis of CSVD.When using EPVS to diagnose CSVD,the anatomical sites need to be distinguished and establish appropriate diagnostic critical value.