Objective To observe the influence of XueShuanXinMaiNing(XSXMN)in the behavior and structures of cerebral cortex and hippocampus of the rats withβamyloid protein(Aβ)-induced Alzheimer’s disease(AD),and to explore its therapeutic effects on the rat AD.Methods 100 male Wistar rats were selected.According to weight, the rats were randomly divided into sham operation group, model group, positive drug group (donepezil hydrochloride,1.75 mg· kg-1 ),XSXMN 1.1 g· kg-1 group and XSXMN 2.2 g· kg-1 group. The rat AD models were made by injecting Aβinto hippocampus.After oral administration for 15 d,Morris water maze test, dark avoidance task and pathology test were performed.Results In Morris water maze test,compared with model group,the latency and swimming distance to platform of the rats in XSXMN 1.1 g·kg-1 group were decreased on the 2nd,4th and 5th day(P<0.05 or P<0.01);in XSXMN 2.2 g·kg-1 group,the latency to platform of the rats were decreased from the 3nd to 6th day(P<0.05 or P<0.01),the swimming distances to platform of the rats were decreased from the 3rd to 5th day(P<0.05 or P<0.01).On the 7th day,in XSXMN groups,the times of passing platform,time of staying on platform,distance of staying on platform,time of staying in effective area, distance of staying in effective area, time of staying on platform/total time, distance of staying on platform/total distance,time of staying on platform/total time were all increased significantly(P<0.05 or P<0.01)within 90 s. In dark avoidance task,compared with model group,the error latency and the error times of the rats in XSXMN groups had no obvious change on the 2nd day.The pathological results showed that there were degeneration nerve cells and necrosis nerve cells in the rat cerebral cortex in XSXMN groups,while in the rat hippocampus there were less number of nerve cells with obscure cell layer and many degeneration and necrosis cells were found;compared with model group,there was no obvious improvement.Conclusion XSXMN can improve the learning and memory function of the AD rats.

Objective:To identify the pathogenic gene of a pedigree with symphalangism and to prove the pathogenicity of this locus in vitro.Methods:The clinical data of patients’families were collected at Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, peripheral blood was collected and genomic DNA was extracted and NOG, FGF9, GDF5 exon regions were amplified by PCR, and the exon gene mutations were detected by first-generation sequencing technique. The structure of noggin-GDF5 protein complex was simulated in silicon. COS-7 cells were transfected with 5 μg empty plasmid, wild type plasmid and V202G mutant plasmid in vitro. Each group of plasmids was transfected into 3 well cells. The experiment was repeated for 3 times, and the expression of noggin protein was detected by Western blotting. C2C12 cells were also transfected with the above plasmids in vitro for osteogenic differentiation. By applying alkaline phosphatase staining and quantitative assay. Relative expression level of osteoblast-related genes Col1α1, ALP and Runx2 were detected by qRT-PCR. Each group of plasmids was transfected into 3 well cells, and the experiment was repeated for 3 times. All statistical analysis were performed by Prism 6 software. The result were shown as mean±standard deviation, and the comparison between groups was done by unpaired t-test. Data were considered statistically significant when P value is less than 0.05. Results:Both the proband and his mother suffered from symphalangism. The result of Sanger sequencing showed that there was a heterozygous missense mutation of NOG gene (p.V202G) in all patients in this pedigree. No disease-related mutations were detected in FGF9 and GDF5. Computer three-dimensional mechanism simulation showed that the site was located at the α helix. The result of Western blotting showed that the expression of mutant protein was significantly lower than that of wild type. Osteogenic differentiation in vitro showed that the inhibitory effect of V202G mutant protein on osteogenic differentiation decreased. The quantitative result of alkaline phosphatase staining showed that the alkaline phosphatase activity in the vector group was (12.3±0.8) U/L, and the alkaline phosphatase activity in the wild type plasmid group was (2.6±0.3) U/L, which was significantly lower than that in the vector group ( t=11.550, P<0.001). The alkaline phosphatase activity in the mutant plasmid group was (10.8±0.3) U/L. There was no significant difference between the mutant group and the vector group ( t=1.830, P=0.141). The mRNA expression level of osteogenesis-related genes was consistent with the above result . Compared with vector group, the expression of osteogenesis-related genes in wild-type noggin group decreased significantly ALP、 Col1α1 and Runx2 ( t=5.987, 4.498, 4.170; P=0.004, 0.011, 0.014). There was no significant difference between mutant plasmid group and blank vector group in ALP、 Col1α1 and Runx2 ( t=0.433, 0.177, 1.159; P=0.688, 0.868, 0.311). Conclusions:NOG gene c. 605T < G p. V202G is a novel mutation in symphalangism, which is located in the α helix of noggin protein, leading to the decrease of the expression of noggin protein and the manifestation of ankylosis.

Objective:To identify the pathogenic gene of a pedigree with symphalangism and to prove the pathogenicity of this locus in vitro.Methods:The clinical data of patients’families were collected at Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, peripheral blood was collected and genomic DNA was extracted and NOG, FGF9, GDF5 exon regions were amplified by PCR, and the exon gene mutations were detected by first-generation sequencing technique. The structure of noggin-GDF5 protein complex was simulated in silicon. COS-7 cells were transfected with 5 μg empty plasmid, wild type plasmid and V202G mutant plasmid in vitro. Each group of plasmids was transfected into 3 well cells. The experiment was repeated for 3 times, and the expression of noggin protein was detected by Western blotting. C2C12 cells were also transfected with the above plasmids in vitro for osteogenic differentiation. By applying alkaline phosphatase staining and quantitative assay. Relative expression level of osteoblast-related genes Col1α1, ALP and Runx2 were detected by qRT-PCR. Each group of plasmids was transfected into 3 well cells, and the experiment was repeated for 3 times. All statistical analysis were performed by Prism 6 software. The result were shown as mean±standard deviation, and the comparison between groups was done by unpaired t-test. Data were considered statistically significant when P value is less than 0.05. Results:Both the proband and his mother suffered from symphalangism. The result of Sanger sequencing showed that there was a heterozygous missense mutation of NOG gene (p.V202G) in all patients in this pedigree. No disease-related mutations were detected in FGF9 and GDF5. Computer three-dimensional mechanism simulation showed that the site was located at the α helix. The result of Western blotting showed that the expression of mutant protein was significantly lower than that of wild type. Osteogenic differentiation in vitro showed that the inhibitory effect of V202G mutant protein on osteogenic differentiation decreased. The quantitative result of alkaline phosphatase staining showed that the alkaline phosphatase activity in the vector group was (12.3±0.8) U/L, and the alkaline phosphatase activity in the wild type plasmid group was (2.6±0.3) U/L, which was significantly lower than that in the vector group ( t=11.550, P<0.001). The alkaline phosphatase activity in the mutant plasmid group was (10.8±0.3) U/L. There was no significant difference between the mutant group and the vector group ( t=1.830, P=0.141). The mRNA expression level of osteogenesis-related genes was consistent with the above result . Compared with vector group, the expression of osteogenesis-related genes in wild-type noggin group decreased significantly ALP、 Col1α1 and Runx2 ( t=5.987, 4.498, 4.170; P=0.004, 0.011, 0.014). There was no significant difference between mutant plasmid group and blank vector group in ALP、 Col1α1 and Runx2 ( t=0.433, 0.177, 1.159; P=0.688, 0.868, 0.311). Conclusions:NOG gene c. 605T < G p. V202G is a novel mutation in symphalangism, which is located in the α helix of noggin protein, leading to the decrease of the expression of noggin protein and the manifestation of ankylosis.