Objective To investigate the clinical manifestations,imaging features,molecular genetic characteristics and possible pathogenic mechanisms of hereditary cerebral small vessel disease (CSVD) caused by heterozygous mutation of HtrA serine protease-1 (HTRA1) gene.Methods The clinical data of a Chinese Han family with CSVD carrying a heterozygous mutation of HTRA 1 gene,which came from the Department of Neurology,Henan Provincial People's Hospital in March 2018,were analyzed retrospectively.The clinical and radiographic features were summarized.Several high-throughput whole exon high-throughput sequencing was used to capture the mutation sites and the Sanger sequencing was used to validate the results.The family diagram was drawn and the 3D model construction and mutation function prediction were performed using silico tools.The relevant literature was reviewed and the pathogenesis was explored.Results The pedigree map showed that the family had an autosomal dominant inheritance pattern.Three generations of the family were investigated,and three family members in the same generation suffered from the disease.The first symptom of the proband was diplopia at the age of 39,accompanied by recurrent stroke,cognitive impairment and mood disorders,without alopecia.Head magnetic resonance imaging revealed bilateral diffuse,symmetric lesions,multiple lacunar infarcts,perivascular space,and microbleeds.The elder sister of the proband developed symptoms of left limb weakness at the age of 46,whose other clinical and imaging features were similar to those of the proband.The proband's mother died at the age of 59 due to repeated strokes.Whole exon sequencing indicated heterozygous missense mutation at c.821G＞A locus of HTRA1 gene in the proband and her 4th elder sibling,which was a new pathogenic mutation after consulting several mutation sites of databases.Function prediction suggested pathogenicity.Conclusions The heterozygous mutation of c.821G＞A in HTRA1 gene may lead to autosomal dominant CVSD.This genetic type should be given clinical attention.

Objective To analyze the clinical and electrophysiological features in a family with spinal muscular atrophy (SMA), and assess the probable causative gene mutations for the family. Methods To identify the nosogenesis of the proband with weakness and atrophy in the double lower proximal limbs, clinical data of his 12 family members were collected, and the proband and his mother were selected for clinical examinations, including laboratory tests, electromyogram (EMG), F-wave, H-reflex, X-ray of the spine and double lower limbs, brain and spinal cord magnetic resonance imaging, etc. Moreover, human whole exome sequencing was performed on blood sample from the proband, then its deleterious effects were assessed according to the Standards and guidelines for the interpretation of sequence variants, a joint consensus recommendation of the American College of Medical Genomics (ACMG) and the Association for Molecular Pathology (AMP). Subsequently, the strong pathogenic mutation was validated by Sanger sequencing. Results Familial investigation showed seven of 12 family members presented with weakness in the double lower proximal limbs. Among them, three had the main manifestation of atrophy in the double lower proximal limbs, one had high arched foot as the main presentation, and the others had weakness in the double lower proximal limbs. EMG studies showed the abnormal results in the anterior horn of the spinal cord. The strong pathogenic mutation in DYNC1H1 gene (exon8, c.2327C＞T, p.P776L) was identified from the proband according to ACMG and AMP guidelines. Sanger sequencing revealed six patients had this variant and it was passed mainly from his maternal grandmother. Conclusions A pathogenic mutation of the DYNC1H1 p.P776L in six Chinese pedigrees which cosegregated with SMA was identified. There existed individual differences in clinical presentations. This finding may have important implications for the study of SMA in Chinese patients.

Objective To investigate the effects of zinc fingers and homeoboxes 3 (ZHX3) silence on expressions of smad3, smad4 and RUNX2 in bone marrow mesenchymal stem cells (BMSCs). Methods ZHX3 low expression vector (ZHX3 silent group) was constructed and was transfected to rat BMSCs. Empty vector was transfected into BMSCs and was used as vehicle control group, and wild type BMSCs was used as the control group. The cell transfection rate was measured under a fluorescence microscope, and then the successful transfection was identified. The immunocytochemistry and immu?noblotting methods were used to detect the expression levels of smad3, smad4 and RUNX2. Results (1) Cells with BMSCs phenotype can be obtained by recovery culturing. (2) After transfection, the green fluorescent protein was found in ZHX3 si?lence group and vehicle control group. Blank control group showed no significant fluorescence. The expression level of ZHX 3 was significantly lower in ZHX3 silence group than that of vehicle control group. (3) Results of immunofluorescence asssay showed that the positive expressions of smad3 and smad4 were located in nucleus and cytoplasm, the positive expression of RUNX2 was mainly located in nucleus. Positive cells were observed in three groups. There was no significant difference in fluorescence intensity between the control group and the vehicle control group, but the fluorescence intensity was significant?ly lower in ZHX3 gene silence group than that of two control groups. (4) There were no significant differences in expressions of smad3, smad4 and RUNX2 betweem control group and the vehicle control group, but they were significantly higher than those of ZHX3 silence group(P < 0.05). Conclusion ZHX3 gene silence can delay vitro osteogenesis of BMSCs, which may play a role by the down-regulated expression levels of smad3, smad4 and RUNX2.

Objective To explore the relationship between restoring Gissane angle and prognosis in calcaneal fracture surgery. Methods Forty patients with single side calcaneal fracture (SandersⅡ-Ⅲ), Gissane angle changed more than 15°and having performed open reduction and internal fixation with steel plate were enrolled. In them, 25 patients (experimental group) recovered Gissane angle in surgery referencing the healthy side with X-rays. Another 15 patients (control group) didn't recover Gissane angle. The ratio of calcaneum height and length was measured at 1 week after surgery. At 6 and 12 months after surgery, the function were valued by Maryland score. Results The ratio of calcaneum height and length in experimental group was significantly higher than that in control group:0.60± 0.04 vs. 0.55±0.05, and there was statistical difference (P<0.05). All the patients were followed up. At 6 and 12 months after surgery, the Maryland score in experimental group were significantly higher than those in control group:(88.9± 5.7) scores vs. (80.5±7.3) scores and (89.5 ±5.5) scores vs. (82.5 ±6.4) scores, and there were statistical differences (P<0.05). Conclusions Restoring Gissane angle is benefitial for prognosis. So in calcaneal fracture surgery, the Gissane angle should be recovered as much as possible referencing the healthy side.

Objective To investigate the effects of different culture methods in vitro mouse zona-free embryonic devel-opment. Methods In various stages of embryonic development rate,blastocyst rate and cell numbers of blastocysts de-veloped were measured as mWOW system (the modified “well of well” system), micro-drops single + group, micro-drops single and micro-drops group culture in vitro. The effects of different methods of were compared in vitro embryo development zona-free. Results Embryonic development rate,blastocyst rate and cell numbers of blastocysts developed were significantly higher in mWOW system culture than micro-drops single + group, micro-drops single and micro-drops group culture. Conclusion Compared with micro-drops single+group,micro-drops single and micro-drops group culture,mWOW system method is more suitable for zona-free cultured in vitro mouse embryos.

Objective To investigate the effect of construct the Notch1 (NICD) eukaryotic expression vector on the proliferation and differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods Rat BMSCs were experimented as the object. NICD eukaryotic expression vector was constructed. pEGFP-N1-NICD expressing plasmids were used to transfect BMSCs. The study included control group (CON group), empty vector group (VEC group) and the trans-fection group (TRA group). After 48-hour transfection, BMSCs were observed for general morphology. The protein expres-sions of NSE, GFAP and Notch1 were detected by real-time PCR and Western blotting assay respectively. The apoptosis, cy-cle distribution and cell proliferation were evaluated by flow cytometry and MTT assay. Results The DNA sequencing con-firmed that the pEGFP-N1-NICD recombinant plasmid was successfully constructed, and both VEC group and TRA group expressed green fluorescence after 48-hour transfection. The relative expression levels of Notch1 and GFAP mRNA and pro-tein were significantly higher in TRA group than those in VEC group and CON group (P<0.05), and there was no significant difference between VEC group and CON group. After 48-hour transfection, the ratio of living cells was significantly lower in TRA group than that of CON group and VEC group, and the early apoptotic rate and late apoptotic rate were significantly higher in TRA group than those of CON group and VEC group (P<0.05). The late apoptotic rate was significantly higher in VEC group than that of CON group. The proportion of G1/G0 cells was significantly higher in TRA group than that of CON group and VEC group, but S and G2/M cells were significantly lower (P<0.05). The value of growth curve was gradually de-creased in TRA group than that of CON group and VEC group (P<0.05). Conclusion The high expression of NICD gene might induce apoptosis of BMSCs, inhibit the proliferation in part, and induce into glial-like cell differentiation.

Aim To investigate the effects and mecha-nisms of theacrine on high fat diet induced hepatic steatosis in mice. Methods The C57BL/6J mice were fed with high fat diet for consecutive 17 weeks to induce hepatic steatosis and given a treatment of theac-rine for 6 weeks. The liver sections were stained with H&E or Sudan IV, and hepatic TG was determined by commercial analysis kits. Expression of SirT3 and phosphorylation of AMPK and ACC were measured by Western blot. Compound C was used to inhibit the phosphorylation of AMPK in HepG2 cells, and the ex-pressions of proteins were determined after the cells were treated with theacrine. Results Theacrine sig-
nificantly decreased hepatic TG content and ameliora-ted hepatic steatosis in mice. Expression of SirT3 and phosphorylation of AMPK and ACC were up-regulated, respectively. And theacrine still could activate SirT3 and up-regulate the phosphorylation of ACC whatever AMPK was inhibited. Conclusions The activation of ACC by theacrine depends on the phosphorylation of AMPK, but the activation of SirT3 by theacrine is in-dependent of the phosphorylation of AMPK. Theacrine ameliorates high fat diet induced hepatic steatosis in mice probably via SirT3/ AMPK/ ACC pathway.

Objective To investigate the effects of Ginsenoside Rg1 on the expression of nitric oxide synthase (NOS) in brain tissue after cerebral arterial thrombosis in adult rats. Methods Thirty rats were randomly divided into the sham-operative group, cerebral ischemia-reperfusion group, Ginsenoside Rg1-L group, Ginsenoside Rg1-M group, Ginsen-oside Rg1-H group and nimodipine group (n=5 for each group). The ischemia-reperfusion rat model was established by mid-dle cerebral artery occlusion. The neurological score after reperfusion was observed. The levels of nitric oxide (NO), neuronal NOS (nNOS) and inducible NOS (iNOS) were detected by nitrate reduction method and colorimetric method. The expressions of nNOS and iNOS after reperfusion were analyzed by immunohistochemistry and Western blot assay. Results (1) The neu-rological scores after cerebral ischemia were significantly lower in Rg1-L group, Rg1-M group and Rg1-H group than those of cerebral ischemia-reperfusion group(2.40±0.55,1.80±0.84, 1.60±0.89 vs 3.20±0.84,P<0.05). (2) Compared with those of model group, serum levels of NO and iNOS were reduced, and nNOS levels increased, in three groups of Rg1. (3) Compared with those of model group, the expression of nNOS was significantly increased,and iNOS expression was significantly re-duced, in three groups of Rg1. Conclusion The preventive effects of Ginsenosides Rg1 on cerebral ischemia-reperfusion injury may be associated with the activation of nNOS and the inhibition of iNOS.

Objective To explore the depression and its related factors of the patients with craniocerebral injury during rehabilitation.Methods 111 patients with craniocerebral injury were investigated with the questionnaire about general condition. They were assessed withHamilton Rating Scale for Depression (HAMD). The relationship between the general condition and the score of HAMD were analyzed. ResultsThere were 27 cases (24.32%) with score of HAMD≤7, 31 cases (27.93%) with the score from 8 to 17 and 53 cases (47.75%) with thescore≥18. The score of HAMD was significantly related to the patients' age, education, profession, economy status, economic compensationand the cause of injury (P<0.05). However, it was not related to the sexuality, marital status, registered residence, level of the injury, thecourse of disease and the coma time (P>0.05). Only economy state (x₁), education (x₂) and economic compensation (x₃) entered into the regressionequation by multiple regression analysis: y=22.782-6.879x₁+2.357x₂+3.54x₃ (F=75.609, P=0.000). Conclusion Patients with craniocerebralinjury generally depress during rehabilitation. The patients with a worse economy state, a higher education and no economic compensationappear more serious depression.

Objective To investigate the role of basic fibroblast growth factor（bFGF）on the vascularization of bone matrix gelatin（BMG） embedded vascular bundles in femoral anteriomedialis in the rabbits.Methods A longitudinal incision was done at the hyper-knee joint of femoral anteriomedialis in the Japan big ears white rabbit.Thirty six rabbits were divided into group A（n =12,arteria saphena and vena saphena were liberated and embeded to the groove of BMG soaked by 2 μg/ml bFGF0,group B（n =12,arteria saphena and vena saphena were liberated and embeded to the groove of BMG untreated）,and group C（n =12,the BMG untreated were directly implanted）.The rabbits were sacrificed at 4 weeks,8 weeks and 12 weeks after the surgery respectively by perfusing ink.After the implants were dislodged,transparent specimens were made and Masson stained for histological observations and quantitative analysis.Results After 12 weeks of operation,the neovascularization arranged in an ordered manner in group A,gradually trended to be orderly in group B,and were cluttered mainly on the edge of the implants in group C.The osteogenic and neovascularization areas of group A were the largest on each time point.Conclusion bFGF could promote the vascularization of BMG embedded vascular bundle.There was a positive correlation between osteogenesis and vascularization.