Objective To explore the effect of dendritic cells (DC) primed by total RNA extracted from human colon cancer Lovo cell on specific cytotoxicity of cytokine-induced killer cells (CIK) in vitro.Methods Cord blood mononuclear cells extracted by Ficoll density gradient centrifuge were induced into CIK and DC cells separately, and their Immunophenotype was detected by Flow cytometer. Trizol harvested total RNA from colon cancer cell Lovo and the RNAs were loaded to DCs obtained from cord blood as tumor anti gens. Effectors were grouped accordingly as CIK cells co-cultured with DCs transfected with Lovo RNA, CIK cells co- cultured with unloaded DCs and CIK cells. Targets was Lovo cells. In vitro cytotoxicity of CHK cells was extured with DCs loaded with Lovo RNA(76.49%±4.21%), DC + CIK group was lower(53.84% ± 2.15%),and CIK cells group possessed the lowest cytotoxicity(32.20% ± 3.07%), showing statistic significance( P ＜0.05). Conclusion Extraction of total RNA from tumor cells is simple and easy for clinical implementation.Total RNAs acted as antigen to pulse DCs can strengthen the specific cytotoxicity of CIK cells, which will have good prospects for clinical application.

Objective To investigate the metabolite changes in systemic lupus erythematosus (SLE) patients with and without neuropsychiatric symptoms using magnetic resonance spectroscopy (MRS) and explore the associations between image findings and clinical variables. Methods Twenty-two SLE patients with neuropsychiatric symptoms (NPSLE), twenty-one SLE patients without neuropsychiatric symptoms (non-NPSLE) and twenty healthy controls (HCs) underwent routine MRI scan and multivoxel magnetic reson-ance spectroscopy (MVS). The absolute metabolite concentrations were measured bilaterally in the posterior cingulate gyrus (PCG), dorsal thalamus (DT), lentiform nucleus (LN) and posterior paratrigonal white matter (PWM) using LCModel and SAGE software. The relationships between metabolite con-centrations and cognitive function scores were analyzed by Spearman rank correlation. Single-factor Chi-square analysis and t-test were used for analysis. Results ① Compared to control subjects, NPSLE patients had significantly lower N-acetylaspartate (NAA) values in bilateral PCG and DT, with the mean differences of -1.504 [95% confidence interval ( CI) (-2.335, -0.672), P=0.001], -1.460 [95%CI (-2.349, -0.570), P=0.002], -1.259 [95%CI (-1.894, -0.625), P=0.000] and -1.022[95%CI (-1.688, -0.356), P=0.003] for RPCG, LPCG, RDT and LDT, respectively. The concentration of total creatinine were observed to decline in RPCG and RDT, with the mean differences of-1.094 [95%CI (-1.845, -0.342), P=0.003], -0.955 [95%CI (-1.630, -0.280), P=0.006], -1.259 [95%CI (-1.894,-0.625), P=0.006] respectively. Glutamine and glutamate-values decreased significantly in RDT [mean difference=-2.586, 95%CI (-4.139, -1.033), P=0.002]. ② Compared to non-NPSLE patients, NPSLE patients had a lower NAA level in LPCG [mean difference=-1.256, 95%CI (-2.146, -0.367), P=0.006]. Positive correlations between mini-mental state examination scores [RPCG: rs=0.312, P<0.05; LPCG: rs=0.355, P<0.01], Montreal cognitive assessment scores (RPCG: rs=0.362, P<0.01; LPCG: rs=0.285, P<0.05) and NAA values in bilateral PCG were detected. Conclusion Both NPSLE and non-NPSLE patients may have metabolite dysfun-ctions in different brain regions. The cognitive disorder in SLE patients may be interpreted by neuronic damage of PCG.

The changes in the tau protein phosphorylation and expression of bcl-2, and bax in rat parietal cortex neurons after focal cerebral ischemia-reperfusion (I/R) were explored, and the relationship between the tau protein phosphorylation and the expression of bax or apoptosis was clarified in order to elucidate the relationship between cerebral infarction and Alzheimer's disease. The rat focal cerebral I/R model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The level of tau protein phosphorylation at Ser396, Ser404, Tyr231, Ser199/202 sites and the expression of bcl-2, bax and total tau 5 in rat parietal cortex during focal cerebral ischemia/reperfusion were detected by Western blot. The relationship between the tau protein phosphorylation and the expression of bax, or apoptosis was examined by TUNEL method and double-labeling immunofluorenscence method. The results showed that the level of tau hyperphosphorylation at Ser199 / 202, Ser396, Ser404, Tyr231 sites and the expression levels of bcl-2, and bax were significantly higher in I/R group than in the sham group, but the ratio of bcl-2/bax was decreased. Neuronal apoptosis, bax expression and the tau protein hyperphosphorylation were co-localized. It is suggested that Alzheimer's disease-like pathological changes occur after cerebral I/R. The highly abnormal phosphorylation of tau protein plays a key role in cerebral I/R-induced apoptosis. The cerebral infarction may contribute to Alzheimer's disease occurrence and development.

Objective To explore value of trephine in pedicle subtraction osteotomy (PSO) for old thoracolumbar compression fracture with kyphotic deformity.Methods Thirty seven patients who underwent surgical treatment for old thoracolumbar kyphotic deformity in our hospital from February 2005 to February 2010 were retrospectively reviewed.Among them,21 patients underwent conventional PSO and 16 patients underwent PSO with trephine.In conventional PSO group,there were 14 males and 7 females,the average age was 55.6±3.7 years and the mean Cobb angle was 45.3°±4.6°.In PSO with trephine group,there were 11 males and 5 females,the average age was 53.3±4.2 years and the mean Cobb angle was 47.6°±5.9°.Results All patients were successfully followed up.The duration of follow-up ranged from 12 to 22 months in conventional PSO group,while 13 to 20 months in PSO with trephine group.The operation time,blood loss,amount of blood transfusion were 224±45 min,1043±234 ml,876±300 ml respectively in conventional PSO group,while 180±31 min,785±163 ml,500±230 ml in PSO with trephine group.Immediately after operation,correction rate of Cobb angle was 91.4％ in conventional PSO group and 90.9％ in PSO with trephine group.At final follow-up,the correction of Cobb angle lost 5.8％ in conventional PSO group and 6.2％ in PSO with trephine group.The improvement rate of JOA score was 81.1％ in conventional PSO group and 83.7％ in PSO with trephine group.The VAS score decreased 7.7±1.1 in conventional PSO group and 7.8±0.8 in PSO with trephine group.One patient in conventional PSO group experienced saddle numbness immediately after operation,which alleviated at final follow up.All patients achieved bony fusion at final follow up.No infection,screw loosening or breakage occurred in both groups.Conclusion The use of trephine in PSO for old thoracolumbar compression fracture with kyphotic deformity can reduce operation time,blood loss and improve efficiency of osteotomy.

Objective To explore the diagnosis of cystinosis.Methods The clinical and biochemical information, and gene detection results in a child with cystinosis was retrospective analyzed.Results Four-year-old female presented with photophobia and corneal crystal was found by ophthalmic examination at 2 years old, bilateral kidney stone was found, accompanied by development delay and rickets at 3 years old. Gas chromatography analysis in urine showed that a variety of amino acids were increased, and urine sugar and urinary micro-protein were also increased, which were in accordance with fanconi syndrome. The blood free carnitine was decreased, ester acyl carnitine spectrum was normal, and multi-amino acids such as lysine, valine and arginine were decreased. Gene analysis showed a homozygous mutation of c.696C>G (p.323 N>K) inCTNS gene, which was a known mutation. Both her parents were carrier of heterozygous mutation of c.696C>G inCTNS gene.Conclusion Child with kidney stone, renal damage, combined by multi-system damage such as eyes, bone, and thyroid should be paid attention to identify the cystinosis.

ObjectiveTo investigate whether Xiayuxue decoction exerts an anti-liver fibrosis effect by inhibiting glial cell line-derived neurotrophic factor (GDNF). MethodsA total of 24 C57BL/6 mice were randomly divided into control group, model group, and Xiayuxue decoction group. The mice in the model group and the Xiayuxue decoction group were given intraperitoneal injection of 10% CCl4, and those in the Xiayuxue decoction group were given 0.4678 g/kg Xiayuxue decoction by gavage since week 4. The liver function parameters alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and liver histopathology was observed. Immunohistochemistry was used to measure the protein expression of alpha-smooth muscle actin (α-SMA) and GDNF. GFP-Col-HSC and human primary hepatic stellate cells (HSCs) were treated with GDNF (10 ng/ml), and HSC activation was measured. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the control group, the model group had significant increases in the levels of ALT and AST, and compared with the model group, the Xiayuxue decoction group had significant reductions in the levels of ALT and AST (all P＜0.01). Liver histopathology showed that the model group had marked inflammatory cell infiltration and formation of fibrous septa by proliferated collagen fibers, and the Xiayuxue decoction group had loose fibrous septa and alleviated inflammatory cell infiltration. Immunohistochemistry showed that compared with the control group, the model group had significant increases in the expression of α-SMA and GDNF (both P＜0.01), which were observed in fibrous septa, and compared with the model group, the Xiayuxue decoction group had significant reductions in the expression of α-SMA and GDNF (both P＜0.05). Western blotting showed that the control group had relatively low expression of GDNF in liver tissue, the formation of liver fibrosis at week 6 of CCl4 modeling, and an around 10-fold increase in the expression of GDNF, and the Xiayuxue decoction group had significantly inhibited protein expression of GDNF (P＜0.01); there were significant increases in the expression of α-SMA and collagen type I α1 (Col1) in mice with liver fibrosis, with significant reductions in α-SMA and Col1 after treatment with Xiayuxue decoction (all P＜0.01). The in vitro experiment showed that GDNF induced the significant increases in the protein expression of α-SMA and Col1 in HSCs, which was significantly inhibited by Xiayuxue decoction (all P＜0.01). ConclusionThe expression of GDNF is significantly upregulated in the formation of liver fibrosis. GDNF can induce HSC activation, and Xiayuxue decoction can exert an anti-liver fibrosis effect by inhibiting GDNF.

The changes in the tau protein phosphorylation and expression of bcl-2,and bax in rat parietal cortex neurons after focal cerebral ischemia-reperfusion(I/R)were explored,and the relationship between the tau protein phosphorylation and the expression of bax or apoptosis was clarified in order to elucidate the relationship between cerebral infarction and Alzheimer's disease.The rat focal cerebral I/R model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method.The level of tau protein phosphorylation at Ser396,Ser404,Tyr231,Ser199/202 sites and the expression of bcl-2,bax and total tau 5 in rat parietal cortex during focal cerebral ischemia/reperfusion were detected by Western blot.The relationship between the tau protein phosphorylation and the expression of bax,or apoptosis was examined by TUNEL method and double-labeling immunofluorenscence method.The results showed that the level of tau hyperphosphorylation at Ser199/202,Ser396,Ser404,Tyr231 sites and the expression levels of bcl-2,and bax were significantly higher in I/R group than in the sham group,bat the ratio of bcl-2/bax was decreased.Neuronal apoptosis,bax expression and the tau protein hyperphosphorylation were co-localized.It is suggested that Alzheimer's disease-like pathological changes occur after cerebral I/R.The highly abnormal phosphorylation of tau protein plays a key role in cerebral I/R-induced apoptosis.The cerebral infarction may contribute to Alzheimer's disease occurrence and development.

Objective:To analyze the clinical features, genetic characteristics, treatment and follow-up results of patients with hydrocephalus caused by methylmalonic acidemia combined with homocysteinuria, and to discuss the optimal strategies for assessing and treating such patients.Methods:From January 1998 to December 2020, 76 patients with hydrocephalus due to methylmalonic acidemia combined with homocysteinuria in the Department of Pediatrics in 11 hospitals including Peking University First Hospital were diagnosed by biochemical, genetic analysis and brain imaging examination. The patients were divided into operation-group and non-operation-group according to whether they underwent ventriculoperitoneal shunt. The clinical features, laboratory examinations, genotype, and follow-up data were retrospectively analyzed. Data were compared between the two groups using rank sum test, and categorical data were compared using χ 2 test. Results:Among the 76 patients (51 male, 25 female), 5 were detected by newborn screening, while 71 were diagnosed after clinical onset, 68 cases (96%) had early-onset, 3 cases (4%) had late-onset. The most common clinical manifestations of 74 cases with complete data were psychomotor retardation in 74 cases (100%), visual impairment in 74 cases (100%), epilepsy in 44 cases (59%), anemia in 31 cases (42%), hypotonia or hypertonia in 21 cases (28%), feeding difficulties in 19 cases (26%) and disturbance of consciousness in 17 cases (23%). Genetic analysis was performed in 76 cases, all of whom had MMACHC gene variations, including 30 homozygous variations of MMACHC c.609G>A. The most common variations were c.609G>A (94, 62.7%), followed by c.658_660del (18, 12.0%), c.567dupT (9, 6.0%) and c.217C>T (8, 5.3%). Therapy including cobalamin intramuscular injection, L-carnitine and betaine were initiated immediately after diagnosis. A ventriculoperitoneal shunt operation was performed in 41 cases (operation group), and 31 patients improved after metabolic intervention (non-operation group). There was no significant difference in the age of onset, the age of diagnosis, the blood total homocysteine, methionine, and urinary methylmalonic acid concentration between the two groups (all P>0.05). The symptoms of psychomotor development, epilepsy, and visual impairments improved gradually after a long-term follow-up in the operation group. Conclusions:Hydrocephalus is a severe complication of methylmalonic acidemia combined with homocysteinuria. The most common clinical manifestations are psychomotor retardation, visual impairment, and epilepsy. It usually occurs in early-onset patients. Early diagnosis and etiological treatment are very important. Hydrocephalus may improve after metabolic intervention in some patients. For patients with severe ventricular dilatation, prompt surgical intervention can improve the prognosis.

Objective To investigate the role of bufalin(BU)in inhibiting M2-type macrophage-mediated colorec-tal cancer metastasis.Methods Human acute leukemia mononuclear cells(THP-1)were differentiated into M0 macrophages using phorbol ester induction(PMA)for 48 hours.The M0 macrophages were then treated with IL-4 and IL-13 medium.Surface markers and morphological changes were observed through ELISA,morphology,and RT-qPCR experiments.RT-PCR and ELISA experiments were conducted to detect the surface markers TGF-β and IL-10 of M2 macrophages.The secretion level of IL-6 in the supernatant of M2 macrophages and colorectal cancer cells HCT116 was compared using ELISA.Additionally,the effect of conditioned medium on colorectal cancer cell HCT116 was assessed through Transwell,Wound healing,RT-qPCR,and Western blot experiments.Subsequent-ly,bufalin was added to the conditioned medium and the changes in AKT/PI3K protein,migration,and epithelial-mesenchymal transition ability in HCT116 were observed using Western blot,Transwell,Wound healing and RT-qPCR experiments.Results THP-1 were successfully differentiated into M2 macrophages.The activation of AKT/PI3K protein in HCT116 cells was induced by the secretion of IL-6 from M2 macrophages,which in turn promoted the migration and epithelial-mesenchymal transition ability of the HCT116 cells.The migration and epithelial-mes-enchymal transition mediated by M2 macrophages in HCT116 cells were effectively inhibited by Bufalin.Conclu-sion The release of IL-6 from M2 macrophages activates the AKT/PI3K signaling pathway in colorectal cancer cells,thereby promoting their migration and epithelial-mesenchymal transition capacity.Moreover,bufalin exhibits inhibitory effects on this effect.

Objectives:To summarize the clinical and genetic characteristics of the patients with isolated methylmalonic acidemia and investigate the strategies for the diagnosis, treatment and prevention.Methods:Three hundred and fourteen patients (180 males, 134 females) with isolated methylmalonic acidemia were ascertained from 26 provinces or cities across the mainland of China during January 1998 to March 2020. Genetic analysis was performed by Sanger sequencing, gene panel sequencing, whole exome sequencing, multiplex ligation-dependent probe amplification or quantitative PCR. According to the age of onset, the patients were divided to early-onset group (≤12 months of age) and the late-onset group (>12 months of age). They were treated by cobalamin, L-carnitine and (or) special diet and symptomatic treatment. Statistical analysis was done using Chi-square test.Results:Fifty-eight of 314 (18.5%) patients were detected by Newborn screening using liquid chromatography tandem mass spectrometry. Five cases (1.6%) had a postmortem diagnosis. Two hundred and fifty-one patients (79.9%) were clinically diagnosed with an age of onset ranged from 3 hours after birth to 18 years. One hundred and fifty-nine patients (71.0%) belonged to early-onset groups, 65 patients (29.0%) belonged to the late-onset group. The most common symptoms were metabolic crises, psychomotor retardation, epilepsy, anemia and multiple organ damage. Metabolic acidosis and anemia were more common in early-onset patients than that in late-onset patients (20.8%(33/159) vs. 9.2% (6/65), 34.6% (55/159) vs. 16.9% (11/165), χ 2=4.261, 6.930, P=0.039, 0.008). Genetic tests were performed for 236 patients (75.2%), 96.2%(227/236) had molecular confirmation. One hundred and twenty-seven variants were identified in seven genes (MMUT, MMAA, MMAB, MMADHC, SUCLG1, SUCLA2, and MCEE), of which 49 were novel. The mut type, caused by the deficiency of methylmalonyl-CoA mutase, was the most common ( n=211, 93%) cause of this condition. c.729_730insTT, c.1106G>A and c.914T>C were the three most frequent mutations in MMUT gene. The frequency of c.914T>C in early-onset patients was significantly higher than that in late-onset patients (8.3% (18/216) vs. 1.6% (1/64), χ 2=3.859, P=0.037). Metabolic crisis was more frequent in mut type than the other types (72.6% (114/157) vs. 3/13, χ 2=13.729, P=0.001),developmental delay and hypotonia were less frequent in mut type (38.2% (60/157) vs. 9/13, 25.5% (40/157) vs. 8/13, χ 2=4.789, 7.705, P=0.030, 0.006). Of the 58 patients identified by newborn screening, 44 patients (75.9%) who were treated from asymptomatic phase developed normally whereas 14 patients (24.1%) who received treatment after developing symptoms exhibited varying degrees of psychomotor retardation. Conclusions:The characteristics of phenotypes and genotypes among Chinese patients with isolated methylmalonic acidemia were analyzed. Expanded the mutation spectrum of the associated genes. Because of the complex clinical manifestations and severe early onset of isolated methylmalonic acidemia, Newborn screening is crucial for early diagnosis and improvement of prognosis. MMUT gene is recommended for carrier screening as an effort to move the test earlier as a part of the primary prevention of birth defects.