Purpose: Invasive ductal carcinoma (IDC) accounts for 90% of triple-negative breast cancer (TNBC). IDC is mainly derived from the breast ductal epithelium which is innervated by the 4th to 6th thoracic sympathetic nerves. However, little is known about the contribution of the interactions between sympathetic nerves and breast cancer cells to the malignant progression of TNBC. Methods: The expression levels of the β2 -adrenergic receptor (β2 -AR, encoded by ADRB2 gene), nerve growth factor (NGF), and tropomyosin receptor kinase A (TrkA) were determined using immunohistochemistry (IHC). NGF expression levels in the serum were compared by enzyme-linked immunosorbent assay (ELISA). Cell proliferation was assessed using the Cell Counting Kit-8 assay. The β2 -AR, NGF, p-ERK, and p-CERB expression levels were determined using western blotting. TNBC cells and neuronal cells of the dorsal root ganglion (DRG) in 2-day-old Sprague Dawley rats were co-cultured. Using norepinephrine (NE), NGF, and β2 -AR, NGF/TrkA blocker pretreatments, the axon growth of each group of DRG neuron cells was detected by immunofluorescence analysis. Results: The sympathetic adrenergic neurotransmitter NE activated the ERK signaling pathway in TNBC cells. NE/β2 -AR signaling promotes NGF secretion. NGF further facilitates the malignant progression of TNBC by increasing sympathetic neurogenesis. In the coculture assay, the sympathetic adrenergic NE/β2 -AR signal pathway also enhanced NGF secretion. NGF binds TrkA in DRG neurons and promotes axonal growth. Conclusion These results suggest that NE/β2 -AR pathway promotes cell proliferation and NGF production in triple-negative breast cancer.

Objective:To summarize the therapeutic effect of deep brain stimulation (DBS) for dystonia.Methods:Detailed clinical information and peripheral blood of children with dystonia at Peking University First Hospital from April 2017 to July 2020 were collected.The motor scores of Burke-Fahn-Marsden Dystonia Rating Scale were recorded of the dystonia before and after the treatment of DBS.Whole-exome sequencing was performed on children with dystonia.Then the effect of DBS was evaluated.Results:A total of 32 cases of patients with dystonia treated with DBS were enrolled, including 16 males and 16 females.Twelve cases were treated with globus pallidus internus DBS, and 20 cases were treated with subthalamic nucleus DBS.Twenty cases (62.5%) with pathogenic gene mutations were detected.Pathogenic variants in PANK2 (9 cases), KMT2B(3 cases), GNAO1 (2 cases), GCDH (2 cases), PINK1(1 case), NDUFAF6(1 case), DYT27(1 case) and ADCY5(1 case) were found.The follow-up period was 1 month to 3 years and 8 months.Only 1 case had local infection due to improper home care.The postoperative improvement was 5.66%-95.92%. Conclusions:All patients have a certain degree of relief after DBS without obvious adverse reactions.DBS is an effective treatment for pediatric dystonia.

Objective To evaluate the role of miR-146a in bone marrow mesenchymal stem cells ( BMSCs)-induced reduction of acute lung injury ( ALI ) in rats. Methods A total of 105 clean-grade healthy male Wistar rats, weighing 170-190 g, were divided into 5 groups ( n=21 each) using a random number table method: control group (group C), phosphate buffer solution group (group P), group ALI, BMSC group ( group B) and BMSC plus miR-146a inhibitor group ( group BM) . ALI was induced by intra-peritoneally injecting 5 mg∕kg lipopolysaccharide ( LPS) 0. 5 ml in anesthetized rats. Phosphate buffer solu-tion 0. 5 ml was injected via the tail vein in group P. In group B, 1×104 cells∕ml BMSC 0. 5 ml was injected via the tail vein after establishing the model. In group BM, miR-146a inhibitor 50 mg∕kg was injected via the tail vein after establishing the model, and 2 h later 1×104 cells∕ml BMSC 0. 5 ml was injected via the tail vein. Group C received no treatment. Blood samples were obtained from the abdominal aorta for blood gas a-nalysis at 6, 24 and 48 h after injection of BMSC ( T1-3 ) , the chest was immediately opened, and the lung tissues were obtained for microscopic examination of pathologic changes and for determination of the wet∕dry lung weight ratio ( W∕D ratio) , expression of IRAK-1, nuclear factor kappa B ( NF-κB) and interleukin-6 ( IL-6) ( by Western blot) and expression of miR-146a and IRAK-1 mRNA ( by quantitative real-time poly-merase chain reaction). Results Compared with group C, the pH value and PO2 were significantly de-creased, PCO2 and W∕D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point ( P<0. 05) , and the pathological changes of lung tissues were aggrava-ted in ALI, B and BM groups. Compared with group P, the pH value and PO2 were significantly decreased, PCO2 and W∕D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point (P<0. 05), and the pathological changes of lung tissues were aggravated in ALI, B and BM groups. Compared with group ALI, the pH value and PO2 were significantly increased, PCO2 and W∕D ratio were decreased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was down-regulated at each time point ( P<0. 05 ) , and the pathological changes of lung tissues were attenuated in group B. Compared with group B, the pH value and PO2 were significantly decreased, PCO2 and W∕D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point ( P<0. 05) , and the pathological changes of lung tissues were aggravated in group BM. Conclusion miR-146a is involved in BMSCs-induced reduction of ALI in rats.

Objective To evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on mammalian target of rapamycin (mTOR) signaling pathways in lung tissues of rats with acute lung injury (ALI).Methods Healthy pathogen-free adult male Sprague-Dawley rats were selected,and the BMSCs were obtained and cultured in vitro.One hundred and five healthy clean adult male SpragueDawley rats,weighing 170-190 g,were divided into 5 groups (n=21 each) using a random number table:control group (group C),PBS group,group ALI,ALI plus BMSC group (group ALI+BMSCs),and ALI plus phosphate buffer solution (PBS) group (group ALI+PBS).Group C received no treatment.PBS 0.5 ml was injected via the tail vein in group PBS.Lipopolysaccharide (LPS,0.5 ml) 5 mg/kg was intraperitoneally injected to establish the model of ALI in group ALI.BMSCs (0.5 ml) 1×104 cells/ml were injected via the tail vein after intraperitoneal injection of LPS in group ALI+ BMSCs.PBS 0.5 ml was injected via the tail vein after intraperitoneal injection of LPS in group ALI+PBS.Arterial blood samples were collected for blood gas analysis at 6,24 and 48 h after injection of BMSCs.Lungs were then removed for determination of wet/dry weight ratio (W/D ratio) and expression of mTOR,nuclear factor kappa B (NF-κB) and tumor necrosis factor-alpha (TNF-o) in lung tissues (by Western blot) and for examination of the pathologic changes of lungs tissues (using haematoxylin and eosin staining).Results Compared with group C,pH value and PaO2 were significantly decreased,PaCO2 and W/D ratio were increased,and the expression of mTOR,NF-κB and TNF-α was up-regulated at each time point in ALI,ALI+BMSCs and ALI+PBS groups (P＜0.05).Compared with group ALI,pH value and PaO2 were significantly increased,PaCO2 and W/D ratio were decreased,the expression of mTOR,NF-κB and TNF-α was down-regulated at each time point (P＜0.05),and the pathologic changes of lungs tissues were significantly attenuated in group ALI+BMSCs.Conclusion The mechanism by which BMSCs reduce ALI may be associated with inhibiting mTOR signaling pathways in lung tissues of rats.

Objective: To study the mutational characteristics of KCNT1 and its clinical features in children with early-onset epileptic encephalopathy. Methods: Retrospective analysis of clinical data of 175 children with early onset epilepsy from the Department of Pediatrics at Peking University First Hospital from January 2012 to December 2017. Gene-based analysis was performed on children with targeted capture second-generation sequencing and the source of mutations was verified by PCR-Sanger. The clinical features of children with KCNT1 mutation were summarized. Results: In 175 infants with early-onset epileptic encephalopathy, 6 children were found to have KCNT1 mutations, all of which were new mutations with an overall mutation rate of 3.4% (6/175). All the mutations were missense mutations. The age of onset was from 2 days to 32 days. Five children were diagnosed with epilepsy of infancy with migrating focal seizure, one case was diagnosed with epilepsy, focal seizures, focal seizures with generalization. A total of 6 children were treated with multi-antiepileptic drugs. The disease in 4 patients were partially controlled, while in 2 patients, the disease was not significantly alleviated. One patient died of "severe pneumonia" at one year and 4 months of age. Then, four cases were treated with quinidine. The seizure frequency had no change in 3 cases, the frequency decreased and then relapsed in 1 case. The case once ketogenic diet and failed. Ketogenic diet treatment was applied to 5 cases, no significant effect was achieved. All the 6 patients had severe developmental delay. They could not sit alone, follow the light and objects and had no language. Conclusions The mutation of KCNT1 gene is mainly de novo. The onset of the disease was early, and mostly occurs in neonate and early infancy. The main seizure type was epilepsy of infancy with migrating focal seizure. Patients usually had severe psychomotor developmental delay. Antiepileptic drugs are ineffective. The efficacy of quinidine was not significant. Though, it still need studies on a large sample.

Objective To investigate the clinical effect of mild hypothermia on neonatal bilirubin encephalopathy, and the value of 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/CT and amplitude integrated electroencephalogram (aEEG) for diagnosis and evaluation of curative effect. Methods From May, 2013 to December, 2014, 29 newborns with bilirubin encephalopathy were divided into conventional group (n=15) and mild hypothermia group (n=14). The conventional group received conventional therapy, and the other group received mild hypothermia in addition. The aEEG and neuron-specific enolase (NSE) were measured before and after treatment, as well as the glucose metabolism rate with 18F-FDG PET/CT after treatment. Results The NSE was lower after treatment in both groups (t>9.670, P<0.001), and was lower in the mild hypothermia group than in the conventional group (F=46.146, P<0.001). After treatment, sleep-wake cycle (SWC), epileptiform activity and the degree of abnormality were obviously improved (P<0.05), and were better in the mild hypothermia group than in the conventional group (P<0.05). The cerebral glucose metabolism rate was significantly better in the mild hypo-thermia group than in the conventional group (t>2.943, P<0.01). The cerebral glucose metabolism rate was negatively correlated with aEEG and NSE (r>0.640, P<0.05). Conclusion Mild hypothermia therapy could further promote the energy metabolism of brain cells in neonatal bilirubin encephalopathy. 18F-FDG PET/CT and aEEG can be used for early diagnosis and therapeutic evaluation.

Aim To observate the effect of chemokine receptor(CXCR4) gene transfection on biological behavior of bone marrow mesenchymal stem cells in vitro.Methods Firstly, bone marrow mesenchymal stem cells were divided into three groups:GFP(transfected GFP into MSCs), CXCR4+(transfected CXCR4+ into MSCs) and CXCR4-(transfected CXCR4-into MSCs) group.Then, their capacity of proliferation, differentiation and migration ability (in vitro) was assessed with immunofluorescence cytochemistry method, flow cytometry assay and Transwell cell chemotaxis test.Results The high or low expression of CXCR4 had no effect on their ability of proliferation and differentiation into lung tissue.Compared with GFP group, however, CXCR4+-MSCs group significantly increased the number of migrating cells, while CXCR4——MSCs group showed no change in the number of migrating cells.Conclusions The proliferation and differentiation capacities are not affected by the high or low expression of CXCR4.The high expression of CXCR4 can significantly enhance the migration ability of MSCs to inflammatory lesions, and the low one has no effect on the migration of the cells.After the transplantation of MSCs, CXCR4′s high expression will access to the lesion area to participate in tissue repairing rapidly and largely, significantly enhancing the therapeutic efficacy.

Objective:To analyze the expression of the four mismatch repair genes protein (hMLH1,hMSH2,hMSH6 and hPMS2) of patients with colorectal cancer and its clinical significance.Methods:177 cases of patients with colorectal caner in the Third Affiliated Hospital of Guangzhou Medical University from January 2013 to December 2015 were randomly selected.Tested the expression of the hMLH1,hMSH2,hMSH6 and hPMS2 by immunohistochemistry,the relationship between protein expression and clinical parameters was analyzed.Results:Among 177 cases ofcolorectal cancer tissue,the deletion rate ofhMLH1 protein was 6.2％ (11/177),the deletion rate of hMLH2 protein was 4.0％(7/177),the deletion rate of hMSH6 protein was 1.7％(3/177),the deletion rate of hPMS2 protein was 8.0％(14/177),the sum of the four values accounted for 19.8％(35/177) of all cases of colorectal cancer.The loss of expression of the four mismatch repair genes protein were correlated to tumor location (P＜0.05),besides,the loss of expression of the hMLH1 and hPMS2 protein were correlated to degree of tumor differentiation (P＜0.05),he loss of expression of the hMSH6 protein were correlated to depth of tumor invasion(P＜0.05);But the loss was not correlated to age,sexes,lymph node metastasis and distant metastasis(P＞0.05).Conclusion:The expression of loss phenomenon with mismatch repair protein appears in part of colorectal cancer,the loss phenomenon with mis match repair protein were correlated to tumor location and degree of tumor differentiation.Mutations of four genes in hMLH1,hPMS2,hMSH6 and bMSH2,to provide a reference value for the clinical judgment of prognosis and to develop a treatment plan.

Objective To construct plvx-cyclooxygenase-2(COX-2)-DsRed and establish breast cancer cell line MCF7 which overexpressed COX-2, to explore the effect of COX-2 on breast cancer cell.Methods The full-length COX-2 PCR product was obtained by total COX-2 PCR primers and COX-2 cDNA vector.After the PCR product and lentiviral vector plvx-DsRed-Monomer-N1 were cut simultaneously by restriction enzyme BamH1 and Xholl, they were connected and sequenced, to get lentiviral vector plvx-COX-2-DsRed.After selected by puromycin, overexpressed COX-2 breast cancer cell line MCF7-plvx-COX-2-DsRed was obtained.The stable cell line was verified by real time PCR and Western blot.The differences of proliferation ability between stable cell line and normal one were compared by colony formation test and Western blot.Results The lentiviral vector plvx-COX-2-DsRed and stable cell line MCF7-plvx-COX-2-DsRed after selecting were obtained.COX-2 expression level of the stable cell line was 75.29 times as high as that of MCF7, and 64.91 times as high as that of cell line MCF7-plvx-DsRed-Monomer-N1 by PCR assay (P ＜ 0.05), which was consistent with the results of Western blot and microscope photo.MTT results showed that cell line MCF7-plvx-COX-2-DsRed had grown faster than cell line MCF7 and MCF7-plvx-DsRed-Monomer-N1 from the 2nd day (P ＜ 0.05), which was accordant with colony formation assay.MCF7-plvx-COX-2-DsRed cell line had higher c-myc expression and lower β-catenin expression than MCF7 cell and cell line MCF7-plvx-DsRed-Monomer-N1 detected by Western blot relative quantification (P ＜ 0.05).Conclusion The plvx-COX-2-DsRed lentiviral vector and cell line MCF7-plvx-COX-2-DsRed are successfully constructed.COX-2 can increase proliferation of MCF7 cells through up-regulating the expression of c-myc.

Objective To evaluate the role of inositol triphosphate receptor (IP3 R) in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.Methods BV-2 microglial cells were seeded in 3.5 cm diameter dishes (5 ml/dish),50 ml culture flasks (8 ml/flask) or 24-well plates (1 ml/hole) with a density of 1 × 105/ml and randomly divided into 5 groups (n =25 each) ∶ control group (group C),fractalkinegroup (group F),CX3C chemokine receptor 1 (CX3CR1) antibody anti-CX3CR1 + fractalkine group (group CF),IP3R antagonist 2-APB + fractalkine group (group AF) and p38 mitogen-activated protease (p38MAPK) inhibitor SB203580 + fractalkine group (group SF).Fractalkine 10 nmol/L was added to the culture medium in groups F,CF,AF and SF.The anti-CX3CR1 15 μmol/L,2-APB 50 μmol/L and SB203580 10 μmol/L were added to the culture medium in groups CF,AF and SF,respectively,1 h before addition of fractalkine.The cells were then cultured for 24 h.The intracellular Ca2+ concentration ([Ca2+]i) was measured during the 10 min incubation with fractalkine.The phosphorylation of p38MAPK was measured at 0,30,60,120 and 240 min of incubation with fractalkine.The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in theculture medium were determined at 24 h of incubation with fractalkine.Results Compared with group C,[Ca2+]i,and the phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly increased in groups F,CF,AF and SF (P ＜ 0.05).[Ca2+]i was significant lower in groups AF and CF and phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly lower in groups CF,AF and SF than in group F (P ＜ 0.05).Conclusion IP3 R is involve in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.