BACKGROUND:Primary cortical neurons and microglial cells play a crucial role in exploring cell therapies for neurological disorders,and most of the current methods for obtaining the two types of cells are cumbersome and require separate extraction.It is therefore crucial to find a convenient and rapid method to extract both types of cells simultaneously. OBJECTIVE:To explore a novel method for simultaneous extraction of primary cortical neurons and microglial cells. METHODS:Newborn suckling SD rats were taken within 24 hours.The brain was removed and placed in a dish with DMEM,and the pia mater was removed for later use.Primary neurons were extracted from the same brain tissue,and then the remaining brain tissue was used to extract microglial cells.The whole process was performed on ice.Extraction and culture steps of primary cortical neurons:The cerebral cortex was taken 2.0-3.0 mm with forceps,and the tissue was digested with papain for 20 minutes.After aborting digestion,the blown tissue presented an adherent tissue suspension.The supernatant cell suspension was obtained,filtered,and dispensed into 15 mL centrifuge tubes.After centrifugation and re-suspension,the cells were inoculated onto 6-well plate crawls coated with L-polylysine.Neuronal morphology was observed at 1-day intervals,and staining could be performed for identification using immunofluorescence staining of MAP2 and β-Tubulin by day 7.Microglia extraction and culture steps:The remaining brain tissue at 8-10 mm thick was subjected to microglial cell extraction,digested by trypsin for 20 minutes.After digestion was stopped,the tissue was blown to a homogenate,and then the homogenate was transferred to the culture bottle for culture.On day 14,the culture flasks were sealed and subjected to constant temperature horizontal shaking for 2 hours.Microglial cells were shed in the supernatant.Purified microglial cells were taken and continued to be cultured for 3 days for identification by Iba1 immunofluorescence staining. RESULTS AND CONCLUSION:(1)After 24 hours of culture,the neurons were adherent to the wall,the cytosol was enlarged,and some neurons developed synapses.After 3 and 5 days of culture,the cytosol was further enlarged,and most of the neurons were in the form of synapses,and some neurons were growing in clusters.On day 7,neuronal synapses were prolonged and thickened,and they were connected with each other to form a network.The neurons were identified by β-Tubulin and MAP2 immunofluorescence staining.(2)The cells grew close to the wall on day 1 of culture.On days 3,5,and 7,the density of microglial cells was small,and the cell morphology was bright oval or round,but the cells basically grew in clumps on the upper layer of other cells.On day 10,the density of microglial cells increased significantly.On day 14,microglial cells grew in dense clumps on the upper layer of other cells,and then they could be isolated and purified.The isolated and purified cells were taken and re-cultured to day 3 and identified as microglial cells by Iba1 immunofluorescence;their purity was greater than 95%.(3)The results show that primary cortical neurons and microglial cells obtained by this method after extraction and culture are of high purity,good morphology,and high viability.

BACKGROUND:Studies have found that activation of nuclear factor-erythroid 2-related factor 2/heme oxidase-1(Nrf2/HO-1)pathway can alleviate oxidative stress caused by cerebral ischemia-reperfusion injury,but whether human bone marrow mesenchymal stem cells(hBMSC)can activate Nrf2/HO-1 pathway to alleviate cerebral ischemia-reperfusion injury is still lacking relevant studies. OBJECTIVE:To investigate whether intracranial transplantation of hBMSC alleviates oxidative stress injury in cerebral ischemia-reperfusion animal models by activating Nrf2/HO-1 pathway. METHODS:Totally 40 male SPF SD rats were randomly divided into sham operation group,model group,hBMSC transplantation group,hBMSC+solvent group and hBMSC+Nrf2 inhibitor group.Each group consisted of eight animals.In the model group and the hBMSC transplantation group,middle cerebral artery occlusion model was prepared by thread embolization method.The thread embolization was removed 1 hour later,and 30 μL PBS or hBMSC cultured to at least passage 5 was injected into the right cortex and striatum of rats.In the hBMSC+Nrf2 inhibitor group and hBMSC+solvent group,the left ventricle was injected with Nrf2 inhibitor Brusatol and its solvent dimethyl sulfoxide respectively 24 hours before model establishment,then the middle cerebral artery occlusion model was prepared,and hBMSC was injected.Relevant indexes were detected 3 days after transplantation. RESULTS AND CONCLUSION:(1)CT and TTC staining showed the same area and volume of cerebral infarction:model group>hBMSC+Nrf2 inhibitor group>hBMSC+solvent group>hBMSC transplantation group>sham operation group.(2)Hematoxylin-eosin staining and Nissl's staining showed that the ischemic brain tissue was intact and the neurons were normal in the sham operation group.Compared with the model group,the pathological morphology and neuronal injury of the hBMSC transplantation group and the hBMSC+solvent group were significantly improved.Compared with the hBMSC+solvent group,the hBMSC+Nrf2 inhibitor group had more serious pathological morphology and neuronal damage.(3)Western blot assay and oxidative stress index detection results showed that compared with the sham operation group,Nrf2 and HO-1 proteins were decreased(all P<0.05),malondialdehyde was increased and superoxide dismutase was decreased(all P<0.05)in the model group.Compared with the model group,the expression levels of Nrf2 and HO-1 proteins were increased(all P<0.05),malondialdehyde was decreased and superoxide dismutase was increased(all P<0.05)in the hBMSC transplantation group and the hBMSC+solvent group.Compared with the hBMSC+solvent group,the expression levels of Nrf2 and HO-1 proteins were simultaneously decreased(all P<0.05),and malondialdehyde was increased and superoxide dismutase was decreased(all P<0.05)in the hBMSC+Nrf2 inhibitor group.(4)These results indicate that hBMSC can alleviate cerebral ischemia-reperfusion injury possibly by activating Nrf2/HO-1 pathway.

Objective:To evaluate clinical efficacy of adjuvant radiotherapy (RT) for central neurocytoma (CN) after surgical resection.Methods:Clinical data of 136 CN patients admitted to Beijing Tiantan Hospital and Xuanwu Hospital from January 2001 to December 2020 were retrospectively analyzed. Preliminary interventions consisted of craniotomy (gross total resection, subtotal resection and partial resection, the latter two belonging to incomplete resection) and postoperative radiotherapy. Three-dimensional conformal or intensity-modulated radiotherapy was adopted, with a median radiotherapy dose of 54 Gy. Post-recurrence treatment included salvage surgery and radiotherapy. The overall survival (OS) and progression-free survival (PFS) were analyzed using the Kaplan-Meier method. Univariate analysis was performed by log-rank test to evaluate the effect of each prognostic factor on OS and PFS. The effects of multiple prognostic factors on PFS and OS were assessed by Cox regression model.Results:The median age was 28 years (range: 6-66 years). The median follow-up was 94.5 months (12-237 months). Among all patients, 79 cases underwent total resection, and 68 of them received adjuvant radiotherapy. Thirty-eight patients underwent subtotal resection, and 37 of them were treated with adjuvant radiotherapy. Sixteen patients received partial resection and adjuvant radiotherapy. Three cases received biopsy and postoperative radiotherapy. Among all patients, 3 cases died, including 2 from tumor recurrence and 1 from postoperative complication. Eight patients had recurrences during follow-up. Among them, 7 patients had recurrences at the primary site,1 had tumor dissemination to the spinal cord. The 5- and 10-year OS rates were 98.5% and 96.8%, and the 5- and 10-year PFS rates were 95.3% and 91.6% for the in the entire cohort. In the gross total resection without radiotherapy group, the 5- and 10-year PFS rates were 90.9% and 90.9%, and 96.6% and 96.6% in the gross total resection + radiotherapy group ( P=0.338). The 5- and 10-year OS rates were 100% and 100% in the gross total resection without radiotherapy group, and 98.5% and 98.5% in the gross total resection + radiotherapy group ( P=0.693). The 10-year PFS rates between the gross total resection±radiotherapy group and the incomplete resection+radiotherapy group was 95.8% vs. 90.3% ( P=0.368), and the 10-year OS rate was 98.6% vs. 94.7% ( P=0.436). Multivariate analysis showed that tumor site, degree of surgical resection, adjuvant radiotherapy and age exerted no significant effects on PFS and OS. A total of 81 patients had late neurotoxicities, including 69 cases at grade 1, 9 cases at grade 2, and 3 cases at grade 3. And 64.2% (52/81 cases) of patients suffered from short-term memory impairment. Conclusions:Gross total resection alone yields high efficacy for CN. Postoperative radiotherapy is not required. Incomplete resection combined with postoperative adjuvant radiotherapy can achieve equivalent clinical efficacy to gross total resection.

Phosphatase and tensin-homolog deleted on chromosome 10 (PTEN) is an important cancer suppressor gene. Its pathogenic mutation leads to PTEN hamartoma tumor syndrome (PHTS), a rare syndrome also known as Cowden syndrome, which is relevant to early-onset hereditary breast cancer (BC). In this paper, we report three patients with unilateral multicentric BC and synchronous and metachronous bilateral BC who harbored PTEN gene mutations, and summarize the clinical manifestations, pathological characteristics, diagnosis, treatment and follow-up outcomes to provide reference for management of PTEN gene mutation-related BC among the Cowden syndrome population.

Objective:To investigate the efficacy and safety of fractionated stereotactic radiotherapy (FSRT) based on linear accelerator for small volume brain metastases.Methods:A total of 21 patients with small volume brain metastases who received FSRT from August 2020 to June 2022 were enrolled as subjects, including 45 lesions. Small-volume brain metastases were defined as ≤3 cm in diameter and ≤6 cm 3 in volume, and the dose/fractionation scheme was 27-30 Gy/3 F or 30-40 Gy/5 F. Three months after radiotherpy, the efficacy of FSRT in small brain metastases and the incidence of radiation brain injury were evaluated, and the incidence of radiation brain injury in subgroup analysis was performed according to the diameter, volume, dose/fractionation scheme, biological effective dose (BED) 10, and location of lesions. Results:Twenty-four lesions (53.33%, 24/45) were evaluated as complete response, another 13 lesions (28.89%, 13/45) were evaluated as partial response, and in the remaining 8 lesions (17.78%, 8/45) were evaluated as stable disease. The local control rate was 100% (45/45), the objective remission rate was 82.22% (37/45), and the intracranial distant progression rate was 23.81% (5/21). During the treatment and follow-up, there were 7 lesions (15.56%, 7/45) of radiation-induced brain injury, and the incidence of symptomatic radiation-induced brain injury was 11.11% (5/45). Subgroup analysis showed that the incidence of radiation brain injury in the group with a lesion diameter of 2-3 cm was higher than that with a lesion diameter of <2 cm group, with a statistically significant difference [80.00% (4/5) vs. 7.50% (3/40), χ2=12.69, P<0.001]; the incidence rate of radiation brain injury in the group with lesion volume of 4-6 cm 3 was higher than that with lesion volume of <4 cm 3 group, with a statistically significant difference [57.14% (4/7) vs. 7.89% (3/38), χ2=7.49, P=0.006]. There was no significant difference in the incidence of radiation brain injury between the dose/fractionation scheme of lesions 27-30 Gy/3 F and 30-40 Gy/5 F [9.52% (2/21) vs. 20.83% (5/24), χ2=0.40, P=0.527]. There was no significant difference in the incidence of radiation brain injury between the BED 10<60 Gy and ≥60 Gy [28.57% (2/7) vs. 13.16% (5/38), χ2=0.22, P=0.641]. There was no significant difference in the incidence of radiation brain injury between the lesions in the same lobe and the single or multiple lesions in different lobes [28.57% (4/14) vs. 9.68% (3/31), χ2=1.38, P=0.240) . Conclusion:FSRT based on linear accelerator is effective for small volume brain metastases. Brain metastases with the diameter <2 cm or volume <4 cm 3 are associated with a lower incidence of radiation brain injury than that of lesions with the diameter of 2-3 cm or volume of 4-6 cm 3.

In growing children, growth plate cartilage has limited self-repair ability upon fracture injury always leading to limb growth arrest. Interestingly, one type of fracture injuries within the growth plate achieve amazing self-healing, however, the mechanism is unclear. Using this type of fracture mouse model, we discovered the activation of Hedgehog (Hh) signaling in the injured growth plate, which could activate chondrocytes in growth plate and promote cartilage repair. Primary cilia are the central transduction mediator of Hh signaling. Notably, ciliary Hh-Smo-Gli signaling pathways were enriched in the growth plate during development. Moreover, chondrocytes in resting and proliferating zone were dynamically ciliated during growth plate repair. Furthermore, conditional deletion of the ciliary core gene Ift140 in cartilage disrupted cilia-mediated Hh signaling in growth plate. More importantly, activating ciliary Hh signaling by Smoothened agonist (SAG) significantly accelerated growth plate repair after injury. In sum, primary cilia mediate Hh signaling induced the activation of stem/progenitor chondrocytes and growth plate repair after fracture injury.
Mice ; Animals ; Hedgehog Proteins/genetics* ; Receptors, G-Protein-Coupled/metabolism* ; Cilia/metabolism* ; Cartilage/metabolism* ; Regeneration

Objective To explore the curative effect of day care unit on the efficacy,safety and satisfaction of elderly patients who underwent a cold snare polypectomy for the treatment colorectal polyps.Methods Clinical data from 454 elderly patients with 824 colorectal polyps(Diameter 4～10 mm)who received a polypectomy from Mar 2020 to Mar 2021 were collected.These patients were classified into three groups.The cold snare polypectomy group and hot snare polypectomy group in day care unit,and the cold snare polypectomy group in general wards.The clinical characteristics,adverse events,recurrence,hospitalization time,and expenses,were compared among three groups.Additionally,the patients'hospitalization satisfaction was investigated and analyzed.Results There were no significant differences in clinical characteristics,histopathology,and rates of postoperative bleeding,perforation,and recurrence among the 3 groups(P＞0.05),but the probability of immediate bleeding was higher in the cold snare polypectomy group.Moreover,coagulation syndrome was unique to the hot snare polypectomy group.The hot snare polypectomy group used the highest amount of endoclips,while the cold snare polypectomy group in the general wards used the least.Furthermore,the hospitalization time and expenses in the day care unit group were significantly lower than in the general wards group.However,the patients'satisfaction survey showed that the day care unit group scored lower than the general wards group(P＜0.05).Conclusions It is safe,cost-effective and effective for elderly patients with colorectal polyps using cold snare polypectomy technique under the day care unit mode,but the lack of communication with the patient's condition in a short period of time rather leads to a decrease in hospital satisfaction.

Patients with Crohn's disease (CD) are often complicated with malnutrition and enteral nutrition is the preferred option for nutritional support. Enteral nutrition is the first-line therapy to induce remission of CD in children, but its application in adults has not been well established. In recent years, studies have found that enteral nutrition can not only improve the nutritional status in adult CD patients, but also induce and maintain CD remission through various mechanisms. Given its favorable nutritional efficacy and safety, enteral nutrition has been considered as the basic treatment for adult CD patients and often used in combination with other treatment approaches. However, any combination therapy should be assessed in terms of benefit and risk. This review aims to describe the efficacy and safety of enteral nutrition combined with other treatment approaches in adult CD patients and enumerate common applicable clinical settings, so as to better inform clinical treatment.

Objective:To explore the performance of the commonly used whole blood C-reactive protein (CRP) detection systems and give related recommendation on the performance requirements of detection systems.Methods:A total of 7 540 venous blood samples from 26 maternal, child and children′s hospitals were collected to conduct this multi-center study on the analytical performance of 5 commonly used whole blood CRP detection systems from March to April in 2019. The blank check, carryover, repeatability, intermediate precision, linearity, sample stability, influence of hematocrit/triglyceride/bilirubin, comparison with SIEMENS specific protein analyzer and trueness were evaluated. The 5 systems included BC-5390CRP autohematology analyzer, AstepPLUS specific protein analyzer, Ottoman-1000 Automated Specific Protein POCT Workstation, i-CHROMA Immunofluorometer equipment Reader and Orion QuikRead go detecting instrument. The 5 systems were labeled as a, b, c, d and e randomly.Results:Within the 5 systems, all values of blank check were less than 1.00 mg/L, the carryovers were lower than 1.00%. The repeatability of different ranges of CRP concentrations including 3.00-10.00, 10.00-30.00 and>30.00 mg/L were less than 10.00%, 6.00% and 5.00%, respectively, and the intermediate precision was less than 10.00%. The linearity correlation coefficients of the 5 systems were all above 0.975, while the slope was within 0.950-1.050. Whole blood samples were stable within 72 hours both at room temperature (18-25 ℃) and refrigerated temperature (2-8 ℃). The CRP results were rarely influenced by high triglyceride or bilirubin, except for the immmunoturbidimetric test based on microparticles coated with anti-human CRP F(ab) 2 fragments. When triglyceride was less than 15.46 mmol/L, the deviation of CRP was less than 10.00%. When bilirubin was less than 345.47 μmol/L, the deviation of CRP was less than 10.00%. CRP was more susceptible to Hct on the systems without Hct correction. The deviation of CRP between different Hct dilution concentration and 40% dilution concentration can reach as high as 67.48%. The correlation coefficients ( r) of 5 systems were all more than 0.975 in the range of 0-300.00 mg/L compared with Siemens specific protein analyzer. All systems passed the trueness verification using the samples with specified values of 12.89 and 30.60 mg/L. Conclusion:The performance of 5 systems can basically meet the clinical needs, but it is suggested that the whole blood CRP detection system without automatic Hct correction should be modified manually.

OBJECTIVE: To explore the genetic etiology for a fetus with congenital orofacial cleft. METHODS: Single nucleotide polymorphism microarray (SNP array) was carried out on skin tissues sampled from the fetus following induced abortion for the detection of copy number variation (CNVs). Pathogenicity of the candidate gene was validated through experiment. RESULTS: SNP array revealed that the fetus has carried a hemizygous 9.23Mb deletion at Xq21.31-q22.1(91 063 807-100 293 555), which was inherited from its mother. The region contained 13 OMIM genes and 1 ncRNA coding gene(MIR548M). Inhibiting of the expression of the MIR548M gene in oral epithelial celllines has resulted in up-regulation of the expression of SUMO1 gene which was known to involve in the pathogenesis of orofacial cleft. CONCLUSION Dosage insufficiency of the MIR548M gene may underlie the etiology of orofacial cleft in this fetus.
Cleft Lip/genetics* ; Cleft Palate/genetics* ; DNA Copy Number Variations/genetics* ; Female ; Fetus ; Humans ; MicroRNAs/genetics* ; Polymorphism, Single Nucleotide ; Pregnancy ; SUMO-1 Protein