Objective:To discuss the effect of cell division cycle protein 20(CDC20)on the proliferation and cell cycle of endometrial carcinoma(EC)cells,and to clarify its mechanism.Methods:Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of CDC20 mRNA and protein in human endometrial stromal T-HESC cell and EC cells(KLE,RL95-2,ZJB-ENC1,and ECC-1 cells).The RL95-2 cells were selected for the subsequent experiments.CDC20 shRNA interference lentivirus was transfected into the RL95-2 cells and the cells were divided into control group,sh-NC group(infected with negative control lentivirus),sh-CDC20 group(infected with CDC20 shRNA interference lentivirus),sh-NC+SM04690 group(infected with negative control lentivirus followed by treatment with 64 nmol·L-1 Wnt/β-catenin signaling pathway inhibitor SM04690 for 48 h),and sh-CDC20+SM04690 group(infected with CDC20 shRNA interference lentivirus followed by treatment with 64 nmol·L-1 SM04690 for 48 h).RT-qPCR and Western blotting methods were used to detect the expression levels of CDC20 mRNA and proteins in the cells in various groups;CCK-8 method was used to detect the proliferation activities of the RL95-2 cells in various groups;BrdU assay was used to detect the percentages of BrdU positive cells in various groups;flow cytometry was used to detect the percentages of the cells at G2/M stage in various groups;Western blotting method was used to detect the expression levels of β-catenin,oncogene c-Myc,and cyclin D1 proteins in the cells in various groups.Results:Compared with T-HESC cells,the expression levels of CDC20 mRNA and protein in the KLE,RL95-2,ZJB-ENC1,and ECC-1 cells were significantly increased(P＜0.05),and the highest expression levels of CDC20 mRNA and protein were observed in RL95-2 cells.Compared with sh-NC group,the proliferation activities and percentages of the BrdU positive cells in sh-CDC20 group and sh-NC+SM04690 group were significantly decreased(P＜0.05),the percentages of the cells at G2/M phase were significantly increased(P＜0.05),and the expression levels of β-catenin,c-Myc,and cyclin D1 proteins were significantly decreased(P＜0.05).Compared with sh-CDC20 group,the proliferation activity and percentage of BrdU positive cells in sh-CDC20+SM04690 group were significantly decreased(P＜0.05),the percentage of the cells at G2/M phase was significantly increased(P＜0.05),and the expression levels of β-catenin,c-Myc,and cyclin D1 proteins in the cells were significantly decreased(P＜0.05).Conclusion:CDC20 is highly expressed in the EC cells.Silencing CDC20 may inhibit the cell proliferation by inducing G2/M phase arrest in the RL95-2 cells through the regulation of Wnt/β-catenin signal transduction.

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

Objective:To retrospectively study the curative effect of Vbeam 595nm pulsed dye laser(PDL)in treating the mid-term infantile hemangiomas(IHs),and analyze and explore the possible influencing factors of that.Methods:A total of 219 IHs patients who underwent PDL treatment in Tangshan Maternal and Child Health Care Hospital from January 2020 to December 2022 were selected,and they received follow-up at least 6 months.The relationship between the treatment effect and the month age,location,thickness of hemangioma and the number of treatments was analyzed.Results:The median month age of the 219 pediatric children was 7 months,and the average month age of them was(4-10)months,and there was a total of 219 lesions.There were no statistically significant differences in the general data such as month age and the characteristics of lesion between different genders(P＞0.05).The overall effective rate of treatment was 92.7%,and the median number of treatments was 3 times,and the average number of treatments was(2-4)times.There were no statistically significant differences in the comparisons of gender,the number of treatments and the treatment outcome(P＞0.05).The curative effects of 6 pediatric children were poor,whose month age was 13(8,19)months.The curative effects of 10 pediatric children were general,whose month age was 13(8,14)months.The curative effects of 31 pediatric children were favorable,whose month age was 8(4,10)months.The curative effects of 172 pediatric children were excellent,whose month age was 6(4,9)months.The difference of month age of pediatric children among different curative effect was significant(Z=24.863,P＜0.001),and the pediatric children with better curative effect had smaller month age.The treatment outcome of pediatric children with hemangioma was correlated with the thickness of lesion,the area size of lesion and the number of treatment(r=-0.32,-0.46,-0.46,P＜0.05),respectively.The effective treatment mainly focused on the treatments of the 2nd-4th times.The adverse reaction caused by treatment was skin erythema,and the rate of the total residual after six months of the complication was 5.48%.Conclusion:The treatment of 595nm PDL combined with dynamic cooling device(DCD)can provide a safe and effective treatment for IHs.Early intervention for IHs can improve the curative effect,and reduce the course of treatment,and further reduce the possibility of corresponding complications.

Objective:To evaluate the effect of extracorporeal shock wave on the phosphoproteomics of the spinal cord in rats with diabetic neuralgia.Methods:Thirty-six healthy male SPF-grade Sprague-Dawley rats, aged 2 months, weighing 200-250 g, were divided into 3 groups ( n=12 each) using the random number table method: control group (group C), diabetic neuralgia group (group D), and extracorporeal shock wave + diabetic neuralgia group (group E). The rats were continuously fed a common diet in group C, while the rats were fed a high-sugar and high-fat diet for 8 weeks in D and E groups. Streptozotocin 35 mg/kg was intraperitoneally injected, and the successful induction of diabetic neuralgia was defined as the blood glucose >14.6 mmol/L and the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) ≤85% of baseline values. Group E received extracorporeal shock wave treatment after developing the model, with 1, 000 shocks per session at a frequency of 10 Hz and an energy of 1.0 bar, once per week for a total of 4 sessions. The MWT and TWL were measured before developing the model (T 0) and at 1, 2, 3 and 4 weeks after developing the model (T 1-T 4). After the last extracorporeal shock wave treatment, the rats were anesthetized and sacrificed, and lumbar spinal cord tissues were obtained for proteomic analysis and for detection of the expression of glial fibrillary acidic protein (GFAP), interleukin-1beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) (by immunohistochemistry). Results:Compared with group C, the MWT and TWL were significantly decreased at T 1-T 4 in D and E groups ( P<0.05). Compared with group D, the MWT and TWL were significantly increased at T 1-T 4 in group E ( P<0.05). The results of phosphoproteomics screening revealed 284 differentially phosphorylated proteins in D and C groups, 282 in E and C groups, and 303 in E and D groups ( P<0.05). The results of immunohistochemistry showed that the expression of GFAP, IL-1β and TNF-α was significantly up-regulated in group D compared with group C ( P<0.05); the expression of GFAP, IL-1β and TNF-α was significantly down-regulated in group E compared with group D ( P<0.05). Conclusions:The mechanism by which extracorporeal shock wave alleviates diabetic neuralgia is related to inhibition of astrocyte activation and excessive phosphorylation of mGluR5 in rats.

[摘 要] 目的：评估细胞分裂周期蛋白20（CDC20）在子宫内膜癌（EC）中的表达，探讨其对EC细胞RL95-2周期和凋亡的影响及可能的机制。方法：从癌症基因组图谱（TCGA）数据库获取EC的mRNA表达矩阵以及患者的临床信息，通过R语言分析CDC20 mRNA的差异表达情况及其与肿瘤分期的相关性，qPCR及WB法检测CDC20在RL95-2细胞中的表达；向RL95-2细胞转染sh-CDC20以敲减CDC20的表达，采用CCK-8法和流式细胞术检测敲减CDC20对RL95-2细胞增殖活力、细胞周期分布和凋亡的影响，WB法分析对Mcl-1/p-Chk1信号活性的影响；建立RL95-2细胞裸鼠移植瘤模型，评估敲减CDC20对肿瘤生长的抑制作用及对移植瘤组织中Mcl-1/p-Chk1信号轴和细胞凋亡的影响。结果：CDC20在EC组织及RL95-2细胞中呈高表达（均P<0.01），且CDC20的高表达与EC的分期有关联。敲减CDC20可显著降低RL95-2细胞增殖活力（P<0.01），阻滞细胞周期于G1期（P<0.01），促进细胞凋亡（P<0.01），抑制细胞中Mcl-1和p-Chk1的表达（P<0.05或P<0.01）。敲减CDC20可显著抑制RL95-2细胞裸鼠移植瘤的生长（P<0.01），降低移植瘤组织内Mcl-1和p-Chk1的表达（P<0.01），促进移植瘤细胞凋亡（P<0.01）。结论：CDC20在EC组织中呈高表达且与肿瘤分期有关联，敲减CDC20能够抑制RL95-2细胞及其裸鼠移植瘤的生长而促进凋亡，这可能与Mcl-1/p-Chk1信号轴有关。

A triple-transgenic (tyrosine hydroxylase/dopamine decarboxylase/GTP cyclohydrolase 1, TH/DDC/GCH1) bone marrow mesenchymal stem cell line (BMSCs) capable of stably synthesizing dopamine (DA) transmitters were established to provide experimental evidence for the clinical treatment of Parkinson's disease (PD) by using this cell line. The DA-BMSCs cell line that could stably synthesize and secrete DA transmitters was established by using the triple transgenic recombinant lentivirus. The triple transgenes (TH/DDC/GCH1) expression in DA-BMSCs was detected using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Moreover, the secretion of DA was tested by enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). Chromosome G-banding analysis was used to detect the genetic stability of DA-BMSCs. Subsequently, the DA-BMSCs were stereotactically transplanted into the right medial forebrain bundle (MFB) of Parkinson's rat models to detect their survival and differentiation in the intracerebral microenvironment of PD rats. Apomorphine (APO)-induced rotation test was used to detect the improvement of motor dysfunction in PD rat models with cell transplantation. The TH, DDC and GCH1 were expressed stably and efficiently in the DA-BMSCs cell line, but not expressed in the normal rat BMSCs. The concentration of DA in the cell culture supernatant of the triple transgenic group (DA-BMSCs) and the LV-TH group was extremely significantly higher than that of the standard BMSCs control group (P < 0.000 1). After passage, DA-BMSCs stably produced DA. Karyotype G-banding analysis showed that the vast majority of DA-BMSCs maintained normal diploid karyotypes (94.5%). Moreover, after 4 weeks of transplantation into the brain of PD rats, DA-BMSCs significantly improved the movement disorder of PD rat models, survived in a large amount in the brain microenvironment, differentiated into TH-positive and GFAP-positive cells, and upregulated the DA level in the injured area of the brain. The triple-transgenic DA-BMSCs cell line that stably produced DA, survived in large numbers, and differentiated in the rat brain was successfully established, laying a foundation for the treatment of PD using engineered culture and transplantation of DA-BMSCs.
Rats ; Animals ; Dopamine ; Parkinson Disease/metabolism* ; Mesenchymal Stem Cells/metabolism* ; Cell Line ; Brain/metabolism* ; Cell Differentiation ; Mesenchymal Stem Cell Transplantation

Objective:To explore the relationship between psychological distress and intimacy in stroke patients and the mediating effect of different dyadic coping style, to provide theoretical guidance for improving the intimate relationship of stroke patients.Methods:A convenient sampling method was used to select 203 patients with stroke who visited the General Hospital of Ningxia Medical University, the People's Hospital of Ningxia Hui Autonomous Region and the Department of Neurology of Yinchuan First People's Hospital from December 2020 to July 2021 as the survey objects. The general information questionnaire, Kessler Psychological Distress Scale, Quality of Relationship Index and Dyadic Coping Inventory were used to investigate patients.Results:The scores of psychological distress, intimate relationship, positive dyadic coping and negative dyadic coping were 19.33 ± 7.46, 32.75 ± 6.79, 86.25 ± 13.22, and 16.46 ± 4.08, respectively. Correlation analysis showed that psychological distress was negatively correlated with intimate relationship and positive dyadic coping ( r=-0.195, -0.204, both P<0.01), psychological distress was positively correlated with negative dyadic coping ( r=0.229, P<0.01). The mediating effect of positive dyadic coping and negative dyadic coping on psychological distress and intimate relationship was 35.87% and 53.26%, respectively. Conclusions:The psychological distress indirectly affects the intimate relationship of stroke patients through positive dyadic coping and negative dyadic coping. Health care workers can guide patients to actively cope with the disease and encourage couples to strengthen communication, so as to enhance the intimacy of couples and promote their physical and mental health development.

Objective:To explore the effect of perceived stress on fatigue and its mechanism, so as to provide theoretical support for relieving the fatigue of the main caregivers of stroke patients.Methods:From December 2020 to August 2021, 311 main caregivers of stroke patients hospitalized in the Department of Neurology of Class Ⅲ Grade A hospitals in Yinchuan were selected by convenient sampling. The caregivers were investigated with the General Information Questionnaire, Perceived Stress Scale, Resourcefulness Scale, Social Support Rating Scale and Fatigue Scale.Results:In this study, a total of 311 questionnaires were distributed, and 308 valid questionnaires were recovered, with an effective recovery rate of 99.04%. The total scores of perceived stress, fatigue, resourcefulness and social support of the main caregivers of stroke patients were (24.98±6.36) , (5.31±2.86) , (83.98±12.85) and (39.76±6.21) respectively. Perceived stress were negatively correlated with resourcefulness and social support ( r=-0.313, -0.274, P<0.01) , and positively correlated with fatigue ( r=0.261, P<0.01) . Fatigue were negatively correlated with resourcefulness and social support ( r=-0.305, -0.268; P<0.01) . There was a positive correlation between resourcefulness and social support ( r=0.210, P<0.01) . The results of intermediary test showed that the direct effect of perceived stress on fatigue was valid, and the respective intermediary effect of resourcefulness and social support between perceived stress and fatigue, and the chain intermediary effect of resourcefulness and social support were also valid, with the amount of intermediary effect being 26.92%, 15.38% and 3.85% respectively. Conclusions:The chain mediation between perceived stress and fatigue is established between the resourcefulness and social support of the main caregivers of stroke patients. In the future, the mental and physical pressure of stroke patients can be alleviated by improving the resourcefulness of their main caregivers and providing social support.

Objective:To evaluate the effect of intravenous infusion of lidocaine on the efficacy of conventional treatment for rheumatoid arthritis.Methods:Forty-four patients with rheumatoid arthritis of either sex, aged 32-85 yr, weighing 40-76 kg, who were admitted to the Department of Pain and Nephrology in our hospital from September 2019 to September 2020, were divided into 2 groups ( n=22 each) according to the random number table method: control group (C group) and lidocaine group (L group). Both groups received conventional treatment.When visual analogue scale (VAS) score ≥5, glucocorticoid (GC) and non-steroidal anti-inflammatory drugs (NSAIDs) were taken orally to maintain the VAS score ≤4.In group L, 0.2% lidocaine hydrochloride injection 3 mg/kg (diluted with 0.9% sodium chloride injection 500 ml) was intravenously infused at a rate of 25 ml/h for 2 h, once a day, for 5 consecutive days, based on the conventional treatment.The VAS score, 28-joint Disease Activity Score (DAS28 score), simplified disease activity index score (SDAI score), consumption of GC and NSAIDs and adverse reactions were recorded before treatment (T 1) and at 1, 4 and 8 weeks after treatment (T 2-4). The temperature of the pain area of the affected joint was evaluated through infrared thermal imaging at T 1 and T 2. Results:Compared with the baseline at T 1, VAS score, DAS28 score and SDAI score were significantly decreased at each time point, and the temperature of the pain area of the affected joint at T 2 was decreased in the two groups ( P<0.05). There were no significant differences in VAS score, DAS28 score and SDAI score at each time point between two groups ( P>0.05). Compared with group C, the consumption of GC and NSAIDs was significantly decreased, and the temperature of the pain area of the dorsum of both hands and the dorsum of right foot at T 2 and incidence of adverse reactions were decreased in group L ( P<0.05). Conclusions:Intravenous infusion of lidocaine can optimize the efficacy of conventional treatment for rheumatoid arthritis.