1.Prognostic analysis of laparoscopic simultaneous radical cystectomy and nephroureterectomy.
Shenmo LI ; Dandan SU ; Jiyu LIN ; Haodong SONG ; Lulin MA ; Xiaofei HOU ; Guoliang WANG ; Hongxian ZHANG ; Jianfei YE ; Shudong ZHANG
Journal of Peking University(Health Sciences) 2025;57(5):961-966
OBJECTIVE:
To investigate the safety and prognostic factors influencing the treatment of upper urinary tract urothelial carcinoma (UTUC) combined with bladder cancer (BCa) by laparoscopic simultaneous radical cystectomy and nephroureterectomy (RCNU).
METHODS:
The clinical data of patients admitted to Peking University Third Hospital for laparoscopic RCNU surgery from January 2009 to September 2023 were analyzed retrospectively. Based on the same gender, age (±5 years), history of uroepithelial tumors, underlying diseases, T-stage, N-stage, M-stage, American Society of Anesthesiologists (ASA) score, Charlson comorbidity index, and body mass index (BMI) (±5), 34 patients with RCNU were matched 1 ∶1 with patients with bladder cancer who underwent laparoscopic radical cystectomy (RC) alone. Kaplan-Meier survival analysis was used to calculate patient survival, and Cox proportional regression risk model was used to analyze clinical factors affecting prognosis.
RESULTS:
Of the 68 patients enrolled, the follow-up rate was 100% with a median follow-up time of 27.0 (11.7, 60.2) months. Comparison of intraoperative conditions (including operation time, estimated intraoperative bleeding, intra-operative blood transfusion, etc.) between the two groups of patients showed no significant difference (P>0.05). Comparison of preoperative creatinine and postoperative creatinine between the two groups of patients showed significant differences (P < 0.05). The perioperative Clavien grade Ⅲ-Ⅳ complication rates were 2.9% (1/34) in the RC group and 5.9% (2/34) in the RCNU group. There was no significant difference in terms of perioperative complications between the two groups. Overall survival was significantly lower in the patients receiving RCNU compared with the matched group receiving RC alone (P < 0.05). Cox regression analysis suggested that two factors, high N stage and high postoperative creatinine, were independent risk factors affecting the prognosis of patients in the 2 groups (P < 0.05).
CONCLUSION
The overall survival prognosis of patients undergoing RCNU surgery was worse compared with laparoscopic RC surgery alone during the same period. There was no clinically significant difference between the two groups in terms of operation time, intraoperative bleeding, and perioperative complications, and there were clinically significant differences in preoperative renal function and post-operative renal function.
Humans
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Laparoscopy/methods*
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Nephroureterectomy/methods*
;
Cystectomy/methods*
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Prognosis
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Male
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Retrospective Studies
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Female
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Urinary Bladder Neoplasms/mortality*
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Middle Aged
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Aged
2.Ultrasonographic findings of intracranial solitary fibrous tumor:a case report
Lin YUAN ; Qiaoying ZHU ; Shudong YANG ; Fengsheng ZHOU
Chinese Journal of Ultrasonography 2025;34(9):813-816
Intracranial solitary fibrous tumor(ISFT)is a rare spindle cell central nervous system tumor originating from mesenchymal tissue. There are few reports of ISFT ultrasound findings in China. In this case,the patient,a 59-year-old man,was admitted to the hospital because of "skull tumor found on the top of the head for more than 2 years". The patient found tumor protrusion on the skull at the top of the head 2 years ago,without pain and other feelings. It was as small as a mung bean at first,and gradually increased during the course of the disease. After admission,ultrasound examination was performed,which suggested solid space occupying lesions in the skull. Considering the possibility of eosinophilic granuloma,malignant tumors were to be excluded.CT scan,three-dimensional imaging of skull and MRI scan and enhancement examination of skull were performed,which suggested that the right frontoparietal skull and skull mass shadow occupied the right side,and meningioma may occur. The brain tumor was resected under neuronavigation,and the solitary fibrous tumor was confirmed by postoperative pathology. The author analyzed the pathophysiology,imaging findings,ultrasound characteristics,differential diagnosis of ISFT in detail,in order to improve the understanding of ISFT in clinical work.
3.Efficacy of rigid ureteroscopic laser lithotripsy combined with N-trap in the treatment of ureteral stones with a maximum diameter equal to or greater than 1.5 cm
Jinghui JI ; Xiushi LIN ; Dameng PAN ; Zhiying WU ; Zixuan XUE ; Xiaojun TIAN ; Shudong ZHANG ; Binshuai WANG ; Min QIU
Journal of Peking University(Health Sciences) 2025;57(4):676-683
Objective:To investigate the efficacy of rigid ureteroscopic laser lithotripsy combined with N-trap(RULL+N-trap),which is scoop-shaped,in the treatment of ureteral stones with a maximum diameter ≥1.5 cm.Methods:This retrospective cohort study included patients diagnosed with ureteral calculi who underwent rigid ureteroscopic lithotripsy(RULL)combined with N-Trap stone entrapment system at the Department of Urology,Peking University Third Hospital,by the same surgical team between June 2021 and September 2024.A total of 364 patients were initially enrolled.After excluding 21 patients due to missing critical outcome variables,two distinct cohorts were established:38 patients with ureteral stones measuring ≥1.5 cm in maximum diameter,and 305 patients with stones<1.5 cm in maximum diameter.To minimize selection bias and control for confounding variables,propensity score matching(PSM)was employed.This resulted in two well-balanced groups:31 patients with stones 1.5 cm in maximum diameter and 31 patients with stones<1.5 cm in maximum diameter,matched on baseline demographic and clinical characteristics.The primary outcomes assessed between the two groups included stone clearance.Secondary outcomes included changes in renal function indicators,specifically serum creatinine(SCr)and estimated glomerular filtration rate(GFR),and other factors like postopera-tive hospital stay and operative time.Results:In the matched cohort,the patients with stones ≥1.5 cm in maximum diameter had significantly longer operative time compared with those with smaller stones:(85.8±28.8)min vs.(62.4±24.6)min(P<0.05).Postoperative length of hospital stay showed no significant difference:(2.26±1.79)d vs.(2.03±0.80)d(P>0.05).The stone clearance on postoperative day one was 90.3%in the study group vs.100.0%in the control group(P>0.05).One month postoperatively,the stone clearance was 93.5%vs.100.0%,respectively(P>0.05).Changes in SCr were(-6.58±16.10)μmol/L vs.(-13.70±12.50)μmol/L,and changes in GFR were(5.92±14.90)mL/(min·1.73 m2)vs.(7.47±11.20)m L/(min·1.73 m2),with no statistically significant differences observed between the two groups for either renal function marker(P>0.05).Conclusion:Ureteroscopic lithotripsy combined with N-trap is an optional method for treating ureteral stones with a maximum diameter ≥1.5 cm.The overall therapeutic efficacy is comparable,with the added benefit of significantly reducing the economic burden on patients.
4.Extraction and subculture of neural stem cells from mouse embryonic spinal cord:comparison and analysis on advantages and disadvantages of three commonly used digestive enzymes
Dan LUO ; Zhilin GE ; Yonghui HOU ; Wanshun WANG ; Jiheng ZHAN ; Yu HOU ; Dingkun LIN ; Shudong CHEN
Chinese Journal of Tissue Engineering Research 2025;29(31):6609-6615
BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLETM Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLETM Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.
5.Short-term outcome study on cervical deep lymph node-venous anastomosis technique in the treatment of Alzheimer’s disease
Cheng GAN ; Zhengdong KONG ; Xiaoye RAN ; Shudong QIAO ; Yixin ZHANG ; Lu YUE ; Yingjie WANG ; Hui BI ; Dong YANG ; Hongtong MA ; Yuan CHEN ; Hongli CHAI ; Ying JIA ; Chenhao MA ; Zixiang CHEN ; Ke LI ; Miao WANG ; Liguo XUE ; Siwen ZHAO ; Ke WEN ; Lin YIN ; Bo DING ; Shan ZHU ; Yuanbo LIU ; Mengqing ZANG
Chinese Journal of Plastic Surgery 2025;41(2):130-143
Objective:To explore the short-term clinical effects of deep cervical lymph node-venous anastomosis in the treatment of Alzheimer’s disease (AD).Methods:A prospective exploratory study was conducted on the treatment of AD patients using the cervical deep lymph node-venous anastomosis technique in Scar and Wound Treatment Department, Plastic Surgery Hospital, Chinese Academy of Medical Sciences, from September to October 2024. The patients underwent high-frequency ultrasound to locate deep cervical lymph nodes and the external jugular vein. Under general anesthesia, bilateral deep cervical lymph node-venous anastomoses were performed. Indocyanine green (ICG) lymphography was conducted via subcutaneous injection behind the ear to visualize lymph nodes in levels Ⅱ and Ⅲ. After making a skin incision along the posterior margin of the sternocleidomastoid muscle, the external jugular vein, internal jugular veins, and associated lymph nodes were exposed. Adjacent veins were selected for anastomosis of lymph node. Using microsurgical techniques, end-to-side or end-to-end anastomosis was completed for lymph nodes in levels Ⅱ and Ⅲ. Preoperative assessments included the mini-mental state examination (MMSE, a higher score indicates better cognitive function), Alzheimer’s disease assessment scale-cognitive subscale (ADAS-Cog, a higher score indicates greater impairment of cognitive function), Alzheimer’s disease cooperative study scale for activities of daily living (ADCS-ADL, a higher score indicates better ability to perform daily activity), and neuropsychiatric inventory (NPI, a higher score indicates more severe behavioral and emotional symptom). Postoperative follow-up included the same scales to observe changes in cognitive function, activities of daily living, and emotional communication.Results:Four patients (1 male, 3 females, aged 58-79 years) with AD were included. All were diagnosed based on cerebrospinal fluid biomarkers. All patients successfully underwent bilateral deep cervical lymph node-venous anastomoses. On average, 4.3 (2-7 per person) anastomoses were performed per patient. Surgical procedures lasted an average of 6.5 h (5.5-8.5 h) with minimal blood loss (less than 50 ml). Patients resumed normal activity within 6 hours postoperatively and were discharged after an average of 4.1 d (3.5-5.0 d). Postoperative complications included one case each of aspiration pneumonia, lower limb venous thrombosis, and transient delirium, all of whom resolved without long-term effects. Clinical symptoms, including memory decline, mood swings, and anxiety, showed varying degrees of improvement. Patients reported enhanced quality of life, emotional stability, and social engagement, confirming the procedure’s safety and potential cognitive benefits. At one month postoperatively, the MMSE scores of the four patients increased by an average of 0.8 points compared to preoperative levels. Additionally, the two patients who completed the ADAS-Cog assessments showed a decrease in their scores (reduced by 1.0 points and 11.3 points, respectively, compared to preoperative scores), indicating a certain degree of improvement in cognitive function during this period. The ADCS-ADL and NPI scores of four patients varied significantly, without showing any clear pattern.Conclusion:Lymphovenous anastomosis of the deep cervical lymph node-venous anastomosis may provide a new surgical intervention approach for AD, but further large-scale studies and long-term follow-up are needed to validate its safety and effectiveness.
6.Influence mechanism of nitrate on the determination of calcium content in pitavastatin calcium by flame atomic absorption spectrometry
Zhijun ZHANG ; Shudong ZHANG ; Lin WANG ; Zhe ZHANG ; Qin HU
Drug Standards of China 2025;26(5):538-542
Objective:To explore the mechanism of the influence of nitrate on the determination of calcium content in pitavastatin calcium by flame atomic absorption spectrometry.Methods:By comparing the absorbance of calci-um standard test solutions using hydrochloric acid and nitric acid as solution media,and measuring the changes in calcium absorbance in calcium ion solutions prepared by adding different concentrations of nitric acid using flame atomic absorption spectrophotometry,the relationship between calcium atomization efficiency and calcium ion con-centration,nitrate concentration,and their concentration ratios was explored.Results:Nitrate had a significant effect on reducing the absorbance value of the calcium standard test solution;The degree of interference varied with the concentration of the element calcium in the sample,and was related to the ratio of the concentration of the ele-ment calcium to the concentration of the interfering substance nitrate.Moreover,the interference of nitrate on calci-um affected the slope of the working curve.Conclusion:The interference mechanism of nitrate in the determination of calcium by flame atomic absorption spectrophotometry is due to the decomposition of calcium nitrate formed by ni-trate and calcium into difficult to melt,evaporate,and effectively dissociate calcium oxide at high temperatures,which reduces the atomization efficiency of calcium atoms and results in low absorbance values in the measurement.It is not advisable to select calcium standard stock solutions containing nitric acid when determining calcium containing samples such as pitavastatin calcium,and it is also necessary to be vigilant against the interference of nitrate ions.
7.Repetitive transcranial magnetic stimulation improves learning memory and enhances drainage efficiency of intracerebral glymphatic system in cerebral amyloid angiopathy model mice
Sijing LI ; Feng YANG ; Guijuan ZHOU ; Limin DENG ; Xuanwei WEN ; Shudong LIN ; Jingming KUANG ; Zijian XIAO
Chinese Journal of Behavioral Medicine and Brain Science 2025;34(2):111-117
Objective:To investigate the effects of repetitive transcranial magnetic stimulation(rTMS) on learning memory and abnormal Aβ deposition in cerebral amyloid angiopathy(CAA) model mice, and further to investigate whether the mechanism involves the transport function of glymphatic system.Methods:Eight-month-old SPF grade Tg-SWDI mice were randomly divided into the CAA group and the rTMS group according to the random number table method with 7 in each group.Seven wild-type mice of the same genetic background and age served as the control group. The mice in rTMS group received two weeks of high-frequency rTMS intervention, and the mice in CAA group and control group were only restrained without rTMS intervention.Learning and memory functions were evaluated using the Morris water maze test.Amyloid-beta deposition, glymphatic system clearance, and aquaporin-4(AQP4) polarization were assessed using immunofluorescence, and AQP4 expression levels were measured by Western blot.Statistical analysis of the data was conducted using SPSS 25.0 and GraphPad Prism 9.5 softwares.Repeated-measures ANOVA was used for data on escape latency, and one-way ANOVA was used for comparisons between multiple groups for other data.Results:(1)In the novel object recognition test, there were statistically significant differences in recognition indices among the three groups of mice ( F=22.59, P<0.05). Compared with the control group, the mice in the CAA group showed a significant decrease in the new object recognition index ( P<0.05).Compared with the CAA group, the mice in the rTMS group showed a significant increase in the new object recognition index ( P<0.05).(2)In the Y-maze, there were statistical differences in the spontaneous alternation rates among the three groups ( F=5.00, P<0.05). Compared with the control group, the spontaneous alternation rate in the CAA group was significantly lower ( P<0.05).And compared with the CAA group, the spontaneous alternation rate in the rTMS group was significantly higher ( P<0.05).(3)In the Morris water maze test, there were significant interactions in escape latency among the three groups ( F=4.05, P=0.02), significant main effects of time ( F=713.22, P<0.01), and significant main effects of group ( F=421.55, P<0.01). There was no significant statistical difference in swimming speed among the three groups ( F=0.19, P>0.05), while the difference of the number of entries into the inner zone and the proportion of time spent were statistically significant( F=71.67, 294.14, both P<0.05).Compared with the control group, the CAA group mice significantly decreased in the number of entries into the inner zone and the proportion of time spent in the middle zone (both P<0.01).(4)Compared with the CAA group, the rTMS group significantly increased in the number of entries into the inner zone and the proportion of time spent in the middle zone (both P<0.01).The result of immunofluorescence test showed that there was a statistically significant difference in the levels of Aβ in the cerebral vessels among the three groups( F=385.76, P<0.01).The levels of Aβ in the cerebral vessels of the CAA group (62.00±2.65) were significantly higher than those in the control group (9.00±1.00, P<0.01).The levels in the rTMS group (51.33±3.21) were significantly lower than those in the CAA group (62.00±2.65, P<0.01). Using the residual fluorescence tracer levels of the control group as a baseline, there were statistically significant differences in the tracer intensities in the corpus callosum and cerebral cortex( F=258.97, 46.44, both P<0.05), the tracer intensities in the corpus callosum (3.57±0.21) and cerebral cortex (4.96±0.79) of the CAA group mice were significantly higher than those in the rTMS group (1.45±0.14, 1.78±0.47, P<0.01). The polarization of AQP4 in the cerebral cortex of rTMS group (0.51±0.07) was significantly higher than that in the CAA group (0.30±0.02, P<0.01). Conclusion:rTMS can alleviate learning memory and abnormal Aβ deposition in CAA model mice by modulating AQP4 polarisation and promoting transport function of glymphatic system.
8.Repetitive transcranial magnetic stimulation improves learning memory and enhances drainage efficiency of intracerebral glymphatic system in cerebral amyloid angiopathy model mice
Sijing LI ; Feng YANG ; Guijuan ZHOU ; Limin DENG ; Xuanwei WEN ; Shudong LIN ; Jingming KUANG ; Zijian XIAO
Chinese Journal of Behavioral Medicine and Brain Science 2025;34(2):111-117
Objective:To investigate the effects of repetitive transcranial magnetic stimulation(rTMS) on learning memory and abnormal Aβ deposition in cerebral amyloid angiopathy(CAA) model mice, and further to investigate whether the mechanism involves the transport function of glymphatic system.Methods:Eight-month-old SPF grade Tg-SWDI mice were randomly divided into the CAA group and the rTMS group according to the random number table method with 7 in each group.Seven wild-type mice of the same genetic background and age served as the control group. The mice in rTMS group received two weeks of high-frequency rTMS intervention, and the mice in CAA group and control group were only restrained without rTMS intervention.Learning and memory functions were evaluated using the Morris water maze test.Amyloid-beta deposition, glymphatic system clearance, and aquaporin-4(AQP4) polarization were assessed using immunofluorescence, and AQP4 expression levels were measured by Western blot.Statistical analysis of the data was conducted using SPSS 25.0 and GraphPad Prism 9.5 softwares.Repeated-measures ANOVA was used for data on escape latency, and one-way ANOVA was used for comparisons between multiple groups for other data.Results:(1)In the novel object recognition test, there were statistically significant differences in recognition indices among the three groups of mice ( F=22.59, P<0.05). Compared with the control group, the mice in the CAA group showed a significant decrease in the new object recognition index ( P<0.05).Compared with the CAA group, the mice in the rTMS group showed a significant increase in the new object recognition index ( P<0.05).(2)In the Y-maze, there were statistical differences in the spontaneous alternation rates among the three groups ( F=5.00, P<0.05). Compared with the control group, the spontaneous alternation rate in the CAA group was significantly lower ( P<0.05).And compared with the CAA group, the spontaneous alternation rate in the rTMS group was significantly higher ( P<0.05).(3)In the Morris water maze test, there were significant interactions in escape latency among the three groups ( F=4.05, P=0.02), significant main effects of time ( F=713.22, P<0.01), and significant main effects of group ( F=421.55, P<0.01). There was no significant statistical difference in swimming speed among the three groups ( F=0.19, P>0.05), while the difference of the number of entries into the inner zone and the proportion of time spent were statistically significant( F=71.67, 294.14, both P<0.05).Compared with the control group, the CAA group mice significantly decreased in the number of entries into the inner zone and the proportion of time spent in the middle zone (both P<0.01).(4)Compared with the CAA group, the rTMS group significantly increased in the number of entries into the inner zone and the proportion of time spent in the middle zone (both P<0.01).The result of immunofluorescence test showed that there was a statistically significant difference in the levels of Aβ in the cerebral vessels among the three groups( F=385.76, P<0.01).The levels of Aβ in the cerebral vessels of the CAA group (62.00±2.65) were significantly higher than those in the control group (9.00±1.00, P<0.01).The levels in the rTMS group (51.33±3.21) were significantly lower than those in the CAA group (62.00±2.65, P<0.01). Using the residual fluorescence tracer levels of the control group as a baseline, there were statistically significant differences in the tracer intensities in the corpus callosum and cerebral cortex( F=258.97, 46.44, both P<0.05), the tracer intensities in the corpus callosum (3.57±0.21) and cerebral cortex (4.96±0.79) of the CAA group mice were significantly higher than those in the rTMS group (1.45±0.14, 1.78±0.47, P<0.01). The polarization of AQP4 in the cerebral cortex of rTMS group (0.51±0.07) was significantly higher than that in the CAA group (0.30±0.02, P<0.01). Conclusion:rTMS can alleviate learning memory and abnormal Aβ deposition in CAA model mice by modulating AQP4 polarisation and promoting transport function of glymphatic system.
9.Efficacy of rigid ureteroscopic laser lithotripsy combined with N-trap in the treatment of ureteral stones with a maximum diameter equal to or greater than 1.5 cm
Jinghui JI ; Xiushi LIN ; Dameng PAN ; Zhiying WU ; Zixuan XUE ; Xiaojun TIAN ; Shudong ZHANG ; Binshuai WANG ; Min QIU
Journal of Peking University(Health Sciences) 2025;57(4):676-683
Objective:To investigate the efficacy of rigid ureteroscopic laser lithotripsy combined with N-trap(RULL+N-trap),which is scoop-shaped,in the treatment of ureteral stones with a maximum diameter ≥1.5 cm.Methods:This retrospective cohort study included patients diagnosed with ureteral calculi who underwent rigid ureteroscopic lithotripsy(RULL)combined with N-Trap stone entrapment system at the Department of Urology,Peking University Third Hospital,by the same surgical team between June 2021 and September 2024.A total of 364 patients were initially enrolled.After excluding 21 patients due to missing critical outcome variables,two distinct cohorts were established:38 patients with ureteral stones measuring ≥1.5 cm in maximum diameter,and 305 patients with stones<1.5 cm in maximum diameter.To minimize selection bias and control for confounding variables,propensity score matching(PSM)was employed.This resulted in two well-balanced groups:31 patients with stones 1.5 cm in maximum diameter and 31 patients with stones<1.5 cm in maximum diameter,matched on baseline demographic and clinical characteristics.The primary outcomes assessed between the two groups included stone clearance.Secondary outcomes included changes in renal function indicators,specifically serum creatinine(SCr)and estimated glomerular filtration rate(GFR),and other factors like postopera-tive hospital stay and operative time.Results:In the matched cohort,the patients with stones ≥1.5 cm in maximum diameter had significantly longer operative time compared with those with smaller stones:(85.8±28.8)min vs.(62.4±24.6)min(P<0.05).Postoperative length of hospital stay showed no significant difference:(2.26±1.79)d vs.(2.03±0.80)d(P>0.05).The stone clearance on postoperative day one was 90.3%in the study group vs.100.0%in the control group(P>0.05).One month postoperatively,the stone clearance was 93.5%vs.100.0%,respectively(P>0.05).Changes in SCr were(-6.58±16.10)μmol/L vs.(-13.70±12.50)μmol/L,and changes in GFR were(5.92±14.90)mL/(min·1.73 m2)vs.(7.47±11.20)m L/(min·1.73 m2),with no statistically significant differences observed between the two groups for either renal function marker(P>0.05).Conclusion:Ureteroscopic lithotripsy combined with N-trap is an optional method for treating ureteral stones with a maximum diameter ≥1.5 cm.The overall therapeutic efficacy is comparable,with the added benefit of significantly reducing the economic burden on patients.
10.Extraction and subculture of neural stem cells from mouse embryonic spinal cord:comparison and analysis on advantages and disadvantages of three commonly used digestive enzymes
Dan LUO ; Zhilin GE ; Yonghui HOU ; Wanshun WANG ; Jiheng ZHAN ; Yu HOU ; Dingkun LIN ; Shudong CHEN
Chinese Journal of Tissue Engineering Research 2025;29(31):6609-6615
BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLETM Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLETM Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

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