1.Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing
Li Ya ZHANG ; Zhe Wen SU ; Chen Rui WANG ; Yan LI ; Feng Jun ZHANG ; Hui Sheng LIU ; He Dan HU ; Xiao Chong XU ; Yu Jia YIN ; Kai Qi YIN ; Ying HE ; Fan LI ; Hong Shi FU ; Kai NIE ; Dong Guo LIANG ; Yong TAO ; Tao Song XU ; Feng Chao MA ; Yu Huan WANG
Biomedical and Environmental Sciences 2024;37(3):294-302
		                        		
		                        			
		                        			Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing. Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing. Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing. Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.
		                        		
		                        		
		                        		
		                        	
2.A multicenter study of neonatal stroke in Shenzhen,China
Li-Xiu SHI ; Jin-Xing FENG ; Yan-Fang WEI ; Xin-Ru LU ; Yu-Xi ZHANG ; Lin-Ying YANG ; Sheng-Nan HE ; Pei-Juan CHEN ; Jing HAN ; Cheng CHEN ; Hui-Ying TU ; Zhang-Bin YU ; Jin-Jie HUANG ; Shu-Juan ZENG ; Wan-Ling CHEN ; Ying LIU ; Yan-Ping GUO ; Jiao-Yu MAO ; Xiao-Dong LI ; Qian-Shen ZHANG ; Zhi-Li XIE ; Mei-Ying HUANG ; Kun-Shan YAN ; Er-Ya YING ; Jun CHEN ; Yan-Rong WANG ; Ya-Ping LIU ; Bo SONG ; Hua-Yan LIU ; Xiao-Dong XIAO ; Hong TANG ; Yu-Na WANG ; Yin-Sha CAI ; Qi LONG ; Han-Qiang XU ; Hui-Zhan WANG ; Qian SUN ; Fang HAN ; Rui-Biao ZHANG ; Chuan-Zhong YANG ; Lei DOU ; Hui-Ju SHI ; Rui WANG ; Ping JIANG ; Shenzhen Neonatal Data Network
Chinese Journal of Contemporary Pediatrics 2024;26(5):450-455
		                        		
		                        			
		                        			Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75%(27/36);boys accounted for 64%(23/36).Among the 36 neonates,31(86%)had disease onset within 3 days after birth,and 19(53%)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61%)had left cerebral infarction and 13(36%)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75%)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72%)and seizures in 10 neonates(34%).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33%,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44%(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.
		                        		
		                        		
		                        		
		                        	
3.4E-BP1 counteracts human mesenchymal stem cell senescence via maintaining mitochondrial homeostasis.
Yifang HE ; Qianzhao JI ; Zeming WU ; Yusheng CAI ; Jian YIN ; Yiyuan ZHANG ; Sheng ZHANG ; Xiaoqian LIU ; Weiqi ZHANG ; Guang-Hui LIU ; Si WANG ; Moshi SONG ; Jing QU
Protein & Cell 2023;14(3):202-216
		                        		
		                        			
		                        			Although the mTOR-4E-BP1 signaling pathway is implicated in aging and aging-related disorders, the role of 4E-BP1 in regulating human stem cell homeostasis remains largely unknown. Here, we report that the expression of 4E-BP1 decreases along with the senescence of human mesenchymal stem cells (hMSCs). Genetic inactivation of 4E-BP1 in hMSCs compromises mitochondrial respiration, increases mitochondrial reactive oxygen species (ROS) production, and accelerates cellular senescence. Mechanistically, the absence of 4E-BP1 destabilizes proteins in mitochondrial respiration complexes, especially several key subunits of complex III including UQCRC2. Ectopic expression of 4E-BP1 attenuates mitochondrial abnormalities and alleviates cellular senescence in 4E-BP1-deficient hMSCs as well as in physiologically aged hMSCs. These f indings together demonstrate that 4E-BP1 functions as a geroprotector to mitigate human stem cell senescence and maintain mitochondrial homeostasis, particularly for the mitochondrial respiration complex III, thus providing a new potential target to counteract human stem cell senescence.
		                        		
		                        		
		                        		
		                        			Mesenchymal Stem Cells/physiology*
		                        			;
		                        		
		                        			Cellular Senescence
		                        			;
		                        		
		                        			Homeostasis
		                        			;
		                        		
		                        			Cell Cycle Proteins/metabolism*
		                        			;
		                        		
		                        			Adaptor Proteins, Signal Transducing/metabolism*
		                        			;
		                        		
		                        			Mitochondria/metabolism*
		                        			;
		                        		
		                        			Electron Transport Complex III/metabolism*
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Cells, Cultured
		                        			
		                        		
		                        	
4.A prospective study on application of human umbilical cord mesenchymal stem cells combined with autologous Meek microskin transplantation in patients with extensive burns.
Tian Tian YAN ; Rong XIAO ; Ying WANG ; Guo An LIN ; Yin ZHENG ; Hui ZHAO ; Wen Jun LI ; Xin Zhi SHANG ; Jin Song MENG ; Dong Sheng HU ; Song LI ; Chao WANG ; Zhi Chen LIN ; Hong Chang CHEN ; Dong Yan ZHAO ; Di TANG
Chinese Journal of Burns 2023;39(2):114-121
		                        		
		                        			
		                        			Objective: To investigate the effects of human umbilical cord mesenchymal stem cells (hUCMSCs) combined with autologous Meek microskin transplantation on patients with extensive burns. Methods: The prospective self-controlled study was conducted. From May 2019 to June 2022, 16 patients with extensive burns admitted to the 990th Hospital of PLA Joint Logistics Support Force met the inclusion criteria, while 3 patients were excluded according to the exclusion criteria, and 13 patients were finally selected, including 10 males and 3 females, aged 24-61 (42±13) years. A total of 20 trial areas (40 wounds, with area of 10 cm×10 cm in each wound) were selected. Two adjacent wounds in each trial area were divided into hUCMSC+gel group applied with hyaluronic acid gel containing hUCMSCs and gel only group applied with hyaluronic acid gel only according to the random number table, with 20 wounds in each group. Afterwards the wounds in two groups were transplanted with autologous Meek microskin grafts with an extension ratio of 1∶6. In 2, 3, and 4 weeks post operation, the wound healing was observed, the wound healing rate was calculated, and the wound healing time was recorded. The specimen of wound secretion was collected for microorganism culture if there was purulent secretion on the wound post operation. In 3, 6, and 12 months post operation, the scar hyperplasia in wound was assessed using the Vancouver scar scale (VSS). In 3 months post operation, the wound tissue was collected for hematoxylin-eosin (HE) staining to observe the morphological changes and for immunohistochemical staining to observe the positive expressions of Ki67 and vimentin and to count the number of positive cells. Data were statistically analyzed with paired samples t test and Bonferronni correction. Results: In 2, 3, and 4 weeks post operation, the wound healing rates in hUCMSC+gel group were (80±11)%, (84±12)%, and (92±9)%, respectively, which were significantly higher than (67±18)%, (74±21)%, and (84±16)% in gel only group (with t values of 4.01, 3.52, and 3.66, respectively, P<0.05). The wound healing time in hUCMSC+gel group was (31±11) d, which was significantly shorter than (36±13) d in gel only group (t=-3.68, P<0.05). The microbiological culture of the postoperative wound secretion specimens from the adjacent wounds in 2 groups was identical, with negative results in 4 trial areas and positive results in 16 trial areas. In 3, 6, and 12 months post operation, the VSS scores of wounds in gel only group were 7.8±1.9, 6.7±2.1, and 5.4±1.6, which were significantly higher than 6.8±1.8, 5.6±1.6, and 4.0±1.4 in hUCMSC+gel group, respectively (with t values of -4.79, -4.37, and -5.47, respectively, P<0.05). In 3 months post operation, HE staining showed an increase in epidermal layer thickness and epidermal crest in wound in hUCMSC+gel group compared with those in gel only group, and immunohistochemical staining showed a significant increase in the number of Ki67 positive cells in wound in hUCMSC+gel group compared with those in gel only group (t=4.39, P<0.05), with no statistically significant difference in the number of vimentin positive cells in wound between the 2 groups (P>0.05). Conclusions: The application of hyaluronic acid gel containing hUCMSCs to the wound is simple to perform and is therefore a preferable route. Topical application of hUCMSCs can promote healing of the autologous Meek microskin grafted area in patients with extensive burns, shorten wound healing time, and alleviate scar hyperplasia. The above effects may be related to the increased epidermal thickness and epidermal crest, and active cell proliferation.
		                        		
		                        		
		                        		
		                        			Female
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Male
		                        			;
		                        		
		                        			Burns/surgery*
		                        			;
		                        		
		                        			Cicatrix
		                        			;
		                        		
		                        			Eosine Yellowish-(YS)
		                        			;
		                        		
		                        			Hyaluronic Acid/therapeutic use*
		                        			;
		                        		
		                        			Hyperplasia
		                        			;
		                        		
		                        			Ki-67 Antigen
		                        			;
		                        		
		                        			Prospective Studies
		                        			;
		                        		
		                        			Umbilical Cord
		                        			;
		                        		
		                        			Vimentin
		                        			;
		                        		
		                        			Young Adult
		                        			;
		                        		
		                        			Adult
		                        			;
		                        		
		                        			Middle Aged
		                        			
		                        		
		                        	
5.Clinical efficacy and safety of venetoclax combined with multidrug chemotherapy in the treatment of 15 patients with relapsed or refractory early T-cell precursor acute lymphoblastic leukemia.
Jin Yu KONG ; Li Hong ZONG ; Yan PU ; Yin LIU ; Xin KONG ; Meng Yun LI ; Jian ZHANG ; Bao Quan SONG ; Sheng Li XUE ; Xiao Wen TANG ; Hui Ying QIU ; De Pei WU
Chinese Journal of Hematology 2023;44(8):649-653
		                        		
		                        			
		                        			Objective: To explore the efficacy and safety of Venetoclax combined with multidrug chemotherapy in patients with relapsed or refractory early T-cell precursor acute lymphoblastic leukemia (R/R ETP-ALL) . Methods: This study retrospectively analyzed 15 patients with R/R ETP-ALL who received Venetoclax combined with multidrug chemotherapy from December 2018 to February 2022. Among them, eight cases were combined with demethylated drugs, four cases were combined with demethylated drugs and HAAG chemotherapy regimen, two cases were combined with demethylated drugs and CAG regimen, and one case was combined with Cladribine. Specific usage and dosage of Venetoclax: 100 mg on day 1, 200 mg on day 2, 400 mg on day 3-28, orally; when combined with azole antifungal drugs, dosage was reduced to 100 mg/d. Results: Fifteen patients (10 males and 5 females) with R/R ETP-ALL were treated with Venetoclax and multidrug chemotherapy with a median age of 35 (12-42) years old. Of 4 refractory and 11 relapsed patients, the efficacy was evaluated on the 21th day following combined chemotherapy: the overall response rate, the complete response (CR) rate, and the CR with incomplete hematological recovery (CRi) rate were 67.7% (10/15), 60.0% (9/15), and 6.7% (1/15), respectively. For the overall study population, the 12-month overall survival (OS) rate was 60.0%, and the median OS was 17.7 months. The disease-free survival (DFS) rate of all CR patients at 12 months was 60.0%, and the median DFS did not reach. About 14 patients had Ⅲ-Ⅳ hematological toxicity, but these adverse reactions were all controllable. No adverse reaction in the nervous system and tumor lysis syndrome occurred in this study, and no adverse reaction of organs above grade Ⅲ occurred. Conclusion: Venetoclax combined with multidrug chemotherapy may be a safe and promising treatment option for patients with R/R ETP-ALL.
		                        		
		                        		
		                        		
		                        			Male
		                        			;
		                        		
		                        			Female
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Adult
		                        			;
		                        		
		                        			Retrospective Studies
		                        			;
		                        		
		                        			Treatment Outcome
		                        			;
		                        		
		                        			Bridged Bicyclo Compounds, Heterocyclic/therapeutic use*
		                        			;
		                        		
		                        			Antineoplastic Combined Chemotherapy Protocols
		                        			;
		                        		
		                        			Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy*
		                        			;
		                        		
		                        			Precursor Cells, T-Lymphoid
		                        			;
		                        		
		                        			Leukemia, Myeloid, Acute/drug therapy*
		                        			
		                        		
		                        	
6.The rationality and surgical errors in countermeasures against difficult removal of screws
Sheng SONG ; Changbao WEI ; Yiwen SHEN ; Yingyan ZHANG ; Ye LU ; Peng WANG ; Qudong YIN
Chinese Journal of Orthopaedic Trauma 2023;25(10):905-909
		                        		
		                        			
		                        			Objective:To investigate the rationality and surgical errors in countermeasures against difficult removal of screws so as to provide reference for standardization of technical procedures.Methods:A retrospective study was conducted to analyze the data of 99 patients who had encountered difficult removal of screws in operative removal of internal fixation at Department of Orthopaedics, Wuxi NO.9 People's Hospital Affiliated to Suzhou University from January 2018 to May 2022. There were 62 males and 37 females with an average age of 38.8±14.7 years. Their internal fixation time ranged from 7 months to 11 years. The irrationality was defined as insufficient preoperative preparation or a countermeasure that failed to follow the surgical indications or scientific principles of minimal injury or priority of simplicity. A surgical error was defined as unnecessary injury, failed removal or complications related to operation. Cases of irrationality and surgical errors were analyzed to find associations between them.Results:The operative removal was successful in 92 cases and failed in 7 cases. Of the patients who experienced difficult removal of screws, irrationality was found in 26.3% (26/99) and a surgical error or errors occurred in 28.3% (28/99). In the patients with countermeasure irrationality, the incidence of surgical errors was 53.9% (14/26) while in those without countermeasure irrationality, the incidence of surgical errors was 19.2% (14/73), showing a statistically significant difference ( χ2=11.360, P<0.001). In the patients with surgical errors, the incidence of countermeasure irrationality was 64.3% (18/28) while in the patients without surgical errors, the incidence of countermeasure irrationality was and 11.3% (8/71), showing a statistically significant difference ( χ2=29.148, P<0.001). In the patients with failed removal, the rate of countermeasure irrationality was 85.71% (6/7) while in those with successful removal, the rate of countermeasure irrationality was 21.7% (20/92), showing a statistically significant difference ( χ2=13.748, P<0.001). Conclusions:Close relationships exist between countermeasure irrationality, surgical errors and failed removal. The higher proportion of countermeasure irrationality, the higher possibility of surgical errors. Therefore, following the rationality principle may avoid or reduce surgical errors in difficult removal of screws.
		                        		
		                        		
		                        		
		                        	
7.Clinical study on quantitative evaluation of proton radiotherapy by off-line PET-CT in vivo biological validation
Fuquan ZHANG ; Zhengshan HONG ; Jian'gang ZHANG ; Yan LU ; Xiangzi SHENG-YIN ; Qing ZHANG ; Dan YOU ; Shaoli SONG ; Guoliang JIANG ; Yun SUN ; Rong ZHOU ; Jingyi CHENG
Chinese Journal of Radiation Oncology 2022;31(11):1017-1021
		                        		
		                        			
		                        			Objective:Patients are breathing freely during adjuvant proton pencil beam radiotherapy after breast conserving surgery. Fluctuation of the thorax may affect the position of the end of the proton beam flow, which needs to be precisely evaluated on a millimeter scale.Methods:For 20 patients with breast cancer treated with proton radiotherapy after breast conserving surgery, PET-CT scan was performed approximately 10 min after the end of proton radiotherapy. The images of PET-CT were processed for ROI determination and sampling line (profile) extraction on a Raystation RV workstation to calculate the actual difference between the predicted and real radioactivity from the same spatial location as obtained by PET acquisition R50. Then, the differences in the spatial location between the actual process of proton irradiation and the planned process were obtained. Depth difference values for each pair of sampling lines were presented. Results:For 20 patients with breast cancer with a median follow-up of 22 months (range 12 - 46 months), all patients survived at the last follow-up, and no radiation pneumonitis was observed during the follow-up period. Among the verification results of 21 cases, the depth difference of evenly distributed was (-0.75±1.89) mm in the primary field and (-0.82±2.06) mm in the secondary field; The depth difference of sequential treatment was (1.81±1.87) mm in the primary field and (1.32±1.74) mm in the secondary field; The depth difference of synchronous addition in the primary field was (-1.47±1.44) mm, and the depth difference in the secondary field was (-1.48±2.11) mm.Conclusion:The results of off-line PET-CT in vivo biological verification show that the accuracy of the dose boundary cut-off was within 3 mm in breast cancer patients, which meets the clinical and physician requirement for the precision in breast cancer treatment.
		                        		
		                        		
		                        		
		                        	
8.Benefits and risks of prolonged dual antiplatelet therapy after percutaneous coronary intervention with drug-eluting stent in patients with stable coronary artery disease and diabetes.
Kong Yong CUI ; Dong YIN ; Lei FENG ; Cheng Gang ZHU ; Wei Hua SONG ; Hua Jian WANG ; Lei JIA ; Dong ZHANG ; Sheng YUAN ; Shao Yu WU ; Ji Ning HE ; Zheng QIAO ; Ke Fei DOU
Chinese Journal of Cardiology 2022;50(5):458-465
		                        		
		                        			
		                        			Objective: To compare the efficacy and safety of prolonged dual antiplatelet therapy (DAPT>1 year) in patients with stable coronary artery disease (CAD) and diabetes who were event-free at 1 year after percutaneous coronary intervention (PCI) with drug-eluting stent (DES) in a large and contemporary PCI registry. Methods: A total of 1 661 eligible patients were selected from the Fuwai PCI Registry, of which 1 193 received DAPT>1 year and 468 received DAPT ≤1 year. The primary endpoint was major adverse cardiac and cerebrovascular event (MACCE) and Bleeding Academic Research Consortium (BARC) type 2, 3 or 5 bleeding, MACCE was defined as a composite of all-cause death, myocardial infarction or stroke. Multivariate Cox regression analysis and inverse probability of treatment weighting (IPTW) Cox regression analysis were performed. Results: After a median follow-up of 2.5 years, patients who received DAPT>1 year were associated with lower risks of MACCE (1.4% vs. 3.2%; hazard ratio (HR) 0.412, 95% confidence interval (CI) 0.205-0.827) compared with DAPT ≤1 year, which was primarily caused by the lower all-cause mortality (0.1% vs. 2.6%; HR 0.031, 95%CI 0.004-0.236). Risks of cardiac death (0.1% vs. 1.5%; HR 0.051, 95%CI 0.006-0.416) and definite/probable ST (0.3% vs. 1.1%; HR 0.218, 95%CI 0.052-0.917) were also lower in patients received DAPT>1 year than those received DAPT ≤ 1 year. No difference was found between the two groups in terms of BARC type 2, 3, or 5 bleeding (5.3% vs. 4.1%; HR 1.088, 95%CI 0.650-1.821). Conclusions: In patients with stable CAD and diabetes who were event-free at 1 year after PCI with DES, prolonged DAPT (>1 year) provides a substantial reduction in ischemic cardiovascular events, including MACCE, all-cause mortality, cardiac mortality, and definite/probable ST, without increasing the clinically relevant bleeding risk compared with ≤ 1-year DAPT. Further well-designed, large-scale randomized trials are needed to verify the beneficial effect of prolonged DAPT in this population.
		                        		
		                        		
		                        		
		                        			Coronary Artery Disease/therapy*
		                        			;
		                        		
		                        			Diabetes Mellitus, Type 2
		                        			;
		                        		
		                        			Drug Therapy, Combination
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		                        			Drug-Eluting Stents
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		                        			Hemorrhage
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		                        			Humans
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		                        			Percutaneous Coronary Intervention
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		                        			Platelet Aggregation Inhibitors/therapeutic use*
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		                        			Risk Assessment
		                        			;
		                        		
		                        			Treatment Outcome
		                        			
		                        		
		                        	
9.Cardiometabolic disease patterns among elderly patients with colorectal cancer in China.
Wang Ping JIA ; Fu Yin KOU ; Hong Li XU ; Ke HAN ; Wen Zhe CAO ; Sheng Shu WANG ; Yang SONG ; Shan Shan YANG ; Ya Feng YAN ; Feng CAO ; Wan Guo XUE ; Ya Bin WANG ; Wen Wen MENG ; Miao LIU ; Yao HE
Chinese Journal of Oncology 2022;44(2):173-177
		                        		
		                        			
		                        			Objective: To explore the distribution patterns of cardiometabolic diseases (CMD) in elderly patients with colorectal cancer, and provide a reference for the prevention and treatment of cardiovascular metabolic diseases in these patients. Methods: Clinical data of 3 894 elderly patients with colorectal cancer from January 2008 to March 2018 admitted in the Chinese PLA General Hospital were recruited and the incidence rate of CMD was retrospectively analyzed. The influence factors of elderly patients with colorectal cancer combined with CMD were analyzed by multivariate Logistic regression model. Results: The morbidity rate of CMD in elderly patients with colorectal cancer is 33.4% (1 301/3 894), among them, the morbidity rate of the male was 31.9% (768/2 409), and that of the female was 35.9% (533/1 485). There was not significant difference between these two sex (P=0.074). The morbidity rates of CMD in patients of 65-74 years, 75-84 years and ≥85 years were 30.6% (754/2 462), 37.0% (479/1 294) and 49.3% (68/138), respectively, with significant differences (P<0.001). Multiple Logistic regression analysis revealed that female (OR=1.213, 95%CI: 1.056-1.394), age (75-84 years group: OR=1.344, 95%CI: 1.164-1.552; ≥85 years group: OR=2.345, 95%CI: 1.651-3.331) and body mass index (BMI 18.5-24.9 kg/m(2) group: OR=1.319, 95%CI: 1.065-1.638; ≥25 kg/m(2) group: OR=2.041, 95%CI: 1.627-2.561) were independent risk factors for elderly colorectal cancer patients with CMD. Conclusion: The morbidity rate of CMD in elderly patients with colorectal cancer increases with age and it is urgent to strengthen multidisciplinary cooperation and develop reasonable treatment plans to extend the survival and life quality of these patients.
		                        		
		                        		
		                        		
		                        			Aged
		                        			;
		                        		
		                        			Aged, 80 and over
		                        			;
		                        		
		                        			Cardiovascular Diseases
		                        			;
		                        		
		                        			China/epidemiology*
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		                        			Colorectal Neoplasms
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		                        			Female
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Male
		                        			;
		                        		
		                        			Retrospective Studies
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		                        			Risk Factors
		                        			
		                        		
		                        	
10.Effects of non-muscle myosin Ⅱ silenced bone marrow-derived mesenchymal stem cells transplantation on lung extracellular matrix in rats after endotoxin/lipopolysaccharide-induced acute lung injury.
Xi YIN ; Wan Fang ZHOU ; Wen Jia HOU ; Ming Zhi FAN ; Guo Sheng WU ; Xiao Bin LIU ; Qi Min MA ; Yu Song WANG ; Feng ZHU
Chinese Journal of Burns 2022;38(5):422-433
		                        		
		                        			
		                        			Objective: To investigate the effects of non-muscle myosin Ⅱ (NMⅡ) gene silenced bone marrow-derived mesenchymal stem cells (BMMSCs) on pulmonary extracellular matrix (ECM) and fibrosis in rats with acute lung injury (ALI) induced by endotoxin/lipopolysaccharide (LPS). Methods: The experimental research methods were adopted. Cells from femur and tibial bone marrow cavity of four one-week-old male Sprague-Dawley rats were identified as BMMSCs by flow cytometry, and the third passage of BMMSCs were used in the following experiments. The cells were divided into NMⅡ silenced group transfected with pHBLV-U6-ZsGreen-Puro plasmid containing small interference RNA sequence of NMⅡ gene, vector group transfected with empty plasmid, and blank control group without any treatment, and the protein expression of NMⅡ at 72 h after intervention was detected by Western blotting (n=3). The morphology of cells was observed by an inverted phase contrast microscope and cells labeled with chloromethylbenzoine (CM-DiⅠ) in vitro were observed by an inverted fluorescence microscope. Twenty 4-week-old male Sprague-Dawley rats were divided into blank control group, ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group according to the random number table, with 5 rats in each group. Rats in blank control group were not treated, and rats in the other 3 groups were given LPS to induce ALI. Immediately after modeling, rats in ALI alone group were injected with 1 mL normal saline via tail vein, rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were injected with 1×107/mL BMMSCs and NMⅡ gene silenced BMMSCs of 1 mL labelled with CM-DiⅠ via tail vein, and rats in blank control group were injected with 1 mL normal saline via tail vein at the same time point, respectively. At 24 h after intervention, the lung tissue was collected to observe intrapulmonary homing of the BMMSCs by an inverted fluorescence microscope. Lung tissue was collected at 24 h, in 1 week, and in 2 weeks after intervention to observe pulmonary inflammation by hematoxylin eosin staining and to observe pulmonary fibrosis by Masson staining, and the pulmonary fibrosis in 2 weeks after intervention was scored by modified Ashcroft score (n=5). The content of α-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), and MMP-9 was detected by immunohistochemistry in 2 weeks after intervention (n=3), the activity of superoxide dismutase (SOD), malondialdehyde, myeloperoxidase (MPO) was detected by enzyme-linked immunosorbent assay at 24 h after intervention (n=3), and the protein expressions of CD11b and epidermal growth factor like module containing mucin like hormone receptor 1 (EMR1) in 1 week after intervention were detected by immunofluorescence staining (n=3). Data were statistically analyzed with one-way analysis of variance, Bonferroni method, and Kruskal-Wallis H test. Results: At 72 h after intervention, the NMⅡprotein expression of cells in NMⅡ silenced group was significantly lower than those in blank control group and vector group (with P values <0.01). BMMSCs were in long spindle shape and grew in cluster shaped like vortexes, which were labelled with CM-DiⅠ successfully in vitro. At 24 h after intervention, cell homing in lung of rats in ALI+NMⅡ silenced BMMSC group was more pronounced than that in ALI+BMMSC group, while no CM-DiⅠ-labelled BMMSCs were observed in lung of rats in blank control group and ALI alone group. There was no obvious inflammatory cell infiltration in lung tissue of rats in blank control group at all time points, while inflammatory cell infiltration in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly less than that in ALI alone group at 24 h after intervention, and alveolar wall turned to be thinner and a small amount of congestion in local lung tissue appeared in rats of the two groups in 1 week and 2 weeks after intervention. In 1 week and 2 weeks after intervention, collagen fiber deposition in lung tissue of rats in ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group was significantly aggravated compared with that in blank control group, while collagen fiber deposition in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly improved compared with that in ALI alone group. In 2 weeks after intervention, modified Ashcroft scores for pulmonary fibrosis of rats in ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group were 2.36±0.22, 1.62±0.16, 1.06±0.26, respectively, significantly higher than 0.30±0.21 in blank control group (P<0.01). Modified Ashcroft scores for pulmonary fibrosis of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were significantly lower than that in ALI alone group (P<0.01), and modified Ashcroft score for pulmonary fibrosis of rats in ALI+NMⅡ silenced BMMSC group was significantly lower than that in ALI+BMMSC group (P<0.01). In 2 weeks after intervention, the content of α-SMA in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were significantly decreased compared with that in ALI alone group (P<0.05 or P<0.01). The content of MMP-2 in lung tissue of rats in the 4 groups was similar (P>0.05). The content of MMP-9 in lung tissue of rats in ALI alone group was significantly increased compared with that in blank control group (P<0.01), and the content of MMP-9 in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI alone group (P<0.01). At 24 h after intervention, the activity of malondialdehyde, SOD, and MPO in lung tissue of rats in ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group were significantly increased compared with that in blank control group (P<0.01), the activity of malondialdehyde in lung tissue of rats in ALI+NMⅡ silenced BMMSC group and the activity of SOD in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were significantly increased compared with that in ALI alone group (P<0.05 or P<0.01), and the activity of SOD in lung tissue of rats in ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI+BMMSC group (P<0.01). The activity of MPO in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI alone group (P<0.01), and the activity of MPO in lung tissue of rats in ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI+BMMSC group (P<0.01). In 1 week after intervention, the protein expression of CD11b in lung tissue of rats in ALI+NMⅡ silenced BMMSC group was significantly increased compared with those in the other three groups (P<0.05 or P<0.01), while the protein expressions of EMR1 in lung tissue of rats in the four groups were similar (P>0.05). Conclusions: Transplantation of NMⅡ gene silenced BMMSCs can significantly improve the activity of ECM components in the lung tissue in LPS-induced ALI rats, remodel its integrity, and enhance its antioxidant capacity, and alleviate lung injury and pulmonary fibrosis.
		                        		
		                        		
		                        		
		                        			Acute Lung Injury/therapy*
		                        			;
		                        		
		                        			Animals
		                        			;
		                        		
		                        			Bone Marrow
		                        			;
		                        		
		                        			Collagen/metabolism*
		                        			;
		                        		
		                        			Endotoxins
		                        			;
		                        		
		                        			Extracellular Matrix
		                        			;
		                        		
		                        			Lipopolysaccharides/adverse effects*
		                        			;
		                        		
		                        			Lung
		                        			;
		                        		
		                        			Male
		                        			;
		                        		
		                        			Malondialdehyde/metabolism*
		                        			;
		                        		
		                        			Matrix Metalloproteinase 2/metabolism*
		                        			;
		                        		
		                        			Matrix Metalloproteinase 9/metabolism*
		                        			;
		                        		
		                        			Mesenchymal Stem Cells/metabolism*
		                        			;
		                        		
		                        			Myosin Type II/metabolism*
		                        			;
		                        		
		                        			Pulmonary Fibrosis
		                        			;
		                        		
		                        			Rats
		                        			;
		                        		
		                        			Rats, Sprague-Dawley
		                        			;
		                        		
		                        			Saline Solution/metabolism*
		                        			;
		                        		
		                        			Superoxide Dismutase/metabolism*
		                        			
		                        		
		                        	
            
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