Objective To explore the transfection efficiency of primary lymphocytes from human peripheral blood by different methods to acquire the method with higher transfection efficiency.Methods Mononuclear cells from human peripheral blood were isolated using Ficoll-Hypaque.Cell viability was detected by Trypan blue staining.Suspending lymphocytes were sucked out and were incubated in 24-well plate after cultured in 6-well plate for 2 h.Activated lymphocytes were transfected by electroporation with plasmid(PEGFP-N1).Resting or activated lymphocytes were transfected by lentivirus vector(LVGFP) single infection or repeated infection,respectively.Green fluorescence protein (GFP) was detected under the fluorescence microscopy and percentage of positive cells was checked by flow cytometry at different time points after infection.At the same time,the effectiveness of lentivirus infection was compared under different conditions.Results Purity of mononuclear cells isolated by Ficoll-Hypaque was 95 ％ and its viability was over 95 ％.The percentage of lymphocytes obtained with a uniform shape was 90 ％-95 ％.Scattered fluorescence was observed by electroporation under the conditions of voltage 2 100 V,pulse width 10 ms,pulse number 1 for lymphocyte,while fluorescent became weaker over time and no green fluorescent was observed after transfection for 72 h.After resting lymphocytes were infected once for 48 h by lentivirus vector,green fluorescent was not found and positive cells were less than 1％.1％-5 ％ of activated lymphocytes could express GFP after single lentivirus infection and the expression levels were enhanced with concentration increasing,while 5 ％-10 ％ of activated lymphocytes showed strong green fluorescent by repeated lentivirus infection.In contrast with electroporation,the fluorescent with lentivirus infection was stronger over time.Conclusion Repeated lentivirus infection could efficiently transfect exogenous genes into activated lymphocytes for stable expression.

Objective To investigate the effects of B lymphocyte-induced maturation protein-1 ( Blimp-1) on the number and function of splenic lymphocytes.Methods The mice with defective Blimp-1 in T cells were generated by cross-breeding B6.Blimp-1flox/flox mice with B6.Lck-Cre mice.The mononuclear lymphocytes isolated from spleen of T cell conditional Blimp-1 knockout (Blimp-1CKO) mice and wild type ( WT) C57/B6 mice were comparatively analyzed.Alterations of CD4+T and CD8+T cell subsets, the secre-tion of cytokines as well as the expression of C-C chemokine receptor type 7 ( CCR7 ) and Sphingosine-1-phosphate receptor 1 (S1P1) in mice from the two groups were analyzed by flow cytometry.The changes of CD19+B cell subsets were also detected.Results Compared with WT mice, the total numbers of mononu-clear cells, T and B lymphocytes were all significantly increased in Blimp-1CKO mice ( P<0.05) .The ab-solute numbers of CD4+T, CD8+T and CD19+CD5+CD1d+B cells in mice form Blimp-1CKO group were higher than those of the control group (P<0.05), however, no significant differences with the percentages of these cell populations were observed between two groups.Higher numbers and percentages of CD19+CD5+B cells were detected in mice from Blimp-1CKO group (P<0.01).The Blimp-1CKO mice showed increased secretion of IFN-γ, TNF-α, IL-17 and IL-2, but decreased expression of CCR7 on CD8+T cells as com-pared with WT mice (P<0.05).No significant differences with the changes of S1P1 were found between the two groups.Conclusion Blimp-1 played an important role in the maintenance of number, phenotype and function of T cells.Furthermore, not only T cells but also B cell subsets in mice were affected by the dele-tion of Blimp-1 in T cells.

Objective Most of the clinical manifestations of aortic dissection and myocardial infarction are chest pain,which can easily lead to misdiagnosis and disastrous consequences.Therefore,this study intends to establish a differential diagnosis model and verify it in order to achieve early accurate prediction.Methods The relevant information of 200 patients with myocardial infarction and 120 pa-tients with aortic dissection diagnosed in the Second Affiliated Hospital of Zhengzhou University was collected,including age,gender,blood routine examination,electrolytes,markers of myocardial necrosis and blood coagulation function at admission.The patients were di-vided into myocardial infarction group and aortic dissection group.The independent risk factors were found out through t-test,AN OVA and binary Logistic regression analysis,and the nomogram was further drawn using R language to develop and validate the differential diag-nosis scoring table.Results The procalcitonin,prothrombin time(PT)、international normalized ratio(INR)、fibrin degradation product(FDP),D-dimer,white blood cell(WBC),percentage of neutrophil,percentage of lymphocyte,absolute value of neutrophil,absolute value of lymphocyte,C-reactive protein,cardiac troponin T(cTNT)、creatine kinase isozyme(CK-MB),systolic blood pressure of pa-tients in the two groups were statistically significant(P＜0.05),There was no significant difference in other indexes(P＞0.05).Binary Logistic regression analysis further showed that procalcitonin,D-dimer,C-reactive protein and systolic blood pressure were independent risk factors for diagnosing aortic dissection,while percentage of lymphocyte and absolute value of lymphocyte were independent risk factors for diagnosing myocardial infarction.According to the validation results of the score table developed by the nomogram,the the area under the receiver operating characteristic curve was 0.978,and the best cut-off value was 40.70 points.The sensitivity and specificity were 92.5％and 96.0％.Conclusion This study confirms that procalcitonin,D-dimer,C-reactive protein and systolic blood pressure are independent risk factors for diagnosing aortic dissection,while percentage of lymphocyte and absolute value of lymphocyte are independent risk factors for diagnosing myocardial infarction.The differential diagnosis scoring table proposed in this study can effectively differentiate patients with aortic dissection and myocardial infarction at an early stage,so as to guide further clinical diagnosis and treatment.

Surgical resection is the only effectively curative option for patients with colorectal liver metastases (CRLM).However,fewer than 20％ of those patients are deemed eligible for surgery when they were initially diagnosed.Conversion therapy,mainly used by chemotherapy,increases the opportunities of surgical treatment for patients with initially unresectable CRLM.Notably,sufficient future liver remnant (FLR)is a key factor for liver resection.The deficiency of FLR could lead to serious complications including postoperative liver failure.Surgical methods,represented by portal vein embolization (PVE),portal vein ligation (PVL) and associating liver partition and portal vein ligation for staged hepatectomy (ALPPS),could increase FLR and subsequently further increase the surgical security for CRLM patients.Most CRLM patients accept systemic chemotherapy before surgery.With extensive research has revealed the chemotherapy-induced liver injury,more attention has been shifted to explore the influence of different chemotherapy regimens,chemotherapy cycles and timing of surgery on the volume of FLR.With this review,we overview the research status of conversion therapy for initial unresectable CELM and the influence of chemotherapy on the volume of FLR.

Objective To evaluate the effect of ditching for drain on the control of the breed of Oncomelania hupensis snails in beaches of Dongting Lake. Methods From November,2009 to November,2012,an O. hupensis snail infested beach of the Yueyang jail and an O. hupensis snail infested beach of Junshan District were selected as research fields in the eastern Dongting Lake area,and the former,as the intervention field,was performed with the ditching for drain by excavators and the latter,as the control field,was not. Results Before the project implemented,the average soil moisture contents on the beaches in dry seasons of the two fields were both about 35.56%. After the project implemented,in the intervention field,the average soil mois?ture content was 26.53%which was significantly lower than that(35.56%)in the control field(F=6.53,P<0.05). The under?ground water levels in different heights in the intervention field were lower than those in the control field (χ2 = 33.33,P <0.05). Before the project implemented,the natural death rates of the snails were 0.98%and 0.89%in the two research fields re?spectively(P>0.05),and after the project implemented(in 2012),no adult and young snails were found in the interventional field,but in the control field,the average densities of living snails and young snails were 29.37 snails/0.1 m2 and 213±108.45 snails/0.1 m2 respectively. Conclusion The intervention of ditching for drain can decrease the soil moisture contents quickly and change the ecological condition,therefore,can control the breed of O. hupensis snails in the beaches of Dongting Lake.

CD5-positive diffuse large B-cell lymphoma (CD5+DLBCL) is a special type of DLBCL, which is characterized with later clinical staging, high-risk of relapse in extranodular tissues like bone marrow and central nervous system (CNS).Combined chemotherapy including rituximab and salvage autotransplantation/ allotransplantation can not significantly improve the prognosis. This article reviews the clinicopathological features, the possible pathogenesis, treatment status and dilemma in order to get the better understanding of CD5+DLBCL and avail the early diagnosis and individualized treatment.

The salary reform of public hospitals has a positive incentive effect on enhancing the public welfare of public hospitals, stimulating the enthusiasm of medical staff, and promoting high-quality development. The authors introduced the practice and exploration of the sequential salary system reform in Union Hospital, Tongji Medical College of Huazhong University of Science and Technology since 2018. It included the reform of physician performance based on medical groups, the reform of nursing performance based on vertical nursing management, the reform of medical technology performance based on resource marginal utilization, and the reform of management performance based on job responsibilities and 360°evaluation. The practice has showed that the salary reform could promote the improvement of medical service ability, improve nurses′ satisfaction, shorten the waiting time for medical technical examination, and steadily improve the performance of " National Examination" and the comprehensive ranking of hospitals. When studying and implementing the policy requirements of salary system reform, hospitals in different regions should take measures in accordance with local conditions, with accurate and safe information systems as the support, and focus on the distribution mode, compensation structure and funding sources.

Objective To evaluate the influence of fluoride on mitochondrial membrane potential of neuroblastoma SH-SY5Y cells,and on the expression levels of mitochondrial proteins mitofusion 1 (Mfn1) and fission 1 (Fis1).Methods A stable and feasible culture method of SH-SY5Y cells in vitro was established with different concentration of sodium fluoride [0.0 (control),0.4,2.0 and 4.0 mmol/L],and various periods exposure of 6,12,24,48 h;the mitochondrial membrane potential of SH-SY5Y cells was detected by mitochondrial membrane potential assay kit (JC-1);and the expression levels of Mfn1 and Fis1 proteins were detected by Western blotting.Results Compared with the control group (1.63 ± 0.18,1.13 ± 0.15,1.30 ± 0.02) for various periods exposure (6,12,48 h),the red/green fluorescence ratios of the mitochondrial membrane potential of SH-SY5Y cells exposed to 2.0 and 4.0 mmol/L of sodium fluoride were decreased significantly (1.01 ± 0.10,0.80 ± 0.04;0.75 ± 0.13,0.62 ± 0.10;0.82 ± 0.01,0.56 ± 0.04,P ＜ 0.05);compared with the control group (0.93 ± 0.03,1.05 ± 0.07,1.17 ± 0.04) for various periods exposure,the expression levels of mitochondrial Mfn1 protein were decreased significantly in 0.4,2.0,4.0 mmol/L sodium fluoride groups (6,12,48 h:0.75 ± 0.02,0.65 ± 0.05,0.57 ± 0.06;0.83 ± 0.06,0.79 ± 0.06,0.69 ±0.06;0.98 ± 0.05,0.73 ± 0.07,0.62 ± 0.09,P ＜ 0.05).Compared with the control group (0.90 ± 0.05) for exposure time 12 h,the expression levels of Fis1 protein were increased significantly in 2.0,4.0 mmol/L sodium fluoride groups (1.14 ± 0.06,1.23 ± 0.06,P ＜ 0.05).Conclusions The mitochondrial membrane potential and the expression levels of mitofusion 1 and fission 1 of SH-SY5Y cells treated with fluoride are abnormal,which might be associated with the theory of nerve cell damage from high oxidative stress.

Objective To investigate the influence of inhibited gene expression of fisson 1 (Fis1) gene on the level of Fis1,mitofusin 1 (Mfn1) and mitochondrial membrane potential in SH-SY5Y cells with fluorine,to study the role of mitochondrial dynamic balance in the pathogenesis of chronic fluorosis.Methods SH-SY5Y cells were cultured in vitro,when adherent cells entered the logarithmic phase,using a group design,they were divided into four groups:blank control group (control),fluoride group [2 mmol/L sodium fluoride (NaF)],fluoride negative control group (2 mmol/L NaF + non-specific siRNA) and the gene-silencing group (2 mmol/L NaF + specific siRNA-Fis1).The protein expression levels of Fis1 and Mfn1 were measured by Western blotting;the mRNA expression levels of Fis1 and Mfn1 were measured by Real-time PCR;and the levels of the mitochondrial membrane potential was detected by mitochondrial membrane potential detection kit.Results Compared with control (1.37 ± 0.18,1.00 ± 0.04;1.57 ± 0.19,1.00 ± 0.04;1.00 ± 0.10),the expression levels of Fisl protein (1.72 ± 0.04) and mRNA (1.48 ± 0.13) in fluoride group were increased,the expression levels of Mfn1 protein (0.87 ± 0.02) and mRNA (0.69 ± 0.07) in fluoride group were decreased,the level of mitochondrial membrane potential (0.76 ± 0.13) was decreased (P ＜ 0.05).Compared with control,the expression levels of Fis1 protein (0.79 ± 0.07) and mRNA (0.06 ± 0.03) in gene-silencing group were decreased,the expression levels of Mfn1 protein (1.71 ± 0.04) and mRNA (1.52 ± 0.05) in gene-silencing group were increased (P ＜ 0.05),the level of mitochondrial membrane potential (0.94 ± 0.01) was decreased.Compared with fluoride group,the expression levels of Fis1 protein and mRNA in gene-silencing group were decreased,the expression levels of Mfn1 protein and mRNA in gene-silencing group were increased,the level of mitochondrial membrane potential in gene-silencing group was increased (P ＜ 0.05).Conclusion Gene expression inhibition of Fis1 gene can reduce the mitochondrial division and damage of mitochondrial membrane potential in SH-SY5Y cells induced by fluoride.

BACKGROUND: Chronic kidney disease is a severe threat to human health with no ideal treatment strategy. Mature mammalian kidneys have a fixed number of nephrons, and regeneration is difficult once they are damaged. For this reason, developing an efficient approach to achieve kidney regeneration is necessary. The technology of the combination of decellularized kidney scaffolds with stem cells has emerged as a new strategy; however, in previous studies, the differentiation of stem cells in decellularized scaffolds was insufficient for functional kidney regeneration, and many problems remain. METHODS: We used 0.5% sodium dodecyl sulfate (SDS) to produce rat kidney decellularized scaffolds, and induce adipose-derived stem cells (ADSCs) into intermediate mesoderm by adding Wnt agonist CHIR99021 and FGF9 in vitro. The characteristics of decellularized scaffolds and intermediate mesoderm induced from adipose–derived stem cells were identified. The scaffolds were recellularized with ADSCs and intermediate mesoderm cells through the renal artery and ureter. After cocultured for 10 days, cells adhesion and differentiation was evaluated. RESULTS: Intermediate mesoderm cells were successfully induced from ADSCs and identified by immunofluorescence and Western blotting assays (OSR1 + , PAX2 +). Immunofluorescence showed that intermediate mesoderm cells differentiated into tubular-like (E-CAD + , GATA3 +) and podocyte-like (WT1 +) cells with higher differentiation efficiency than ADSCs in the decellularized scaffolds. Comparatively, this phenomenon was not observed in induced intermediate mesoderm cells cultured in vitro. CONCLUSION: In this study, we demonstrated that intermediate mesoderm cells could be induced from ADSCs and that they could differentiate well after cocultured with decellularized scaffolds.
Animals ; Blotting, Western ; Fluorescent Antibody Technique ; Humans ; In Vitro Techniques ; Kidney ; Mesoderm ; Nephrons ; Rats ; Regeneration ; Renal Artery ; Renal Insufficiency, Chronic ; Sodium Dodecyl Sulfate ; Stem Cells ; Ureter