BACKGROUND:The research of dental stem cells in the fields of regenerative medicine and tissue engineering has been deepening,bringing hope for the repair of tooth-related tissues and the treatment of systemic diseases.However,there is a lack of systematic research and analysis on the biological characteristics of dental stem cells in different age groups. OBJECTIVE:To explore the biological characteristics of the human deciduous tooth and permanent tooth pulp stem cells cultured in umbilical cord blood platelet lysate to provide a reliable basis for human platelet lysates to replace fetal bovine serum. METHODS:The pulp tissues of deciduous teeth,juvenile permanent teeth and adult permanent teeth were taken out and cultured in DMEM/F-12 medium supplemented with 10%fetal bovine serum or different concentrations(5%,10%and 15%)of human platelet lysates.Cell proliferation in the four groups was detected by cytometry.The optimal concentration of human platelet lysates was selected for subsequent experiments.Under the optimal concentration of human platelet lysates,human deciduous tooth and juvenile and adult permanent tooth pulp stem cells were cultured in vitro.The cell growth status was observed under the microscope.The specific antigen on the cell surface was detected by flow cytometry.The cell proliferation ability was tested by the cell counting method and CCK-8 assay.The cell differentiation ability in vitro was observed by a three-line differentiation assay. RESULTS AND CONCLUSION:(1)The cell proliferation rate of the 10%human platelet lysate group was the highest.(2)In all three groups,fusiform fibrous cells grew and expanded from around the tissue block.There was no significant difference between deciduous teeth and juvenile permanent tooth cells,but the adult permanent tooth cells were larger than the deciduous and juvenile permanent tooth cells of the same generation.(3)The results of flow cytometry showed that deciduous teeth,juvenile permanent teeth and adult permanent teeth conformed to the phenotypic characteristics of mesenchymal stem cells.(4)The proliferative capacity of adult permanent dental pulp stem cells was significantly lower than those of deciduous teeth and juvenile permanent dental pulp stem cells(P<0.01).(5)mRNA expressions of osteoblast-related genes alkaline phosphatase and bone morphogenetic protein 2,lipoprotein lipase and peroxisome proliferator-activated receptor γ2,mRNA expressions of chondroblast related gene type II collagen α1 and cartilage oligomeric matrix protein in adult pulp stem cells of permanent teeth were significantly lower than those of deciduous teeth and juvenile permanent teeth pulp stem cells(P<0.01).(6)Compared with adult dental pulp stem cells,human deciduous teeth and juvenile permanent teeth dental pulp stem cells have the stronger proliferative capacity and multidirectional differentiation potential,and are more suitable for clinical research and disease treatment.

Objective To compare the early embryonic developmental potential and clinical outcomes of oocytes matured in vivo and those matured by modified in vitro maturation(LVM)technology during the same controlled ovarian hyperstimulation(COH)cycle,and to explore the clinical application of melatonin-supplemented IVM technology.Methods 159 patients were recruited into the study.920 mature oocytes were collected during their COH cycles processed for conventional IVF/ICSI protocols,while 1 283 immature oocytes from the same cycles were matured in a melatonin-supplemented IVM medium before ICSI was performed.A retrospective analysis was conducted to compare the impact of conventional assisted reproductive technology and improved IVM technology on the outcomes of assisted reproductive therapy and pregnancy outcomes.Results Compared with mature oocytes collected from COH cycles treated with conventional IVF/ICSI,oocytes promoted by improved melatonin-supple-mented IVM technology had a lower rate of high-quality blastocyst formation.However,after embryo transfer,there was no significant difference in the clinical outcomes of mature oocytes obtained through two methods,including clinical pregnancy rate,full-term birth rate,neonatal length,and neonatal Apgar score.Conclusion The applica-tion of melatonin-supplemented IVM significantly increases the utilization of immature oocytes collected from COH cycles,improving the pregnancy outcomes of patients assisted by assisted reproductive technology.

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.
Humans ; Apolipoprotein E4/genetics* ; Cytosine ; Mutation ; Blastocyst ; Heterozygote ; Gene Editing ; CRISPR-Cas Systems

Researches have shown that lung injury due to excessive spontaneous breathing effort, that is, patient self-inflicted lung injury (P-SILI), may be the important manifestation and possible mechanism of ventilation-associated lung injury and ventilation-induced diaphragmatic dysfunction in acute respiratory distress syndrome (ARDS) patients who were mechanically ventilated with intense spontaneous breathing. This paper reviews the concept of P-SILI, possible occurrence mechanism, clinical significance, and prevention and treatment, in order to provide more ideas for clinical ARDS management.

Objective To investigate the clinical therapeutic effect of sigmoid neobladder after laparoscopic radical cystectomy in the treatment of invasive bladder cancer .Methods 21 patients with invasive bladder cancer accepted sigmoid neobladder after laparoscopic radical cystectomy and pelvic lymphadenectomy .The postoperative complications and clinical therapeutic effect were analyzed .Results All surgeries were completed successfully .The operation time was (245 ±125) min,blood lost was (98 ±45) mL.All the 21 patients were followed up for 3-24 months,mean of 12 months.Both side mild hydronephrosis in 6 cases (28.6%) and unilateral hydronephrosis in 1 case(0.5%).Average bladder reservoir was (548.8 ±221.6)mL,the maximum bladder pressure during the new bladder urine storage period was (2.26 ±0.24) kPa,the maximum bladder pressure during voiding period was (6.18 ±0.29) kPa,volume of residual urine was (87 ±52) mL.The mean maximum uroflow rate was (83.2 ± 5.6)mL/s.The continence of micturition was 90.4% in day-time and 76.1% in night-time.1 case had urethral stricture and got better after the urethral dilatation .Conclusion The sigmoid neobladder after laparoscopic radical cystectomy has more reliable results,without serious postoperative complications and patients with high quality of life after operation.This operation may be applied in clinical practice .

Background Studies showed that microRNA (miR)-375 suppresses the growth,apoptosis,migration and adhesion of tumor cells,and it plays a regulation to the changes of vascular endothelial growth factor (VEGF) in tumor tissue to arrest neovascularization.However,whether miR-375 intervenes the formation of new blood vessel in eyes is unelucidated.Objective This study was to explore the effects of miR-375 on human retinal capillary endothelial cells (HRCECs) function induced by hypoxia.Methods HRCECs were cultured using IMDM and divided into normal control group,CoCl2 model group,CoCl2 +miR-375 mimic group,CoCl2 +miR-375 mimic control group,CoCl2+miR-375 inhibitor group and CoCl2 +miR-375 inhibitor control group,and hypoxia cell models were created by adding 200 μmol/L CoCl2.MiR-375 and frizzled 4 (FZD4) small interfering RNA (siRNA) were transfected into the cells by 50 nmol/L miRNA lipidosome for 48 hours.The proliferation of the cells was detected by MTT assay;migrated number of the cells was examined by Transwell chamber assay;ELISA was employed to detect the concentrations of VEGF and VE-cadherin in the medium;the expression of β-catenin,cyclinD1,matrix metalloproteinase-2 (MMP2) and VEGF proteins were analyzed by Western blot;tube length of vessel formation was evaluated by Matrigel assay.Cultured cells were divided into normal control group,CoCl2 model group,CoCl2 +mock group and CoCl2 + FZD4 siRNA group,the relative expression of FZD4,a miR-375 targeted gene,was detected by luciferase reporter.Results The relative expression of miR-375 mRNA was significantly increased in the CoCl2 +miR-375 mimic group compared with the CoCl2 + miR-375 mimic control group and reduced in the CoCo2 + miR-375 inhibitor group compared with the CoCo2 + miR-375 inhibitor control group (t =-19.237,8.764,both at P＜0.01),with a higher transfected efficacy for miR-375.The cell proliferative fold,migrated cell number,VEGF and VE-Cadherin contents in the medium and the tube length were significantly different among the CoCl2 model group,CoCl2 +miR-375 mimic group,CoCl2 +miR-375 mimic control group,CoCl2 +miR-375 inhibitor group and CoCl2 +miR-375 inhibitor control group (F =24.324,26.776,14.113,19.225,15.040,all at P＜0.001),and those in the CoCl2 +miR-375 mimic group were evidently reduced in the CoCl2 +miR-375 mimic group compared with the CoCl2 +miR-375 mimic control group,while those in the CoCl2 +miR-375 inhibitor group were considerably elevated in comparison with the CoCl2 +miR-375 inhibitor control group (all at P＜0.01).The expressions of β3-catenin,cyclinD1,MMP2 and VEGF protein were significantly different among the normal control group,CoCl2 model group,CoCl2 +miR-375 mimic group,CoCl2+miR-375 mimic control group,CoCl2 +miR-375 inhibitor group and CoCl2 +miR-375 inhibitor control group (F=11.753,13.283,16.770,10.334,all at P＜0.001).In addition,the cell proliferative fold,migrated cell number and the tube length were significantly increased in the CoCl2 model group and CoCl2+mock group,and those in the CoCl2+FZD4 siRNA group were decreased in comparison with the CoCl2 +mock group (all at P＜0.05).Conclusions MiR-375 inhibits the growth,migration and tube formation ability of HRCECs in hypoxic status probably by regulating the activation of Wnt pathway via directly targeting FZD4.

Objective To compare the clinical efficacy of subinguinal microsurgical and single -port laparoscopic high ligation in the treatment of varicocele.Methods 218 patients with varicocele were enrolled in this study.According to the digital table,they were randomly divided into two group.148 cases were treated by subinguinal microscopic varicocelectomy(microscopic group),70 cases were treated by single -port laparoscopic high ligation varicocele(single -port laparoscopic group).Postoperative follow up was 3 -24months.The operative duration,length of hospital stay,hospitalization expense,postoperative complications and semen quality parameters were compared between the two groups.Results There were statistically significant differences in operative duration and hospitalization expense between the two groups(all P <0.05).There was no statistically significant difference in length of hospital stay (P >0.05).In the 218 followed -up patients,the sperm concentration and motility (grade a + b sperm)all significantly improved,which of the microscopic group and single -port laparoscopic group preoperation were (19.1 ± 8.2)×106 /mL,(18.2 ±7.9)×106 /mL and (22.7 ±7.8)%,(21.6 ±8.9)% respectively,which at 3 -6 months after operation were (56.2 ±10.8)×106 /mL,(45.8 ±12.9)×106 /mL and (58.8 ±9.7)%,(44.6 ±10.7)%, there were statistically significant differences compared with preoperation (t =6.227,9.579,all P <0.05 ). Conclusion The surgical methods in the treatment of varicocele can improve the quality of patients,but microscopic group is obviously better than single -port laparoscopic group in improvement of semen quality parameters,safety, patient -based compliance and economy.

Objective To explore the effect and potential mechanism of suppressors of cytokine signaling 3 (SOCS3) knockout on cell viability and apoptosis of retinal ganglion cells (RGCs) after optic nerve injury.Methods The optic nerve transection was used to construct optic nerve injury model of rats,and RGCs were isolated after optic nerve injury.The experimental animals were divided into optic nerve transection injury (ONT) group and sham-operation (Sham) group.The expression of SOCS3 in RGCs was detected by Western blot and RT-PCR in each group.Subsequently,SOCS3 siRNA was transfected into RGCs of Sham group and ONT group,and the experimental were further subdivided into blank control group,negative control group and SOCS3 silence groups.Cell viability was measured by CCK8 and MTr methods.Apoptosis was detected by Hoechst 33342 staining and flow cytometry.Furthermore,the mTOR siRNA and SOCS3 siRNA were co-transfected into RGCs,and cell viability and apoptosis were detected.Results The expression of SOCS3 was dramatically increased at 3 days after injury in the ONT group when compared with Sham group (P =0.049),and it showed an increased tendency gradually along with the extension of injury time.Compared with the blank control in the ONT group,SOCS3 silence markedly promoted cell viability [(49.47 ± 7.35) ％ vs.(73.24 ± 8.70) ％],reduced cell growth inhibition [(27.25 ±0.75)％ vs.(10.96 ± 1.07)％] and apoptosis [(23.06 ± 1.43)％ vs.(10.65 ± 1.77)％].The result of Hoechst 33342 staining indicated that SOCS3 silence ameliorated the cell apoptosis induced by ONT.In addition,SOCS3 silence significantly improved pS6 expression at 2 weeks after injury,and mTOR and SOCS3 co-silence reduced cell viability,increased cell growth inhibition and apoptosis compared with SOCS3 silence group after injury.Conclusion SOCS3 silence promotes injury-induced cell viability of RGCs and suppresses injury-induced apoptosis of RGCs via up-regulating mTOR activity in the later period of injury.

Objective To investigate the expression of gastric and intestinal phenotypic markers in Siewert typeⅡand Ⅲ early gastroesophageal junction(GEJ) cancer, and to explore its correlation with clinic-pathological features.Methods From April 2010 to July 2015, 53 cases diagnosed as early GEJ cancer were enrolled.The gastric and intestinal phenotypic markers such as mucin5AC(MUC5AC),mucin6(MUC6),mucin2(MUC2),caudal related homeodomain transcription 2(CDX2) and cluster of differentiation 10(CD10) were detected, and then the patients were divided into gastric type, gastrointestinal type, intestinal type and non-classified type according to the results of immunohistochemical staining.Combined with Siewert classification the clinicopathological features were analyzed.Chi square test or Fisher′s exact test was performed for statistical analysis.Results In the cancer tissues of 47 patients with Siewert type Ⅱand Ⅲ early GEJ cancer, the case numbers of positive expression of MUC5AC,MUC6,MUC2, CDX2 and CD10 were 21(44.7%),19(40.4%),31(66.0%),27(57.4%) and 17(36.2%),respectively;the case numbers of gastric type, gastrointestinal type, intestinal type and non-classified type were 11(23.4%),14(29.8%),21(44.7%) and one(2.1%), respectively.The positive expression rates of MUC5AC and MUC6 in Siewert typeⅡwere 55.9%(19/34) and 50.0%(17/34),which were higher than those of Siewert typeⅢ(2/13), and the positive expression rate of MUC2 was 55.9%(19/34), which was lower than that of Siewert typeⅢ(12/13), and the differences were statistically significant (x2=6.240,4.679 and 4.053;all P<0.05).In Siewert typeⅡ, the proportion of intestinal type was 32.4%(11/34), which was lower than that of Siewert typeⅢ(10/13), and the differences were statistically significant (x2=7.142,P=0.010).In patients with Siewert typeⅡand Ⅲ early cancer, males predominated in intestinal type which were mostly well differentiated type with less submucosal carcinoma.The maximum diameter of tumor was less than those of gastric type and gastrointestinal type.In paracancerous mucosal tissues, the incidences of intestinal metaplasia in gastrointestinal type and intestinal type were 11/14 and 81.0%(17/21), which were higher than that of gastric type (3/11);the incidences of atrophy in gastrointestinal type and intestinal type were 12/14 and 85.7%(18/21),which were higher than that of gastric type (4/11),and the differences were statistically significant (Fisher′s exact test,all P<0.05).Conclusions Siewert typeⅡand Ⅲ early GEJ cancer can directly originated not only from gastric mucosa, but also from gastrointestinal and intestinal mucosa.Atrophy and intestinal metaplasia could exist before cancer genesis.

Objective To investigate the micro -channel percutaneous nephrolithotomy under ultrasound guidance in clinical studies of renal calculi in non -dilated collecting system.Methods Clinical data of 126 patients with renal calculi in non -dilated collecting system treated by ultrasound -guided percutaneous nephrolithotomy microchannel were retrospectively analyzed.Ureteroscopy up to the renal pelvis or ureter segment,zebra guide wire was placed along the guide wire indwelling ureteral catheter end connected to the blood transfusion needle while external saline suspension 100cm.Ultrasound -guided puncture target cup,established 16 -18F channel percutaneous nephro-lithotomy,ureteroscopy downlink percutaneous nephrolithotomy lithotripsy.The clinical data of operation time,stone clearance rate,blood loss,complications were analyzed.Results 124 patients with successful puncture,puncture fail-ure in two patients,conversion to open surgery.118 cases of successful implementation of a single -channel stone, nine cases of postoperative residual stones was found,again two stone surgery in 6 cases,3 cases refused surgery again;six cases were due to stones larger load,operation time exceeded 2h,to 2 stage of stone surgery in 4 cases, 2 cases changed to standard channel.Operation time was 30 -150min,average 78min.A stone clearance rate was 87.3%,summarize stone clearance rate was 95.2%(118 /124).Postoperative hemoglobin level decreased to (16.0 ± 7.6)g/L,7 patients with postoperative bleeding more blood transfusion 400 -800mL,none embolization.4 cases of postoperative fever,septic shock in 1 case,1 case of pneumothorax.Conclusion The micro -channel percutaneous nephrolithotomy under ultrasound guidance in the treatment of renal calculi in non -dilated collecting system is safe and feasible,it is the preferred method of treating renal calculi in non -dilated collecting system.