BACKGROUND: It is conceivable that bone marrow stem cells can differentiate into smooth muscle cells (SMCs) and contribute to neointimal formation in atherogenesis. However, the mechanism remains unknown. The "milieu-induced-differentiation" hypothesis focuses on the key role of cell-to-cell contact and cytokine on the differentiation of stem cells. Bone marrow mesenchymal stem cells (MSCs) have the potential to differentiate into SMCs.OBJECTIVE: To induce MSCs into SMCs in vitro, and investigate the influence of the differentiated SMCs or cell factors on MSCs differentiation.DESIGN: Controlled experiment in vitro with repeated observation and measurement based on cells.SETTING: Department of Cardiology, First Hospital of Xi'an Jiaotong University.MATERIALS: The experiment was accomplished in the Laboratory of Cardiology, First Hospital of Xi'an Jiaotong University between May 2003 and May 2004. SD rats of either gender were provided by the Animal Center of Xi'an Jiao Tong University, 60-80 g, 90-110 g. The following antibodies were used: Mouse anti human SM-α-actin (NeoMarkers),Mouse anti human Calponin (NeoMarkers), TRITC-coupled goat anti mouse IgG antibody (SBA). Mouse anti rat CD34 conjugated FITC (Santa Cruz), Mouse anti rat CD71 conjugated FITC (Oxford Biotechnology), Mouse anti rat anti-CD90 conjugated PE (Oxford Biotechnology). Lipofectamine 2000 (Invitrogen). PEGFP-N3 (the laboratory).METHODS: Bone marrow mesenchymal stem cells were obtained from rat bone marrow by using percoll density gradient centrifugation. SMCs were isolated by using tissue explantation method. Flow cytometer was used to detect the immunofluorescence stain. Then MSCs and SMCs were identified. MSCs were transfected with pEGFP-N3 by Lipofectamine 2000, while untransfected MSCs were taken as controls. Conditioned culture of MSCs and SMCs: ①MSCs at passage 3 were seeded on chamber slides in a 12-well culture plate. The medium was DMEM containing 0%, 5%,7.5% fetal bovine serum (FBS) and SMCs conditioned medium containing 0%, 5%, 7.5% FBS, respectively. The cells were cultured for 10-14 days and immunofluorescence analysis was performed by using monoclonal antibodies against SM-α-actin, calponin.②Indirect co-culture of MSCs with SMCs were established using a semi-permeable membrane cell culture insert. The inserts were plated into culture well. SMCs were cultured on the inside of inserts while MSCs were added to the outside of inserts, respectively. MSCs were culture alone in medium containing 3%, 7.5% FBS and immunofluorescence analysis was performed by using monoclonal antibodies against SM-α-actin, calponin.③MSCs were transfected with pEGFP-N3. After 24 hours, the MSCs were cocultivated with SMCs at an equal density for 7-14 days.As a control, MSCs were cultured alone. MSCs co-cultured were stained with antibodies against calponin, SM-α-actin. MAIN OUTCOME MEASURES: ①Identification of MSCs by floe cytometer.②cytoplasmic antigen expression of SMCs. RESULTS: ①Immunofluorescence analysis showed that MSCs expressed SM-α-actin, but did not express calponin. As a control, SMCs expressed both SM-α-actin and calponin.②Flow cytometry showed that MSCs expressed CD71 of low level, CD90 of high level and no expression of CD34. ③The MSCs transfected with green fluorescence protein continued to express for 2-3 weeks. ④MSCs grew well in SMCs conditioned medium or different concentrations of FBS. Cell growth was FBS concentration dependent in indirect co-culture system of MSCs and SMCs. Several double-positive cells in direct co-culture system were detected enhanced green fluorescence protein and antibodies against calponin, SM-α-actin. CONCLUSION: ①SMCs conditioned medium and cell factor only promote MSCs growth and cytoplasmic granules increase. But these do not induce MSCs differentiate into SMCs. ②The cell-to-cell contact is essential for MSCs differentiation to SMCs.

Objective To investigate safety and efficacy of hysteroscopy in treatment of cesarean scar pregnancy(CSP).Methods From Aug.2003 to Dec.2011, 33 cases with CSP treated by hysteroscopy guided by transabdominal ultrasound or laparoscopy were studied retrospectively in Women's Hospital,School of Medicine, Zhejiang University.The clinical characteristics including gestational age, myometrial thickness anterior to the CSP, β-hCG level before treatment,success rate, cure rate, operative time, blood loss, time of serum β-hCG resolution and CSP mass clearance, and complication were collected and analyzed.Results Median gestational age was 54 days (range, 37 - 140 days).Median level of β-hCG before treatment was 15 000 U/L( range,3.3 - 151 747 U/L).Mean thickness of anterior myometrium was 3.3 mm.Twenty-nine cases underwent uterine artery embolism (UAE) before hysteroscopy.Pouch in the anterior uterine isthmus with gestation masses implanted were observed in 30 cases (91%, 30/33 ).CSP masses progressed toward the pouch or uterine cavity in all cases was removed by cutting wire loop electrode combined with curettage.The mean operative time was (34 ± 10) minutes.Both success rate and cure rate were 94% ( 31/33 ) .Salvage methotrexate ( MTX ) therapy was administrated in one case.Complication occurred in three cases (9%, 3/33 ).Both massive hemorrhage rate and hysterectomy rate were performed in two cases (6%, 2/33).No uterine perforation occurred.The mean time of hCG resolution was (22 ± 10)days.The mean time of CSP mass clearance was (21 ± 12) days.Four pregnancies were achieved in four cases:one term pregnancy and three abortions.No recurrent CSP occurred.Conclusion Management of CSP by hysteroscopy combined with UAE is safe and effective.

Objective:To investigate the effect of tissue engineered bladder patch constructed by double-layer silk scaffold and adipose-derived stem cells (ADSCs) in the repair and reconstruction of bladder.Methods:This study was conducted from May 2020 to March 2021. The silk fibroin (SF) aqueous solution was obtained from silkworm cocoons, and a double-layer silk scaffold composed of silk fibroin film and silk fibroin sponge was further prepared. The rat ADSCs were isolated, cultured, and the ADSCs surface markers (CD29, CD90, CD45, CD106) were identified by flow cytometry. The ADSCs were planted on a double-layer silk scaffold to construct a tissue-engineered bladder patch. Thirty-six male SD rats were randomly divided into three groups: tissue engineered bladder patch group (SF-ADSCs group, n=15), double-layer silk scaffold group (SF group, n=15), control group ( n=6). The tissue engineered bladder patch (SF-ADSCs group) and double-layer silk scaffold (SF group) were wrapped on the omentum to promote vascularization. The vascularization was evaluated by HE and immunofluorescence staining. The wrapped tissue engineered bladder patch and double-layer silk scaffold were used to repair the defective bladder. In the control group (six rats), the incision was closed immediately after the bladder tissue fully exposed. At 4 weeks and 12 weeks after operation, the general morphology of bladder tissue and cystography were performed to evaluate the recovery of bladder morphology. After the graft was harvested, HE and Masson's trichrome staining and immunofluorescence staining were used to observe the regeneration of bladder wall tissue. Urodynamics was used to assess the recovery of bladder function at 12 weeks after operation. Results:The flow cytometry results confirmed that the isolated cells positively expressed CD29 and CD90, and there was no significant expression of CD45 and CD106. Gross observation and scanning electron microscope confirmed that the preparation of double-layer silk scaffold not only had a pore structure that was conducive to cell planting, but also had good toughness and was facilitated to surgical suture. The number (43.50±2.66) and area (0.73±0.03)% of vascular-like structures in the SF-ADSCs group after the omentum encapsulation was significantly higher than that in the SF group [(24.50±3.51), (0.55±0.05)%], and the difference was statistically significant ( P<0.05). At 4 weeks after bladder repair, the histological staining of the grafts in the SF-ADSCs and SF groups showed a large number of degraded fragments of double-layer silk scaffold. At 12 weeks, the morphology of the graft in the SF-ADSCs group showed uniform bladder morphology, which was similar to that of normal bladder tissue. Immunofluorescence staining showed that the continuous urothelial layer, abundant smooth muscle tissue, vascular structure and regenerated neurons could be observed in the SF-ADSCs group. Urodynamic test showed that the bladder maximum volume (0.74±0.03)ml and compliance (16.68±0.44)μl/cm H 2O in the SF-ADSCs group, which were better than that in the SF group [(0.47±0.05)ml, (14.89±0.37)μl/cm H 2O], but lower than that in the control group [(1.12±0.08)ml, (19.34±0.45)μl/cm H 2O], and the difference was statistically significant ( P<0.05). Conclusions:The tissue engineered bladder patch constructed with double-layer silk scaffolds and ADSCs could promote the morphological repair of bladder tissue, the regeneration of bladder wall structure and the recovery of bladder physiological function.

Objective:To prepare the whole bladder acellular matrix (BAM) using the self-designed perfusion decellularization system, and evaluate the feasibility of constructing the tissue engineering bladder with the adipose-derived stem cells (ADSCs).Methods:This study was conducted from October 2020 to April 2021. The self-designed perfusion decellularization system was used, and four different decellularization protocols (group A, group B, group C and group D) were formulated, according to the flow direction of the perfusate and the action time of different decellularization solutions. Among them, the urethral orifice of the bladder tissue was used as the outflow tract of the perfusion fluid in groups A and B. The top of the bladder was cut off and used as the outflow tract of the perfusion fluid in groups C and D. In groups A and C, 1% Triton X-100 was treated for 6 h, and 1% sodium dodecyl sulfate (SDS) was treated for 2 h. In groups B and D, 1% Triton X-100 was treated for 7 h, and 1% sodium dodecyl sulfate (SDS) was treated for 1 h. In addition, the tissue in the normal bladder group was directly obtained from the natural bladder tissue, which did not require perfusion, cryopreservation and thawing. The fast and efficient decellularization protocol was screened out through HE, DAPI, Masson trichrome and Alcian Blue staining and quantitative analyses to prepare the whole bladder scaffold. The prepared BAM was used as the scaffold material, and the ADSCs were used as the seeding cells to construct the tissue engineering bladder. HE and DAPI staining were used to observe the distribution of ADSCs on the BAM.Results:HE and DAPI staining showed that there was no obvious nuclear residue in the group C. Masson trichrome and Alcian Blue staining showed that the collagen structure and glycosaminoglycan were well preserved in the group C. There was no significant difference in bladder wall thickness between the group C and the normal bladder group [(975.44±158.62)μm vs.(1 064.49±168.52)μm, P > 0.05]. The DNA content in the group C [(43.59 ±4.59) ng/mg] was lower than that in the normal bladder group, group A, group B and group D [(532.50±26.69), (135.17±6.99), (182.49±13.69) and(84.00±4.38)ng/mg], and the difference was statistically significant ( P<0.05). The collagen content [(10.98 ± 0.29)μg/mg] and glycosaminoglycan content [(2.30±0.18)μg/mg] in group C were not significantly different with those in the normal bladder group [(11.69±0.49) and (2.36±0.09)μg/mg, P>0.05]. Scanning electron microscopy showed that a large number of pore structures could be observed on the surface of the prepared BAM in groups A-D and were facilitated to cell adhesion. The isolated and cultured ADSCs were identified by flow cytometry to confirm the positive expression of CD90 and CD29, and the negative expression of CD45 and CD106. Live/dead staining and CCK-8 detection confirmed that the prepared BAM in the group C had no cytotoxicity. HE and DAPI staining showed that a large number of ADSCs were distributed on the surface and inside of the tissue engineering bladder. Conclusions:The whole bladder shape BAM prepared by the self-designed perfusion decellularization system could be used as the scaffold material for bladder tissue engineering, and the constructed tissue engineering bladder could be used for bladder repair and reconstruction.

Objective To investigate the safety and efficacy of hysterosopic management of typeⅡcesarean scar pregnancy (CSP) and the value of prophylactic uterine artery embolization (UAE). Methods Totally 104 patients with typeⅡCSP treated with hysteroscopic surgery at the Women′s Hospital,School of Medicine, Zhejiang University, during Jan. 2009 to Jun. 2016 were analyzed retrospectively, 67 patients combined with UAE (UAE group) and 37 patients without combined with UAE (non-UAE group). Laparoscopy or sonography guidance was conducted simultaneously.The following clinical parameters were compared, including: primary cure rate, uterine packing rate, uterine perforation rate, hemoglobin level change,the time for the mass absorption and the return of β-hCG to normal,complications,hospital days and hospital stay cost.Results Median gestational age,size of mass,thickness of the anterior myometrium and β-hCG level in UAE group versus non-UAE group were 47 versus 47 days,30 versus 30 mm,2 versus 2 mm, 36 524 versus 32 226 U/L(all P>0.05).Out of 104,100 patients were managed successfully with hysteroscopic surgery, and 4 patients transformed to laparoscopic or laparotomy surgery. Hysteroscopic surgery was effective in 63 out of 67 patients(94%)in UAE group and 34 out of 37 patients(92%)in non-UAE group(P>0.05). There was no significant differences regarding uterine perforation rate, uterine packing rate, hemoglobin change and recovery time between UAE group and non-UAE group (all P>0.05). The median hospital day was 7 days in UAE group versus 5 days in non-UAE group(P<0.01).The median hospital stay cost was 13 654 yuan in UAE group versus 9 108 yuan in non-UAE group (P<0.01). Serious complication occurred in 4 patients (6%, 4/67) in UAE group and 2 patients (5%, 2/67) in non-UAE group (P=0.906). Conclusions Hysteroscopic surgery is effective and safe for patients with typeⅡCSP in the first trimester with size≤30 mm in diameter and gestation age<7 weeks.The value of prophylactic UAE is uncertain.

Objective:To explore the application effect of situation comedy teaching combined with peer teaching in clinical practice teaching of undergraduate nursing students.Methods:A total of 96 undergraduate nursing students who practiced in a first-class hospital at grade 3 in 2019 were randomly divided into the observation group and the control group, with 48 nursing students in each group. The control group adopted clinical teaching mode, including group theory teaching and group operation demonstration. On the basis of this, the observation group additionally adopted the clinical teaching mode of situation comedy teaching combined with peer teaching. The differences between the two groups in the assessment of nursing knowledge and skills assessment, independent learning ability and teaching satisfaction were observed. SPSS 20.0 software was used for t-test. Results:After the implementation of clinical teaching, the scores of nursing comprehensive ability of nursing students in the observation group (87.71 ± 5.11) were higher than those in the control group (78.47±6.24) ( P < 0.05). The independent learning ability of nursing students in the observation group (98.80±10.61) was significantly higher than that in the control group (74.47±9.83), and the difference was statistically significant ( P < 0.05). The score of teaching satisfaction in the observation group (2.83±7.07) was significantly higher than that in the control group (50.17±6.75), and the difference was statistically significant ( P < 0.05). Conclusion:The application of situation domedy teaching combined with peer teaching in the clinical teaching of undergraduate nursing students can improve their independent learning ability and clinical practice ability. Meanwhile, the process of teachers and students participating in situational experience and peer analysis and discussion can increase the teacher-student interaction, and improve the satisfaction of nursing students with clinical teaching.

Objective To observe the effect of Xiaochaihutang on ammonia-induced edema of astrocytes in rats and explore the mechanism of Xiaochaihutang in the treatment of cerebral edema based on NF-κB signaling pathway.Methods Astrocytes were isolated from the cerebral cortex of SD rats 1-2 days old.When the cell content was more than 95%,the cells could be subcultured and divided into three groups:Vehicle group(10%blank control group serum,Vehicle),Model group(10%blank control group serum+5 mmol·L-1 ammonium chloride,Model),and Xiaochaihutang group(10%serum+5 mmol·L-1 ammonium chloride,XCHT).The expression of AQP4 was detected by immunofluorescence.The levels of AQP4,GFAP,and TNF-α were detected by RT-PCR and Western blot.NF-κB P65 was measured by Western blot.Results ① Ammonium chloride increased the expression of AQP4 in astrocytes(P<0.01)and decreased the expression of GFAP(P<0.05,P<0.01),however,the expression of AQP4 in astrocytes decreased(P<0.01)while GFAP increased(P<0.05)after the intervention of serum containing Xiaochaihutang.② Compared with the Vehicle group,the expression level of TNF-α and phosphorylation of NF-κB P65 in the Model group was significantly increased(P<0.05),while after Xiaochaihutang serum medicated treatment,TNF-α and phosphorylation of NF-κB P65 content lower(P<0.05).Conclusion Xiaochaihutang can improve the edema of astrocytes induced by ammonia and enhance the activity of astrocytes.Its mechanism may be related to inhibition of NF-κB signaling pathways,and reduce inflammation medium(especially TNF-α)produced and released.

OBJECTIVE: To determine the efficacy of second generation endometrial ablation (NovaSure) combined with levonorgestrel-releasing intrauterine system (Mirena) in the treatment of adenomyosis. METHODS: Clinical data of patients with adenomyosis admitted in Women's Hospital, Zhejiang University School of Medicine from January 2015 to December 2018 were retrospectively analyzed. Among 66 patients, 44 received Mirena placement only (control group) and 22 received Mirena placement and NovaSure treatment (study group). The menstruation blood loss, dysmenorrhea score, uterine size, expulsion rate of Mirena and the patients' satisfaction rate were assessed in two groups. RESULTS: There was a significant reduction in menstruation blood loss (<0.05) and significant improvement in dysmenorrhea (<0.05) after the treatment in both groups. The patients in study group had more marked improvement in menstruation blood loss than those in control group (<0.05). The patients' satisfaction was higher and the expulsion rate of Mirena was lower in study group than that in control group (all <0.05). The score of dysmenorrhea and the size of uterine had no significant difference between two groups (all >0.05). CONCLUSIONS NovaSure can improve the efficacy of Mirena in treatment of adenomyosis.
Adenomyosis ; therapy ; Dysmenorrhea ; Endometrial Ablation Techniques ; Female ; Humans ; Levonorgestrel ; administration & dosage ; Organ Size ; Retrospective Studies ; Uterus ; anatomy & histology