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:The aim of this study was to investigate the association of mRNA expressions of ERCC1 (excision repair cross-complementing group 1) and BRCA1 (breast cancer 1) with chemosensitivity to cisplatin in malignant pleural and peritoneal effusions.Methods:Malignant pleural and peritoneal effusions were collected from 46 patients diagnosed with stage Ⅳ malignant tumor, prospectively. The tumor cells were isolated and the sensitivity of tumor cells to cisplatin was detected by adenosine triphosphate-bioluminescence assay (ATP-TCA). Real-time quantitative PCR was used to determine the mRNA expressions of ERCC1 and BRCA1. Results:The expression level of ERCC1 mRNA was negatively correlated with sensitivity of non-small cell lung cancer (NSCLC) to cisplatin (P= 0.001, r=0.685). BRCA1 mRNA expression level had negative correlation with sensitivity to cisplatin in both NSCLC (P=0.014, r=0.541) and gastric cancer (P=0.002, r=0.625). A significant interaction was found between the effects of ERCC1 and BRCA1 mRNA expressions on sensitivity to cisplatin (P=0.010 for all patients;P=0.027 for gastric cancer patients).Conclusion:ERCC1 and BRCA1 mRNA expression levels correlated with ex vivo chemosensitivity of tumor cells to cisplatin in malignant pleural and peritoneal effusions. Detection of both ERCC1 and BRCA1 may have a higher reliability in predicting the sensitivity of tumor cells to cisplatin than detection of single ERCC1 or BRCA1 expression.

Objective To understand how SIRT1 differently regulates oncogenesis in hepatocellular carcinoma (HCC) with wild type and mutant type p53.Methods HCC cell line PLC5 cells (249 site mutated p53),and HepG2 cells (wild type p53) were infected with lentivirus containing shSIRT1.Western blotting was used for signaling pathway detection.Cell growth and proliferation assay,colony formation assay and tumor xenograft assay were performed to test the tumor growth ability of HepG2 cells,HepG2-shSIRT1 cells,PLC5 cells and PLC5-shSIRT1 cells respectively.Results SIRT1 silencing resulted in significant inhibition of cell proliferation in HepG2 cells but stimulating cell proliferation in PLC5 cells (t =3.595,P ＜0.01).Acetylation of p53 was found in HepG2 (HepG2-shSIRT1) and p21 was up-regulated,however,in PLC5 (PLC5-shSIRT1) cells,acetylation of p53 was found but p21 was not induced despite of p53 activation.Silence of SIRT1 resulted in no change of AMPK function in HepG2 cells but a lower activity of AMPK in PLC5 cells (t =4.268,P ＜ 0.01).Conclusions In HCC cell lines the function following SIRT1 activation is largely determined by p53 mutant status.

Objective To observe the anesthetic effect of Sumianxin Ⅱcombined with chloral hydrate in plastic surgical operation on rabbit and explore the fast , safe and effective anesthetic methods .Methods A total of 82 Rabbits for operation were randomly divided into A , B and C groups.The A group was injected with SumianxinⅡintramuscularly (0.35 mL/kg by weight).The B group was injected with 10%chloral hydrate intravenously (2.5~3 mL/kg by weight).The C group was injected with Sumianxin Ⅱintramuscularly ( 0.2 mL/kg by weight ) combined with 10% chloral hydrate intravenously(1 ~1.5 mL/kg by weight).The anesthetic effect of induction and recovery time of anesthesia ,anesthesia maintaining time ,and anesthesia mortality rate were observed and compared .Results The anesthetic effect of induction of anesthesia,recovery time of C groups fall in between which of A and B groups ( P <0.05).However, as for anesthesia maintaining time , there was no significant difference between A and C group and both the two groups were longer than B group.Conclusion SumianxinⅡcombined with chloral hydrate possess the advantages of fast anesthesia induction and recovery effect , longer anesthesia maintaining time and low mortality .Therefore it is an easy to operate , expeditious , safe and effective anesthetization for plastic surgical operation on rabbit .

Objective To study serum albumin levels in children with severe sepsis and to correlate serum albumin levels with patient outcome and to identify the causes inducing hypoalbuminemia and its effective countermeasures.Methods Seventy-five children admitted to PICU of Provincial Hospital Affiliated to Shandong University for severe sepsis were included in the study from Aug 2010 to Sep 2011.According to their serum albumin levels within 24 hours and on the third and the seventh day of admission to PICU,75 children were divided into hypoalbuminemia group and normal serum albumin group.Then hypoalbuminemia group was divided into instant hypoalbuminemia group and continuous hypoalbuminemia group according to the duration of hypoalbuminemia.The correlation between the occurring and duration of hypoalbuminemia with patients' prognosis,the etiopathogenisis of hypoalbuminemia and its effective countermeasures were analyzed.Results (1) Sixty-three cases (84.0％) proceeded hypoalbuminemia and their mortality was 33.3％ (21/63),while 12 cases (16.0％) showed normal serum albumin level and their mortality was 0.(2) In 63 patients with hypoalbuminemia,26 cases showed continuous hypoalbuminemia and their mortality was 46.0％,while 37 cases proceeded instant hypoalbuminemia and their mortality was 15.4％.There was significant difference (x2 =5.116,P ＜ 0.05) between their mortality.(3) In the 63 cases with hypoalbuminemia,32 cases presented with hepatic injury and their mortality was 37.5％,13 cases proceeded capillary leakage and their mortality was 23.1％,and 18 cases displayed hepatic injury complicated with capillary leakage and their mortality was 33.3％.There was significant difference (x2 =7.812,P ＜ 0.05) between the mortality with different causes.Conclusion Hypoalbuminemia influenced the prognosis of children with severe sepsis,the longer duration correlated with the worse prognosis.Hepatic injury and capillary leakage were two main causes inducing hypoalbuminemia.Active treatment of hypoproteinemia aimed directly at different causes could improve their prognosis significantly.

Objective To investigate the clinical,imaging,intestinal pathological characteristics and prognosis of gluten ataxia (GA).Methods The clinical data,treatment and prognosis in a patient with GA that was confirmed by pathology and hospitalized in the Department of Neurology,China-Japan Friendship Hospital in July 2018,were analyzed retrospectively.The related literature was reviewed and the clinical feature was summarized.Results The patient is a 41-year old man.He suffered from progressive cerebellar ataxia,and the brain magnetic resonance imaging exhibited diffused cerebellar atrophy.Serum human leukocyte antigen (HLA) tests showed that the patient carried HLA-DQ2 genotype.IgA type anti-gliadin antibody was positive (39.39 RU/ml).Duodenoscopy biopsy revealed mild villus atrophy and lymphocytic infiltration,indicating celiac disease.The diagnosis of GA was established then and the patient was administered gluten-free diet combined with intravenous immunoglobulin,which markedly improved the cerebellar symptoms and signs of cerebellar speech,walk capability and daily living activities.He could do long distance driving independently two months later.Conclusions GA is one of immune-mediated reversible acquired cerebellar ataxia caused by gluten sensitivity.The genotype,serologic features,and clinical phenotype of GA in Chinese mainland population might be similar with those in European and American countries.

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:This study aimed to investigate the physical properties, biocompatibility, and 3D printing performance of a novel hybrid bioink composed of gelatin methacrylated (GelMA) and chitin nanocrystal (ChiNC).Methods:The study was conducted from May 2021 to December 2022, four different bioinks were prepared by adding varying amounts of ChiNC to GelMA bioink. The GelMA concentration in all four bioinks was 100 mg/ml, while the ChiNC concentrations were 0 mg/ml (no ChiNC added), 5 mg/ml, 10 mg/ml, and 20 mg/ml, respectively, named as GC0, GC5, GC10, and GC20 bioinks. The cross-sectional morphology of the hydrogels formed after photocuring the four bioinks was observed using scanning electron microscopy, and the porosity was calculated. Weighing the hydrogels before and after swelling, and then calculate the equilibrium swelling rate. HUVECs were seeded on the surfaces of the hydrogels prepared from the four bioinks and cultured in medium. Cell proliferation was assessed using CCK-8 assays at 1d, 3d, and 7d to compare the proliferation rates of cells on the four hydrogels. HUVECs were added to the four bioinks, and grid-like scaffolds were printed and cultured in medium. Live-Dead staining was performed at 1d and 7d to observe cell viability. Compare the printing effect of bioinks by observing its forming continuous threads properties during extrusion. Finally, tissue-engineered bladder patches simulating the mucosal layer, submucosal layer, and muscular layer anatomical structures of the bladder wall were 3D bioprinted using the optimized bioink composition, and the stability and fidelity of the printed structures were observed to further validate the feasibility of printing multi-layered complex structures with the bioink.Results:Scanning electron microscopy revealed that the porosity of the GC0, GC5, GC10, and GC20 hydrogels were (51.43±6.23)%, (51.85±6.47)%, (50.55±4.59)%, and (42.49±2.20)%, respectively. The differences in porosity between the GC0 group and the other three groups were not statistically significant ( P=0.9994, P=0.9948, P=0.1200). The equilibrium swelling ratio of the other three groups [(8.81±0.41), (7.95±0.19), (7.71±0.14)] was significantly lower than that of the GC0 group (9.37 ± 0.49), and the differences were statistically significant ( P=0.0457, P<0.01, P<0.01). CCK-8 assay showed no significant difference in absorbance value between the GC10 group (0.360±0.009) and the GC0 group (0.357±0.007), GC5 group (0.350±0.012), and GC20 group (0.345±0.018) on the first day ( P=0.9332, P=0.5464, P=0.4937). However, on the third day, the absorbance value of the GC10 group (0.755±0.012) was significantly higher than that of the GC0 group (0.634±0.010), GC5 group (0.704±0.009), and GC20 group (0.653±0.015) ( P<0.01, P=0.0033, P=0.0002). On the seventh day, the absorbance value of the GC10 group (1.001±0.031) was significantly higher than that of the GC0 group (0.846±0.026), GC5 group (0.930±0.043), and GC20 group (0.841±0.024)( P=0.0012, P=0.1390, P=0.0010). The addition of human umbilical vein endothelial cells (HUVECs) into the four groups of hydrogels enabled the printing of grid-like scaffolds, and Live-Dead staining was performed on day 1 and day 7. The cell viability of HUVECs in the four groups on day 1 was (90.13±1.63)%, (90.6±2.45)%, (92.58±2.15)%, and (91.40±3.17)%, respectively. There were no statistically significant differences between the GC0 group and the other three groups ( P=0.9869, P=0.3093, P=0.8008). On day 7, the cell viability was (89.97±3.10)%, (92.18±2.21)%, (92.05±2.25)%, and (90.12±1.97)% for the four groups, respectively. There were no statistically significant differences between the GC0 group and the other three groups ( P=0.3965, P=0.4511, P=0.9995). Bioink extrusion test showed that the GC0 hydrogel could be extruded continuously and form threads at temperatures between 24℃ and 25℃, while the GC10 hydrogel could be extruded continuously and form threads at temperatures between 24℃ and 27℃. Printing tissue engineered bladder patches simulating the anatomical structure of the bladder mucosal layer, submucosal layer, and muscular layer using GC10 bioink, and the printed patches were stable, without collapse, and had high fidelity. Conclusions:Adding ChiNC to GelMA promotes cell adhesion, proliferation, and expands the printing window of GelMA bioink. The biocompatibility of the mixed bioink prepared by adding 10 mg/ml ChiNC in GelMA is good, capable of printing tissue-engineered bladder patches that mimic the anatomical structure of natural bladder walls.

Objective To investigate the effect of long?chain non?coding RNA Fez family zinc finger protein 1 antisense RNA1 ( lncRNA FEZF1?AS1) on the biological function of hepatocellular carcinoma (HCC). Methods SMMC771 and BEL?7402 cells were transfected with sh?FEZF1?AS1 and OE?FEZF1?AS1, respectively. The expression of lncRNA FEZF1?AS1 was detected by real?time quantitative PCR. Cell proliferation was detected by Cell Counting Kit?8 ( CCK?8), and apoptosis was detected by flow cytometry. The effects of lncRNA FEZF1?AS1 on invasion and migration were detected by Transwell and wound healing assays. The expression levels of adhesion molecules were detected by Western blot. The effect of lncRNA FEZF1?AS1 on the in vivo growth was verified by nude mice xenograft experiments. Results The silencing or ectopic expression of lncRNA FEZF1?AS1 inhibited or promoted the proliferation of hepatocellular carcinoma cells. CCK?8 assay showed that the proliferation abilities of SMMC7721 and BEL?7402 cells in sh?FEZF1?AS1 transfection group significantly decreased, achieving (35.43± 4.06)% and ( 34.68± 3.97)%, respectively, on the fifth day. There were significant differences between sh?FEZF1?AS1 group and sh?NC group [52.21 ± 8.46)% and (53.76 ± 7.64)%] ( all P＜0.05). In contrast, the proliferation ability of SMMC7721 and BEL?7402 cells transfected with OE?FEZF1?AS1 was significantly increased, achieving (83.49±6.92)% and (80.31 ± 3.13)%, respectively, on the fifth day. There were significant differences between OE?FEZF1?AS1 and OE?NC group [53.03 ± 8.84)% and ( 55.11 ± 7.09)%] ( all P＜0.05). The subsequent flow cytometry results showed that cell apoptotic rates of SMMC7721 and BEL?7402 cells transfected with sh?FEZF1?AS1 were ( 13.02 ± 1.38)% and ( 11.88 ± 1.29)%, respectively, which were significantly higher than those in sh?NC groups [(5.57±1.46)% and (8.06±1.42)%, respectively, all P＜0.05]. In contrast, the apoptotic rates of SMMC7721 and BEL?7402 cells transfected with OE?FEZF1?AS1 were (3.01 ± 0.39)% and ( 3.22 ± 0.43)%, which were significantly lower than those in OE?NC groups [(6.68±0.96)% and (6.63±0.45)%, all P＜0.05]. In addition, knockdown or overexpression of lncRNA FEZF1?AS1 expression inhibited or enhanced the migration and invasion abilities as well as the levels of adhesion molecules in hepatocellular carcinoma cells.After 30 days of feeding under the same conditions, the tumor volumes of sh?FEZF1?AS1 and sh?NC SMMC7721 cells xenograft mice models were (0.26±0.03) cm3 and (0.63±0.06) cm3, respectively, showing significant difference (P＜0.05). The tumor volumes of sh?FEZF1?AS1 and sh?NC BEL?7402 cells were (0.31±0.02) cm3 and (0.72±0.08) cm3, and the difference was statistically significant ( P＜0.05). Conclusion lncRNA FEZF1?AS1 may strengthen the growth, migration and invasion of hepatocellular carcinoma cells.