【摘要】 目的 通过比较两种原代人脐静脉内皮细胞的分离培养方法并对细胞特异性抗原进行鉴定,探索提高原代内皮细胞体外培养存活率及纯化率的方法。 方法 采用一次性无菌注射器向人脐静脉灌注消化液,消化液的浓度和消化时间分别025%(质量体积比)胰蛋白酶,10 min和01%(质量体积比)胶原酶Ⅱ,15 min。通过在倒置显微镜下观察细胞的形态特点和用免疫荧光染色的方法对细胞进行鉴定,比较两种消化方法的优劣。 结果 01%胶原酶Ⅱ,15 min的消化方法较025%胰蛋白酶,10 min对原代人脐静脉内皮细胞有更好的分离效果,活细胞数量多且细胞纯度较高。免疫荧光染色结果表明细胞内有Ⅷ因子相关抗原表达。结论 胶原酶Ⅱ可以有效分离脐静脉内皮细胞,最佳消化条件是01%胶原酶Ⅱ,37℃,15 min。【Abstract】 Objective To explore the optimal method for primary culture of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were prepared from human umbilical cords by 01% collagenase Ⅱ digestion for 15 minutes and 025 trypsinase digestion for 10 minutes,respectively. HUVECs were observed under inverted microscope and identified by immunofluorescence.The two methods of digestion were compared. Results More HUVECs were harvested through the method of 01% collagenase Ⅱ for 15 minutes,which expressed Ⅷ related antigen. Conclusion The method of 0.1% collagenase Ⅱ digestion for 15 minutes is a better choice to isolate HUVECs.
ObjectiveTo investigate whether exosomes derived from miR-27a-overexpressing human umbilical vein endothelial cells (HUVECs)—exo (miR-27a) can promote bone regeneration and improve glucocorticoids (GC) induced osteonecrosis of femoral head (ONFH) (GC-ONFH).MethodsThe exo (miR-27a) were intended to be constructed and identified by transmission electron microscopy, nanoparticle tracking analysis, Western blot, and real-time fluorescent quantitative PCR (qRT-PCR). qRT-PCR was used to evaluate the effect of exo (miR-27a) in delivering miR-27a to osteoblasts (MC3T3-E1 cells). Alkaline phosphatase staining, alizarin red staining, and qRT-PCR were used to evaluate its effect on MC3T3-E1 cells osteogenesis. Dual-luciferase reporter (DLRTM) assay was used to verify whether miR-27a targeting Dickkopf WNT signaling pathway inhibitor 2 (DKK2) was a potential mechanism, and the mechanism was further verified by qRT-PCR, Western blot, and alizarin red staining in MC3T3-E1 cells. Finally, the protective effect of exo (miR-27a) on ONFH was verified by the GC-ONFH model in Sprague Dawley (SD) rats.ResultsTransmission electron microscopy, nanoparticle tracking analysis, Western blot, and qRT-PCR detection showed that exo (miR-27a) was successfully constructed. exo (miR-27a) could effectively deliver miR-27a to MC3T3-E1 cells and enhance their osteogenic capacity. The detection of DLRTM showed that miR-27a promoted bone formation by directly targeting DDK2. Micro-CT and HE staining results of animal experiments showed that tail vein injection of exo (miR-27a) improved the osteonecrosis of SD rat GC-ONFH model.Conclusionexo (miR-27a) can promote bone regeneration and protect against GC-ONFH to some extent.
Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results Therecombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.
The clinical experiences in the appieation of umbilical-thoracic skin flap in the coverage of the defect of the forearm in 9 cases were reported. The flap was supplied by the branches of inferior epigastric artery.The biggest flap was 8.5×28cm,the smallest one was 7× 16cm.All flaps surviVed.The results were satisfactory. The advantages of the flap were:(1)potients felt comfortable when the upper extremity was immobilized at the side of the they;(2)the size of skin taken from the do...
ObjectiveTo prepare polyurethane (PU) microspheres and evaluate its physicochemical properties and biocompatibility for biomedical applications in vitro. MethodsThe PU microspheres were prepared by self-emulsification procedure at the emulsification rates of 1 000, 2 000, 3 000, and 4 000 r/min. The molecular structure was tested by Fourier transform infrared spectrometer and the surface and interior morphology of PU microspheres were observed by scanning electron microscopy (SEM). PU microspheres prepared at best emulsification rate were selected for the subsequent experiment. The human umbilical vein endothelial cells (HUVECs) were cultured and seeded on the materials, then cell morphology and adhesion status were observed by calcein-acetoxymethylester/pyridine iodide (Calcein-AM/PI) staining. The cells were cultured in the H-DMEM containing 10%FBS with additional 1% phenol (group A), in the extracts of PU prepared according to GB/T 16886.12 standard (group B), and in H-DMEM containing 10%FBS (group C), respectively. Cell counting kit 8 (CCK-8) assay was used to detect the cell viability. The blood compatibility experiments were used to evaluate the blood compatibility, the PU extracts as experimental group, stroke-physiological saline solution as negative control group, and distilled water as positive control group. The hemolytic rate was calculated. ResultsThe SEM results of PU microspheres at the emulsification rate of 2 000 r/min showed better morphology and size. The microstructure of the PU was rough on the surface and porous inside. The Calcein-AM/PI staining showed that the HUVECs attached to the PU tightly and nearly all cells were stained by green. CCK-8 assays demonstrated that group B and group C presented a significantly higher cell proliferative activity than group A (P<0.05), indicating low cytotoxicity of the PU. The absorbance value was 0.864±0.002 in positive control group and was 0.015±0.001 in negative control group. The hemolysis rate of the PU extracts was 0.39%±0.07% (<5%), indicating no hemolysis. ConclusionThe PU microspheres are successfully prepared by self-emulsification. The scaffold can obviously promote cell attachments and proliferation and shows low cytotoxicity and favorable blood compatibility, so it might be an ideal filler for soft tissue.
目的 探讨利用常规腹腔镜器械完成经脐单孔腹腔镜结直肠手术的可能性和技术要点。方法 收集中国医科大学附属盛京医院微创外科于2009年4月至2010年1月期间施行的12例经脐单孔腹腔镜结直肠手术的临床资料。阑尾炎8例，均为女性，平均年龄40岁； 回盲部肿物2例，均为女性，其中1例为回盲部淋巴水瘤（68岁），另1例为回盲部溃疡性结肠炎（47岁）； 乙状结肠息肉1例，女，55岁； 直肠癌1例，男，52岁。 12例均于脐部行2.5~3.0 cm长单切口，利用常规腹腔镜手术器械完成手术。结果 8例阑尾手术，手术时间20~50 min，出血量均少于10 ml； 2例回盲部切除术手术时间分别为60 min和90 min，出血量分别为10 ml和20 ml； 1例乙状结肠切除术用时120 min，术中出血约50 ml，术后4 d拔除引流管； 直肠癌手术时间210 min，术中出血少于200 ml，术后1周拔除引流管并出院。结论 利用常规腹腔镜手术器械完成经脐单孔腹腔镜结直肠手术安全可行。
ObjectiveTo compare clinical efficacy between transumbilical three-port laparoscopic surgery (TU-TPLS) and transumbilical single-incision laparoscopic surgery (TU-SILS) in repair of acute peptic ulcer perforation. MethodsThe patients with acute peptic ulcer perforation who underwent TU-TPLS or TU-SILS in Chengdu Second People’s Hospital Affiliated to Sichuan University from January 2022 to December 2024 were retrospectively collected, and then were divided into the TU-TPLS group and TU-SILS group. The operation time, postoperative 24 h incision pain score (visual analogue scale) , postoperative hospital stay, total hospitalization cost, incision scar score (Vancouver scar scale), comprehensive satisfaction, and postoperative complications were compared between the two groups. ResultsA total of 105 patients met the inclusion criteria were enrolled, comprising 50 patients in the TU-TPLS group and 55 patients in the TU-SILS. There were no statistically significant differences in baseline characteristics between the two groups, such as gender, age, body mass index, perforation site, perforation diameter, and Boey score (all P>0.05). Postoperatively, the TU-TPLS group demonstrated significantly lower visual analogue scale pain score at 24 h compared to the TU-SILS group [(2.34±0.63) score vs. (3.22±1.05) score, P<0.001] and significantly higher comprehensive satisfaction score [(7.60±0.86) score vs. (7.02±1.01) score, P=0.002]. However, no statistically significant differences were observed between the TU-TPLS group and TU-SILS group regarding operative time [(71.84±10.51) min vs. (69.78±7.98) min, P=0.257], postoperative hospital stay [(10.35±2.08) d vs. (9.96±1.75) d, P=0.310], or total hospitalization costs [(20 856.23±4 095.73) yuan vs. (19 988.83±2 933.43) yuan, P=0.212]. The incidence of umbilical wound infection was 1 case in the TU-TPLS group and 3 cases in the TU-SILS group (P=0.619). Postoperative residual intra-abdominal infection occurred in 2 cases in the TU-TPLS group and 1 case in the TU-SILS group (P=0.604). Incisional bleeding occurred in 0 case in the TU-TPLS group and 1 case in the TU-SILS group (P>0.999). Furthermore, there was no statistically significant difference in the scar assessment score between the TU-TPLS group and TU-SILS group [(3.11±1.13) score vs. (2.92±0.70) score, P=0.301] at the 2-month postoperative follow-up. ConclusionsBoth TU-TPLS and TU-SILS have achieved good therapeutic effects in treatment of acute peptic ulcer perforation. However, TU-TPLS has more advantages over TU-SILS. TU-TPLS causes milder incision pain, leads to higher patient satisfaction, and does not require special equipment.