【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.
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...
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。
Objective To study the effect of allogeneic canine cord blood mesenchymal stem cells（cbMSC）transplantation on the distribution of CD4+T and CD8+T in infracted area of hearts. Methods Mononuclear cells of cord blood were isolated by density gradient centrifugation and amplified by adherent culture. 36 adult male dogs were divided into experimental group and control group. Animal models of acute myocardial infarction were established by ligating anterior descending coronary artery. The fourth generations of mesenchymal stem cells (MSC) were transplanted into infarcted area of hearts by left anterior descending coronary artery after 72h induced by 5-aza and transfected by LacZ. The survival of transplanted cells in hearts can be confirmed by βgal expression. CD4+T and CD8+T cells distributed in infarcted area were detected by immunohistochemical staining method. The ImagePlus 5.1 software was used to analyze the images. Results Cells transplanted into infarcted area could survive for a long time. 2, 4, 8 weeks after transplantation, the IOD of CD4+T in experimental group were 44.35±7.03, 19.29±4.11 and 20.27±3.51 respectively, and the CD4+T/CD8+T ratios were 0.63±0.12, 0.51±0.15 and 0.66±0.08. In control group, the IOD of CD4+T at 2, 4, 8 weeks after transplantation were 65.78±10.27, 28.02±2.59, 29.79±6.83, and the CD4+T/CD8+T ratios were 1.28±0.20, 1.34±0.09 and 1.50±0.16. The IOD of CD4+T and CD4+T/CD8+T ratio in experimental group were significantly lower than that in control group. In experimental group the IOD of CD8+T at 2, 4, 8 weeks after transplantation were 69.88±7.84 , 37.80±8.83 and 30.81±7.42, higher than that in control group which were 51.28±10.01, 20.87±4.50 and 19.91±2.87. Conclusion The preliminary results indicated that allogeneic cbMSC transplanted in infarcted area can escape from immune rejection, its mechanism may be associated withdecreasing the amount of CD4+T cells infiltrated in periphery of infarcted area and maintaining CD4+T/CD8+T ratios at a lower level.
Objective To evaluate which is better method zymogen or low temperature frozen in removing vascular endothelial cell so as to lay a foundation for creating a kind of brace which is not to be rejected and the same as own blood vessel. Methods Fresh and not damaged umbilical blood vessel was collected from natural labour women, human umbilical blood vessel was remove carefully from normal foetus, then was put into disinfectant at 37℃ for 24 hours. They were divided into 3 groups:normal group(NG),zymogen group(ZG) and low temperature frozen group(LG). ZG: 0.1% collagenⅡ enzyme was addedin umbilical blood vessel and closed the both sides and the vascular endothelialcell was removed in 37℃ water. LG:Umbilical blood vessel was put into liquidnitrogen for 24 hours after frozened step by step, and then it was put into 37℃ water for 30-60 s and the vascular endothelial cells were washed away by normal saline. NG:Umbilical blood vessel was kept into 4℃ Kerb’s liquid. The bacteria were culturedin each group. The samples were stained by HE,elastic fiber and collagen fiberwere observed by light and scanning electron microscope. The difference of compliance was compared. Human leukocyte antigen ABC(HLA-ABC) and HLA-DR were observed by immunohistochemical method and the expression of antigen of umbilical blood vessel was analysed. Results In LG, umbilical vascular endothelial cells were removed completely; artery showed vertical smooth muscle and vein showed elastic membrane. InZG, umbilical vascular endothelial cells were removed completely after 20 minutes;artery showed vertical smooth muscle cells and vein showed lower endothelial layer. The vascular compliance in LG was higher than that in NG, and the latter was also higher than that in ZG,but showing no significant differences (Pgt;0.05). The compliance of umbilical vein was 2-3 times as much asthat of umbilical artery.The expression of HLA-ABC and HLA-DR in LG andZG were lower than that in NG, showing significant differences (Plt;0.01). Conclusion Low temperature frozen methodand zymogen method(0.1% collagen Ⅱ enzyme for 20 min) can remove vascular endothelial cells of human umbilical blood vessel completely.Low temperature frozenmethod was better than zymogen method.
Objective To explore the effectiveness of preperitoneal herniorrhaphy with Ultrapro Plug (UPP) mesh for umbilical hernia repair in adults. Methods Between September 2011 and June 2015, 71 patients with umbilical hernia underwent preperitoneal herniorrhaphy with UPP mesh. There were 26 males and 45 females, aged 19-92 years (mean, 54.3 years). The disease duration was 45 days to 30 years (median, 18 months). Umbilical hernia was diagnosed through physical examination, ultrasound, and other relevant auxiliary examination. According to American Society of Anesthesiologists (ASA) classification, 12 cases were rated as grade Ⅰ, 34 cases as grade Ⅱ, 21 cases as grade Ⅲ, and 4 cases as grade Ⅳ. The operation time, postoperative hospitalization time, complication, and recurrence were recorded. Results The diameter of hernia ring ranged 0.5-3.0 cm (mean, 1.8 cm). There was no vessel or intestine injury. The operation time was 12-35 minutes (mean, 22.4 minutes); postoperative hospitalization time was 12-48 hours (mean, 16.3 hours). Fat liquefaction of incision occurred in 2 cases, and primary healing of incision was obtained in the other cases. Sixty-nine patients were followed up 8-51 months (median, 28 months). Hernia recurrence and patch infection occurred in 1 case respectively during follow-up. No postoperative foreign body sensation and chronic pain occurred. Conclusion Repairing umbilical hernia in adults with UPP mesh should be safe and reliable, because it has the advantages of short operation time, short hospital stay, less complication, and lower incidence of recurrence.
ObjectiveTo investigate the multi-directional differentiation potential and other biological characteristics of chicken umbilical cord mesenchymal stem cells (UMSC), as well as their reparative effects on bleomycin (BLM)-induced lung injury in mice. MethodsAn acute lung injury model in mice was established by injecting BLM into the bronchus. UMSC were then transplanted via the tail vein. The reparative effects of UMSC on lung injury were evaluated through pathological section observation, survival and differentiation of transplanted cells in mice, and detection of hydroxyproline (HYP) content, among other indicators. ResultsThe UMSC successfully isolated in this study positively expressed specific surface markers CD29, CD44, CD90, and CD166, while the expression of CD34 and CD45 was negative. Induced UMSC could differentiate into adipocytes, osteocytes, chondrocytes, and alveolar epithelial cells. Animal experiments revealed that BLM-treated mice exhibited damaged alveolar structures, significant inflammatory cell infiltration, abnormal collagen deposition, and pulmonary fibrosis. However, after UMSC transplantation, the extent and severity of lung damage were reduced, and the HYP content in lung tissue decreased but remained higher than that of the control group. ConclusionUMSC can continuously proliferate and maintain their biological characteristics under in vitro culture conditions. They possess the ability to migrate to damaged sites and undergo directional differentiation, demonstrating a certain reparative effect on BLM-induced acute lung injury in mice.
Objective To investigate the feasibility of differentiating human umbilical cord blood stem cells into hepatocytes. Methods Thirty-six BALB/c nude mice were randomly divided into experimental group and control group(18 in each of the group), and experimental group was again randomly divided into group A， B and C (six in each of the group). The mice in experimental group and control group were exposed to 350 cGy radiation produced by 60Ｃｏ. After 3 h, karyocytes at different concentrations in the fresh human umbilical cord blood were injected into the mice in experimental group A, B, C via their tail veins, and the equal volume of normal sodium (NS) was also injected into control group via tail veins. After one month, carbon tetrachloride (CCl4) was injected into experimental group A, B and control group via abdominal cavity, and the equal volume of normal sodium was injected into experimental group C. After two months, immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect the expressions of human cytokeratin-18 (CK18), cytokeratin-19 (CK19) and albumin (ALB) in liver tissues of all mice. Results The expressions of CK18, CK19 and ALB in injured liver tissues were all positive, and the expressions of experimental group B were higher than those of experimental group A （P＜0.05）, but the expressions of CK18, CK19 and ALB in the liver tissues of control group and experimental group C, whose were not injured with CCl4, were all negative. Conclusion Human umbilical cord blood-derived stem cells can differentiate into hepatocytes and express ALB under special microenvironment after liver injured by CCl4 , and the expression level of ALB maybe directly related to the number of human umbilical cord blood stem cells.