靶向STAT3的慢病毒表达载体的构建及其对血管平滑肌细胞增殖凋亡的影响

目的 构建大鼠STAT3基因的shRNA慢病毒表达载体,并观察其对大鼠血管平滑肌细胞增殖和凋亡的影响.方法 针对STAT3基因的不同部位设计4对shRNA的寡核苷酸片段,克隆到慢病毒载体PLKO.1中,构建靶向STAT3基因的慢病毒载体PLK0.1-STAT3-shRNA,检测并筛选最佳抑制效率的shRNA干扰载体.并将其转染大鼠血管平滑肌细胞,用噻唑蓝法和流式细胞仪检测沉默STAT3基因后对血管平滑肌细胞增殖和凋亡能力的影响.结果　靶向STAT3慢病毒表达载体构建成功.转染PLKO.1-STAT3-shRNA后,STAT3蛋白表达明显下降,其中以PLKO.1-STAT3-S1最为明显,达到90%以上;转染PLKO.1-STAT3-S1的细胞增殖能力(A值=0.25±0.05)明显低于未转染组(A值=0.62±0.12)和阴性对照组细胞(A值=0.59±0.11)(P＜0.05);而早期细胞凋亡率(26.9±2.8)%和晚期细胞凋亡率(9.5±1.6)%均明显高于未转染组和阴性对照组(P＜0.01).结论　成功构建并筛选最佳抑制效率的靶向STAT3慢病毒表达载体PLKO.1-STAT3-S1,该载体能有效抑制大鼠血管平滑肌细胞增殖,并促进细胞凋亡.

Abstract：

Objective To construct a recombinant short hairpin RNA (shRNA) lentiviral vector carrying STAT3 gene in rats, and to investigate its effects on proliferation and apoptosis of vascular smooth muscle cells by silencing STAT3. Methods Four oligonucleotides targeting STAT3 gene were synthesized and cloned into lentivirus vector PLKO. 1. The shRNA lentiviral vector with best transfection efficiency was detected and identified, which was transfected into vascular smooth muscle cells in rats, and its effects on proliferation and apoptosis of vascular smooth muscle cells were measured by MTT and flow cytometry after silencing STAT3. Results The recombinant lentivirus vector PLKO. 1-STAT3-shRNA was constructed successfully. PLKO. 1-STAT3-shRNA knocked down the expression of STAT3 protein dramatically, especially PLKO. 1-STAT3-S1, whose transfection efficiency was more than 90%. The proliferation capacity of vascular smooth muscle cells transfected with PLKO. 1-STAT3-S1 (A value =0. 25 ±0. 05 ) was significantly lower than no-transfected group (A value =0. 62 ±0. 12) and negative control group (A value =0. 59 ±0. 11 )(P ＜ 0. 05). Meantime the early apoptosis rate (26. 9 ± 2. 8 ) % and late apoptosis rate (9. 5 ± 1.6 ) % in PLKO. 1-STAT3-shRNA-transfected group were significantly higher than in no-transfected group and negative control group (P ＜ 0. 01 ). Conclusion The recombinant lentivirus shRNA vector targeting STAT3,PLKO. 1-STAT3-S1, with best transfection efficiency, is constructed successfully. PLKO. 1-STAT3-S1 can inhibit the proliferation of vascular smooth muscle cells, and promote the cell apoptosis. This study lays the foundation for further studying on targeting treatment of vascular restenosis.     

颅面部CT三维模型复杂结构辅助定点法

目的：介绍一种在CT三维模型复杂结构上辅助定点的方法，解决这些部位定点困难的问题。方法：间接使用计算机辅助设计（Computer—aided design，CAD）点与被测点重合的方法，通过读取CAD点的坐标值反映被测点的精确坐标。结果：辅助定点法能够精确地对复杂的组织结构例如髁突顶点甚至是骨组织内部结构定点，误差小于0．3mm。结论：此方法简单易学，重复性好，信度高，是一种确实可行的精确定点方法。     

软骨细胞周基质对软骨细胞作用的研究进展

软骨细胞周基质(pericellular matrix,PCM)是软骨细胞周围的狭窄基质条带, 与其包绕的软骨细胞共同组成软骨单位。大量研究表明PCM富含蛋白多糖、胶原蛋白和纤维蛋白, 且在调节软骨细胞代谢微环境方面发挥着重要作用。用微管吮吸技术直接检测软骨单位发现, PCM与软骨细胞和软骨细胞外基质的力学特性明显不同。然而, 关于PCM的功能还不是十分明确, 仍需进一步研究。     

兔膝关节软骨单位微管吸吮黏弹性力学分析

目的 探讨体外急性消化软骨单位的生物力学特性.方法 成年8月龄新西兰白兔8只,随机分为两组,各4只.无菌条件下剖取双膝关节全层软骨,一组采用常规质量浓度0.4%的pronase酶和质量浓度0.025%的Ⅱ型胶原酶依次消化为软骨细胞;另一组采用质量浓度0.3%的dispase酶和质量浓度0.2%的Ⅱ型胶原酶联合搅拌消化3 h为软骨单位.利用微管吸吮结合半无限体细胞力学模型定量分析急性消化软骨细胞及软骨单位黏弹性力学特性,包括平衡模量(E∞)、瞬间模量(E0)和表观黏性(μ)等黏弹性参数.结果 成年软骨细胞在0.2-0.4 kPa恒定微管负压下,表现为典型黏弹性固体特征,即在微管中产生瞬间微小变形,随后发生形率单调减小的蠕变过程,其到达平衡状态时间为(110±18)s.成年软骨单位在微管吸吮负压提高到1.0～1.2 kPa时,与软骨细胞发生同样的黏弹性蠕变行为,但其瞬间吸入微管内的长度明显减少,且到达平衡状态的时间缩短为(36.5±4.5)s.同时,软骨单位黏弹性参数平衡模量(E∞)、瞬间模量(E0)和表观黏性(μ)均明显高于软骨细胞.结论 与软骨细胞相比,成年软骨单位同样表现为黏弹性固体特征,但其黏弹性力学特性明显提高.

Abstract：

Objective To characterize the biomechanical behavior and properties of the chondrons enzymatically isolated from rabbit knee articular cartilage in virto. Methods Eight months old New Zealand white rabbits were randomly divided into chondroctye and chondron groups (4 rabbits in each group). In chondrocyte groups, the full articular cartilages from both knees were enzymatically isolated to chondrocytes by 0.4% pronase and 0.025% collagenase type-Ⅱ in turn. In chondron groups, chondrons were obtained from articular cartilage using the mixture of 0.3% dispase (a neutral protease) and 0.2% collagenase type-Ⅱin at 37C for 3 h. The micropipette aspiration was used to quantify changes in biomechanical properties of chondrons and chondrocytes and the viscoelastic parameters, including K1, K2, E∞ (equilibrium modulus), E0(instantaneous modulus), and μ (apparent viscosity), were calculated coupled with standard linear half-space viscoelastic solid model. Results In response to a constant negative pressure of 0.2-0.4 kPa, the chondrocytes exhibited standard linear viscoelastic solid properties. Namely, the cells showed an initial elastic response followed by a viscoelastic creep response. then cells continued to enter into the micropipette with a monotonically decreasing rate of deformation, until reaching equilibrium within about (110±18) s. Comparing with chondrocytes, the chondrons exhibited significant viscoelasticity under a greater negative pressure of 1.0-1.2 kPa. But the instantaneous length deformed into the micropipette significantly reduced, and the equilibrium time reduced to (36.5±4.5) s. The equilibrium modulus (E∞), the instantaneous modulus (E0) and the apparent viscosity (μ) of chondrons were significantly higher than the those of chondrocytes. Conclusion Comparing with chondrocytes, the chondrons exhibited significant viscoelastic properties, and viscoelastic properties of chondrons have increased in vitro.     

A2B腺苷受体在6%羟乙基淀粉130/0.4降低脓毒症大鼠肺毛细血管通透性中的作用

目的 探讨A2B腺苷受体(A2BAR)在6%羟乙基淀粉(HES)130/0.4降低脓毒症大鼠肺毛细血管通透性中的作用.方法雄性SD大鼠50只,体重250～300 g,随机分为5组(n=10):假手术组(S组)、脓毒症组(CLP组)、低剂量HES组(H1组)、中剂量HES组(H2组)和高剂量HES组(H3组).CLP组、H1组、H2组和H3组采用盲肠结扎穿孔术(CLP)制备脓毒症模型,S组仅开腹后缝合.盲肠结扎穿孔术后4 h时,H1组、H2组及H3组分别经2 h输注6%HES 130/0.4 7.5、15.0、30.0 ml/kg,CLP组给予生理盐水30 ml/kg.CLP后6 h时处死动物,取肺组织,测定肺毛细血管通透性、A2BAR表达、环磷酸腺苷(cAMP)、蛋白激酶A(PKA)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)的含量.结果与S组比较,其余各组肺毛细血管通透性和A2BAR表达上调,CLP组肺组织cAMP、IL-6和TNF-α含量升高,H1组、H2组和H3组肺组织cAMP、PKA、IL-6、IL-10和TNF-α含量升高(P＜0.05或0.01);与CLP组比较,H1组、H2组和H3组肺毛细血管通透性均降低,A2BAR表达均上调,肺组织cAMP、PKA和IL-10含量升高,而肺组织IL-6和TNF-α含量降低(P＜0.05或0.01);6%HES 130/0.4降低肺毛细血管通透性及上调A2BAR表达的效应呈剂量依赖性(P＜0.05或0.01);6%HES 130/0.4 15.0ml/kg升高肺组织cAMP和PKA含量及抑制炎性反应的效应最明显(P＜0.05或0.01).结论 6%HES 130/0.4可上调脓毒症大鼠肺组织A2BAR表达,从而降低肺毛细血管通透性.     

IL-6/STAT3信号通路在结直肠癌发展和治疗中的进展

结直肠癌(colorectal cancer,CRC)是我国常见的恶性肿瘤,发病率逐年增长,探究CRC的发展机制及治疗方案尤为重要.IL-6/STAT3信号通路是CRC发展的关键途径之一,可通过参与肠癌细胞的抗凋亡、增殖及肿瘤血管生成等多种恶性表型促进肿瘤发展.此外,临床研究表明,针对IL-6/STAT3信号通路的靶向...     

经脐双孔法腹腔镜治疗小儿斜疝750例的疗效评价

目的 探讨经脐双孔法腹腔镜治疗小儿腹股沟斜疝的临床应用价值.方法 总结经脐双孔法腹腔镜治疗750例小儿腹股沟斜疝的手术方法及临床疗效.结果 手术全部在腹腔镜下完成,每侧疝手术时间5～15 min,平均7 min.715例患儿得到随访,随访率为95.3%,术后随访6～24个月,发现阴囊积气3例,脐戳孔大网膜疝3例,术后伤口线头异物反应4例,术后疝复发6例,无死亡病例.结论 经脐双孔法腹腔镜治疗小儿腹股沟斜疝是一种安全、简易、有效的手术方法,其美容效果极好,手术并发症发生少,值得临床推广应用.     

模拟微重力下三维培养的大鼠甲状旁腺细胞与睾丸塞尔托利细胞联合移植

目的 观察模拟微重力条件下三维培养的大鼠甲状旁腺细胞与同系睾丸塞尔托利细胞联合移植后的分泌功能,以及移植物存活情况.方法 取SD大鼠的甲状旁腺细胞,分别进行常规培养和模拟微重力条件下三维培养;取SD大鼠睾丸塞尔托利细胞,进行常规培养.取Wistar大鼠作为移植受者,制备甲状旁腺功能低下症模型.用随机数字表法将建模成功的Wistar大鼠分为3组:A组大鼠接受常规培养的单纯甲状旁腺细胞移植;B组大鼠接受常规培养的甲状旁腺细胞联合睾丸塞尔托利细胞移植;C组大鼠接受经三维培养的甲状旁腺细胞联合睾丸塞尔托利细胞移植.移植后观察各组移植物的存活情况,并检测移植物的细胞成分,淋巴细胞凋亡及甲状旁腺细胞功能.结果 A组、B组和C组移植物的存活时间依次延长,分别为(17.3±1.6)d、(43.2±2.4)d和(52.5±1.5)d,3组间两两比较,差异均有统计学意义(P＜0.05).C组移植物内甲状旁腺细胞已长入支架材料内部,并与之紧密结合,甲状旁腺激素分泌旺盛;还可见大量散在的、Fas配体表达阳性的睾丸塞尔托利细胞;在移植物与肾实质交界处可见大量淋巴细胞凋亡.结论 模拟微重力条件下三维培养技术与具有免疫赦免作用的睾丸sertoli细胞共同应用于甲状旁腺细胞移植,可以提高移植后甲状旁腺细胞的分泌功能,延长移植物的存活时间.

Abstract：

Objective To observe the function and survival of parathyroid cells cultured under simulated microgravity condition after cotransplanation of syngeneic allogeneic testicular sertoli cells.Methods Parathyroid cells in SD rats were assigned to flask-culture or bioreactor-culture.Allogeneic testicular sertoli cells in SD rats were cultured by using routine method.The recipients of hypoparathyroidism Wistar rat models were divided into 3 groups randomly:group A,receiving parathyroid cells(cultured with routine method)transplantation only;group B,receiving parathyroid cells and allogeneic testicular sertoli cells(cultured with routine method)transplantation;group C,receiving parathyroid cells(cultured under simulated microgravity condition)and allogeneic testicular sertoli cells transplantation.Allograft survival,change in cell components,apoptosis of infiltrative lymphocytes and parathyroid cells function were analyzed after transplantation respectively.Results The average survival time in group A,B and C was(17.3±1.6),(43.2±2.4)and (52.5±1.5)days,respectively.There was significant difference among group (P<0.05).In group C,parathyroid cells with strong secreting function grew into scaffold materials and adhered to them.FasL-expressing testicular cells and apoptotic lymphocytes were quite evident between allograft and kidney parenchyma.Conclusion Parathyroid cell cultured under simulated microgravity condition enhances its survival and function after cotransplanation of allogeneic testicular sertoli cell with immune privilege.     

下肢动脉硬化性闭塞症介入治疗围手术期并发症分析

目的 总结下肢动脉硬化性闭塞症介入治疗围手术期相关并发症的防治经验.方法 对207例下肢动脉硬化件闭塞症行介入治疗,统计围手术期各种并发症的发生情况,分析并发症的发生率与泛大西洋介入协会(transAtlantic inter-society consensus,TASC)分型分级之间的关系以及糖尿病和冠心病对重要脏器并发症发生率的影响.结果 本组207例,介入手术成功190例.17例失败,其中13例为介入操作失败、4例死于术后重要脏器并发症.围手术期并发症包括穿刺点出血12例、假性动脉瘤4例、消化道出血2例、动脉破裂6例、脑梗塞8例、急性心衰9例、呼衰13例、肾衰6例、支架内血栓形成5例和蓝趾综合征1例.在本组患者的TASC分型中,股腘动脉病变介入治疗并发症的发生率为39.84%(51/128),高于主髂型的18.99%(15/79)(P<0.05);患有糖尿病以及合并糖尿病和冠心病患者重要脏器并发症的发生率分别为27.66%(13/47)和24.49%(12/49),高于无合并症患者的5%(2/40)(P<0.05).结论 下肢动脉硬化性闭塞症介入治疗围手术期并发症发生率较高可能与术中操作不当、病变类型复杂及合并糖尿病和冠心病有关,术前注意合并症的处理及术中选择合理治疗方式、缩短手术时间等可减少并发症的发生.     

腘动脉瘤外科治疗25例

目的 总结腘动脉瘤的外科诊治经验.方法 回顾性分析1988年1月至2012年1月间25例腘动脉瘤患者的临床资料,其中男21例,女4例,年龄13 ～72岁,平均(56±16)岁.病因为动脉粥样硬化18例,外伤6例,腘动脉陷迫综合征1例.本组症状主要为腘窝部搏动性肿物、患肢疼痛、急慢性远端缺血、患肢肿胀等.结果 25例患者中23例行手术治疗,其中17例行动脉瘤切除、自体大隐静脉移植术,4例行动脉瘤切除、人工血管移植术,1例行动脉瘤结扎术,1例行动脉瘤切除、对端吻合术.23例手术患者围手术期无死亡,1例患者术后第45天发生吻合口血栓形成,导致远端缺血坏死行膝上截肢术.术后21例(84％)获得随访,平均随访(6.5±0.5)年,1例双侧腘动脉瘤术后4年又发现右锁骨下动脉瘤,手术成功切除.另2例保守治疗.结论 腘动脉瘤可发生破裂或肢体远端缺血,积极手术治疗可获得良好治疗效果,并需加强术后随访.