转染GFP对结肠直肠癌细胞遗传物质稳定性的影响

为探究绿色荧光蛋白(GFP)对结肠直肠癌细胞遗传物质稳定性是否存在影响,选取3种常用的结肠直肠癌细胞系HCT116,SW480,DLD-1;采用脂质体转染法把GFP转入细胞,统计几种细胞系微核、核芽的比例;分别比较各种细胞系自发微核、核芽和GFP组的差异.结果发现,HCT116细胞中对照组与GFP组的微核细胞率分别为3...     

Exendin -4在大肠杆菌中的串联表达、纯化及活性鉴定

目的:完成钝尾毒蜥Exendin -4基因在大肠杆菌中的串联高效表达.方法:按照大肠杆菌偏爱密码子设计Exendin -4基因片段,利用同尾酶构建多拷贝串联的表达载体,于大肠杆菌BL21( DE3)中诱导表达,SDS - PAGE鉴定结果,表达产物Ni柱纯化后肠激酶切割,高效液相色谱及四级杆-飞行时间质谱纯化、鉴定,确定目标产物,作用胰岛瘤细胞INS -1检测胰岛素释放活性.结果:成功构建重组载体pET28a -(Exendin -4)n(n=2,4,6),且均获得可溶性表达产物,重组表达蛋白经切割、纯化、鉴定和制备,最终得到纯度98%的Exendin -4,其具有与标准品相似的促葡萄糖刺激的胰岛素释放活性.结论:试验成功利用串联表达方式制备有活性的Exendin -4,为下一步2型糖尿病治疗药物的研究奠定了基础.     

草鱼促性腺激素受体的克隆及表达分析

利用RT-PCR技术从草鱼性腺中克隆出两种促性腺激素受体基因(FSHR和LHR),采用N-J法构建了系统进化树。通过RT-PCR及Real-time PCR技术对FSHR和LHR在成体草鱼不同组织中的时空表达进行分析,发现FSHR和LHR在草鱼卵巢、精巢中表达明显,表达量随着卵母细胞的发育逐渐增高,以上结果为进一步探讨GTHR在鱼类生殖周期中的生理功能奠定了基础。     

人类ZNF434基因的克隆、真核表达和组织表达谱分析

目的:克隆人类ZNF434基因并构建其真核表达质粒,鉴定ZNF434基因的组织表达谱。方法:实时定量PCR检测ZNF434基因在人体10种组织中的表达情况;克隆ZNF434基因的全长开放读码框区并连入真核表达载体pIRES2-EGFP,磷酸钙沉淀法转染HEK293T细胞,荧光显微镜和Western blot鉴定ZNF434蛋白的表达情况。结果:ZNF434基因在检测的人体组织中均有表达,其中骨髓和睾丸表达最高,成功克隆了ZNF434基因并构建了该基因的真核表达质粒ZNF434-pIRES2-EGFP,转染HEK293T细胞可观察到绿色荧光蛋白表达,Western blot可检测出分子量56kDa的Flag-ZNF434融合蛋白表达。结论:明确了人类ZNF434基因的组织表达谱,构建了该基因的真核表达载体,为该基因的进一步研究奠定了基础。     

奥拉西坦联合高压氧治疗重度一氧化碳中毒的疗效及对血清LPA、H-FABP及认知功能的影响

目的:探讨奥拉西坦联合高压氧治疗重度一氧化碳中毒的疗效及对血清溶血磷脂酸(LPA)、心型脂肪酸结合蛋白(H-FABP)及认知功能的影响。方法:选择2016年1月-2018年1月我院收治的重度一氧化碳中毒患者90例进行研究,以随机数表法分为观察组(n=48)和对照组(n=42)。对照组给予高压氧治疗,观察组在对照组的基础上加用奥拉西坦治疗。比较两组患者的临床疗效、血清LPA、H-FABP、认知能力、住院时间、迟发性脑病发生率。结果:两组总有效率分别为93.75%、76.19%,差异显著(P0.05);治疗前,两组血清LPA、H-FABP水平无显著性差异;治疗后,两组血清LPA、H-FABP均显著下降,且观察组血清LPA、H-FAB P水平低于对照组,(P0.05);治疗前,两组患者认知功能无显著性差异;治疗后,两组认知功能较治疗前均显著升高(P0.05),且观察组定向力、记忆力、注意力、语言能力水平均明显高于对照组(P0.05);观察组患者住院时间、迟发性脑病发生率均显著低于对照组,差异显著(P0.05)。结论:在重度一氧化碳中毒患者中应用奥拉西坦联合高压氧效果显著,可有效改善患者血清LPA、H-FABP水平及认知功能。     

Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes

The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.     

刺五加雌花蜜腺的显微形态和超微结构观察

利用光学显微镜、扫描电镜及透射电镜对刺五加雌花蜜腺结构进行了观察,结果表明:(1)刺五加雌花蜜腺为花盘蜜腺,由表皮和泌蜜组织构成;(2)蜜腺表面几乎均匀地分布着大量变态的气孔,蜜汁从气孔泌出;(3)蜜腺发育过程中有淀粉粒的积累和水解过程,液泡也伴随规律性变化;(4)蜜腺分泌方式为渗透型,或者以渗透型为主胞吐型为辅的分泌方式;(5)金胺O染色说明蜜腺表面具有角质层,可观察到微通道从中穿过,可能是蜜汁向外界分泌的通道之一.     

胶质瘤组织中PTEN、p57／Kip2蛋白的表达及临床意义研究

探讨PTEN、p57／Kip2在脑胶质瘤中的表达情况和临床意义。选取54例胶质瘤手术切除标本,采用sP法进行免疫组织化学染色。PTEN、p57／Kip2阳性表达率分别为48．1％（26／54）和44．4％（24／54）,两者在高度恶性组（Ⅲ～Ⅳ级）的表达强度明显低于低度恶性组（Ⅰ—Ⅱ级）,且随着胶质瘤的恶性程度不同表现出明显的统计学差异（P〈0．05）。PTEN与p57／Kip2的低表达可能参与肿瘤的生长分化和进展,可能提示预后不良。     

Post-conditioning protects cardiomyocytes from apoptosis via PKCε-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum–mitochondria crosstalk

The intracellular Ca²⁺ concentration ([Ca²⁺]_i) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca²⁺]_i, results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca²⁺]_i via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP₃) to increase the release of intracellular Ca²⁺ from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca²⁺]_i through an interaction of PKC with CaR to alleviate [Ca²⁺]_ER depletion and [Ca²⁺]_m elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC_ε translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca²⁺]_i, the [Ca²⁺]_ER was not reduced and [Ca²⁺]_m was not increased in the PC and GdCl₃–PC groups. Furthermore, there was no evident ?ψ_m collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl₃–PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC_ε interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the apoptotic pathway.     

生长发育期上颌牙列严重拥挤患者30例未拔牙治疗机制探讨

目的:探讨生长发育期牙弓宽度宽变化的可塑性的限度及规律,尽可能减少正畸治疗中拥挤患者的拔牙比率.方法:通过回顾性分析选出我科十年来治疗完成的、采用未拔牙手段治疗成功的病例100例.从中选出30例严重拥挤病例,其中男18例,女12例,平均年龄11.2岁,对其治疗前后牙弓宽度长度及拥挤部位进行比较分析.结果:尖牙唇侧错位造成间隙不足的患者,通过牙弓前段和中段宽度的增加来解除拥挤排齐牙列,治疗前后比较,有显著性差异;牙齿腭侧错位造成拥挤的患者,通过拥挤对应区段长度的增加来解除拥挤,治疗前后比较,差异有显著性.结论:生长发育期严重拥挤惠者可通过牙弓长度或宽度的生长适应性改建而非绝对拔牙手段采解除拥挤.