蛙血清小分子肽对大鼠脑缺血再灌注细胞凋亡的影响

目的 探讨蛙血清小分子肽对大鼠脑缺血再灌注损伤后细胞凋亡及凋亡相关蛋白的作用,分析其可能的作用机制.方法 将雄性SD大鼠100只,按随机数字表法分为假手术组、模型组和蛙血清小分子肽高(90 mg/kg)、中(30 mg/kg)、低(10 mg/kg)剂量组,每组20只.采用改良线栓法栓塞大鼠大脑中动脉建立大鼠缺血再灌注损伤模型,缺血2 h再灌注24 h,进行神经行为评分,后处死;采用TUNEL法检测细胞凋亡;采用免疫组化法检测B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl相关x蛋白(Bax)、细胞色素c(Cytc)、半胱氨酰天冬氨酸特异性蛋白酶3(Caspase-3)的表达情况.结果 在神经行为评分上,假手术组为(0.00±0.00)分,模型组为(2.38±0.78)分,小分子肽高剂量组为(1.47±0.41)分,中剂量组为(1.68±0.52)分,低剂量组为(2.01±0.66)分,小分子肽高、中剂量组与模型组比较差异均有统计学意义(P<0.05);在凋亡细胞计数上,假手术组为(3.37±1.10)个,模型组为(42.80±3.54)个,小分子肽高剂量组为(28.00±2.28)个,中剂量组为(32.40±3.26)个,低剂量组为(41.40±1.20)个,小分子肽高、中剂量组与模型组比较差异均有统计学意义(P<0.05);在凋亡相关蛋白Bax、Cytc、Caspase-3和Bcl-2表达上(个/高倍视野),假手术组分别为(3.01±1.12)、(2.58±1.74)、(2.34±1.37)、(65.42±3.65),模型组分别为(70.67±3.06)、(58.31±5.04)、(68.04±5.85)、(31.26±2.81),小分子肽高剂量组分别为(40.56±4.52)、(33.65±3.44)、(41.56±4.52)、(55.64±5.49),中剂量组分别为(47.29±5.04)、(38.09±4.24)、(47.29±5.04)、(48.33±4.26),低剂量组分别为(53.20±4.70)、(44.53±4.39)、(53.20±4.70)、(40.35±3.17),小分子肽高、中、低剂量组均能抑制Bax、Cytc、Caspase-3蛋白表达,增加Bcl-2蛋白表达,与模型组比较差异有统计学意义(P<0.05),以高剂量组效果最优,且存在量效依赖关系.结论 蛙血清小分子肽是通过增强Bcl-2蛋白表达和抑制Bax、Cytc、Caspase-3蛋白表达来减少神经元细胞凋亡数目,改善神经功能症状,保护脑缺血再灌注损伤.     

褪黑激素对增生性瘢痕成纤维细胞增殖的影响及机制研究

目的：探讨褪黑激素对人增生性瘢痕成纤维细胞增殖的影响及作用机制。方法收集人皮肤增生性瘢痕标本,原代培养人皮肤增生性瘢痕成纤维细胞(HSFBs)。将HSFBs细胞根据不同的处理方法分为空白对照组、低浓度褪黑激素组、中浓度褪黑激素组、高浓度褪黑激素组、NVP-BEZ235处理组、NVP-BEZ235+褪黑激素组、褪黑激素+siRNA-PTEN组、褪黑激素+siRNA-scramble组。MTT法检测各组细胞的增殖情况,qRT-PCR和Western blot检测各组细胞中磷酸酶与张力蛋白同源物(PTEN)、CyclinD1和Caspase-3的表达以及PI3k/AKt/mTOR通路相关蛋白p-Akt和p-mTOR的蛋白水平。结果与空白对照组比较,从低到高浓度褪黑激素均能够减少HSFBs细胞同一时间点OD值,上调PTEN和Caspase-3表达量,同时抑制CyclinD1、p-Akt和p-mTOR表达水平,组间差异具有统计学意义(P<0.05)。同时与高浓度褪黑激素组比较,NVP-BEZ235+褪黑激素组中细胞增殖显著被抑制,差异具有统计学意义(P<0.05);沉默PTEN后拮抗褪黑激素对HSFBs细胞增殖和PI3K/Akt/mTOR信号通路有抑制作用,差异具有统计学意义(P<0.05)。结论褪黑激素通过上调PTEN表达,抑制PI3K/Akt/mTOR信号通路活化从而抑制HSFBs细胞增殖。     

445例体表淋巴结针吸细胞学误诊分析

目的：分析体表淋巴结针吸细胞学镜下特点及误诊原因。方法经术后组织病理学证实,回顾性总结德阳市人民医院及旌阳区中医院2010年1月至2015年5月445例体表淋巴结针吸细胞学诊断准确率及误诊原因。结果445例针吸细胞学诊断正确率为95.51%(425/445),误诊率为4.49%(20/445),误诊的主要原因有坏死严重未见有效成分、过诊断、漏穿、肿瘤细胞少未见典型病变及肿瘤细胞异型性小。结论体表淋巴结针吸细胞学诊断准确率较高,不断总结误诊原因可以提高体表淋巴结针吸细胞学诊断准确率。     

靶向S1PR3基因shRNA慢病毒表达载体构建及功能鉴定

目的：构建靶向小鼠S1PR3基因的shRNA慢病毒表达载体,并验证其对内皮祖细胞迁移能力的影响。方法依照shRNA设计原则,合成对应的shRNA序列,将其克隆到PHBLV载体中,使用三载体系统进行病毒包装,收集制备成功的病毒,并用荧光显微镜法测定病毒滴度,使用病毒感染EPC后,测定各组细胞中S1PR3蛋白表达情况,选择合适的病毒感染EPC后,测定其对EPC迁移能力的影响。结果成功将shRNA序列克隆到慢病毒表达载体PHBLV-U6-RFP,表达质粒与辅助质粒共转染293细胞后48 h就可以看到细胞中成功表达红色荧光。收集培养成功病毒感染小鼠EPC,可以不同程度干扰细胞中S1PR3蛋白表达,S1PR3蛋白表达受到抑制后,EPC的迁移能力明显减弱。结论我们成功构建了可以有效干扰S1PR3表达的慢病毒载体,并初步观察了其对EPC细胞的迁移能力的影响,为后续进一步调控S1PR3相关的细胞功能的研究奠定了基础。     

肺动脉平滑肌细胞上TMEM16A的表达及其阻滞剂对细胞增殖的作用

目的：研究TMEM16A在大鼠肺动脉平滑肌细胞(PASMCs)上的表达及其阻滞剂(T16Ainh-A01)对PASMCs增殖的作用。方法组织块贴壁法分离正常大鼠PASMCs,原代培养。光镜下观察细胞形态,细胞免疫化学和细胞免疫荧光进行鉴定。细胞免疫荧光检测TMEM16A在PASMCs上表达。原代培养的3~6代PASMCs分别在10μmol/L T16Ainh-A01、20μmol/L T16Ainh-A01和50μmol/L T16Ainh-A01下培养24 h,用CCK-8法检测细胞增殖情况。结果倒置显微镜下PASMCs以长梭形为主,细胞核卵圆形,部分细胞重叠区域呈典型的“峰-谷”状结构。免疫学检测显示胞浆内与细胞长轴平行的肌丝被染成棕黄色,标记肌丝的红色荧光也与细胞长轴平行,标记细胞膜上的TMEM16A的绿色荧光可见表达,而细胞核不染色。CCK-8实验检测,相对于对照组,10μmol/L T16Ainh-A01组细胞活力均数为(96.74±0.92)%,20μmol/L T16Ainh-A01组均数为(92.21±1.25)%,50μmol/L T16Ainh-A01组均数为(87.41±3.27)%,差异均具有统计学意义(P<0.01)。结论正常大鼠PASMCs表达TMEM16A;T16Ainh-A01可抑制PASMCs增殖,其抑制作用呈浓度依赖性;TMEM16A可能参与调控PASMCs增殖。     

Human amnion epithelial cells expressing HLA-G as novel cell-based treatment for liver disease

Despite routine liver transplantation and supporting medical therapies, thousands of patients currently wait for an organ and there is an unmet need for more refined and widely available regenerative strategies to treat liver diseases. Cell transplants attempt to maximize the potential for repair and/or regeneration in liver and other organs. Over 40 years of laboratory pre-clinical research and 25 years of clinical procedures have shown that certain liver diseases can be treated by the infusion of isolated cells (hepatocyte transplant). However, like organ transplants, hepatocyte transplant suffers from a paucity of tissues useful for cell production. Alternative sources have been investigated, yet with limited success. The tumorigenic potential of pluripotent stem cells together with their primitive level of hepatic differentiation, have limited the use of stem cell populations.Stem cell sources from human placenta, and the amnion tissue in particular are receiving renewed interest in the field of regenerative medicine. Unlike pluripotent stem cells, human amnion epithelial (AE) cells are easily available without ethical or religious concerns; they do not express telomerase and are not immortal or tumorigenic when transplanted. In addition, AE cells have been reported to express genes normally expressed in mature liver, when transplanted into the liver. Moreover, because of the possibility of an immune-privileged status related to their expression of HLA-G, it might be possible to transplant human AE cells without immunosuppression of the recipient.     

Endoplasmic reticulum stress participates in uric acid - induced phenotypic transformation of renal tubular epithelial cells

Objective To explore the effect of endoplasmic reticulum stress (ER stress) in uric acid-induced phenotypic change in renal tubular epithelial cells (HK-2). Methods (1) HK-2 cells were cultured with 0, 75, 150, 225, 300 mg/L uric acid for 24 h in vitro. (2) The cells were divided into normal control group, ER stress inhibitor 4-PBA (5 μmol/L) group, uric acid (150 mg/L) group and 4-PBA+uric acid group for 24 h. Morphological changes of HK-2 cells were observed under inverted microscope. MTT assay was used to detect the proliferation of HK-2 cells treated with 150 mg/L uric acid for 24, 48 and 72 h. The protein expressions of α-smooth muscle actin (α-SMA), vimentin, snail, glucose regulated protein 78 (GRP78) and the phosphorylation of eukaryotic initiation factor 2α (p-eIF2α) in HK-2 cells were measured by Western blotting. Results Compared with the control group, HK-2 cells in uric acid groups (150, 225, 300 mg/L) showed fibroblast-like appearance. The protein expressions of α-SMA, vimentin, snail, GRP78 and p-eIF2α in 150 mg/L and 225 mg/L uric acid groups were higher than those in the control group (all P＜0.05). The proliferation of HK-2 cells in 150 mg/L uric acid group was lower than that in control group at 48 and 72 h (all P＜0.01). Compared with the uric acid group, the cell morphology in 4-PBA+uric acid group was improved, and the protein expressions of α-SMA, vimentin, snail, GRP78 and p-eIF2α were decreased (all P＜0.05). Conclusions Uric acid may induce the phenotype transformation of renal tubular epithelial cell, and ER stress is involved. 4-PBA may inhibit the uric acid-induced ER stress response and phenotypic transformation, and may be beneficial in attenuating uric acid-induced renal tubular damage.     

Effects of hypoxia-inducible factor-2α on the expression of tight junction proteins and permeability in rat glomerular endothelial cells under hypoxia condition

Objective To investigate the role of hypoxia-inducible factor-2α (HIF-2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia condition. Methods The expressions of the HIF-2α and tight junction proteins such as occludin and ZO-1 of rGENCs were examined after exposed to 5% oxygen at different treatment time periods (0 h, 12 h, 24 h and 48 h). Then lentiviral transfection was used to knock down HIF-2α expression in rGENCs. The cells were split into four groups, including i) control group where rGENCs were cultured under normal oxygen conditions, ii) hypoxia group, iii) negative control group where rGENCs were infected with a negative vector, iv) HIF-2α lentivirus transfection group. Group ii, iii and iv were kept in hypoxic chamber (5% O2, 5% CO2 and 90% N2) for 24 h. The expressions of occludin, ZO-1 and HIF-2α were assessed by Western blotting. The permeability of rGENCs was measured using trans-epithelium electrical resistant (TEER) by Millicell? ERS voltohmmeter. Results With the elongation of hypoxia time, the expression of HIF-2α was increased gradually, while the occludin expression was decreased, there was statistically significance difference in each group (all P＜0.01). The expression of ZO-1 also decreased gradually under hypoxia circumstance, but no statistically significant was found between 24 h and 48 h groups (all P＞0.05). And a dramatic decrease in TEER of hypoxia cells was detected as compare with control cells (P＜0.01). After knockdown of HIF-2α expression, both expressions of occludin and ZO-1 were increased significantly compared with hypoxia cells (P＜0.01), and TEER elevated at the same time (P＜0.01). Above indexes had no statistical difference between hypoxia cells and negative control cells (all P＞0.05). Conclusion Hypoxia may promote HIF-2α expression, which could increase the permeability of rGENCs by reducing the expression of occludin and ZO-1.     

嗅鞘细胞移植联合跑步训练对改善脊髓损伤大鼠后肢运动功能的研究

目的 :研究嗅鞘细胞移植联合跑步训练对改善脊髓损伤大鼠后肢运动功能的效果,同时探索嗅鞘细胞移植联合跑步训练改善大鼠运动功能的可能机制。方法:选用雄性SD大鼠80只,随机分为4组,每组20只,均进行脊髓损伤造模后进行如下分组处理:生理盐水注射组(A组)、嗅鞘细胞移植组(B组)、生理盐水注射联合跑步训练组(C组)和嗅鞘细胞移植联合跑步训练组(D组)。建模3 d后C、D两组进行跑步训练,1周后B、D两组进行嗅鞘细胞移植(每只大鼠注射总量为4μl,细胞浓度为1.0×106/μl),A、C两组给予相应剂量盐水。观察时间为4周,每组每周测量BBB评分,利用免疫组化染色观察Bax、Bcl-2及NF-200的表达,采用Mallory磷钨酸苏木素染色观察神经纤维数量,使用TUNEL染色观察神经元凋亡。结果:(1)BBB评分:嗅鞘细胞联合跑步训练组与其他3组在第4周差异有统计学意义(P0.05)。(2)在Bcl-2蛋白表达上,嗅鞘细胞移植联合跑步训练有交互作用,两者相互促进(P0.05);在Bax蛋白的表达上,嗅鞘细胞移植可以明显降低其表达,与跑步训练交互作用不明显(P0.05);TUNEL染色显示,嗅鞘细胞移植、跑步训练与时间因素三者具有交互作用,显著抑制细胞凋亡(P0.05)。(3)在Mallory磷钨酸苏木素染色及NF-200免疫组化染色上,嗅鞘细胞移植、跑步训练与时间因素三者具有交互作用(P0.05),促进神经再生。结论 :嗅鞘细胞移植联合跑步训练能够显著改善大鼠后肢运动功能,这可能通过以下途径实现:嗅鞘细胞移植与跑步训练能够相互协同,明显上调Bcl-2基因的表达,从而显著抑制神经元凋亡;同时能明显促进神经元轴索再生,提高神经纤维数量,并且这种效果可以随时间的延长更加显著。     

以细胞筛为载体的玻璃化冷冻和慢速程序化冷冻保存人卵巢组织的效果比较

目的比较以细胞筛为载体的玻璃化冷冻与慢速程序化冷冻用于人卵巢组织冷冻的效果。方法人卵巢组织取皮质切块后,随机分为3组:新鲜组织对照组(F组)、慢速程序化冷冻组(S组)及以细胞筛为载体玻璃化冷冻组(V组)。卵巢组织冷冻复苏后,固定切片后行苏木素-伊红染色,观察卵泡形态;使用TdT介导的dUTP缺口末端标记技术,观察卵泡凋亡情况;部分卵巢组织体外培养,隔日采集培养液后检测雌二醇(E2)浓度。比较三组卵泡正常形态率、卵泡凋亡率及E2浓度。结果与F组原始卵泡正常形态率(91.1%)相比,V组原始卵泡正常形态率(88.1%)无显著差异(P0.05),而S组原始卵泡正常形态率(79.6%)显著下降(P0.001);V组初级卵泡正常形态率(74.2%)与S组(73.6%)相似,但两组均低于F组(89.0%)(P0.05);凋亡检测中,3组凋亡率无显著差异(P0.05);体外培养2周,各组E2浓度持续上升,F组E2浓度显著高于S组、V组(P0.001),而S组与V组E2浓度无显著差异(P0.05)。结论以细胞筛为载体的玻璃化冷冻能较好地保存人卵巢组织,复苏后组织体外培养后,还可持续分泌E2。