兔骨髓间充质干细胞的体外分离培养、低温冻存及复苏研究

目的寻求体外培养、冻存、复苏兔骨髓间充质十细胞（bone marrow mesenehymal stein cell,BMMSC）的方法,观察体外培养BMMSC的形态及生长特性,研究低温冻存对兔BMMSC生长的影响,为兔BMMSC进一步的实验研究打下基础。方法取新西兰白兔胫骨穿刺获取骨髓液,密度梯度离心法联合贴壁培养法体外纯化扩增,并行液氮冻存3个月。通过倒置显微镜观察其生物学表现,并进行细胞增殖活性分析．绘制生长曲线。结果兔BMMSC为贴壁生长,形态为均匀成纤维细胞样,增殖能力强,传代及冻存后细胞仍然保持其生物学特性。结论兔BMMSC可采取密度梯度离心法联合贴壁培养法进行较好地纯化和扩增,并町长期保存于液氮中,在一定的条件下可复苏存活,冻存不影响细胞的增殖能力。     

补阳还五汤对缺血性脑卒中大鼠神经元自噬的保护作用及机制分析

目的探究补阳还五汤中药对大脑中动脉闭塞(MCAO)大鼠再灌注24 h后抗神经元缺血损伤的治疗效果,并分析其改善神经元自噬相关的可能作用机制。方法采用MCAO法对成年雄性SD大鼠进行大脑中动脉闭塞处理2 h,再灌注24 h后成模,分为假手术组、模型组和治疗组。模型组和治疗组大鼠在MCAO造模手术后进行24 h再灌注,治疗组腹腔注射补阳还五汤方剂,模型组和假手术组大鼠均腹腔注射接受等体积的生理盐水,共计14 d。再灌注损伤后1 d、7 d、14 d进行神经系统损伤严重程度(NSS)评分;第14天进行平衡木行走试验;采用苏木精-伊红染色法测定大脑梗死体积;采用免疫组化分析皮质缺血区存活神经元密度;采用免疫印迹法对自噬相关蛋白表达水平(Beclin1,LC3)和线粒体动力学蛋白水平(Parkin,DRP1,OPA1)。结果与假手术组比较,模型组在MACO术后1 d、7 d、14 d NSS评分显著升高;第7天模型组和治疗组间NSS评分无显著差异(P> 0. 05),第14天时治疗组大鼠NSS评分显著低于同时间点的模型组大鼠评分(P <0. 01)。与假手术组相比,模型组大鼠通过平衡木的时间均显著延长(P <0. 01);而与模型组相比,治疗组大鼠通过平衡木的时间显著缩短(P <0. 01)。第14天后,与假手术组相比,模型组大鼠脑梗死面积百分比显著升高,缺血皮质缺血区神经元密度显著下降(P <0. 01);与模型组比较,治疗组大鼠脑梗死面积百分比显著降低,皮质区神经元丢失程度显著缓解(P <0. 01)。与假手术组相比,模型组大鼠皮质区Beclin1、LC3-II、Parkin、DRP1和OPA1水平显著均下降(P <0. 001),而与模型组相比,治疗组皮质区Beclin1、LC3-II、Parkin、DRP1和OPA1蛋白表达下降程度显著缓解(P <0. 01)。结论补阳还五汤通过调节大鼠脑卒中后皮质缺血区域自噬,稳定线粒体功能,发挥神经保护作用。     

Influence of hypoxia on mitochondrial function and energy status in CCl4-induced cirrhotic rat liver

The influence of hypoxia on hepatic mitochondrial function and energy status was studied in normal and carbon tetrachloride (CCl4)-induced cirrhotic rats. Under hypoxemia of 50 mm Hg-PaO2, hepatic energy status was suppressed both in normal and cirrhotic rats. After the reversal of hypoxia, it was completely restored in normal rats concomitant with a rapid elevation of hepatic mitochondrial redox state (overshoot phenomenon) and increase in the mitochondrial oxidative phosphorylative activity. By contrast, in cirrhotic rats, such an enhancement of mitochondrial function was not observed. It was clarified that cirrhotic liver mitochondrial function was not observed. It was clarified that cirrhotic liver mitochondria have little capacity to respond to the hypoxic stress. A lower resistance to hypoxic episode in cirrhotics might be attributable to the absence of mitochondrial enhancement which is a compensatory mechanism for the deranged energy metabolism of the liver.     

丁酸钠联合电离辐射对肺癌A549细胞凋亡的影响及其机制

研究组蛋白去乙酰化酶抑制剂（HDACi）丁酸钠（NaBt）单独或联合X射线照射对人非小细胞肺癌（NSCLC）A549细胞凋亡的影响,并阐明其作用机制。     

Roles of AKAP1 in high-glucose induced mitochondrial fission in podocytes

Objective To investigate the roles of A kinase anchoring protein1(AKAP1)in high-glucose induced mitochondrial fission in podocytes. Methods Conditionally immortalized human podocytes were cultured in serum-free medium for 24 hours, and then exposed to different glucose concentration conditions in different time periods. The protein expressions of AKAP1 were observed by immunofluorescence, and AKAP1, dynamin related protein1 (Drp1) and phospho Ser 637-Drp1 (p-Drp1) were analyzed by Western blotting. AKAP1 siRNA was transfected to block AKAP1 expression.Podocytes were then divided into normal control group (5 mmol/L glucose), hypertonic group (30 mmol/L mannitol+5 mmol/L glucose), high glucose group (35 mmol/L glucose), and high glucose+AKAP1 siRNA group. Mitochondrial morphological changes were assessed by mitotracker red staining. Podocyte apoptosis was assessed by flow cytometry. Results Compared with normal group, high-glucose induced more podocytes apoptosis (P＜0.05), more mitochondrial fission with decreased aspect ratio and form factor (all P＜0.05). Upregulated AKAP1 protein level, and increased ratio of p-Drp1/Drp1 (all P＜0.05) in time and concentration dependent manners were also observed. Compared with high glucose group, transfection of AKAP1 siRNA showed less apoptosis (P＜0.05), less mitochondrial fission with increased aspect ratio and form factor (all P＜0.05), and down-regulated AKAP1 protein level as well as p-Drp1/Drp1 ratio (all P＜0.05). Conclusion High glucose induced mitochondrial fission might be induced through AKAP1-Drp1 pathway.     

Drp1介导线粒体能量代谢拮抗大鼠缺血再灌注肾损伤

目的　探讨线粒体动力相关蛋白1（Drp1）介导线粒体能量代谢参与缺血再灌注肾损伤的分子机制。方法　实验时间2020年10月14日。8～10周龄雄性Wistar大鼠随机分为正常对照组、假手术组、模型组和Drp1抑制剂组,每组5只,每只体质量为0.25～0.30 kg。通过手术建立大鼠肾脏缺血再灌注急性肾损伤模型,Drp1抑制剂组大鼠腹腔注射线粒体分裂抑制剂（mitochondrial division inhibitor 1,Mdivi-1）20 mg/kg体质量,其余各组大鼠注射等体积生理盐水,造模24 h留取血清及肾脏组织。比较组间大鼠血肌酐水平、肾组织病理学改变、肾组织细胞凋亡情况、线粒体超微结构、线粒体ATP酶活力,并建立体外HK-2细胞缺氧复氧模型,经处理后JC-1染色法检测细胞线粒体膜电位变化。采用方差分析。结果　与假手术组比较,模型组大鼠血肌酐明显升高［（41.12±1.895）µmol/L比（48.68±2.065）µmol/L］,肾小管损伤评分升高［（1.29±0.426）分比（6.50±0.577）分］,每高倍镜视野下大鼠肾组织细胞凋亡数量增加［（2.40±0.547）个比（10.20±1.095）个］,电镜下线粒体损伤更明显,线粒体ATP酶活力降低［（6.38±0.321）U/mg prot比（4.18±0.198）U/mg prot］,HK-2细胞缺氧复氧模型中,模型组较假手术组线粒体膜电位降低的细胞比例增多［（9.81±0.251）%比（4.24±0.598）%］,差异均有统计学意义（均P<0.05）。与模型组相比,Drp1抑制剂组大鼠血肌酐下降［（48.68±2.065）µmol/L比（43.28±0.895）µmol/L］,肾小管损伤评分降低［（6.50±0.577）分比（4.50±0.578）分］,每高倍镜视野下大鼠肾组织细胞凋亡数量减少［（10.20±1.095）个比（6.60±1.140）个］,线粒体结构损伤减轻,细胞线粒体ATP酶活力增加［（4.18±0.198）U/mg prot比（5.16±0.628）U/mg prot］,HK-2细胞缺氧复氧模型中,Drp1抑制剂组较模型组线粒体膜电位降低的细胞比例减少［（5.90±0.360）%比（9.81±0.251）%］,差异均有统计学意义（均P<0.05）。结论　选择性抑制Drp1可通过抑制线粒体分裂,有效改善能量代谢障碍,减少细胞凋亡,减轻肾缺血再灌注损伤。     

Mitochondrial transplantation as a promising therapy for mitochondrial diseases

Mitochondrial diseases are a group of inherited or acquired metabolic disorders caused by mitochondrial dysfunction which may affect almost all the organs in the body and present at any age. However, no satisfactory therapeutic strategies have been available for mitochondrial diseases so far. Mitochondrial transplantation is a burgeoning approach for treatment of mitochondrial diseases by recovery of dysfunctional mitochondria in defective cells using isolated functional mitochondria. Many models of mitochondrial transplantation in cells, animals, and patients have proved effective via various routes of mitochondrial delivery. This review presents different techniques used in mitochondrial isolation and delivery, mechanisms of mitochondrial internalization and consequences of mitochondrial transplantation, along with challenges for clinical application. Despite some unknowns and challenges, mitochondrial transplantation would provide an innovative approach for mitochondrial medicine.     

Early effector maturation of naïve human CD8+ T cells requires mitochondrial biogenesis

The role of mitochondrial biogenesis during naïve to effector differentiation of CD8⁺ T cells remains ill explored. In this study, we describe a critical role for early mitochondrial biogenesis in supporting cytokine production of nascent activated human naïve CD8⁺ T cells. Specifically, we found that prior to the first round of cell division activated naïve CD8⁺ T cells rapidly increase mitochondrial mass, mitochondrial respiration, and mitochondrial reactive oxygen species (mROS) generation, which were all inter‐linked and important for CD8⁺ T cell effector maturation. Inhibition of early mitochondrial biogenesis diminished mROS dependent IL‐2 production – as well as subsequent IL‐2 dependent TNF, IFN‐γ, perforin, and granzyme B production. Together, these findings point to the importance of mitochondrial biogenesis during early effector maturation of CD8⁺ T cells.     

红景天苷对小鼠原代T淋巴细胞体外行为的影响

目的:分析红景天苷(Sal)对小鼠原代T淋巴细胞体外行为的影响。方法:无菌分离并培养BALB/c近交系小鼠淋巴结细胞,MTT比色法检测并分析Sal对小鼠T淋巴细胞活力的影响;荧光染料标记的单克隆抗体染色结合流式细胞术(FCM)检测刀豆蛋白A(Con A)诱导作用下Sal对T淋巴细胞活化抗原CD69表达水平的影响。羧基二乙酸荧光素琥珀酰亚胺酯(CFDA-SE)染色分析T淋巴细胞体外增殖情况。2',7'-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光染色检测小鼠T淋巴细胞胞内活性氧簇(ROS)生成。Di OC6(3)染色检测Sal对T淋巴细胞胞内线粒体活性的影响以及地塞米松(DEX)诱导作用下Sal对T淋巴细胞线粒体膜电位的影响。制备小鼠胸腺T淋巴细胞悬液,FCM检测DEX诱导作用下胸腺T淋巴细胞的凋亡率。结果:终浓度为80、160和320μmol/L的Sal均能提高T淋巴细胞活化抗原CD69的表达水平(P0.05)。Sal能促进Con A诱导72 h条件下T淋巴细胞体外增殖(P0.01),且能减少T淋巴细胞胞内ROS生成量(P0.05),对T淋巴细胞线粒体膜电位有保护作用(P0.01)。Sal能明显降低DEX诱导条件下的胸腺T淋巴细胞凋亡率(P0.01)。结论:Sal能够促进T淋巴细胞的体外活化和增殖,减少T淋巴细胞内ROS的产生,维持细胞内线粒体膜电位稳定性,抑制DEX诱导条件下胸腺T淋巴细胞的凋亡。