S波段高功率微波辐射对原代培养乳鼠心肌细胞的影响

目的研究S波段高功率微波（HPM）辐射后乳鼠心肌细胞的损伤效应及其机制。方法采用平均功率密度为5～30mW/cm^2的S波段HPM模拟源辐射原代培养的乳鼠心肌细胞，应用流式细胞术、原子力显微镜观察辐射后心肌细胞凋亡和坏死率、[Ca^2＋]。及细胞膜形态。结果10—30mW／cm^2的S波段HPM辐射后6h，心肌细胞凋亡和坏死率增加（P〈0．05或P〈0．01），且随辐射剂量增大，坏死细胞增多；30mW/cm^2 HPM辐射后即刻[Ca^2＋]i升高（P〈0．01），细胞膜发生穿孔。结论10—30mW/cm^2的S波段HPM辐射可造成心肌细胞凋亡和坏死，[Ca^2＋]；升高以及细胞膜穿孔，是S波段HPM辐射致心肌细胞损伤的重要机制。     

新生大鼠原代心肌细胞分离方法的改进

目的对传统的原代乳鼠心肌细胞分离方法进行改良,获得活性好、纯度高、反应性好的原代乳鼠心肌细胞。方法利用断头开胸取心法取新生24 h内的Wistar大鼠心肌组织,无菌剪碎后用0.25%不含EDTA胰酶混合II型胶原酶消化法,采用自制消化组合装置分离消化细胞。逐日在倒置相差显微镜下观察细胞状态。采用心肌细胞特异性蛋白α-横纹肌肌动蛋白(α-actin)单克隆抗体对培养的心肌细胞进行免疫荧光鉴定。另外使用异丙肾上腺素和去甲肾上腺素作用于细胞,观察细胞反应情况。结果培养后24 h即可观察到部分细胞搏动,可初步确定为心肌细胞;48 h后细胞逐渐展开,连续观察20 d,发现心肌细胞在3~14 d期间搏动频率与形态无明显变化。对比观察发现断头开胸取心法获取的原代心肌细胞中红细胞数量明显减少。免疫荧光鉴定发现,本方法获得的心肌细胞纯度高达95%。此外,分离的原代乳鼠心肌细胞对异丙肾上腺素和去甲肾上腺素反应灵敏。结论断头开胸取心法配以混合酶液消化心脏组织分得的原代乳鼠心肌细胞反应性好、纯度高、有活力,可满足大多数实验的要求。     

基于神经细胞活性评价高功率微波照射安全性研究

目的研究平均功率密度5mW／cm^2的高功率微波（HPM）对神经细胞活性的影响，为制定微波照射的安全标准提供参考数据。方法出生后12h内Wistar大鼠乳鼠皮层神经细胞常规培养7d后，进行HPM照射。采用细胞内ATP酶活性测定法，检测HPM对原代培养神经细胞总ATP酶、钙镁ATP酶和钠钾ATP酶活性的影响，并用MTT法检测照射后细胞琥珀酸脱氢酶（SDH）活性的变化，观察时间点分别为HPM照射后1和6h，1、3和7d。结果原代培养神经细胞经平均功率密度5mW／cm^2的HPM连续照射6min后，同一时间点内HPM照射组和对照组之间细胞SDH活性和各ATP酶活性均无显著差异。结论平均功率密度为5mW／cm^2的HPM照射对原代神经细胞的活性无显著影响，提示就神经细胞活性而言，该功率密度的HPM照射是安全的。     

电磁脉冲对培养大鼠大脑皮层神经元细胞内和培养上清LDH及培养上清CHE、K+、Na+浓度的影响

目的 :以体外原代培养的乳鼠皮层神经元为研究对象 ,测定电磁脉冲辐照神经细胞前后细胞内LDH和培养上清中LDH、CHE、K+、Na+浓度与时间的关系 ,探讨电磁脉冲对大脑神经细胞膜损伤的机制及其时相性。方法 :Wistar大鼠大脑皮层神经细胞在 6孔板中进行原代培养 ,在培养第 12～ 14天时 ,用高场强EMP模拟源 (场强为 6× 10 4 V·m-1,脉冲上升时间为 2 0ns,脉宽为 3 0 μs) ,2min内辐照 5次。并于辐照后 0 (即刻 )、1、6、12和 2 4h应用中生公司生化检测试剂盒测定细胞内和培养上清中LDH及培养上清中CHE、K+、Na+浓度。结果 :电磁脉冲辐照后即刻、1、6和 12h培养上清LDH明显升高 ;辐照后各时间点细胞内LDH明显降低 ,而培养上清中CHE、K+和Na+明显升高 ;辐照后 2 4h所有上述指标基本恢复。结论 :电磁脉冲辐照后可能损伤大鼠皮层神经元细胞膜     

高功率微波对原代培养心肌细胞的影响

目的：观察高功率微波(high Power microwave，HPM)对心肌细胞的影响及细胞内钙高于浓度的变化，探讨HPM对心肌细胞损伤的发生机制。方法：以功率密度950mW／cm^2的HPM辐照心肌细胞后，用荧光染料Fluo—3／AM负载，于激光扫描共聚焦显微镜下观察心肌细胞形态和[Ca^2 ]i的变化；用FITC—膜联蛋白(annexin)V／PI双染色法，于流式细胞仪检测心肌细胞的坏死和凋亡。结果：HPM辐照后，细胞内钙高于浓度明显降低，与对照组相比差异显著(P＜0．01)。心肌细胞发生坏死和凋亡，与对照组相比差异显著(P＜0.01)。结论：HPM辐照可引起心肌细胞[Ca^2 ]i明显降低，提示HPM辐照可引起心肌细胞膜的损伤，从而造成[Ca^2 ]i的大量漏出和细胞的死亡。     

乳鼠心肌细胞的体外培养及生物学特性研究

目的 探讨乳鼠心肌细胞的体外培养方法并对其生物学特性进行观察.方法 1～3日龄新生SD大鼠10只,用无酶消化法行含血清培养基原代心肌细胞培养,连续观察细胞生长及传代情况40天.α-横纹肌肌动蛋白抗体标记培养细胞,以流式细胞仪行心肌细胞纯度鉴定,Giemsa染色观察培养心肌细胞形态,吖啶橙-碘化丙啶(AO-PI)染色法检测培养细胞活性.记录培养心肌细胞总数,计算单个乳鼠心脏细胞产量.结果 原代组织块和单个心肌细胞在接种后24～48h贴壁,其中部分出现搏动并持续至观察期末.培养细胞群鉴定心肌细胞纯度为94.1%,活细胞比例95.3%,95%可信区间(CI)为91.6%～99.0%.平均每个乳鼠心脏单细胞产量为3.3×106个,95% CI为2.1×106～4.5×106个.结论 无酶消化法培养的乳鼠心肌细胞纯度、活力及产量能够满足心肌细胞研究的需要.     

高功率微波辐照对心肌细胞和心脏间质细胞生存能力和细胞活力影响的研究

目的：研究HPM对心肌细胞和心脏间质细胞生存能力和细胞活力的影响。方法：HPM辐照体外培养乳鼠心肌细胞和心脏间质细胞，用流式细胞仪检测细胞凋亡和坏死；用MTT实验测定细胞活力。结果：HPM可以对心肌细胞和间质细胞的功能和形态产生明显影响，引起二者的凋亡和坏死。结论：心脏可能是HPM损伤的易感器官之一，并提示在探讨HPM对心脏的损伤时，应同时关注对心脏间质细胞的研究。     

新生SD大鼠心肌细胞分离培养的快速简单方法及鉴定

目的：探讨心肌组织块体外原代培养新生大鼠心肌细胞的方法。方法：将剪成1mm^3左右小块心肌组织，采用体外贴块种植原代培养新生大鼠心肌细胞。结果：成功地培养出原代心肌细胞，并能够传代；倒置相差显微镜下证实培养的细胞为心肌细胞，免疫组化染色鉴定为心肌细胞特异性蛋白cTnT表达阳性，同时具有心肌细胞节律收缩特性。结论：心肌组织块体外原代培养操作简单，可以获得较多单个心肌细胞，所获得的细胞具有良好的功能状态。     

高功率微波辐照对大鼠血睾屏障通透性的影响

目的 探讨高功率微波（HPM）辐照大鼠睾丸对血睾屏障的影响。方法 雄性SD大鼠35只，随机分为7组，每组5只。6组为辐照组，1组为对照组。于辐照后2、6、12、4．8、96h和8d活杀大鼠后取双侧睾丸组织各约1mm^3，镧-醛固定液固定后制备电镜标本，在透射电镜下进行观察。结果 对照组可见硝酸镧主要沉积在曲细精管基膜部和精原细胞周围，未越过紧密连接；辐照后2-6h电镜下所见与对照组大致相同；辐照后12-96h，从基底部到管腔面的各层生精细胞之间、支持细胞内都有大量的镧颗粒沉积；辐照后8d，仍可见细胞间隙增大，部分区域靠近精子表面的镧颗粒沉积甚至比前面时间点的更为严重。结论 高功率微波辐照能够改变细胞的通透性，破坏血睾屏障的保护作用。     

高功率微波辐照对大鼠肝脏形态学及血清氧自由基的影响

目的：观察不同剂量高功率微波辐照对sD大鼠肝脏的损伤作用及血清氧自由基的影响。方法：分别用1万（Ⅰ组）、10万（Ⅱ组）、40万脉冲（Ⅲ组）剂量高功率微波辐照大鼠,辐照后14,24,48及72h观察肝脏在体及光镜、电镜下形态学改变,以生化法测定血清总超氧化物歧化酶（T—SOD）、丙二醛（MDA）含量。结果：1万、10万脉冲组在体肝脏未见明显改变,但40万脉冲组肝叶边缘有多条毛细血管扩张,部分大鼠肝叶表面有点片状出血,光镜及电镜下改变也以40万脉冲组最严重,部分可见肝细胞轻度变性坏死。与正常对照组相比,辐照组T—SOD活性降低（P〈0．01）,而MDA含量升高（P〈0．01）,尤其在40万脉冲组更为明显。结论：高功率微波可能通过氧自由基引起大鼠肝脏损伤,损伤程度与辐射剂量有一定的关系,辐射剂量越大,损伤越严重。     

高功率微波对大鼠心肌组织结构损伤及凋亡相关基因表达的研究

目的：研究高功率微波（high power microwave,HPM)对心肌组织结构、超微结构和凋亡相关蛋白表达的影响，以探讨HPM对心肌损伤的剂量效应关系，基本病变规律和分子病理机制。方法：5个不同功率密度的HPM辐照大鼠，于伤后1h,6h,24h,7d,14d及28d采取心肌组织，通过光镜、电镜观察其病变规律，免疫组化检测Bax,Bcl-2,c-Fos和p53蛋白的表达，结果：HPM辐照后心肌纤维早期出现细胞水肿，水变性，中后期可见收缩带，透明样变，损伤存在剂量效应关系。bcl-2/bax,c-fos和p53参与了HPM引起的心肌细胞凋亡。     

高功率微波对兔眼辐射损伤的研究

目的：采用高功率微波(HPM)辐照青紫蓝兔，长期动态观察角膜、晶状体及视网膜的病理变化，探讨HPM对视觉系统的影响。方法：6种功率密度的HPM辐照35只青紫蓝兔，并于照射后30，90，180和360d应用裂隙灯、检眼镜、光镜和电镜观察角膜、晶状体和视网膜的病理变化。结果：眼部各组织对HPM辐射损伤以晶状体最为敏感，角膜次之，视网膜轻微。辐照后30d，晶状体上皮细胞以变性为主。照后90～360d，晶状体囊膜增厚，上皮细胞增生，晶状体后皮质水肿、空泡形成，白内障发生。损伤具有剂量-效应相关性。结论：HPM可导致晶状体后皮质混浊、白内障发生，辐射功率与病理改变呈正相关。     

高功率微波辐照后大鼠卵巢组织Bcl-2及C-myc蛋白的表达

 目的检测高功率微波(High power microwave,HPM)与细胞凋亡相关基因Bcl-2和C-myc蛋白的关系.方法健康成年雌性SD大鼠100只,随机分为20组,每组5只.以S波段平均功率密度分别为20 mW/cm2的HPM辐照实验组大鼠10min,40mW/cm2辐照5min、10min.照后6 h、24h、48h、72 h、5 d取卵巢组织,应用免疫组织化学S-P法检测Bcl-2和C-myc蛋白表达的改变.结果HPM辐照后,24h组Bcl-2和C-myc蛋白表达升高,40mW/m2组二者升高较20mW/cm2组升高明显,差异有显著性意义(P＜0.01),正常对照组无表达.结论HPM辐照可促进Bcl-2和C-myc蛋白的活化和表达,可能是HPM诱导凋亡的机制之一.     

Effects of multiple doses of ionizing radiation on cytokine expression in rat and human cells

PURPOSE: To determine the effect of daily fractionated irradiation on the expression of growth factors and cytokines in different cardiac and vascular cell types. MATERIALS AND METHODS: Cell cultures of rat cardiac myocytes, fibroblasts, a rat cardiac microvascular endothelial cell line and human artery endothelial cells were irradiated with doses of 2 Gy, given daily during 5 consecutive days. Twenty-four hours after each fraction, gene expression was determined by competitive or semiquantitative polymerase chain reaction. Protein secretion into culture media was determined by enzyme-linked immunoabsorbant assay. RESULTS: Of all investigated mRNA levels, transforming growth factor (TGF)-ss1 and fibroblast growth factor (FGF)-2 were slightly upregulated in the rat cardiac endothelial cell line after irradiation. TGF-ss1 protein secretion by these cells was slightly, but non-significantly, elevated. Interleukin 1ss protein levels in myocyte culture media were decreased in control cultures at days 3 and 4 compared with day 2. No significant changes were observed in expression of FGF-2 in either of the four cell types. Moreover, no changes were observed in gene expression of platelet-derived growth factors A, B and interleukin 8 in the human artery endothelial cells. CONCLUSIONS: Fractionated irradiation leads to minor changes in the expression of specific cytokines in cardiac myocytes, fibroblasts and endothelial cells.     

高功率微波辐射对大鼠心肌、血浆心钠素和内皮素的影响

探讨高功率微波（HPM）辐射后大鼠心肌、血浆心钠素和内皮素的改变及其意义。方法：采用平均功率密度为10—100mW／cm^2的HPM模拟源辐射90只二级Wiatar雄性大鼠，应用光镜、图像分析技术和放射免疫法检测HPM辐射后6h，1、3、7及14d大鼠变性心肌、血浆心钠素和内皮素变化。结果：HPM辐射后6h～7d内变性心肌范围呈进行性扩大并加重，7d见心肌肌浆凝聚，横纹消失，且随辐射剂量增加，损伤效应加重，14d病变减轻。大鼠血浆心钠素浓度于1～7d升高（P〈0．01或P〈0．05），且与辐射剂量呈正相关，即辐射剂量越大，效应越明显，14d恢复正常水平。血浆内皮素浓度于辐射后7d内呈升高趋势，10和100mW／cm^2组1d升高有统计学差异（P〈0．01或P〈0.05），14d恢复正常水平。结论：一定功率密度的HPM辐射可造成心肌纤维变性，心脏内分泌功能受损。参与了HPM辐射所致心脏损伤的病理生理过程。     

Irradiated versus nonirradiated endothelial cells: effect on proliferation of vascular smooth muscle cells.

PURPOSE: Endovascular radiation therapy is a promising strategy for the prevention of restenosis. Radiation prevents proliferation of vascular smooth muscle cells, thereby reducing the incidence of restenosis, but may also affect the remaining endothelial cells. For this reason, a comparison was made between irradiated and nonirradiated endothelial cells and their effects on the proliferation of vascular smooth muscle cells in a coculture system was evaluated. MATERIALS AND METHODS: A coculture system was used, in which both endothelial cells and vascular smooth muscle cells were grown on opposite sides of a semipermeable membrane. After a period of growth arrest, the proliferation of vascular smooth muscle cells was measured during four subsequent days. RESULTS: The presence of endothelial cells stimulated the proliferation of vascular smooth muscle cells during the first days of analysis but had an inhibitory effect during the subsequent days (P <.5). gamma-irradiation of endothelial cells resulted in a complete blockage of the proliferation of these cells. However, irradiated endothelial cells affected the proliferation of vascular smooth muscle cells in coculture in a fashion comparable to nonirradiated endothelial cells (P >.5). CONCLUSION: The results suggest that, in endovascular radiation therapy, irradiation of endothelial cells does not change their effects on the proliferative behavior of vascular smooth muscle cells.     

UV or X-Irradiation Increases the Cytoplasmic Accumulation of Rhodamine 123 in Various Cancer Cell Lines

PURPOSE: Previous studies indicated that ATP-binding cassette (ABC) membrane transporters protect against UV-induced apoptosis. We investigated the effect of UVB and X-ray irradiation on the export function of these ABC transporters in primary lymphocytes and various cancer cell lines. MATERIAL AND METHODS: We used rhodamine accumulation assays in various human malignant cell lines and peripheral blood lymphocytes (PBL). Cells were irradiated with up to 960 mJ/cm2 and up to 50 Gy of UVB and X-ray, respectively. RESULTS: We demonstrated that UVB as well as X-ray irradiation inhibit the export function of the ABC transporters in a dose-dependent fashion. For PBL, this effect did not correlate with an apoptotic phenotype. In the case of the tumor cell lines, even though the irradiation-induced inhibition of membrane transporters was accompanied by phosphatidylserine exposure, only a minority of cells had lost their mitochondrial membrane potential during the observation period. Furthermore, we demonstrated that the inhibition of membrane transporters is not a general feature of apoptosis. CONCLUSION: Irradiation inhibits the export function of ABC transporters. Although some of the irradiated cells undergo apoptosis following irradiation, the inhibition is an unique feature accompanying irradiation and not a general hallmark of apoptotic cell death. The inhibition of drug export by irradiation may offer new potential for reverting multidrug resistance of cancer cells.     

Effects of multiple doses of ionizing radiation on cytokine expression in rat and human cells

Purpose: To determine the effect of daily fractionated irradiation on the expression of growth factors and cytokines in different cardiac and vascular cell types.

Materials and methods: Cell cultures of rat cardiac myocytes, fibroblasts, a rat cardiac microvascular endothelial cell line and human artery endothelial cells were irradiated with doses of 2?Gy, given daily during 5 consecutive days. Twenty‐four hours after each fraction, gene expression was determined by competitive or semiquantitative polymerase chain reaction. Protein secretion into culture media was determined by enzyme‐linked immunoabsorbant assay.

Results: Of all investigated mRNA levels, transforming growth factor (TGF)‐ß1 and fibroblast growth factor (FGF)‐2 were slightly upregulated in the rat cardiac endothelial cell line after irradiation. TGF‐ß1 protein secretion by these cells was slightly, but non‐significantly, elevated. Interleukin 1ß protein levels in myocyte culture media were decreased in control cultures at days 3 and 4 compared with day 2. No significant changes were observed in expression of FGF‐2 in either of the four cell types. Moreover, no changes were observed in gene expression of platelet‐derived growth factors A, B and interleukin 8 in the human artery endothelial cells.

Conclusions: Fractionated irradiation leads to minor changes in the expression of specific cytokines in cardiac myocytes, fibroblasts and endothelial cells.