Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73

Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.

Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.

Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37?°C after heat treatment at 44?°C for 15?min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3?h of incubation at 37?°C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3?h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.

Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.     

蛇床子水提取液抑瘤作用的实验研究

目的 :研究蛇床子水提取液的体内抗肿瘤作用。方法 :通过 S180 肉瘤移植建立荷瘤小鼠模型 ,给予不同剂量蛇床子水提取液后观察 S180 荷瘤小鼠肿瘤生长曲线、血清唾液酸 (SA)、瘤重及小鼠生存天数的变化。结果 :蛇床子水提取液能明显抑制肿瘤生长 ,降低荷瘤小鼠血清 SA水平 ,0 .0 6mg/(g· d) ,0 .1 1 mg/(g· d)、0 .2 1 mg/(g· d)剂量组平均瘤重低于肿瘤对照组 (P<0 .0 5 ) ,抑瘤率依次为 2 3 .2 %、2 9.1 %和 2 4 .8%,且能延长荷瘤小鼠生存天数 (P<0 .0 1 ) ,动物生命延长率依次为 :2 6 .9%、3 4 .8%和 2 6 .6 %。结论 :蛇床子水提取液具有较强的抗肿瘤效应 ,有很好的利用前景 ,值得对其进行深入研究。     

CD80mAb协同imDC诱导同种异体大鼠胰十二指肠移植免疫耐受

目的 观察共刺激分子阻断剂CD80单克隆抗体（CD80mAb）在协同未成熟树突细胞（imDC）诱导同种异体大鼠胰十二指肠移植免疫耐受中的作用。方法 建立糖尿病大鼠胰十二指肠移植动物模型；4E5杂交瘤细胞株BABIMC小鼠腹腔注射，抽取腹水，分离纯化后获得CD80mAb；分离供体大鼠骨髓来源DC细胞前体，经GM—CSF、IL-4体外刺激后。再加入IL-10共培养，鉴定为imDC；移植前7d，将2×10^6imDC经静脉途径注射至受体体内，同时分别给予生理盐水1ml、CD80mAb5mg连续14d。结果 四组受体大鼠移植后中位生存时间分别为12．7d、32．4d、50．2d、92．0d，实验组存活时间明显延长；组织学观察发现移植后7dCD80mAb＋imDC组移植物形态尚完整，淋巴细胞浸润减少；混合淋巴细胞反应证实移植后7dCD80mAb＋imDC组供受体间呈低反应性。结论 共刺激分子阻断剂CD80mAb能够协同imDC诱导受体T细胞对移植物的免疫耐受，降低宿主对移植物的急、慢性排斥反应，延长移植物的存活时间。     

一种西门子新型X线机的综合性能与安装

本文叙述西门子型号为Polydoros 80/100的新型X线主机,该主机采用微电脑控制技术、光纤维传输技术及中频技术,X射线剂量精度高,影像质量优良,故障率低,是一种技术较为成熟的X线主机,也是当今西门子公司产品的主流机种。该机的安装调试具有一定难度,本文同时针对这一问题进行讨论,并总结归纳出一套安装调试全过程。     

RNA干扰阻断BT／CD28共刺激通路在小鼠心脏移植中的抗排斥作用

目的探讨RNA干扰（RNAi）技术阻断B7／CD28共刺激通路对小鼠异体心脏移植排斥反应的影响及其机制。方法经体外转录合成针对CD80 mRNA和CD86 mRNA序列特异性小片段干扰RNA（siRNA），转染供者骨髓来源的树突状细胞（DC），半定量逆转录聚合酶链反应、流式细胞仪检测DC转染CD80siRNA、CD86siRNA前后CD80 mRNA和CD86 mRNA的表达水平以及细胞表面CD80及CD86的表达情况。在小鼠异位心脏移植前7d，经静脉给受者输注经siRNA干扰后的DC（干扰DC组），同时设立同种异体对照组、环孢素A（CsA）治疗组（术后皮下注射CsA 5mg／d）、同系移植对照组和未干扰DC组（移植前输注未转染DC），观察各组移植心脏的存活时间，对移植物的排斥反应进行病理分级，并测定移植物组织中白细胞介素2（IL-2）、γ干扰素（IFN-γ）及IL-10的mRNA表达水平。结果siRNA转染DC后，其CD80 mRNA及CD86 mRNA的表达受到明显抑制，CD80、CD86的阳性率分别由84％和67％下降至35％和30％。与同种异体对照组和未干扰DC组比较，干扰DC组移植心脏存活时间明显延长（P〈0．01），组织排斥反应病理分级显著降低（P〈0．01），移植心脏组织中IL-2 mRNA和IFN-γ mRNA的表达水平明显降低（P〈0．01），而IL-10 mRNA的表达水平明显升高（P〈0．01）。结论利用RNAi敲减供者骨髓来源的DC表面B7分子的表达，以阻断BT／CD28共刺激通路，具有抑制小鼠心脏移植排斥反应的作用，其机理可能是通过诱导T淋巴细胞无能并使T辅助细胞分化向Tn2型方向偏移。     

Mast cell activation involves plasma membrane potential- and thapsigargin-sensitive intracellular calcium pools

Summary— The regulation and role of the intracellular Ca²⁺ pools were studied in rat peritoneal mast cells. Cytosolic free calcium concentration ([Ca²⁺]i) was monitored in fura-2 loaded mast cells. In the presence of Ca²⁺ and K+, compound 48/80 induced a biphasic increase in [Ca²⁺]i composed of a fast transient phase and an apparent sustained phase. The sustained phase was partially inhibited by the addition of Mn²⁺. DTPA, a cell-impermeant chelator of Mn²⁺, reversed this inhibition, suggesting that a quenching of fura-2 fluorescence occurs in the extracellular medium. In the absence of extracellular Ca²⁺, the transient phase, but not the sustained one, could be preserved, provided that mast cells were depolarized. The transient phase was completely abolished by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor. Maximum histamine release induced by either compound 48/80 or antigen was obtained in the absence of added Ca²⁺ only when mast cells were depolarized. These histamine releases were inhibited by low doses (< 30 nM) of thapsigargin. Thapsigargin at higher doses induced histamine release which was unaffected by changing the plasma membrane potential, but was completely dependent on extracellular Ca²⁺, showing that a Ca²⁺ influx is required for thapsigargin-induced exocytosis. Together, these results suggest that the mobilization of Ca²⁺ from thapsigargin sensitive-intracellular pools induced by compound 48/80 or antigen is sufficient to trigger histamine release. The modulation of these pools by the plasma membrane potential suggest their localization is close to the plasma membrane.     

蛛网膜下腔注射神经降压素对大鼠血压和心率的影响

本文研究了蛛网膜下腔注射神经降压素（ｎｅｕｒｏｔｅｎｓｉｎ，ＮＴ）对大鼠血压和心率的影响，结果表明：注射ＮＴ后血压立即下降，继而有所回升，但不及正常水平，然后再次降低，持续６０ｍｉｎ；心率无明显改变。Ｈ１受体阻断剂苯海拉明能部分阻断ＮＴ的降血压效应，Ｈ２受体阻断剂甲氰咪胍无翻转作用。注射组胺释放剂Ｃｏｍｐｏｕｎｄ４８／８０使血压下降，待血压恢复正常水平后，再注射ＮＴ，其降压效应减弱。结果提示：蛛网