妊娠早期小鼠子宫内膜热休克蛋白70的免疫组化研究

目的 :研究妊娠早期小鼠子宫内膜热休克蛋白 70的表达。方法 :采用免疫组织化学方法。结果 :热休克蛋白 70主要存在于子宫内膜固有层的基质细胞及蜕膜细胞 ,内膜上皮、腺上皮中未见表达。与未孕小鼠相比 ,孕鼠热休克蛋白 70免疫反应阳性细胞显著增多 (P <0 .0 1 )且随妊娠日龄的增加而增加 (P <0 .0 1 )。结论 :热休克蛋白70可能参与了子宫内膜蜕膜反应中基质细胞的增殖 ,与蜕膜反应密切相关     

热休克预处理对大鼠缺血再灌注心肌保护作用机制的探讨

目的:观察心肌缺血再灌注时P-选择素（Ps）表达情况；探讨热休克蛋白(HSP)对缺血再灌注心肌Ps及细胞凋亡表达的影响。 方法:成年雌性Wistar（n=40）大鼠随机分为3组。热休克组全麻后高热处理造成热休克动物模型，对照组及假手术组仅予全麻处理。24 h后热休克组及对照组结扎左冠状动脉前降支(LAD)1 h，再灌注2 h造成心肌缺血再灌注动物模型。假手术组只于LAD处穿线而不结扎。术毕测心梗范围、HSP70、Bax、Bcl-2、Ps、凋亡细胞及血清CK-MB。 结果:热休克组HSP70表达高于对照组及假手术组(P<0.05)，后两组无明显差别(P>0.05)；热休克组心梗范围小于对照组(P<0.05)，CK-MB值低于对照组(P<0.01)，凋亡细胞、Bax及Ps表达低于对照组(P<0.05)，两组Bcl-2表达无显著差别(P>0.05)；假手术组无Ps表达。 结论:HSP70可抑制缺血再灌注诱导的心肌细胞凋亡，抑制Bax表达致Bax/Bcl-2比值下降为其机制之一；Ps参与心肌缺血再灌注损伤；HSP70可能有抑制心肌Ps表达的作用，这或许是热休克预处理对大鼠缺血再灌注心肌的另一保护机制。     

榄香烯或热休克对人肝癌细胞HepG2HSP70膜表达及多种HSP基因表达的影响

目的 :研究榄香烯或热休克对人肝癌细胞HepG2 HSP70膜表达及多种HSP基因表达的影响。方法 :免疫荧光和FCM观察榄香烯 (5 0 μg ml,1h)或热休克 (42℃ ,1h)处理后肿瘤细胞HSP70的膜表达 ,放线菌素D阻断基因转录。应用人类基因表达谱芯片分析经榄香烯或热休克处理的人肝癌细胞HepG2 多种热休克蛋白基因及与热休克蛋白调控相关基因表达谱的改变。结果 :榄香烯或热休克处理 1h后 ,肿瘤细胞膜表面HSP70的表达均有增高 ,而以榄香烯处理为明显 ,放线菌素D的加入在两种处理中均增高了HSP70膜表达的阳性率。两种处理均使细胞的HSP70HP(EnhancerProtein)基因表达增高 ,而以热休克处理为更明显 ,HSPA2的表达均有所下降 ,也以热休克处理更明显 ,HSF1基因在热休克处理为上调 ,而在榄香烯处理则为下调 ,与肿瘤免疫密切相关的HSP70、HSP72、HSP75及HSP90、gp96基因的表达则没有变化。结论 :榄香烯较热休克处理早期能更多地促进HepG2 细胞HSP70的膜表达 ,其机制可能是与其改变胞内已存在的HSP70的分布 ,和 或促进HSP70mRNA的翻译有关。两种处理均能改变与HSPs调控相关基因的表达 ,并可引起HSPA2基因表达下调。     

VP-16诱导细胞凋亡及其细胞核基质中热休克蛋白表达的研究

目的　研究VP 16诱导HL 6 0细胞凋亡的变化特点以及凋亡细胞及核基质中热休克蛋白 (HSP)表达的变化。　方法　琼脂糖凝胶电泳观察凋亡细胞基因组DNA断裂 ;TUNEL法观察凋亡细胞的形态学变化 ;用免疫印迹方法显示凋亡细胞核基质蛋白、细胞总蛋白中HSP表达的差异。　结果　VP 16作用HL 6 0细胞 0 5h出现早期凋亡特征。作用 4h可见明显的DNA梯状条带。与未凋亡的细胞比较 ,凋亡细胞核基质蛋白中HSP70和HSC70表达明显上调 ;VP 16作用 2、3及 4h细胞总蛋白中HSP70的表达随时间延长略显增加 ;诱导 2 2h比诱导 4h时去除VP 16后孵至 2 2h凋亡细胞总蛋白HSP70表达量增强了 3 4倍。　结论　 1 HL 6 0细胞早期凋亡形态出现在DNA梯状条带形成之前。 2 凋亡细胞核基质中HSP70、HSC70的表达明显增加 ,经 2、3及 4h作用的细胞总蛋白中HSP70表达差异不显著。这提示细胞凋亡后HSP70的活性位点主要位于核基质上。 3 VP 16作用细胞时间增长至2 2h ,HSP的保护作用可能消失     

大鼠局灶性脑梗死后细胞凋亡及热休克蛋白70表达的变化

目的 探讨大鼠局灶性脑梗死后热休克蛋白70(HSP70)及细胞凋亡的变化。方法采用光化学法诱导制作大鼠局灶性脑梗死模型，冰冻切片行HSP70免疫组化染色，并用TdT-介导duTP-生物缺口末端标记(TUNEL)法检测凋亡细胞。结果免疫组化显示对照组和假手术组动物无HSP70免疫反应；局灶性脑梗死组12h即有：HSP70的表达，48h时HSP70表达至高峰，阳性反应局限于半暗带。TUNEL结果显示，对照组和假手术动物无凋亡细胞；局灶性脑梗死组6h时在半暗区凋亡细胞出现，3d达高峰。结论局灶性脑梗死可诱导HSP70的表达、神经细胞凋亡，两者的分布一致，但HSP70的表达高峰明显早于细胞凋亡。     

SHR和WKY大鼠心肌细胞凋亡、HSP70、iNOS水平的比较研究

目的:检测自发性高血压大鼠(SHR)和正常血压大鼠(WKY)心肌细胞凋亡和诱导型一氧化氮合成酶、热休克蛋白70水平变化,并探讨其机制。方法:透射电镜、TUNEL法检测SHR和WKY大鼠心肌细胞凋亡;免疫组化检测其iNOS、HSP70蛋白表达。结果:透射电镜示SHR组可见凋亡特征的心肌细胞;TUNEL法示SHR组凋亡明显高于WKY组(P<0.01);免疫组化示SHR组iNOS、HSP70蛋白表达明显高于WKY组(P<0.01)。结论:SHR心肌细胞凋亡在自发性高血压病发病过程中起重要作用;SHR的iNOS、HSP70蛋白表达增加与细胞凋亡同时存在,可能也参与自发性高血压心肌细胞凋亡的调控。     

URSA小鼠模型中NKT细胞功能失调研究

利用原因不明复发性自发性流产 (URSA )小鼠模型 (CBA雌鼠×DBA/ 2J雄鼠 ) ,测定免疫性流产孕鼠不同孕期子宫蜕膜及胎盘内NKT细胞的数量及其分泌的细胞因子的格局 ,以调查NKT细胞数量和功能失调在URSA中的可能作用。研究结果显示 ,与正常孕鼠相比 ,流产模型鼠妊娠早期子宫和蜕膜中NKT细胞数量减少 ,分泌IFN γ能力降低。我们推测 ,妊娠早期子宫胎盘和蜕膜NKT细胞数量和功能失调可能是导致URSA的机制之一。     

热休克蛋白在高温处理后鼠胚心脏的表达

目的：探讨热休克蛋白在高温处理后金黄地鼠胚心脏的表达变化。方法：将金黄地鼠孕鼠于受孕第8d置42℃水浴持续20min，定时剖腹取胎，制备石蜡切片。利用免疫组织化学(SABC法)染色，观察心脏正常发育和异常分化过程中热休克蛋白(HSP70和HSP90)的表达。结果：高温处理后8h，实验组鼠胚内皮细胞及心胶质HSP70和HSP90的表达较对照组明显增强；16h，表达最强；24h，仍较对照组强；48h，和对照组的表达无显著性差异。结论：高温诱导金黄地鼠胚心脏热休克蛋白表达增强，对心脏的异常发育具有保护作用。     

子宫内膜基质细胞AQP7的表达以及卵巢激素对AQP7的调控作用

目的:探讨水通道蛋白7(Aquaporin 7,AQP7)在小鼠子宫内膜及在体外诱导蜕膜化模型中的表达,卵巢激素对AQP7的调控,以探讨AQP7在子宫蜕膜化中的作用.方法:分离孕4-8天小鼠子宫基质细胞(Uterine stromal cells,ESCs),实时定量PCR(RT-PCR)检测基质细胞AQP7表达.收集孕4～8天小鼠子宫,免疫组织化学染色检测子宫内膜组织AQP7的表达.分离原代小鼠基质细胞,建立体外诱导蜕膜化模型,RT-PCR检测AQP7在ESCs中的表达.在体外培养的孕5天小鼠子宫基质细胞中分别加入雌二醇(E2)、孕酮(P4)以及E2和P4联合处理.用雌激素受体(ER)的拮抗剂ICI 182,780和孕激素受体(PR)拮抗剂RU486预处理基质细胞,再用E2+P4处理.收集各组ESCs,RT-qPCR检测卵巢激素对基质细胞中AQP7的调控作用.结果:AQP7 mRNA在体外分离的孕4～8天的子宫基质细胞中的表达逐渐升高.从孕5～8天,随着蜕膜化进程,子宫基质细胞中AQP7表达也逐渐增加.在体外诱导蜕膜化模型中,小鼠基质细胞中AQP7 mRNA表达显著升高(P<0.01).在E2和P4单独作用下,基质细胞中AQP7表达无明显变化;而与对照组比较,E2和P4联合处理组在E2和P4联合作用24小时后基质细胞中AQP7表达显著升高(P<0.01);且在E2和P4联合作用后、基质细胞中AQP7的升高能够被雌激素受体的拮抗剂ICI 182,780和孕激素受体拮抗剂RU486阻断(P<0.01).结论:随着蜕膜化进程,子宫基质细胞中AQP7表达逐渐增加.卵巢雌、孕激素联合作用可上调基质细胞中AQP7表达.子宫基质细胞中AQP7在蜕膜化和胚胎着床中发挥着重要作用.     

自然流产模型小鼠蜕膜细胞凋亡及相关基因的表达

目的 通过比较正常妊娠模型小鼠及自然流产模型小鼠蜕膜细胞凋亡及Bcl-2、Bax、Fas、FasL蛋白的表达,从细胞及分子水平探讨自然流产的发病机制.方法建立正常妊娠模型CBAXBALB/c和自然流产模型CBAXDBA/2.用免疫组化SABC法测定两组模型孕13 d蜕膜细胞Bcl-2、Bax、Fas、FasL蛋白的表达,并通过MIAS-2000医用彩色病理图像免疫组化测量系统对其表达进行半定量分析,其结果用平均灰度值表示;同时应用DNA缺口原位末端标记技术(TUNEL)测定两组模型孕13天蜕膜细胞凋亡情况.结果与正常妊娠模型相比,自然流产模型蜕膜细胞Bcl-2蛋白的表达降低(P＜0.01);Bax蛋白的表达明显升高(P＜0.01);FasL的表达明显升高(P＜0.01);Fas的表达两组比较无明显差异(P＞0.05).蜕膜细胞凋亡指数(AI),自然流产模型明显高于正常妊娠模型(P＜0.01).结论 早孕期蜕膜组织细胞凋亡异常是自然流产的机制之一,Bcl-2/Bax,Fas/FasL途径可能是诱导早孕期蜕膜细胞凋亡的重要因素.     

Histopathologic changes of uterus following gossypol treatment during early pregnancy in rats

Intramuscular injections of gossypol acetic acid (25 mg in 10% EtOH/kg/day beginning on day 2 of diestrus) disrupted early pregnancy in rats as determined by light and electron microscopy. As in pregnant controls, in the uteri of treated rats increased glandular secretion, stromal hyperemia, and decidual tissue formation were noted at days 3-5 of pregnancy. At day 6, extreme hyperemia and stromal hemorrhage had occurred around well-developed decidual tissue with foci of denuded mucosal surface. There was extravasation of blood into the uterine lumen, which was absent in controls. At days 5 and 6 of pregnancy, electron microscopy revealed shorter and fewer microvilli on the uterine glandular cells in the treated versus the control uterus. Luminal epithelial cells had not undergone the normal changes of pregnancy. These results imply that gossypol administered under our conditions neither prevented nor delayed implantation and formation of decidual tissue in the rat uterine endometrium but continuing development of the endometrium was disrupted at day 6 of pregnancy. This disruption of pregnancy may have resulted from a luteolytic action by gossypol that would not permit full structural differentiation in the rat uterus after implantation.     

白细胞介素-18在大鼠胚泡植入前后子宫的分布及表达

目的 系统研究了白细胞介素-18(IL-18)在大鼠胚泡植入前后子宫中的分布与表达,以探讨IL-18在胚泡植入过程中可能的生理作用.方法 免疫组织化学SP法,图像分析法.结果 IL-18在胚泡植入前后子宫均有表达,其主要分布于子宫内膜或蜕膜的腔上皮细胞、腺上皮细胞及子宫基质细胞或蜕膜细胞,子宫肌层及部分血管内皮细胞也有微弱表达.与非妊娠大鼠相比,妊娠大鼠子宫IL-18的表达明显增加(P<0.05).随着妊娠的进行,子宫IL-18的表达逐渐递增,并且IL-18在胚泡植人后期的表达显著高于植入前期(P<0.05).结论 IL-18可能参与胚泡植入,与妊娠的建立和维持有关,在妊娠中起重要作用.     

热休克蛋白70、90在子宫内膜癌中的表达

目的:探讨子宫内膜癌组织中热休克蛋白(HSP)70、90的表达及意义。方法:用免疫组化Envision法及图象分析仪,检测 30例正常子宫内膜、30例增生过长子宫内膜和53例子宫内膜癌中HSP70、HSP90的表达。结果:子宫内膜癌中HSP70表达的灰度值为(209.06±5.36),明显高于正常内膜[(145.21±4.09),P<0.01]和增生过长内膜[(148.59±4.23),P<0.01];子宫内膜癌中HSP90表达的灰度值为(166.98±5.71),明显低于正常子宫内膜[(208.57±31.14),P<0.05]和增生过长子宫内膜[(249.73±4.94),P<0.01]。子宫内膜癌中HSP70的表达随肿瘤病理分级的增加而增强(P<0.01),非内膜样癌(229.90±3.77)较内膜样癌表达强[(198.37±3.15),P<0.01];子宫内膜癌中HSP90表达随肿瘤病理分级的升高而表达减弱,非内膜样癌(140.21±3.22)较内膜样癌表达减弱[(176.59±2.79),P<0.01]。子宫内膜癌中HSP70、90的表达与肌层浸润深度、临床分期及淋巴结转移未见显著相关性(P>0.05)。结论:HSP70、90可能与子宫内膜癌的发生及预后有关。     

Immunohistochemical localization of the growth hormone in human endometrium and decidua

PROBLEM: Recent evidence of growth hormone (GH) receptor expression in rat endometrium and human myometrium have focused our attention on the role of the GH in endometrial development. We tested the expression of GH in the human endometrium throughout the menstrual cycle and during pregnancy. METHOD OF STUDY: Immunohistochemical study was performed on endometrial specimens of fertile women in different periods of the menstrual cycle and in decidua of pregnant women. RESULTS: Glandular cells of the human endometrium were positive for GH in the mid and late luteal phase. Furthermore, the glandular cells of decidua showed intense staining for GH, while the stromal cells were negative. No immunostaining was expressed in the proliferative or early luteal phase. The intensity levels of staining for GH in decidual specimens were significantly higher than in glandular cells of secretory endometrium specimens (P < 0.01). CONCLUSIONS: The glandular cells of the human endometrium express GH from the late luteal phase throughout pregnancy in the decidual tissue. We suppose that GH plays an important role in blastocyst implantation.     

Perlecan and syndecan-4 in uterine tissues during the early pregnancy in mice

PROBLEM: During early pregnancy in mice, there is recruitment of specific immune cells, remodeling of the endometrium, cell differentiation and synthesis of new molecules. METHOD OF STUDY: Immunohistochemistry was used to determine the distribution of perlecan and syndecan-4 in the uteri before and after embryo implantation. RESULTS: During pre-implantation, perlecan was identified in basement membranes and extracellular spaces of the endometrial stroma. In contrast, expression of syndecan-4 was quite weak. In the peri-implantation period, perlecan remained in the basement membranes, and it was no longer observed in the stroma and it was identified in the embryonic cells. On day 4 of pregnancy, syndecan-4 increased in the fibroblasts of the subepithelial stroma. After implantation, syndecan-4 was pronounced in pre-decidual and mature decidual cells. CONCLUSIONS: The coordinate balance between the pre- and post-implantation periods suggests a role of these two molecules in the adaptive modification of the uterine microenvironment to receive and implant the embryo.     

热休克蛋白在子宫内膜异位症中的表达及意义

目的 探讨热休克蛋白27（HSP27）和HSP70在子宫内膜异位症发病中的作用。方法 采用免疫组化链霉菌抗生素蛋白－过氧化物酶染色法（SP法），检测子宫内膜异位症中56例异位内膜与在位内膜（30例）中HSP27和HSP70的表达。采用原位杂交，检测HSP70mRNA的水平，并与正常子宫内膜相比较。结果 免疫组化结果显示，异位内膜组织中HSP27与HSP70均呈高表达，与正常内膜组的表达差异显著（P＜0．01），而且失去其在正常内膜组织中表达的周期性变化。卵巢子宫内膜异位症组（OEM）与子宫腺肌症组（AM）组中，HSP27和HSP70的表达无显著差异（P＞0．05）。原位杂交结果显示，HSP70 mRNA（分别为P＜0．01和P＜0．05）。HSP27与HSP70的表达水平，同OEM的严重性无关。结论 异位内膜组织中的HSP70基因被激活，转录增加。HSP27和HSP70呈高表达，可能在EM的发病中起重要作用。     

Concerted upregulation of CLP36 and smooth muscle actin protein expression in human endometrium during decidualization

The human endometrium prepares for implantation of the blastocyst by reorganization of its whole cellular network. Endometrial stroma cells change their phenotype starting around the 23rd day of the menstrual cycle. These predecidual stroma cells first appear next to spiral arteries, and after implantation these cells further differentiate into decidual stroma cells. The phenotypical changes in these cells during decidualization are characterized by distinct changes in the actin filaments and filament-related proteins such as alpha-actinin. The carboxy-terminal LIM domain protein with a molecular weight of 36 kDa (CLP36) is a cytoskeletal component that has been shown to associate with contractile actin filaments and to bind to alpha-actinin supporting a role for CLP36 in cytoskeletal reorganization and signal transduction by binding to signaling proteins. The expression patterns of CLP36, alpha-actinin and actin were studied in endometrial stroma cells from different stages of the menstrual cycle and in decidual stroma cells from the 6th week of gestation until the end of pregnancy. During the menstrual cycle, CLP36 is only expressed in the luminal and glandular epithelium but not in endometrial stroma cells. During decidualization and throughout pregnancy, a parallel upregulation of CLP36 and smooth muscle actin, an early marker of decidualization in the baboon, was observed in endometrial decidual cells. Since both proteins maintain a high expression level throughout pregnancy, a role of both proteins is suggested in the stabilization of the cytoskeleton of these cells that come into close contact with invading trophoblast cells.     

Leukocyte function-associated antigen-1 expression on decidual natural killer cells in patients with early pregnancy loss.

A large number of CD56(bright) natural killer (NK) cells, which comprise a very small fraction of peripheral blood lymphocytes, appear in the endometrium during the late secretory phase and early pregnancy. These cells are thought to immunologically maintain or inhibit pregnancy. However, the details regarding their contribution to the immuno-elimination systems of embryonic cells or decidual stromal cells remain unclear. Recently, leukocyte function-associated antigen-1 (LFA-1) was shown to play a critical role in the regulation of NK cytolysis in peripheral blood lymphocytes. We speculated that LFA-1 on the decidual CD56(bright) NK may be involved in the regulation of pregnancy. The expression of LFA-1 on the decidual CD56(bright) NK cells was analysed using flow cytometry with fluorescent monoclonal antibodies; CD56 (NKH1), CD16 (Fcg-R3) and CD11a (LFA-1 alpha-chain). In comparison with non-pregnant endometrium during the late secretory phase, the subpopulation of CD56(bright)CD16(-) cells in decidual lymphocytes was significantly increased during normal pregnancy, but was less than that in early pregnancy loss (P < 0.05). Furthermore, the number of CD56(bright) NK cells expressing LFA-1 was significantly higher in early pregnancy loss, and the late secretory phase, than during normal pregnancy (P < 0.05). Our results indicate that up-regulation of LFA-1 on CD56(bright) NK cells is related to spontaneous abortion or the onset of menstruation.     

Moesin is involved in the cytoskeletal remodelling of rat decidual cells

The extensive actin cytoskeletal remodelling of the uterine stroma during early pregnancy involves changes in actin-binding proteins. This study provides the first detailed localisation of the actin-binding protein, moesin, in rat uterine endometrium during this period. Western blot analysis and immunofluorescence microscopy showed that the amount of moesin in the uterus peaked at the time of implantation, corresponding to the presence of intensely immunolabelling decidual cells. Furthermore, moesin increased in active membrane/cytoskeleton bound protein at the time of implantation, concomitantly decreasing in cytosolic protein. The increase of moesin at decidualisation corresponds with the appearance of alpha smooth muscle actin (alphaSMA) suggesting that decidual cells have contractile abilities that may aid in containing an invasive trophoblast. The results of this study suggest that moesin is important in developing a specialised cytoskeleton and increased adhesiveness of decidual cells, possibly functioning to bridge adhesion molecules to the underlying cytoskeleton.