饰胶蛋白聚糖对大鼠肾系膜细胞生长的抑制作用及其信号转导途径的研究

目的 探讨饰胶蛋白聚糖(DCN)对大鼠系膜细胞(MsC)生长的抑制作用及其信号转导分子MAPKs和p21表达的影响&#65377;方法 经脂质体介导将DCN基因转染体外培养的大鼠MsC,筛选和鉴定后,收集阳性细胞克隆的培养上清(DCN上清),将其加入正常MsC的培养液中, 采用流式细胞仪检测细胞周期&#65377;用Western 印迹法分别检测MAPKs,包括细胞外调节激酶(ERK)1/2&#65380;应激活化蛋白激酶(SAPK)/氨基末端激酶(JNK)和p38和p21蛋白表达;用免疫荧光法观察p21在细胞中的表达&#65377;结果 DCN上清明显抑制正常MsC的增殖, G2-M期细胞数明显减少,仅为对照组的35%(P < 0.05); 磷酸化ERK1/2及SAPK/JNK表达增强, 分别为对照组的2.2倍&#65380; 1.4倍及1.7倍&#65380; 1.8倍;磷酸化p38无明显变化&#65377;DCN抗体呈浓度依赖性抑制磷酸化ERK1/2&#65380; SAPK/JNK的表达上调&#65377;DCN上清还可使细胞p21的表达明显增强,而DCN抗体也同样呈浓度依赖性地抑制其上调表达的作用, ERK1/2 及SAPK/JNK通路抑制剂U0126和circumin均可抑制其上调表达的作用,分别为对照组的64%和61%;而p38通路抑制剂SB203580则对其无影响&#65377;结论 DCN对肾MsC的生长有抑制作用,其机制可能经ERK1/2&#65380;SAPK/JNK和p21蛋白介导&#65377;     

Stemming Resistance to HER-2 Targeted Therapy

Although the development of trastuzumab and lapatinib has improved the outlook for women with HER-2 positive breast cancer, resistance to HER-2 targeted therapy is a growing clinical dilemma. Recent evidence indicates that the HER-2 pathway may play an important role in the maintenance of cancer stem cells (CSCs). The success of HER-2 targeted therapies may, in part, be explained by their direct activity against HER-2 positive CSCs. Our understanding of the mechanisms involved in resistance to trastuzumab, including loss or blockade of the trastuzumab binding site, activation of alternative signaling pathways, and induction of epithelial–mesenchymal transition (EMT), suggests that CSCs may be at the root of resistance of HER-2 targeted therapy. A variety of novel HER-2 targeted approaches have demonstrated promising preliminary clinical activity. Future clinical trials should involve the integration of technologies to assess the impact of novel HER-2 targeted therapies on HER-2 positive CSCs.     

Stereological and biochemical analysis of muscular and connective tissue components in the penile corpus cavernosum adjacent to the fibrous plaque of Peyronie's disease

OBJECTIVE

To investigate the structural organization of the connective tissue in the corpus cavernosum (CC) adjacent to the fibrous plaque in Peyronie’s disease (PD) using stereological and biochemical techniques, as most studies on PD have focused on the analysis of the fibrous plaque that forms in the tunica albuginea (TA). Because this fibrotic reaction is mediated by various inflammatory soluble factors, adjacent connective tissues might also be affected and this secondary effect might explain, for example, the erectile dysfunction that occurs in PD.

PATIENTS AND METHODS

During surgery biopsies were taken from the CC adjacent to the fibrous plaque and from the plaque itself in seven patients with PD (mean age 48.3 years). All the patients had normal erections. Control samples were similarly located samples from ‘normal’ penises obtained during autopsy of five men (mean age 52.3 years). Tissue samples were stained with Weigert’s stain (elastic fibres), Van Gieson’s stain (connective tissue), and Sirius red (collagen). Stereological analysis was done using a 42‐point grid to determine volumetric densities (Vv). Total collagen content was estimated as micrograms of hydroxyproline per milligram dry CC.

RESULTS

The Vv of elastic fibres was significantly reduced in PD by 17.3% compared with controls, at a mean (sd ) of 19.49 (3.27)% vs 23.56 (1.87)% (P < 0.05). While in PD the Vv of smooth muscle at 34.46 (2.06)% and connective tissue at 35.39 (6.15)% were not significantly different from those of controls at 38.38 (3.17)% and 38.02 (5.03)%, respectively. The Vv of elastic fibres in the fibrous plaque was decreased by 38.3% compared with the normal TA, at 20.25 (5.49)% vs 32.81 (4.75)% (P < 0.02). The mean (sd ) collagen concentration in the CC from controls was 77.94 (24.26) µg/mg and in the patients with PD was 66.57 (19.39) µg/mg, which did not differ significantly. Sirius red‐stained sections under polarized light showed that, in the normal CC, collagen‐associated colours were homogeneously distributed. However, in the PD samples, stained collagen had a disrupted orientation and had a more heterogeneous birefringence, implying looser collagen bundles.

CONCLUSIONS

The quantitative analyses indicated that collagen in the CC close to the fibrous plaque was not affected, although its organization was noticeably altered. The CC elastic fibres were reduced though, and there was a similar change in the fibrous plaque of the TA. These results suggest that, although occurring primarily in the TA, the PD fibrous plaque may induce changes in the adjacent CC.     

不同浓度葡萄糖对大鼠间皮细胞增殖及细胞外基质产生的影响

观察了不同浓度葡萄糖对膜腹间皮细胞增殖和产生细胞外基质的作用，发现：①不同浓度的高渗葡萄糖可以抑制大鼠腹膜间皮细胞的增殖，引起间皮细胞的死亡，增强层粘连蛋白和Ⅳ型胶原的产生；②葡萄糖浓度与在此环境下培养的活细胞百分比呈负相关，但与层粘连蛋白的产生呈正相关，且活细胞百分比与层粘连蛋白的产生呈负相关。提示：培养液中葡萄糖的浓度在大鼠腹膜间皮细胞的增殖和细胞外基质的产生中起重要作用。     

Ultra-microstructural changes in iliac artery after bare and magnetic stent implantation in rabbits

Objective To investigate the preventive effect of magnetic stent on coronary restenosis after percutaneous arterial stenting. Methods Twenty rabbits were divided randomly into 2 groups.Bare stent (BS group,n=10) or magnetic stent (MS group,n=10) was implanted in the left iliac artery of the rabbits of the 2 groups,respectively.Aspirin (25rag,qd) was administered orally to the rabbits of both groups from 3 days before stenting until the rabbits were executed.Unfractionated heparin (2500u,qd) was delivered subcuta- neously after stenting for 7 days.Five rabbits of each group were randomly selected to be executed at 7 or 30 days.Structural changes in the injured arteries were studied by optical microscopey,transmissive electronic microscopey and immunohistochemistry.Results At 7 days,more myofibroblasts were found migrating from adventitia to tunica media and intima in BS group than in MS group.Inside the media and intima,large amount of smooth muscle cells of synthetic type were observed.At 30 days after stenting,in magnetic group, most uascular smooth muscle cells (SMCs) under the intima had transformed to contractile type and only little extracellular matrix (ECM) was observed around the SMCs;whereas,in BS group,the SMCs remained to be synthetic type and large amount of ECM was observed around the SMCs,which was composed mainly ofproteoglycans and glycoproteins.Conclusions Magnetic stent can inhibit proliferation and migration of SMCs and reducing the production of ECM,and therefore,may prevent restenosis after coronary stenting.     

蛋白激酶对内毒素休克后内皮细胞骨架的作用

目的　探讨内毒素休克发病机制中蛋白激酶G(PKG)的作用。　方法　用脂多糖 (LPS)刺激培养的内皮细胞 ,通过细胞裂解和离心获得细胞裂解液 ,用放射性同位素法标记法检测PKG的活性。同时采用特异性荧光染色法检测LPS刺激后细胞内肌动蛋白微丝 (F actin)的结构和分布变化。用PKG特异性抑制剂KT5 82 3预处理细胞后 ,再检测LPS介导的细胞内PKG活性和F actin的变化。以空白组为阴性对照 ,以PKG激动剂 8 Br cGMP刺激细胞作为阳性对照组。　结果　LPS分别刺激 5、10、30和 6 0min后细胞内PKG活力呈时间依赖性的增高 (与空白组相比P <0 .0 1) ,细胞内的F actin出现极性分布 ;而KT5 82 3预刺激 2 0min后再用LPS刺激没有出现上述变化。PKG的激动剂 8 Br cGMP刺激细胞后的变化与LPS的刺激相似。　结论　LPS可以介导血管内皮细胞PKG的激活和F actin的应力性变化 ;内毒素休克后内皮细胞通透性增高与cGMP PKG通路的激活有关。     

磷酸化ERK在扩张皮肤组织中的分布和表达

目的：观察细胞外信号调节激酶（ERK1／2）在扩张皮肤表皮中的分布情况，探讨皮肤扩张机理。方法：利用免疫组织化学技术ABC法，对人不同扩张区皮肤和正常皮肤磷酸化ERK的分布和表达进行定性测定，并对结果进行图像分析。结果：（1）扩张皮肤和正常皮肤表皮基底层都有磷酸化ERK的表达和分布，但扩张皮肤中的磷酸化ERK的分布和表达较明显，染色较深且密，部分阳性细胞呈多层排列，有散在的增殖闭区；（2）扩张皮肤的顶部和侧部差别不十分明显，而侧部和顶部与基底部有一定的差别，基底部染色较深，多为细胞核着色。（3）经图像分析不同部位的正常皮肤和扩张皮肤以及扩张皮肤的不同部分的相对灰度值和阳性细胞密度，进一步证实了上述观察结果。结论：扩张皮肤表皮细胞磷酸化ERK增加，推测磷酸化ERK可能在扩张皮肤细胞增殖过程中发挥一定的作用，在扩张刺激的作用下，表皮细胞内的ERK被大量激活，随即激活下游分子，进而引起细胞的增殖，最终实现皮肤的扩张。     

人DNase I的表达、纯化及降解NETs活性研究

为获得纯度较高的人脱氧核糖核酸酶Ⅰ(DNase I)以研究其对中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)的降解作用,构建基因工程表达菌E.coli Rosetta(DE3)/pET32a-His-DNase I,乳糖诱导表达,经镍柱亲和纯化获得融合蛋白His-DNase I。提取小鼠中性粒细胞,用佛波酯PMA刺激形成NETs,SytoxGreen及荧光显微镜检测融合蛋白对NETs的降解活性。结果表明,成功构建人DNase I基因克隆并在原核细胞中实现高效表达,纯化的His-DNase I具备较高的核酸酶活性。本研究为进一步探究DNase I的临床应用奠定了理论基础。     

ERK1/2信号通路介导PDGF-CC诱导的鼠心肌纤维化及其机制

目的 研究ERK1/2通路在血小板源性生长因子(PDGF)-CC诱导的鼠心肌纤维化中的作用及其可能的机制.方法 取SD大鼠乳鼠心脏组织,差速贴壁法分离并纯化心肌成纤维细胞.按不同药物处理随机分成对照组(CON组)、PDGF-CC(P组)、PDGF-CC+ERK1/2抑制剂U0126(PU组).MTT法检测心肌成纤维细胞的增殖,qRT-PCR法检测mRNA含量,Western blot法分析蛋白表达量.结果 MTT法显示P组细胞数较CON组显著增加(P＜0.01),而PU组细胞数较P组明显减少(P＜0.01).qRT-PCR法显示P组中PDGF-α受体(PDGFR-α)、ERK1、ERK2、Ⅰ型和Ⅲ型胶原蛋白(Col Ⅰ、ColⅢ)的mRNA表达水平均明显高于CON组(P ＜0.001),PDGFR-β的mRNA表达量在P组与CON组间差异无统计学意义.与P组相比,PU组中PDG-FR-α、ERK1、ERK2、Col Ⅰ和ColⅢmRNA表达均明显下降(P＜0.01).Western blot法显示P组中磷酸化PDGFR-α(p-PDGFR-α)、ERK1/2、p-ERK1/2、Col Ⅰ和ColⅢ的表达量均明显高于CON组(P ＜0.001),PU组中p-PDGFR-α、ERK1/2、p-ERK1/2、Col Ⅰ和ColⅢ蛋白表达量均显著低于P组(P＜0.001).结论 PDGF-CC可能通过结合PDGFR-α激活ERK 1/2信号通路,诱导鼠心肌成纤维细胞过量增殖伴胶原蛋白的合成,参与心肌纤维化的发生.     

细胞外信号调节蛋白激酶在前列腺上皮内瘤组织中的表达及意义

目的 探讨细胞外信号调节蛋白激酶（ERK）在前列腺上皮内瘤（PIN）组织中表达的意义。方法 应用免疫组织化学SP法检测12例PIN、11例前列腺癌（PCa)及16例BPH组织标本中ERK的表达。结果 12例PIN标本均有表达,其中6例为高表达;11例PCa组织10例为阳性,但多为弱表达;16例BPH组织12例为阳性,表达也较弱。PIN组织中ERK表达与PCa组和BPH相比ERK在PIN组织中呈高表达,与早期前列腺癌的发生有密切关系。