结直肠癌中MEK2/ERK信号传导通路的研究

目的　研究丝裂原激活化蛋白激酶 (MAPK )中MEK2 /ERK信号传导通路在结直肠癌发生发展中的作用。方法　 ( 1)采用Westernblot检测 5 2例结直肠癌组织及其邻近肠黏膜中MEK 2蛋白的表达。 ( 2 )用丝裂原细胞外激酶 (MEK )抑制剂作用于结肠癌细胞系SW 480 ,然后以MTT法检测细胞增殖状态 ;用Westernblot检测MEK2 ,p ERK及其靶基因产物C myc的表达。结果　结直肠癌组织中MEK2蛋白表达水平明显高于邻近的肠黏膜 (P <0 .0 5 ) ,且与肿瘤的分化、Dukes分期及淋巴结转移有关 (P <0 .0 5 )。应用MEK的抑制剂后SW 480细胞中MEK2 ,p ERK ,C myc表达水平随作用时间延长而下降。结论　MEK2活性增高与结直肠癌细胞侵袭力有关 ,阻断MEK2 /ERK信号传导通路可以抑制结肠癌细胞的增殖 ,促进其凋亡。     

表皮生长因子对人小细胞肺癌细胞Survivin表达的影响及其机制

目的 探讨表皮生长因子(EGF)对人小细胞肺癌NIC-H446细胞中凋亡抑制因子Survivin表达的影响及其调控Survivin的机制.方法 噻唑蓝(MTT)法测定EGF对细胞增殖率的作用.逆转录-聚合酶链反应(RT-PCR)和免疫印迹(Western blot)方法测定EGF对NIC -H446细胞Survivin表达的影响.Western blot方法测定EGF对NIC-H446细胞p38丝裂原活化激酶(p38MAPK)、磷酸化p38MAPK(p-p38MAPK)、c-Jun氨基端激酶(JNK)、磷酸化JNK (p-JNK)、细胞外信号调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)信号通路蛋白的表达影响.结果 EGF可促进NIC-H446细胞增殖,具有浓度-时间依赖性.与对照组(0.36±0.06、0.57 ±0.15)比较,EGF组Survivin mRNA(0.69±0.12)和蛋白(0.89±0.19)表达明显升高(P＜0.01).与对照组(0.29±0.08)比较,EGF组p-p38MAPK蛋白表达水平(0.68±0.27)明显上升(P＜0.01),其他蛋白表达均无明显变化.EGF+ SB203580组Survivin蛋白表达(0.56±0.17)、p-p38MAPK蛋白表达(0.41±0.11)显著低于EGF组(0.92 ±0.21、0.72 ±0.19,P＜0.01),与对照组比较差异无统计学意义.结论 EGF通过活化p38MAPK信号通路上调小细胞肺癌细胞中Survivin的表达,促进细胞增殖,这可能是小细胞肺癌发展的重要机制.     

氧化应激介导的Ras-ERK信号通路活化参与醛固酮诱导的肾小球系膜细胞增殖

目的 探讨氧化应激介导的Ras-胞外信号调节激酶(ERK1/2)信号通路活化在醛同酮( ALDO)诱导的系膜细胞增殖中的作用.方法 体外培养人肾小球系膜细胞,应用3H-胸腺嘧啶(3H-TdR)掺入法和细胞计数测定系膜细胞增殖;Western印迹检测Ki-RasA、c-Raf、MEK1/2和ERK1/2活化.结果 ALDO可显著促进系膜细胞增殖,抗氧化剂乙酰半胱氨酸(NAC)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)显著抑制ALDO诱导的系膜细胞增殖(均P＜ 0.01).ALDO刺激系膜细胞3h,活化的Ki-RasA、c-Raf、MEK1/2和ERK1/2表达显著增强,分别是对照组的4.05倍、3.62倍、4.52倍和3.40倍(均P＜0.01).抗氧化剂NAC几乎完全阻断ALDO诱导的Ki-RasA、c-Raf、MEK1/2和ERK 1/2活化(均P＜0.01).Ki-RasA siRNA可呈浓度依赖性降低系膜细胞Ki-RasA表达,并显著抑制ALDO诱导的Ki-RasA活化及系膜细胞增殖(P＜0.01).c-Raf抑制剂GW5074和MEK1/2抑制剂PD98059亦显著抑制ALDO诱导的系膜细胞增殖,其抑制率均达到65％(P＜0.01).Ki- RasA siRNA不能降低ALDO诱导的磷酸肌醇-3激酶( PI3K)磷酸化.联合应用PI3K抑制剂LY294002和MEKl/2抑制剂PD98059可完全阻断ALDO诱导的系膜细胞增殖(P＜0.01).结论 ALDO可通过氧化应激活化Ki-RasA-c-Raf-MEK-ERK信号通路.同时阻断ERK1/2和PI3K信号通路可完全抑制ALDO诱导的系膜细胞增殖.     

有丝分裂蛋白激酶及其上游调节信号在人肝癌组织中的表达

目的本研究检测了肝癌和癌旁肝组织中ERK1、ERK2、JNK1、p38及其上游MEK1、MEK2的蛋白表达量.方法手术切除16例肝癌及癌旁肝组织标本,Western印迹检测ERK1、ERK2、JNK1、p38和MEK1、MEK2蛋白含量.结果每例患者肝癌组织中ERK1、ERK2、p38蛋白表达显著高于癌旁肝组织,在肝癌和癌旁肝组织中ERK1积分光密度值(integralopticdensity,IOD)分别为300±98和98±48(P＜0.01);ERK2的IOD值分别为587±83和232±96(P＜0.01);p38的IOD值分别为270±85和107±87(P＜0.01);JNK1的IOD值分别为111±93和292±109(P＜0.01);肝癌组织中JNK1蛋白表达显著低于癌旁肝组织,MEK1、MEK2蛋白表达显著高于癌旁肝组织,MEK1的IOD值在肝癌组织中JNK1蛋白表达显著低于癌旁肝组织,分别为1*!418±244和805±90(P＜0.01),MEK2的IOD值分别为1*!041±122和468±40(P<0.005).结论在肝癌组织中细胞分裂增殖的信号传导通路ERK1、ERK2、p38、MEK1、MEK2激酶处于高表达,JNK1激酶在肝癌组织中处于低表达,它们的失衡是导致肝癌细胞生长失控和无限增殖的重要原因之一.     

有丝分裂蛋白激酶及其上游调节信号在人肝癌组织中的表达

目的探讨有丝分裂原蛋白激酶(MAPK)通路在调控细胞的增殖和凋亡过程中所起到的作用.方法手术切除16份肝癌及癌旁组织标本,Western印迹检测ERK1、ERK2、JNK1、p38和MEK1、MEK2的蛋白含量.结果在本组的16例患者中,每例患者肝癌组织中的ERK1、ERK2、JNK1、p38蛋白表达显著高于癌旁组织,ERK1积分光密度值(Integral optic density IOD)在肝癌和癌旁组织中分别为300±98和98±48,差异有显著意义(P＜0.01).ERK2的IOD值在肝癌和癌旁组织中分别为587±83和232±96,差异有显著意义(P＜0.01);p38的IOD值在肝癌和癌旁组织中分别为270±85和107±88,差异有显著意义(P＜0.01);肝癌组织中的JNK1蛋白表达显著低于癌旁组织;JNK1的IOD值在肝癌和癌旁组织中分别为111±93和292±109,差异有显著意义(P＜0.01);MEK1、MEK2蛋白表达显著高于癌旁组织,MEK1的IOD值在肝癌和癌旁组织中分别为1418±244和806±90,差异有显著意义(P＜0.01),MEK2的IOD值在肝癌和癌旁组织中分别为1041±122和468±40,差异有显著意义(P＜0.05).结论在肝癌组织中细胞分裂增殖的信号传导通路ERK1、ERK2、MEK1、MEK2激酶处于高表达,JNK1激酶在肝癌组织中处于低表达,它们的失衡导致肝癌细胞生长失控和无限增殖的重要原因之一.JNK1和p38在肝癌组织中可能存在不同的激活途径.     

MAPK信号传导通路在髓核细胞研究进展

丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)是将细胞外刺激信号转换成广泛的细胞内反应的一类丝氨酸/苏氨酸蛋白激酶,在许多生理过程中发挥着重要的细胞信号传导的作用[1].在真核细胞生物中,MAPK信号通路在基因表达、细胞增殖、分化、凋亡、新陈代谢等生理病理过程中发挥着重要的作用.在哺乳类动物中,MAPK家族基因有10来个,目前研究发现常见的MAPK信号通路有:细胞外信号调节激酶(ERK1/2),c-Jun氨基末端激酶(JNK1/2/3),p38激酶(α,β,γ,δ)以及大丝裂原活化蛋白激酶(ERK5/BMK1) [2-4].每一个常见的信号通路都存在三级激酶级联,细胞受外部信号刺激后通过MAPKKK转化为细胞内的信号并进一步磷酸化激活MAPKK,最后MAPKK通过双重磷酸化进一步激活MAPK,进而引起一系列细胞内信号的改变,在细胞增殖及功能上发挥着重要的作用.研究腰椎间盘退变过程中髓核细胞MAPK信号通路的信号变化将有助于人类对退行性腰椎间盘病进一步认识,并可能从中找到治疗退行性腰椎间盘病的方法.本文将综述MAPK常见信号通路在髓核细胞的研究进展.     

烟雾吸入性损伤大鼠肺组织丝裂原活化蛋白激酶通路的变化

目的 了解烟雾吸入性损伤大鼠肺组织丝裂原活化蛋白激酶(MAPK)通路及炎性细胞因子含量的变化,探讨其损伤机制. 方法建立密闭舱内烟雾吸入性损伤模型,将30只SD大鼠分为烟雾吸入性损伤后1、6、24、72 h及7 d组,另设正常对照组(6只).取各组大鼠肺组织行病理学观察,检测肺组织匀浆液中肿瘤坏死因子α(TNF-α)、巨噬细胞炎性蛋白2(MIP-2)和白细胞介素1β(IL-1β)含量,用蛋白质印迹法检测肺组织p38MAPK、c-Jun氨基末端激酶(JNK)、细胞外信号调节激酶1/2(ERK1/2)及各酶磷酸化水平.收集大鼠支气管肺泡灌洗液(BALF),检测TNF-α、MIP-2、IL-1β含量并行粒细胞分类、计数. 结果烟雾吸入使大鼠产生急性肺损伤样病理改变.伤后1 h组大鼠肺组织及BALF中TNF-α和IL-1β含量均高于正常对照组(P<0.01).各组肺组织MIP-2水平与正常对照组接近(P>0.05),伤后1 h组BALF中MIP-2水平高于正常对照组(P<0.01).各致伤组p38MAPK、ERK1/2、JNK水平接近正常对照组,但此3种酶的磷酸化水平在伤后不同时相组有高表达.伤后1 h组大鼠BALF粒细胞总数为(0.36±0.08)×106个/L,较正常对照组(0.61±0.09)×106个/L明显减少(P<0.05),伤后7 d组粒细胞总数[(1.71±0.67)×106个/L]却高于正常对照组(P<0.05).伤后6 h～7 d组中性粒细胞数多于正常对照组(P<0.05).伤后1 h组巨噬细胞数少于正常对照组(P<0.05),但6 h～7 d组逐渐增多.各组大鼠淋巴细胞数量接近(P>0.05).结论 密闭舱室内非金属材料燃烧释放的毒性气体能诱导肺组织产生明显的炎性反应,激活细胞MAPK通路中重要激酶的表达,这可能是毒性混合气体导致肺损伤的重要机制之一.     

戊乙奎醚预处理对脓毒症小鼠肺损伤时MAPK信号转导通路的影响

目的 探讨戊乙奎醚(PHC)预处理对脓毒症小鼠肺损伤时丝裂原活化蛋白激酶(MAPK)信号转导通路的影响.方法 健康雌性昆明小鼠105只,体重20～25 g,随机分为3组(n=35):假手术组(S组)、脓毒症(CLP)组和戊乙奎醚(PHC)组.采用盲肠结扎并穿孔法制备脓毒症模型.PHC组于造模前1 h腹腔注射戊乙奎醚0.45 mg/kg,s组和CLP组于造模前1 h注射等容量生理盐水.于造模后即刻测定肺微血管通透性;造模后12 h时进行动脉血气分析,观察肺组织病理结果,测定肺组织丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性和磷酸化的p38丝裂原活化蛋白激酶(p38MAPK)、细胞外信号调节激酶(ERK1/ERK2)和c-jun氨基末端蛋白激酶(JNK)表达.结果 与S组比较,CLP组PaO2、PaO2/FiO2和pH值降低,肺微血管通透性和肺组织MDA含量升高,SOD活性降低,磷酸化的p38MAPK、ERK1/ERK2和JNK表达上调(P<0.05或0.01);与CLP组比较,PHC组PaO2、PaO2/FiO2和pH值升高,肺微血管通透性和肺组织MDA含量降低,SOD活性升高,磷酸化的p38MAPK和ERK1/ERK2表达下调(P<0.05或0.01).结论 戊乙奎醚预处理可通过抑制MAPK信号转导通路(p38MAPK和ERK1/ERK2)的激活,从而减轻脓毒症小鼠肺损伤.     

MAPK信号通路与勃起功能障碍关系的研究进展

MAPK信号通路在细胞的分化、增殖及凋亡等过程中起着十分关键的作用。MAPK家族的信号通路主要包括细胞外信号调节蛋白激酶(ERK),应激活化蛋白激酶(JNK)、p38丝裂原活化蛋白激酶(p38MAPK)等。从目前的研究来看,ERK、JNK、p38MAPK 3条信号通路与ED的发生发展紧密联系。本文根据相关文献对MAPK信号通路与勃起功能障碍作一综述。     

The effects of genistein on tyrosine protein kinase-mitogen activated protein kinase signal transduction pathway in hypertrophic scar fibroblasts

OBJECTIVE: To investigate the inhibitory effects of genistein on tyrosine protein kinase (TPK)-mitogen activated protein kinase (MAPK) signal transduction pathway in hypertrophic scar fibroblasts (HSFb), in order to explore the molecular mechanism of inhibition of scar hyperplasia by genistein. METHODS: HSFbs were isolated from human hypertrophic scar tissues and cultured in vitro. The cells were treated by genistein in different concentrations (25, 50, 100 micromol/L, respectively), followed by basic fibroblast growth factor (bFGF) stimulation. The activity of TPK was assessed with [gamma-32P] ATP substrate incorporation. The phosphorylation protein expression levels of main signal molecules in TPK-Ras-MAPK pathway including c-Raf, MEKl/2, extracellular signal regulated kinase (ERK), p38MAPK, c-Jun N-terminal kinase (JNK) were determined by Western blot. HSFbs treated with dimethylsulfoxid (DMSO) were used as control group. RESULTS: After being treated with genistein in concentration of 25, 50, 100 micromol/L, the activity of TPK in HSFbs was depressed significantly [(7.15 +/- 0.35) x 10(5), (5.62 +/- 0.88) x 10(5), (5.62 +/-0.88) x 10(5) 10(5) pmol x min(-1) x mg(-1), respectively] when compared with that in control group [(8.92 +/- 0.28) x 10(5) pmol x min(-1) x mg(-1), P < 0.05]. Compared with those in control group,the phosphorylation protein expression levels of c-Raf, MEK1/2, ERK1/2 and p38 MAPK were lowered in different degree (P < 0.05 or P < 0.01) after genistein treatment. The phospho-JNK levels after treatment with genistein were similar to that of control group. Under the condition of pretreatment with genistein, the activities of TPK and signal pathway protein expressions in HSFb showed a downward trend after stimulation with bFGF. CONCLUSION: Genistein can effect the proliferation and activation of HSFb by inhibiting the phosphorylation of receptor of TPK signal transduction pathway (TPK --> Raf --> MEK --> ERK/p38).     

Genistein inhibits proliferation and functions of hypertrophic scar fibroblasts

Hypertrophic scarring is abnormal proliferation of dermal fibroblasts and excessive deposition of extracellular matrix. To date, despite many studies, treatments have not been satisfactory. Genistein, a potent, specific inhibitor of tyrosine protein kinases (TPKs), has been proved to inhibit many kinds of tumour and some fibrotic diseases. The purpose of this study was to investigate the effects of genistein on the proliferation and functions of hypertrophic scar fibroblasts (HSFBs) and the mechanism by which genistein inhibits TPK signal transduction. The first effect was observed by methyl-thiazol-diphenyl-tetrazolium assay and the second by [gamma-(32)p] adenosine triphosphate incorporation assay. The results demonstrated that genistein inhibits the proliferation and function of HSFBs and changes the TPK signal transduction pathway, which can provide an experimental basis for treating HS with genistein.     

Aldosterone stimulates proliferation of mesangial cells by activating mitogen-activated protein kinase 1/2, cyclin D1, and cyclin A

Recently, attention has been focused on the role of aldosterone in the pathophysiology of hypertension and cardiovascular disease. Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. However, the molecular mechanisms of the effects of aldosterone in signal transduction and the cell-cycle progression of mesangial cells are not well known. For determining the signaling pathway of aldosterone in cultured mesangial cells, the effects of aldosterone on the mitogen-activated protein kinase 1/2 (MAPK1/2) pathway and the promoter activities of cyclin D1, cyclin A, and cyclin E were investigated. First, it was shown that the mineralocorticoid receptor (MR) was expressed in rat mesangial cells and glomeruli and that aldosterone stimulated the proliferation of mesangial cells via the MR and MAPK1/2 pathway. Next, it was demonstrated that aldosterone stimulated Ki-RasA, c-Raf kinase, MEK1/2, and MAPK1/2 in rat mesangial cells. Aldosterone induced cyclin D1 and cyclin A promoter activities and protein expressions, as well as the increments of CDK2 and CDK4 kinase activities. The presence of CYP11B2 and 11beta-HSD2 mRNA in rat mesangial cells also was shown. In conclusion, aldosterone seems to exert mainly MR-induced effects that stimulate c-Raf, MEK1/2, MAPK1/2, the activities of CDK2 and CDK4, and the cell-cycle progression in mesangial cells. MR antagonists may serve as a potential therapeutic approach to mesangial proliferative disease.     

Inhibition of microsomal triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus flavonoid naringenin and by insulin involves activation of the mitogen-activated protein kinase pathway in hepatocytes

Microsomal triglyceride transfer protein (MTP) is necessary for hepatocyte assembly and secretion of apolipoprotein (apo)B100-containing lipoproteins. The citrus flavonoid naringenin, like insulin, decreased MTP expression in HepG2 cells, resulting in inhibition of apoB100 secretion; however, the mechanism for naringenin is independent of insulin receptor substrate-1/2. Recently, it was reported that insulin decreased MTP expression in HepG2 cells via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (MAPK(erk)) pathway. We hypothesized that naringenin acts via a similar mechanism. Inhibition of MAPK kinase (MEK) 1/2 in HepG2 cells significantly attenuated the naringenin- and insulin-induced reduction in MTP expression. Both naringenin and insulin increased ERK1/2 phosphorylation, which was completely inhibited by MEK1/2 inhibition and enhanced by inhibition of MAPK(p38), a negative regulator of MAPK(erk) activity. Inhibition of MEK1/2 significantly attenuated both the naringenin- and insulin-induced decrease in apoB100 secretion demonstrating a direct link between MAPK(erk) activation and apoB100 secretion. Furthermore, both compounds increased MAPK(p38) activation, and therefore inhibition of MAPK(p38) amplified thenaringenin- and insulin-induced decrease in apoB100 secretion. We conclude that MAPK(erk) signaling in hepatocytes is critical for inhibition of apoB100 secretion by naringenin and insulin. Therefore, naringenin may prove useful for activating insulin-signaling pathways important for regulation of hepatocyte lipid homeostasis.     

Mitogen-activated protein kinase pathway regulates cell proliferation in venous ulcer fibroblasts

Venous ulcer fibroblasts have been demonstrated to have low growth rates in response to platelet-derived growth factor (PDGF). Mitogen-activated protein kinase (MAPK) is an important signal transduction mechanism that regulates growth, differentiation, and apoptosis in eukaryotic cells. PDGF binds PDGF receptors that activate a multitiered signaling cascade involving MAPK. We hypothesize that the growth regulation in venous ulcer fibroblasts is dependent on the MAPK extracellular signal-regulated kinase (ERK) pathway in the presence of PDGF. Fibroblasts (fb) were isolated from 8 patients with venous ulcers (w-fb) and the normal skin (n-fb) of the ipsilateral thigh via punch biopsies. Fb were plated at 1,500 cells/dish and treated with PDGF-AB (10 ng/mL) for 15 days. Growth rates were determined. Immunoblot analysis of MAPK ERK for n-fb and w-fb were analyzed. To determine if PDGF-stimulated w-fb and n-fb utilized the MAPK ERK pathway in a dependent manner, the upstream kinase MAPK kinase 1 (MEK 1) was inhibited by PD 98059. In addition, fb were treated with chronic venous ulcer wound fluid (WF) to study its effect on MAPK ERK. In the presence of PDGF, growth rates were substantially lower in w-fb than in n-fb, and MAPK was activated in 6/8 w-fb and in only 2/8 n-fb. Fibroblasts expressing MAPK had significantly reduced cell proliferation compared to fibroblasts not expressing MAPK (p = 0.023). PD 98059 significantly inhibited w-fb and n-fb cell proliferation from basal level, which was reversible with addition of PDGF. In neonatal fibroblasts WF demonstrated inhibition of MAPK ERK over time and addition of PD98059 was not additive. This study suggests that the MAPK ERK pathway is important for cell proliferation in venous ulcer fibroblasts. In the presence of PDGF, fibroblasts with decreased growth rate express MAPK, and proliferation is further abrogated with addition of MEK 1 inhibitor, suggesting the importance of the MAPK ERK pathway regulating w-fb and n-fb proliferation. Although the majority of w-fb activated the MAPK ERK pathway in the presence of PDGF, proliferation was significantly attenuated, indicating that other MAPK inhibitory pathways are competing. Venous ulcer wound fluid directly inhibits the MAPK ERK pathway, suggesting that the venous ulcer wound environment has negative trophic factors that effect fibroblasts proliferation and ulcer healing.     

饲服环磷酰胺对兔耳早期增生性瘢痕组织的影响

目的 通过饲服环磷酰胺观察其对兔耳早期瘢痕的影响,以探讨临床应用口服免疫抑制剂防治早期瘢痕的可行性.方法 以32只新西兰白兔兔耳建立增生性瘢痕动物模型,并将其随机分成4组,即蒸馏水组(A组)、5mg·kg-1·d-1环磷酰胺组(B组)、10 mg·kg-1·d-1组(C组)及30mg·kg-1·d-1(D组).在制作模型前及灌胃后14、28 d时分别采血观察白细胞、淋巴细胞变化.28 d时取组织HE染色、VG染色法观察瘢痕增生指数(HI)、成纤维细胞密度(NA)、胶原纤维面密度(AA)数值变化.各组数据(HI、NA、AA)比较采用方差分析,方差不齐时作秩和检验.结果 14 d时,A、B、C、D组白细胞数量分别为(8.62 +0.58)×109/L、(4.48±0.41)×109/L、(2.7±0.26)×109/L、(1.33±0.27)×109/L,淋巴细胞数量分别为(3.11±0.21)×109/L、(1.67 +0.16)×109/L、(0.42 +0.10)×109/L、(0.40 +0.09)×109/L;28 d时,A、B、C、D组白细胞数量分别为(8.63±0.53)× 109/L、(5.10±0.27)×109/L、(3.10±0.26)× 109/L、( 1.98±0.20)×109/L,淋巴细胞数量分别为(3.06±0.16)×109/L、( 2.08±0.14)×109/L、(0.96±0.19)×109/L、(0.14±0.07)×109/L,实验各剂量组(B、C、D组)白细胞数量和淋巴细胞数量下降,且随药物剂量的增加而下降更明显,呈负相关,各组间比较差异有统计学意义(P＜0.05).A、B、C、D组HI值分别为3.02±0.24、2.59±0.43、2.06±0.19、1.63±0.11,AA值分别为40.49±2.07、35.29±1.99、28.36±1.87、24.99±1.82,NA值分别为4570.5±259.3、4222.5±199.6、3540.3±170.3、3341.4±228.8,对照组与实验各剂量组的HI、AA、NA数值之间比较差异有统计学意义(P＜0.01),且随剂量的增加,除NA值C和D组比较差异无统计学意义外,其余实验各剂量组HI、AA、NA值变小更明显,呈负相关,各组之间比较差异有统计学意义(P＜0.05).结论 饲服环磷酰胺在抑制白细胞和淋巴细胞的同时能明显抑制瘢痕增生,对兔耳早期增生性瘢痕有明显的防治作用.     

增生性瘢痕成纤维细胞中分泌型凋亡相关蛋白1相互作用蛋白的研究

目的 寻找与分泌型凋亡相关蛋白1 （secreted apoptosis-related protein1,SARP1）存在相互作用的蛋白质分子,分析其分子机制.方法 成功构建的重组SARP1腺病毒载体Ad-SARP1,转染进入原代培养的增生性瘢痕成纤维细胞（HSFb）.免疫共沉淀法沉淀出蛋白,经聚丙烯酰胺凝胶电泳（SDS-PAGE）分离后,用考马斯亮蓝染色,对SDS-PAGE胶上切取的电泳蛋白条带进行酶解与质谱分析,根据质谱分析获得的肽序列谱图自动进行数据库搜索.结果 在阳细胞对照组和导入Ad-SARP1感染的细胞当中,均共沉淀出7条蛋白带,相对分子质量分别约为93×103、43×103、40×103、37×103、31×103、26×103和12×103;在未加SARP1抗体组则没有沉淀出蛋白条带.分析蛋白条带得到6个可能与SARP1有相互作用的蛋白,分别为骨膜蛋白前体（periostin precursor或OSF-2）、牙周韧带相关蛋白1（asporin precursor或PLAP1）、磷酸甘油激酶1（phosphoglycerate kinase 1）、未知蛋白rCG50690、载脂蛋白（apolipoprotein A-I,Apo-AI）、硫氧还蛋白1（thioredoxin 1,TRX1）.结论 通过免疫共沉淀法结合液相色谱/质谱联用离子阱检测技术,可以获得SARP1的相互作用蛋白,为研究SARP1调控HSFb凋亡信号通路的作用机制提供了新的线索.     

重组人内皮型一氧化氮合酶基因转染对增生性瘢痕成纤维细胞的影响

目的 了解重组人内皮型一氧化氮合酶(eNOS)基因转染人增生性瘢痕成纤维细胞(HSFb)的可行性,以及转染后一氧化氮(NO)的生成和Ⅰ、Ⅲ型胶原的合成情况. 方法体外构建人eNOS真核表达载体.取体外培养的人HSFb进行转染实验,根据细胞培养液中所加质粒不同分为pcDNA3.0空载体组、pcDNA3.0-eNOS转染组.另设未转染组,细胞按常规培养.采用实时荧光定量PCR法检测各组细胞eNOS及Ⅰ、Ⅲ型胶原mRNA表达.硝酸还原酶法测定NO含量. 结果细胞转染后eNOS显著表达,pcDNA3.0-eNOS转染组eNOS mRNA相对表达量为5.92±0.21,明显高于pcDNA3.0空载体组(0.98±0.13,P<0.05);pcDNA3.0-eNOS转染组Ⅰ型胶原mRNA相对表达量为0.76±0.15,Ⅲ型胶原mRNA相对表达量为0.79±0.08,均明显低于pcDNA3.0空载体组(0.98±0.15、1.02±0.12,P<0.05).pcDNA3.0-eNOS转染组NO含量为(36.1±0.8)μmol/L,明显高于pcDNA3.0空载体组(28.4±1.0)μmol/L和未转染组[(27.7±1.3)μmol/L,P<0.01]. 结论 HSFb可作为eNOS基因转染的靶细胞,转染的细胞能表达eNOS并产生NO,并且对细胞的胶原合成功能有抑制作用.     

澳洲茄边碱对前列腺癌细胞增殖的抑制作用及机制研究

目的：研究澳洲茄边碱(solamargine,SM)对人前列腺癌激素非依赖细胞增殖的影响及可能的作用机制.方法用不同浓度 SM(0、1、2、4、6、8、10μmol/L)处理 DU145和 PC3细胞, MTT法检测 SM 对细胞生长的影响,Western blot 检测 SM 对相关信号通路蛋白 p38 MAPK、ERK1/2 MAPK、MUC1表达的影响.结果6μmol/L SM作用24 h后DU145和PC3细胞活力分别为(52.53±9.05)％、(56.28±2.36)％,并具有时间和剂量依赖;10μmol/L SM作用24 h后细胞活力分别为(27.36±2.72)％、(32.07±2.53)％.流式细胞术分析显示不同浓度 SM(0、4、6、8μmol/L)处理 PC3细胞能引起 PC3细胞阻滞在 G1期,G1期细胞比例分别为(52.61±0.50)％、(52.96±1.49)％、(66.16±2.84)％和(69.03±2.38)％.且能激活 MAPK信号通路减少下游蛋白 MUC1的表达.结论 SM能明显抑制DU145和PC3细胞生长,该作用可能与 MAPK信号通路的激活以及下游 MUC1蛋白表达下调有关.