Differential expression of autocrine motility factor receptor (AMFR) mRNA in normal and cancer cells detected by in situ hybridization

The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH) detection of AMFR mRNA in both paraffin-embedded surgical sections and cultured cells using either biotinylated oligonucleotide probes or digoxigenin-labeled RNA probes. The results showed that the AMFR mRNA was expressed at an enhanced level in hyperplaJstic and malignant tissues of breast and prostate cancer patient surgical specimens, indicating that the elevated AMFR expression was associated with the tissue malignancy Moreover, AMFR mRNA was detected in both normal and earcinoma cells when cultured at a subconfluent density. However, AMFR expression was inhibited in confluent normal (3T3-A31 murine fibroblast and FHs738BL human bladder) cells while it continued to express in carcinoma (J82 human bladder)and metastatic (3T3-M murine fibroblast) cells irrespective of cell density This suggested a cell-cell contact downregulation of AMFR mRNA expression in normal but not in cancer cells. The ISH data obtained in this study are closely consistent with the AMFR protein expression pattern previously reported, implying that the differential expression of AMFR gene may be regulated and controlled at the transcriptional level.

Protease activated receptor 2 and epidermal growth factor receptor are involved in the regulation of human sperm motility

Aim： To investigate mechanisms of tryptase-induced reduction of sperm motility and explore whether epidermal growth factor receptor （EGF-R） and protease activated receptor 2 （PAR-2）- associated pathways are involved. Methods： Fresh semen was collected from healthy donors （n = 15）. Semen parameters and quality were assessed in accordance with the World Health Organization （WHO） criteria. Swim-up sperm were fixed and subjected to immunocytochemistry and immunoelectronmicroscopy with specific antibodies directed against PAR-2 and EGF-R. Protein extractions from swim-up spermatozoa were analyzed by Western blotting with antibodies for both receptors. Motility of spermatozoa was evaluated by computer-assisted semen analysis. Results： Immunocytochemistry found PAR-2 and EGF-R in approximately 30% of examined human ejaculated spermatozoa. Both receptors were localized in the plasma membrane. Like tryptase, the PAR-2 synthetic agonist SLIGKV reduced sperm motility, and this effect was inhibited by application of two specific EGF-R pathway blockers （AG1478 and PD168393）. Conclusion： The observed reduction of sperm motility by tryptase through the PAR-2 receptor involves EGF-R pathways.

SIMULTANEOUS OVER-EXPRESSION OF INSULIN-LIKE GROWTH FACTOR- Ⅱ (IGF- Ⅱ ) AND IGF- Ⅱ RECEPTOR(IGF- Ⅱ R) GENES IN HUMAN PRIMARY CANCER-IMPLICATION OF AUTOCRINE AND PARACRINE MECHANISM IN AUTONOMOUS GROWTH OF HEPATIC CANCER

This is first report about the simultaneous over-expression of both Insulin-like growth factor (IGF- I ) and its receptor (IGF- I R) at mRNA level in human primary hepatic Cancer (PHC). In 10 PHC samples from China, IGF-I and IGF- I R were both over-expressed, whereas only a background signal was detected in normal liver. In 5 pairs of PHC and its non- tumorous adjacent liver tissues from South Africa, IGF- I and IGF- I R were also over-expressed in PHC. mRNA expression of IGF- I in all 5 cases and IGF- I R in 4 of 5 cases were higher in cancer than non- tumorous adjacent liver tissues. These results strongly implicate that an autocrine and/ or paracrine mechanism might be Involved in formation and progression of PHC.

CO-EXPRESSION OF MACROPHAGE COLONY-STIMULATING FACTOR WITH ITS RECEPTOR IN HUMAN HEPATOMA CELLS AND ITS POTENTIAL ROLES

Objective: To investigate the potential role of macrophage colony-stimulating factor (M-CSF) and macrophage colony-stimulating factor receptor (M-CSF-R) on the growth of human hepatoma cells. Methods: Specimens of different origin, including tissues of human hepatocellular carcinoma (HCC), human fetal liver (FL) and normal liver (NL), the hepatoma cell lines, as well as the peripheral blood mononuclear cells (PBMC) from patients with HCC or liver metastatic tumor (LMT), were used to detect the expression levels of M-CSF and M-CSF-R by ABC immunohistochemistry staining and reverse transcription polymerase chain reaction methods the expression levels of M-CSF and M-CSF-R. Influence of monoclonal antibody against M-CSF (B5) or M-CSF-R (RE2) on proliferation ability of hepatoma cell linesin vitro was also studied. Results: The results showed that hepatoma tissues produced elevated levels of both M-CSF and M-CSF-R compared with those of fetal liver (P<0.001). The M-CSF/M-CSF-R expression levels of PBMC from hepatoma patients were higher than those of LMT patients (P<0.01,P<0.05) and the normal people (P<0.001). The hepatoma cell lines showed strong positive for M-CSF and M-CSF-R production. Both B5 and RE2 displayed a dose-dependent inhibitory effect on the growth and proliferation of hepatoma cells. Conclusion: The study indicates a co-expression model for M-CSF-R in hepatoma cells, suggesting an involvement of M-CSF/M-CSF-R in growth signaling of those malignant cells. The M-CSF/M-CSF-R seems to function through an autonomy mechanism in human hepatoma.

Deficiency of platelet-derived growth factor receptor-α-positive cells in Hirschsprung's disease colon

AIM: To investigate whether the expression of platelet-derived growth factor receptor-α-positive (PDGFRα+)-cells is altered in Hirschsprung’s disease (HD).METHODS: HD tissue specimens (n = 10) were collected at the time of pull-through surgery, while colonic control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 10). Immunolabelling of PDGFRα+-cells was visualized using confocal microscopy to assess the distribution of these cells, while Western blot analysis was undertaken to quantify PDGFRα protein expression.RESULTS: Confocal microscopy revealed PDGFRα+-cells within the mucosa, myenteric plexus and smooth muscle in normal controls, with a marked reduction in PDGFRα+-cells in the HD specimens. Western blotting revealed high levels of PDGFRα protein expression in normal controls, while there was a striking decrease in PDGFRα protein expression in the HD colon.CONCLUSION: These findings suggest that the altered distribution of PDGFRα+-cells in both the aganglionic and ganglionic HD bowel may contribute to the motility dysfunction in HD.

Effect of exogenous hydrogen sulfide in the nucleus tractus solitarius on gastric motility in rats

BACKGROUND Hydrogen sulfide(H2S)is a recently discovered gaseous neurotransmitter in the nervous and gastrointestinal systems.It exerts its effects through multiple signaling pathways,impacting various physiological activities.The nucleus tractus solitarius(NTS),a vital nucleus involved in visceral sensation,was investigated in this study to understand the role of H2S in regulating gastric function in rats.AIM To examine whether H2S affects the nuclear factor kappa-B(NF-κB)and transient receptor potential vanilloid 1 pathways and the neurokinin 1(NK1)receptor in the NTS.METHODS Immunohistochemical and fluorescent double-labeling techniques were employed to identify cystathionine beta-synthase(CBS)and c-Fos co-expressed positive neurons in the NTS during rat stress.Gastric motility curves were recorded by inserting a pressure-sensing balloon into the pylorus through the stomach fundus.Changes in gastric motility were observed before and after injecting different doses of NaHS(4 nmol and 8 nmol),physiological saline,Capsazepine(4 nmol)+NaHS(4 nmol),pyrrolidine dithiocarbamate(PDTC,4 nmol)+NaHS(4 nmol),and L703606(4 nmol)+NaHS(4 nmol).RESULTS We identified a significant increase in the co-expression of c-Fos and CBS positive neurons in the NTS after 1 h and 3 h of restraint water-immersion stress compared to the expressions observed in the control group.Intra-NTS injection of NaHS at different doses significantly inhibited gastric motility in rats(P<0.01).However,injection of saline,first injection NF-κB inhibitor PDTC or transient receptor potential vanilloid 1(TRPV1)antagonist Capsazepine or NK1 receptor blockers L703606 and then injection NaHS did not produce significant changes(P>0.05).CONCLUSION NTS contains neurons co-expressing CBS and c-Fos,and the injection of NaHS into the NTS can suppress gastric motility in rats.This effect may be mediated by activating TRPV1 and NK1 receptors via the NF-κB channel.

The expression of the products of IGF-Ⅱ,IGF-Ⅱ receptors and CSF-Ⅰ receptors in human primary hepatocellular carcinoma and juxtacancerous liver tissue

The expression of the products of IGF-Ⅱ,IGF-Ⅱ receptors(IGF-Ⅱ-R)and CSF-Ⅰ re-ceptors(CSF-Ⅰ-R)was observed in 17 cases of human primary hepatocellular carcinoma(PHC)and the juxtacancerous liver tissue with immunohistochemistry(ABC),Western blot and North-ern blot technique,It was found that the expression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-Ⅰ-R was signif-icantly higher in PHC than in normal liver tissue and the expression of IGF-Ⅱ and IGF-Ⅱ-R wasremarkably higher in the juxtacancerous liver tissue from PHC patients than in PHC proper.Itwas noteworthy that the expression of IGF-Ⅱ in both the cancer proper and the juxtacancerousliver tissue was characterized by its fetal type.Besides,the expression of CSF-Ⅰ-R was signifi-cantly higher in PHC than in the juxtacancerous liver tissue.It is believed that the abnormal ex-pression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-I-R in PHC and the juxtacaneerous liver tissue might berelated to the autocrine mechanism of human PHC.

阻断转化生长因子β_1自分泌环基因治疗骨肉瘤的机制研究

目的阻断骨肉瘤细胞TGF-β1自分泌环能抑制肿瘤细胞的增殖活性,从而达到基因治疗骨肉瘤的目的,探讨阻断TGF-β1自分泌环基因治疗骨肉瘤的机制。方法将反义TGF-β1基因转染骨肉瘤细胞MG-63后,采用RT-PCR的方法检测肿瘤细胞BMP-2、IGF-1、bFGF基因表达的变化。结果阻断TGF-β1自分泌环后,骨肉瘤细胞BMP-2、IGF-1、bFGF基因表达明显降低或消失。结论通过阻断自分泌环以降低骨肉瘤细胞表达的TGF-β1后,肿瘤细胞其他各种细胞因子的表达明显抑制,肿瘤细胞生物学活性降低。研究阻断TGF-β1自分泌环抑制肿瘤生长和发展的机制,将为开辟骨肉瘤基因治疗的新方向奠定基础。

大鼠下颌下腺内自分泌运动因子及其受体的定位

目的:探讨自分泌运动因子及其受体在大鼠下颌下腺中的表达特点,为进一步研究自分泌运动因子及其受体对下颌下腺是否有功能意义提供依据。方法:应用HE和免疫组织化学SABC法染色。结果:大鼠下颌下腺颗粒曲管、纹状管及小叶间导管上皮细胞呈自分泌运动因子及其受体免疫反应阳性。免疫反应产物分布于胞质,胞核为免疫反应阴性。下颌下腺的腺泡上皮细胞均呈自分泌运动因子及其受体免疫反应阴性。结论:正常大鼠下颌下腺仅导管上皮有自分泌运动因子及其受体的分布,提示下颌下腺产生的自分泌运动因子对下颌下腺及机体可能有重要调节意义。

Autotaxin对儿童难治性肺炎支原体肺炎的预测价值及其与炎性细胞因子的相关性

目的探讨血清和支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)自分泌运动因子(autotaxin)水平对儿童难治性肺炎支原体肺炎(refractory Mycoplasma pneumoniae pneumonia,RMPP)的预测价值及其与白细胞介素-6(interleukin-6,IL-6)、白细胞介素-8(interleukin-8,IL-8)及C-反应蛋白(C-reactive protein,CRP)的相关性。方法回顾性选取2019年1月至2021年12月收治的MPP患儿238例为研究对象,根据疾病严重程度分为RMPP组(82例)和普通肺炎支原体肺炎(general Mycoplasma pneumoniae pneumonia,GMPP)组(156例)。比较各组血清和BALF中autotaxin、IL-6、IL-8及CRP水平,分析血清和BALF中autotaxin水平对儿童RMPP的预测价值,以及RMPP患儿autotaxin水平与IL-6、IL-8及CRP水平的相关性。结果RMPP组血清和BALF中autotaxin、IL-6、IL-8及CRP水平均高于GMPP组(P<0.05)。RMPP患儿急性期血清和BALF中autotaxin、IL-6、IL-8及CRP水平均高于恢复期(P<0.05)。受试者工作特征曲线显示,血清和BALF中autotaxin水平对儿童RMPP均具有较好的预测价值(P<0.05),曲线下面积分别为0.874(95%CI:0.816~0.935)、0.862(95%CI:0.802~0.924)。相关分析显示,RMPP患儿血清和BALF中autotaxin水平与IL-6、IL-8及CRP水平均呈正相关(P<0.001)。结论RMPP患儿血清和BALF中autotaxin水平明显升高,且与病情恢复程度和炎性细胞因子相关,可作为儿童RMPP的预测指标。

集落刺激因子1及其受体的单抗对人肝癌细胞在裸鼠体内生长的抑制作用

探讨集落刺激因子┐１及其受体在人原发性肝癌中的过量表达及其意义。方法抗ＣＳＦ１Ｒ和抗ＣＳＦ１单克隆抗体对裸鼠移植性人肝癌７７２１细胞的生物学作用。结果两个单抗分别以７４．８８％和７７．９３％的体积抑瘤率在裸鼠体内抑制７７２１细胞的生长。结论ＣＳＦ１Ｒ和ＣＳＦ１可能是一新发现的肝癌自泌和旁泌系统。

人肝癌细胞株VEGF/VEGFR的检测及意义探讨

目的:筛选血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)高表达肝癌细胞株,为进一步研究VEGF在肝癌治疗中的作用提供理论依据,并探讨VEGF受体在肝癌细胞株的表达及意义。方法:ELISA法及Westernblot法分别检测人肝癌细胞株培养上清及细胞内VEGF蛋白的表达,免疫细胞化学方法检测VEGFR在人肝癌细胞中的表达。结果:5株肝癌细胞株均见VEGF蛋白表达,其中SMMC-7721的VEGF表达量最高,VEGF特异性受体Flt-1在HepG2、HHCC、SMMC-7721、Bel-7402见阳性表达,KDR在HepG2、HHCC、Bel-7402、QGY-7701见阳性表达。结论:肝癌细胞株中VEGF表达量不尽一致,VEGF在肝癌发生发展中可能存在自分泌作用方式。

急性髓细胞白血病患儿骨髓单个核细胞中自分泌运动因子受体表达的检测

目的:通过检测自分泌运动因子受体(AMFR)在急性髓细胞白血病(AML)患儿骨髓单个核细胞中的表达情况,探讨其与AML化疗缓解率的关系。方法:采用RT-PCR及Western blot方法检测试验组(63例AML患儿)与对照组(50例非AML患儿)骨髓单个核细胞中AMFR mRNA及蛋白的表达水平,其中试验组包括AML初治患儿35例、完全缓解患儿18例、复发患儿10例,分析AMFR mRNA表达与缓解率的关系。结果:1AMFR mRNA及蛋白表达水平在对照组、缓解组、初治组及复发组中逐渐升高(F=13.105、10.010,P均<0.001),组间两两比较差异均有统计学意义(P均<0.05)。2AMFR mRNA高表达患儿的缓解率为15%,低于AMFR mRNA低表达患儿的缓解率84%(χ2=21.254,P<0.001)。结论:AMFR在儿童AML中存在高表达,AMFR可作为化疗敏感性及预后判断的指标。

自分泌运动因子及其受体与神经系统的关系研究进展

自分泌运动因子(autocrine motility factor,AMF)能刺激细胞的迁移和运动,与肿瘤发生、转移、血管生成密切相关。近年来人们发现有3种AMF:ATX-t、ATX-m和PD-Ialpha,其中PD-Ialpha在脑中特异表达,在神经发育、生长和神经分化、神经外伤修复与神经性疼痛以及神经退行性疾病中具有重要的作用,提示AMF及其受体与神经系统的病理生理过程密切相关。该文就AMF及其受体在神经系统中的作用和作用机制作一综述,为相关的研究提供参考。

自分泌运动因子及受体mRNA表达与肝癌临床分期的关系

目的探讨自分泌运动因子(AMF)、自分泌运动因子受体(AMFR)mRNA表达与肝癌临床分期的关系。方法对96例肝癌患者进行分期,采用逆转录实时荧光定量PCR测定患者肝癌组织中AMF、AMFR mR-NA表达。结果Ⅱ、Ⅲ期肝癌患者AMF、AMFR mRNA水平显著高于Ⅰ期,Ⅲ期肝癌患者AMF/AMFR mRNA水平显著高于Ⅱ期。肝癌转移组中AMF、AMFRmRNA表达明显高于未转移组。结论 AMF、AMFR mRNA水平与肝癌严重程度密切相关,可能对临床判断肝癌的严重程度具有一定的预测价值。

转化生长因子调控表皮生长因子受体在雄激素非依赖型前列腺癌细胞中表达的意义

目的 :探讨转化生长因子 (TGF)α对前列腺癌雄激素非依赖型细胞系中表皮生长因子受体 (EGFR)表达的调控。 方法 :采用逆转录 多聚酶链式反应和Western印迹法对TGFα刺激前列腺癌雄激素非依赖型细胞系PC3、ARCaP后EGFRmRNA表达及其蛋白水平进行定量分析。 结果 :TGFα引起PC3、ARCaP的EGFRmRNA表达升高 ,分别为 5 .0 1± 0 .4 5和 9.0 5± 0 .6 3,明显高于对照组 (P <0 .0 5 )。PC3细胞系TGFα治疗后EGFR蛋白水平为 2 .2 8±0 .5 3,明显高于对照组 (P <0 .0 5 ) ;而ARCaP细胞系TGFα治疗后EGFR仅为 1 .2 4± 0 .2 2 ,和对照组比较无差别 (P >0 .0 5 )。 结论 :TGFα可以明显提高前列腺癌细胞的EGFR表达量 ;TGFα

自分泌运动因子对EC9706、Eca109及EC-1细胞丝切蛋白磷酸化的影响

目的:探讨自分泌运动因子(AMF)对食管鳞状细胞癌细胞中丝切蛋白磷酸化水平的影响。方法:构建真核表达载体pEGFP-C1-AMF和pcDNA3.1(+)-AMF,脂质体法转染食管鳞状细胞癌细胞EC9706、Eca109及EC-1,荧光显微镜观察EGFP-AMF融合蛋白在细胞中的定位,RT-PCR和Western Blot法检测转染细胞中AMF mRNA和蛋白的表达,Western Blot检测转染前后细胞中磷酸化丝切蛋白表达的变化。结果:EGFP-AMF融合蛋白均定位于3种细胞的胞质中;转染pcDNA3.1(+)-AMF、未转染及转染pcDNA3.1(+)的EC9706、Eca109及EC-1细胞中AMF mRNA表达(F分别为411.516、475.759和144.857,P均<0.001)及磷酸化丝切蛋白的表达(F分别为40.482、67.125和498.219,P均<0.001)差异均有统计学意义,转染pcDNA3.1(+)-AMF的细胞中AMF mRNA及磷酸化丝切蛋白的表达均较未转染和转染pcDNA3.1(+)的细胞显著增高(P<0.05)。结论:AMF能够提高食管鳞状细胞癌细胞株丝切蛋白磷酸化水平。

自分泌运动因子及其受体在乳腺癌中的表达

目的 了解自分泌运动因子 (autocrinemotilityfactor,AMF)及其受体 (autocrinemotilityfactorreceptor,AMFR)在乳腺癌组织中的表达及其与乳腺癌临床病理特征之间的关系。方法 应用免疫组化SABC方法研究 2 5例正常乳腺组织、2 3例乳腺上皮增生组织、71例乳腺癌组织中AMF、AMFR的表达。结果 AMF与AMFR阳性结果完全一致。正常乳腺组织、乳腺上皮增生组织AMF、AMFR阳性率均低于癌组织 (P <0 .0 5 )。 71例原发性乳腺癌组织中有 4 5例 (6 3% )阳性。浸润性导管癌AMF、AMFR水平明显高于导管内癌 (P =0 .0 0 1)。AMF、AMFR高表达与乳腺癌组织学分级、TNM分期有关 (P <0 .0 5 ) ,与年龄、肿块大小、淋巴结是否转移无关 (P >0 .0 5 )。结论 AMF、AMFR表达水平高与乳腺癌的进展有关 ,具有判断预后的价值。

肝癌、癌旁肝组织IGF-Ⅱ、IGF-Ⅱ受体及CSF-1受体/c-fms癌基因产物的表达

本文采用免疫组织化学(ABC)法、Western印迹法和Northern印迹法从细胞、蛋白及转录水平观察17例肝癌及癌旁肝组织中IGF-Ⅱ、IGF-Ⅱ受体和CSF-1受体/c-fms癌基因产物的表达。结果表明,肝癌组织中3种基因产物的表达水平显著高于正常肝组织;癌旁肝组织中IGF-Ⅱ和IGF-Ⅱ受体的表达水平显著高于肝癌组织;肝癌及癌旁肝组织IGF-Ⅱ基因呈胚胎型表达特点。但是,肝癌组织中CSF-1受体基因表达则显著高于癌旁肝组织。由此提示3种基因产物异常的过量表达与肝癌细胞自分泌生长刺激机制有关。

RNA干扰TGF-β信号通路对人膀胱癌T24细胞迁移和侵袭能力的影响

目的探讨过表达的TGF-β1和作用于TGF-β受体Ⅰ的干扰RNA(TsiRNA)在体外对人膀胱癌T24细胞迁移、侵袭能力的影响。方法采用Transwell迁移试验、划痕试验以及Transwell侵袭实验观察过表达的TGF-β1和TsiRNA在体外对人膀胱癌T24细胞迁移、侵袭能力的影响,RT-PCR技术和蛋白免疫印迹法(Western blot)检测TGF-β1及TsiRNA处理后TGF-β受体Ⅰ基因与蛋白表达水平的变化。结果过表达的TGF-β1可以显著提高膀胱癌T24细胞的迁移、侵袭能力,而TsiRNA可以完全降低由TGF-β1引起的T24的迁移、侵袭能力的变化。结论膀胱癌细胞的迁移、侵袭能力与过表达的TGF-β1密切相关,TsiRNA可以降低T24细胞的迁移、侵袭能力。