P2X7受体蛋白在胰腺癌中的表达及其对侵袭迁移影响的实验研究

目的:探讨P2X7受体蛋白在胰腺癌中的表达及其与临床病理之间的关系,探讨P2X7受体蛋白表达对胰腺癌侵袭迁移的影响及机制。方法:免疫组织化学法分析人胰腺癌组织及癌旁组织中P2X7受体蛋白的表达情况,分析其与胰腺癌临床病理特点之间的关系;选取胰腺癌细胞系PANC-1细胞,分别经P2X7受体激动剂Bz ATP(200μmol/L)及抑制剂A740003(20μmol/L)处理后,Transwell实验、细胞划痕实验检测PANC-1的侵袭迁移能力变化,Western blot实验检测侵袭及迁移相关蛋白MMP2、MMP9的表达变化。结果:P2X7受体蛋白在胰腺癌组织中高表达,在癌旁组织中低表达;且P2X7受体蛋白表达水平与胰腺癌的临床肿瘤分化程度及淋巴结转移呈显著性相关(P<0.05)。P2X7受体蛋白可通过上调MMP2、MMP9蛋白的表达促进胰腺癌细胞的侵袭迁移。结论:P2X7受体蛋白在胰腺癌中高表达并参与调控胰腺癌的侵袭及迁移,可能成为胰腺癌治疗的潜在分子靶点。

P2X7受体蛋白表达对胰腺癌的发生及发展的影响

目的探讨胰腺癌组织中P2X7受体蛋白表达及与患者临床病理学特征之间的关系。方法选取本院病理科收集的手术后胰腺癌组织80例为病例组、既往病理科收集的20例正常胰腺组织石蜡块为对照组,收集时间2013年1月-2017年1月,应用免疫组织化学染色法(SP)检测两组标本中的P2X7受体蛋白表达水平,并分期P2X7受体蛋白表达与患者临床分期、肿瘤分化、淋巴结转移等特征的关系。结果病例组胰腺癌组织中的P2X7受体蛋白阳性表达率66.25%显著的高于对照组胰腺组织中的10.00%,差异具有统计学意义(P<0.05);UICC分期(Ⅲ期)胰腺癌组织的P2X7受体蛋白阳性表达率、低分化程度的P2X7受体蛋白阳性表达率、发生淋巴结转移的P2X7受体蛋白阳性表达率均分别显著的高于UICC分期(Ⅰ期+Ⅱ期)、高+中分化、未发生淋巴结转移的胰腺癌组织,差异具有统计学意义(P<0.05);经Logistic回归分析,UICC分期为Ⅲ期、组织学低分化、P2X7受体蛋白阳性表达是胰腺癌患者12个月随访不良结局的独立危险因素(P<0.05)。结论胰腺癌组织中P2X7受体蛋白表达上调,并且与UICC分期、肿瘤分化及发生淋巴结转移密切相关。

PCPA失眠大鼠大脑皮质星形胶质细胞GFAP和P2X7受体表达的变化及酸枣仁汤的干预作用

目的:研究PCPA诱导大鼠失眠后大脑皮质星形胶质细胞GFAP和P2X7R表达的变化及SZRD的干预作用。方法:采用连续3d腹腔注射PCPA的方法复制失眠模型,大鼠随机分为对照组,模型组,中药治疗组和中药对照组,中药治疗组和中药对照组给予SZRD灌胃7d。各实验组分别在第4、5、6、8、10天取大鼠大脑皮质前额叶,采用免疫荧光双染法检测各组大鼠不同时间大脑皮质GFAP和P2X7R蛋白表达,用实时荧光定量PCR法测定各组大鼠不同时间大脑皮质P2X7R基因mRNA表达。结果:与对照组比较,模型组大鼠不同时间大脑皮质GFAP、P2X7R蛋白及mRNA表达均明显上调(P<0.01);与模型组比较,第5、6、8、10天中药治疗组GFAP表达逐渐下降(P<0.05,P<0.01),第6、8、10天中药治疗组P2X7R蛋白表达逐渐降低(P<0.05,P<0.01),第8、10天中药治疗组P2X7RmRNA表达水平逐渐下调(P<0.05,P<0.01)。结论:SZRD干预PCPA所致失眠的机制可能与调控大鼠大脑皮质星形胶质细胞GFAP和P2X7R的表达有关。

Presenilin 2 deficiency facilitates Aβ-induced neuroinflammation and injury by upregulating P2X7 expression

Accumulating evidence suggests that β-amyloid （Aβ）-induced neuroinflammation plays a prominent and early role in Alzheimer＇s disease （AD）. In this study, we demonstrated that Presenilin 2 （PS2） deficiency facilitates Aβ-induced neuroinflammation and injury by upregulating P2X7 expression both in vitro and in vivo. PS2 knockout mice demonstrated increased cognitive impairments and cerebral injury. PS2 deficiency increased the expression of P2X7 both in neurons and microglial cells. Furthermore, extracellular ATP also increased in both Aβ-treated and untreated PS2 knockout microglial cells. Notably, Aβ-induced classical proinflammatory cytokines such as IL-113, IL-1α and TNF-α were increased in PS2 knockout microglial cells, suggesting a potential role for PS2 in the regulation of neuroinflammation. The expression of P2X7 clearly increased in PS2 knockdown BV2 cells. Consistent with in vivo data, Aβ-induced IL-1βproduction was also clearly enhanced in PS2 knockdown BV2 cells. Additionally, expression of the transcription factor Sp was increased in PS2 knockdown cells. When we treated PS2 knockdown ceils with the specific Spl inhibitor MIT, we observed that enhanced P2X7 expression was significantly rescued. Taken together, these data suggests that PS2 plays a protective role during Aβ-induced neuroinflammation and injury through down-regulation of P2X7 expression.

Effects of moxibustion on the P2X7R/STAT3/VEGF pathway in rats with colitis-associated colon cancer

Objective:To observe the effects of herb-partitioned moxibustion and ginger-partitioned moxibustion on the growth of colon tumors in rats with colitis-associated colon cancer(CACC),and explore the mechanism of moxibustion intervening CACC through the purinergic receptor P2X ligand-gated ion channel 7(P2X7R)/signal transducer and activator of transcription 3(STAT3)/vascular endothelial growth factor(VEGF)pathway.Methods:A total of 26 male Sprague-Dawley rats were selected.According to the random number table method,6 rats were selected as the normal group.The remaining 20 rats were injected intraperitoneally with azoxymethane(AOM)combined with oral dextran sodium sulfate(DSS)to prepare the CACC model.After the model was successfully established,2 rats were randomly selected for model identification.The remaining 18 rats which were successfully modeled were randomly divided into a model group,a herb-partitioned moxibustion group and a ginger-partitioned moxibustion group,with 6 rats in each group.Moxibustion intervention was performed in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group at Qihai(CV 6)and bilateral Tianshu(ST 25).Moxibustion was performed twice at each point each time,once a day,at a 1-day interval after 6 consecutive interventions,for a total of 30 interventions.After intervention,the colon tumor load,pathological change and histopathological score were observed.Immunohistochemistry was used to detect the expressions of VEGF,P2X7R,phospho-STAT3(p-STAT3),and nuclear factor-kappa B p65(NF-κB p65)proteins in rat colon tissue.Western blot was used to detect the levels of p-STAT3 and NF-κB p65 proteins in rat colon tissue.Results:Compared with the normal group,the colon tumor load and histopathological score in the model group were significantly increased(both P<0.001),and different grades of dysplasia were observed in colon tissue from the model group,reaching the degree of adenocarcinoma;the expression level of P2X7R protein in colon tissue was significantly decreased(P<0.001),and the expression levels of p-STAT3,NF-κB p65 and VEGF proteins were significantly increased(all P<0.001)in the model group.Compared with the model group,the colon tumor load,colon histopathological score and the levels of p-STAT3,NF-κB p65 and VEGF proteins in colon tissue were significantly decreased(all P<0.05)in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group while the expression levels of P2X7R protein in colon tissue were significantly increased(both P<0.05).Conclusion:Both herb-partitioned moxibustion and ginger-partitioned moxibustion can reduce the colon tumor load in CACC rats and delay the progression of colon adenomas.The mechanism may be mediated by the P2X7R/STAT3 pathway to inhibit STAT3 phosphorylation,thereby reducing VEGF protein expression.

非诺贝特对糖尿病视网膜病变小鼠视网膜神经细胞的保护作用及机制

目的探讨嘌呤受体P2X7-NOD样受体蛋白-3(Nod-like receptor pyrin domain 3,NLRP3)炎性相关信号转导通路在糖尿病小鼠视网膜神经细胞中的表达情况,初步研究非诺贝特对糖尿病视网膜病变(diabetic retinopathy,DR)的保护作用。方法健康雄性小鼠60只,随机分为实验对照组(N组)、糖尿病组(D组)、非诺贝特治疗组(F组),D组及F组通过腹腔内注射链脲佐菌素制备DR模型,分别于造模后每天记录血糖情况,并于4周、8周、12周进行三组间血糖水平的比较。自造模成功后第2天起,F组每天口服非诺贝特100 mg·kg^-1连续喂养12周,4周后免疫荧光化学法观察视网膜神经胶质细胞及视网膜神经节细胞(retinal ganglion cell,RGC)的活化情况,Western blot检测视网膜组织中P2X7、NLRP3蛋白的表达情况。结果与N组相比,D组血糖水平在造模后4周、8周、12周随时间推移均有不同程度增长,差异均有统计学意义(均为P<0.05)。N组中,GFAP少量表达;D组4周时GFAP的表达主要分布于内界膜(inner limiting membrane,ILM)、RGC层和内丛状层(inner plexiform layer,IPL);非诺贝特治疗后,F组小鼠视网膜GFAP主要分布于ILM和RGC层,且密度与D组相比明显减弱。Western blot检测结果显示:N组中,视网膜P2X7和NLRP3蛋白仅有少量表达;D组中二者的表达水平在4周时明显升高,与N组相比,差异均有统计学意义(均为P<0.05);而此时F组经非诺贝特治疗后小鼠视网膜组织中P2X7和NLRP3蛋白的表达水平较D组明显降低,但仍高于N组,差异均有统计学意义(均为P<0.05)。结论非诺贝特可通过下调糖尿病小鼠视网膜组织中P2X7和NLRP3蛋白的表达从而发挥视网膜保护作用。