Molecular Cloning,Genomic Organization and Functional Analysis of the Ribosomal Protein S30(RPS30) Gene from Arachis hypogaea

The ribosomal proteins are crucial for the maintenance of ribosomal translational efficiency and fidelity.In the study,we characterized the ribosomal protein S30(RPS30)gene from Arachis hypogaea that has been isolated through Genefishing analysis during defense responses to Ralstonia solanacearum.The cDNA of RPS 30 contained a 189 base pair(bp)open-reading frame encoding 62 amino acids.The genomic DNA consists of 272 bp containing two exons and one 83 bp intron.The RPS 30 mRNA transcript was mainly expressed in roots and leaves.The expression level of the RPS 30 mRNA transcripts was up-regulated sharply 6 h after bacterial challenge and was 12 times greater than that of the control group.The phylogenetic analysis for genes encoding proteins showed that RPS30 were conserved within dicotyledonous and monocotyledonous plants.d S extremely exceeded d N in all branches of the tree(d N/d S<1.0),indicating that functional constraint have acted on RPS 30 throughout evolution.

A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson s disease,but the regulato ry mechanism remains elusive.Prohibitin-2(PHB2)is a newly discove red autophagy receptor in the mitochondrial inner membrane,and its role in Parkinson’s disease remains unclear.Protein kinase R(PKR)-like endoplasmic reticulum kinase(PERK)is a factor that regulates cell fate during endoplasmic reticulum stress.Parkin is regulated by PERK and is a target of the unfolded protein response.It is unclear whether PERK regulates PHB2-mediated mitophagy thro ugh Parkin.In this study,we established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced mouse model of Parkinson’s disease.We used adeno-associated virus to knockdown PHB2 expression.Our res ults showed that loss of dopaminergic neurons and motor deficits were aggravated in the MPTP-induced mouse model of Parkinson’s disease.Ove rexpression of PHB2 inhibited these abnormalities.We also established a 1-methyl-4-phenylpyridine(MPP+)-induced SH-SY5Y cell model of Parkinson’s disease.We found that ove rexpression of Parkin increased co-localization of PHB2 and microtubule-associated protein 1 light chain 3,and promoted mitophagy.In addition,MPP+regulated Parkin involvement in PHB2-mediated mitophagy through phosphorylation of PERK.These findings suggest that PHB2 participates in the development of Parkinson’s disease by intera cting with endoplasmic reticulum stress and Parkin.

MPTP对小鼠多巴胺能神经元细胞MN9D凋亡的影响

目的:探讨MPTP对小鼠多巴胺能神经元细胞MN9D凋亡的影响及其可能机制。方法:MN9D细胞分别以0、1μmol/L MPTP处理(对照组和MPTP组),或分别以pcDNA4、pcDNA4-BACE2转染(对照组和BACE2组),采用Western blot法检测BACE2、Cleaved Caspase-3和内源性DSG2蛋白的表达。MN9D细胞分为DSG2组、DSG2+BACE2组和DSG2+BACE2+BACE抑制剂组,分别转染pENTER-DSG2+pcDNA4、pENTER-DSG2+pcDNA4-BACE2以及转染pENTER-DSG2+pcDNA4-BACE2后用1μmol/L的BACE抑制剂处理,采用Western blot法检测DSG2全长以及DSG2各片段的表达。结果:与对照组相比,MPTP组中BACE2及Cleaved Caspase-3表达均上升(P<0.05);与对照组相比,BACE2组Cleaved Caspase-3表达增加,内源性DSG2表达减少(P<0.05)。与DSG2组相比,DSG2+BACE2组中全长DSG2表达下降,DSG2羧基末端片段以及胞外DSG2氨基末端片段均增多(P<0.05),而DSG2+BACE2+BACE抑制剂组中全长DSG2及各DSG2片段的表达与DSG2组相似(P>0.05)。结论:MPTP可通过上调BACE2促进MN9D细胞凋亡,其机制可能与BACE2对DSG2的剪切有关;BACE2和DSG2可能参与了帕金森病的发病。

Overexpression of 14-3-3 protein protects pheochromocytoma cells against 1-methyl-4-phenylpyridinium toxicity

Objective To investigate the effects of 14-3-3 protein overexpression on the 1-methyl-4-phenylpyridinium （MPP^＋） induced pheochromocytoma （PC12） cell death and the potential mechanisms. Methods pcDNA3.1（＋）-14-3-3 plasmids, which could be expressed in mammalian cell, were constructed and transfected into PC 12 cells with Lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein, and BAD protein were determined by western blot. 3-（4,5- dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, microplate reader, and flow cytometric analysis were used to measure cell viability, the caspase activity, and apoptotic ratio respectively. Results （1） The expression of 14-3-3 protein increased significantly three weeks after pcDNA3.1（＋）-14-3-3 plasmids transfected into PC 12 cells. （2） MPP^＋ caused a decrease of cell viability in a dose-dependent manner. At 100μmol/L MPP^＋, cell viability reduced approximately 50%. （3） The caspase activity increased along with the MPP^＋ concentrations rising and reached its maximum value （0.34 μmol/mg protein） at 100 μmol/L MPP＊. However caspase activity decreased significantly when the MPP^＋ concentration exceeded 100 μmol/L. （4） Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC 12 cells treated with 100μmol/L MPP^＋ from 26.5% to 8.6%. （5） Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC 12 cells with 100 μmol/L MPP^＋. Overexpression of 14-3-3 protein significantly increased the cellular level of Bcl-2 protein and decreased that of BAD protein. Conclusion Overexpression of 14-3-3 protein may reduce MPP^＋-induced apoptotic cell death in PC12 cells by up-regulating the Bcl-2 expression and down-regulating the BAD expression. These results may provide a promising target for treatment of Parkinson＇s disease.

C1q/肿瘤坏死因子相关蛋白3、脂蛋白相关磷脂酶A2、可溶性髓样细胞触发受体-1对脑血管狭窄患者术后支架内再狭窄预测价值

目的探讨血清补体C1q/肿瘤坏死因子相关蛋白3(CTRP3)、脂蛋白相关磷脂酶A2(Lp-PLA2)、可溶性髓样细胞触发受体-1(sTREM-1)水平在预测脑血管狭窄人群的颅脑动脉支架术后支架内再狭窄(ISR)方面的价值。方法选取2020年5月至2022年6月廊坊市人民医院收治的107例接受颅脑动脉支架成形术的患者。根据患者术后6个月内是否发生ISR分为ISR组(n=26)与NISR组(n=81)。比较两组患者的低密度脂蛋白胆固醇(LDL-C)、支架数量、球囊扩张支架的占比、狭窄程度、残余狭窄程度,以及CTRP3、Lp-PLA2、sTREM-1水平。结果ISR组LDL-C、支架数量、球囊扩张支架的占比、狭窄程度、残余狭窄程度、Lp-PLA2水平、sTREM-1水平均高于NISR组,差异有统计学意义(P<0.05);ISR组CTRP3水平低于NISR组,差异有统计学意义(P<0.05)。支架数量多、狭窄程度及残余狭窄程度高、CTRP3水平降低、Lp-PLA2、LDL-C、sTREM-1水平上升为脑血管狭窄病患血管成形术后出现ISR的独立危险因素(P<0.05)。CTRP3、Lp-PLA2、sTREM-1三者联合在脑血管狭窄病患颅脑血管成形术后出现ISR的预测曲线下面积分别为0.840、0.784、0.809、0.899,三者联合预测价值显著高于单一指标。结论CTRP3水平降低,Lp-PLA2、sTREM-1水平升高与脑血管狭窄患者颅脑动脉支架术后发生ISR有关,联合指标检测对脑血管狭窄患者颅脑动脉支架术后发生ISR具有一定预测价值。

核糖体蛋白L34和STAT3表达与宫颈癌临床病理特征及预后的关系

目的探讨核糖体蛋白L34(RPL34)和信号转导和转录激活因子3(STAT3)表达与宫颈癌临床病理特征及预后的关系。方法回顾性收集2019年1月至2022年6月台州市中心医院(台州学院附属医院)手术切除的30例宫颈癌和30例正常宫颈(对照)新鲜组织,以及2017年1月至2022年6月手术切除的76例宫颈癌和40例正常宫颈(对照)组织石蜡标本。采用qRT-PCR法检测新鲜组织中RPL34和STAT3 mRNA相对表达量,采用免疫组化法检测石蜡标本中RPL34和STAT3蛋白表达水平,分析RPL34和STAT3在不同临床病理特征宫颈癌患者中的表达差异,采用Spearman秩相关分析RPL34和STAT3表达的相关性,绘制Kaplan-Meier生存曲线分析RPL34和STAT3表达的预后意义。结果宫颈癌组织中RPL34 mRNA相对表达量和蛋白表达水平均显著低于对照组织(均P<0.01),STAT3 mRNA相对表达量和蛋白表达水平均显著高于对照组织(均P<0.01)。不同国际妇产科学联盟(FIGO)分期、浸润深度及淋巴结转移患者宫颈癌组织中RPL34表达水平比较,差异均有统计学意义(均P<0.01),不同FIGO分期、浸润深度、淋巴结转移及分化程度患者宫颈癌组织中STAT3表达水平比较,差异均有统计学意义(均P<0.01)。宫颈癌组织中RPL34表达和STAT3表达呈显著负相关(P<0.05)。与阴性组患者相比,RPL34阳性组患者术后3年总生存率显著升高(P<0.05),STAT3阳性组患者术后3年总生存率显著降低(P<0.05)。结论宫颈癌组织中RPL34 mRNA和蛋白呈低表达,STAT3 mRNA和蛋白呈高表达,RPL34和STAT3表达与宫颈癌的浸润深度、淋巴结转移等临床病理特征有关,并影响患者3年总生存率。RPL34和STAT3有望成为宫颈癌预后判断的标志物。

梓醇-川芎嗪方调控STAT3改善阿尔茨海默病神经可塑性的机制研究

目的:探讨梓醇-川芎嗪(CT)方对阿尔茨海默病(AD)的作用及转录激活蛋白3(STAT3)的影响。方法:将野生型C57BL/6J小鼠作为对照组(Control),同时将粉样前体蛋白/早老素1双转染小鼠按照随机数字表法随机分为模型组(Model)、低剂量梓醇-川芎嗪方组(CT-L)、高剂量梓醇-川芎嗪方组(CT-H),每组6只。CT-L组和CT-H组分别给予梓醇-川芎嗪方药50、100 mg/kg灌胃处理。Control组和Model组均给予等体积的生理盐水灌胃。每日1次,连续灌胃8周。水迷宫实验探索梓醇-川芎嗪对AD小鼠学习记忆能力的影响,免疫组化检测海马组织中生长相关蛋白43(GAP-43)的表达。海马神经元HT-22细胞分为空白对照组(Control组)、模型组(Model组)、低剂量梓醇-川芎嗪方组(CT-L组)、高剂量梓醇-川芎嗪方组(CT-H组)、STAT3敲除+梓醇-川芎嗪方组(CT+siSTAT3组)。通过Aβ_(1-42)构建体外AD模型,通过转染siSTAT3构建STAT3沉默的神经元模型,并加入高、低剂量的梓醇-川芎嗪方培养24 h,细胞活性检测(CCK-8)检测细胞活力,蛋白免疫印迹法(Western Blot)检测Cyclin D1、Ki-67、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、NF200、GAP-43、STAT3和磷酸化的STAT3(p-STAT3)蛋白表达水平,Brdu检测细胞增殖,流式细胞术和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)染色检测细胞凋亡。结果:梓醇-川芎嗪方可以明显缩短APP/PS1双转染小鼠的逃避潜伏期,提高目标象限停留时间和目标象限停留次数,上调海马组织GAP-43的表达,其中高剂量的梓醇-川芎嗪方作用更显著(P<0.05或P<0.01)。梓醇-川芎嗪方可以恢复AD模型细胞的活力和增殖能力,上调Cyclin D1、Ki-67、NF200、GAP-43和STAT3蛋白的表达,下调凋亡相关蛋白Bax的表达并恢复了抗凋亡Bcl-2蛋白的水平,其中高剂量梓醇-川芎嗪方的作用更显著(P<0.05或P<0.01)。而沉默STAT3逆转了梓醇-川芎嗪方对AD神经元增殖和对NF200、GAP-43蛋白表达的促进作用,阻断了梓醇-川芎嗪方对AD神经元的抗凋亡作用(P<0.05或P<0.01)。结论:梓醇-川芎嗪方可以显著改善AD模型小鼠的认知和记忆能力,且其效应具有剂量依赖性。其作用机制可能与促进神经元细胞的增殖,降低凋亡,调控STAT3的表达从而促进神经元的可塑性有关。

The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells

AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS: The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722 x 10(-9)M (Bmax=12810 sites per cell) and 8.931 x 10(-8)M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION: The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene.

Role of microRNA-21 in uveal melanoma cell invasion and metastasis by regulating p53 and its downstream protein

AIM： To reveal the insight mechanism of liver metastasis in uveal melanoma, we investigated cell functions of microRNA-21 in three different uveal melanoma cell lines and analyze the relationship of target gene p53 and its downstream targets which been found significant expression in our previous study.METHODS： Quantitative real-time polymerase chain reaction （qRT-PCR） was used to detect microRNA-21 expression in normal uveal tissue and uveal melanoma cell lines. Lenti-virus expression system was used to construct OCM-1, MuM-2B and M619 cell line with stable overexpression and inhibition of microRNA-21. In vitro cell function tests such as cell proliferation, cell apoptosis, cell circle and abilities of migration and invasion were examined by MTT, BrdU assay, flow cytometry, transwell assay and Matrigel invasion assay respectively. The target gene was predicted by bioinformatics and confirmed by using a dual luciferase reporter assay. The expression of p53 and its suspected downstream targets LIM and SH3 protein 1 （LASP1） and Glutathione S Transferase pi （GST-Pi） were determined by qRT-PCR in mRNA level and western blotting analysis in protein level. Finally, the effect of microRNA-21 in a xenograft tumor model was assessed in four-week-old BALB/c nude mice. RESULTS： Compared to normal uveal melanoma, expressions of microRNA-21 were significantly higher in uveal melanoma cell lines. Overexpression of microRNA-21 promoted proliferation, migration, and invasion of OCM-1, M619 and MuM-2B cells, while inhibition of microRNA-21 reveal opposite effects. Wild type p53 was identified as a target gene of microRNA-21-3p, and proved by dual luciferase reporter assay. Up-regulated microRNA-21 inhibited the expression of wild type p53 gene, and the increased expression of LASP1 in mRNA level and protein level, while down-regulated microRNA-21 presented opposite way. However, GST-pi showed the potential pattern as expected, but relative mRNA level showed no statistically significant difference in OCM-1 cells. Furthermore, the mRNA expression of GST-pi was decreased in microRNA-21 overexpressing MuM-2B, and increased in M619 cells with inhibition of microRNA-21. In vivo, inhibition of microRNA-21 reduced tumor growth with statistically significant difference.CONCLUSION： These findings provide novel insight into molecular etiology of microRNA-21 in uveal melanoma cell lines, and suggest that microRNA-21 might be a potential candidate for the diagnosis and prognostic factor of human uveal melanoma in future.

miR-15b-5p targeting amyloid precursor protein is involved in the anti-amyloid eflect of curcumin in swAPP695-HEK293 cells

Curcumin exerts a neuroprotective effect on Alzheimer’s disease;however,it is not known whether microRNAs are involved in this protective effect.This study was conducted using swAPP695-HEK293 cells as an Alzheimer’s disease cell model.swAPP695-HEK293 cells were treated with 0,0.5,1,2,5,and 10μM curcumin for 24 hours.The changes in miR-15b-5p,miR-19a-3p,miR-195-5p,miR-101-3p,miR-216b-5p,miR-16-5p and miR-185-5p expression were assessed by real-time quantitative polymerase chain reaction.The mRNA and protein levels of amyloid precursor protein,amyloid-β40 and amyloid-β42 were evaluated by quantitative real-time polymerase chain reaction,western blot assays and enzyme-linked immunosorbent assays.swAPP695-HEK293 cells were transfected with miR-15b-5p mimic,or treated with 1μM curcumin 24 hours before miR-15b-5p inhibitor transfection.The effects of curcumin on amyloid precursor protein,amyloid-β40 and amyloid-β42 levels were evaluated by western blot assays and enzyme-linked immunosorbent assay.Luciferase assays were used to analyze the interaction between miR-15b-5p and the 3′-untranslated region of amyloid precursor protein.The results show that amyloid precursor protein and amyloid-βexpression were enhanced in swAPP695-HEK293 cells compared with HEK293 parental cells.Curcumin suppressed the expression of amyloid precursor protein and amyloid-βand up-regulated the expression of miR-15b-5p in swAPP695-HEK293 cells.In addition,we found a negative association of miR-15b-5p expression with amyloid precursor protein and amyloid-βlevels in the curcumin-treated cells.Luciferase assays revealed that miR-15b-5p impaired the luciferase activity of the plasmid harboring the 3′-untranslated region of amyloid precursor protein.These findings indicate that curcumin down-regulates the expression of amyloid precursor protein and amyloid-βin swAPP695-HEK293 cells,which was partially mediated by miR-15b-5p via targeting of the 3′-untranslated region of amyloid precursor protein.

Role of Tyro3, Axl, and Mer Receptors and Their Ligands (Gas6, and Protein S) in Patients with Hepatocellular Carcinoma

The Tyro3, Axl and Mer (TAM) receptor tyrosine kinases are activated by endogenous ligands, protein S1 (PROS1) and growth arrest-specific gene 6 (Gas6), and those have important effects on cell biology. These receptors (Rs) can be shad from the cell membrane and their soluble(s) forms can be found in plasma. We investigated the fluctuation and interactive role of sTAMRs and its ligands in patients with hepatocellular carcinoma (HCC), hepatitis groups, and healthy normal adult controls (NC). The measurement cases were 45 patients with HCC group (stage 1 in 4, stage 2 in 8, stage 3 in 16, and stage 4 in 17), 4 patients with fulminant hepatitis (FH), 14 patients with acute hepatitis (AH), 10 patients with chronic hepatitis (CH), 16 patients with liver cirrhosis (LC), and 20 NCs matched by age. Plasma levels of three sTAMRs and their ligands were measured by ELISA. In comparison with NCs, Gas6, des-γ-carboxy Gas6, and sTAMRs levels were significantly higher in HCC patients, but free PROS1 levels were significantly lower. The sTyro3 and sAxl levels peaked HCC stages 2 and 3 respectively, and gradually decreased afterwards while maintaining high levels. sMer levels increased with the progression of HCC. Gas6 and des-γ-carboxy Gas6 levels gradually increased, and PROS1 levels decreased with the progression of HCC. Gas6 levels were positively correlated with sAxl levels, whereas sMer levels were negatively correlated with free PROS1 levels. sTAMRs and Gas6 levels increased in parallel to the progression of HCC fibrosis. Through the progression of HCC, Axl played the major role in TAMRs activation. However, sTYro3 continued increasing rapidly from the early stage, and that of Mer increased throughout the progression. Roles of Axl may be changed in Mer, because des-γ-carboxy Gas6 levels increasing with Gas6 in the advanced stage of HCC cannot send a signal to Axl.

血清α-突触核蛋白、微管相关蛋白1轻链3、载脂蛋白A1与帕金森病认知功能、疾病进展的关系

目的检测α-突触核蛋白(α-synuclein)、微管相关蛋白1轻链3(LC3)、载脂蛋白A1(Apo A1)水平在帕金森病(PD)患者血清中的表达情况,以及三者与PD患者发生认知功能障碍(CI)、疾病进展的相关性。方法选取96例PD患者作为研究组,另外选取同期体检健康者90例为对照组。根据蒙特利尔认知评估量表(MoCA)评分将患者分为CI组(42例)和认知功能正常组(54例)。根据Hoehn-Yahr(H-Y)分期量表将PD患者分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ期,其中Ⅰ~Ⅱ期PD患者纳入早期PD组(57例),Ⅲ~Ⅴ期PD患者纳入中晚期PD组(39例)。测定PD患者血清中α-synuclein、LC3、Apo A1水平。分析α-synuclein、LC3、Apo A1与H-Y分期的相关性。采用多因素Logistic回归分析筛选PD患者发生CI的影响因素。结果研究组α-synuclein水平高于对照组,LC3、Apo A1水平低于对照组,差异有统计学意义(P<0.05);早期PD组α-synuclein水平低于中晚期PD组,LC3与Apo A1水平高于中晚期PD组,差异有统计学意义(P<0.05)。血清α-synuclein水平与H-Y分期呈正相关(P<0.05)。三者联合预测PD患者发生CI的曲线下面积(AUC)为0.925,大于α-synuclein、LC3、Apo A1单独预测的AUC(P<0.05)。α-synuclein、LC3、Apo A1、高血压史是PD患者发生CI的影响因素(P<0.05)。结论α-synuclein在PD合并CI患者血清中呈高表达,LC3、Apo A1呈低表达,三者与PD认知功能、疾病进展的关系密切。

胃癌组织RRBP1、LAMB3的mRNA表达水平及其与患者临床病理特征、预后的关系

目的探讨胃癌组织内质网核糖体结合蛋白1(RRBP1)、层粘连蛋白亚基β3(LAMB3)的mRNA表达水平及其与患者临床病理特征、预后的关系。方法收集373例胃癌患者的胃癌组织标本和癌旁组织标本。采用实时荧光定量PCR检测胃癌组织和癌旁组织中RRBP1和LAMB3的mRNA表达水平,分析RRBP1和LAMB3的mRNA表达水平与胃癌患者临床病理特征的关系,绘制生存曲线分析胃癌组织中RRBP1和LAMB3的mRNA表达水平与患者预后的关系,采用COX回归模型分析胃癌患者预后的影响因素。结果与癌旁组织相比,胃癌组织中RRBP1和LAMB3的mRNA表达水平升高(P<0.05)。肿瘤直径≥4 cm、分化程度为低或未分化、浸润深度为T_(3)~T_(4)、发生淋巴结转移及远处转移的胃癌患者RRBP1 mRNA表达水平更高(P<0.05);分化程度为低或未分化、浸润深度为T_(3)~T_(4)、发生淋巴结转移及远处转移的胃癌患者LAMB3 mRNA表达水平更高(P<0.05)。RRBP1高表达组和LAMB3高表达组的3年生存率分别低于RRBP1低表达组和LAMB3低表达组(P<0.05)。COX回归分析结果显示,RRBP1 mRNA高表达、LAMB3 mRNA高表达、低或未分化程度、T_(3)~T_(4)浸润深度和发生淋巴结转移均是影响胃癌患者预后的独立危险因素(P<0.05)。结论RRBP1和LAMB3 mRNA在胃癌组织中高表达,且与患者临床病理特征和预后密切相关。

DYRK1A调控NLRP3激活在帕金森病模型中的研究

目的 通过在帕金森病模型中分析双特异性酪氨酸调控激酶1A(DYRK1A)与NOD样受体热蛋白结构域相关蛋白3(NLRP3)激活的关系,探讨帕金森病中神经炎症的治疗靶点。方法 (1)采用MPP^(+)处理BV2细胞(小鼠小胶质细胞)后,使用免疫印迹法(Western blot)检测DYRK1A及NLRP3蛋白的表达水平;加用DYRK1A抑制剂(Harmine)处理帕金森细胞模型,使用CCK8法检测小胶质细胞活性,免疫印迹法检测NLRP3蛋白表达水平。(2)MPTP慢性PD动物模型中,腹腔注射DYRK1A抑制剂(Harmine),免疫组织荧光观察小鼠中脑黑质区小胶质细胞数目,免疫印迹法检测小鼠中脑黑质区NLRP3蛋白表达水平。结果 (1)不同浓度MPP^(+)处理小胶质细胞后,DYRK1A及NLRP3的蛋白水平较对照组均有升高(P<0.05);Harmine(浓度为10、15μmol/L)时可改善MPP^(+)对BV2细胞活性的损伤(P<0.05);相比MPP^(+)组,Harmine+MPP^(+)组NLRP3蛋白水平明显下降(P<0.05)。(2)与对照组相比,模型组小鼠中脑黑质的小胶质细胞数量增多;与模型组相比,模型中浓度抑制剂组小鼠中脑黑质的小胶质细胞数量减少;模型组小鼠中脑黑质区NLRP3的蛋白水平较对照组升高,与模型组比较,模型中浓度及高浓度抑制剂组小鼠中脑黑质中NLRP3蛋白表达下降明显(P<0.05)。结论 抑制DYRK1A可减轻帕金森病模型中NLRP3炎症蛋白的激活,为帕金森病的治疗和研究提供新的思路。

Inhibiting 5-hydroxytryptamine receptor 3 alleviates pathological changes of a mouse model of Alzheimer's disease

Extracellular amyloid beta(Aβ) plaques are main pathological feature of Alzheimer’s disease.However,the specific type of neuro ns that produce Aβ peptides in the initial stage of Alzheimer’s disease are unknown.In this study,we found that 5-hydroxytryptamin receptor 3A subunit(HTR3A) was highly expressed in the brain tissue of transgenic amyloid precursor protein and presenilin-1 mice(an Alzheimer’s disease model) and patients with Alzheimer’s disease.To investigate whether HTR3A-positive interneurons are associated with the production of Aβ plaques,we performed double immunostaining and found that HTR3A-positive interneurons were clustered around Aβ plaques in the mouse model.Some amyloid precursor protein-positive or β-site amyloid precursor protein cleaving enzyme-1-positive neurites near Aβ plaques were co-localized with HTR3A interneurons.These results suggest that HTR3A-positive interneurons may partially contribute to the generation of Aβ peptides.We treated 5.0-5.5-month-old model mice with tro pisetron,a HTR3 antagonist,for 8 consecutive weeks.We found that the cognitive deficit of mice was partially reversed,Aβ plaques and neuroinflammation we re remarkably reduced,the expression of HTR3 was remarkably decreased and the calcineurin/nuclear factor of activated T-cell 4 signaling pathway was inhibited in treated model mice.These findings suggest that HTR3A interneurons partly contribute to generation of Aβ peptide at the initial stage of Alzheimer’s disease and inhibiting HTR3 partly reve rses the pathological changes of Alzheimer’s disease.

阿尔茨海默病患者血清C-X-C基序趋化因子配体12和几丁质酶3样蛋白1的表达水平及其临床意义

目的分析阿尔茨海默病(AD)患者血清C-X-C基序趋化因子配体12(CXCL12)、几丁质酶3样蛋白1(CHI3L1)的表达水平及其临床意义。方法选择113例AD患者作为AD组,60例血管性痴呆(VD)患者作为VD组,60例健康体检者作为对照组,根据简易精神状态检查(MMSE)量表评分结果将AD组患者分为轻度组(n=27)、中度组(n=51)、重度组(n=35)。比较AD组、VD组、对照组之间及不同病情严重程度的AD患者之间的血清CXCL12、CHI3L1表达水平。采用Pearson检验分析AD患者血清CXCL12、CHI3L1表达水平与MMSE量表评分的相关性,采用受试者工作特征(ROC)曲线分析血清CXCL12、CHI3L1表达水平对AD的诊断效能。结果与对照组相比,AD组和VD组的血清CXCL12表达水平降低,血清CHI3L1表达水平升高,且AD组的血清CXCL12表达水平低于VD组,血清CHI3L1表达水平高于VD组(P<0.05);不同病情严重程度AD患者血清CXCL12、CHI3L1表达水平差异有统计学意义,其中血清CXCL12表达水平为重度组<中度组<轻度组,血清CHI3L1表达水平为重度组>中度组>轻度组(P<0.05)。Pearson相关分析结果显示,AD患者血清CXCL12表达水平与MMSE量表评分呈正相关,血清CHI3L1表达水平与MMSE量表评分呈负相关,血清CXCL12表达水平与血清CHI3L1表达水平呈负相关(P<0.05)。ROC曲线分析结果显示,血清CXCL12、CHI3L1表达水平单独及联合诊断AD的曲线下面积分别为0.862、0.831、0.915,二者联合诊断的效能优于单独诊断(P<0.05),且血清CXCL12和CHI3L1表达水平的最佳截断值分别为2.03 ng/mL和211.08 ng/mL。结论AD患者血清中CXCL12表达下调、CHI3L1表达上调,二者表达异常与AD病情严重程度密切相关。二者或可作为辅助诊断AD及判断其病情严重性的生物学标志物。

Au-Fe_(3)O_(4) dumbbell-like nanoparticles based lateral flow immunoassay for colorimetric and photothermal dual-mode detection of SARS-CoV-2 spike protein

Lateral flow immunoassay(LFIA)has become popular in laboratories,at-home testing,and medical diagnostics due to its minimal cost and user-friendliness.Nevertheless,conventional test strips based on colloidal gold can only obtain qualitative or semi-quantitative results with low sensitivity.In this work,AuFe_(3)O_(4) dumbbell-like nanoparticles were synthesized and used as the LFIA labelling marker for highly sensitive colorimetric-photothermal dual-mode detection of SARS-CoV-2 spike(S)protein.The unique dumbbell structure of Au-Fe_(3)O_(4) NPs makes it possible to combine the best features of both Au NPs and Fe_(3)O_(4) NPs.The increased surface area of these NPs enhances their LSPR effect and photothermal effect,which achieves signal amplification to increase sensitivity.The Au-Fe_(3)O_(4) NPs modified with S protein antibody could identify S protein in samples,which were recognized and accumulated on T-line by another antibody,generating color band for qualitative colorimetric detection.The T-line was irradiated by laser to obtain temperature change for quantitative detection of photothermal.In optimized conditions,the detection limit was 1.22 pg/m L,three orders of magnitude more sensitive than colorimetric detection.Finally,the approach was performed on SARS-CoV-2 pseudovirus samples and outperformed traditional colloidal gold strips.This LFIA platform exhibits significant promise for practical implementation,as it can satisfy the need for low-cost,high-sensitivity,and home-based quantitative detection for respiratory infectious diseases.

七氟醚与丙泊酚复合麻醉对心内直视术患者S-100β蛋白、NSE和认知功能影响的比较

目的 比较七氟醚与丙泊酚复合麻醉对心内直视术患者S-100β蛋白、神经元特异性烯醇化酶（NSE）和认知功能的影响,为减少心内直视术后脑损伤提供参考.方法 择期行心脏二尖瓣置换术患者60例,性别不限,年龄24-63岁,体质指数16.5-25.8 kg/m2,ASA分级Ⅱ或Ⅲ级,随机分为两组（n=30）：七氟醚复合麻醉组（S组）和丙泊酚复合麻醉组（P组）.麻醉诱导：静脉注射咪达唑仑0.05-0.1 mg/kg,依托咪酯0.3-0.5 mg/kg,瑞芬太尼0.5-1 μg/kg,顺苯磺酸阿曲库铵0.15 mg/kg.麻醉维持：S组持续吸入1.0%-3.0%七氟醚（体外循环期间通过体外循环旁路吸入1.0%-3.0%七氟醚）,P组靶控输注2-4 μg/mL丙泊酚;两组均静脉输注瑞芬太尼0.2-0.4 μg/（kg·min）和顺苯磺酸阿曲库铵0.1 mg/（kg·h）至手术结束.于麻醉诱导前（T0）、体外循环开始后30 min（T1）、体外循环结束时（T2）、手术结束时（T3）、术后12 h（T4）及术后24 h（T5）时采集颈内静脉血测定血清S-100β蛋白和NSE浓度,于术前1 d和术后2 d用简易精神状态检查表（MMSE）评估患者的认知功能.结果 与P组比较,S组T1-T4时点血清S-100β蛋白和NSE浓度降低（P〈0.05）,术后2 d时MMSE评分升高（P〈0.05）,术后认知功能障碍（POCD）发生率下降（P〈0.05）.结论 在心内直视术中,与丙泊酚复合麻醉比较,七氟醚复合麻醉可降低血浆S-100β蛋白和NSE浓度,减少术后POCD的发生,可推荐为预防心内直视术后脑损伤的麻醉方法.

苦瓜核糖体失活蛋白诱导HL-60细胞发生凋亡及其分子机制的研究

目的 探讨苦瓜核糖体失活蛋白 (Ribosomalinactivatingprotein ,RIP)诱导HL 60细胞凋亡的活性 ,以及对HL 60细胞Bcl 2、Bax、caspase 3表达变化的影响及其分子机制。方法 以一定浓度苦瓜RIP处理HL 60细胞 2 4～ 72h ,用TUNEL法检测细胞凋亡。运用RT PCR和Westernblot免疫印记法检测Bcl 2、Bax、caspase 3mRNA和蛋白表达的变化。结果 苦瓜RIP在一定作用浓度下可使HL 60细胞发生凋亡 ,且随着作用时间的延长凋亡细胞比例逐渐升高。在此过程中 ,HL 60细胞的caspase 3mRNA的表达及其胞内活性亚单位水平显著升高 (P <0 .0 5 ) ,Bcl 2mRNA表达变化虽不显著 ,但其蛋白水平明显降低 (P <0 .0 5 ) ,与此同时 ,BaxmRNA和蛋白表达均有所上调。结论 在苦瓜RIP诱导的HL 60细胞凋亡中 ,caspase 3发挥了极为重要的作用 ,而Bcl 2和Bax可能通过其表达量的变化。

丁苯酞软胶囊改善帕金森痴呆患者认知功能和日常生活能力的效果及其对相关因子的影响

目的观察丁苯酞软胶囊改善帕金森痴呆(PDD)患者认知功能和日常生活能力的效果。方法 2013年6月—2015年6月湖北医药学院附属十堰市人民医院神经内科收治帕金森痴呆患者84例,按照随机数字表法分为观察组及对照组各42例。2组均予常规治疗,对照组加用盐酸多奈哌齐口服,观察组在对照组基础上加服丁苯酞软胶囊,治疗时间均为12周。观察2组治疗效果;记录2组治疗前后帕金森病评定量表(UPDRS)、简易精神状态量表(MMSE)、蒙特利尔认知评估量表(MoCA)及日常生活能力(ADL)评分;同时测定2组治疗前后血清C-反应蛋白(CRP)、重组人帕金森病蛋白7(PARK7)、神经营养因子-3(NT-3)水平。结果治疗后,观察组总有效率为95.24%,显著高于对照组的76.19%(X^2=6.222,P<0.05);观察组UPDRS总评分低于对照组[(20.32±5.26)分vs.(35.12±5.11)分,P<0.05],而MMSE、MoCA、ADL评分均显著高于对照组[分别为(28.92±4.78)分、(27.89±4.36)分、(75.39±4.58)分和(26.02±5.02)分、(24.12±2.78)分、(62.58±5.78)分],差异均有统计学意义(P<0.05);观察组CRP、PARK7均低于对照组[分别为(3.98±0.36)mg/L、(14.32±2.15)μg/L和(6.35±0.48)mg/L、(25.26±4.63)μg/L],而NT-3水平高于对照组[(32.25±3.78)μg/L vs.(24.98±4.23)μg/L],差异均有统计学意义(P<0.05)。结论丁苯酞软胶囊能有效改善PDD患者肢体功能运动及脑神经功能,可明显降低患者血清CRP、PARK7水平,提高NT-3水平,对PDD具有良好治疗效果,值得临床应用。