Biological markers play a pivotal role in the early and accurate diagnosis of Alzheimer’s disease, enabling precise identification and monitoring of therapeutic interventions. The detection of central β-amyloid and Tau proteins has become an indispensable tool in clinical trials. Recent years have witnessed substantial progress in the development of readily accessible and cost-effective blood biomarkers. This comprehensive article provides a comprehensive overview of the clinical applications of blood biomarkers, encompassing β-amyloid, phosphorylated Tau protein, neurofilament light chain protein, and glial fibrillary acidic protein, all of which have demonstrated clinical relevance in Alzheimer’s disease diagnosis. Notably, phosphorylated Tau protein exhibits superior diagnostic efficacy. The incorporation of blood biomarkers facilitates early screening, accurate diagnosis, and efficacious treatment of Alzheimer’s disease.
目的 研究磷脂酰肌醇3-激酶（PI3K）和磷酸化蛋白激酶B（p-Akt）在人膀胱尿路上皮癌組織中的表達特征及臨床意義。 方法 2005年6月－2010年7月，采用免疫組織化學法檢測40例膀胱尿路上皮癌組織及10例正常膀胱組織PI3K與p-Akt的表達，并對結果進行統計學分析。 結果 PI3K和p-Akt在正常膀胱黏膜組織陽性表達率均低于膀胱尿路上皮癌組織中，差異均有統計學意義（P＜0.05）。同一標本中PI3K和p-Akt的表達不具有相關性（r=0.051，P=0.747）。 結論 PI3K、p-Akt在膀胱尿路上皮癌中高表達，兩者在膀胱尿路上皮癌中共同促其發展，但其在膀胱尿路上皮癌的預后和進展中的作用尚不明確。
Objective To study the expression of thymidine phosporylase (TP) and the counts of lymph vessels in pancreatic cancer and chronic pancreatitis tissues, and to explore their clinicopathologic significances and correlation in the course of pancreatic cancer. Methods SP immunohistochemical method was used to detetct the expression of TP and the locations of lymph vessels on the routinely paraffin-embedded sections of the specimens from 51 cases pancreatic cancer and 10 cases of chronic pancreatitis. Results The positive rate of TP and the counts of lymph vessels were significantly higher (P＜0.05 and P＜0.01 respectively) in pancreatic cancer 〔54.9%, (12.5±4.3)/HP〕 than those in chronic pancreatitis 〔20.0%，(5.2±2.4)/HP〕. The positive rate of TP and the counts of lymph vessels were significantly lower （P＜0.05, P＜0.01） in well-differentiated adenocarcinoma cases and cases without metastasis compared with poor-differentiated adenocarcinoma cases and cases with metastasis. The counts of lymph vessels were significantly higher in the positive cases of TP than those in the negative ones in pancreatic cancer 〔(13.8±3.4)/HP vs (10.9±3.2)/HP〕, P＜0.01.Conclusion The expression of TP and counts of lymph vessels might be important markers reflecting the progression, biological behaviors, metastatic status and prognosis of pancreatic cancer. TP might promote lympoangiogenesis in pancreatic cancer tissues.
The intracellular domain of clusters of differentiation 44 (CD44) binding to the FERM (protein 4.1-ezrin-radixin-moesin) domain of ERM (ezrin/radixin/moesin) proteins and furthermore triggering the recruitment of spleen tyrosine kinase (Syk) are very important in the process of tumor cell adhesion, migration and proliferation. At first, it was found that CD44/FERM structure was stable by observing CD44/FERM complex conformation and analyzing the interaction of interface residues both in static crystal structure and in equilibrium process. Meanwhile, unconventional immunoreceptor tyrosine-based activation motif (ITAM-like), and phosphorylation sites Y191 and Y205 were buried in FERM domain, which would hinder the phosphorylation of ERM proteins, the recruitment of Syk and subsequent signal transduction. Then, steered molecular dynamics simulation was applied to simulate the interaction between CD44 and FERM domain in the mechanical environment. The results showed that mechanical signal could induce the exposure of the ITAM-like motif and phosphorylation site Y205 by tracking and analyzing CD44/FERM complex conformational changes and the solvent-accessible surface area. This study revealed how the force regulates the activation of downstream signal through CD44 intracellular domain for the first time, and would be useful for further understanding the adhesion and migration pathway of cancer cells and the design of antitumor drugs.
The present study is to explore the change process and distribution of phosphorylated DARPP-32 (p-DARPP-32) in rat brain including cortex, hippocampus and striatum and to further deduce whether p-DARPP-32 was possibly involved in epilepsy induced by repetitive low doses of pentylenetetrazol (PTZ). PTZ-induced epilepsy model in rat was established with 30 male SD rats randomly divided into 6 groups, control group and five trial groups [PTZ 1 h,PTZ 6 h,PTZ 24 h,PTZ 48 h and PTZ 72 h respectively, after onset of status epilepticus (SE)]. Immunohistochemistry and immunofluorescence double-labeling were used to detect the temporal time change and distribution of p-DARPP-32 expression and to analyze the coexpression of DARPP-32 and p-DARPP-32 in rat brain after the onset of PTZ-induced generalized SE. The results showed that there was a temporal time change of p-DARPP-32 expression in rat brain after the onset of SE. The number of p-DARPP-32-positive cells increased significantly and reached the peaks at the ends of 1 hour and 6 hours after the onset of SE, but decreased at the end of 24 hours. The moderate to strong p-DARPP-32-immunopositive neurons were observed in cortex, hippocampus and striatum, and located in cell cytoplasm and cell nucleus. Further immunofluorescence double-labeling revealed that denser colocalization of p-DARPP-32 and DARPP-32 in the neurons existed in the area mentioned above. Therefore, PTZ-induced SE may cause phosphorylation of DARPP-32 in rat brain. The temporal time change and distribution of p-DARPP-32 suggest that phosphorylation of DARPP-32 may be involved in PTZ-induced epilepsy in rat brain including cortex, hippocampus and striatum, and p-DARPP-32 may play a central role in the onset of SE.
ObjectiveTo detect the expression level of phosphate and tension homolog deleted on chromsome ten(PTEN) and its downstream signal molecules phosphorylated protein kinase B (p-AKT) in liver cells of rats during intermittent hypoxia,to investigate the effect of PTEN and p-AKT of liver cells on insulin resistance which intermittent hypoxia is relevant. MethodsA total of 24 healthy male SD rats were selected and divided into 3 groups randomly,ie.CIA (chronic intermittent air) group,CIH4 (chronic intermittent hypoxia for 4 weeks) group,and CIH8 (chronic intermittent hypoxia for 8 weeks) group. The fasting blood glucose,fasting insulin,PTEN and p-AKT expressions in the liver cells were detected. The insulin resistance was evaluated systematically by the insulin sensitive index (ISI) and homeostasis model assessment of insulin resistance (HOMA-IR). Average gray value was used to represent the protein expressions of PTEN and p-AKT. ResultsCompared with CIA group,the decline of ISI in CIH4 group and CIH8 group was significant (P<0.05). Furthermore,the decline in CIH8 group was more significant than that in CIH4 group (P<0.05). Compared with CIA group,the rise of HOMA-IR in CIH4 and CIH8 groups was statistically significant (P<0.05). In addition,the rise in CIH8 group was more significant than that in CIH4 group (P<0.05). Compared with CIA group,there was a significant rise in the protein expressions of PTEN in CIH4 and CIH8 groups (P<0.05). Compared with CIH4 group,the rise of the protein expressions of PTEN in CIH8 group was still statistically significant (P<0.05). Compared with CIA group,there was a significant decline in the protein expressions of p-AKT in CIH4 and CIH8 groups (P<0.05). Compared with CIH4 group,the decline of protein expression of p-AKT in CIH8 group was still of statistical significance (P<0.05). There was a significantly increasing trend for the expression of PTEN in the liver cells of rats with intermittent hypoxia along with the decline of ISI and rise of HOMA-IR. The expression increased significantly with the longer duration of intermittent hypoxia. The expression of p-AKT in liver cells of rats with intermittent hypoxia decreased along with the decline of ISI and rise of HOMA-IR. Furthermore,the decline tendency was more significant with the long duration of intermittent hypoxia. ConclusionThe fasting blood glucose of rats and insulin level increase due to the chronic intermittent hypoxia,resulting in the insulin resistance. The degree of insulin resistance increases with the longer duration of intermittent hypoxia. The expression of PTEN protein increases with intermittent hypoxia,and that of p-AKT protein decreases,which is obviously correlated with ISI and HOMA-IR. It is indicated that the PTEN protein possibly play an important role in the mechanism of insulin resistance for rats with intermittent hypoxia.
ObjectiveTo analyze the effects of miR-451a on the proliferation and apoptosis of human pancreatic cancer BxPc3 cells, and to explore its molecular mechanisms.MethodsThe liposome transfection mimics of miR-451a were established in the BxPc3 cells, which were used as the research objects, and different concentrations (25, 50, 100 and 200 μmol/L) of miR-451a and blank control group were set up respectively. The expression of miR-451a mRNA in the BxPc3 cells after the transfection was detected by the qRT-PCR method. The effects of miR-451a at different concentrations on the proliferation, cell clone number, cell cycle and apoptosis, and the expressions of the macrophage migration inhibitory factor (MIF), calcium binding protein 39 (CAB39), phosphorylated phosphatidylinositol-3-kinase (p-PI3K) and phosphorylated protein kinase B (p-AKT) proteins in the BxPc3 cells were detected by the MTT assay, plate cloning assay, flow cytometry, and Western blot, respectively.ResultsThe expressions of miR-451a mRNA in the transfected BxPc3 cells were significantly higher than in the blank control BxPc3 cells (P<0.050). The miR-451a could inhibit the proliferation of BxPc3 cells in a time- and concentration-dependent manner significantly (P<0.050), block the differentiation of BxPc3 cells in the G0/G1 phase, and induce the apoptosis with a concentration-dependent manner (P<0.050). The expressions of MIF, CAB39, p-PI3K, and p-AKT proteins in the BxPc3 cells were down-regulated with a concentration-dependent manner (P<0.050).ConclusionFrom results of this study, miR-451a could inhibit proliferation and induce apoptosis of BxPc3 cells in a concentration-dependent manner, and its mechanisms might be related to inhibition of PI3K/AKT signaling pathway.
Objective To study the function of platelet-derived growth factor (PDGF) in inducing phosphorylation extracellular signalregulated kinase 1/2 (pERK1/2) localization in osteoblasts. Methods Primary osteoblasts were isolated and cultured from cranial bone of 10 mice atthe age of 3 days, weighting 6-9 g without limitation in male and female.The sixth passage osteoblasts were incubated in 1% serum for 12 hours and divided into 2 groups: treated with DMSO(control group) or with PP2(experimentalgroup) for 30 minutes. Each group was further divided into 2 subgroups according to with or without PDGF (20ng/ml) stimulation for 10 minutes. pERK1/2 localization was analysized by immunofluorescence staining in osteoblasts pretreated with or without Src inhibitor PP2. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or with PP2 (experimental group) for 30 minutes. Each group was further divided into two subgroupsaccording to with or without PDGF (20 ng/ml) stimulation for 10 mintues. The ability of osteoblast migration was determined by wound healing assay. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or 10 μmol/L PP2 (experimental group) for 30 mintues. Each group was further divided into 2 subgroups according to with or without PDGF (20 ng/ml) stimulation.The pERK1/2 was determined by Western blot in osteoblastic cytoskeleton inducedby PDGF. Results Immunofluorescence staining showed pERK1/2 localization in osteoblastic nuclears and focal adhesions after PDGF stimulation. PP2 significantly inhibited ERK1/2 localization in focal adhesions, but not in nuclears. The wound healing assay results showed that PP2 significantly inhibited osteoblast migration induced by PDGF. The result of Western blot demonstrated that pERK1/2 in osteoblastic cytoskeleton was significantly inhibitedSrc activation is required for pERK1/2 translocalization to focal adhesions and osteoblasts migration.
Objective To investigate the changes of autophagy after spinal cord injury (SCI) in rats and its relationship with multisite phosphorylation of B-cell lymphoma-2 (Bcl-2) protein. Methods Forty male Sprague-Dawley rats aged 8 weeks were used to prepare SCI models by modified Allen method, and the SCI model were prepared successfully in 36 rats. The 36 SCI models were randomly divided into SCI group, autophagy inhibitor group, and autophagy promoter group, with 12 rats in each group. Another 12 rats were selected as sham operation group with only laminectomy and no spinal cord injury. At the end of modeling, the autophagy inhibitor group and the autophagy promoter group were intrathecally injected with 20 μL of 600 nmol/L 3-methyladenine and 25 nmol/L rapamycin, respectively, once a day for 4 weeks. The sham operation group and the SCI group were injected with only 20 μL of normal saline at the same time point. The motor function of rat in each group was evaluated by the Basso-Beattie-Bresnahan (BBB) score at 1 day and 1, 2, 4 weeks after modeling. The rats in each group were sacrificed at 24 hours after the last injection and the spinal cord tissues were taken. ELISA assay was used to detect the levels of inflammatory factors in spinal cord tissues, including myeloperoxidase (MPO), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β); the morphological changes of spinal cord were observed by HE staining; the autophagy of mitochondria in spinal cord tissues was observed by transmission electron microscopy; the expressions of Beclin1 and microtubule-associated protein light chain 3 (LC3) were detected by immunofluorescence staining; neuronal apoptosis in spinal cord tissues were observed by TUNEL staining; LC3/TUNEL positive cells were calculated by immunofluorescence double staining; the expressions of Bcl-2 associated X protein (Bax), Bcl-2, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) were detected by Western blot. Results Compared with sham operation group, BBB score of SCI group decreased at each time point, while the levels of MPO, TNF-α, and IL-1β increased; peripheral space of nerve cells enlarged, cells swelled, vacuoles appeared, and autophagic bodies appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins, and apoptotic rate of neurons significantly increased; the LC3/TUNEL positive cells significantly increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased; all showing significant differences (P<0.05). Compared with SCI group, BBB score in autophagy inhibitor group decreased at each time point, while the levels of MPO, TNF-α, and IL-1β increased; a few autophagic vesicles appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins decreased and the apoptotic rate of neurons increased significantly; the LC3 positive cells decreased and the TUNEL positive cells increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased. The results of autophagy promoter group were opposite to those of autophagy inhibitor group; all showing significant differences between groups (P<0.05). Conclusion Induction of autophagy after SCI in rats can reduce neuronal apoptosis and protect spinal cord function, which may be related to the inhibition of Bcl-2 protein multisite phosphorylation.
ObjectiveTo investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment. MethodsThe bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin. ResultsMTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P > 0.05), indicating no effect of 1×10-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P < 0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P < 0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P < 0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P < 0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P < 0.05). ConclusionSimvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.