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1.
目的:探讨活化转录因子 6(ATF6)对骨肉瘤细胞 MCA205 免疫原性的影响,初步解析其调控机制。方法:利用CRISPR-Cas9技术敲除MCA205细胞的Atf6基因,借助CCK-8实验、细胞能量代谢检测、流式细胞术、ATP检测试剂盒、干扰素刺激响应元件(ISRE)-荧光素酶报告细胞实验和qPCR法分别检测野生型(WT)和Atf6-/- MCA205细胞在PBS或衣霉素(Tm)处理后的活力、线粒体耗氧速率(OCR)和胞外酸化速率(ECAR)、磷脂酰丝氨酸外翻和细胞膜通透性、胞内钙离子动员、胞内外ATP浓度、IFN-a/b分泌和干扰素刺激基因(ISG)的表达水平。在免疫系统健全的小鼠皮下接种WT或Atf6-/- MCA205细胞,比较两者的成瘤速度、肿瘤组织基因转录图谱、局部抗肿瘤效应T细胞活化有无差异。将WT和Atf6-/- MCA205细胞分别接种于nu/nu小鼠背部两侧皮下,或将Atf6-/- MCA205细胞分别接种于免疫系统健全小鼠和Ifnar-/-小鼠皮下,记录肿瘤生长曲线。分别用Tm预处理的WT和Atf6-/- MCA205细胞对na?ve小鼠(未经免疫刺激的小鼠)进行初次免疫,使肿瘤抗原特异性T细胞发生初次活化(prime),随后收集引流淋巴结细胞并进行体外再刺激(boost),分析特异性T细胞再次活化有无差异。按照不同的效靶比,将NK细胞与染料标记的WT和Atf6-/- MCA205细胞共培养,流式细胞术检测细胞杀伤情况。结果:PBS或Tm处理后,WT和Atf6-/-骨肉瘤细胞活力和增殖、氧化磷酸化和糖酵解、离子霉素触发的胞内钙离子动员、胞内外ATP和IFN-a/b分泌均无显著差异。Tm处理后,Atf6-/-细胞死亡比例低于WT细胞(P<0.01)。在免疫系统健全的小鼠体内,Atf6-/-肿瘤的生长速度明显低于WT肿瘤(P<0.05)。然而,在缺乏T细胞的nu/nu小鼠体内,两种肿瘤生长速度的差异显著缩小。与免疫系统健全的小鼠相比,Ifnar-/-小鼠体内Atf6-/-肿瘤的生长速度略快(P<0.05)。Atf6-/-肿瘤内免疫应答相关基因的表达、效应T细胞的活化均显著高于WT肿瘤(P<0.05)。与WT MCA205细胞相比,Atf6-/- MCA205细胞与NK细胞共培养后死亡比例更高(P<0.05)。在Tm刺激后,Atf6-/- MCA205细胞比WT细胞表达更多的Irf3和Irf7,前者在prime-boost实验中可刺激T细胞分泌更多的IFN-g。结论:阻断ATF6信号通路能显著增强MCA205细胞的免疫原性,促进免疫监视,阻碍肿瘤进展。  相似文献   
2.
Conventional dendritic cells (cDCs) are specialized in antigen presentation. In the mouse spleen, cDCs are classified in cDC1s and cDC2s, and express DEC205 and DCIR2 endocytic receptors, respectively. Monoclonal antibodies (mAbs) αDEC205 (αDEC) and αDCIR2 have been fused to different antigens to deliver them to cDC1s or cDC2s. We immunized mice with αDEC and αDCIR2 fused to an antigen using Poly(I:C) as adjuvant. The initial immune response was analyzed from days 3 to 6 after the immunization. We also studied the influence of a booster dose. Our results showed that antigen targeting to cDC1s promoted a pro-inflammatory TH1 cell response. Antigen targeting to cDC2s induced TFH cells, GCs, and plasma cell differentiation. After boost, antigen targeting to cDC1s improved the TH1 cell response and induced TH1-like TFH cells that led to an increase in specific antibody titers and IgG class switch. Additionally, a population of regulatory T cells was also observed. Antigen targeting to cDC2s did not improve the specific antibody response after boost. Our results add new information on the immune response induced after the administration of a booster dose with αDEC and αDCIR2 fusion mAbs. These results may be useful for vaccine design using recombinant mAbs.  相似文献   
3.
《Vaccine》2019,37(35):4963-4974
Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms.In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8+ memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited.Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches.  相似文献   
4.
BackgroundIndividuals with HIV have ~2-fold increased risk of developing pulmonary fibrosis. The mechanism(s) by which this occurs has yet to be determined. HIV-1 protein gp120 activates CXCR4 in the lymphocyte, promoting a variety of intracellular signaling pathways including those common to TGFβ1 associated with lung fibroblast-to-myofibroblast transdifferentiation. We hypothesized that gp120 promotes pulmonary fibrotic changes via activation of CXCR4 in the lung fibroblast.MethodsMouse primary lung fibroblasts (PLFs) were cultured ± gp120, then analyzed for α-SMA expression and stress fiber formation. In parallel, PLFs were cultured ± gp120 ± AMD3100 (a CXCR4 antagonist), and α-SMA, pan and phospho-Akt, and total and phospho-MAPK (or ERK1/2) protein expression was quantified. Finally, lungs and PLFs from wild-type and HIV-1 transgenic mice were analyzed for hydroxyproline and α-SMA content.Resultsgp120 treatment increased α-SMA expression and myofibroblast differentiation in PLFs. gp120 treatment activated phosphorylation of ERK1/2, but not PI3K-Akt. Pretreatment with AMD3100 inhibited gp120-induced ERK1/2 phosphorylation and gp120-induced α-SMA expression. In parallel, there was a significant increase in hydroxyproline content in lungs from older HIV-1 transgenic mice and a >3-fold increase in α-SMA expression in PLFs isolated from HIV-1 transgenic mice.Conclusionsgp120 induces α-SMA expression and fibroblast-to-myofibroblast transdifferentiation by activating the CXCR4-ERK1/2 signaling pathway in mouse PLFs. Lungs of older HIV-1 transgenic mice contain higher hydroxyproline content and their PLFs have a striking increase in α-SMA expression. These results suggest a mechanism by which individuals with HIV are at increased risk of developing pulmonary fibrotic changes as they age.  相似文献   
5.

Background

Robot-assisted thoracoscopic lobectomy has been shown to be a safe approach to pulmonary lobectomy. This study sought to define, mathematically, the learning curve for RATS lobectomy.

Methods

Patients undergoing robot-assisted thoracoscopic lobectomy at a single institution from 2010 through 2016 were considered. Covariates included patient demographics, comorbidities, operating time, length of stay, estimated blood loss, and postoperative complications. A cumulative sum analysis of operating time was performed to define the learning curve.

Results

A total of 101 patients were included. Three distinct phases of the learning curve were identified: cases 1–22, cases 23–63, and cases 64–101. There was a statistically significant difference in operating time and estimated blood loss between phases 1 and 2 (P < .05, P?=?.016, respectively) and between phases 1 and 3 (P < .05, P?=?.006, respectively). There was no statistically significant difference in comorbidities, chest tube duration, length of stay, postoperative complications, or conversion rate across the learning curve.

Conclusion

Based on operating time, the learning curve for robot-assisted thoracoscopic lobectomy is 22 cases, with mastery achieved after 63 cases. No differences in length of stay, chest tube duration, conversion rate, or complication rate were observed in the learning curve. Other factors not measured in this study may play a role in the learning process and warrant further study.  相似文献   
6.
BackgroundPrimary brain tumors are relatively rare malignancy, with high-grade gliomas (glioblastoma multiforme and anaplastic gliomas) are the most common types. We aimed to evaluate the prognostic value of Prognostic Nutritional Index (PNI), which is calculated by lymphocyte count and albumin, in recurrent glioblastoma patients treated with systemic treatment.MethodsData of 64 patients with recurrent glioblastoma who received systemic treatment and followed in our clinic between 2012 and 2018 was retrospectively collected and analyzed. PNI was calculated as: [(10 × serum albumin (g/dL)) + (0.005 × total lymphocyte count)]. Patients were categorized according to the median PNI value. We investigated the prognostic role of PNI groups, and survival outcomes.ResultsMedian value of PNI was 45.7, and median follow-up duration was 9 months (1–68 months). Median overall survival (OS) was 7.9 months (95%CI: 5.5–10.4). Median OS was significantly longer in patients with PNI > 45.7 compared to patients with PNI  45.7 (13.9 months (95%CI: 10.5–17.4), and 4.6 months (95%CI: 2.5–6.8), p < 0.001, respectively). In multivariate analysis, PNI was found to be an independent prognostic factor for OS [HR:0.41 (95%CI:0.22–0.74), p = 0.03)].ConclusionIn our study, the PNI was found to be an independent prognostic biomarker in patients with recurrent glioblastoma, but further prospective trials are necessary to validate its prognostic role.  相似文献   
7.
ObjectiveThe aim of this study was to investigate the relation between health-related physical fitness and weight status in 13- to 15-year-old Latino adolescents.MethodThe final sample consisted of 73,561 adolescents aged 13–15 years (35,175 girls) from Chile (n = 48,771) and Colombia (n = 24,790). Cardiorespiratory and musculoskeletal fitness were measured using 20-m shuttle run (relative peak oxygen uptake – VO2peak) and standing broad jump test (lower body explosive strength), respectively. The International Obesity Task Force definition was used to define weight status (i.e., underweight, normal weight, overweight, and obese).ResultsThe present study found an inverted J-shape relationship between body mass index, cardiorespiratory fitness, and musculoskeletal fitness in both genders and all age groups (p < 0.01). Results also suggest that underweight adolescents, and not just overweight and obese adolescents, have lower odds of having a healthy cardiorespiratory fitness (based on new international criterion-referenced standards) profile when compared with their normal weight peers, except in girls aged 14 (p = 0.268) and 15 years (p = 0.280).ConclusionsThe present results indicate low cardiorespiratory fitness and musculoskeletal fitness levels in underweight, overweight, and obese adolescents when compared with their normal weight peers. The findings appear to suggest that exercise programs should to decrease fat mass in overweight/obese adolescents and increase muscle mass in underweight adolescents.  相似文献   
8.
目的 研究子宫内膜癌组织中微小RNA(microRNA miR-205-5p)、程序性细胞坏死因子5(programmed cell necrosis factor 5,PDCD5)、Beclin1的表达情况及三者之间的相关性。 方法 选择75例子宫内膜癌组织和距离癌组织边缘5 cm癌旁组织。通过免疫组织化学、实时荧光定量法检测miR-205-5p、PDCD5、Beclin1的表达情况,分析miR-205-5p、PDCD5、Beclin1的相关性及对子宫内膜癌的诊断价值。 结果 癌组织中miR-205-5p表达高于癌旁组织,PDCD5、Beclin1表达低于癌旁组织(P<0.05)。不同组织学分级、临床分期、淋巴结转移、肌层浸润子宫内膜癌患者miR-205-5p、PDCD5、Beclin1表达差异有统计学意义(P<0.05),Ⅲ~Ⅳ期、中低分化、有淋巴结转移、≥1/2肌层浸润子宫内膜癌患者miR-205-5p表达升高,PDCD5、Beclin1表达降低,差异有统计学意义(P<0.05)。不同年龄、病理类型子宫内膜癌患者miR-205-5p、PDCD5、Beclin1表达差异无统计学意义(P>0.05)。miR-205-5p、PDCD5之间呈负相关,miR-205-5p、Beclin1之间呈负相关,PDCD5、Beclin1之间呈正相关(P<0.05)。三项联合诊断子宫内膜癌的价值高于miR-205-5p、PDCD5、Beclin1单项诊断(P<0.05)。 结论 miR-205-5p表达升高,PDCD5、Beclin1表达降低对子宫内膜癌的发展起到了促进作用,参与子宫内膜癌的演变进程,临床可根据上述指标对子宫内膜癌进行预测。  相似文献   
9.
Introduction and ObjectivesMyocardial performance may be impaired in cytokine-mediated immune reactions. The myocardial performance index (MPI) is a practical parameter that reflects systolic and diastolic cardiac function. We aimed to assess the MPI in patients with COVID-19.MethodsThe study population consisted of 40 healthy controls and 40 patients diagnosed with COVID-19 who had mild pneumonia and did not need intensive care treatment. All participants underwent echocardiographic examination. First, the MPI and laboratory parameters were compared between healthy controls and patients in the acute period of infection. Second, the MPI and laboratory parameters were compared between the acute infection period and after clinical recovery.ResultsCompared with healthy controls, patients with COVID-19 had a significantly higher MPI (0.56±0.09 vs. 0.41±0.06, p<0.001), longer isovolumic relaxation time (IRT) (112.3±13.4 vs. 90.6±11.2 ms, p<0.001), longer deceleration time (DT) (182.1±30.6 vs. 160.8±42.7 ms, p=0.003), shorter ejection time (ET) (279.6±20.3 vs. 299.6±34.7 ms, p<0.001) and higher E/A ratio (1.53±0.7 vs. 1.21±0.3, p<0.001). Statistically significantly higher MPI (0.56±0.09 vs. 0.44±0.07, p<0.001), longer IRT (112.3±13.4 vs. 91.8±12.1 ms, p<0.001), longer DT (182.1±30.6 vs. 161.5±43.5 ms, p=0.003), shorter ET 279.6±20.3 vs. 298.8±36.8 ms, p<0.001) and higher E/A ratio (1.53±0.7 vs. 1.22±0.4, p<0.001) were observed during the acute infection period than after clinical recovery. Left ventricular ejection fraction was similar in the controls, during the acute infection period and after clinical recovery.ConclusionsSubclinical diastolic impairment without systolic involvement may be observed in patients with COVID-19. This impairment may be reversible on clinical recovery.  相似文献   
10.
《Pancreatology》2020,20(6):1139-1148
Background/aimsStudies have found that LncRNA CYTOR is an important regulator of cancer. However, the function of lncRNA CYTOR in pancreatic cancer (PC) is unclear. This study amid to explore the regulation of lncRNA CYTOR in PC.MethodsThe expression of CYTOR and miR-205-5p in PC was detected by RT-qPCR. CCK-8 assay, colony formation assay and scratch test were conducted to detect the effects of CYTOR and miR-205-5p on proliferation and migration of PC cells. Target gene prediction and screening and luciferase reporter assays were used to verify downstream target genes of CYTOR and miR-205-5p. The expression of Cyclin-dependent protein kinase 6 (CDK6) was detected by Western blotting. The tumor growth in mice was detected by in vivo experiments in nude mice.ResultsThe expression of LncRNA CYTOR was significantly elevated in PC. Knockdown of CYTOR significantly inhibited cell proliferation and migration of PC cells. In vivo animal studies showed that CYTOR promoted tumor growth. MiR-205-5p was a direct target of CYTOR, and the expression levels of miR-205-5p were significantly reduced in PC cell lines. Furthermore, co-transfection of shCYTOR with miR-205-5p inhibitor partially abolished the effect of shCYTOR on cell proliferation and migration. In addition, CYTOR was negatively correlated with the expression of miR-205-5p. CDK6 was a direct target of miR-205-5p, and miR-205-5p mimic and sh CYTOR significantly reduced the expression levels of CDK6.ConclusionCYTOR can promote PC progression by modulating the miR-205-5p/CDK6 axis, which may be a potential therapeutic target for PC.  相似文献   
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