Inhibition of Cyclin F Promotes Cellular Senescence through Cyclin-dependent Kinase 1-mediated Cell Cycle Regulation

Objective Kidney renal clear cell carcinoma(KIRC)is a common renal malignancy that has a poor prognosis.As a member of the F box family,cyclin F(CCNF)plays an important regulatory role in normal tissues and tumors.However,the underlying mechanism by which CCNF promotes KIRC proliferation still remains unclear.Methods Bioinformatics methods were used to analyze The Cancer Genome Atlas(TCGA)database to obtain gene expression and clinical prognosis data.The CCK8 assay,EdU assay,and xenograft assay were used to detect cell proliferation.The cell senescence and potential mechanism were assessed by SA-β-gal staining,Western blotting,as well as ELISA.Results Our data showed that CCNF was highly expressed in KIRC patients.Meanwhile,downregulation of CCNF inhibited cell proliferation in vivo and in vitro.Further studies showed that the reduction of CCNF promoted cell senescence by decreasing cyclin-dependent kinase 1(CDK1),increasing the proinflammatory factors interleukin(IL)-6 and IL-8,and then enhancing the expression of p21 and p53.Conclusion We propose that the high expression of CCNF in KIRC may play a key role in tumorigenesis by regulating cell senescence.Therefore,CCNF shows promise as a new biomarker to predict the clinical prognosis of KIRC patients and as an effective therapeutic target.

Expression of cyclin-dependent kinase inhibitor 2A 16,tumour protein 53 and epidermal growth factor receptor in salivary gland carcinomas is not associated with oncogenic virus infection

It is known that human papillomavirus （HPV） infection can cause squamous cell neoplasms at several sites, such as cervix uteri carcinoma and oral squamous carcinoma. There is little information on the expression of HPV and its predictive markers in tumours of the major and minor salivary glands of the head and neck. We therefore assessed oral salivary gland neoplasms to identify associations between HPV and infection-related epidermal growth factor receptor （EGFR）, cyclin-dependent kinase inhibitor 2A （CDKN2A/p16） and tumour protein p53 （TP53）. Formalin-fixed, paraffin-embedded tissue samples from oral salivary gland carcinomas （n=51） and benign tumours （n=26） were analysed by polymerase chain reaction （PCR） analysis for several HPV species, including high-risk types 16 and 18. Evaluation of EGFR, CDKN2A, TP53 and cytomegalovirus （CMV） was performed by immunohistochemistry. Epstein-Barr virus （EBV） was evaluated by EBV-encoded RNA in situ hybridisation. We demonstrated that salivary gland tumours are not associated with HPV infection. The expression of EGFR, CDKN2A and TP53 may be associated with tumour pathology but is not induced by HPV. CMV and EBV were not detectable. In contrast to oral squamous cell carcinomas, HPV, CMV and EBV infections are not associated with malignant or benign neoplastic lesions of the salivary glands.

Expression of cyclin-dependent protein kinase 5 in the hippocampus of vascular dementia mice after cerebral ischemia and reperfusion

BACKGROUND： The p25-activated cyclin-dependent protein kinase 5 （Cdk5） may induce neuronal cell death and cause the development of dementia following cerebral ischemia and reperfusion. OBJECTIVE： To observe changes in the expression of Cdk5 and p25 in hippocampal tissue of vascular dementia mice at different time points following cerebral ischemia and reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed in the clinical trial center of Hebei Provincial People＇s Hospital between September 2007 and October 2008. MATERIALS： Cdk5 rabbit anti-mouse polyclonal antibody, p35 rabbit anti-mouse polyclonal antibody, and β-actin mouse monoclonal antibody were purchased from Santa Cruz Biotechnology, Inc., USA; horseradish peroxidase-labeled goat anti-rabbit IgG and horseradish peroxidase-labeled goat anti-mice IgG were offered by Beijing Zhongshan Geldenbridye Biotechnology Co.,Ltd., China; the protein quantitative kit was produced by Applygen Gene Technology Corp., Beijing, China; cDNA reverse transcription and PCR amplification reagents were products of TianGen＆ Biotech （Beijing） Co.,Ltd., China. METHODS： One hundred and sixty male Kunming mice were randomly divided into two groups： a sham-operated group （n = 65） and a model group （n = 95）. Vascular dementia was induced with three periods of transient ischemia and reperfusion of the bilateral common carotid arteries. In the sham-operated group, the bilateral common carotid arteries were not blocked. MAIN OUTCOME MEASURES： Behavioral tests were done at four and six weeks post surgery. Pathological changes in the hippocampal CA1 region were observed with hematoxylin-eosin staining Cdk5 mRNA expression was examined by RT-PCR, and Western blots were used to evaluate Cdk5 and p25 expression. Learning and memory performance were assayed using the Morris water maze. RESULTS： Vascular dementia reduced learning and memory performance at 4 and 6 weeks post surgery. Vascular dementia also caused severe, time-dependent neuronal damage and death in the hippocampal CA1 region. Dementia induction also increased mRNA and protein expression of Cdk5 and p25 at both 4 and 6 weeks after surgery. CONCLUSION： Cdk5/p25 is involved in the development of vascular dementia in mice following cerebral ischemia and reperfusion.

Cyclin-dependent kinase 5 is required for suppressing D1-dependent signaling mediated through muscarinic 4 in isolated medium spiny neurons

OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.

Can cyclin-dependent kinase 4/6 inhibitors convert inoperable breast cancer relapse to operability? A case report

BACKGROUND Pathological complete response(pCR) is rare in hormone receptor-positive(HR+)HER2-negative breast cancer(BC) treated with either endocrine therapy(ET) or chemotherapy. Radical resection of locoregional relapse, although potentially curative in some cases, is challenging when the tumor invades critical structures.The oral cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with ET has obtained a significant increase in objective response rates and progression-free survival in patients with advanced BC and is now being evaluated in the neoadjuvant setting. We present a clinical case of a patient with an inoperable locoregional relapse of HR+ HER2-negative BC who experienced p CR after treatment with palbociclib.CASE SUMMARY We report the clinical case of a 60-year-old patient who presented with an inoperable locoregional relapse of HR+, HER2-negative BC 10 years after the diagnosis of the primary tumor. During a routine follow-up visit, breast magnetic resonance imaging and positron emission tomography/computed tomography revealed a 4-cm lesion in the right subclavicular region, infiltrating the chest wall and extending to the subclavian vessels, but without bone or visceral involvement. Treatment was begun with palbociclib plus letrozole, converting the disease to operability over a period of 6 mo. Surgery was performed and a p CR achieved. Of note, during treatment the patient experienced a very uncommon toxicity characterized by burning tongue and glossodynia associated with dysgeusia, paresthesia, dysesthesia, and xerostomia. A reduction in the dose of palbociclib did not provide relief and treatment with the inhibitor was thus discontinued, resolving the tongue symptoms. Laboratory exams were unremarkable. Given that this was a late relapse, the tumor was classified asendocrine-sensitive, a condition associated with high sensitivity to palbociclib.CONCLUSION This case highlights the potential of the cyclin-dependent kinase 4/6 inhibitor plus ET combination to achieve pCR in locoregional relapse of BC, enabling surgical resection of a lesion initially considered inoperable.

Expression of cyclin-dependent kinases in HL-60 cells during differentiation induced by retinoic acid

This study was designed to investigate the relationship of the expression of cyclin-dependent kinases (CDKs) with theeffects of all-trans retinoic acid (ATRA) on the proliferation of HL-cells. HL-60 cells were treated with ATRA for 1-4 d. Then thecapacity of DNA Synthesis was evaluated with 3H-TdR incorporation and the expression of cyclin E, cyclin D, CDK2 and CDK4protein determined with immunocytochemical staining. In addition, the expression Of CDC2, CDK2 and CDK4 mRNA was deter-mined with in situ hybridization. It was found that ATRA suppressed the proliferation of HL-60 cells and decreased their capacityof DNA synthesis to result in a down-regulation of the expression of cyclin E, cyclin D and CDC2 without comcomittant suppressionon the expression of CDK2 and CDK4. It is concluded that the effects of ATRA on the proliferation of HL-60 cells may be relatedto the down-regulation of the expression of cyclin E, cyclin D and CDC2.

AR、SKP2、SOX10、PD-L1及TIL表达在三阴性乳腺癌中的意义

目的:探索雄激素受体(androgen receptor,AR)、S期激酶相关蛋白2(S-phase kinase-associated protein 2,SKP2)、性别决定区Y相关的HMG盒含因子10(sry-related HMG box-containing factor 10,SOX10)、程序性死亡配体1(programmed death-ligand 1,PD-L1)及肿瘤浸润性淋巴细胞(tumor infiltrating lymphocyte,TIL)在三阴性乳腺癌(triple negative breast cancer,TNBC)表达与临床病理特征和预后的关系。方法:根据苏木精-伊红染色(hematoxylineosin, HE)染色切片评判109例TNBC瘤巢内TIL的比例,采用Leica Bond-Max全自动免疫组化仪检测TNBC组织中AR、SKP2、SOX10、PD-L1的表达。分析以上各生物指标与临床病理特征间的关系,并采用kaplan-Meier、Log-rank进行生存分析。结果:95例患者获得随访,中位随访时间为48个月,中位无病生存时间(disease-free survival, DFS)为42个月,中位总生存时间(overall survival, OS)48个月。在TNBC中,AR阳性表达与淋巴结转移阴性(P=0.009)、肿瘤最大径<2 cm(P=0.008)相关,TIL高表达与低级别TNBC相关(P=0.007),SKP2阳性表达与神经/脉管侵犯阳性(P=0.011)、高级别TNBC相关(P=0.002),SOX10阳性表达与淋巴结转移阳性(P=0.022)、高级别TNBC(P=0.005)相关,PD-L1阳性表达与淋巴结转移阳性(P=0.020)、神经/脉管侵犯阳性(P=0.006)、高级别TNBC(P=0.042)相关。生存分析显示,SKP2、SOX10阳性表达与更差的DFS(P=0.007、P<0.001)和OS(P=0.013、P<0.001)相关,TIL高表达与更好的DFS(P=0.016)及OS(P=0.004)相关。在生物表志物的联合表达中,AR+/SKP2-、AR+/SOX10-与更好的DFS(P=0.004、P<0.001)及OS(P=0.007、P=0.001)相关,SOX10+/低TIL、PD-L1+/低TIL与更差的DFS(P<0.001、P=0.008)及OS(P=0.001、P=0.002)相关,AR-/低TIL者具有更差的OS(P=0.014)。SKP2(HR=4.143,95%CI为1.578~10.875)、SOX10(HR=7.578,95%CI为2.067~27.782)的阳性表达是影响TNBC患者DFS的独立预后因子,SKP2(HR=3.758,95%CI为1.400~10.084)、SOX10(HR=5.131,95%CI为1.316~20.000)及TIL(HR=0.375,95%CI为0.154~0.917)的阳性表达是TNBC患者OS的独立预后因子(P均<0.05)。结论:在TNBC中,AR阳性、TIL高表达与具有更好预后的临床病理特征相关,SKP2、SOX10和PD-L1与具侵袭性的临床病理特征相关。SKP2、SOX10及TIL表达与TNBC预后相关,提示这些生物指标可能成为TNBC新的预后因子,同时它们也有可能成为潜在的治疗靶点。

流行性出血热患者血清CK-MB、IL-6、IL-10水平与病情程度、预后的关系研究

目的:分析流行性出血热(EHF)患者血清肌酸激酶同工酶(CK-MB)、白细胞介素-6(IL-6)及白细胞介素-10(IL-10)水平与病情程度、预后的关系。方法:回顾性选择2019年6月—2022年6月于南昌市第九医院进行治疗的EHF患者60例作为本次研究对象,按照病情严重程度分为以下三组:轻症组(n=28,轻型、中型患者)、重症组(n=19,重型患者)及危重症组(n=13,危重型患者)。检测其入院后第1、3、7天的CK-MB、IL-6及IL-10水平,分析以上3项实验室检查指标与患者病情严重程度的相关性,并依据90 d预后情况分为存活组与死亡组,分析CK-MB、IL-6、IL-10与EHF患者预后的关系。结果:轻症组入院第1、3、7天血清CK-MB、IL-6、IL-10水平均低于重症组及危重症组(P<0.05),重症组以上指标水平均低于危重症组(P<0.05)。血清CK-MB、IL-6、IL-10水平与病情严重程度成正相关(rs=0.562、0.355、0.301,P<0.05)。60例患者随访90 d发现存活54例,死亡6例,存活组入院后第1、3、7天血清CK-MB、IL-6及IL-10水平均明显低于死亡组(P<0.05);受试者操作特征(ROC)曲线分析显示,入院第1天血清CK-MB、IL-6、IL-10单独及联合检测均对EHF患者预后有预测价值(P<0.05),其中联合检测的预测效能最高,其AUC为0.849,敏感度为98.14%,特异度为59.16%。结论:EHF患者血清CK-MB、IL-6及IL-10水平与病情发展呈正相关,可用于评估病情严重程度及预后,且三者联合时的效能最高。

Long non-coding RNA H19 promotes proliferation inhepatocellular carcinoma cells via H19/miR-107/CDK6 axis

Hepatocellular carcinoma (HCC) is the leading cause of cancer death worldwide;nevertheless, currenttherapeutic options are limited or ineffective for many patients. Therefore, elucidation of molecular mechanisms inHCC biology could yield important insights for the intervention of novel therapies. Recently, various studies havereported dysregulation of long non-coding RNAs (lncRNAs) in the initiation and progression of HCC, including H19;however, the biological function of H19 in HCC remains unclear. Here, we show that knockdown of H19 disruptedHCC cell growth, impaired the G1-to-S phase transition, and promoted apoptosis, while overexpression of H19yielded the opposite results. Screening for expression of cell cycle-related genes revealed a significant downregulationof CDK6 at both RNA and protein levels upon H19 suppression. Bioinformatic analysis of the H19 sequence and the3′ untranslated region (3′ UTR) of CDK6 transcripts showed several binding sites for microRNA-107 (miR-107), andthe dual luciferase reporter assay confirmed their direct interaction with miR-107. Consistently, blockage of miR-107activity alleviated the growth suppression phenotypes induced by H19 downregulation, suggesting that H19 serves asa molecular sponge for miR-107 to promote CDK6 expression and cell cycle progression. Together, this studydemonstrates a mechanistic function of H19 in driving the proliferation of HCC cells and suggests H19 suppressionas a novel approach for HCC treatment.

CXC趋化因子配体10对肝细胞癌SMMC-7721细胞增殖和迁移的影响及其机制

目的:探讨外源性CXC趋化因子配体10 (CXCL10)对肝细胞癌(HCC) SMMC-7721细胞增殖和迁移的影响,并阐明其作用机制。方法:按照CXCL10作用浓度,将人HCC SMMC-7721细胞分为0 mg·L^(-1)CXCL10组、10 mg·L^(-1)CXCL10组和30 mg·L^(-1)CXCL10组。上述部分细胞给予细胞外调节蛋白激酶(ERK)抑制剂PD98059 (80μmol·L^(-1))后,将SMMC-7721细胞分为0 mg·L^(-1)CXCL10+PD98059组、 10mg·L^(-1)CXCL10+PD98059组和30mg·L^(-1)CXCL10+PD98059组。CCK-8法检测各组SMMC-7721细胞增殖率,EdU法检测各组SMMC-7721细胞中EdU阳性表达率,Transwell小室实验检测各组SMMC-7721细胞迁移率,Western blotting法检测各组SMMC-7721细胞中ERK、磷酸化ERK (p-ERK)和细胞周期蛋白D1 (Cyclin D1)蛋白表达水平。结果:CCK-8法检测,培养24h后,与0mg·L^(-1)CXCL10组比较,10mg·L^(-1)CXCL10和30mg·L^(-1)CXCL10组SMMC-7721细胞增殖率升高(P<0.05或P<0.01)。EdU法检测,与0mg·L^(-1)CXCL10组比较,10mg·L^(-1)CXCL10和30mg·L^(-1)CXCL10组SMMC-7721细胞中EdU阳性表达率升高(P<0.01);Transwell小室实验检测,培养48 h后,与0 mg·L^(-1)CXCL10组比较,10 mg·L^(-1)CXCL10和30 mg·L^(-1)CXCL10组SMMC-7721细胞迁移率升高(P<0.01)。Western blotting法检测,细胞培养24 h后,采用CXCL10溶液处理24h,与0mg·L^(-1)CXCL10组比较,10mg·L^(-1)CXCL10组和30 mg·L^(-1)CXCL10组SMMC-7721细胞中ERK、p-ERK及Cyclin D1蛋白表达水平升高(P<0.01)。CCK-8法检测,加入ERK抑制剂PD98059后,与0 mg·L^(-1)CXCL10组比较,10 mg·L^(-1)CXCL10+PD98059组和30 mg·L^(-1)CXCL10+PD98059组SMMC-7721细胞增殖率降低(P<0.05);与10 mg·L^(-1)CXCL10组比较,10 mg·L^(-1)CXCL10+PD98059组SMMC-7721细胞增殖率降低(P<0.05);与30 mg·L^(-1)CXCL10组比较,30 mg·L^(-1)CXCL10+PD98059组SMMC-7721细胞增殖率降低(P<0.05)。结论:CXCL10能够促进HCCSMMC-7721细胞增殖和迁移,其作用机制与上调ERK/p-ERK/Cyclin D1通路蛋白表达有关。

微RNA-132-3p靶向第10号染色体缺失的磷酸酶和张力蛋白同源物调控葡萄膜黑色素瘤细胞增殖与凋亡

目的 研究微RNA(miR)-132-3p在葡萄膜黑色素瘤细胞增殖、凋亡中的作用及其作用机制。方法 该研究起止时间为2018年2月至2019年7月。定量聚合酶链反应(qPCR)检测正常葡萄膜上皮细胞ARPE-19和葡萄膜黑色素瘤细胞SP6.5、M23中miR-132-3p表达。SP6.5细胞中转染miR-132-3p干扰质粒(anti-miR-132-3p)、第10号染色体上缺失的磷酸酶和张力蛋白同源物(PTEN)过表达质粒(pcDNA3.1-PTEN)或共转染anti-miR-132-3p和PTEN干扰质粒(si-PTEN),MTT法和流式细胞术分别检测细胞增殖与凋亡,蛋白质印迹法检测PTEN、细胞周期蛋白D1(cyclin D1)、周期素依赖激酶抑制剂p21(P21)、B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达,生物信息学预测结合双萤光素酶报告实验分析miR-132-3p与PTEN的靶向关系。结果 与ARPE-19细胞相比,SP6.5、M23细胞中miR-132-3p表达量(0.26±0.02比0.94±0.09、0.81±0.08)明显升高(P<0.05)。与anti-miR-132-3p阴性对照(anti-miR-NC)组相比,anti-miR-132-3p组24 h、48 h、72 h的细胞活性(0.51±0.05比0.30±0.03、0.97±0.09比0.45±0.05、1.40±0.14比0.76±0.07)、cyclin D1、Bcl-2蛋白表达量显著降低(P<0.05),细胞凋亡率[(8.03±0.68)%比(21.51±2.06)%]、P21、Bax水平明显提高(P<0.05),与过表达PTEN相同。miR-132-3p与PTEN之间有靶向调控关系。抑制PTEN能逆转抑制miR-132-3p对SP6.5细胞增殖的抑制作用及对细胞凋亡的促进作用。结论 miR-132-3p通过直接靶向PTEN调控葡萄膜黑色素瘤细胞增殖与凋亡。

Novel insights into D-Pinitol based therapies:a link between tau hyperphosphorylation and insulin resistance

Alzheimer’s disease is a neurodegenerative disorder characterized by the amyloid accumulation in the brains of patients with Alzheimer’s disease.The pathogenesis of Alzheimer’s disease is mainly mediated by the phosphorylation and aggregation of tau protein.Among the multiple causes of tau hyperphosphorylation,brain insulin resistance has generated much attention,and inositols as insulin sensitizers,are currently considered candidates for drug development.The present narrative review revises the interactions between these three elements:Alzheimer’s disease-tau-inositols,which can eventually identify targets for new disease modifiers capable of bringing hope to the millions of people affected by this devastating disease.

白介素-10抑制TNF-α诱导的血管平滑肌细胞增殖

研究观察了重组人白介素 10 (rhIL 10 )对肿瘤坏死因子 (TNF α)刺激的离体大鼠胸主动脉血管平滑肌细胞增殖、细胞周期及对p4 4 /p4 2丝裂素活化蛋白激酶的影响。实验培养大鼠主动脉血管平滑肌细胞 ,采用MTS/PES法确定血管平滑肌细胞 (vascularsmoothmusclecells,VSMCs)的增殖状态 ;应用流式细胞术测定细胞周期 ;利用p4 4 / 4 2磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白表达。结果显示 :( 1)TNF α处理组与对照组相比 ,TNF α对VSMC增殖具有明显的刺激作用 (P <0 0 5 )。rhIL 10单独应用对VSMCs生长没有影响 (P >0 0 5 )。在TNF α刺激下 ,低至 10ng/ml的rhIL 10可抑制VSMCs的生长 (P <0 0 5 )。流式细胞术测定的结果显示 ,rhIL 10分别可使TNF α作用下的VSMC大部分处于G0 /G1期 ,与对照组相比有明显差异 (P <0 0 1)。 ( 2 )TNF α对p4 4 /p4 2MAPK蛋白表达有显著的增强作用 ,此作用可被rhIL 10抑制。结果提示 ,rhIL 10可抑制TNF α诱导的VSMC增殖及p4 4 /p4

乳鼠心肌细胞中C反应蛋白对基质金属蛋白酶-10表达调控的研究

目的:研究在乳鼠心肌细胞中C反应蛋白诱导基质金属蛋白酶-10(MMP-10)表达的作用及其分子机制。方法:培养乳鼠心肌细胞,以炎性因子C反应蛋白诱导MMP-10的表达。分别应用酶谱法和实时定量反转录—聚合酶链反应检测培养上清中MMP-10的活性和细胞中MMP-10信使核糖核酸(mRNA)表达水平的变化,应用蛋白免疫印迹杂交技术观察胞外调节激酶(ERK)通路以及转录因子活化蛋白-1的蛋白水平的变化。实验分为4组:对照组,C反应蛋白组,C反应蛋白+抑制剂组和抑制剂组。结果:心肌细胞给予C反应蛋白(5μg/ml)刺激后24 h,细胞培养上清中MMP-10活性较对照组升高了9.23倍(P<0.01);细胞MMP-10 mRNA水平与0 h相比,6 h增加了0.83倍(P<0.05),12 h达最高,增加了2.67倍(P<0.05),24 h略有下降,增加了1.92倍(P<0.05),差异均有统计学意义。C反应蛋白可激活心肌细胞p-ERK1和p-ERK2,从15 min开始,可持续到240 min;C反应蛋白刺激细胞4 h,活化蛋白-1的蛋白水平与0 h相比,增加了3.66倍(P<0.01),差异有统计学意义;ERK抑制剂PD98059预处理心肌细胞,可阻断C反应蛋白引起的活化蛋白-1的蛋白水平的上调作用以及MMP-10活性的增加及MMP-10 mRNA的表达。结论:C反应蛋白通过ERK通路,上调转录因子活化蛋白-1的蛋白水平,从而对心肌细胞中MMP-10基因的转录和表达进行调节。

丝裂原活化蛋白激酶10及整合素α6预测声门上型喉癌对TPF诱导化疗方案的敏感性

目的预测丝裂原活化蛋白激酶(mitogenactivated protein kinase10,MAPK10)及整合素α6(Integrinα6)在声门上型喉癌中对紫杉醇+顺铂+5氟尿嘧啶(TPF)诱导化疗方案敏感性的价值。方法将2014年9月~2016年9月在我科治疗的57例声门上型喉鳞状细胞癌患者纳入研究。所有患者接受2个周期的TPF方案诱导化疗,并将患者按照疗效分为化疗敏感组和化疗耐受组。采用免疫组化对所有患者病理标本中MAPK10及Itga6表达进行检测,并分析mRNA与蛋白表达一致性,以及蛋白表达与患者临床病理资料之间的关系。结果 MAPK10在敏感组中表达率较高为90.48%,Itga6在耐受组中表达率较高为83.33%,两组之间差异有统计学意义。MAPK10在耐受组中表达水平低于敏感组,Itga6 mRNA高于敏感组,差异有统计学意义。一致性检验表明MAPK10及Itga6在耐受组之中mRNA表达及与蛋白表达一致性较高。MAPK10及Itga6的表达与年龄、性别、肿瘤直径无关;MAPK10的表达与临床分期、病理分级相关,阳性率越低患者病情越重。Spearman等级相关分析结果表明MAPK10与Itga6表达水平之间存在负相关关系,在敏感组喉癌组织中MAPK10与Itga6表达水平之间同样存在负相关关系。结论 MAPK10在对化疗耐受的喉癌组织中表达下调,而Itga6表达上调,且表达水平之间存在负相关关系,因此有可能成为喉癌诱导化疗疗效预测的分子标志物。

皮肌炎患者外周血CD4^+ T细胞中TGF-β_1、IL-10 mRNA的表达及临床意义

目的:检测活动期皮肌炎(DM)患者外周血CD4+T细胞转化生长因子β1(TGF-β1)、白介素10(IL-10)的表达以及血清肌酸激酶(CK)含量。初步探讨CD4+T细胞TGF-β1、IL-10在DM发病机制中的作用。方法:收集15例活动期DM患者和15例健康对照者外周血,采用免疫磁珠分离获得CD4+T细胞,通过逆转录聚合酶链法(RT-PCR)检测出各组TGF-β1、IL-10表达水平。观察CD4+T细胞中TGF-β1、IL-10表达与血清CK含量的相关性。结果:活动期DM患者外周血CD4+T细胞中TGF-β1和IL-10表达量显著高于健康对照,血清CK含量与CD4+T细胞TGF-β1表达量呈中度正相关。结论:活动期DM患者外周血CD4+T细胞中TGF-β1和IL-10 mRNA表达显著升高,可能与DM疾病发生有关。

miR-365-3p抑制CDK-10表达对口腔鳞癌细胞增殖、凋亡和周期的影响

目的探讨miR-365-3p通过周期素依赖性激酶-10(CDK-10)影响口腔鳞癌(OSCC)细胞增殖、凋亡与周期的机制。方法收集在本院就诊的40例口腔鳞癌患者肿瘤组织样本及癌旁正常组织样本,检测其miR-365-3p和CDK-10表达水平。随机将人舌鳞癌细胞系(CAL-27)分为空白组、对照组及miR-365-3p三组。对照组与miR-365-3p组分别转染miR-365-3p NC与miR-365-3p mimic,空白组不进行转染。采用实时荧光定量PCR(qRT-PCR)法检测miR-365-3p表达,CCK-8法及流式细胞术(FCM)检测细胞周期和细胞凋亡。qPCR和Western blot检测CDK-10的表达水平。结果相较于对照组与空白组,miR-365-3p在miR-365-3p组的表达水平显著升高(P<0.05),细胞增殖指数显著降低(P<0.05);细胞凋亡水平显著升高(P<0.05)。转染72 h后,相较于空白组与对照组,miR-365-3p组G1期比例显著降低(P<0.05),S期+G2期比例则显著增加(P<0.05)。转染48 h与72 h后,相较于空白组与对照组CDK-10蛋白在miR-365-3p组的相对表达量显著降低(P<0.05)。结论miR-365-3p在口腔鳞癌细胞中的高表达能够抑制CDK-10表达,从而抑制细胞增殖,调节细胞周期,促进细胞凋亡。

王浆酸对人结肠癌SW620细胞增殖的抑制作用及其网络药理学分析

目的:基于网络药理学探讨王浆酸(10-HDA)对人结肠癌SW620细胞增殖和迁移的影响,阐明其相关分子机制。方法:利用中药系统药理学(TMSCP)数据库和中医药综合数据库(TCMID)以关键词“蜂王浆”进行检索得到10-HDA等活性成分及对应靶点,采用Swiss Target Prediction数据库预测小分子靶点。采用Gene Cards数据库和在线人类孟德尔遗传(OMIM)数据库以关键词“Colon Cancer”获得靶点,利用String数据库和Cytoscape 3.8.0软件构建蛋白-蛋白互作(PPI)网络,筛选核心靶点;利用Metascape数据库对基因本体(GO)功能富集和京都基因与基因组百科全书(KEGG)信号通路进行富集分析,筛选特有成分10-HDA进行体外活性实验。将生长状态良好的人结肠癌SW620细胞分为对照组和不同剂量(1、5、10、15和20 mmol·L^(-1))10-HDA组,采用MTT法检测各组细胞活性并计算细胞存活率。SW620细胞分为对照组、低剂量(5 mmol·L^(-1))10-HDA组、中剂量(10mmol·L^(-1))10-HDA组和高剂量(15mmol·L^(-1))10-HDA组,Hoechst33342染色法观察各组细胞形态表现,细胞划痕实验检测各组细胞划痕愈合率,流式细胞术检测各组不同细胞周期细胞百分率,生化法检测各组细胞中总抗氧化能力(T-AOC)和超氧化物歧化酶(SOD)活性,Western blotting法检测各组细胞中B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)、含半胱氨酸的天冬氨酸蛋白水解酶9(Caspase-9)、糖原合成酶激酶3β(GSK3β)、β-连环蛋白(β-catenin)和细胞周期蛋白D1(Cyclin D1)蛋白表达水平。结果:TCMSP数据库筛选得到蜂王浆6种活性成分,10-HDA治疗结肠癌核心靶点28个。GO功能富集分析主要涉及细胞增殖和细胞凋亡等信号通路;KEGG信号通路富集分析涉及细胞周期、前列腺癌、细胞衰老和p53等信号通路,GSK3β/β-catenin信号通路与细胞周期有密切关联。与对照组比较,5、10、15和20 mmol·L^(-1)10-HDA组细胞存活率呈剂量依赖性降低(P<0.05或P<0.01)。与对照组比较,不同剂量10-HDA组细胞中凋亡细胞数明显增多,细胞划痕愈合率明显降低(P<0.05或P<0.01),中和高剂量10-HDA组细胞中S期细胞百分率明显升高(P<0.05或P<0.01),不同剂量10-HDA组细胞中T-AOC和SOD活性明显降低(P<0.05或P<0.01)。与对照组比较,低剂量10-HDA组细胞中Bcl-2蛋白表达水平明显降低(P<0.01),GSK3β蛋白表达水平明显升高(P<0.05);与对照组比较,中和高剂量10-HDA组细胞中Bax、Caspase-3、Caspase-9和GSK3β蛋白表达水平明显升高(P<0.05或P<0.01),Bcl-2、β-catenin和Cyclin D1蛋白表达水平明显降低(P<0.01)。结论:10-HDA可明显抑制结肠癌细胞增殖和迁移,并可促进结肠癌细胞的凋亡和氧化水平,其作用机制可能与激活GSK3β/β-catenin信号通路有关。

Rho激酶联合生脉注射液对浅二度烧伤患者外周血TNF-α、IL-10、ICAM-1及CD11/CD18水平影响

目的探讨Rho激酶联合生脉注射液对浅二度烧伤患者外周血肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、白细胞介素-10(interleukin-10,IL-10)、细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)及粘附分子CD11/CD18水平的影响。方法收集三峡大学仁和医院收治的浅二度烧伤住院患者48例,根据用药不同分为对照组和实验组,每组各24例,2组患者均给予抗伤口感染、保暖、抗休克、止痛、促进伤口愈合等对症治疗,对照组患者在此基础上给予Rho激酶糖治疗,实验组在对照组的基础上给予生脉注射液,连续治疗2周。治疗结束后,对患者血清TNF-α、IL-10、ICAM-1及CD11/CD18水平进行比较。结果与治疗前相比,治疗后2组患者的血清TNF-α、IL-10、ICAM-1、CD11/CD18水平均显著降低(P<0.05)。治疗后,与对照组相比,实验组患者的TNF-α、IL-10、ICAM-1、CD11/CD18水平较低(P<0.05)。结论 Rho激酶联合生脉注射液能够显著降低浅二度烧伤患者的TNF-α、IL-10、ICAM-1以及CD11/CD18水平,对临床有指导意义。

泛素特异性蛋白酶10在低氧性肺动脉高压中的表达及对USP10-AMPK信号通路的调控作用

目的研究泛素特异性蛋白酶10(UPS10)在低氧性肺动脉高压(PAH)中表达的意义及对USP10-单磷酸腺苷活化蛋白激酶(AMPK)信号通路的调控作用。方法40只大鼠随机分为4组,空载组与沉默组分别经气管滴入USP10-lentivirus、NC-lentivirus,对照组与模型组滴入等量生理盐水。3 d后除对照组外均建立低氧PAH模型。以PowerLab压力记录分析系统检测各组平均肺动脉压(MPAP)、右心室收缩压(RVSP),计算肺血管管壁相对厚度指数(RTI)、右心肥大指数(RVHI),对比各组MPAP、RVSP、RTI、RVHI。实时荧光定量PCR技术检测USP10、AMPK、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)、内皮细胞型一氧化氮合成酶(eNOS)mRNA,比较各组USP10、AMPK、PI3K、Akt、eNOS mRNA相对表达量。蛋白质印迹法(Weston blot)检测USP10、AMPK、PI3K、Akt、eNOS蛋白及AMPK、PI3K、Akt、eNOS蛋白磷酸化表达情况,对比USP10蛋白相对表达量及p-AMPK/AMPK、p-PI3K/PI3K、p-Akt/Akt、p-eNOS/eNOS蛋白比值。结果沉默组、模型组与空载组大鼠均出现精神状态变差,活动量减少,毛发暗淡,口唇、眼眶发紫,沉默组更为严重。肺动脉血管组织HE染色观察,沉默组、模型组与空载组管壁增厚、管腔变窄,伴有平滑肌和弹力纤维层增厚,沉默组变化更为明显。与对照组比较,其余3组的MPAP、RVSP、RTI、RVHI均增加(P<0.05),沉默组均大于模型组与空载组(P<0.05);与对照组比较,其余3组的USP10 mRNA、蛋白相对表达量及p-AMPK/AMPK、p-PI3K/PI3K、p-Akt/Akt、p-eNOS/eNOS蛋白比值均下降(P<0.05),沉默组均低于模型组与空载组(P<0.05)。结论USP10在低氧性PAH大鼠肺组织中表达低于正常肺组织,且其在肺组织中的表达下调刺激了PAH的发展,推测可能与抑制AMPK/PI3K/Akt/eNOS信号通路有关。